Dominique Brault

Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies.

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.

Comparative mutagenicity of 7H-dibenzo[c,g]carbazole and two derivatives in Muta™Mouse liver and skin

7H-Dibenzo[c,g]carbazole (DBC) is an environmental pollutant that produces DNA adducts and tumors in mouse liver and skin following subcutaneous injection and topical application. The two synthetic derivatives 5,9-dimethyl-DBC (DMDBC) and N7-methyl-DBC (NMDBC) induce tissue-specific lesions. DNA adducts and tumors are observed only in liver following exposure to DMDBC and only in skin following exposure to NMDBC. We used the positive selection MutaMouse model to measure the induction of mutations in the two target organs, 28 days after a single subcutaneous injection or topical application of DBC, DMDBC and NMDBC. In liver, DBC and DMDBC induced 30- to 50-fold increases in mutant frequency (MF), while NMDBC had only a weak effect, regardless of the route of administration. After topical application, DBC and NMDBC produced 3.4- to 7.9-fold increases in MF in skin, while DMDBC had a weak effect. After subcutaneous injection, the three compounds had no or weak effect in skin. This study shows gene mutations arise in the respective target organs in which primary DNA damage and tumors are observed. These results illustrate the relevance of the MutaMouse model for testing organ-specific mutagens.

Effect of ethylnitrosourea and methyl methanesulfonate on mutation frequency in Muta™Mouse germ cells (seminiferous tubule cells and epididymis spermatozoa)

As part of the Germ Cell Collaborative Study, we used the positive-selection Muta Mouse model to evaluate the effects of two direct alkylating agents, ethylnitrosourea (ENU) and methyl methanesulfonate (MMS), on male germ cells. The LacZ mutation frequency in seminiferous tubule cells and epididymis spermatozoa was measured 3, 14, 25 and 50 days after a single intraperitoneal (i.p.) administration of 150 mg/kg ENU and 3 and 14 days after a single i.p. administration of 40 mg/kg MMS. Three and 14 days after ENU treatment, the mutation frequency was slightly but significantly increased in seminiferous tubule cells (3.5- and 3.6-fold, respectively), while it remained unchanged in epididymis spermatozoa. After 25 and 50 days, time-dependent increase in the mutation frequency was observed in seminiferous tubule cells (8.9- and 14.3-fold, respectively) and epididymis spermatozoa (3.4- and 7.9-fold, respectively), confirming the sensitivity of premeiotic cells to the mutagenic activity of ENU. Three and 14 days after MMS administration, the mutation frequency remained unchanged in seminiferous tubule cells and epididymis spermatozoa. The inability of Muta Mouse model to reveal the mutagenic activity of MMS was confirmed in bone marrow cells, 14 days after treatment. These data indicate that the Muta Mouse model can be used to detect the induction of gene mutations but not chromosome damage in germ cells.

Kinetics of induction of DNA damage and lacZ gene mutations in stomach mucosa of mice treated with β‐propiolactone and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, using single‐cell gel electrophoresis and MutaTMMouse models

β‐Propiolactone (BPL) and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) are two direct alkylating agents that induce multiple genetic lesions and tumors in the rodent stomach. We measured the kinetics of the induction of DNA damage by using the single‐cell gel electrophoresis assay (SCGE) and the induction of gene mutations by using the Muta™Mouse model in the glandular stomach mucosa of mice exposed to a single oral administration of BPL or MNNG. The aims were to determine the optimal sampling time and to investigate the cause‐effect relationship between DNA damage and gene mutations. The induction of comets, evaluated in individual cells with the tail moment, was analyzed 1, 2, 4, 24, and 72 hr after a single oral administration of 25 mg/kg BPL or 20 mg/kg MNNG. The effects of both compounds were most intense at the earlier sampling times (1–2 hr), tailing off 4 hr after treatment and becoming undetectable at 72 hr. The lacZ mutant frequency (MF) was measured 3, 7, 14, 28, and 50 days after a single oral administration of 150 mg/kg BPL or 100 mg/kg MNNG, and 3 and 14 days after a single administration of 25 mg/kg BPL or 20 mg/kg MNNG. The MF was strongly enhanced at the highest doses and all sampling times, the most marked effects being observed 14 days (11.1‐fold) and 28 days (19.0‐fold) after BPL and MNNG administration, respectively. At the lowest doses, only a small increase in MF (∼2.5‐ to 3.5‐fold) was found at both sampling times. Primary DNA damage detected with SCGE shortly after treatment (1–2 hr) was rapidly (3 days) transformed into stable gene mutations that remained detectable for 50 days. These results illustrate the ability and complementarity of the SCGE and Muta™Mouse models to assess the genotoxicity of direct alkylating agents in the mouse gastric mucosa in vivo. Environ. Mol. Mutagen. 34:182–189, 1999