Universal Toxicity Gene Signatures for Early Identification of Drug-Induced Tissue Injuries in Rats.

A new safety testing paradigm that relies on gene expression biomarker panels was developed to easily and quickly identify drug-induced injuries across tissues in rats prior to drug candidate selection. Here, we describe the development, qualification, and implementation of gene expression signatures that diagnose tissue degeneration/necrosis for use in early rat safety studies. Approximately 400 differentially expressed genes were first identified that were consistently regulated across 4 prioritized tissues (liver, kidney, heart, and skeletal muscle), following injuries induced by known toxicants. Hundred of these "universal" genes were chosen for quantitative PCR, and the most consistent and robustly responding transcripts selected, resulting in a final 22-gene set from which unique sets of 12 genes were chosen as optimal for each tissue. The approach was extended across 4 additional tissues (pancreas, gastrointestinal tract, bladder, and testes) where toxicities are less common. Mathematical algorithms were generated to convert each tissue's 12-gene expression values to a single metric, scaled between 0 and 1, and a positive threshold set. For liver, kidney, heart, and skeletal muscle, this was established using a training set of 22 compounds and performance determined by testing a set of approximately 100 additional compounds, resulting in 74%-94% sensitivity and 94%-100% specificity for liver, kidney, and skeletal muscle, and 54%-62% sensitivity and 95%-98% specificity for heart. Similar performance was observed across a set of 15 studies for pancreas, gastrointestinal tract, bladder, and testes. Bundled together, we have incorporated these tissue signatures into a 4-day rat study, providing a rapid assessment of commonly seen compound liabilities to guide selection of lead candidates without the necessity to perform time-consuming histopathologic analyses.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function.

The Predictive Safety Testing Consortium's first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Diagnosis of drug-induced renal tubular toxicity using global gene expression profiles.

Toxicogenomics can measure the expression of thousands of genes to identify changes associated with drug induced toxicities. It is expected that toxicogenomics can be an alternative or complementary approach in preclinical drug safety evaluation to identify or predict drug induced toxicities. One of the major concerns in applying toxicogenomics to diagnose or predict drug induced organ toxicity, is how generalizable the statistical classification model is when derived from small datasets? Here we presented that a diagnosis of kidney proximal tubule toxicity, measured by pathology, can successfully be achieved even with a study design of limited number of training studies or samples. We selected a total of ten kidney toxicants, designed the in life study with multiple dose and multiple time points to cover samples at doses and time points with or without concurrent toxicity. We employed SVM (Support Vector Machine) as the classification algorithm for the toxicogenomic diagnosis of kidney proximal tubule toxicity. Instead of applying cross validation methods, we used an independent testing set by dividing the studies or samples into independent training and testing sets to evaluate the diagnostic performance. We achieved a Sn (sensitivity) = 88% and a Sp (specificity) = 91%. The diagnosis performance underscores the potential application of toxicogenomics in a preclinical lead optimization process of drugs entering into development.

A low volume, high-throughput forward mutation assay in Salmonella typhimurium based on fluorouracil resistance.

A forward mutation assay based on 5-fluorouracil (FU) resistance has been developed using a strain of Salmonella typhimurium derived from the Ames strain TA100. The sensitivity of the assay benefits from the genetic characteristics present in the standard Ames strain that enhances the response to genotoxic agents. A mutation conferring resistance to 5-fluorouridine was also introduced into the test strain to avoid unwanted toxicity resulting from cross-feeding of 5-fluorouridine between wild-type and FU-resistant (FU(R)) cells during selection with FU. In the mutation assay 1 ml aliquots of exponentially growing bacteria are exposed to the test agent for 2 h in the presence and absence of a rat-liver S-9 metabolizing system. The aliquots are then diluted, allowed to grow for 3 h, and assessed for treatment-related toxicity/inhibition by optical density. The cultures are diluted a second time and grown overnight to permit full recovery from toxicity and to allow expression of the FU(R) phenotype. Samples of the cultures are then plated in 384-well microtiter dishes in the presence of 2 microg/ml FU and the pH-sensitive indicator bromcresol purple. Three days later positive wells containing FU(R) colonies are detected by their yellow color and enumerated. Results were obtained using a variety of 45 compounds to validate the assay. Of the 25 mutagens and 20 non-mutagens tested, the results correlated 100% with those collected using the battery of standard Ames reversion strains, further supporting the use of the FU Assay as an alternative screen to the Ames assay. The use of a single strain exposed in liquid suspension permits assessment of high concentrations of test compound but with a low overall compound requirement (30 mg total). The highly parallel nature of culture handling/dilution and use of standard microtiter plates also offers the possibility of assay automation.

Induced mutation spectra at the upp locus in Salmonella typhimurium: response of the target gene in the FU assay to mechanistically different mutagens.

A novel forward mutation assay has been developed in Salmonella typhimurium based on resistance to 5-fluorouracil (FU). The mutational target in the FU assay was determined to be the uracil phosphoribosyl transferase (upp) gene. To validate the upp gene as a suitable target for monitoring a variety of induced mutations, the mutational specificity was determined for five mechanistically different mutagens. The mutagens included a polycyclic hydrocarbon (benzo[a]pyrene, B[a]P), SN1 and SN2 alkylating agents (N-nitroso-N-methylurea, MNU, and methyl methanesulfonate, MMS, respectively), a frameshift mutagen (ICR-191), and an oxidative-damaging agent (hydrogen peroxide, H2O2). Induced mutation frequencies were measured in the presence and absence of the plasmid pKM101 (strain FU100 and FU1535, respectively). pKM101 renders FU100 more susceptible to induced mutation by providing error-prone replicative bypass of DNA adducts. B[a]P, MMS, and H2O2 failed to induce the mutant frequency in FU1535, demonstrating the dependence of pKM101 on induced mutations with these agents. ICR-191 and MNU were not dependent on pKM101, and did significantly induce mutations in FU1535. In contrast to FU1535, all agents significantly induced mutations in FU100. Approximately 60 independent mutants were sequenced for each agent that significantly induced the mutant frequency above background. The resulting mutational spectra illustrated predictable molecular fingerprints based on known mutagenic mechanisms for each agent. The predominant mutations observed were G:C to T:A transversions for B[a]P, A:T to T:A and G:C to T:A transversions for MMS, G:C to T:A transversions and A:T frameshifts for H2O2, G:C frameshifts for ICR-191, and G:C to A:T transitions for MNU. It can be concluded that the upp gene in the FU assay is a sensitive and suitable target to monitor a variety of induced mutations in Salmonella.

5-fluorouracil forward mutation assay in Salmonella: determination of mutational target and spontaneous mutational spectra.

A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and -2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.

Genotoxicity of naturally occurring indole compounds: correlation between covalent DNA binding and other genotoxicity tests.

3-Methylindole (3MI), melatonin (Mel), serotonin (Ser), and tryptamine (Tryp) were evaluated in vitro for their potential to induce DNA adducts, DNA strand breaks, chromosomal aberrations (Abs), inhibition of DNA synthesis, and mutations. All compounds produced DNA adducts in calf thymus DNA in the presence of rat liver S9. In cultured rat hepatocytes, all produced DNA adducts but none induced DNA strand breaks. In Chinese hamster ovary cells, 3MI and Mel produced DNA adducts, Abs, and inhibition of DNA synthesis with and without S9, except that Mel without S9 did not form adducts. Ser formed DNA adducts, was an equivocal Abs inducer, and suppressed DNA synthesis. Tryp induced neither adducts nor Abs, but did suppress DNA synthesis with S9. Ser and Tryp were less cytotoxic than 3MI and Mel. Mel, Ser, and Tryp failed to induce mutations in Salmonella and E. coli strains with or without S9. 3MI and Mel produced DNA adducts but not mutations in Salmonella TA100 with S9. 3MI and its metabolite indole 3-carbinol also did not induce mutations in a shuttle vector system in human cells. The lack of correlation between DNA adducts and other genotoxicity endpoints for these indole compounds may be due to the higher sensitivity of the (32)P-postlabeling adduct assay or it may indicate that the indole-DNA adducts per se are not mutagenic and are not able to induce strand breaks or alkali-labile lesions. The indole-induced Abs may result from cytotoxicity and suppression of DNA synthesis with minimal if any contribution from DNA adducts.