Alkaline comet assay in liver and stomach, and micronucleus assay in bone marrow, from rats treated with 2-acetylaminofluorene, azidothymidine, cisplatin, or isobutyraldehyde.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative international validation study of the in vivo rat alkaline comet assay (comet assay), we examined the ability of the assay to determine the genotoxicity of 2-acetylaminofluorene (AAF), azidothymidine (AZT), cisplatin (CPN), and isobutyraldehyde (IBA) in liver and glandular stomach of male Sprague-Dawley rats. Rats were given oral doses of test compound or control once daily for three days. High dose levels were approximately maximum tolerated doses and were based on preliminary range-finding studies. Tissues were harvested 3h after the final dose (48h after the initial dose). A bone marrow micronucleus assay (MN) was also conducted on the rats treated with AZT, CPN, and IBA. Acute toxic effects of treatment were determined primarily through histomorphologic analysis of liver and stomach but also by body weight and serum liver enzyme changes. The comet assay was conducted on fresh tissue preparations but frozen samples from two studies were also assayed. Statistically significant dose-related differences in comet % DNA in tail were found in liver and stomach for the genotoxin AZT and in liver for the genotoxin CPN, but not in liver or stomach for the non-genotoxin IBA. Statistically significant differences in % DNA in tail were measured in liver for the low and mid dose of the genotoxin AAF, but not the high dose. The comet assays of frozen liver suspensions from CPN- and AAF-treated rats yielded comparable results to the assays of fresh preparations. There were no indications of significant toxicity induced by any treatment. The micronucleus assay was positive for CPN and AZT and negative for IBA. In conclusion, the in vivo comet assay is capable of detecting genotoxic effects of a variety of chemicals and may fill an important role in the genotoxicity test battery.

Occupational hazard evaluation of p-bromobenzyl bromide from tests for genotoxicity.

As part of an occupational hazard evaluation, p-bromobenzyl bromide (p-BBB) was evaluated for genotoxic activity in the Ames microbial mutagenicity assay, the alkaline elution assay for DNA strand breaks in rat hepatocytes and the in vitro chromosome aberration assay in Chinese hamster ovary cells. The compound produced equivocal results in the microbial mutagenicity assay but was negative in the alkaline elution assay for DNA strand breaks in rat hepatocytes. The compound produced weakly positive results in the in vitro chromosome aberration assay. There was substantial cytotoxicity in all three assays. It is concluded that p-BBB is weakly genotoxic.

Revalidation of the in vitro alkaline elution/rat hepatocyte assay for DNA damage: improved criteria for assessment of cytotoxicity and genotoxicity and results for 81 compounds.

The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals, we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (K+) content, tetrazolium dye reduction (MTT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks; DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also evaluated 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysis/rinse fraction and the gamma-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Ames assay with no, or limited, other evidence of genotoxicity, and 33 genotoxins, most of which are also carcinogens, were evaluated. The results showed that DNA degradation as measured by a 1-h PACE (Programmed Autonomously Controlled Electrodes)/PFGE assay was a sensitive indicator of cytotoxicity which correlated well with results of the other cytotoxicity indicators. The delayed TBDE (after a 3-h recovery), intracellular potassium and ATP assays as well as the gamma-intercept parameter were also shown to be sensitive and in some cases complementary measures of cytotoxicity. Using new criteria based on these data of an induced slope (treatment slope-negative control slope) of 0.020 for the 3- to 9-h elution period and cytotoxicity limits of 70% relative viability for the delayed TBDE assay and 50% for intracellular ATP content, the assay scores the genotoxicity of these 61 reference compounds with an overall accuracy of 92%. Test results using these new criteria are provided for an additional 20 compounds (5 non-genotoxic carcinogens and 15 compounds whose genotoxic and carcinogenic potential are unknown or equivocal).

Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays.

The use of genetic toxicology tests for hazard identification is complicated by the fact that some in vitro tests using cultured mammalian cells are subject to potential artifacts that can make it difficult to distinguish between direct, chemically-induced genotoxicity, and DNA damage that occurs secondary to chemically-induced cytotoxicity (e.g., mediated by endogenous nucleases). Recently, we demonstrated that cytotoxicity-induced DNA double strand breaks (dsb) can produce artifacts in the in vitro alkaline elution/rat hepatocyte assay [Elia et al., 1993]. To explore this further, we used pulsed field gel/DNA dsb assays to characterize the relationship between chemically-induced cytotoxicity and the degradation of genomic DNA to high molecular weight fragments. Two sets of compounds were tested: 17 cytotoxic agents judged to be neither genotoxic nor carcinogenic, and 10 known genotoxic carcinogens. We found a close correlation between chemically-induced cytotoxicity and the rapid degradation of DNA to high molecular weight, double-stranded fragments. In contrast, the classic genotoxic chemicals tested generally did not trigger DNA dsb fragmentation at doses that were genotoxic but not immediately cytotoxic. These data indicate that pulsed field gel/DNA dsb assays can be used with in vitro genetic toxicology assays to help distinguish between genotoxic and cytotoxic mechanisms of DNA damage.