WO2001032928A2

WO2001032928A2 - Methods of determining individual hypersensitivity to an agent

Info

Publication number: WO2001032928A2
Application number: PCT/US2000/030474
Authority: WO
Inventors: Spencer Farr
Original assignee: Phase-1 Molecular Toxicology
Priority date: 1999-11-05
Filing date: 2000-11-03
Publication date: 2001-05-10
Also published as: AU1466001A; WO2001032928A9; WO2001032928A3

Abstract

Methods of identifying hypersensitivity in a subject by obtaining a gene expression profile of multiple genes associated with hypersensitivity of the subject suspected to be hypersensitive, and identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity are disclosed. The gene expression profile of the subject may be compared with the gene expression profile of a normal individual and a hypersensitive individual. The gene expression profile of the subject that is obtained may comprise a profile of levels of mRNA or cDNA. The gene expression profile may be obtained by using an array of nucleic acid probes for the plurality of genes associated with hypersensitivity. The expression of the genes predetermined to be associated with hypersensitivity is directly related to prevention or repair of toxic damage at the tissue, organ or system level. Gene databases arrays and apparatus useful for identifying hypersensitivity in a subject are also disclosed.

Description

METHODS OF DETERMINING INDIVIDUAL HYPERSENSITIVITY TO AN AGENT

TECHNICAL FIELD The invention generally relates to methods, compositions and devices for identifying individuals who are hypersensitive to a given agent.

BACKGROUND OF THE INVENTION Individuals exhibit a high degree of variability in their response to chemicals, including pharmaceutical compounds. A major concern of pharmaceutical manufacturers is that a subset of the patient population may display significant toxic side effects that cannot be predicted from preclinical studies. In many cases this hypersensitivity results in extreme, and even lethal, responses. The incidence of serious and lethal adverse reactions to drags among hospitalized patients in the United States causes at least 100,000 deaths per year. This makes adverse responses to therapeutic drags the fifth main cause of death in the United States.

The existence of a hypersensitive sub-population is usually only discovered after a compound has been broadly prescribed and a population base of sufficient size has had exposure to the compound. These same drags are generally safe for the majority of individuals and most respond favorably to the desired effects of the drags. In many cases, the same drag that may elicit severe toxic responses in a subset of the population is the best drag for the particular disease being treated. For example, clozapine is a very effective drag for treating moderate to severe depression and with the majority of patients shows no toxic side effects at the recommended doses. Yet at the same dose (usually 300 mg), approximately 1% of the patient population develop agranulocytosis, a severe blood disorder. Many compounds have either been withdrawn from the market altogether or severely restricted in use due to severe adverse responses by a subset of the patient population. In some instances, it is known that a subset of the population is hypersensitive, and physicians are advised to be alert for indications of extreme toxic response in such patients. Exemplary compounds include tienilic acid, halothane, dihydrazine, diclofenac,

T A T"l_ Λ fialuridine, carbamazepine, Trovan (frovafloxacin), Seldane (terfenadine), hismanol, dihydrolazine, warfarin, phenytoin, omeprazole, diazepam, haloperidol, perphenazine, perhexiline, phenformin, tolbumamide, penicillin, clozapine, aminopurine, quinidine and remoxipide. Unfortunately, in the vast majority of these cases, there is no way of identifying a hypersensitive individual before prescribing the drags or exposing the worker to the compound. Hypersensitive individuals are discovered the hard way; they exhibit toxic side effects that most people do not. Furthermore, since the mechanisms of toxicity are specific and usually different for each drug or compound, the hypersensitive populations are also different and specific for each drag or compound.

When an approved drag is found to elicit serious toxicity in only a subset of the population the manufacturer is usually required to tightly restrict access to the drag, carefully monitor all patients who receive the drag for toxic side effects, or withdraw it from the market altogether. A high number of compounds also fail in the late stages of development because of serious toxicity in a subset of the clinical trial population. When a drag is found to cause severe toxicity in a sub-population, besides the trauma and pain for such hypersensitive individuals, there is great financial loss inclined by the manufacturer. The cost to the manufacturer of withdrawing or restricting a compound can be billions of dollars in lost market capitalization, legal liabilities and unrecoverable research and development expenses. Adverse reactions are becoming the main challenge for pharmaceutical research and development. (Drag Discovery Today) 4:393-395 (1999). In addition, very effective drags are often pulled from the market and thus become unavailable to those who would benefit greatly from them.

It would save lives, decrease pain and suffering and save pharmaceutical manufacturers and consumers a great deal of money if there were a way to determine in advance which individuals were likely to experience severe toxic responses to a drag.

SUMMARY OF THE INVENTION

Disclosed herein are methods, gene databases, gene anays, protein anays, and devices that may be used to determine the hypersensitivity of individuals to a given agent, such as a drag or other chemical, in order to prevent toxic side effects.

In one embodiment, the invention relates to a method of identifying hypersensitivity in a subject by obtaining the gene expression profile of specific genes associated with hypersensitivity of the subject suspected to be hypersensitive and identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity. The gene expression profile of the subject may be compared with the gene expression profile of individuals who have an acceptable response and compared with other hypersensitive individuals. The embodiment also includes, for example, identifying hypersensitivity to an agent in a subject, where the agent may be a pharmaceutical agent, industrial, household or other chemical or compound. Exemplary pharmaceutical agents are disclosed in Table 1.

The gene expression profile of the subject that is obtained may comprise a profile of levels of mRNA or cDNA. The gene expression profile may be obtained by using an anay of nucleic acid probes complementary to the genes associated with hypersensitivity. The genes used may comprise at least two genes, at least 3, 4, 6, 7, 8, or 9 genes predetermined to be associated with hypersensitivity, and may also comprise at least 5, at least 10, at least 25, at least 50, at least 100, at least 250 or more genes determined to be associated with hypersensitivity.

Genes associated with hypersensitivity and used in this invention may, for example, comprise genes from a variety of different cell types, including, but not limited to, genes from multiple types of tissues, organs or systems or genes from a single type of tissue, organ or system. Exemplary organs and tissues include the liver, kidneys, heart, brain, thyroid, lung, pancreas, muscle, brain, testes, ovaries, spleen, stomach, intestines, colon, rectum, eyes, muscle, skin, and bone. Exemplary types of cells include liver cells such as, Kupfer cells, sinusoidal cells, ito cells, hepatocytes, bile duct epithelial cells, hepatic venule endothelial cells and sinusoidal epithelial cells.

A further embodiment encompasses the expression profile of the genes predetermined to be associated with hypersensitivity where expression of the genes is related to prevention or repair of toxic damage at the nucleotide, protein, macromolecule, organelle, cell, tissue, organ or system level. In another embodiment, the gene expression profile may comprise a profile of protein expression levels, where the proteins are encoded by genes associated with hypersensitivity. The level of expression of the proteins may be directly related to the prevention or repair of toxic damage at the protein, nucleotide, macromolecule, organelle, cell, tissue, organ or system level. An additional embodiment includes protein expression profiles, where the proteins are encoded by genes associated with hypersensitivity, and the expression of the genes is, for example, associated with response to the presence of an agent, such as a toxic agent. Exemplary agents that can induce a characteristic profile of protein expression associated with hypersensitivity include those agents listed in Table 1.

The gene expression profile may be obtained from a sample from the subject, which sample may be from a cell or tissue sample and may comprise cells of different cell types.

For gene expression, the sample may comprise, for example, white blood cells, skin, spinal fluid or organ biopsy material. For protein expression analysis, the sample may comprise, for example, blood, tissue, urine, spinal fluid or serum.

In another embodiment, cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. These primary cell cultures are then exposed to the agent. Co-cultures are also encompassed in the invention and are grown from two or more cell types that reflect, for example, the cell types involved in systemic toxicity. These co-cultures would then be exposed to the agent of interest.

In another embodiment, the gene expression profiles of samples from normal individuals, hypersensitive individuals or cell cultures are determined for individual agents using the methods herein described to determine drag-drug interactions. The gene expression profiles are compared to determine whether the multiple agents, for example two or more agents, elicit the same or similar gene expression profiles in the samples. The expression of the same or similar pattem(s) of toxic response genes for two or more compounds in either normal or hypersensitive individuals, is indicative that a drag-drag interaction, also described as a synergistic toxic effect, can be present if the agents are administered together, for example, during the same time period or in the same dose.

The genes used in the gene expression profile may include, but are not limited to, genes, and the proteins which they encode, which are associated with toxic outcomes affecting the pulmonary system, cardiovascular system, nervous system, digestive system, immune system, reproductive system, endocrine system, vision or skin. Exemplary types of toxicity include cardiotoxicity, blood toxicity, liver (hepatic) toxicity, kidney (renal) toxicity, neural toxicity, skin toxicity, immunotoxicity, and pulmonary toxicity. Exemplary genes associated with specific organ or system toxic outcomes are disclosed in Table 5.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, altered lipid metabolism, altered thyroid function, organ hypertrophy, skin initation, skin sensitization, tumor formation, dementia, inflammation, myelosuppression, peripheral neuropathy, necrosis, signal refractivity, spreading, transformation, retinopathy or optic atrophy.

The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the digestive system or the organs and tissues which comprise the digestive system, for example, the liver, kidneys, colon, bladder, pancreas, stomach, intestines, rectum, or gallbladder.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with exemplary toxic outcomes such as, but not limited to, proteinuria, glomerulitis, nephritis, renal damage, renal failure, liver weight change, cholestasis, pancreatitis, liver steatosis, hyperplasia, fatty liver, jaundice, hepatitis, mutagenesis, or altered bile flow.

The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the pulmonary system or the organs and tissues which comprise the pulmonary system, for example the lungs or trachea.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, lung fibrosis, pulmonary edema or lung airway reactivity.

The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the cardiovascular and circulatory systems or the organs, fluids and tissues which comprise the cardiovascular and circulatory systems, for example, the heart, spleen, arteries, blood vessels, blood or blood cells, including genes associated with toxic outcomes associated with bone manow. The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with exemplary toxic outcomes such as, but not limited to, tachycardia, anhythmia, leukemia, neutropenia, hematological alteration, hypotension, hypertension or agranulocytosis. The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the nervous system or the organs and tissues which comprise the nervous system, for example, the brain, spinal cord or nerves.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, neurodegeneration or neurotoxicity.

The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the immune system or the organs and tissues which comprise the immune system, for example, the thymus, lymph nodes or lymph glands.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, a change in thymic weight or immunosuppression.

The genes used in the gene expression profile may include, but are not limited to genes, and the proteins which they encode, which are associated with toxic outcomes affecting the reproductive system or the organs and tissues which comprise the reproductive system, for example the testes, ovaries, fallopian tubes or uterus.

The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with toxic outcomes such as, but not limited to, teratogenesis, loss of fertility, alteration in sperm count, alteration in testes weight or alteration in testosterone levels. The genes used in the gene expression profile include those genes, and the proteins which they encode, associated with cellular manifestations of toxicity such as, but not limited to, apoptosis, cell adhesion, autophagocytosis, cell division, chemotaxis, cell cycle anest, circadian rhythm, cytokine release, differentiation, de-differentiation, mitochondrial damage, migration, mutation, oncosis, recombination, senescence, peroxisome proliferation, polyploidy, signal refractivity, spreading, transformation or necrosis.

The genes involved, and the proteins which they encode, may also include those associated with a specific ethnic group, sex or age group.

The genes or proteins used in the expression profile may also include the genes, and the proteins or amino acids which they encode, which are selected from the genes disclosed in (or genes comprising sequences disclosed in) Table 3, Table 4, Table 5, Table 6, Table 8, Table 10 and Table 11.

In another embodiment, the method includes obtaining a gene expression profile of genes comprising different cell types, of the subject, determining if the gene expression profile of the subject comprises a pattern of gene expression associated with hypersensitivity to an agent, and withholding that agent from those subjects who are hypersensitive or altering the therapy and closely monitoring the subjects who are hypersensitive for toxic effects.

In another embodiment, a method of identifying a plurality of genes associated with hypersensitivity to an agent is provided, comprising comparing the gene expression profile of cells treated with an agent with the gene expression profile of cells not treated with the agent and identifying genes that have altered expression due to exposure to the agent in the treated cells. The cells may comprise, for example, a number of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent. The cells may also comprise cells from of different cell types where all the cell types are derived from a single type of tissue, organ or system. The organs or tissues from which cell types may be derived include, but are not limited to, the kidneys, liver, lungs, heart, brain, spleen, thyroid, bone, muscle, intestine, stomach, pancreas, testes, ovaries, colon or skin.

The invention also relates to a method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent by comparing the gene expression profile of one or more cell types, for example, at least 2, at least 3, at least

4, at least 5, at least 10, at least 50, at least 100 or at least 250, ofa subject known to be hypersensitive to the agent with the gene expression profile of the cell types in an individual known not to be hypersensitive to the agent and identifying genes from the two or more cell types which exhibit a pattern of differential gene expression associated with hypersensitivity to the agent.

In an alternative embodiment, the method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent comprises comparing the gene expression profile of one or more cell types, for example, at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100 or at least 250, of a subject known to be hypersensitive to the agent before treatment with the agent with the gene expression profile of the one or more cell types of the subject after treatment with the agent and identifying genes from the cell types having a pattern of differential gene expression associated with hypersensitivity to the agent.

In an alternative embodiment, the method of identifying proteins having a pattern of differential protein expression indicative of hypersensitivity to an agent comprises comparing the protein expression profile of one or more cell types ofa subject known to be hypersensitive to the agent before treatment with the agent with the protein expression profile of the one or more cell types of the individual after treatment with the agent and identifying proteins from the cell types having a pattern of differential protein expression associated with hypersensitivity to the agent.

In another embodiment, there is provided an anay for the identification ofa gene expression profile indicative of a hypersensitivity to an agent which comprises gene probes, for example, nucleic acid sequences which comprise a gene sequence associated with hypersensitivity to the agent, associated with the hypersensitivity to the agent. The genes are selected from the genes identified by methods disclosed herein or are selected from those genes disclosed in whole or in part in Table 3, Table 4, Table 5, and Tables 6, 8, 10 and 11. The anay comprises for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 250 different gene probes. Exemplary anays include, for example, gene probes supported on glass slides or nylon membranes with fluorescent or radio labels, amplified fragment length polymorphism (AFLP) methods or Northern Blots. The invention further encompasses a database of genes associated with hypersensitivity to an agent. The genes are those identified by methods disclosed herein or are selected from those genes disclosed in whole or in part in Table 3 and Table 4, Table 5 and Tables 6, 8, 10 and 11. The database of genes may comprise, for example, genes associated with altered lipid metabolism, cholestasis, immunosuppression, pancreatitis, agranulocytosis, tumor formation, teratogenesis, liver steatosis, apoptosis, cell adhesion, autophagocytosis, cell cycle anest, circadian rhythm, cytokine release, differentiation, migration, oncosis, recombination, senescence, signal refractivity, spreading, transformation, peroxisome proliferation, necrosis, glomerulitis, nephritis, anhythmia, hypotension, hypertension, leukemia, neutropenia renal damage, renal failure, pulmonary edema, neurotoxicity or retinopathy. The invention further encompasses a method for identifying individuals who may be hypersensitive to the toxic side effects of drags such as those listed in Table 1 or industrial compounds such as those listed in Table 2.

An additional embodiment includes an apparatus for identifying hypersensitivity in a subject comprising means for obtaining a gene expression profile of a number of genes associated with hypersensitivity of the subject suspected to be hypersensitive; and means for identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity, thereby to identify hypersensitivity in the subject.

Also provided are methods of determining hypersensitivity of an individual to an agent, such as a pharmaceutical drag, such as penicillin, by detecting a gene expression or protein expression profile of the individual, wherein the genes or proteins encoded therefrom are selected from genes listed in whole or in part in Tables 10 and 11. The pattern of expression may be detected in a cell, such as an immune cell, such as a leukocyte, e.g. a lymphocyte.

Also provided are devices for detection of gene expression profiles comprising nucleic acid sequences for detecting expression of the nucleic acids disclosed in the Tables, for example by hybridization. Such devices include, for example, immobilized nucleic acid anays.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating gene expression changes associated with toxicity caused by sfreptozotocin.

Figure 2 is a graph illustrating co-induction of genes for hepatocyte growth factor receptor and glutathione transferase.

Figure 3 is a graph illustrating a portion ofa gene expression profile from heart muscle tissue after exposure to the cardiotoxin, doxombicin.

Figure 4 is a graph illustrating a portion ofa gene expression profile from liver tissue after exposure to the peroxisome proliferation caused by WY 14,643.

Figure 5 is a graph illustrating a portion ofa gene expression profile from liver tissue after exposure to the anti-neoplastic compound, carbamazapine. Figure 6 is a chart illustrating the result of testing for penicillin hypersensitivity amongst a group of penicillin sensitive and penicillin refractive individuals by using a 180 gene penicillin anay. Figure 7 is a chart illustrating the result testing for penicillin hypersensitivity amongst a group of penicillin sensitive and penicillin refractive individuals by using a 20 gene penicillin anay.

Figure 8 is a chart illustrating 20 discriminator genes analyzed for co-regulation.

Figure 9 is a graph illustrating the results of a Taqman® assay in a penicillin sensitive person.

Figure 10 is a graph illustrating the results of a Taqman® assay in a penicillin refractive person.

BRIEF DESCRIPTION OF THE TABLES

Table 1 is a list of pharmaceutical agents which potentially can cause greatly heightened toxic responses in some individuals.

Table 2 is a list of industrial agents which potentially can cause greatly heightened toxic responses in some individuals.

Table 3 is a list of genes, altered expression patterns of which can indicate and render an individual hypersensitive to drugs and chemical agents.

Table 4 is a list of genes, altered expression patterns of which can indicate and render an individual hypersensitive to drags and chemical agents. Table 5 is a list of genes associated with specific manifestations of organ or system toxicity.

Table 6 is a list of genes that can be associated with specific cellular manifestations of toxicity.

Table 7 lists compounds for which gene expression data in either human cells, rats or both has been generated.

Table 8 lists genes whose expression was measured when rats were exposed to the cardiotoxin doxombicin. Table 9 lists cell types in organs of toxicity.

Table 10 lists the characterization of genes which were isolated and sequenced from gel bands.

Table 11 lists the genes that are useful discriminator genes.

DETAILED DESCRIPTION OF THE INVENTION

Provided are methods, compositions and apparatus for identifying hypersensitivity in an individual. In one embodiment, hypersensitivity in a subject is determined by obtaining from the subject a sample from which can be determined the gene expression profile of genes associated with hypersensitivity, and identifying in the gene expression profile the presence or absence of a pattern of gene expression of the genes associated with hypersensitivity, thereby to identify hypersensitivity in the individual.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A

Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Handbook of Experimental Immunology (D.M. Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller & M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); The Immunoassay Handbook (David Wild, ed., Stockton Press NY, 1994); Antibodies: A Laboratory Manual (Harlow et al., eds., 1987) and Methods of Immunological Analysis (R. Masseyeff, W.H. Albert, and N.A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993).

Definition of Terms

As used herein, the terms 'gene', 'polynucleotide', 'nucleotide' and 'nucleic acid' are interchangeable and refer to polynucleotide sequences, which for example, encode protein products and encompass mRNA, cDNA, single stranded DNA, double stranded DNA and fragments thereof. The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. It also may be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, myristylation, acetylation, alkylation, phosphorylation or dephosphorylation. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids) as well as other modifications known in the art.

The terms 'stress gene', 'toxicity gene' and 'toxic response gene' as used herein are interchangeable. A toxic response gene can be defined as a gene whose message or protein level is altered by adverse stimuli. The specific set of genes that cells induce is dependent upon the type of damage or toxic threat caused by the agent and which organs are most threatened. In addition to the up-regulation of genes which respond to specific toxic threat, genes which encode functions not appropriate under conditions of toxic injury may be down-regulated. As used herein, 'toxic outcome' refers to the microscopic or macroscopic symptoms, physiological, morphological or pathological changes which are observed as a result of exposure to an agent. A 'toxic response' as used herein refers to a cellular, tissue, organ or system level response to exposure to an agent and includes, but is not limited to, the differential expression of genes and/or proteins encompassing both the up- and down-regulation of such genes; the up- or down-regulation of genes which encode proteins associated with the repair or regulation of cell damage; or the regulation of genes which respond to the presence of an agent.

A 'gene expression profile associated with hypersensitivity' as used herein refers to the pattern of relative levels of gene expression found to be associated with hypersensitivity. Gene expression profiles may be measured in a sample, such as samples comprising a variety of cell types and may, for example, comprise blood, urine, spinal fluid or serum.

A 'protein expression profile associated with hypersensitivity' is defined as the pattern of relative levels of protein expression where said proteins are encoded by genes determined to be associated with hypersensitivity. For each gene expression profile that is determined, a conesponding 'protein expression profile associated with hypersensitivity' may be determined.

The terms 'up-regulation' and 'induction' are used interchangeably herein and refer to the regulation of gene expression, specifically the turning on of a particular gene(s). Similarly, the terms 'down-regulation' and 'repression' are used interchangeably herein and refer to the suppression of expression of a particular gene(s).

An 'agent' to which an individual is hypersensitive is defined as any substance to which an individual may be hypersensitive and includes, but is not limited to, drags, household chemicals, industrial chemicals and other chemicals and compounds to which individuals may be exposed. 'Hypersensitivity', as used herein, refers to the exaggerated micro- or macroscopic responses of cells, tissues, organs or systems to low or average doses of an agent. These responses may lead to observable symptoms such as dizziness or nausea and can also result in toxic outcomes. Hypersensitivity often results in toxic side effects that are different, in either degree or kind, from the response of the majority of patients at the recommended dose. Hypersensitivity may be characterized by, but is not limited to, the differential expression of genes when compared to the response ofa similar individual who is not hypersensitive to a given agent. Hypersensitive individuals do not have normal gene expression patterns of key toxicologically relevant genes either prior to, or after, exposure to an agent.

'Differential expression' as used herein refers to the change in expression levels of genes, and/or proteins encoded by said genes, in cells, tissues, organs or systems upon exposure to an agent. As used herein, differential gene expression includes differential franscription and translation, as well as message stabilization. Differential gene expression encompasses both up- and down-regulation of gene expression.

The term 'individual' is used interchangeably with the term 'subject' and 'patient' and refers to a mammal, preferably the primate, more preferably the human. The term 'normal individual' or 'normal subject' refers to individuals who exhibit the same or similar dose response curves to an agent as does the majority of the exposed population. Most drags at high enough dosages will cause a toxic response, therefore a 'normal toxic response' refers to the toxic response elicited in an average or normal individual at high doses of an agent. The term 'sample' as used herein refers to samples for testing or analysis. The samples may comprise cells or tissue samples and may be, for example, blood, urine or serum. Samples are characterized in a prefened embodiment by comprising at least two different genes and may also include genes from multiple cell types. Samples include, but are not limited to, those of eukaryotic, mammalian or human origin. As used herein, "anay" and "microanay" are interchangeable and refer to an anangement ofa collection of nucleotide sequences in a centralized location. Anays can be on a solid substrate, such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any permutations thereof. The nucleotide sequences can also be partial sequences from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences. "Penicillin sensitive" refers to individuals who exhibit hypersensitivity to penicillin, for example, a higher than average immune response to penicillin. The immune response can be a hypersensitive response of any type, for example Type I, II, III, or IV. Hypersensitive reactions can include but are not limited to anaphylaxis, skin rash, and hives. Hypersensitive responses also include hypertoxicity. "Penicillin refractive" or "penicillin insensitive" or "penicillin non-sensitive" as used herein refers to individuals who exhibit a normal or non-hypersensitive response to penicillin.

Isolating DNA/RNA from human PBL Nucleotide sequences from human peripheral blood lymphocytes (PBL) are isolated using any number of commercially available kits i.e. from Qiagen, GenHunter, Promega, etc.

In one embodiment, total RNA is isolated from tissue samples using the following materials: Qiagen RNeasy midi kits, 2-mercaptoethanol, liquid N₂, tissue homogenizer, dry ice. It is important to take precautions to minimize the risk of RNA degradation by RNases by wearing gloves at all times and to inhibit RNase activity in work areas and equipment by treating with an RNase inhibitor such as with "RNase Zap" (Ambion® Products, Austin, TX). Autoclaving tips and microfiige tubes does not necessarily eliminate RNase enzymes and its RNA degradation activities. Samples are kept on ice when specified . Protocol which can be used is based on Qiagen® RNeasy® midi kit. This total RNA isolation technique is used for RNA isolation from human PBL and can be modified readily by one of skill in the art to accommodate different amount of human PBLs. The human PBL is preferably taken from circulating blood of a human donor. However, human PBL can also be obtained from lymph nodes, spleen, and other tissues into which human PBLs circulate. If tissue containing human PBL is used, then the tissue needs to be microdissected. One way is to physically break the tissue by placing it on a double layer of aluminum foil which is then placed within a weigh boat containing a small amount of liquid nifrogen. The aluminum foil is folded around the tissue and then the tissue is stmck by a small foil- wrapped hammer to administer mechanical stress forces.

To preserve integrity of the RNA, all tissues are kept on dry ice when other samples are being weighed. A buffer is added to the sample to aid in the homogenization process. An example of a buffer which can be used is RLT (Qiagen®) buffer. The tissue is homogenized using any type of commercially available homogenizer (i.e. IKA Ultra Tunax T25 homogenizer, Virtishear Cyclone 750W rotor/stator homogenizer (Virtis item # 278077, etc.) can be used with the 7 mm microfine sawtooth shaft and generator (195 mm long with a processing range of 0.25 ml to 20 ml, item # 372718). After homogenization, samples are stored on ice until all samples are homogenized. The homogenized tissue sample can then be spun to remove nuclei thus reducing DNA contamination. The supernatant of the lysate is then transfened to a clean container containing an equal volume of 70% EtOH in DEPC treated H₂O and mixed. In the event that a stringy white material comes out of solution, it may then be removed. RNA is isolated by putting the supernatant through an RNeasy spin column, washed, and subsequently eluted.

In another embodiment, DNA or RNA is isolated from human PBLs obtained from a human donor. Generally, lymphocytes can be isolated from blood by separating the blood over a gradient, for example a sucrose gradient or Percoll™ or Ficoll™ gradient. Lymphocytes can be distinguished from non-lymphocyte contaminates by morphology, size and scatter by flow cytometry, or by cell surface markers such as CD2, CD3, CD4, or

CD8. In general, lymphocytes which are cultured in vitro are non-adherent but in some instances, lymphocytes can be adherent or non-adherent depending on several factors, for example, activation state of lymphocytes, receptors expressed on lymphocytes, and culture media contents. In some aspects, adherent cells are more problematic than non-adherent cells because of the necessity of an extra step to separate the adherent cells from the tissue culture container. However, a skilled artisan may solve this problem by treating the cells with cold PBS/EDTA solutions or an equivalent and use any number of commercially available kits, for example, from Qiagen or Ambion, to isolate the DNA or RNA from the cells. In one embodiment, total RNA of high quality and high purity can be isolated from cultured cells by using Qiagen RNeasy midi kits and 2-mercaptoethanol. This embodiment is exemplified in Example 2 infra. Precautions should be taken to minimize the risk of RNA degradation by RNases by wearing gloves, treating work areas and equipment with an

RNase inhibitor, for example RNase Zap (Ambion® Products, Austin, TX), and keeping samples on ice. Using a Qiagen® RNeasy® midi kit (50), this total RNA isolation technique can be used for any type of cell, including but not limited to human lymphocytes and cell derived from particular organs such as kidney, liver, lung, breast, neuronal cells, skin, intestine, such as HepG2, Caco-2, MCF-7, Jurkat, Daudi, HL-60, MCL-5, SKBr-3,

SKOV-3, PC-3, WISH, and HeLa.

To practice this embodiment, cells are checked under the microscope to confirm viability. Cells are then dosed with an agent, which can be a drag, chemical, or pharmaceutical composition, when they reach confluence. In a prefened embodiment, the cells are at least about 20% confluent, more preferably at least about 40% confluent, even more preferably at least about 60% confluent, and even more preferably about 80% confluent. It is preferable to avoid isolating RNA from flasks that have reached 100% confluence because the cells are no longer growing in log phase.

The adherent cells are washed and freshly prepared buffer, for example RLT buffer (RLT buffer requires the addition of 10 μl beta mercaptoethanol for each 1.0 ml RLT), is added directly to the cell culture flask. The amount of RLT buffer differs with tissue container size. Enough RLT buffer is added to cover the surface area in which the adherent cells are growing such that most of the adherent cells come into contact with the RLT buffer. In one embodiment, T-75 flasks receive about 3 ml RLT buffer and T-175 flasks receive about 5 ml RLT buffer. It is preferable to lightly agitate the flasks at this point. Cells exposed to RLT buffer become a gelatinous layer. The cells are allowed to sit for 4 minutes, then fluid is withdrawn and is placed into and RNase-free tube. An equivalent volume of 70% ethanol is added to each tube and vortexed to distribute evenly. In the event that a precipitate with a string-like appearance forms, it is acceptable to remove and discard this string-like precipitate. The fluid is applied to a spin column, centrifuged, and the column is washed and subsequently eluted for RNA samples. The elution can be precipitated using the LiCl precipitation protocol and resuspended in RNA storage buffer for future storage. The yield can be between 200-400 μg of total RNA from a T-75 flask with greater than 50% confluency.

The isolated DNA or RNA is amplified to generate a product which can be attached to a subsfrate. In a prefened embodiment, the substrate is a solid subsfrate (i.e. glass slide). The amplification process involves using primers which have a reactive group (i.e. amine group or derivative thereof) on one end of the primer, which is incorporated into the amplification product. One example of reactive primers that can be used is Amine Primers from Synthegen. The gene fragments which are attached to the glass slide can vary in length. The more nucleotides ofa gene that are in the anay, the tighter the binding and the greater the specificity in binding can occur. However, it is important to consider that longer fragments are more difficult to amplify and may contain point mutations or other enors associated with amplification. Therefore, the desired length ofa gene or a fragment thereof that is to be included in the anay should take into consideration the balance between a high specificity of binding obtained with a long (i.e. >1 kb) gene sequence with the high mutational rate associated with a longer fragment. The gene fragments attached to the glass slide are at least about 50 base pairs (bp) in length, more preferably at least about 100 bp in length, more preferably at least about 200 bp, even more preferably at least about 300 bp, even more preferably at least about 400 bp, even more preferably at least about 500 bp in length. In a prefened embodiment, the gene fragments are about 500 bp in length. The region of a gene that is used to attach to a solid substrate to generate an anay can be any portion of the gene, coding, non-coding, 5' end, 3' end, etc. In a prefened embodiment, about 500 base pairs of the 3' end of canine gene related to toxicological responses are selected to be included in an anay.

Several techniques are well-known to a skilled artisan for attaching a gene or a fragment thereof to a solid substrate such as a glass slide. One method is to attach an amine group, a derivative of an amine group, another group with a positive charge or another group which is reactive to one end of a primer that is used to amplify a gene or a gene fragment to be included in the anay. Subsequent amplification ofa PCR product will then incoφorate this reactive group onto one end of the product. The amplified product is then contacted with a solid substrate, such as a glass slide, which is coated with an aldehyde or another reactive group which will form a covalent link with the reactive group that is on the amplified PCR product and become covalently attached to the glass slide.

Other methods using amino propryl silicane surface chemistry are disclosed by Corning Company at <http://www.cmt.coming.com.> other methods for making microaπays which are readily accessible at <http://cmgm.stanford.ecu pbrownl>

In one embodiment of the invention, fluorescence-labeled single strand (or "first strand") cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microanay slides spotted with DNA specific for toxicologically relevant genes. This is exemplified in Example 8-14. The materials needed to practice this embodiment are: total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice. The Cy™ dyes may be obtained from Amersham. The embodiment may also be practiced with equivalents of the materials listed above, for example, Superscript II may be replaced with an equivalent enzyme and Cy5 and Cy3 may be replaced with another fluorescent dye.

In one embodiment, a discrete amount of RNA, for example 20μg of total RNA or 2μg of mRNA, is used to generate cDNA. In a prefened embodiment, the volume is no more than 14 μl. If RNA is too dilute, the samples are concentrated to a volume of less than 14 μl in a centrifuge with vacuum (i.e. Speedvac) without heat. The Speedvac should be capable of generating a vacuum of 0 Milli-Ton so that samples can freeze dry under these conditions. It is preferable for the tubes containing RNA to be kept on ice to avoid RNA degradation until the next step is ready to proceed. Following standard techniques well-known in the art, cDNA samples are amplified from RNA templates. A mixture of fluorescent dyes is made for labeling the cDNA samples. A variety of dyes can be used. In one embodiment, Cy3 dye, which is pink-red, and Cy5 dye, which is blue, are used. The Cy dyes are light sensitive, therefore, any solutions or samples containing Cy-dyes should be kept out of light, i.e. cover with foil. Example 9-12 discloses prefened mixtures and methods of using Cy3 and Cy5 dyes for labeling cDNA samples and purification steps therewith.

In an embodiment wherein the sequences of toxicologically relevant genes are not known and canine cells are divided into two groups, untreated and treated, to identify toxicologically relevant genes as disclosed infra, Cy3 dye mixture is incubated with the cDNA of each treated sample and Cy5 dye mixture is incubated with the cDNA of each confrol sample. Following the methods disclosed in Example 9, a visible pellet can be seen which is pink/red for cDNA incubated with Cy3 and blue for cDNA incubated with Cy5. It is recommended that the tubes are centrifuged at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the cDNA sample (or cDNA probe) is seen spread on one side of the tube instead of a tight pellet. If the pellet is white (no pink/red or blue), it is likely that the reaction has not occuned to maximal efficiency. Purification of fluorescent probes

Purification of fluorescence-labeled first strand cDNA probes can be achieved in one embodiment using the following materials: Millipore MAHV N45 96 well plate, v- bottom 96 well plate (Costar), Wizard DNA binding Resin, wide orifice pipette tips for 200 to 300 μl volumes, isopropanol, and nanopure water. It will be evident to a skilled artisan that equivalent products may be substituted in other embodiments, i.e. other types of tissue culture plates, binding resin from other commercially available sources, for example Qiagen. Plate alignment during centrifugation is important since misaligned plates can lead to sample cross contamination and/or sample loss. In one embodiment, probes are purified by binding to a resin. The binding resin can be obtained by itself or from a kit provided by any number of commercial sources, i.e. Qiagen, Promega, etc.

Fluorescence Readings of cDNA Probe

Incorporation of fluorescence into cDNA probes can be achieved by using a number of methods. In one embodiment, the following material is used: 384 well, 100 μl assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent). Prior to use as a cDNA probe in hybridization, cDNA probes are purified and concentrated as exemplified in Example 10.

It is preferable that a consistent amount of cDNA is pipeted into the plate wells because readings can vary with volume. Controls or identical samples can be pooled at this step, if required or desired. The Cy-3 and Cy-5 fluorescence are analyzed using a fluorimeter, luminometer, flow cytometer, or any equivalent device which can detect different fluorescent dyes at different wavelengths. In a prefened embodiment, the Wallac 1420 workstation programmed for reading Cy3-Cy-5 is used. A typical range for Cy-3 (20μg) is 250-700,000 fluorescence units. A typical range for Cy-5 (20μg) is 100-250,000 fluorescence units. Prefened settings for the Wallac 1420 fluorescence analyzer are as follows: Cy3

CW lamp energy = 30445 Lamp filter = P550 slot B3 Emission filter= D572 dysprosium slot A4

Emission aperture = normal Count time = 0.1 s

CW lamp energy = 30445

Lamp filter = D642 samarium slot B7 Emission filter= D670 slot A8 Emission aperture = normal Count time = 0.1 s

After detection of the cDNA probes, it is important to concenfrate the cDNA probes so that they can be resuspended in hybridization buffer at an appropriate volume for hybridizing to the anay. Internal normalization is achieved by taking into consideration the ratio of Cy5 fluorescence to Cy3 fluorescence in the treated and untreated canine cell groups, respectively.

Microanay Hybridization

Hybridization of labeled cDNA probes to single stranded, covalently bound DNA target genes on glass slide microaπays can be accomplished by a variety of methods. In one embodiment, exemplified in Example 7, the following material are used: formamide,

SSC, SDS, 2 μm syringe filter, salmon sperm DNA, hybridization chambers, incubator, coverslips, parafilm, and heat blocks. It is preferable that the anay is completely covered to ensure proper hybridization. Hybridization buffer is prepared with consideration towards stringency. Stringency can be varied by increasing or decreasing the amount of SSC and detergent (i.e. SDS, Triton, etc.). Stringency can also be varied by the temperature at which the hybridization occurs. A higher temperature tends towards high stringency conditions. A skilled artisan can determine, in a stepwise fashion, the stringency of the hybridization buffer desired. Clean slides and coverslips are desirable and can be obtained using N₂ stream. Hybridization buffer is added to dried probe and mixed in the dark at room temperature and then brought to a higher temperature in a heat block. Each probe can remain in a heat block until it is ready for hybridization. The probe is applied to a slide or to a coverslip and then covered with slide. It is highly preferable to avoid the material at the bottom of the tube and to avoid generating air bubbles. This may mean leaving some residual volume in the pipette tip. Slides are then placed in a hybridization chamber, wrapped to prevent the liquids from desiccating. One problem that can occur with overly dried slides is increased fluorescence on the edge of the spot containing the target gene fragment to which the labeled cDNA probe binds. In an alternative, the hybridization chamber can have a built-in humidity gauge to avoid desiccation of the slides.

In a prefened embodiment, the slides are placed in a 42°C humidity chamber in a 42°C incubator for 18 to 24 hours. It is preferable to avoid probes or slides sitting at room temperature for long periods.

Post-Hybridization Washing

To obtain single stranded cDNA probes on the anay, all non-specifically bound cDNA probe should be removed from the anay. In one embodiment, removal of all non- specifically bound cDNA probe can be accomplished by washing the anay using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. Equivalents of SSC and SDS may also be used as substitutes. It is highly preferable that great caution be used with the standard wash conditions since deviations can affect data significantly.

In one embodiment, glass buffer chambers and glass slide holders are filled with heated SSC buffer with sufficient volume to submerge the microanays. It is important to exercise caution in heating of the SSC buffer since a high temperature may strip off the probes, preferably the temperature is at most about 60°C, more preferably at most about 50°C, even more preferably at most about 40°C, and even more preferably at most about 35°C. A skilled artisan can vary the concentration of SSC in the buffer according to the stringency desired. The slides are placed in buffer which may contain SSC and/or detergent (i.e. SDS, Triton, etc.) and the coverslips are dislodged and fall off the slide within several minutes of submersion. In the event that the coverslips do not fall off within several minutes of submersion, very gentle agitation may be administered to the chamber in which the wash is being conducted to dislodge the coverslips. The slides with the hybridized probes are subjected to several rounds of washes with different conditions. In one embodiment, a detergent (i.e. SDS) is added to the wash buffer in different concentrations and the slides are washed in this buffer before a final wash in nanopure water. The slides are dried in a manner that will minimize background signal of the array.

A prefened method of drying is to use a folded paper towel underneath the slide and a gently dabbing motion on the slide with a tissue. It is important that the slides do not air dry since this will lead to increased background.

Gene Expression Profiles

The pattern of gene expression characteristic of hypersensitivity is predetermined, and is, for example, provided in a database. By comparing the gene expression profile of the subject with the predetermined pattern of gene expression of multiple genes characteristic of hypersensitivity, the hypersensitivity of the subject can be conveniently and rapidly determined. Advantageously, the invention provides a large number of predetermined gene expression patterns of genes associated with hypersensitivity, for example in a database, so that a large number of genes can be rapidly analyzed and compared in the subject. Analysis of information about expression of a wide spectrum of genes associated with hypersensitivity facilitates the rapid determination of hypersensitivity of a subject to an agent, or multiple agents.

For example, the differential gene expression profile associated with a given agent can be determined for a given agent using, for instance, eukaryotic or mammalian cells or cell lines or animal models and exposing a population of the eukaryotic or mammalian cells or cell lines or animal models to an agent and comparing their gene expression to the same type of eukaryotic or mammalian cells or cell lines or animal models from an untreated population to determine the gene expression profile associated with hypersensitivity. Hypersensitivity to an agent, for example, a pharmaceutical drag or household, industrial or other chemical, can be rapidly determined with samples from an individual or group of individuals by treating the sample(s) with an agent and comparing the gene expression profile with the gene expression profile associated with hypersensitivity determined previously for a particular agent and, for instance, stored in a database and accessed and compared with associated software.

Table 1 lists approximately 200 drags sold in the U.S. and Europe. There are individuals who are hypersensitive to the toxic side effects of each of these drags. Table 2 lists at least 100 major industrial chemicals for which there is documented evidence of toxicity due to occupational exposure. For each of these chemicals there are individuals whose toxic response is heightened compared to the majority of the population.

In a prefened embodiment, multiple genes are analyzed. Preferably, the number of genes, associated with hypersensitivity, whose expression levels are determined and which comprise the gene expression profile is large; for example, one or more, at least 2, at least 3, at least 4, at least 5, at least 10, at least 50, at least 100, or at least 250. The present invention also encompasses gene expression profiles where the number of genes is greater than 400, 500, 600 or more.

In another embodiment, the genes, whose expression levels comprise the gene expression profile, are drawn from a variety of cell types.

For example, the genes, whose expression levels comprise the gene expression profile, are drawn from cells of a number of different tissues or organs.

In another embodiment, cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. These primary cell cultures are then exposed to the agent. Cell cultures established from the appropriate tissues of hypersensitive individuals are more sensitive to the toxic effects of the agent than cultures established from normal individuals. This hypersensitivity is reflected in the gene expression patterns elicited from the cell cultures. In another embodiment, cells or tissues derived from an individual are used to establish primary cell cultures, for example fibroblasts, hepatocytes, and other examples known in the art. Co-cultures would be grown from two or more cell types that reflect the cell types involved in systemic toxicity. These co-cultures are then exposed to the agent of interest. Cell co-cultures established from the appropriate tissues of hypersensitive individuals are more sensitive to the toxic effects of the compound than co-cultures established from normal individuals. This hypersensitivity is reflected in the gene expression patterns elicited from the cell co-cultures.

In another embodiment, the gene expression profile consisting of the expression levels of multiple genes includes genes drawn from a single cell, tissue or organ type, and the profile is examined to determine the association of the gene expression profile with hypersensitivity.

In addition to the determination of absolute levels of expression for the genes in the gene expression profile associated with hypersensitivity, the relative expression levels of two or more genes in the gene expression profile associated with hypersensitivity can be determined and can be relevant to a determination of hypersensitivity. Hypersensitive individuals will have profiles of expression of relevant toxicity genes that are distinct from individuals who are not hypersensitive.

In another embodiment, gene expression profiles from normal individuals, hypersensitive individuals or cell cultures are established for individual agents to determine possible toxic drag-drag interactions when patients (normal or hypersensitive individuals) are treated with multiple drags. There are hundreds of combinations of compounds that are more toxic when taken together than when taken singly. Usually these toxic drag-drug interactions are discovered as clinical manifestations once the drags reach market. Examples of compounds that cause severe toxicity when taken together include cyclosporin A and trimethoprim, Walworth et al. (Lancet) 1 :336(1983); and Clonidine and Tricyclic antidepressants such as Amoxapine. Briant et al. (Br J Pharmacol) 46:563(1972). The expression of the same pattern of toxic response genes for two or more compounds in either normal or hypersensitive individuals, indicates that the two or more compounds, taken together, will often show a synergistic toxic effect. Gene expression profiles for each compound, determined in vitro or in vivo, allows prediction of the severe toxicity if the two compounds were taken together. In another embodiment, the gene expression profile of genes associated with certain disease states is analyzed. Normal individuals can become temporarily hypersensitive to the toxicity of certain drags because of disease states. Hypersensitivity is present in normal individuals when toxic defense mechanisms are temporarily compromised. For example, an individual who suffers from AIDS-induced immunosuppression will be hypersensitive to the toxic effects of immunosuppressive compounds such as cyclosporin A. An individual suffering from pulmonary edema due to viral infection will be temporarily hypersensitive to compounds such as bleomycin which elicit pulmonary edema as a toxic side-effect.

In another embodiment, the method includes obtaining a protein expression profile of a number of proteins encoded by genes of the subject, determining if the protein expression profile of the subject comprises a pattern of protein expression associated with hypersensitivity to an agent, and withholding the agent from those individuals or altering the therapy or dosage and closely monitoring the individual for toxic effects. In addition, a method of identifying a number of genes associated with hypersensitivity to an agent is provided comprising comparing the protein expression profile, where the proteins are encoded by the genes identified as associated with hypersensitivity to the agent, of cells treated with the agent with the protein expression profile of cells not treated with the agent and determining proteins that have altered expression due to the exposure to the agent in the treated cells. The cells may comprise, for example, a variety of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent, and the protein encoded by gene. An additional embodiment includes a method of identifying a number of genes associated with hypersensitivity to an agent which comprises comparing the protein expression profile, where the proteins are encoded by the genes identified as associated with hypersensitivity to the agent, of cells freated with the agent with the protein expression profile of the same type of cells from the same subject not treated with the agent and determining proteins that have altered expression due to exposure to said agent in the treated cells. The cells may comprise, for example, a variety of different cell types and each cell type may comprise a gene associated with hypersensitivity to the agent, and the protein encoded by the gene.

In a further embodiment, the gene expression profile of multiple genes associated with cellular response to toxic agents are analyzed to determine the association with hypersensitivity of the genes in the profile.

Using the methods, compositions and devices disclosed herein, rapid, accurate and inexpensive tests of an individual can be conducted in order to confirm whether the individual is hypersensitive to an agent. For example, an individual can be screened for hypersensitivity to a drag before the drug is administered. Such screenings avoid incidents of hypersensitivity in individuals to whom a drug might otherwise be administered. Alternately, the drag can be given in lower doses to hypersensitive individuals and/or those individuals considered at risk may be closely monitored for adverse reactions to the agent. Avoiding exposing hypersensitive individuals to any given drag or compound, or to a higher than necessary dose or level of the drag or compound, provides cost savings to manufacturers who may produce the drag or compound with an assurance that hypersensitivity reactions will be avoided. Those who are not hypersensitive may safely receive the drag or compound and receive its benefits, while those who are hypersensitive may safely avoid the drag or be prescribed a different drug or in the case where the toxicity is due to exaggerated pharmacological effects, a smaller, but just as effective dose. The invention also encompasses using the methods, composition and devices disclosed herein for rapid, accurate and inexpensive tests that can be used, for instance, to determine the causative agent in an individual exhibiting symptoms consistent with or indicative of a toxic response or hypersensitivity to various agents. By ascertaining the gene profile of a number of genes associated with particular cells, tissues, organs or systems, the agent eliciting the toxic response or hypersensitivity may be determined and thereon avoided. In one embodiment, gene expression analysis might be used to determine the nature of the toxic insult and thus provide treatment. For example, analysis of expression of tox-response genes might aid in the effective diagnosis and treatment of an unconscious child suspected of having been inappropriately exposed to a drag or chemical agent. Gene expression patterns could be useful in determining if the unconscious state were the result of exposure to a soporific agent or one that inhibited mitochondrial function, the treatments of which would be quite distinct.

Exemplary genes associated with hypersensitivity whose expression may be screened in order to determine hypersensitivity are provided in whole or in part in Tables 3, 4, 5, 6, 8, 10 and 11. Also provided herein are methods of identifying genes associated with hypersensitivity.

Genes

Tables 3, 4, 5, 6, 8, 10 and 11 provide a list of exemplary genes from which genes associated with hypersensitivity to a particular agent may be selected. Genes selected from Table 3 and Table 4 are responsive to toxic stimuli and important to the defense or repair of toxic damage. Individuals with significantly altered expression levels of two or more of the genes in Tables 3, 4, 5, 6, 8, 10 and 11 can also show different toxic responses from normal individuals. For a given agent, the expression profile of two or more genes, for example, selected from Tables 3, 4, 5, 6, 8, 10 and 11 can be obtained from a cell, tissue or organ and, a pattern of gene expression predetermined to be associated with hypersensitivity can be established. Genes such as those selected from Tables 3 and 4 are evaluated for differential gene expression, for example in the major toxic target organs in humans and/or rats and mice. Examples of genes in which differential expression is indicative of toxicity or hypersensitivity in specific organs or systems such as liver (hepatic), kidney (renal), lung (pulmonary), central nervous system (neural), heart (cardio) and immune system are shown in Table 5.

As an example, Figure 1 shows the pattern of gene expression of approximately 250 genes in the liver when the subject received a relatively high dose of streptozotocin. Samples, including for instance, blood, urine, serum or tissue, from individuals known to be hypersensitive to streptozotocin can be obtained after the subject is treated with streptozotocin. Alternately, for example, samples may be from untreated individuals known to be hypersensitive to streptozotocin and the samples may then be treated in vitro with streptozotocin. The samples are then examined to identify genes associated with hypersensitivity. This may show, for example, highly exaggerated expression of toxic response genes and/or patterns of induction or repression of genes in treated individuals or upon in vitro treatment of the sample with streptozotocin compared to individuals who are not hypersensitive or sample which is not treated with streptozotocin. As streptozotocin is an example of a bulky alkylating agent, individuals who are hypersensitive to streptozotocin may be tested for hypersensitivity to compounds with similar toxic properties, such as bulky alkylating agents, such as merbarone and carmustine. Genes whose levels of expression change in response to toxic stimuli may be evaluated. Examples of genes with expression changes in response to toxic stimuli are listed in Tables 3 and 4. The genes in Table 3 and Table 4 have been shown to be induced in either cell lines, primary cells, tissues or tissue slices, from human or animal origin. For example, the GADD 153 gene has been shown to be induced in many human cell lines upon exposure to radiation. The environmentally important compound trichloroethylene was recently demonstrated to cause induction of several genes, including c-Myc and c-Jun in mice exposed to low toxic levels for 24 hr. Tao et al.(J Biochem Mol Toxicol) 13(5):

231-7 (1999). In primates, closely related to humans, hyperoxia causes increased expression of the genes encoding thioredoxin and thioredoxin reductase gene expression in lungs. Das et al., (Chest) 116(1 Suppl): 101S (1999).

Many of the genes in Tables 3 and 4 are known to be involved in the prevention or repair of damage to DNA, cells or tissue in response to toxic agents (several examples are provided by the following references: Kegelmeyer et al. (Mol. Carcinog.) 20(3): 288-97 (1997); Koerber et al. (Mol. Reprod. Dev.) 49(4): 394-9 (1998); Kuhn (Nufr. Rev.) 56:11- 9, discussion 54-75 (1998); Lu et al. (Mol. Carcinog.) 20(2): 204-15 (1997); Muhlenkamp et al. (Toxicol. Appl. Pharmacol.) 148(1): 101-8 (1998); Melhus et al. (Biochem. Mol. Biol. Int.) 43(5): 1145-50 (1997); Pentecost (Steroid Biochem. Mol. Biol.) 64(l-2):25-33 (1998);

Quattrochi et al. (Arch. Biochem. Biophys.) 349(2):251-60 (1998); Rout et al. (Cell Calcium) 22(6): 463-74 (1997); Sadekova. et al (Int. J. Radiat. Biol.) 72(6): 653-60 (1997); Yuan et al. (J. Biol. Chem.) 273(7):3799-802 (1998); Zhao et al. (Oncogene) 16 (3):409-15 (1998). Table 6 shows a set of genes associated with specific types of cellular toxicity.

Studies of single gene expression have shown over- or under- expression of certain of these genes, to affect the sensitivity of the cell or organism to toxic stimuli and are described in the art. Advantageously, the expression levels of all of these genes can be measured simultaneously. Individuals hypersensitive to an agent can be identified by measuring the expression patterns of the toxicity genes specific to that agent. Tables 3, 4, 5 and 6 are non-limiting examples of such toxicity genes. Agents

Many compounds are toxic at a high enough concentration. For example, while most individuals might experience extreme tachycardia after receiving a very high dose - 20 times normal - of a drag, they experience no such effects at recommended doses. The hypersensitive individual would experience extreme tachycardia at the recommended dose or at a lower than normal dose. A hypersensitive individual might also experience a qualitatively distinct toxic response to a compound, not just the same response that a normal individual would experience at high doses. For example, the hypersensitive patient might experience extreme dizziness, a side effect not reported by individuals even at high doses.

Agents to which individuals may be hypersensitive, and for which hypersensitivity can be determined, may include, for example, drags, industrial chemicals, household or other chemicals, including those in the workplace. Examples of drugs and industrial chemicals for which a sub-population is hypersensitive are listed in Tables 1 and 2. As a further example, individuals who are employed in manufacturing or other environments which expose them to a variety of agents may be screened for agents to which they might come into contact. Individuals, or for example, a subset of workers, who are hypersensitive to the agents can then be identified. Hypersensitivity to other agents also may also be determined, such agents including, but not limited to biological agents such as naturally occurring organic compounds, including proteins, saccharides and lipids.

Exemplary pharmaceutical agents include, for example, tienilic acid, halothane, dihydrazine, diclofenac, fialuridine, carbamazepine, Trovan™ (trovafloxacin), Seldane™ (terfenadine), hismanol, dihydrolazine, warfarin, phenytoin, omeprazole, diazepam, haloperidol, perphenazine, perhexiline, phenformin, tolbumamide, penicillin, clozapine, aminopurine, quinidine and remoxipide. Table 1 lists additional agents for which there are individuals who demonstrate hypersensitivity. Examples of other chemicals include industrial chemicals, such as paint, volatile organic compounds (VOCs), solvents, adhesives, pesticides, herbicides, perfumes, aerosols, cleaning compounds and synthetic polymers such as textiles.

Identification of Genes

Genes initially suspected of being associated with hypersensitivity and hence potentially useful in the present invention are identified, for example, by conducting extensive literature searches; investigating known biochemical pathways with toxicological relevance; and measuring gene expression from toxin-exposed animals, humans or cell lines. Hypersensitivity to an agent, such as a drag, may also be determined based on the ability to identify the underlying molecular basis for the toxicity of specific drags. Hypersensitivity can also be determined by examining the gene expression of hypersensitive and normal individuals.

In one embodiment, methods are provided wherein literature reports on the expression levels of single genes in response to a single agent are collected, for example, in a database, and then analyzed to establish patterns of expression that can be conelated to hypersensitivity. Advantageously, large amounts of data can be collected and analyzed, for example by software means. For example, Matrix Express , and Chem Profiler (Phase- 1 Molecular Toxicology, Santa Fe, NM) accommodate capture and analysis of gene expression profiles. For example, it allows identification of induced genes from the total set of genes measured using a number of criteria; for example, statistical significance, twofold, and 1.5 X the standard deviation. The software also allows the search of other profiles and determines the commonality between subsets, ranking profiles by several measures of similarity, for example, using all or a subset of the genes. Experiments include both in vivo and in vitro responses to agents, for example, the exposure of eukaryotic, mammalian or human cells, and animals to agents listed in Table 7. One ultimate benefit of this exercise is to reduce the need for animal testing. Each agent is tested at several concentrations and time points.

The toxicology of an agent is evaluated by measuring toxic insult by detecting observable changes in organ or system appearance and/or function, at the micro- or macroscopic levels. For example, a drag may cause changes in fatty acid metabolism in liver hepatocytes. This in turn causes observable changes in liver appearance, such as a specific toxicological outcome refened to as fatty liver. In order for cells, and thus tissues and organs, to undergo observable morphological changes due to toxic insult, they generally express a subset of genes differently than untreated cells. Thus, manifestations of toxic injury frequently require differential gene expression. Such genes that are differentially expressed in response to toxic injury are evaluated for use as genes associated with hypersensitivity in accordance with the present invention.

Thus, the expression of genes that are differentially expressed in total across cell, organ and tissue types in humans, in particular in response to toxic insult, may be evaluated to determine which genes have expression that is linked to hypersensitivity in an individual.

Individuals who do not properly express the appropriate toxicity response genes for a specific compound will be hypersensitive to the toxic effects of that compound.

Organs are composed of tissues, which in rum are composed of various cell types. There is a core set of genes whose products are involved in functions essential to all cells, and whose expression is shared by most human cell types. In addition to these common core genes, each cell type expresses a set of genes that is unique to that cell type. When animals, including humans, are exposed to chemicals that cause damage to one or more organs, cells that comprise those organs attempt to mitigate or repair that damage by turning on genes that encode toxic-damage defense or repair proteins. The specific set of genes that cells induce is dependent upon the type of damage or toxic threat caused by the compound and upon which organs are most threatened. In addition to the genes that are induced to deal with the specific toxic threat, there may be genes which encode functions that are not needed nor appropriate under conditions of toxic injury. Therefore, both the up- and down-regulation of genes can be measured in order to understand the molecular response to that compound, and the linkage of gene expression to hypersensitivity. The pattern of differential gene expression within the toxic target organs can be limited to a relatively small number of genes, and may be very specific to both the organ being threatened and the type of damage. Such genes may be analyzed to determine which genes are responsible for hypersensitivity, for example, within a certain organ. Such genes may be analyzed to identify subsets of genes that are associated with hypersensitivity to certain agents. The measurement of gene expression patterns is useful because many factors can affect the level of transcripts of toxicity genes, including mutations in the regulatory regions of genes, mutation in transcription factor that control the gene(s) of interest, and gene duplications and deletions. Examples of genes whose expression may be screened for association with hypersensitivity to certain agents are further discussed herein. Genes associated with changes in expression levels due to adverse stimuli or toxic insult include, for example, genes which respond to the presence ofa compound, and genes which respond to damage caused by a compound at, for example, the protein, nucleotide, macromolecular, membrane, cell, tissue, organ or system level. For example, certain proteins either prevent or repair toxic cellular injury. Individuals who do not express the appropriate gene profile will suffer greater damage from toxic compounds through a lack of repair enzymes.

Toxic responses can be measured by pathological changes, for example, at the protein, nucleotide, cell, tissue, organ or system level. These pathological changes can be associated with differential gene expression of at least two genes. In addition, and the conespondence between the pathological change and the differential gene expression can be established. At the concentration where pathological outcomes are observable, gene expression changes are specific and causally related to the outcome. For example, compounds that cause peroxisome proliferation as observed in the electron microscope, such as WY 14,643 (Sigma Chemicals; St. Louis, MO), a common toxicological compound known in the art, turn on genes causally related to peroxisome proliferation (See Figure 4). Compounds that cause DNA damage as manifested by increased mutations and cell-cycle disraption turn on genes required to alter the cell cycle and repair the damage (See Figure 5 below). Furthermore, since most drags elicit pleiotropic effects, and are metabolized differently, there is a specific gene expression pattern for each compound, even though there may be a sub-pattern with all compounds that, for example, alkylate DNA at the O-4 position of thymine. Genes associated with hypersensitivity also may be identified by examination of the gene expression profile of hypersensitive individuals differing from normal gene expression patterns of the genes associated with differential gene expression either before or after exposure to the particular drag in question.

Genes which may be identified and tested for their association with hypersensitivity to a certain agent include a variety of genes known in the art that are induced in mammalian or eukaryotic cells or cell lines exposed to high concentrations of chemicals. Genes associated with toxicological response that can be identified for predicting different types of hypersensitivity to different agents include, for example, those genes described in: Cattell (Semin. Nephro.) 19(3):277-87 (1999); Schnabel, M. et al. (Int. J. Mol. Med.) l(3):593-5 (1998); Cruse et al. (Carcinogenesis) 20(5) 817-824 (1999); Fogg, S. et al.

(Am. J. Respir. Cell Mol. Biol.) 20(4):797-804 (1999); Aoki et al. (FEBS Lett.) 333:114- 118 (1993); Feuerstein et al. (Can. J. Physiol. Pharmacol.) 75(6):731-4 (1997); Rodrigo et al. (Scand. J. Gastroenterol.) 34(3):303-307 (1999); Schmidt et al. (Biochem. Biophys. Res. Commun.) 242: 529-533 (1996); Rockett et al. (Eur. J. Drag Metab. Pharmacokinet.) 22: 239-233 (1997); Rudat et al. (Int. J. Radiol. Bio.) 73: 325-330 (1998); Buters et al.

(Proc. Natl Acad. Sci USA) 96(5): 1977-1982 (1999); Wang et al. (Cardiovasc. Res.) 35 .414-421 (1997); Pang et al.( Ann. Hum. Genet.) 62(3): 271-4 (1998); and Henlich, et al. (Biol. Chem.) 378(11) :1217-29 (1997).

Many toxic response genes are induced to higher levels of expression only when needed. An individual can show a defective or hypersensitive response if a cracial protein is defective or is not produced in sufficient abundance when needed. Thus individuals who do not synthesize sufficient amounts of key proteins or produce defective proteins required to minimize the toxic damage from a given agent will suffer from greater toxic injury.

Altered levels of the gene products of the genes listed in Table 3, Table 4, Table 5 and Table 6 are likely to render the cell or organism hypersensitive to toxic stimuli, and there is great variability among the population in basal and induced levels of these genes.

There have been many studies of some of these individual genes in the literature, some of which are discussed below. For example, a mouse knock-out mutant for the DNA repair gene PARP was shown to be hypersensitive to the toxicity and genetic damage caused by gamma-inadiation and MNU. Tracco C. et al. (Mol Cell Biochem) 193(1-2): 53-60 (1999). Humans with low basal or induced expression of the PARP gene will be hypersensitive to gamma-inadiation, MNU and all radiomimetic agents.

In another example, it was recently shown that DNA repair methylfransferase (Mgmt) knockout mice are hypersensitive to the toxic effects of several chemotherapeutic alkylating agents. Glassner et al. (Mutagenesis) 14(3): 339-47 (1999). Individuals with decreased expression of the Mgmt gene will be hypersensitive to the same compounds.

In another example, a 'temporary' knock-out of the cyclophilin-A gene in mice was made by injecting an anti-sense RNA against the cyclophilin A gene in rat neonatal cardiomyocytes. The expression level of the cyclophilin A gene was reduced by 93% and animals freated were hypersensitive to the toxic effects of t-butylhydroperoxide. Doyle et al. (Biochem. J.) 341( l):127-32 (1999). Humans who show depressed levels of cyclophilin A gene expression are expected to be hypersensitive to the toxic effects of t- butylhydroperoxide and other compounds that form active oxygen radicals. Polymorphisms occur in the human population for the gene encoding serum paraoxonase (PON1). The PON1 gene product plays a major role in the detoxification of organophosphate (OP) compounds. One polymorphism (Argl92 isoform) hydrolyzes diazoxon, soman and sarin slowly. Costa et al. (Chem. Biol. Interact) 119-120: 429-38 (1999).

Genes associated with hypersensitivity can be selected from those in Table 3, which are induced by toxic damage and have important physiological roles in responding to toxic stimuli. For example, Rettie et al. (Epilepsy Res.) 35(3):253 (1999) showed that humans carrying a polymorphism that decreases expression of the CYP2C9 gene are very sensitive to compounds such as phenytoin and (S)-warfarin. The data demonstrate that the

CYP2C9*3 polymorphism gene product retains only 4-6% of the metabolic efficiency of the wild-type protein CYP2C9*1 towards phenytoin and (S)- warfarin. Individuals who show dramatically reduced expression of the normal CYP2C9*1 could show the same hypersensitivity to these drags. Several factors can affect the basal and induced levels of expression of these genes. For example, mutations or polymorphisms that affect the promoter region of tox- response genes can cause hypersensitivity to compounds. For example, several polymorphisms have been identified in the promoter region of the human HLA-DQAl gene that affect the levels of mRNA and thus protein levels of the HLA haplotype. Indovina, P. et al. (Hum. Immunol.) 59(12): 758-67 (1998). Polymorphisms in the regulatory region of the genes encoding plasminogen activating inhibitor increase the risk for developing coronary heart disease (Grenett et al. (Arterioscler. Thromb. Vase. Biol.) 19(11):1803-1809 (1998). The polymorphisms mentioned above for human plasminogen activating inhibitor are in the regulatory region of the gene and result in altered expression of the gene. This risk of developing coronary heart disease, and likely increased risk to drags with cardiotoxic properties, is increased specifically as a function of the altered expression levels. Many toxic stimuli induce or repress TGF-βl levels. Individuals who overexpress TGF-βl show heightened levels of apoptosis and fibrosis seen with mycotoxin-induced liver injury. Cruse et al. (Carcinogenesis) 20(5):817-824 (1999).

Polymorphisms in the gene encoding the vitamin D receptor change differential expression of many downstream genes and render the individual likely to develop drag induced psoriasis. Park et al. (Arterioscler Thromb Vase Biol) 19(11):1803-1809 (1999).

Expression levels of the gene for cytochrome P450 CYPIBI have a strong effect on the susceptibility to 7, 12-dimethylbenz[a]anthracene-induced lymphomas. Thus individuals who do not express appropriate levels of the P450 CYPIBI gene would be at enhanced risk for toxic side effects of compounds like 7, 12-dimethylbenz[a]anthracene that are metabolized by that P450 protein. Butlers et al. (Proc. Natl. Acad. Sci. USA) 96(5):1977-

1982 (1999). Classic quotidian fever was found to be associated with significantly lower levels of plasma IL-6. The published evidence shows that there is a genetically determined difference in the degree of the IL-6 response to stressful stimuli between individuals (Coulthard et al. (Blood) 92(8): 2856-62 (1998). Thus individuals with genetically linked quotidian fever are likely to be at enhanced risk for a number of drags that elicit IL-6 expression as part of their inherent toxicity.

The level of expression of the enzyme thiopurine methylfransferase is an important determinant of the metabolism of thiopurines used in the treatment of acute lymphoblastic leukemia and acute myeloid leukemia. TPMT expression displays genetic polymorphism with 10% of individuals having intermediate and one in 300 undetectable levels.

Individuals who do not express TPMT are at extreme risk of severe cardiotoxicity when treated with compounds such as azathioprine (Collie-Duguid et al. (Pharmacogenetics) 9(l):37-42 (1999); Coulthard. et al. (Blood) 92(8):2856-62 (1998). In another example where altered expression of tox-response genes affects the response to specific drags, experiments have recently demonstrated that overexpression of the human HAP1 protein sensitizes cells to the lethal effect of bioreductive drugs. Prieto- Alamo et al. (Carcinogenesis) 20(3):415-9 (1999). Altered expression can come from many causes besides mutations in the promoter region. These include, include mutations in the transcription factors or receptors that regulate a gene and gene duplications. While cDNA sequence analysis ofa normal sequence that had been duplicated would not detect any change in the coding regions of the genes of interest, gene expression analysis would. For example, two active copies of the X- linked gene spermidine/spermine Nl-acetyltransferase (SSAT) in a female lung cancer cell line have been associated with an increase in sensitivity to an anti-tumor polyamine analogue. Mank-Seymour et al. (Clin. Cancer Res.) 4(8): 2003-8 (1998). Duplications in the CYP2D6 or CYP2C19 genes have been shown to be linked with sensitivity to a number of drags including warfarin, codeine and clofenac. Lundqvist et al. (Gene) 226 (2): 327-38

(1999); Yasar et al. (Biochem. Biophys. Res. Commun.) 254 (3): 628-31 (1999).

There are numerous examples where expression polymorphisms comprise a significant percentage of the population. For example, a genetic polymorphism in the metabolism of the anticonvulsant drag S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large intenacial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. Ibeanu et al. (J. Pharmacol. Exp. Ther.) 286(3): 1490-5 (1998). Several individuals showing poor metabolic capacity to coumarin and (+)-cis-3,5-dimethyl- 2-(3-pyridyl)thiazolidin-4-one hydrochloride show very low levels of the CYP2A6 gene product. See Nunoya et al. (Pharmacogenetics) 8(3):239-49 (1998).

There are multiple additional examples of reported variation in genes known to be important in toxic responses, but clinical investigation has not yet been performed to determine their relative susceptibility to specific drags. The human UDP- glucuronosyltransferase (UGTl A) locus is regulated in a tissue specific fashion in liver and extrahepatic tissues. Activity assays demonstrated 2- to 4-fold inter-individual differences in UGT activity and qualitative differences between individuals. The polymorphic regulation of UGTl A gene products in gastric tissue may be the biological basis that determines inter-individual differences in extrahepatic microsomal drag metabolism.

Strassburg et al. (Mol. Pharmacol.) 54(4):647-54 (1998).

Very importantly, it is likely that many mutations in single genes result in altered expression of many more genes, an amplification effect. A knock-out mutant has been created in mice that destroyed the function ofa single gene, the au-beta 6 gene. The resulting animals showed altered basal expression of 101 genes in lung epithelial cells.

Kaminski et al. (New York Academy of Sciences meeting, Toxicology for the Next

Millenium, Airlie VA, USA) September 1999.

Single mutations in any one of hundreds of key toxicity genes can potentially cause differential basal levels of expression of many additional genes. It may be the altered expression of these genes that render the cell, or organism sensitive to toxic stress, not the initial mutation by itself.

Gene expression analysis has been used to predict who will respond beneficially to the therapeutic effects of treatments. The levels of Bax and Bcl-2 expression after radiotherapy have been used as prognostic markers in patients with human cervical carcinoma. Harima et al. (J Cancer Res Clin) Oncol 124(9): 503-10. (1998). In acute myeloid leukemia, coexpression of at least two proteins, including P-glycoprotein, the multi-drag resistance-related protein, bcl-2, mutant p53, and heat-shock protein 27, have been reported to be predictive of the response to chemotherapy. Kasimir-Bauer et al. (Exp Hematol) 26(12): 1111-7 (1998). The work by Kasmir-Bauer et al. shows that gene expression profiling can be used to predict who will benefit from the therapeutic effects of a drag; it does not address the question as to who will suffer enhanced toxicity ofa drag. All of the above examples show that altered levels of gene expression of a certain set of tox-response genes are associated with qualitatively or quantitatively distinct responses to the toxic effect of different drags. Many of the examples show that DNA sequence polymorphisms would not be sufficient to predict hypersensitive individuals.

Finally, the disclosure and examples herein show that measurement of a multiple set of tox- response genes will reveal patterns of gene expression that will identify hypersensitive individuals for specific types of toxicity.

Experimental Identification of Genes Genes associated with hypersensitivity to an agent may be identified in a variety of ways experimentally. Generally the expression of genes that are differentially expressed in total across cell, organ and tissue types in humans, in particular in response to toxic insult is evaluated to determine genes associated with hypersensitivity in an individual. In one embodiment, a method of identifying genes associated with hypersensitivity to an agent is provided, that comprises comparing the gene expression profile of cells treated with an agent with the gene expression profile of untreated cells, and determining genes in the treated cells that have altered expression due to the treatment, thereby to identify one or more genes associated with hypersensitivity to the agent. The cells may comprise one or more different cell types, wherein each said cell type comprises a gene associated with hypersensitivity to the agent. Alternately, the cell types are derived from a single tissue or organ.

Exemplary cell types are those derived from a specific organ , cell or tissue, such as kidney, liver, lung, heart, breast, lymphocytes, neuronal cells, skin, or intestine, such as HepG2, Caco-2, MCF-7, Jurkat, Daudi, HL-60, MCL-5, SKBR-3, SKOV-3, PC-3, WISH and HeLa.

Another method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent comprises comparing the gene expression profile of multiple cell types of an individual known to be hypersensitive to an agent with the gene expression profile of said cell types in an individual known not to be hypersensitive to the agent; and identifying genes from said multiple cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent. An alternative to this method comprises, comparing the gene expression profile of multiple cell types of an individual known to be hypersensitive to an agent before treatment with the agent with the gene expression profile of multiple cell types of the hypersensitive individual after treatment with the agent, and identifying genes from the multiple cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent.

When normal or hypersensitive animals, humans or cells are exposed to a selected agent, gene expression changes can be analyzed in genes such as those listed in Table 3, 4, 5 , 6, 8, 10 and 11. Different types of toxic insult lead to different patterns of gene expression changes in normal, as well as in hypersensitive individuals. Since substantially all compounds elicit toxicity at a high enough dose, the mechanisms of drag toxicity in normal individuals has been well examined. Genes that cells induce to combat the toxic effect of various compounds are important for anti-toxicity for each compound. Patterns of gene expression of these genes in individuals who show hypersensitivity to a given compound that differ from the pattern of differential expression of normal individuals, with or without treatment can be identified. Using these methods, sets of genes that have characteristic expression in hypersensitive individuals that differs from normal individuals may be identified.

Subsets of genes and expression profiles thereof that can be used to identify hypersensitive individuals are identified as follows. A technique such as amplified fragment length polymorphism (AFLP) or serial analysis of gene expression (SAGE), which are known in the art, is used to compare gene expression profiles from treated and untreated human cells. The agent is administered at a toxic dose. This procedure identifies all candidate genes within the cells that respond to the toxic stimuli posed by a particular agent. The method further comprises using a technique, such as AFLP or SAGE, which are known in the art, to compare the gene expression profiles from treated and untreated normal cells. This step would identifies all genes within an individual that respond to that agent. It also permits investigators to understand the normal expression range of individuals who are not hypersensitive. A technique, such as AFLP or SAGE, is used to compare gene expression profiles for samples from treated and untreated hypersensitive individuals or cell cultures derived therefrom. This step identifies all genes within hypersensitive individuals that respond to the treatment by that agent. It also allows investigators to understand the expression range of hypersensitive individuals. This permits identification of the genes that were differentially expressed in all of the above experiments, thus eliminating genes associated with therapeutically beneficial effects and individual variation in expression of genes unrelated to the compound. The expression of these genes can then be measured in a larger population of normal and hypersensitive individuals using, for example gene anays, RT-PCR or other techniques known in the art to confirm the conelation between those genes identified in the above procedures and hypersensitivity observed in particular individuals.

Gene expression responses to toxic stimuli can be analyzed using a database of information. The first method is to determine which genes are induced and what is their function. For example, if all genes induced by a compound are regulated by DNA damage, the interpretation is that the compound causes DNA damage. This interpretation requires a database about the function and regulation of all genes in the database. Another method of interpretation is to determine whether the gene expression pattern induced by a second compound is similar to that induced by a compound, the toxicity of which is well- characterized. This approach to interpretation requires an extensive database of gene expression profiles generated from well-characterized compounds. Table 7 shows a partial list of well-characterized compounds for which gene expression data has been generated.

The methods of gene expression analysis discussed herein can be performed using a computer system with computer code suitable for accessing and comparing the gene expression profile determined according to the methods of this invention. Suitable software will also rank the results of these analyses. Computer code suitable for these purposes can be programmed by a person skilled in the art. Exemplary software and a gene expression profile database related to toxicology are commercially available from Phase- 1 Molecular Toxicology, (Santa Fe, NM), for example, Chem Profiler™ and Matrix Express™.

Examples of Genes Associated with Hypersensitivity Several drags have been shown to elicit allergic reactions in a subset of the population. The more extreme form of these allergic reactions can be quite severe and involve extensive damage of significant portions of the skin covering the body. Many patients die from dehydration and infection. The extreme form of these allergic reactions have the names Steven Johnson Syndrome and TEN (Toxic Epidermal Necrosis). Drags known to elicit Steven Johnson Syndrome and TEN and less severe forms of skin allergy include navirapine, dapsone, acebutolol, trimethoprim, sulfasalozine, sulfacetamide, sulfadiazine, sulfamethoxizole, sulfasoxazole, sulfamethizole cotrimoxazole, amoxacillin, phenytoin, sulfonamide and penicillin.

Gene expression data suggests that the expression levels ofa relatively small number of genes can identify who will develop allergic reactions to these drags. The genes whose expression in CD8 T Cells and keratinocytes is likely to identify hypersensitive individuals include: inducible NOS, Ki-67, Transglutaminase-1, IL-1, FASL, TNF -alpha, CD 1 lb/CDl 8, p75-R-TNF (TNF Receptor), IL-6 receptor, G-CSF receptor, HSP-70, INF- gamma, ICAM-1, VCAM-1, ECAM-1, and TGF-beta.

While not being limited to any theory, it is believed that there is a similar molecular mechanism for both Steven Johnson Syndrome and TEN. The invention provides a method to determine who will develop these syndromes prior to taking these drags. All publications, patents, and patent applications refened to herein are incorporated herein by reference.

The following examples are intended to illustrate but not to limit the invention. EXAMPLES

Example 1: Identification of Genes Associated with Hypersensitivity and Screening of Subjects Prior to Drag Administration

Initially, one or more compounds predetermined to cause blood toxicity, such as agranulocytosis, in at least 4% of the patient population, are chosen, e.g. Haldol™ (haloperidol). The differential gene expression profile associated with Haldol™

(haloperidol) is determined in neufrophils from both normal and hypersensitive subjects when exposed to high concentrations of Haldol™ (haloperidol). The gene expression profile from untreated and treated cells is compared using for example, AFLP, a microanay of the genes listed in Tables 3 and 4, or SAGE, to identify genes that vary as a function of toxicity and vary as a function of hypersensitivity to the Haldol™ (haloperidol). Next, gene expression from clinical samples from a patient population exposed to Haldol™ (haloperidol) or a placebo is measured. The clinical samples are provided by the manufacturer of Haldol™ (Hoechst Marion Roussel). Genes are identified that co-varied with the hypersensitivity status. Additional clinical samples are blinded and provided by the manufacturer which includes samples from normal and hypersensitive subjects. Using the present invention, prediction of the hypersensitivity status is based upon gene expression profiles. The level of accuracy of the prediction or conect identification is determined by unblinding the compounds.

In the final stage, gene expression analysis of the key set of genes would be performed on a prospective basis with new patients just beginning treatment with Haldol.

The level of accuracy of the prediction or conect identification of hypersensitivity is determined by monitoring patients over time to see if those predicted to develop agranulocytosis indeed did so. This empirical approach is then be extended to other drags and other drag manufacturers.

Example 2: cDNA Probe Production A fluorescent dye labeled cDNA probe complementary to the mRNA component of cellular RNA harvested from cells exposed to toxicologic challenge is produced by this protocol, which is designed to produce sufficient Cy3 labeled probe from one experimental sample, and Cy5 labeled probe from one confrol sample, to develop one microanay slide. The procedure is scalable to easily accommodate, for example, 16 samples. This will produce sufficient probe mixtures for at least 8 microanay slides. General procedures as described, for example, in Gerard et al. (Focus®) 14:91 (1992); Kotewitcz et al. (Gene) 35: 249 (1985); and Gerard et al. (DNA) 5: 271 (1986) are utilized. cDNA probes may be used in an assay for detecting expression of genes associated with hypersensitivity to an agent. In one embodiment, microanay slides are provided that contain ssDNA sequences, or targets, from a number of toxicologically relevant genes. The microanay slides, for example, may be 3"x 1" glass microscope slides comprising an anay of micron-scale spots of ssDNA sequences on the upper face. The DNA may be bound to the slide using covalent linkage chemistries known in the art.

Total RNA from cells contains mRNA species that are homologous to these sequences. "Total RNA (high quality)" refers to substantially total cellular RNA. As RNA is very labile, special care must be taken to insure that it is of sufficient integrity at the time of use as template in the production of probe. The level of these mRNA species is proportional to the degree of induction of the gene by the agent under study. This protocol describes the production of fluorescent labeled cDNA probe from the total RNA of cells which have either been exposed to the agent under study, or are serving as a non-treated control. These probes are then pooled and hybridized to the microanay slide. The experimental and control probes are distinguishable because the Cy3 and Cy5 labels fluoresce at different wavelengths. The degree to which each probe binds to a specific gene sequence on the slide reveals the level of induction of that gene in the cells exposed to the agent under study.

The following materials are used:

Material Amount Exemplary Source

DEPC treated water 10ml Ambion®

Alk Water (pH 7.5 with NaOH) lml

Total RNA (of high quality) or lOμg/sample

Messenger RNA (of high quality) 2μg/sample First strand buffer 4μl/sample 0.1 M DTT 2μl/sample Sigma® 1 :8 dilution Cy 3 dCTP 1 μl/exp. Sample Amersham®

(3-amino-propargyl-2'-deoxycytidine 5'-triphosphate)

(i.e., 0.125mM Cy3 dCTP) 1:10 dilution Cy5 dCTP 1 μl/control sample Amersham®

(3-amino-propargyl-2'-deoxycytidine 5'-tiphosphate) (i.e., 0.1mM Cy5 dCTP) Superscriptll (RT) 1 μl/sample Life Technologies, Inc. ANTI-RNase 1 μl/sample Ambion® 7.5 M ammonium acetate 34μl/sample Sigma® 70% EtOH 1 ml/sample J. T. Baker® 95% EtOH 220μl/sample J. T. Baker® Nucleotide Mix "3" 1 μl/exp. sample

0.5 mM dATP/dGTP/dTTP

0.125 mM dCTP

Nucleotide Mix "5' 1 μl/control sample

0.5 mM dATP/dGTP/dTTP

0.15 mM dCTP

Stock anchored oligo dT: 4 μl/sample

0.25μg/μl of each oligo dT

(in Water @ -20°C)

RNase Zap (1) bottle The RNA Co. ™

Wet ice (1) bucket

Qiagen Qiaquick PCR purification kit (l) ea Qiagen®

PE/ETOH (1) bottle

(100 ml PE buffer + 400 ml >96% EtOH)

EB Buffer 10 ml (10 mM Tris-HCl pH 8.5) General Protocol

Steps are performed at room temperature unless otherwise specified. Work areas are cleaned and swabbed with RNase Zap. Gloves are worn at all times. RNase (RNA specific endo-and exo-nucleases) is a ubiquitous and very stable enzyme. Standard cleaning and/or autoclaving will not remove or inactivate it. Therefore all materials contacting the samples must be known RNase-free. All water, including for buffers, must be DEPC-treated. DEPC treatment consists of an autoclaved solution of 0.1% Diethyl pyrocarbanate in de-ionized water.

Preparation of RNA template in water is implemented by adjusting mRNA to a concenfration of 2μg/7μl or total RNA to a concenfration of 10μg/7μl for each sample in a standard microfuge tube. If concentration adjustment requires dehydration in the Speedvac™, 1 μl Anti-RNase is added prior to dehydration. The reaction solution is prepared by adding 4 μl of stock anchored oligo dT per tube, heating at 70°C for 10 minutes in a heat block, spinning 5 seconds in microfuge, and placing on ice for 2 minutes. The following is then added to each tube:

4 μl 5x First Strand Buffer for Superscriptll 2 μl 0.1 M DTT and either (for treated samples): l μl Nucleotide Mix "3" 1 μl of 1 :8 dilution of Cy3 or (for control samples):

1 μl Nucleotide Mix "5" 1 μl of 1 :10 dilution of Cy5.

The tube then is incubated at room temperature for 10 min. The dCTP is added to limit the concentration of Cy dCTPs incoφorated. Due to the size of the Cy dCTP, the polymerase will fall off the template if more than two are incoφorated in a row. To perform the reaction, 1 μl SuperScriptll is added to each tube, and the contents mixed gently. The tube then is incubated for 1.5-2 hr. at 45°C in a heat block, keeping the reaction protected from light. The fluorescent dyes Cy3 and Cy5 are sensitive to light. Excessive exposure during processing will reduce the intensity of emission upon final scanning.

To collect the labeled cDNA probe, ethanol precipitation is implemented by adding to each tube 46μl of water, 34μl of 7.5M ammonium acetate and 220μl of 95% EtOH, and then incubating at -80°C for 15-20 min. If desired, procedure may be interrupted at this point. The sample may be stored at -80°C for up to 7 days. The tubes are loaded in centrifuge with orientation of lid noted, centrifiiged for 15 min at 20800 x g, and the supernatant discarded, to obtain a visible pellet (pink for Cy3, blue for Cy5). The pellet is washed by adding 750μl 70% EtOH per tube and vortexing briefly, centrifuging at 20800 x g for 10 min, decanting and discarding the supernatant, centrifuging the pellet and optionally gently removing remaining EtOH with a pipette, while being careful not to loosen the pellets. The pellet is allowed to dry for 10 min. at room temp, but not over drying by using a vacuum, and resuspended in 40μl water. cDNA/mRNA hybrid is denatured by incubating at 95 °C for 5 min. in a heat block. The tube then is spun 5 seconds in microfuge.

The labeled cDNA probe is purified in an adaptation of the procedure described on page 18 of the QIAquick Spin Handbook, (1997) Qiagen®. To bind the cDNA probe to a column, 200 μl of Buffer PB is added to each 40μl probe solution, the QIAquick spin columns are placed in 2 ml collection tubes, and the samples are applied to the QIAquick columns and centrifiiged at 10,000 x g for 2 min. The flow-through is discarded and QIAquick columns replaced into the same tubes. To wash bound cDNA probe, 750μl Buffer PE/ETOH is added to each column, and the column incubated for 1 min. at room temp. The column is centrifiiged at 10,000 x g for 2 min., and the supernatant discarded. The wash is repeated. QIAquick columns are placed back in the same tubes, and centrifiiged for an additional 1 min at maximum speed with tube lids open. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

QIAquick columns are placed in clean 1.5ml microfuge tubes. To elute the cDNA probe, 40μl (+/- 1 Oμl) Alk. Water is added to the center of each column. The tubes are incubated for 1 min, centrifuge at 6000 x g for 1 min., and the elution steps repeated once into same tube. The elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound cDNA.

To quantify the cDNA probe, each sample is put in ~80μl of EB buffer, and transfened to one well of a 384 well plate. Scanning, including the measurement and recording of the type and degree of fluorescence from each spot on a processed microanay slide, is accomplished in a confocal laser scanning fluorimeter. The fluorimeter is set to the appropriate excitation emission frequencies and records the level of emission for the sample. The exposure time and intensity is controlled, because exposure of the label to strong light incrementally reduces its fluorescent activity. Values from this procedure are the result of many variable factors. Therefore it is preferable to compare to an archive of values produced from the same procedure and equipment.

To prepare the final probe mixture, the Cy3 labeled experimental probe is combined with the Cy5 labeled confrol probe. If a control requires multiple reactions, they are combined prior to aliquoting equal amounts to the experimental samples. The combined probes are concentrated to ~lμl in a Speedvac at a temperature not exceeding 45°C. If the probe is not used immediately, 10 μl water is added and it is stored at 4°C.

Example 3 : Determination Of Gene Expression Changes Associated With Toxicity To determine genes useful for identifying patterns of genes associated with toxicity, animals were exposed to concentrations of selected compounds that elicit peroxisome proliferation, a type of liver toxicity. Treatments were with WY 14,643, gemfibrozil and clofibrate in Sprague Dawley rats. Each compound was administered in 1% carboxymethycellulose/0.2% Tween 80 by oral gavage daily for 14 days. Administered doses were to three animals per dose per time point as follows; WY 14,643, 40 mg/kg/day; gemfibrozil, 24 mg/kg/day and 100 mg/kg/day, and clofibrate 40 mg/kg/day and 250 mg/kg/day.

Gene induction was measured using microanays consisting of 250 toxicologically relevant rat genes using the hybridization protocol described above. As illustrated in Figure 4, several genes were induced by the treatment, example given is for WY14,643. This figure shows a gene expression profile showing the relative induction levels compared to untreated controls. As shown in Figure 4, the genes refened to in Figure 4 as A

(Cytochrome p450 4A CYP4A, B (Enoyl Co-A Hydratase), C (3-ketoacyl CoA thiolase 2), D (Acyl CoA Oxidase), and E (Ketoacyl CoA thiolase type 1), had enhanced expression in comparison to the confrol after treatment with the compound. These genes were found to be induced by a variety of other peroxisome proliferating agents including gemfibrozil, clofibrate, fenofibrate and DEHP. This set of genes was thus empirically shown to be induced by a variety of compounds that exhibit a specific type of hepatotoxicity, peroxisome proliferation. By way of example, individuals who display hypersensitivity to these types of compounds should show altered expression of this set of genes.

Example 4: Probe For Hepatocyte Growth Factor

New genes associated with and predictive of toxicity were identified. Different types of damage to the liver cause the formation of dead and dying hepatocytes, which the liver replaces to maintain its function. Induction of the hepatocyte growth factor receptor gene by toxic stimuli in both rats and humans was examined. When several nitrosoureas including streptozotocin, carmustine and MNU were used to determine gene expression profiles, all of these compounds induced several genes in common. These compounds are all known to form covalent adducts to the DNA in liver and liver cells. All compounds, for example induce both the hepatocyte growth factor receptor gene and the glutathione fransferase gene. Exemplary data is provided in Figure 1 which shows the gene expression profile in the liver of male Sprague-Dawley rats when freated with the hepatotoxicant streptozotocin. The probe for the hepatocyte growth factor receptor gene was created by cloning at least a 250 base-pair section from the 3' coding region of the gene starting with total genomic DNA. The fragment was derived by PCR from genomic DNA using two primer with appropriate linkers for insertion into a plasmid vector. A single stranded probe complementary to the cDNA sequence was attached to a glass slide anay using a polyamine attachment.

In more detail, an example of creation of a specific probe for the hepatocyte growth factor receptor is as follows. The first step in the process is obtaining the sequence for the gene. The search for gene sequence, either by gene name or accession number, is performed using the NIH National Center for Biotechnology Information website using Genbank (http://www2.ncbi.nlm.nih.gov/genbank/query_form.html). The accession number for the rat hepatocyte growth factor receptor gene is X96786. When the sequence of interest is located, the sequence information is copied to a Microsoft Word file. Intron sequences are then removed, if present, as well as numbers and white spaces. The resulting condensed sequence is then submitted to a PCR primer design software program, such as Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Primers are selected that optimally have a T_m in the range of 60°-63°C. The optimal length of the gene fragment is 500 bp. Shorter fragments are chosen if the starting sequence is shorter than 500 bp. Once the primers are designed, the sequence that is flanked by the primers is submitted to a BLAST search. BLAST (Altschul et al (Nucleic Acid Res) 25: 3389-3402. (1997)) is a sequence analysis software program supported by the NIH. The BLAST search software searches for other DNA sequences that are homologous to the target sequence and ranks these sequences according to the amount of homology. This ensures that the chosen gene fragment sequence will not cross-hybridize with a gene sequence other than the desired sequence. PCR primers are ordered and an attempt is made to isolate the gene fragment from a cDNA library that is created by reverse transcription of RNA from either a cell line(H4IIE) or rat tissue. Upon identification of a PCR band of the conect size, the PCR product is cloned into a vector (TA cloning vector, Invitrogen Coφ., Carlsbad, CA).

Following cloning, a bacterial mini-prep is performed to amplify and isolate the plasmid containing the gene fragment of interest. The region of the plasmid containing the gene fragment is then sequenced. If this sequence matches the original target sequence, the target sequence of this clone is amplified by PCR, purified (Wizard system, Promega Coφ., Madison, WI), quantified, and used for spotting.

The probe refers to a population of cDNAs bearing fluorescently active ligands which are produced from the mRNA of the cells under examination, while "probe mixture' refers to a mixture of two or more populations of cDNA. The cDNAs may also be labeled with a variety of ligands, such as fluorescently active ligands, radioisotope ligands or biotinylated ligands.

Example 5: Glutathione Transferase Positive Foci

Enhanced gene expression and co-induction of genes associated with the formation glutathione transferase positive foci was identified.

Certain types of toxic liver damage produce glutathione transferase positive foci Lemmer et al. (Carcinogenesis) 20:817-824 (1999) which are cells that are in the late stages of dying. In response, neighboring hepatocytes must replicate in order to replace the dying cells and induce expression of hepatocyte growth factor so that they are 'primed' for growth hormone signals. Co-induction of the glutathione transferase and hepatocyte growth factor receptor genes was determined by hybridization to microanays containing at least 300 human toxicologically relevant genes using the hybridization protocols described above.

Figure 2 is a graph showing the results, which indicated a very strong conelation between the induction of the glutathione fransferase and hepatocyte growth factor receptor genes. Co-induction thus shows conelation to focal cell death occuning in the liver.

Example 6: Heart Muscle Tissue Gene Expression Profile

Animals were exposed to doxombicin, a cardiotoxin. Male Sprague-Dawley rats were treated with 1 mg/kg doxombicin in 5% saline for 6 ho 24 hours, and 7 days and 6 weeks with one dose per day and a 6 week recovery period. A gene expression profile of heart muscle tissue cells was then obtained. RNA was isolated and the gene expression profile was analyzed as described below. Gene expression of all genes listed in Table 8 was determined. The results of the gene expression of the first 66 genes is shown in Figure 3.

As can be seen from Figure 3 and Table 8, several genes, including activating transcription factor 4, activin receptor type II, ataxia telangeictasia, c-jun, carnatine palmitoyl-CoA transferase, DNA Dependent helicase, Epozide hydrolase, famesol receptor, Gadd 45, Interleukin 6, MDM-2, Ribonucleotide reductase subunit Ml and at least 10 others were differentially expressed at significant levels. Many of these genes, including

Carnatine Palmitoyl fransferase, Epoxide hydrolase, Famesol receptor, Lipoprotein lipase precursor, and MDM-2 have never been reported or previously known to be induced by cardiotoxicity.

Thus a profile of gene expression characteristic of the cardiotoxin, doxombicin was obtained. Genes thus identified as having altered expression in the presence of cardiotoxin are significant, because individuals with diminished or altered expression of the induced genes may potentially be hypersensitive to the toxicity of doxombicin. Such hypersensitivity could manifest itself at the molecular level as altered induction of these genes as well as a shift in the dose-response curve such that the same genes would be induced at lower concentrations.

Example 7 Determining genes associated with hypersensitive reaction to penicillin

Three different methods, differential display, microanay technology, and Taqman® assay were used to determine genes associated with hypersensitive reaction to penicillin. Seven self-described penicillin-sensitive individuals and six individuals self-described to have normal reaction to penicillin were tested by differential display. Six self-described penicillin-sensitive individuals and six individuals self-described to have normal reaction to penicillin were tested by microanay technology.

1. Lymphocyte culture Six individuals self-described as penicillin sensitive and seven individuals self- described as having normal reaction to penicillin were used to determine potential hypersensitive reactions to penicillin in humans. Peripheral blood leukocytes (PBL) were isolated from a population of individuals, cultured with PHA at a standard concentration for culturing lymphocytes for 24 hours, washed, cultured for another 24 hours without PHA, and divided into two groups. One group was exposed to penicillin in vitro for 24 hours and the other group was not exposed to penicillin as a control group. At a non-toxic dose of 1250 μg/ml, penicillin G is known to elicit an immune response in peripheral blood of individuals with proven penicillin G allergy.

2. Isolation of RNA from cultured lymphocytes

RNA from select individual from both groups (treated and untreated) of cultured lymphocytes was isolated as follows. Total RNA of high quality and high purity is isolated from cultured cells by using Qiagen QIAamp® RNA blood mini kit and 2- mercaptoethanol. RNA degradation by RNases is not desirable when synthesizing fluorescent cDNA for hybridization with the penicillin anay. Precautions are taken to minimize the risk of RNA degradation by RNases by wearing gloves, treating work areas and equipment with a RNase inhibitor, for example, RNase Zap (Ambion® Products, Austin, TX) and keeping samples on ice. This total RNA isolation technique is based on a Qiagen QIAamp®RNA blood mini kit and is used with some modification for human lymphocyte cells in a T-75 flask.

Cells are checked under the microscope to make sure that they are viable. Cells are dosed with penicillin on the third day in culture (48 hours after introduction of the cells into culture).

Cells are scraped from the flask and poured into a 50 ml conical tube. The flask is then rinsed with 10 ml of room temperature PBS. The PBS wash is removed with a pipette. The tube is then spun for 10 minutes at 1,000 φm and the supernatant pipeted off. The remaining pellet is resuspended in 600μl of freshly prepared RLT buffer (RLT buffer requires the addition of lOμl of beta mercaptoethanol for each 1.0 ml RLT) by vortexing.

The resuspended pellet is pipeted into a QIAshredder® column and centrifiiged for 2 minutes at 14,000 φm in a Eppendorf® 5417C centrifuge. The QIAshredder® column is discarded and 600 μl of 70% ethanol added to the lysate. The lysate is then pipeted into a QIAamp® spin column sitting in a 2 ml collection tube and centrifiiged for 15 seconds at 14,000 φm. Any remaining lysate is placed on the same column and the centrifugation is repeated. The QIAamp® spin column with the RNA bound to the column is transfened to a new 2 ml collection tube. 700μl of Qiagen® RW1 buffer is added to wash the column and centrifiiged for 15 seconds at 14,000 φm. The QIAamp® spin column is transfened to a new 2 ml collection tube. 500μl of Qiagen® RPE buffer is added to the column and centrifiiged for 15 seconds at 14,000 φm. The QIAamp® spin column is transfened to a new 2 ml collection tube. 500μl of Qiagen® RPE buffer is added to the column and centrifiiged for 3 minutes at 14,000 φm. The QIAamp® spin column is transfened to a new 2 ml collection tube and centrifiiged for 1 minute at 14,000 φm. The QIAamp® column is transfened to 1.5 ml microcentrifuge tube and 50 μl of RNase-free water is added to the column and centrifiiged for 1 minute at 14,000 φm. An additional 50 μl of RNase-free water is added to the column and centrifiiged for another 1 minute at 14,000 φm.

To measure the yield, the O.D. reading is taken at 260nm on a Beckman DU®350 UV vis spectrophotometer. 1.0 μl RNA is added to 49 μl of sterile nanopure water and the O.D. reading is taken and calculated as follows: (Absorbance) x (dilution factor) x (40)/ 1000 = amount of RNA in μg/μl Example: absorbance = 0.45

Dilution factor = 50

(0.45) x 50 x 40 = RNA concentration in μg/μl 1000 The sample is stored in -80°C freezer.

3. MessageClean® of Total RNA

It is important that total RNA that is used to make mRNA differential display is absolutely free of DNA contamination. Regardless of the method used for RNA isolation, a cleaning step is important to ensure the removal of DNA contamination, especially if the differential display banding pattern on the denaturing polyacrylamide gel is independent of the reverse franscription step. MessageClean® from GenHunter (Nashville, TN) was used to clean total RNA. Components for twenty RNA sample cleanings included the following materials: 140 μl lOx Reaction Buffer, 20 μl GH-DNase I (RNase free, 10 units/μl), 140 μl 3M NaOAc, and 1 mL DEPC-freated H₂0. For DNase I digestion, the following materials were added in order: 50 μl total RNA (10-50 μg), 5.7 μl lOx Reaction Buffer, 1 μl DNase I

(10 units/μl) for a total volume of 56.7 μl. The materials were mixed well and incubated at 37 degrees for 30 minutes. A 3:1 phenol/chloroform mixture is used to ensure removal of protein contamination and DNase I from the RNA. About 40 μl of phenol/chloroform is added to the mixture, vortexed for 30 seconds, and allowed to sit on ice for about 10 minutes. Then the mixture was spun in an Eppendorf centrifuge at 4 degrees for 5 minutes at maximum speed and the upper phase of the mixture is collected. Ethanol precipations was performed as follows. About 5 μl of 3M NaOAc and 200 μl of 100% ethanol was added to the upper phase that was collected. This was placed at -80 degrees for more than 1 hour and then spun for 10 minutes at 4 degrees. The supernatant was removed, the RNA pellet was washed with 0.5 mL of 70% ethanol (in DEPC-freated water), and spun for 5 minutes to remove the ethanol. The tube containing the materials were spun again and the residual liquid was removed. The RNA was re-dissolved in 10-20 μl DEPC-treated water.

The RNA was quantitated by reading on a spectrophotometer at OD₂₆₀. RNA that is diluted for any puφose, such as quantisation, should not be re-used after freezing and thawing. The integrity of the RNA can be checked by running a few micrograms on a 7% formaldehyde agarose gel and looking for the clear appearance of 28S and 18S rRNA bands.

4. Reverse franscription

In a tube, the following ingredients are added: 9.4 μl dH₂0, 4.0 μl 5x RT buffer, 1.6 μl dNTP (250 μM), 2.0 μl of 0.1 μg/μl freshly diluted total RNA that is DNase-free, 2.0 μl H-TπM (2 μM) for a total volume of 19 μl. The ingredients are mixed well and incubated at 65°C for 5 minutes, 37°C for 60 minutes, 75°C for 5 minutes, and held at 4°C. After the tubes had been at 37°C for 10 minutes, and 1 μl of Superscript II reverse transcriptase (Life Technologies Inc.) is added to each reaction, and quickly mixed by finger tapping the tubes before the incubation continued. At the end of the reverse transcription, the tubes are spun briefly to collect condensation. The tubes are set on ice for PCR or stored at -20°C for later use. 5. PCR to amplify gel band

The following is an exemplary protocol for PCR. The following ingredients are used: 10 μl dH₂0, 2 μl lOx PCR buffer, 1.6 μl DNTP (25 μm), 2 μl of 2 μm H-AP primer, 2 μl of 2 μm H-TπM, 2 μl RT-mix described above (must contain the same H-TπM used for PCR), 0.2 μl α-³³p DATP (2000 ci/mmole), 0.2 μl TAQ DNA polymerase from PE

Biosystems for a total volume of 20 μl. The tube containing all these ingredients are mixed well by pipeting up and down and placed in a thermocycler at 95°c for 5 minutes and then amplified for 40 cycles under the conditions of 94°c for 30 seconds, 40°c for 2 minutes, 72°c for 30 seconds and finally held at 4°c until the samples are removed from the thermocycler.

6. Gel electrophoresis

RNA was analyzed by gel electrophoresis to identify possible candidate genes. A 6% denaturing polyacrylamide gel in TBE is prepared and allowed to polymerize for at least 2 hours before using. Then the gel is run for about 30 minutes before any samples are loaded. It is important for all the sample wells in the gel to be flushed and cleared of all urea prior to loading any samples in the wells. About 3.5 μl of each sample is mixed with 2 μl of loading dye and incubated at 80°C for 2 minutes immediately before loading onto the 6% gel. In this example, the loading dye is xylene and after the gel is loaded with the samples obtained from the rounds of PCR, the gel is run at 60 watts of constant power until the xylene dye is about 6 inches from the bottom of the gel. Once the power is turned off, the gel is blotted onto a large sheet of exposed autoradiograph film. The gel is covered with plastic wrap and under dark conditions, the gel is placed in a large autoradiograph cassette with a new sheet of unexposed film, marked for orientation, and the film is allowed to be exposed to the gel at -80°C. The exposure period can be anywhere from overnight to

72 hours. Once the film has been developed, bands of interest, which show differential expression between penicillin sensitive and normal individuals, are identified by alignment with the developed film and subsequently isolated by cutting the band of interest out of the polyacrylamide gel with a clean scalpel blade. The isolated band is placed in 100 μl of water and boiled at 95% for 5 minutes.

7. Cloning re-amplified PCR products for differential display

The following procedure was used to clone re-amplified PCR products from differential display. Material which may be used include the PCR-TRAP® Cloning System (GenHunter®). For a 20ul Ligation reaction, add in order: lOul dH2O; 2ul 10X ligase buffer; 2ul Insert-ready PCR-TRAP® Vector; 5ul PCR product; lul T4 DNA ligase. The reaction is mixed well by finger tipping and is briefly spun. Then the reaction is ligated overnight at 16°C. The reaction can then be used directly for transformation or stored at -20°C. For transformation, the GH-competent cells are thawed in ice water slush for 15 minutes. While the cells are melting, the appropriate number of 1.5ml microfuge tubes are labeled and set on ice. The cells are quickly mixed by finger tipping and are divided into lOOul aliquots into each 1.5ml microfuge tube. The remaining competent cells are immediately re-frozen for future use. The ligation tubes are spun briefly to collect condensation. About lOul of each ligation mix is added to an above tube containing the competent cells and mixed well by finger tipping and incubated on ice for 2 minutes. About 0.4ml of LB medium is added and the cells are incubated at 37°C for 1 hour. It is important that no Tetracycline be in the LB during this step because the bacteria with recombinant plasmids need time to express the Tetracycline resistance gene. It is recommended that the LB-Tet plates are warmed at 37°C for 1 hour before plating. After vortexing briefly, about 200ul of cells are plated on an LB-Tet plate (containing lOug/ml of tetracycline). For the lacZ control insert, about 200ul of cells are added to the plate. Then 30ul of X-gal is added to the middle of the cells and the cells are immediately spread onto the LB-Tet plate. Unplated cells can be stored at 4°C if replating is needed within 1 week. Once the plate surface is dry, the plate is incubated upside-down overnight at 37°C. The Tet colonies are scored and the plate is save upside-down at 4°C. Three individual Tet resistant colonies are picked for each clone with a lOul pipette tip, placed in labeled sterile culture tube containing 3ml of LB broth and grown overnight at 37°C.

8. Screening colonies for inserts

Plasmid DNA was isolated using the Qiagen Qiaprep Miniprep kit. PCR was used to check for inserts in the plasmids. For each colony the following PCR reaction mixture was set up: dH2O lOμl lOxPCR buffer 2μl dNTPs (250μM) 1.6μl

Left primer 2μl

Right primer 2μl

Plasmid DNA 2μl Taq DNA Polymerase 0.2μl

The PCR parameters were 94°C for 30 sec, 52°C for 40 sec, 72°C for 1 min for 30 cycles followed by 5 min extension at 72°C and a final incubation at 4°C. All 20μl of the PCR product was analyzed on a 1.5% agarose gel with ethidium bromide staining. Once the positive colonies were identified, they were sequenced by standard methods well-known to a skilled artisan. The sequences were compared to known sequences to determine if the sequence was already known.

As indicated at the beginning of Example 7, either differential display or microanay techniques were used to further determine genes related to penicillin hypersensitivity.

9. Genes Identified By gel electrophoresis, about 260 candidate genes were identified and about 220 were cloned and sequenced to identify genes that predict hypersensitivity to penicillin. A summary of the genes associated with penicillin hypersensitivity is summarized in Table 10. Several new genes were identified that did not match any sequence listing in GenBank. Novel sequences which did not match any BLAST searches or GenBank searches are indicated in Table 10 under the "Identification " column as "no significant match to anything". Thus, provided herein are nucleic acids comprising said novel sequences and fragments thereof as well as amino acid sequences encoded therefrom and fragments thereof. Also provided are nucleic acids that hybridize to said novel sequences under stringent conditions. Such stringent conditions include conditions ofa hybridization reaction that allow nucleic acid duplexes to be distinguished based on their degree of mismatch. Means for adjusting the stringency ofa hybridization reaction are well-known to those of skill in the art. See, for example, Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Ausubel, et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1996 and periodic updates; and Hames et al, NUCLEIC ACID HYBRIDIZATION: A PRACTICAL APPROACH, IRL Press, Ltd., 1985. In general, conditions that increase stringency (i.e., select for the formation of more closely-matched duplexes) include higher temperature, lower ionic strength and absence of solvents; lower stringency is favored by lower temperature, higher ionic strength, and higher concentrations of solvents (for example, formamide or dimethyl sulfoxide).

The following are some of the genes identified using the methods disclosed herein (GenBank identification numbers in parenthesis): hypothetical protein (HSPC004), UBA3 (UBA3) mRNA, clone CTA-732E4 on chromosome 22ql2.1, ribosomal protein S7 (RPS7), myosin-binding protein C, cardiac (MYBPC3), CGI-51 protein mRNA, latexin mRNA,

NADH oxidoreductase subunit MWFE, jun B proto-oncogene (JUNB), KIAA0787 protein, fatty acid synthase, polymerase (RNA) II (DNA directed) polypeptide B (140 kD), UbA52 gene coding for ubiquitin-52 amino acid fusion protein, small nuclear ribonucleoprotein 70kD polypeptide (RNP antigen) (SNRP70), isocitrate dehydrogenase 3 (NAD+) gamma (IDH3G), clone 565E6 on chromosome 1 Iql2-lq22.2, hypothetical protein FLJ20436 (FLJ20436), c-Cbl-interacting protein L7a (RPL7A), ribosomal protein L7a (RPL7A), ribosomal protein S21 (RPS21 ), sorting nexin 6 (SNX6), TNF-inducible protein CGI 2- 1

(CG12-1), BRCA2 gene region chromosome 13ql2-13, CGI-128 protein mRNA, Tu translation elongation factor, mitochondrial (TUFM), KIAA0787 protein, ribosomal protein L13 (RPL13), ribosomal protein L19 (RPL19), clone 245M18 on chromosome 6p21.32- 22.3, clone TCBA00781, chromosome 19 cosmid R26529, tumor suppressing subfransferable candidate 1 (TSSC1), transfeπin receptor (TFRC), ubiquitin-conjugating enzyme E2D 3 (UBE2D3), putative DNA-directed RNA polymerase III Cl 1 subunit, myosin-binding protein C (cardiac) (MYBPC3), tapasin (NGS-17), CoREST protein (COREST) (KIAA0071 protein), dynamitin (dynactin complex 50 kD subunit) (DCTN-50), alpla-L-fucosidase, metallothionein-IG (MT1G), Familial Cylindromatosis cyld gene, cDNA FLJ10589 fis (clone NT2RP2004389), eukaryotic translation elongation factor 1 delta (guanine nucleotide exchange protein) (EEF1D), chromosome 16 BAC clone CIT987SK-A-67A1, proteasome (prosome, macropain) subunit beta type 8 (large multifunctional protease 7) (PSMB8), and lectin galactoside-binding soluble 9 (galectin 9). An unexpected result that was found was that there were apparently no p450 genes or metabolism genes that were gene candidates for penicillin hypersensitivity.

10. Gene Conelations

Gene expression profiles comprised of 180 genes on the penicillin anay were compared for similarity between six penicillin-normal individuals and six self-identified penicillin- sensitive individuals. Three of the penicillin-sensitive profiles were repeat samples taken at different times. As shown in Figure 6, Samples 6005, 6015, and 6042 are from one individual, and samples 6041 and 6043 are from another individual. Using all genes for comparison, sensitive individuals tend to resemble one another while non-sensitive individuals have little discemable pattern. The one exception is non-sensitive individual 6002, whose profile has some resemblance to the sensitive individuals. In an exploratory analysis, independent-samples t-tests were performed to suggest which genes were differentially expressed between penicillin-sensitive and penicillin-insensitive individuals. Twenty genes in which the p-value of the t-test showed a statistically significant difference between the two classes at a level of 0.005 or less were identified as indicated in Table 11. Using the 20 genes identified as "discriminator" genes, the conelation between normal individuals and the discriminator genes were calculated as well as the conelation between sensitive individuals and the discriminator genes. The discriminator conelations are shown in Figure 7. Using the 20 discriminator genes and relevance network grouping, a similar conelation resulted. At a 0.9 conelation level, the only group that reveals itself is among the sensitive individuals. At a lower similarity level of 0.8, non-sensitive individual #2, who appeared to be borderline hypersensitive, joins the group of sensitive individuals. Methods of analyzing expression data statistically which are known in the art may be used, such as those described in "Family- Wise Enor Rate", Glass, G. and Hopkins, K., Statistical Methods in Education and Psychology (1984), Prentice- Hall; and "Relevance Networks", Butte, A. J. and Kohane, I. S. (2000), Mutual Information Relevance Networks: Functional Genomic Clustering Using Pairwise Entropy Measurements. PSB00, 5:415-426.

Figure 8 shows that the 20 discriminator genes were analyzed for co-regulation, revealing several co-varying groups, as shown in both the similarity matrix and the relevance network grouping.

10. Preparation of penicillin anays

In addition to differential display, microanay techniques were utilized to determine genes related to penicillin hypersensitivity. The following are methods that were used to prepare microanay for testing for penicillin hypersensitivity. Of 260 potential gel band, 220 were cloned and sequenced. About 180 genes were put on a penicillin anay, made as described below, and 20 discriminator genes (Table 11) were selected related to penicillin hypersensitivity.

Large Scale PCR (in 96-well plates)

For 1000 PCR reactions, 4X Master Mix can be made with the following materials:

10X PCR buffer 10 ml dATP 200ul (lOOmM) dGTP 200ul (lOOmM) dCTP 200ul (lOOmM) dTTP 200ul (lOOmM)

Amine-linked vector primer (= "3X") 900ul (Forward or Reverse) lug/ul

Taq Polymerase 1 ml 5U/ul

H₂O 12.3 ml

25 ml total

About 2.5ml aliquots are put into 15ml conical tubes and store at -20°C. One tube is enough for 1 96-well plate of PCR. Alternatively, about 12.5ml aliquots can be used in 50ml conical tubes, which is enough for 5 plates of PCR. dNTPs was obtained from Pharmacia Ultrapure dNTP set, cat#27-2035-02 (set contains all 4, 1ml each) and Taq Polymerase was obtained from Perkin Elmer N808-0155 (comes with 10X buffer).

Template and gene-specific primer mix was made for 2 rows, or 16 wells by utilizing the following materials: 400ul H₂O, 2.5ul plasmid, 15ul of lug/ul gene specific primer.

To perform PCR, the following steps were performed:

1. Take one tube of PCR master mix and add 2 volumes of water (i.e., add 5ml water to 2.5ml MM).

2. Using a multichannel pipette, distribute 75ul MM to each well ofa 96-well plate.

3. Add 25ul H₂O to 2 wells of the plate to serve as negative controls.

4. Add 25ul template and gene-specific primer mix to appropriate wells. 5. Seal all wells with strip caps.

6. Plates can be stored at 4°C for up to 48 hours (maybe more) before cycling.

7. Run PCR using program TKB (95° for 5min, 95° for 15s, 50° for 30s, 72° for 30s, go to step of 95° for 15s and repeat 34 times, 72° for lOmin., 4° until PCR needed for subsequent steps

8. Run product on 1.5% agarose gel and check insert size. (Only need to check 1 well of each "gene.")

9. Clean PCR products using any commercially available kit for cleaning PCR products.

Gene purification

The Anaylt™ kit from TeleChem, International, Inc. Sunnyvale, CA was used for gene purification. The following protocol was used:

1. Position a SuperFilter 100 on a 96-well vacuum manifold. Make sure the SuperFilter is properly fitted to allow a tight seal for vacuum filtration.

2. Add 500 μl of Array It™ Binding Buffer to each well of the SuperFilter 100 using a 12- channel pipetting device set for 500 μl. Pipetting should be performed as quickly as possible (within 1 minute per plate) to minimize the loss of the Binding Buffer due to gravity flow. Avoid splashing the contents from well to well. 3. Quickly add 100 μl per well of PCR sample for a 96-well plate to the coreesponding well of the SuperFilter 100. Transfer the PCR samples to the SuperFilter 100 as quickly as possible (within 1 minute per plate) to minimize the loss of the Binding Buffer due to gravity flow. 4. Immediately mix the Binding Buffer and the PCR sample thoroughly by pipetting up and down 10 times with an automatic pipetting device. Mixing should be completed as quickly as possible (within 5 minutes after adding the Binding Buffer to the SuperFilter 100) to minimize the loss of the Binding Buffer due to gravity flow, void splashing the contents from well to well.

5. Apply a gentle vacuum such that a little trickle flows from the SuperFilter 100 to allowing binding of the PCR product to the SuperFilter 100 membrane. Primers, nucleotides, single-stranded products, salts, and other impurities pass through the

SuperFilter 100 into the waste reservoir at the bottom of the vacuum filtration block.

6. Shut off the vacuum and add 800 μl of Wash Buffer to eachwell of the SuperFilter 100 with a 12-channel pipetting device. Apply a gentle vacuum until all of the Wash Buffer has passed through the SuperFilter 100 membrane. The 800 μl of Wash Buffer used in the first wash step is necessary to remove Binding Buffer and PCR sample that adheres to the walls of the SuperFilter 100 during mixing.

7. Shut off the vacuum and add 100 μl per well of Wash Buffer to the SuperFilter 100 with a 12-channel pipetting device. Apply a gentle vacuum until all of the Wash Buffer has passed through the membrane. Repeat this step with an additional 100 μl of Wash Buffer. The second and third wash steps remove additional trace contaminants from the bound PCR Product.

8. Apply a full vacuum for 3 minutes to dry the SuperFilter membrane. This removes small amounts of Wash Buffer that may interfere with the elution step and assists in fixing the DNA to the filter prior to elution. 9. Remove the SuperFilter 100 from the vacuum manifold and place it on an unmarked 96- well microplate. 10. Centrifuge the two plates for 5 minutes at ambient temperature in a microplate centrifuge (~500xg) to remove trace amounts of Wash Buffer. This step aids in eluting the DNA from the SuperFilter and improves yield. 11. Discard the unmarked microplate containing the residual wash Buffer.

12. Transfer the SuperFilter 100 containing the bound PCR product onto a marked 96-well microplate. 13. Re-hydrate the Anay It™ SuperFilter by adding 75 μl per well of H₂0 (ph=8.0) with an automatic pipetting device. For maximal DNA recovery, be sure to add the 0.1 X TE directly onto the surface of the SuperFilter membrane. The mild elution buffer (ImM TrisCl, 0.1 mM EDTA) is used to minimize the interference of the buffer in downstream applications.

Attaching hypersensitivity relevant genes to glass slide

The genes to be attached to the glass slides are amplified as provided herein. An important modification to the amplification process is the inclusion of amine primers, which can be obtained from any commercial source, i.e. Synthegen, such that a reactive amine group, a derivative thereof, or another reactive group is included in the amplified product. The amplified product is purified by any number of methods disclosed herein and immobilized or "spotted" onto a solid substrate, such as a glass slide, which can react with the amine group on the amplified product and form a covalent linkage. An MD Generation II Anay Spotter main instrument (Molecular Dynamics, 928 East

Arques Avenue, Sunnyvale CA 04-86-4520) was used for spotting the hypersensitive genes according to following parameters:

MD ARRAY SPOTTER OPERATION The terminology and equipment used in this example comprised the following:

Spotter: MD Generation II Anay Spotter main instrument

Spotting Chamber: Area of spotter enclosed in glass which houses the pins, plates, trays and most spotter machinery.

Controller: Dedicated Dell Computer and Monitor to right of Spotter Unit Pins: (6) fine tubes in the Spotter Unit which pick-up and spot the Target

Slides: Std. size glass microscope slides with a special coating on one side

Plates: Plastic 96 well plates which hold the Target solution to be spotted

Target: A solution of PCR product which the spotter deposits on the slides.

N2 Tank: 5 ft. high steel gas tank labeled "Nitrogen, Compressed" N2 : The N2 gas from the N2 tank

Air Conditioner: Kenmore air conditioner installed in window of spotting chamber

Humidifier 1 : Essick 2000 Evaporative Cooler against the window Humidifier 2: Bemis Airflow with white flexible duck into the Spotter Unit Humidifier 3: Bemis Airflow against the wall Humidifier 4: Kenmore QuietComfort 7 Vacuum Pump: Gast Laboratory Oilless Piston Vacuum Pump Dampbox: The plastic sealable container containing an NaCl / water slurry

Materials used for reagent solutions are: Nanopure water, 0.2 M KC1 (1/10 dilution of Stock 2M KCL in water), and 95% EtOH Reagent. The temperature control is adjusted to 60°. The spotter chambers are adjusted to be greater than 39 % relative humidity and less than 65° C. The spotting pins are pre-washed for 20 cycles.

Slide Preparation/Loading:

When the pre-wash is completed, the slides are first each blown with N₂ gas for about 2 seconds per side. The slides are inserted into the Spotter following Anay Spotter Run Values. The slides are aligned using a clean nanow rod orienting it on the center right edge of the slide and gently pushed to the left until the slide is aligned vertically against the metal pins. After slides are loaded and straightened, a visual check is done to make sure no more debris had fallen. The humidity is confirmed to be greater than 39% relative humidity. The MD spotter recognizes 16 plates as a maximum for a run and will pause automatically after 8 plates. The MD spotter also advances sequentially to plates in an invariable order and is not programmable to accommodate unique plate sourcing scheme. Therefore, it is important to manually rotate (or shuffle) plates to accomplish the spotting for the canine anays.

Blocking (Slide Preparation post-spotting)

This blocking procedure is important because it reduces the non-specific background signals. The amounts provided in this protocol are for 19 slides, however, a skilled artisan may make modifications accordingly. More staining dishes and slide racks will be required if more than 19 slides are to be blocked. A clean glass container is obtained and filled with Nanopure H20. The container is placed on a hot plate and heated to a high temperature. A blocking solution is made by adding 2.5 ml of 20% SDS to 500mL blocking solution bottle. The blocking solution is warmed in microwave for 2.5 minutes and checked to determine if the temperature had reached 50°C. If the temperature of the solution is not at yet 50°C, then the solution is warmed in the microwave at 10 second intervals until it reached the desired temperature. One staining dish is placed on an orbital shaker with 4x SSC solution and turned to an agitation speed of 75 φm. Slides are placed in metal racks and placed in boiling water for several minutes (i.e. 2 minutes). The slides are taken out of boiling water and allowed to cool briefly. The slides are then transfened to staining container containing 4x SSC solution on orbital shaker for several minutes (i.e. 2 minutes), rinsed with nanopure water in a staining container, and then briefly placed in blocking solution for about 15 minutes. After 15 minutes, the slides are taken out of the blocking solution and rinsed three times by dipping into three separate containers with nanopure water each time. The tops of the slides are dabbed lightly with a tissue and the slides are placed in a centrifuge for about 5 minutes at a speed of 1000 φm.

Microanay RT Reaction

An exemplary procedure for labeling the probes is as follows. Fluorescence-labeled first strand cDNA probe is made from total or mRNA by first isolating RNA from control and treated cells, disclosed supra. This probe is hybridized to microanay slides spotted with DNA specific for hypersensitivity relevant genes. The materials needed to practice this example are: total or messenger RNA, primer, Superscript II buffer, dithiothreitol (DTT), nucleotide mix, Cy3 or Cy5 dye, Superscript II (RT), ammonium acetate, 70% EtOH, PCR machine, and ice. The volume of each sample that would contain 20μg of total RNA (or 2μg of mRNA) is calculated. The amount of DEPC water needed to bring the total volume of each RNA sample to 14 μl is also calculated. If RNA is too dilute, the samples are concentrated to a volume of less than 14 μl in a speedvac without heat. The speedvac must be capable of generating a vacuum of 0 Milli-Ton so that samples can freeze dry under these conditions. Sufficient volume of DEPC water is added to bring the total volume of each RNA sample to 14 μl. Each PCR tube is labeled with the name of the sample or control reaction. The appropriate volume of DEPC water and 8 μl of anchored oligo dT mix (stored at -20°C) is added to each tube.

Then the appropriate volume of each RNA sample is added to the labeled PCR tube. The samples are mixed by pipeting. The tubes are kept on ice until all samples are ready for the next step. It is preferable for the tubes to kept on ice until the next step is ready to proceed. The samples are incubated in a PCR machine for 10 minutes at 70°C followed by

4°C incubation period until the sample tubes are ready to be retrieved. The sample tubes are left at 4°C for at least 2 minutes.

The Cy dyes are light sensitive, so any solutions or samples containing Cy-dyes should be kept out of light as much as possible (i.e. cover with foil) after this point in the process. Sufficient amounts of Cy3 and Cy5 reverse transcription mix are prepared for one to two more reactions than would actually be run by scaling up the following protocols:

For labeling with Cy3

8 ul 5x First Strand Buffer for Superscript II 4 ul 0.1 M DTT 2 ul Nucleotide Mix

2 ul of 1:8 dilution of Cy3 (i.e., 0.125mM Cy3 dCTP). 2 ul Superscript II

For labeling with Cy5

8 ul 5x First Strand Buffer for Superscript II 4 ul 0.1 M DTT

2 ul Nucleotide Mix

2 ul of 1:10 dilution of Cy5 (i.e., O.lmM Cy5 dCTP).

2 ul Superscript II

About 18 μl of the pink Cy3 mix is added to each treated sample and 18 μl of the blue Cy5 mix is added to each control sample. Each sample is mixed by pipeting. The samples are placed in a PCR machine for 2 hours at 45°C followed by 4°C until the sample tubes are ready to be retrieved. The samples are transfened to Eppendorf tubes containing 600 μl of ethanol precipitation mixture. Some of the EtOH precipitation mixture is used to rinse the PCR tubes. The tubes are inverted to mix. Samples are placed in -80°C freezer for at least 20-30 minutes. If desired, samples may be left at -20°C overnight or over the weekend.

The samples are centrifiiged for 15 minutes at 20800 x g (14000 φm in Eppendorf model 5417C) and carefully the supernatant is decanted. A visible pellet is seen (pink red for Cy3, blue for Cy5). It is a preferable to centrifuge the tubes at a fixed position so the pellet will be at a known area in the tube. In some rare instances, the probe is seen spread on one side of the tube instead of a tight pellet. If the pellet is white or nonexistent, the reaction has not occuned to maximal efficiency.

Ice cold 70% EtOH (about 1 ml per tube) is used to wash the tubes and the tubes are subsequently inverted to clean tube and pellet. The tubes are centrifiiged for 10 minutes at 20800 x g (14000 φm in Eppendorf model 5417C), then the supernatant is carefully decanted. The tubes are flash spun and any remaining EtOH is removed with a pipet. The tubes are air dried for about 5 to 10 minutes, protected from light. The length of drying time will depend on the natural humidity of the environment. For example, an environment in Santa Fe would require about 2 to 5 minutes of drying time. It is preferable that the pellet are not overdried. When the pellets are dried, they are resuspended in 80 ul nanopure water. The cDNA mRNA hybrid is denatured by heating for 5 minutes at 95 °C in a heat block and flash spun.

To purify fluorescence-labeled first strand cDNA probes, the following materials are used: Millipore MAHV N45 96 well plate, v-bottom 96 well plate (Costar), Wizard DNA binding Resin, wide orifice pipette tips for 200 to 300 μl volumes, isopropanol, nanopure water. It is highly preferable to keep the plates aligned at all times during centrifugation. Misaligned plates lead to sample cross contamination and/or sample loss. It is also important that plate carriers are seated properly in the centrifuge rotor.

The lid of a "Millipore MAHV N45" 96 well plate is labeled with the appropriate sample numbers. A blue gasket and waste plate (v-bottom 96 well) is attached. Wizard DNA Binding Resin (Promega cat#Al 151 ) is shaken immediately prior to use for thorough resuspension. About 160 μl of Wizard DNA Binding Resin is added to each well of the filter plate that is used. If this is done with a multi-channel pipette, wide orifice pipette tips would have been used to prevent clogging. It is highly preferable not to touch or puncture the membrane of the filter plate with a pipette tip. Probes are added to the appropriate wells (80 μl cDNA samples) containing the Binding Resin. The reaction is mixed by pipeting up and down -10 times. It is preferable to use regular, unfiltered pipette tips for this step. The plates are centrifiiged at 2500 φm for 5 minutes (Beckman GS-6 or equivalent) and then the filtrate is decanted. About 200 μl of 80% isopropanol is added, the plates are spun for 5 minutes at 2500 φm, and the filfrate is discarded. Then the 80% isopropanol wash and spin step is repeated. The filter plate is placed on a clean collection plate (v-bottom 96 well) and 80 μl of Nanopure water, pH 8.0-8.5 is added. The pH is adjusted with NaOH. The filter plate is secured to the collection plate with tape to ensure that the plate did not slide during the final spin. The plate sat for 5 minutes and is centrifiiged for 7 minutes at 2500 φm. If there are replicates of samples they should be pooled. To semi-quantitatively assess the incoφoration of fluorescence into cDNA probes and to concentrate probes prior to hybridization, the following material is used: 384 well, 100 μl assay plate (Falcon Microtest cat#35-3980) and Wallac Victor 1420 Multilabel counter (or equivalent).

It is preferable that a consistent amount of cDNA is pipeted into the 384-well plate wells because readings will vary with volume. Controls or identical samples should be pooled at this step, if required. The probes are transfened from the Millipore 96 well plate to every other well of a 384 well assay plate (Falcon Microtest). This is done using a multi- channel pipette. For replicate samples that have been pooled, 60 μl aliquots are transfened into wells of the assay plate.

The Cy-3 and Cy-5 fluorescence is analyzed using the Wallac 1420 workstation programmed for reading Cy3-Cy-5 in the 384-well format and the data is saved to disk. The typical range for Cy-3 (20μg) is 250-700,000 fluorescence units. The typical range for

Cy-5 (20μg) is 100-250,000 fluorescence units. Settings for the Wallac 1420 fluorescence analyzer are as follows:

C 3

CW lamp energy = 30445 Lamp filter = P550 slot B3

Emission filter= D572 dysprosium slot A4 Emission aperture = normal Count time = 0.1 s

C >

CW lamp energy = 30445

Lamp filter = D642 samarium slot B7

Emission filter= D670 slot A8

Emission aperture = normal Count time = 0.1 s

The dry-down process of the probes is as follows. Concenfration of the cDNA probes is highly preferable so that they can be resuspended in hybridization buffer at the appropriate volume. The volume of the control cDNA (Cy-5) is measured and divide by the number of samples to determine the appropriate amount to add to each test cDNA (Cy- 3). Eppendorf tubes are labeled for each test sample and the appropriate amount of control cDNA is allocated into each tube. The test samples (Cy-3) are added to the appropriate tubes. These tubes are placed in a speed-vac to dry down, with foil covering any windows on the speed vac. At this point, heat (45°C) may be used to expedite the drying process. Time will vary depending on the machinery. The drying process takes about one hour for 150 μl samples dried in the Savant. Samples may be saved in dried form at -20°C for up to

14 days. To hybridize labeled cDNA probes to single stranded, covalently bound DNA target genes on glass slide microanays, the following material are used: formamide, SSC, SDS, 2 μm syringe filter, salmon sperm DNA, hybridization chambers, incubator, coverslips, parafilm, heat blocks. It is preferable that the anay is completely covered to ensure proper hybridization.

About 30 μl of hybridization buffer is prepared per sample. Slightly more than is what is needed should be made since about 100 μl can be lost during filtration.

Hybridization Buffer: for 100 μl: • 50% Formamide 50 μl formamide

• 5X SSC 25 μl 20X SSC

• 0.1% SDS 25 μl 0.4% SDS

The solution is filtered through 0.2 μm syringe filter, then the volume is measured. About 1 μl of salmon sperm DNA (lOmg/ml) is added per 100 μl of buffer. Materials used for hybridization are: 2 Eppendorf tube racks, hybridization chambers (2 anays per chamber), slides, coverslips, and parafilm. About 30 μl of nanopure water is added to each hybridization chamber. Slides and coverslips are cleaned using N₂ stream. About 30 μl of hybridization buffer is added to dried probe and vortexed gently for 5 seconds. The probe remained in the dark for 10-15 minutes at room temperature and then is gently vortexed for several seconds and then is flash spun in the microfuge. The probes are boiled for 5 minutes and centrifuged for 3 min at 20800 x g (14000 φm, Eppendorf model 5417C). Probes are placed in 70 °C heat block. Each probe remained in this heat block until it is ready for hybridization. Pipette 25 μl onto a coverslip. It is highly preferable to avoid the material at the bottom of the tube and to avoid generating air bubbles. This may mean leaving about 1 μl remaining in the pipette tip . The slide is gently lowered, face side down, onto the sample so that the coverslip covered that portion of the slide containing the anay. Slides are placed in a hybridization chamber (2 per chamber). The lid of the chamber is wrapped with parafilm and the slides are placed in a 42°C humidity chamber in a 42°C incubator . It is preferable to not let probes or slides sit at room temperature for long periods. The slides are incubated for 18-24 hours. To obtain single stranded cDNA probes on the anay, all non-specifically bound cDNA probe should be removed from the anay. Removal of all non-specifically bound cDNA probe is accomplished by washing the anay and using the following materials: slide holder, glass washing dish, SSC, SDS, and nanopure water. It is highly preferable that great caution be used with the standard wash conditions as deviations can greatly affect data.

Six glass buffer chambers and glass slide holders are set up with 2X SSC buffer heated to 30-34°C and used to fill up glass dish to 3/4th of volume or enough to submerge the microanays. It is important to exercise caution in heating of the 2X SSC buffer since a temperature of greater than 35°C might strip off the probes. The slides are removed from chamber and placed in glass slide holders. It is preferable that the slides are not allowed dry out. The slides are placed in 2X SSC buffer but it is recommended that no more than 4 slides be placed per dish. Coverslips should fall off within 2 to 4 minutes. In the event that the coverslips do not fall off within 2 to 4 minutes, very gentle agitation may be administered. The stainless steel slide earners are placed in the second dish and filled with 2X SSC, 0.1%SDS. Then the slides are removed from glass slide holders and placed in the stainless steel holders submerged in 2X SSC, 0.1%SDS and soaked for 5 minutes. The slides are transfened in the stainless steel slide carrier into the next glass dish containing 0.1X SSC and 0.1%SDS for 5 minutes. Then the slides are transfened in the stainless steel canier to the next glass dish containing only 0.1X SSC for 5 minutes. The slides, still in the slide canier, is transfened into nanopure water (18 megaohms) for 1 minute.

To dry the slides, the stainless steel slide earners are placed on micro-canier plates with a folded paper towel underneath. The top of the slides are gently dabbed with a tissue. Then the slides are spun in a centrifuge (Beckman GS-6 or equivalent) for 5 minutes at 1000 φm. It is very important that the slides do not air dry, as this will lead to increased background.

When the examples are practiced by a skilled artisan as disclosed, an analysis of a toxicological response to an agent, for example, cadmium chloride, can be obtained.

Preparation of cDNA

The following materials were used to prepare cDNA from RNA: total or messenger RNA; 3DNA™ Submicro™ Expression Anay Detection Kit (Genisphere 3DNA 14 Phillips Parkway Montvale, NJ 07645; Kit numbers: K20F00-41 and K20F00-31); Linear

Acrylamide (Ambion); RNase free water (Ambion); 0.5M NaOH/50mM EDTA;1M Tris- HD1, pH 7.5; lOmM Tris pH 8/lmM EDTA; 3M Ammonium Acetate; 70% Ethanol (Aldrich); 100% Ethanol (Aldrich); Denhardt's Salmon Sperm DNA (Sigma); RNase Zap (Ambion); Thermal Cycler; -80°C Freezer; Heat block; 4°C Microfuge; SpeedVac; MicroAnay slides; Coverslips; Hybridization Chamber; 42°C Humidity Chamber;

Parafilm.

For synthesis of cDNA, prepare 2 separate identical reactions for each sample. In a PCR or 1.5ml tube combine: 1.5ug lymphocyte RNA in 7ul DEPC treated water (if sample is too dilute, concentrate it in the SpeedVac at room temperature), and 3ul RT Primer. Separate tubes for freated and untreated RNA. Heat mixture to 80°C for 10 minutes, 4°C for 2 minutes. Place samples on ice and add the following: 4ul 5X RT buffer, lul dNTP mix, 4ul RNase free water, and lul Reverse transcriptase enzyme. Gently mix and centrifuge the contents of the tube. Incubate at 42°C for 1.5 to 2 hours. Stop the reaction by adding 3.5ul of 0.5M NaOH/50nM EDTA. Incubate at 65°C for 10 minutes to denature the DNA/RNA hybrids. Neutralize the reaction with 5ul of 1 M Tris-HCL, pH 7.5.

Transfer to 1.5ml tube if in PCR tube and add 38.5ul of lOmM Tris, pH8/lmM EDTA. Precipitate by adding the following to each tube: 4ul Linear acrylamide, 175ul 3M Ammonium Acetate, and 625ul 100% Ethanol. Incubate at -80°C for 30 minutes. Centrifuge at 13,000 φm in 4°C centrifuge for 15 minutes. Carefully decant supernatant.

11. Taqman® RT Reaction Taqman® technology from Roche Molecular System was used in the following manner. The mRNA was converted to cDNA using 3μg total RNA and 1.5μl random hexamer primers. After a 10 minute incubation at 70°C the following components were added to the reaction mixture: 6μl of 5x first strand buffer, 3μl 0.1 DTT, 1.5μl lOmM dNTPs, 1.5μl Superscript enzyme and 6.5μl DEPC-treated water. The reaction is incubated for two hours at 45°C and 1 μl of this reaction is used for the Taqman® assay.

For the Taqman® assay 50μl reactions were set up with Rnase-free water, Taqman® Universal PCR Master Mix, target and control primers /probes and cDNA.

Real time PCR can be performed using the Taqman® assay . The method measures PCR product accumulation with a dual-labeled fluorogenic probe. The probes are labeled with 6-FAM on the 5' end and TAMRA on the 3' end. TAMRA is a quencher dye. This assay exploits the 5 '-3' exonuclease property of Taq polymerase. When the probe hybridizes to its target the reporter dye (FAM) is cleaved by the 5' exonuclease activity of the Taq polymerase and can emit a fluorescent signal. With increasing cycles of amplification more signal is emitted and detected using an ABI 7700 sequence detector. For each gene, a set of two primers and a fluorogenic probe are designed and synthesized.

For quantitation of mRNA the best design for probes and primers requires primers to be positioned over exon-intron junctions. This mles out amplification of contaminating genomic DNA. For initial studies, primer and probe sets have been designed for 13 genes that were up- or down-regulated by penicillin in differential display experiments. The probes and primer sets were tested for their ability to amplify genomic DNA. If genomic

DNA was amplified, the probes and primers for that particular gene were not used for the Taqman® assay. Figure 9 and 10 show results obtained with a penicillin sensitive person as well as a penicillin refractive person. The genes in these figures are as follows: 1 A is Inhibitor of apoptosis protein- 1, 76B is cyclin D2, 142B is Fc-gamma-receptorllA (FCGR2A), 167B is chromosome 16 clone, RP11-296110 198 A is ribosomal protein S24 (RPS24a), 198B is ribosomal protein S24 (RPS24a). The Y-axis refers to levels of gene expression based on ABI Prism 7700 Realitive Quantification Software, in which cDNA levels are measured based on Ct (Cycle Threshold) values between control and treated samples.

Example8: Differential Protein Expression in Penicillin Treated and Untreated Human Lymphocytes from Penicillin Sensitive and Refractive Individuals

Protein expression in lymphocytes was studied using two technologies, SDS Polyacrylamide Electrophoresis (SDS-PAGE) and Surface Enhanced Laser Desoφtion/Ionization Time-of-Flight Mass Specfrometry (SELDI-TOF) of proteins applied to ProteinChips. Differences in protein profiles, treated and untreated, for sensitive and refractive samples were observed using both techniques. The following methods were used: Cell Preparation

For these experiments, blood was drawn from four refractive (control) individuals and two penicillin-sensitive individuals. White blood cells were isolated and cultured for 24 hours, using standard cell culture conditions. The cultures were split, half the cells were treated with penicillin, and all cells were grown for an additional 24 hours. Media was removed by centrifugation. Cells were then subjected to hypotonic lysis in nanopure water, followed by centrifugation to remove solid cellular debris. The supernatants were frozen prior to protein experiments. Cell lysates were concentrated by vacuum centrifugation prior to SDS-PAGE and ProteinChip experiments.

SDS-PAGE Proteins were electrophoresed using a Bio-Rad MiniProtean gel apparatus, on ReadyGel Precast 4-20% acrylamide gels, using the standard method of Laemmli. For each concentrated lysate, 20 ul sample was mixed with 5 ul 5X SDS sample buffer. The samples were boiled for 10 minutes in the presence of 2-mercaptoethanol and half of each sample was loaded into conesponding wells on two identical gels. Two stains were used to visualize proteins in the replicate gels, Coomassie Blue and Ruby SYPRO (BioRad). Bands were observed directly for Coomassie stained gels, and by fluorescence scanning (Hitachi Scanner) for Ruby stained gels. All gels were dried in cellophane membranes as permanent records stored in (the laboratory notebook).

ProteinChip/SELDI-TOF

ProteinChips were obtained from Ciphergen Biosystems. Chips containing spots with hydrophobic (H4) and normal phase (NP) chromatographic surfaces were used. For the H4 surface, 1 ul acetonitrile was pipetted onto each spot to pre- wet the C- 18 surface. Nanopure water was used to to wet the normal phase chip. Three microliters of concentrated lysate was added to each spot on replicate chips, with eight spots/samples per chip. The spots were dried at room temperature, then washed with 10% acetonitrile and nanopure water, for the H4 and NP chips, respectively. Washes were performed by pipetting 5 ul wash solution onto each spot, allowing a 5 minute incubation to resolubilize non-specifically bound biomolecules, and pipetting in and out five times prior to removing the wash buffer. Spots were dried under a 100 Watt bulb (placed 2 feet above benchtop). Each spot was then treated with 0.5 ul sinapinic acid (saturated in 50% acetonitrile, 0.5% trifluoroacetic acid), which acts as an energy absorbing "mafrix" to assist laser ionization of proteins. Proteins were detected directly from the chips using a PBS-II mass spectrometer (Ciphergen Biosystems). Spectra were electronically stored in poweφoint files.

Results Using both techniques, differences were observed in the protein profiles of treated and untreated, sensitive and refractive samples. The SDS-PAGE 1-D data is low resolution, but clearly shows increased production of at least four proteins in penicillin- treated sensitive cells, compared with the controls. Sensitivity was comparable for SELDI- TOF on ProteinChips is a more sensitive technique, and showed hundreds of peaks in each profile. The differences in protein spectra were striking, showing that refractive cells exhibit protein induction that is different than the induction in sensitive cells. While many differences were observed (at least 5-10 proteins), the similarities in the overall profiles was striking, and permits reasonable difference comparison by providing internal standards.

TABLE 1

atovaquone atropine attapulgite azathioprine azelastine azithromycin aztreonam bacampicillin baclofen beclomethasone belladonna benazepril benazepril benzodiazepines benzoyl peroxide benztropine beta carotene betamethasone betamethasone betamethasone valerate bethanechol bisacodyl bismuth subsalicylate plus bisoprolol/HCTZ bleomycin bradykinin antagonist bromfenac brominide tartrate bromocriptine bronchodilators buclizine budesonide bumetanide bupropion HCL buspirone busulfan calcipotriene calcitonin salmon calfactant candesartan cilexetil capsaicm captopril carbamazapine carbenicillin carbidopa carboplatin carisoprodol carmustine carved ilol cefaclor cefepime cefprozil ceftibuten cefuroxime celecoxib cephalexin cephalosporins cerivastatin cetirizine chenodiol chlophedianol chloral hydrate chlorambucil chloramphenicol chloroquine chlorpropamide chlorthalidone chlorzoxazone cholestyramine cimetidine cinoxacin ciprofloxacin

(+)-cis-3,5-dimethyl-2-(3- pyridyl)thiazolodin-4 cisapride cisplatin citalopram clarithromycin clavulanate clavulanate clavulanic acid clidinium clindamycin clofibrate clomiphene clonazepam clonidine clotrimoxazole cloxacillin clozapine codeine colchicine colestipol collagen-alginate conjugated estrogens copolymer-1 cortisone courmaπn cromolyn cyclacillin cyclandelate cyclizine cyclobenzaprine cyclopegic cyclopentolate cyclophosphamide cycloserine cyclosporine cyclosporine A cytoxin dalteparin injection danazol dantrolene dapsone daunomycin daunorubicin dehydrocholic acid desmopressin desogestrel dexamethasome dextromethorphan dextrothyroxine diazepam diazoxon dichloralphenazone diclofenac diclofenacdihydrazine dicloxacillin dicyclomine didanosine difenoxin digitalis glycosides digoxin dihydrazine dihydroergotamine mesylate dihydrolazine diltiazem dimethyl sulfoxide dinoprostone dione diphenidol diphenoxylate dipyridamole dipyridamole disopyramide disulfiram divalproex divalproex sodium docusate sodium dolasetron mesylate donepezil doxazosin doxercalciferol doxombicin doxycycline enalapril enoxapaπn entacapone ephedrine epirubicin eptifibatide ergoloid mesylates ergonovine erythromycin estradiol estramustine etanercept ethacrynic acid ethchlorvynol ethinamate ethinyl estradiol ethinyl estradiol ethionamide etidronate etoposide etretinate exemestane famciclovir famotidine felbamate felodipine felodipine SR fenofibrate fenoldopam mesylate fentanyl citrate fexofenadine fialuridine finasteride flavoxate flecainide acetate flosequinan fluconazole flunisolide fluoroquinolones fluorouracil fluoxetine flutamide fluticasone fluticasone fluticasone propionate fluvastatin fluvoxamine maleate foscarnet sodium fosinopril fosphenytoin furazolidone furosemide gabapentin ganciclovir ganirelix acetate gemcitabine gemfibrozil glimepiride glipizide glucagon glyburide glycopyrrolate gold compounds gold sodium thiomalate granisetron grepafloxacin griseofulvin guaifenesin guanabenz guanadrel guanethidine guanfacine haloperidol heparin hismanol hydantoin hydralazine hydrochlorothiazide hydrocodone hydrocortisone hydroxychloroquine hydroxy urea hydroxyzine hyoscine hyoscyamine hyoscyamine hyperozia ibuprofen ibutilide fumarate imiglucerase injection imiquimod 5% cream inactivated hepatitis A vaccine indapamide indinavir indomethacin insulin interferon-beta-1a (recombinant) interferon-beta-1b (recombinant) iodinated glycerol iodoquinol ipecac iphosphamide ipratropium irbesartan irinotecan isometheptene isoniazid isoproterenol isosorbide mononitrate S.A. isotretinoin isoxsuprine isradipine itraconazole kanamycin ketoconazole ketorolac lactulose lamivudine, 3TC lamotrigine lansoprazole latamoxef atanoprost eflunomide etrozole eucovonn euprolide evamisole evetiracetam evobupivacaine evocabastine evocarnitine evodopa evofloxacin evonorgestrel evothyroxine idocaine incomycin iposomal amphotericin B isinopril ispro insulin ithium

^■norgestrel

•norgestrel/ethinyl estradiol omustine operamide oracarbef oratadine

Loratidine/Pseudoephedrine orazepam osartan ovastatin oxapine magnesium sulfate maprotiline masoprocol mazindol mecamylamine mechlorethamine meclizine medroxyprogesterone medroxyprogesterone mefloquine melatonin melphalan menotropin meprobamate merbarone mercaptopunne meropenem mesalamine metformin methenamine methicillin methotrexate methylcellulose methyldopa methylergonovine methylphenidate methylprednisolone methyprylon methysergide metoclopramide metoprolol metoprolol metronidazole metyrapone metyrosine mexiletine mibefradil miconazole cream 2% miglitol minocycline minoxidil misoprostol misoprostol mitotane mixed amphetamines moclobemide molindone mometasone moricizine moxifloxacin mupirocin nabilone nabumetone nafarelin nafcillin nalidixic acid naltrexone naproxen naratriptan natamycin navirapme nedocromil nefazodone neomycin

Neomycin/Polymx/HC neostigmine nicardipine nicorandil nicotine nifedipine nimodipine nitrofurantoin nitroglycerin nizatidine norethindrone norethindrone/ ethinyl estradiol norgestimate norgestimate/ethinyl estradiol norgestrel norgestrel/ethinyl estradiol nylidrin nystatin ofloxacin olsalazine omeprazole orphenadrine oxacillin oxaprozin oxtriphylline oxybutynin oxycodone oxymetazoline paclitaxel pancreatin pancrelipase papaveπne paraldehyde paramethasone paregoric paroxetine pediculisides pemoline penicillamine penicillin pentamidine pentoxifylline pepsin pergolide perhexiline perindopril perphenazine pexiganan acetate phenazopyridine phendimetrazine phenformin phenobarbital phenolphthalein phenothiazines phentermine phenylephrine phenylephrine phenylpropanolamine phenylpropanolamine phenytoin pilocarpine pioglitazone piroxicam podophyllum poloxamer 188 polycarbophil calcium polyethylene glycol polythiazide potassium chloride potassium iodide potassium phosphates pramipexole pravastatin prazosin prednisolone prednisone pπmaquine primethamine primidone probenecid probucol procainamide procarbazine progestins promethazine propafenone propantheline propoxyphene propranolol propulsid pseudoephedhne psoralens psyllium pyridostigmine pyridoxine (vitamin b-6) quinacrine quinapril quinidine quinine rabeprazole raloxifene ramipril ranitidine recombinant clotting factor VIII recombinant interferon alpha-2b recombinant OspA remoxipide reserpine rezulin ribavirin rifampin rimantadine risedronate risperidone ritodrine rosiglitazone salicylates salmeterol saquinavir scopolamine seldane selegiline sertraline sibutramine sildenafil citrate simethicone simvastatin s-mephenytoin sodium ferric gluconate soman somatostatin sotalol spironolactone stanol esters streptozotocin succinimide sucralfate sulfacytine sulfadoxine sulfamethoxazole sulfamethoxazole sulfasalazine sulfinpyrazone sulfisoxazole sumatriptan

(s)-warfarin tacrine tamoxifen tamsulosin telmisartan temazepam terazosin terbinafine HCl terbutaline sulfate terfenadine terpin hydrate testolactone tetracycline HCl tetracyclines theophylline thiamine thiazide thioguanine thiopurine thiothixene tiagabine ticlopidine tienilic acid timolol tiopronin tirofiban tobramycin tobramycin/dexamethasone tocainide tolbumamide tolcapone tolterodine topotecan toremifene tramadol trandolapril trastuzumab trazodone tretinoin triamcinolone triamterene/HCTZ triamterine triamterine triazolam trihexyphenidyl trilostane trimeth/sulfameth trimethobenzamide trimethoprim troglitazone trovafloxacin urokinase ursodiol valproic acid valsartan vancomycin venlafaxine verapamil vincristine warfarin xanthine xylometazoline zafirlukast zalcitabine zidovudine zolpidem

TABLE 2

TABLE 3

Gene Name Genbank

Accession No.

Ataxia telangeictasia U33841

ATF4 (activating trxn factor 4) D90209

ATP-dep. Helicase II (70kDa) M32865

ATP-dep. Helicase II (Ku80) M30938

Bax (alpha) L22473

Bcl-xL Z23115 c-Abl M 14752 c-Fos K00650

Chk1 AFO 16582 c-H-Ras J00277 c-Jun J04111

Clusterin (serum protein 40) X14723 c-Myc X00364

Connexin 32 (gap junction protein) X04325

Cyclin G D78341

Cytochrome P-1-450 (cyp1A1) K03191

DNA binding protein inhibitor ID-2 D13891

DNA dependent helicase L36140

DNA dependent protein kinase U47077

DNA ligase IV X83441

DNA polymerase alpha X06745

DNA repair protein (Rad 50) U63139

DNA repair protein XRCC1 M36089

DNA topoisomerase I J03250

ERCC1 (excision repair protein) M13194

DNA repair helicase II ERCC-3 M31899

Excision repair ERCC-5 L20046

Gadd153 S40706

Gadd45 M60974

Glutathione Peroxidase M21304

HDLC1 U32944

Hsp70 M11717

Hsp90 X15183

ICE Rel II U28014

Mdm-2 U33199

Mdr-1 M 14758

MnSOD Y00985

Mut S homologue (hMSH2) U04045

MUTL homolog=hMLH1 U07418

Poly (ADP-ribose) polymerase (PARP) M32721/X56140

Prolifer.cell nuclear antigen (PCNA) J04718

RAD L24564 RAD51 homolog D 13804

RNA-dependent Helicase (DEAD-box protein p72) U59321

SQM1 M33374

Stress activated protein kinase JNK1 L26318

UV Excision repair protein RAD23 (XP-C) D21090

Vascular cell adhesion molecule 1 (VCAM-1) M73255

Alpha-Tubulin K00558

Beta-Actin X00351

Glucose-6-phosphate dehydrogenase (G6PD) X03674 cytochrome p-4504A L04751 connexin 40 L34954

Bak U16811

Collagenase, type I interstitial X54925

G T mismatch binding protein U28946

Mismatch repair/binding protein (hMSH3) U61981

DNA mismatch repair protein (hPMS2) U 14658

Apolipoprotein A-ll M29882

Acyl CoA dehydrogenase U 12778

Carnitine palmitoyl CoA transferase M58581

Hepatic lipase J03540

Ornithine decarboxylase M 16650

Superoxide dismutase Cu/Zn (SOD) K00065

Ref-1=redox factor S43127

Thioredoxin J04026

Glutathione synthetase L42531

Glutathione reductase X15722

Thymidine kinase K02581

Bag-1=bcl-2 S83171

BRCA1 U 14680

Phenol sulfotransferase U26309

Aldehyde dehydrogenase 1 (ALDH-1) K03000

Aldehyde dehydrogenase 2 (ALDH-2) K03001

12-lipoxygenase M58704

Phospholipase A2 M86400

Calnexin M94859

Apolipoprotein CHI. X01388

Branched chain Acyl-CoA Oxidase X95190

Cyclin dependent kinase 4 (cdk4) M 14505

ERp72 J05016

MCL-1 L08246

HMG CoA reductase M11058

Lipopolysaccharide binding protein M35533

Lysyl oxidase M94054

Famesol Receptor U68233

Osteopontin J04765

P38 mitogen activated protein (MAP) kinase L35253

Peroxisomal acyl-CoA oxidase X71440 Uncoupling protein 2 (UCP2) U82819

Very-long-chain acyl-CoA dehydrogenase D43682

Vimentin X56134

EGR1 X52541

GRP94 X15187

P53 K03199

Defender against cell death-1 D15057

Hypoxanthine-guanine phosphonbosyltransferase V00530

Aspartate aminotransferase, mitochondrial M22632

Creatine kinase B L47647

Peroxisome assembly factor-1 M86852

T-cell cyclophilin Y00052

Transferrin M 12530

UDP-glucuronosyltransferase 2B AFO 16492

Octamer-binding protein 1 X13403

E-cadherin L08599

Catalase X04076

11 beta-hydroxysteroid dehydrogenase type II U14631

Bilirubin UDP-glucuronosyltransferase isozyme 1 M57899

Calreticulin M84739

Calcineurin-B M30773

Catechol-O-methyltransferase M58525

Fas antigen M67454

DNA repair and recombination homologue (RAD52) L33262

Flavin-containing monooxygenase 1 M64082

Gamma-glutamyl transpeptidase L20490

Insulin-like growth factor binding protein 1 M31145

Oxygen-regulated protein 150 U65785

Thymidylate synthase X02308

Biliverdin reductase U34877

Adenine nucleotide translocator 1 J02966

Hepatocyte nuclear factor 4 X76930

RANTES M21121

Phosphoglycerate kinase V00572

PAPS synthetase Y10387

Plasminogen activator inhibitor 2 M 18082

Enolase alpha M 14328

Interferon inducible protein 15 M 13755

Insulin-like growth factor I M37484

Platelet/endothelial cell adhesion molecule-1 M28526

60S ribosomal protein L6 X69391

FosB L49169

Alpha-catenin D 13866

FEN-1 (endonuclease) L37374

GOS24 (zinc finger transcriptional regulator) M92843

Caspase 8 (FLICE) U58143

Caspase 3 (CPP32-beta) U 13738 Caspase 7 (Mch3-alpha) U37448

Intercellular adhesion molecule-3 X69819

Phosphoenolpyruvate carboxykinase X92720

Alpha-1 acid glycoprotein M 13692

IkB-a M69043

Protein-tyrosine phosphatase M83738

Ubiquitin conjugating enzyme (Rad6 homolog) M74524

Alpha-2-macroglobulin M11313

Zinc finger protein 37 AF022158

Cyclin-dependent kinase inhibitor p27kip1 U 10906

Caspase 1 U 13697

Organic anion transporter 1 AF057039

Alcohol dehydrogenase 2 M24317

Alchohol dehydrogenase 4 M 15943

Annexin V M21731

Calbindin-D (28kDa) X06661

Colony-stimulating factor-1 M37435

Hypoxia-inducible factor 1 alpha U22431

Growth arrest-specific protein 1 L13698

Inhibitor of apoptosis protein-1 AF070674

Nucleic acid binding protein U 19765

OX40 ligand X79929

Retinoic acid receptor gamma 1 M38258

Cytochrome c oxidase subunit IV M34600

Glutathione S-transferase theta-1 X79389

Survivin NM001168

STAT 3 AJ012463

Growth arrest-specific protein 3 L03203

Cyclin D3 M92287

ID-1 X77956 lnterleukin-1 beta X02532 lnterleukin-8 Y00787

Monocyte chemotactic protein-1 S69738

Phenylalanine hydroxylase K03020

Prohibitin S85655

Cathepsin L M20496

Transthyretin X59498

Stromelysin-1 X05232

Spermidine/spermine N1-acetyltransferase (SSAT) M55580

Ferritin H-chain L20941

Transferrin receptor M11507

Ceruloplasmin M 13699

Glucosylceramide synthase D50840

Leukemia inhibitory factor (LIF) X13967

Integrin beta-1 X07979

Vascular endothelial growth factor receptor 1 (flt-1 ) X51602

Urokinase plasminogen activator receptor (uPAR) U08839 c-fms X03663 c-erb B-2 X03363

C5a anaphylatoxin receptor M62505

FYN proto-oncogene NM002037

Peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA L07077 dehydrogenase

Nucleoside diphosphate kinase beta isoform X73066

Myelin basic protein M 13577

Peroxisomal 3-oxoacyl-CoA thiolase (=rat peroxisomal X12966

3-ketoacyl-CoA thiolase 2)

Prostaglandin H synthase S36271

Retinoid X receptor alpha NM002957 lnterleukin-13 X69079

Tryptophanyl-tRNA synthetase (WRS) M61715

Silencer of death domains AF111116

Mannose receptor J05550

Death receptor 5 (DR5) AF016268

Neomycin U55761

TABLE 4

(clone hKvBeta3) K+ channel beta subunit

APO-1 cell surface antigen

11-beta hydroxysteroid dehydrogenase type II

12-lipoxygenase

17-beta hydroxysteroid dehydrogenase

25-hydroxyvitamin D3-1 alpha-hydroxylase

60S ribosomal protein L6

6-C-kine

6-O-methylguanine-DNA methyltransferase acety I hydrolase IB beta-subunit

Acid ceramidase actin-binding protein (filamin) (ABP-280)

Activating transcription factor 2

Activating transcription factor 3

Activating transcription factor 4

Activin beta E

Activin receptor type II

Acyl - CoA dehydrogenase

Acyl CoA Carrier Protein

Adenine nucleotide translocator 1

Adenylyl cyclase-associated protein (CAP)

Adhesion protein (SQM1)

Alanine aminotransferase

Alcohol dehydrogenase 1

Alcohol dehydrogenase 2

Alcohol dehydrogenase 3

Alcohol dehydrogenase 4

Alcohol dehydrogenase 7

Aldehyde dehydrogenase 1

Aldehyde dehydrogenase 2

Aldehyde dehydrogenase 3

Aldose reductase

Alpha 1-antitrypsin

Alpha 1-inhibitor III

Alpha interferon

Alpha(l)procollagen

Alpha-1 acid glycoprotein

Alpha-1 antichymotrypsin

Alpha-2 macroglobulin

Alpha-2 microglobulin

Alpha-catenin

Alpha-tubulin

Amyloid protein homologue

Androgen receptor

Annexin V Antiquitin, 26g turgor protein homolog

Aorta caldesmon

APC gene

Apolipoprotein A1

Apolipoprotein All

Apolipoprotein CHI

Apolipoprotein E

Aryl hydrocarbon receptor

Aspartate aminotransferase, mitochondrial

Ataxia telangeictasia

ATP Synthase 6

ATP-dependent helicase II (70kDa)

ATP-dependent helicase II (Ku80)

Atrial natiuretic factor

BAG-1

BAK

Bax (alpha)

Bcl-2

Bcl-3

Bcl-xL

Beta-actin

Beta-chemokine I-309

Bile salt export pump (sister of p-glycoprotein)

Biliary glycoprotein

Bilirubin UDP-glucuronosyltransferase isozyme 1

Biliverdin reductase

B-myb

Bone morphogenetic protein-4

Bone sialoprotein gene

Brain-derived neurotrophic factor

Branched chain acyl-CoA oxidase

BRCA1

BR-cadherin

Breast basic conserved gene (ribosomal protein

L13)

Breast cancer resistance protein (BCRP)

C10 beta-chemokine

C4b-binding protein

C5a anaphylatoxin receptor c-abl

Calbindin-D (28kDa)

Calbindin-D (9K)

Calcineurin-B

Calnexin

Calprotectin

Calreticulin canalicular multispecific organic anion transporter Carbonic Anhydrase III

Carcinoembryonic antigen (CD66e)

Carcinoembryonic antigen family member 2 cardiac gap junction protein

Carnitine palmitoyl-CoA transferase

Casein kinase 1 delta

Caspase 1

Caspase 2 (Nedd2)

Caspase 3 (CPP32-beta)

Caspase 5 (ICE rel-lll)

Caspase 6 (Mch2-alpha)

Caspase 7 (Mch3-alpha)

Caspase 8 (FLICE)

Catalase

Catechol-O-methyltransferase

Cathepsin G

Cathepsin L

Caveolin-1

CCAAT/enhancer-binding protein alpha

CCAAT/enhancer-binding protein epsilon

CCR-5

CD44 (metastasis suppressor gene)

CD64 (Fc gamma)

Cell division cycle protein 2

Cell division cycle protein 25

Cellular retinoic acid binding protein 1

Cellular retinoic acid binding protein 2 c-erb B-2 c-erbA-1

Ceruloplasmin (ferroxidase) c-fms (CSF-1 receptor) c-fos

CHD2

Checkpoint kinase-1

Cholesterol esterase c-H-ras

CIG49 (cig49) c-jun

Clone 22 mRNA, alternative splice variant alpha-

1

CLP

Clusterin c-myb c-myc binding protein

Collagen type II

Colony-stimulating factor-1

Complement component C3 Connexin 30

Connexin-32 (aka gap junction protein)

Connexin-40

Corticosteroid binding globulin

Corticotropin releasing hormone

C-reactive protein

Creatine kinase B

Csa-19

CTCF

CXCR4

Cyclin A1

Cyclin D1

Cyclin D3

Cyclin dependent kinase 1

Cyclin dependent kinase 2

Cyclin dependent kinase 4

Cyclin dependent kinase inhibitor 1A

Cyclin E

Cyclin G

Cyclin-dependent kinase 4 inhibitor B (P16)

Cyclin-dependent kinase inhibitor P27Kip1

Cyclooxygenase 2

Cysteine protease CPP32 isoform alpha

Cystic fibrosis transmembrane conductance regulator

Cytochrome c oxidase subunit III

Cytochrome c oxidase subunit IV

Cytochrome P450 11A1

Cytochrome P450 17A

Cytochrome P450 1A1

Cytochrome P450 1A2

Cytochrome P450 1 B1

Cytochrome P450 2A1

Cytochrome P450 2A3

Cytochrome P450 2A6

Cytochrome P450 2B1

Cytochrome P450 2B10

Cytochrome P450 2B2

Cytochrome P450 2C11

Cytochrome P450 2C12

Cytochrome P450 2C19

Cytochrome P450 2C9

Cytochrome P450 2D6

Cytochrome P450 2E1

Cytochrome P450 2F2

Cytochrome P450 3A1

Cytochrome P450 3A4

no Cytochrome P4504A

Cytochrome P4504A1 cytoskeletal gamma-actin

Damage-specific DNA binding protein p48 subunit

Death receptor 5 (DR5)

Defender against cell death-1

Deleted in colorectal cancer

Delta-like protein

Diacylglycerol kinase zeta

Dihydrofolate reductase

Disulfide isomerase related protein (ERp72)

DNA binding protein inhibitor ID2

DNA dependent helicase

DNA dependent protein kinase

DNA ligase I

DNA ligase III

DNA ligase IV

DNA mismatch repair protein (MLH1)

DNA mismatch repair protein (PMS2)

DNA mismatch repair/binding protein (MSH3)

DNA polymerase alpha

DNA polymerase beta

DNA repair and recombination homologue (RAD

52)

DNA repair helicase II ERCC-3

DNA repair protein (RAD 50)

DNA repair protein (XRCC1)

DNA replication factor C (36kDa)

DNA topoisomerase I

DNA topoisomerase II

DNA-binding protein (APRF)

DOC-2

Dopamine beta-hydroxylase

Dopamine receptor D2

DRA

Dynamin (DNM)

Dynein light chain 1

E2F-1

Early growth regulated protein 1

E-Cadherin

ECE-1 (endothelin converting enzyme)

ELAV-like neuronal protein-2 Hel-N2

Elongation factor 1 -alpha 1 (PTI-1)

Endothelin-1

Enolase alpha enteric smooth muscle gamma-actin

in Eosinophil-derived neurotoxin

Eotaxin

Epidermal growth factor

Epoxide hydrolase

ERA-B

ERCC 1 (excision repair protein)

ERCC 3 (DNA repair helicase II)

ERCC 5 (excision repair protein)

ERCC 6 (excision repair protein)

Erythrocyte membrane protein

Erythropoietin

Erythropoietin receptor

E-Selectin

Estrogen receptor

Extracellular-signal-regulated kinase 1

Famesol receptor

Fas antigen

Fas associated death domain (FADD)

Fas ligand

Fas/Apo1 receptor

Fatty acid synthase

Fatty acyl-CoA oxidase

Fatty acyl-CoA synthase

FEN-1 (endonuclease)

Ferritin H-chain

FGF-1

FGF-7

Fibrinogen gamma chain

Fibronectin receptor

FIC1

Filaggrin

Flavin containing monooxygenase 1

Flavin containing monooxygenase 3 for gamma-interferon inducible early response gene (with homology to platelet proteins)

FosB

Fra-1

Fucosyl transferase (alpha-1 ,2- fucosyltransferase)

Fyn proto-oncogene

Gadd153

Gadd45

Galanin

Gamma glutamylcysteinyl synthetase

Gamma-glutamyl hydrolase (hGH)

Gamma-glutamyl hydrolase precursor

Gamma-glutamyl transpeptidase Garg-16

GAS-7

GCLR

GCLS

Gelsolin

Glucocorticoid receptor

Glucose-6-phosphate dehydrogenase

Glucose-regulated protein 170

Glucose-regulated protein 58

Glucose-regulated protein 78

Glucose-regulated protein 94

Glucosylceramide synthase

Glutamic-oxaloacetic transaminase

Glutamic-pyruvic transaminase

Glutamine synthetase

Glutaredoxin

Glutathione peroxidase

Glutathione reductase

Glutathione S-transferase alpha subunit

Glutathione S-transferase theta-1

Glutathione S-transferase Ya

Glutathione synthetase

Glyceraldehyde 3-phosphate dehydrogenase

Gonadotropin (alpha subunit)

GOS24 (zinc finger transcriptional regulator)

Granulin

Granulocyte-macrophage colony-stimulating factor

Growth arrest-specific protein 1

Growth arrest-specific protein 3

GT mismatch binding protein

Hamartin (TSC1)

H-cadherin

Heat shock protein 12

Heat shock protein 27

Heat shock protein 47

Heat shock protein 70

Heat shock protein 90

Helicase-like transcription factor

Heme binding protein 23

Heme oxygenase-1

Hemopexin

Hepatic lipase

Hepatocyte growth factor

Hepatocyte growth factor activator

Hepatocyte nuclear factor 4

Histamine N-methyltransferase Histidine decarboxylase

Histone 2A

Histone 2B

Histone deacetylase 1 (HDAC-1) hMEF2C, myocyte enhancer-binding factor 2

HMG CoA reductase

HMG-I protein isoform mRNA (HMGI gene), clone 7C

Hydroxysteroid sulfotransferase a

Hypoxanthine-guanine phosphonbosyltransferase

Hypoxia-inducible factor 1 alpha

ICE-rel II (Caspase 4)

ID-1

IkB-a immunoglobulin lambda heavy chain

Immunophilin homolog ARA9

Inhibitor of apoptosis protein 1

Inhibitor of apoptosis protein 2

Insulin-like growth factor binding protein 1

Insulin-like growth factor binding protein 2

Insulin-like growth factor binding protein 5

Insulin-like growth factor binding protien 3

Insulin-like growth factor I

Insulin-like growth factor II

Integrin alpha

Integrin alpha L

Integrin betal

Integrin beta2

Integrin beta-4

Intercellular adhesion molecule-1

Intercellular adhesion molecule-2

Intercellular adhesion molecule-3

Interferon gamma

Interferon inducible protein 10

Interferon inducible protein 15

Interferon stimulatory gene factor-3 lnterleukin-1 alpha lnterleukin-1 beta lnterleukin-10 lnterleukin-12 lnterleukin-13 lnterleukin-18 lnterleukin-2 lnterleukin-3 lnterleukin-4 lnterleukin-5 lnterleukin-6 lnterleukin-8

Involucrin

IRF-7

Iron permease (FTR1)

ISG-15

Jagged 1

Jagged 2

JNK1 stress activated protein kinase

JunB

JunD

K+ channel beta 2 subunit

KAI1 metastasis suppressor gene (CD82)

K-cadherin

Keratin 4

Keratin 6 isoform K6e (KRT6E)

Keratin K17

Keratinocyte growth factor

Ki67

Ku autoimmune antigen gene (p80)

L09604

Lactate Dehydrogenase-B

Lactoferrin

Leukemia inhibitory factor (LIF)

Lipopolysaccharide binding protein

Lipoprotein lipase precursor

Liposin

Liver fatty acid binding protein

L-myc long-chain acyl-CoA synthetase

Low density lipoprotein receptor

Lung cancer antigen NY-LU-12 variant A

Luteinizing hormone

Lymphoid enhancer-binding factor-1 (LEF-1)

Lysyl hydroxylase

Lysyl oxidase macropain subunit zeta

Macrophage inflammatory protein-1 alpha

Macrophage inflammatory protein-1 beta

Macrophage inflammatory protein-2 alpha

Macrophage inflammatory protein-3 alpha

Macrophage-stimulating protein (MST1)

Macrostatin

MAD-related protein 2

Major acute phase protein alpha-1

Major basic protein

Malic enzyme Mannose receptor

MAP kinase kinase

Matrix metalloproteinase-1

Matrix metalloproteinase-2

MDM-2

MET proto-oncogene

Metallothionein 1

Metallothionein 2

Metal-regulatory transcription factor-1

Metastasis-associated mtal

Methionine adenosytransferase (MAT2A)

MHC class I

MHC class II

MHC class II transactivator

Mim

Mitochondrial ATP Synthase Subunit E mitochondrial short-chain enoyl-CoA hydratase

Mitochondrial transcription factor 1

Mitogen activated protein kinase (P38)

Mitogen inducible gene (mig-2)

MOAT-B (MRP/organic anion transporter)

Monoamine oxidase A

Monoamine oxidase B

Monocyte chemotactic protein-1

Monocyte chemotactic protein-1 receptor (CCR2)

Mr 110,000 antigen

MSH3 gene mss4, Zn2+ binding protein/guanine nucleotide exchange factor

Multidrug resistance-associated protein

Multidrug resistant protein-1

Multidrug resistant protein-2

Multidrug resistant protein-3 = cMOAT2

MUTL homologue (MLH1)

MutS Homologue (MSH2)

Myelin basic protein

Myeloid cell differentiation protein-1

Myeloid cell leukemia-1 (MCL-1)

Myeloperoxidase

Na/taurocholate cotransporting polypeptide

NADPH cytochrome P450 reductase

NADPH quinone oxidoreductase-1 (DT-

Diaphorase)

Natural killer cell-enhancing factor B

N-cadherin

Neural cell adhesion molecule (N-CAM)

Neurofibromin (NF1 tumor suppressor) neuropathy target esterase

NF-E2

NF-kappaB (p65)

Nidogen

Nitric oxide synthase-1 , inducible

NMB

Non-specific cross-reacting antigen

Notch 1

Nucleic acid binding protein

Nucleoside diphosphate kinase beta isoform nucleosome assembly protein

O-6-alkylguanine-DNA-alkyltransferase

OB-cadherin 1

Octamer binding protein 1

Octamer binding protein 2

Oncostatin M

Organic anion transporter 1

Organic anion transporter 3

Organic anion transporter K1

Organic anion transporting polypeptide 1

Organic cation transporter 1

Organic cation transporter 2

Organic cation transporter N1

Organic cation transporter N2

Ornithine decarboxylase

Osteocalcin

Osteopontin

Osteoprotegerin (TRAIUApo2L receptor)

OTK27

OX40 ligand

Oxygen regulated protein 150

Oxysterol-binding protein (OSBP)

Oxytocin receptor p190-B (p190-B)

P311 HUM (3.1) p53 p55CDC p70 ribosomal protein S6 kinase alpha-1

Pancreatitis-associated protein

PAPS synthetase

PBX2 mRNA

P-cadherin

PCDH7 (BH-Pcdh)c

PDGF associated protein

PEG3

Perlecan

Peroxisomal 3-ketoacyl-CoA thiolase 1 Peroxisomal 3-ketoacyl-CoA thiolase 2 Peroxisomal enoyl-CoA hydratase: 3- hydroxyacyl-CoA dehydrogenase Peroxisomal fatty acyl-CoA oxidase Peroxisome assembly factor 1 Peroxisome assembly factor 2 Peroxisome biogenesis disorder protein-1 Peroxisome biogenesis disorder protein-11 Peroxisome biogenesis disorder protein-4 Peroxisome hydratase

Peroxisome proliferator activated receptor alpha Peroxisome proliferator activated receptor gamma

Phenol sulfotransferase Phenylalanine hydroxylase Phosphatase 2A B56-alpha (PP2A) Phosphoenolpyruvate carboxykinase Phosphoglyceride kinase Phospholipase A2 Phosphomannomutase (PMM2) Pim1 proto-oncogene Plasma cell membrane glycoprotein plasma gelsolin

Plasminogen activator inhibitor 2 Platelet derived growth factor B Platelet/endothelial cell adhesion molecule-1 Poly(ADP-ribose) polymerase polyA binding protein Presenilin-1 Prion protein (PrP) pro-cathepsin L (major excreted protein MEP) Progesterone receptor Prohibitin Prolidase

Proliferating cell nuclear antigen gene Proliferation-associated gene A (natural killer- enhancing factor A) prolyl 4-hydoxylase beta subunit (EC 1.14.11.2) (procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase, 4-hydroxylating) Prostacyclin-stimulating factor (IGFBP-7) Prostaglandin H synthase Prostate-specific antigen protein disulfide isomerase Protein kinase C alpha Protein tyrosine phosphatase alpha Protein-tyrosine phosphatase Psoriasin 1 (S100 calcium-binding protein A7)

PTEN/MMAC1

Putative cyclin G1 interacting protein

Quinone reductase (zeta-crystallin)

RAD

RAD 51 homologue

RANTES

RAP1A (ras-related protein)

Recombination activating gene 1 (RAG-1)

Ref-1

RelB

Replication factor C, 40-kDa subunit (A1)

Replication protein A (70 kDa subunit)

Retinoblastoma

Retinoblastoma related protein (P107)

Retinoic acid receptor beta

Retinoic acid receptor gamma-1

Retinoid X receptor alpha

Retinoid X receptor beta

Retinoid X receptor gamma

Ribonucleotide reductase M1 subunit

Ribosomal protein L13A

Ribosomal protein L34 (RPL34)

Ribosomal protein L37a (RPL37A) ribosomal protein S12

Ribosomal protein S4 (RPS4X) isoform

Ribosomal protein S9

RNA-dependent helicase

SAA-3

S-adenosylmethionine synthetase

Sarcoplasmic reticulum calcium ATPase

Sarcosin

Sec23B isoform, 2450bp

Senescence marker protein-30

Serine kinase

Serum amyloid A1

Serum amyloid A2-alpha

Serum response factor

Silencer of death domains (SODD)

Small proline-rich protein (sprl)

SMT3A protein

SMT3B protein snRNP polypeptide B

Sodium/bile acid cotransporter

Sonic hedgehog gene

Sorbitol Dehydrogenase

SoxS SPARC (secreted protein acidic and rich in cysteine)

Spermidine/spermine N1-acetyltransferase

(SSAT)

Sphingomyelinase (neutral)

STAT 1

STAT 2

STAT 3

Stem cell factor

Steroid hormone receptor Ner-I

Sterol carrier protein 2

Sterol regulatory element binding protein-2

Stromelysin-1

Superoxide Dismutase Cu/Zn

Superoxide dismutase Mn

Supressor of cytokine signaling 1 (SOCS-1)

Supressor of cytokine signaling 3 (SOCS-3)

Survivin

Synapsin I

Synaptophysin II

Synaptotagmin I

Syntaxin 3

Tau protein

T-cell activation gene 3

T-cell cyclophilin

T-cell mRNA for glycyl tRNA synthetase

T-cell receptor

Tenascin

Thiol-specific antioxidant protein mRNA

Thiopurine methyltransferase

Thioredoxin

Thrombin receptor (PAR- 1)

Thrombomodulin

Thrombospondin 2

Thymidine kinase

Thymidylate synthase

Thymosin beta-10

Tight junction protein Zo-1

Tissue factor

Tissue factor pathway inhibitor

Tissue inhibitor of metalloproteinases-1

Tissue inhibitor of metalloproteinases-3

Tissue transglutaminase

TNF receptor-1 associated protein (TRADD) transcription elongation factor S-ll, hS-ll-T1

Transcription factor I ID transcriptional activator hSNF2b Transferrin

Transferrin receptor

Transforming growth factor-beta 3

Transthyretin

Tropoelastin

Tryptophan hydroxylase

Tryptophanyl-tRNA synthetase ts11 gene encoding a G-1 progression protein

Tumor necrosis factor associated factor 2

(TRAF2)

Tumor necrosis factor receptor 1

Tumor necrosis factor receptor 2

Tumor necrosis factor receptor-1 associated protein (TRADD)

Tumor necrosis factor-alpha

Tumor necrosis factor-beta

Type 1 interstitial collagenase

Tyrosine aminotransferase

Tyrosine hydroxylase

Tyrosine protein kinase receptor (UFO)

U1 small nuclear RNP-specific C protein

Ubiquitin

Ubiquitin conjugating enzyme (Rad 6 homologue)

Ubiquitin conjugating enzyme G2 (UBE2G2)

Ubiquitin-homology domain protein PIC1

UDP-glucuronosyltransferase 2

UDP-glucuronosyltransferase 2B

Uncoupling protein 1

Uncoupling protein 2

Uncoupling protien 3

Urate oxidase

Urokinase plasminogen activator receptor

(uPAR)

UV excision repair protein RAD 23 (XP-C)

Vascular cell adhesion molecule 1 (VCAM-1)

Vascular endothelial growth factor

Vascular endothelial growth factor D

Vascular endothelial growth factor receptor 1 (fit—

1 )

Very long-chain acyl-CoA dehydrogenase

Vesicle-associated membrane protein-2 (VAMP-

2)

Vesicular acetylcholine transporter (VAChT)

Vesicular monoamine transporter (VMAT)

Vimentin

Visinin-like peptide 1 homolog

Vitellogenin Waf1

Wnt-13 mRNA

X13694

Zinc finger protein ZNF134

Zinc finger protein

Zinc-finger DNA-binding motifs (IA-1)

Zinc-finger protein-37

Zipper protein kinase (ZPK)

Serum paraoxonase

TABLE 5

Renal Toxicity Neural Toxicity

Alpha-2 microglobulin Acid ceramidase

Bile salt export pump (sister of p- Ataxia telangeictasia glycoprotein)

Calbindin-D (28kDa) Brain-derived neurotrophic factor

Calbindin-D (9K) Brain-derived neurotrophic factor

Calcineurin-B Choline kinase

Calnexin Cystic fibrosis transmembrane conductance regulator

Cholesterol esterase Dopamine beta-hydroxylase endothelin-1 Dopamine receptor D2

FGF-1 Dopamine transporter

FGF-7 Endothelin-1

Gamma glutamylcysteinyl synthetase Glial fibrillary acidic protein

Gamma-glutamyl hydrolase precursor Glutamine synthetase

Gamma-glutamyl transpeptidase Myelin basic protein

Heat shock protein 90 Nerve growth factor

Kidney injury molecule-1 Nerve growth factor receptor

NMB Neural cell adhesion molecule

Organic anion transporter 1 Neuropathy target esterase

Organic cation transporter 1 Synapsin I p-glycoprotein (MDR-1) Synaptophysin

Phosphoenolpyruvate carboxykinase Synaptotagmin I

Sphingomyelinase, neutral Tau protein

Vimetin Vesicular acetylcholine transporter

MOAT-B (MRP/organic anion Vesicular monoamine transporter transporter)

Organic anion transporter 1 Norepinephrine transporter

Organic anion transporter 3 Serotonin N-acetyltransferase

Organic anion transporter K1 Serotonin transporter (SERT)

Organic anion transporting polypeptide 1 Sphingomyelinase (neutral)

Organic cation transporter 1

Organic cation transporter 2

Organic cation transporter 3

Osteopontin

Renal organic anion transporter Hepatic Toxicity Immunotoxicity

11-beta hydroxysteroid dehydrogenase type II

12-lipoxygenase 6-C-kine

15-hydroxyprostaglandin dehydrogenase Complement component C3

17-beta hydroxysteroid dehydrogenase Cyclooxygenase 2

25-hydroxyvitamin D3-1 alpha-hydroxylase Eosinophil-derived neurotoxin

Alanine aminotransferase Eotaxin

Alcohol dehydrogenase 1 Granulocyte-macrophage colony- stimulating factor

All Cytochrome P450 genes IkB-a

Alpha 1-antitrypsin Interferon gamma

Bile salt export pump (sister of p-glycoprotein) Interferon inducible protein 10

Bilirubin UDP-glucuronosyltransferase isozyme Interferon inducible protein 15

1

Biliverdin reductase Interferon stimulatory gene factor-3

Branched chain acyl-CoA oxidase lnterleukin-1 alpha

Canalicular multispecific organic anion lnterleukin-1 beta transporter

Carnitine palmitoyl-CoA transferase lnterleukin-10

Catechol-O-methyltransferase lnterleukin-12

Cholesterol esterase lnterleukin-13

Corticosteroid binding globulin lnterleukin-18

Enoyl CoA hydratase lnterleukin-2

Epoxide hydrolase lnterleukin-8

Fatty acid synthase lnterleukin-4

Fatty acyl-CoA oxidase lnterleukin-5

Fatty acyl-CoA synthase lnterleukin-6

Flavin containing monooxygenase 1 Macrophage inflammatory protein-1 alpha

Focal adhesion kinase (pp125FAK) Macrophage inflammatory protein-1 bet< Gamma glutamylcysteinyl synthetase Macrophage inflammatory protein-2 alpha

Gamma-glutamyl hydrolase precursor Macrophage inflammatory protein-2 bet Gamma-glutamyl transpeptidase Macrophage inflammatory protein-3 alpha

Glucose-regulated protein 58 Macrophage inflammatory protein-3 bet. Glutamic-oxaloacetic transaminase Macrophage metalloelastase Glutamic-pyruvic transaminase MHC class 1 Glutathione S-transferase Ya MHC class 2 Hepatic lipase MHC class 2 transactivator Hepatocyte growth factor Monocyte chemotactic protein receptor

(CCR2)

Hepatocyte growth factor receptor Monocyte chemotactic protein-1 Hydroxysteroid sulfotransferase a Neutrophil elastase Na/taurocholate cotransporting polypeptide Phospholipase A2 Senescence-marker protein-30 Suppressor of cytokine signaling 1

Hepatocyte growth factor activator Suppressor of cytokine signaling 3

Lipopolysaccharide binding protein T-cell activation gene 3

Liver fatty acid binding protein T-cell cyclophilin

Major acute phase protein alpha-1

NADPH cytochrome P450 reductase

Peroxisomal 3-ketoacyl-CoA thiolase 1

Peroxisomal 3-ketoacyl-CoA thiolase 2

Peroxisomal acyl-CoA oxidase

Peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase

Peroxisomal fatty acyl-CoA oxidase

Peroxisome assembly factor 1

Peroxisome assembly factor 2

Peroxisome biogenesis disorder protein-1

Peroxisome biogenesis disorder protein-11

Peroxisome biogenesis disorder protein-4

Peroxisome hydratase

Peroxisome proliferator activated receptor alpha

Peroxisome proliferator activated receptor gamma

Serum amyloid A1

Serum amyloid A2-alpha

Transthyretin

Cardiotoxicity Pulmonary Toxicity

Adrenomedullin GARG-16

Atrial natiuretic factor GAS-7

Endothelin-1 IRF-7

Glucose transporter 1 ISG-15

Nitric oxide synthase-1 , inducible Lipocalin

Osteopontin Liposin

Protein kinase C - beta 1 Macrostatin

RhoA MME

Sarcoplasmic reticulum calcium MRP14

ATPase

Vascular endothelial growth factor MRP-8

Osteopontin

SAA-1

SAA-3

Tenascin

Tropoelastin TABLE 6

TABLE 7

1 -chloro-2-nitrobenzene dt-5-fluorouracil

2,4-dinitrophenol erythromycin

2-acetylaminofluorene ethyl methanesulfonate

2-azido-2-deoxycytidine etoposide

2-azido-2-deoxyu rid i ne fenofibrate

4-acetamidofluorene flufenamic acid

5-azacytidine gemfibrozil

5-chlorouracil guanine

5-fluorouracil hdp527

6-mercaptopurine hydroxy urea

6-thioguanine icrf acetamidofluorene icrf/doxorubicin acetaminophen indomethacin acetylsalicylic acid iodoacetamide acrid ine isonicotinic acid actinomycin M077 allyl alcohol mechlorethamine aminopterin melatonin aminotriazole melphalan antimycin a methotrexate antipyrine methyl methanesulfonate benz[a]pyrene mitomycin c bleomycin mitoxantrone busulfan n-nitroso-n-ethylurea caffeine n-nitroso-n-methylurea camptothecin naloxone carbamazepine naproxen carbon tetrachloride nicotine carboplatin nitrofurantoin carmustine o-toluidine chlorambucil oligomycin chloroquine paclitaxel cimetidine PGU693 cisplatin phenobarbital clenbuterol phorbol 12-myristate 13-acetate diester clofibrate prednisone clozapine proflavin colchicine progesterone corticosterone puromycin cycloheximide rezulin cyclophosphamide rifampicin cyclosporin rosiglitazone cytosine arabinoside sodium azide dacarbazine streptozotocin dexamethasone tacrine diethylhexylpthalate tamoxifen diethylstilbestrol thioguanine diflunisol transplatin digitoxin triethylenemelamine dimethylhydrazine triethylenethiophosphoramide dmso verapamil doxombicin wy 14,643

TABLE 8

TABLE 9

CELL TYPES IN THE HEART:

Myocytes (cardiac muscle cells)

Vascular endothelial cells

Purkinje cells - regulate rate and rhythm of the heart

CELL TYPES IN THE LUNG:

Columnar, ciliated epithelial cells - line trachea, bronchi, bronchioles

Goblet cells - secrete mucus

Neuroendocrine cells - contain serotonin, calcitonin and gastrin-releasing peptide

Capillary endothelial cells

Interstitial fibroblast cells

Smooth muscle cells

Mast cells - in pulmonary interstitium

Type I alveolar (epithelial) cells - compose 90% of alveolar surface

Type II alveolar (epithelial) cells - secrete surfactant and mediate repair of alveolar epithelium

Alveolar macrophages

Serous cells - produce a fluid to dissolve mucus

Bmsh cells (Type III epithelial cells)

Clara cells - highly metabolic

Parenchymal cells (connectivity tissue cells)

CELL TYPES IN THE KIDNEY:

Capillary endothelial cells

Visceral epithelial cells (podocytes) - form glomerualr barrier

Parietal epithelial cells - line Bowman's space

Mesangial cells - have contractile abilities to reduce amount o glomerular surface available for filtration

Tubular epithelial cells

Juxtaglomemlar cells (modified granulated smooth muscle cells; also called granular cells)

Lacis cells (non-granular cells)

Fibroblast-like cells

Macula densa cells (specialized tubular epithelial cells)

CELL TYPES IN THE BRAIN:

Neurons

Asfrocytes - found in gray and white matter; responsible for repair and scar formation

Oligodendrocytes - main component of white matter; produce and maintain CNS myelin

Microglia cells - serve as macrophage-type cells

Ependyma cells (columnar epithelial-like cells with a ciliated border) - line the ventricular system

Fibroblasts

Capillary endothelial cells

Meningeal fibroblast cells

Leptomeningeal (mesenchymal) cells

Purkinje cells

Meningothelial cells Macrophages

CELL TYPES IN THE LIVER:

Kupfer cells - resident macrophages

Sinusoidal endothelial cells

Ito cells (lipocytes) - synthesize collagen and store vitamin A

Hepatocytes (parenchymal cells) - majority of cells in the liver are of this type

Bile duct epithelial cells

Hepatic venule endothelial cells

Sinusoidal epithelial cells

Page 1

Page 2

-4

Page 3

Page 4

Page 5

4-

O

Page 6

Page 7

Page 8

Page 9

Page 10

Page 11

Page 12

Page 13

Page 14

Page 15

Ul

©

Page 16

Page 17

Page 18

Page 19

Page 20

Ul Ul

Page 21

Ul

9\

Page 22

-4

Page 23

oe

Page 24

Ul yo

Page 25

Page 26

Page 27

Page 28

Page 29

Page 30

Page 31

Page 32

-4

Page 33

Page 34

Page 35

-4

©

Page 36

Page 37

-4

Page 38

Page 39

Page 40

-4 Ul

Page 41

-4

Page 42

-4 -4

Page 43

-4 oe

Page 44

Page 45

oe ©

Page 46

Page 47

Page 48

oe

Uι

Page 49

oe

4-

Page 50

oe

Ul

Page 51

Page 52

oe

-4

Page 53

oe oe

Page 54

oe yo

Page 55

Page 56

Page 57

Page 58

Page 59

Page 60

yo Ul

Page 61

Page 62

Page 63

Page 64

Page 65

© ©

Page 66

Table 11 Class Discriminator Genes

Claims

1. A method of identifying hypersensitivity in a subject, the method comprising: obtaining a gene expression profile of genes associated with hypersensitivity ofa subject suspected to be hypersensitive; and detecting in the gene expression profile of the subject a predetermined pattern of gene expression of genes associated with hypersensitivity.

2. The method of claim 1 wherein the pattern of gene expression associated with hypersensitivity is obtained by comparing the gene expression profile ofa hypersensitive individual with the gene expression profile of an individual who is not hypersensitive.

3. The method of claim 1 , wherein the genes associated with hypersensitivity comprise at least 2 genes associated with hypersensitivity.

4. The method of claim 1, wherein the genes comprise at least 5 genes associated with hypersensitivity.

5. The method of claim 1, wherein the genes comprise at least 10 genes associated with hypersensitivity.

6. The method of claim 1, wherein the gene expression profile of the subject that is obtained comprises a profile of levels of mRNA or cDNA.

7. The method of claim 1 , wherein the gene expression profile comprises a profile of levels of protein expression.

8. The method of claim 1, wherein expression of the genes predetermined to be associated with hypersensitivity is directly related to prevention or repair of toxic damage at a protein, nucleotide, macromolecule, organelle, tissue, organ or system level.

9. The method of claim 1, wherein the gene expression profile is a profile of nucleic acid expression obtained from a cell or tissue sample, or a protein expression profile derived from cells, tissues, blood, urine or serum.

10. The method of claim 1, wherein the gene expression profile is obtained from a blood, urine or serum sample.

11. The method of claim 1 , wherein the method comprises identifying hypersensitivity in the subject to an agent.

12. The method of claim 11 , wherein the agent is a pharmaceutical agent.

13. The method of claim 11 , wherein the agent is selected from the group consisting of pharmaceutical agents listed in Table 1.

14. The method of claim 1, wherein the genes comprise genes associated with tissues or cells within the digestive system, comprising the liver, pancreas, intestines, colon, rectum, stomach, gallbladder, kidneys or bladder.

15. The method of claim 14, wherein the genes are genes associated with liver toxicity including altered lipid metabolism, fatty liver, cholestasis, jaundice, hepatitis, steatosis, necrosis, hyperplasia, mutagenesis, tumor formation or peroxisome proliferation.

16. The method of claim 1 , wherein the genes are genes associated with tumor formation, teratogenesis, immunosuppression, pancreatitis, or agranulocytosis.

17. The method of claim 1, wherein the genes are genes associated with cellular manifestations of toxicity.

18. The method of claim 17, wherein the plurality of genes comprises genes associated with apoptosis, cell adhesion, autophagocytosis, cell cycle arrest, circadian rhythm, cytokine release, de-differentiation, differentiation, mitochondrial damage, migration, mutation, oncosis, peroxisome proliferation, recombination, senescence, signal refractivity, spreading, or transformation.

19. The method of claim 1 , wherein the plurality of genes are genes associated with renal toxicity.

20. The method of claim 19, wherein the genes comprise genes associated with, necrosis, glomerulitis, nephritis, tumor formation, hyperplasia, proteinuria, renal damage or renal failure.

21. The method of claim 1 , wherein the genes are genes associated with cardiotoxicity, blood toxicity, skin toxicity, eye toxicity or neurotoxicity.

22. The method of claim 21, wherein the plurality of genes comprises genes associated with tachycardia, arrhythmia, hypotension, hypertension, leukemia, neutropenia, agranulocytosis, peripheral neuropathy, dementia, inflammation, irritation, sensitization, myelosuppression or retinopathy.

23. The method of claim 1 , wherein the genes associated with hypersensitivity are associated with a specific ethnic group, sex or age group.

24. The method of claim 1 , wherein the genes are selected from the group consisting of the genes listed in Tables 3, 4, 5, 6, 8, 10 and 11.

25. The method of claim 24, wherein the genes comprises at least 5 genes.

26. The method of claim 1, wherein the genes are selected from any one of the group consisting of the genes listed in Table 4.

27. The method of claim 26, wherein the genes comprise at least 5 genes.

28. The method of claim 1, wherein the genes are expressed in one or more different cell types within a single tissue or organ.

29. The method of claim 28, wherein the tissue or organ is selected from the group consisting of liver, kidney, lung, heart, pancreas, muscle, brain, testes, ovaries, spleen, stomach, intestines, colon, rectum, eye, and bone.

30. The method of claim 29 wherein the cell types are selected from the group of liver cells consisting of Kupfer cells, sinusoidal cells, ito cells, hepatocytes, bile duct epithelial cells, hepatic venule endothelial cells and sinusoidal epithelial cells.

31. The method of claim 28 wherein the cell types are selected from the group consisting of the cells listed in Table 9.

32. A method of identifying a number of genes associated with hypersensitivity to an agent, the method comprising: comparing the gene expression profile of cells treated with the agent with the gene expression profile of cells not treated with the agent; and determining the genes that have altered expression due to exposure to the agent in the treated cells, thereby to identify the genes associated with hypersensitivity to the agent.

33. The method of claim 32, wherein the cells comprise cells of one or more different cell types, and wherein each said cell type comprises a gene associated with hypersensitivity to the agent.

34. The method of claim 33, wherein said cell types are derived from a single type of tissue or organ.

35. The method of claim 34, wherein said cell types are derived from an organ or a tissue selected from the group consisting of kidney, liver, lung, heart, brain, spleen, thyroid, bone, muscle, intestine, stomach or skin.

36. A method of identifying genes having a pattern of differential gene exoression indicative of hvϋersensitivitv to an aeent. the method comnrisine: comparing a gene expression profile of one or more cell types ofa subject known to be hypersensitive to the agent with the gene expression profile of said cell types in an individual known not to be hypersensitive to the agent; and identifying the genes from said one or more cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent.

37. A method of identifying genes having a pattern of differential gene expression indicative of hypersensitivity to an agent, the method comprising: comparing the gene expression profile of one or more cell types of a subject known to be hypersensitive to the agent before treatment with the agent with the gene expression profile of the one or more cell types of the subject after treatment with the agent; and identifying genes from said cell types having a pattern of differential gene expression, wherein the pattern of differential gene expression is associated with hypersensitivity to the agent wherein the samples of multiple individuals are compared in at least six individuals and wherein the results of gene expression profiles are compared statistically using computer software.

38. An array for the identification ofa gene expression profile indicative ofa hypersensitivity to an agent, the array comprising at least 25 different gene probes, each probe comprising a nucleic acid sequence of a gene associated with the hypersensitivity to the agent, wherein said gene is selected from the group consisting of genes listed in Tables 3, 4, 5 and 6.

39. An array of claim 38 wherein the array comprises at least 100 different gene probes.

40. An apparatus for identifying hypersensitivity in a subject comprising: a detector for obtaining a gene expression profile of a number of genes associated with hypersensitivity of the subject suspected to be hypersensitive; and a second dectector for identifying in the gene expression profile of the subject a pattern of gene expression of the genes associated with hypersensitivity, thereby to identify hypersensitivity in the subject.

41. The method of claim 11, wherein the method comprises identifying hypersensitivity in a subject to multiple agents administered together.

42. A method of determining hypersensitivity of a subject to an agent, the method comprising: obtaining a cell from a subject; culturing said cell to obtain a cell culture; exposing said cell culture to an agent; obtaining a gene or protein expression profile of a cell or cells of said exposed culture; and detecting in said gene or protein expression profile a predetermined pattern of expression associated with hypersensitivity to the agent.

43. The method of claim 42, wherein the subject is a human being.

44. The method of claim 42, wherein the expression profile of at least 20 genes or proteins is obtained.

45. The method of claim 44, wherein the expression profile of at least 50 genes or oroteins is obtained.

46. The method of claim 42, wherein the cell obtained from the subject is a leukocyte.