Clustering of hepatotoxins based on mechanism of toxicity using gene expression profiles.

Microarray technology, which allows one to quantitate the expression of thousands of genes simultaneously, has begun to have a major impact on many different areas of drug discovery and development. The question remains of whether microarray analysis and gene expression signature profiles can be applied to the field of toxicology. To date, there are very few published studies showing the use of microarrays in toxicology and important questions remain regarding the predictability and accuracy of applying gene expression profiles to toxicology. To begin to address these questions, we have treated rats with 15 different known hepatotoxins, including allyl alcohol, amiodarone, Aroclor 1254, arsenic, carbamazepine, carbon tetrachloride, diethylnitrosamine, dimethylformamide, diquat, etoposide, indomethacin, methapyrilene, methotrexate, monocrotaline, and 3-methylcholanthrene. These agents cause a variety of hepatocellular injuries including necrosis, DNA damage, cirrhosis, hypertrophy, and hepatic carcinoma. Gene expression analysis was done on RNA from the livers of treated rats and was compared against vehicle-treated controls. The gene expression results were clustered and compared to the histopathology findings and clinical chemistry values. Our results show strong correlation between the histopathology, clinical chemistry, and gene expression profiles induced by the agents. In addition, genes were identified whose regulation correlated strongly with effects on clinical chemistry parameters. Overall, the results suggest that microarray assays may prove to be a highly sensitive technique for safety screening of drug candidates and for the classification of environmental toxins.

[1]  R. Custer,et al.  Hepatotoxicity in Wistar rats following chronic methotrexate administration: a model of human reaction. , 1977, Journal of the National Cancer Institute.

[2]  A. Gartel,et al.  p53-independent induction of p21WAF1/CIP1 expression in pericentral hepatocytes following carbon tetrachloride intoxication. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[3]  H. Glatt,et al.  Development of hydroxysteroid sulfotransferase-deficient lesions during hepatocarcinogenesis in rats. , 1993, Carcinogenesis.

[4]  T. Hughes,et al.  Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. , 2000, Science.

[5]  M. Wright,et al.  Methapyrilene hepatotoxicity is associated with oxidative stress, mitochondrial disfunction and is prevented by the Ca2+ channel blocker verapamil. , 1998, Toxicology.

[6]  Roger Williams,et al.  Immunolocalization of regenerating cells after submassive liver necrosis using PCNA staining , 1992, The Journal of pathology.

[7]  R. O. Recknagel Carbon tetrachloride hepatotoxicity. , 1967, Pharmacological reviews.

[8]  L. Schiaffonati,et al.  Gene expression in liver after toxic injury: analysis of heat shock response and oxidative stress-inducible genes. , 2008, Liver.

[9]  D. Wilson,et al.  DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells. , 2000, Toxicology and applied pharmacology.

[10]  M. Broggini,et al.  Accumulation of DNA strand breaks in cells exposed to methotrexate or N10-propargyl-5,8-dideazafolic acid. , 1988, Cancer research.

[11]  A. Campello,et al.  Methotrexate: pentose cycle and oxidative stress , 1998, Cell biochemistry and function.

[12]  E. Gallagher,et al.  The effects of diquat and ciprofibrate on mRNA expression and catalytic activities of hepatic xenobiotic metabolizing and antioxidant enzymes in rat liver. , 1995, Toxicology and applied pharmacology.

[13]  T. Massey,et al.  Investigation of the role of oxidative stress in amiodarone-induced pulmonary toxicity in the hamster. , 1994, Canadian Journal of Physiology and Pharmacology.

[14]  D. Botstein,et al.  A gene expression database for the molecular pharmacology of cancer , 2000, Nature Genetics.

[15]  S. Chakrabarti,et al.  Effect of four-day treatment with carbamazepine at different dose levels on microsomal enzyme induction, drug metabolism and drug toxicity. , 1988, Pharmacology & toxicology.

[16]  Yudong D. He,et al.  Functional Discovery via a Compendium of Expression Profiles , 2000, Cell.

[17]  J. Popp,et al.  Effects of methapyrilene on rat hepatic xenobiotic metabolizing enzymes and liver morphology. , 1985, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[18]  R. Dunn,et al.  Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[19]  S. Iwase,et al.  Concentration-dependent variable effects of etoposide on the cell cycle of CML cells. , 2000, Anticancer research.

[20]  G. Church,et al.  RNA expression analysis using a 30 base pair resolution Escherichia coli genome array , 2000, Nature Biotechnology.

[21]  Christian A. Rees,et al.  Systematic variation in gene expression patterns in human cancer cell lines , 2000, Nature Genetics.

[22]  L. Schook,et al.  Acute hepatotoxicant exposure induces TNFR-mediated hepatic injury and cytokine/apoptotic gene expression. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[23]  J. Waring,et al.  The impact of genomics-based technologies on drug safety evaluation. , 2000, Annual review of pharmacology and toxicology.

[24]  M. Higgins,et al.  Micronuclei in mice treated with monocrotaline with and without phenobarbital pretreatment , 1995, Environmental and molecular mutagenesis.

[25]  R. Lubet,et al.  Induction of cytochrome P450 and other drug metabolizing enzymes in rat liver following dietary exposure to Aroclor 1254. , 1991, Toxicology and applied pharmacology.

[26]  J. Mesirov,et al.  Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Fujita,et al.  PCBs: structure–function relationships and mechanism of action , 1985, Environmental health perspectives.

[28]  Curtis D. Klaassen,et al.  Mechanisms of toxicity. , 1969, British medical journal.

[29]  R. Jolly,et al.  Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants. , 1991, Free radical biology & medicine.

[30]  J. A. Hartigan,et al.  A k-means clustering algorithm , 1979 .

[31]  S. Belinsky,et al.  Mechanism of hepatotoxicity to periportal regions of the liver lobule due to allyl alcohol: role of oxygen and lipid peroxidation. , 1986, The Journal of pharmacology and experimental therapeutics.

[32]  Curtis D. Klaassen,et al.  Casarett and Doull's Toxicology. The Basic Science of Poisons , 1981 .

[33]  Z. Ilic,et al.  Cell kinetics of repair after allyl alcohol‐induced liver necrosis in mice , 1996, International journal of experimental pathology.

[34]  D. Gerhold,et al.  Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays. , 2001, Physiological genomics.

[35]  J. F. Brown,et al.  Comparative Carcinogenicity in Sprague-Dawley Rats of the Polychlorinated Biphenyl Mixtures Aroclors 1016, 1242, 1254, and 1260 , 1998, Toxicological Sciences.

[36]  C. Mackerer,et al.  The effects of a polychlorinated biphenyl mixture (Aroclor 1254) on liver gluconeogenic enzymes of normal and alloxan-diabetic rats. , 1975, Toxicology.

[37]  Lindén Cj Toxicity of intraperitoneally administered antitumour drugs in athymic rats. , 1989 .

[38]  C. D. Klaasen Casarett & Doull's Toxicology: The Basic Science of Poisons , 1980 .

[39]  U. Alon,et al.  Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[40]  R. Young,et al.  Biomedical Discovery with DNA Arrays , 2000, Cell.

[41]  M. Comporti Three models of free radical-induced cell injury. , 1989, Chemico-biological interactions.

[42]  W. J. Donarski,et al.  Effects of novel antioxidants on carbon tetrachloride-induced lipid peroxidation and toxicity in precision-cut rat liver slices. , 1990, Toxicology and applied pharmacology.

[43]  C. V. Smith,et al.  Sex differences in diquat-induced hepatic necrosis and DNA fragmentation in Fischer 344 rats. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[44]  R. Somogyi,et al.  Gene Expression Microarray Data Analysis for Toxicology Profiling , 2000, Annals of the New York Academy of Sciences.

[45]  P. Ganey,et al.  Depletion of neutrophils and modulation of Kupffer cell function in allyl alcohol-induced hepatotoxicity. , 1995, Toxicology.

[46]  J. Mesirov,et al.  Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. , 1999, Science.

[47]  T. Thum,et al.  Induction of nuclear transcription factors, cytochrome P450 monooxygenases, and glutathione S-transferase alpha gene expression in Aroclor 1254-treated rat hepatocyte cultures. , 2001, Biochemical pharmacology.

[48]  R. Gebhardt,et al.  Diethylnitrosamine exposure-responses for DNA damage, centrilobular cytotoxicity, cell proliferation and carcinogenesis in rat liver exhibit some non-linearities. , 1996, Carcinogenesis.

[49]  R. Davis,et al.  Independent regulation of JNK/p38 mitogen-activated protein kinases by metabolic oxidative stress in the liver. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[50]  J. Sarma,et al.  Role of Oxidative Stress in Amiodarone-induced Toxicity , 1997, Journal of cardiovascular pharmacology and therapeutics.

[51]  P. H. Bach,et al.  Effects of arsenite on hepatic mixed-function oxidase activity in rats. , 1992, Xenobiotica; the fate of foreign compounds in biological systems.

[52]  C. Byus,et al.  Activation of 3':5'-cyclic AMP-dependent protein kinase and induction of ornithine decarboxylase as early events in induction of mixed-function oxygenases. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[53]  K. Hostetler,et al.  Comparative evaluation of amiodarone-induced phospholipidosis and drug accumulation in Fischer-344 and Sprague-Dawley rats. , 1996, Toxicology.

[54]  J. Borlakoglu,et al.  Evidence for the induction of fatty acid desaturation in proliferating hepatic endoplasmic reticulum in response to treatment with polychlorinated biphenyls. Are fatty acid desaturases cytochrome P-450-dependent monooxygenases? , 1991, The International journal of biochemistry.

[55]  E. Yu,et al.  Time course of cell cycle-related protein expression in diethylnitrosamine-initiated rat liver. , 1998, Journal of hepatology.

[56]  D. Waxman,et al.  Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes. , 1991, Xenobiotica; the fate of foreign compounds in biological systems.

[57]  L. Bengochea,et al.  An experimental model of liver damage and portal hypertension induced by a single dose of monocrotaline. , 1999, Hepato-gastroenterology.

[58]  G. Sherlock Analysis of large-scale gene expression data. , 2000, Current opinion in immunology.

[59]  K. Mahaffy CONCURRENT EXPOSURE OF LEAD, CADMIUM AND ARSENIC: EFFECTS ON TOXICITY AND TISSUE METAL CONCENTRATION IN RAT , 1981 .

[60]  E. Glover,et al.  Studies on the mechanism of action of halogenated aromatic hydrocarbons. , 1985, Clinical physiology and biochemistry.