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.

Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity.

A rate-limiting step that occurs in the drug discovery process is toxicological evaluation of new compounds. New techniques that use small amounts of the experimental compound and provide a high degree of predictivity would greatly improve this process. The field of microarray technology, which allows one to monitor thousands of gene expression changes simultaneously, is rapidly advancing and is already being applied to numerous areas in toxicology. However, it remains to be determined if compounds with similar toxic mechanisms produce similar changes in transcriptional expression. In addition, it must be determined if gene expression changes caused by an agent in vitro would reflect those produced in vivo. In order to address these questions, we treated rat hepatocytes with 15 known hepatoxins (carbon tetrachloride, allyl alcohol, aroclor 1254, methotrexate, diquat, carbamazepine, methapyrilene, arsenic, diethylnitrosamine, monocrotaline, dimethyl-formamide, amiodarone, indomethacin, etoposide, and 3-methylcholanthrene) and used microarray technology to characterize the compounds based on gene expression changes. Our results showed that gene expressional profiles for compounds with similar toxic mechanisms indeed formed clusters, suggesting a similar effect on transcription. There was not complete identity, however, indicating that each compound produced a unique signature. These results show that large-scale analysis of gene expression using microarray technology has promise as a diagnostic tool for toxicology.

Overexpression of fragile X gene (FMR-1) transcripts increases cAMP production in neural cells.

Fragile X syndrome results from amplification of an unstable trinucleotide (CGG) repeat in the first exon of FMR-1, the "fragile X gene." This mutation silences the gene, resulting in loss of expression of the FMR proteins (FMRP), a series of RNA-binding proteins generated by alternative splicing of FMR-1 transcripts. We have shown that cAMP production is diminished in cells from patients with fragile X syndrome. To establish a direct relationship between FMR-1 expression and cAMP metabolism, FMRP isoforms 1 and 7 were overexpressed in the neurotumor hybrid cell line HN2. Cyclic AMP production in clonal HN2 lines overexpressing FMRP was significantly higher than in nonoverexpressing control lines and increased with increasing total FMR-1 mRNA on Northern blots and FMRP signal on Western blots. These data support a role for FMRP in the regulation of cAMP signal transduction by increasing intracellular cAMP, perhaps through a mechanism involving binding and enhanced translation of mRNA(s) for cAMP cascade proteins. Diminished cAMP production in the absence of FMR-1 may provide one neurochemical mechanism through which FMR-1 influences cognitive function.

Reduced cyclic AMP production in fragile X syndrome: cytogenetic and molecular correlations.

The cAMP cascade is an intracellular signal transduction system thought to be important for neuronal regulation and information storage. cAMP production is reduced in platelets from patients with fragile X syndrome. In the present study we assayed cAMP metabolism, Xq27.3 fragile site percentages, size of amplification mutation in fragile X mental retardation-1 gene (FMR-1), and FMR-1 mRNA levels in 21 lymphoblastoid cell lines (LCL) from fragile X patients. cAMP production was diminished in fragile X LCL relative to controls (n = 20) when cells were assayed in prostaglandin E1 (74%, p < 0.02) and in forskolin (64%, p < 0.1) although the difference was statistically significant only in prostaglandin E1. The length of the FMR-1 amplification mutation correlated with measures of cAMP production which were unassociated with receptor activation (r = -0.53, p = 0.02, and r = -0.48, p = 0.03, for unstimulated and forskolin-stimulated cAMP production, respectively). In fragile X LCL, fragile site percentages did not correlate with any measure of cAMP production. All fragile X LCL showed absence of FMR-1 mRNA. These data suggest that diminished cAMP production in fragile X tissues may be linked to the fragile X amplification mutation, either as a result of influences of the mutation on FMR-1 expression or on transcription of other genes downstream from FMR-1.