Mammalian toxicological response markers

Details Mammalian toxicological response markers Mammalian toxicological response markers

1999-11-19

2002-04-16

Ponnaluri, Padmashri (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S004000, C435S007100, C435S091500, C435S091500, C435S091500, C536S063000, C436S501000

Reexamination Certificate

active

06372431

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to mammalian nucleic acid and protein molecules, and methods for their use in diagnostic and therapeutic applications including detecting metabolic and toxicological responses, and in monitoring drug mechanism of action.
BACKGROUND OF THE INVENTION
Toxicity testing is a mandatory and time-consuming part of drug development programs in the pharmaceutical industry. A more rapid screen to determine the effects upon metabolism and to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays of various kinds may be produced using full length genes or gene fragments. These arrays can then be used to test samples treated with the drug candidates to elucidate the gene expression pattern associated with drug treatment. This gene pattern can be compared with gene expression patterns associated with compounds which produce known metabolic and toxicological responses.
Benzo(a)pyrene is a known rodent and likely human carcinogen and is the prototype of a class of compounds, the polycyclic aromatic hydrocarbons (PAH). It is metabolized by several forms of cytochrome P450 (P450 isozymes) and associated enzymes to form both activated and detoxified metabolites. The ultimate metabolites are the bay-region diol epoxide, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) and the K-region diol epoxide, 9-hydroxy benzo(a)pyrene-4,5-oxide, both of which induce formation of DNA adducts. DNA adducts have been shown to persist in rat liver up to 56 days following treatment with benzo(a)pyrene at a dose of 10 mg/kg body weight three times per week for two weeks (Qu and Stacey (1996) Carcinogenesis 17:53-59).
Acetaminophen is a widely-used analgesic. It is metabolized by specific cytochrome P450 isozymes with the majority of the drug undergoing detoxification by glucuronic acid, sulfate and glutathione conjugation pathways. However, at supratherapeutic doses, acetaminophen is metabolized to an active intermediate, N-acetyl-p-benzoquinone imine (NAPQI) which can cause hepatic and renal failure. NAPQI then binds to sulhydryl groups of proteins causing their inactivation and leading to subsequent cell death (Kroger et al. (1997) Gen. Pharmacol. 28:257-263).
Clofibrate is an hypolidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly and an increase in hepatic peroxisomes (peroxisome proliferation). Peroxisome proliferators (PPs) are a class of drugs which activate the PP-activated receptor in rodent liver, leading to enzyme induction, stimulation of S-phase, and a suppression of apoptosis (Hasmall and Roberts (1999) Pharmacol. Ther. 82:63-70). PPs include the fibrate class of hypolidemic drugs, phenobarbitone, thiazolidinediones, certain non-steroidal anti-inflammatory drugs, and naturally-occurring fatty acid-derived molecules (Gelman et al. (1999) Cell. Mol. Life Sci. 55:932-943). Clofibrate has been shown to increase levels of cytochrome P450 4A. It is also involved in transcription of &bgr;-oxidation genes as well as induction of PP-activated receptors (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). Peroxisome proliferation that is induced by both clofibrate and the chemically-related compound fenofibrate is mediated by a common inhibitory effect on mitochondrial membrane depolarization (Zhou and Wallace (1999) Toxicol. Sci. 48:82-89).
Toxicological effects in the liver are also induced by other compounds. These can include carbon tetrachloride (a necrotic agent), hydrazine (a steatotic agent), &agr;-naphthylisothiocyanate (a cholestatic agent), 4-acetylaminofluorene (a liver mitogen), and their corresponding metabolites, which are used in experimental protocols to measure toxicological responses (Waterfield et al. (1993) Arch. Toxicol. 67:244-254).
The present invention provides mammalian nucleic acid and protein molecules, their use in diagnostic and therapeutic applications including detecting metabolic and toxicological responses, and in monitoring drug mechanism of action.
SUMMARY OF THE INVENTION
The invention provides a method for detecting or diagnosing the effect of a test compound or molecule associated with increased or decreased levels of nucleic acid molecules in a mammalian subject. The method comprises treating a mammalian subject with a known toxic compound or molecule which elicits a toxicological response, measuring levels of a plurality of nucleic acid molecules, selecting from the plurality of nucleic acid molecules those nucleic acid molecules that have levels modulated in samples treated with known toxic compounds or molecules when compared with untreated samples. Some of the levels may be upregulated by a toxic compound or molecule, others may be downregulated by a toxic compound or molecule, and still others may be upregulated with one known toxic compound or molecule and be downregulated with another known toxic compound or molecule. The selected nucleic acid molecules which are upregulated and downregulated by a known toxic compound or molecule are arrayed upon a substrate. The method further comprises measuring levels of nucleic acid molecules in the sample after the sample is treated with the toxic compound or molecule. Levels of nucleic acid molecules in a sample so treated are then compared with the plurality of the arrayed nucleic acid molecules to identify which sample nucleic acid molecules are upregulated and downregulated by the test compound or molecule. In one embodiment, the nucleic acid molecules are hybridizable array elements of a microarray.
Preferably, the comparing comprises contacting the arrayed nucleic acid molecules with the sample nucleic acid molecules under conditions effective to form hybridization complexes between the arrayed nucleic acid molecules and the sample nucleic acid molecules; and detecting the presence or absence of the hybridization complexes. In this context, similarity may mean that at least 1, preferably at least 5, more preferably at least 10, of the upregulated arrayed nucleic acid molecules form hybridization complexes with the sample nucleic acid molecules at least once during a time course to a greater extent than would the probes derived from a sample not treated with the test compound or molecule or a known toxic compound or molecule. Similarity may also mean that at least 1, preferably at least 5, more preferably at least 10, of the downregulated arrayed nucleic acid molecules form hybridization complexes with the sample nucleic acid molecules at least once during a time course to a lesser extent than would the sample nucleic acid molecules of a sample not treated with the test compound or a known toxic compound. In one aspect, the arrayed nucleic acid molecules comprise SEQ ID NOs: 1-47 or fragments thereof.
Preferred toxic compounds are selected from the group consisting of hypolipidemic drugs, n-alkylcarboxylic acids, n-alkylcarboxylic acid precursors, azole antifungal compounds, leukotriene D4 antagonists, herbicides, pesticides, phthalate esters, phenyl acetate, dehydroepiandrosterone (DHEA), oleic acid, methanol and their corresponding metabolites, acetaminophen and its corresponding metabolites, benzo(a)pyrene, 3-methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, their corresponding metabolites, and the like, carbon tetrachloride, hydrazine, &agr;-naphthylisothiocyanate, 4-acetylaminofluorene, and their corresponding metabolites. Preferred tissues are selected from the group consisting of liver, kidney, brain, spleen, pancreas and lung.
The arrayed nucleic acid molecules comprise fragments of messenger RNA transcripts of genes that are upregulated-or-downregulated at least 2-fold, preferably at least 2.5-fold, more preferably at least 3-fold, in tissues treated with known toxic compounds when compared with untreated tissues. Preferred arrayed nucleic acid molecules are selected from the group consisting of SEQ ID NOs: 1-47 or fragments thereof, some of whose expression is upregulated following treatment with a toxic compound

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