Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for ...

Environmental Health Centre

We have isolated and characterized a stable epithelial cell line from Muta Mouse lung that is a suitable complement to the in vivo assay system. The cells are contact inhibited, forming a flat monolayer, and retain several epithelial/pulmonary characteristics. The genome is stable across more than 50 generations, with a modal chromosome number of 78. Spontaneous rates of micronuclei (19.2 +/- 1.4 per 1,000), sister chromatid exchanges (0.25 +/- 0.004 per chromosome), and chromosome aberrations ( approximately 4%) are lower than, or comparable to, other transgenic cell lines currently used in mutagenicity research. Fluorescence in situ hybridization analyses showed that 80% of cells contain three lambdagt10lacZ loci. Slot-blot analyses indicated that the average cell contains approximately 17 transgene monomers. Spontaneous mutant frequency at the lacZ transgene is stable (39.8 +/- 1.1 x 10(-5)), and the direct-acting mutagens N-ethyl-N-nitrosourea and ICR-191 yielded increases in mutant frequency of 6.3- and 3.2-fold above control, respectively. Benzo[a]pyrene (BaP) exposure increased mutant frequency more than 25-fold above control and did not require an exogenous metabolic activation mixture. Inhibition of Cyp1A1 by 5 microM alpha-naphthoflavone eliminated BaP mutagenesis. Activation and mutation induction by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine required a low concentration (0.05% v/v) of exogenous rat liver S9. High activity of alpha, micro, and pi glutathione-S-transferase isozymes appears to confer resistance to the cytotoxic effects of xenobiotics. The cell line is a suitable complement to the in vivo Muta Mouse assay, and provides an opportunity for routine in vitro mutagenicity testing using an endpoint that is identical to that employed in vivo.

Development and characterization of a stable epithelial cell line from Muta Mouse lung.

There is ongoing concern that an assay for germ cell effects in female animals is not available. While transgenic mutation detection systems provide unprecedented access to numerous rodent tissues, studies on the induction of gene mutations in oocytes are still not possible because sufficient numbers of cells cannot be harvested. However, following stimulation of an ovarian follicle, the granulosa cells contained therein divide rapidly, increasing substantially in numbers. Since these granulosa cells share the same environment as the ovum, they may serve as suitable surrogates for the study of exposure of female germ cells to mutagens. Female lacZ transgenic mice (MutaMouse) were treated by intraperitoneal injection of N-ethylnitrosourea (ENU) and subsequently with pregnant mare serum gonadotropin (PMSG, 5IU/animal, i.p.) to induce follicular growth. Animals were sacrificed 48 h after the administration of PMSG and granulosa cells and bone marrow were harvested. A comparable dose-related increase in the mutant frequency (MF) of both granulosa and bone marrow cells was observed. The highest dose caused a decrease in the MF of granulosa cells, but not in the bone marrow, suggesting possible greater susceptibility of granulosa cells to ENU toxicity. Doubling dose estimates for bone marrow and granulosa cells were lower than those derived from the literature on oocyte mutation frequency using the Russell specific locus assay, suggesting that both cell types are more sensitive to ENU-induced mutation than oocytes. The results indicate that transgene mutations in granulosa cells may provide a sensitive pre-screening tool for potential genotoxic germ cell effects of exposed oocytes.

A lacZ transgenic mouse assay for the detection of mutations in follicular granulosa cells.

Carbon black and quartz are relatively inert solid particulate materials that are carcinogenic in laboratory animals. Quartz is a human carcinogen, whereas data on carbon black are contradictory, and there are few data on mammalian mutagenesis. We determined the mutant frequency following eight repeated 72-hr incubations with 75 mug/ml carbon black (Printex 90) or 100 mug/ml quartz (SRM1878a) particles in the FE1 Muta Mouse lung epithelial cell line. For carbon black exposed cells, the mutant frequency was 1.40-fold (95% CI: 1.22-1.58) for cII and 1.23-fold (95% CI: 1.10-1.37) for lacZ compared with identically passaged untreated cells. Quartz did not significantly affect the mutant frequency. Carbon black also induced DNA strand breaks (P = 0.02) and oxidized purines (P = 0.008), as measured by the Comet assay. Quartz induced marginally more oxidized purines, but no change in strand breaks. We detected five (phenanthrene, flouranthene, pyrene, benzo[a]anthracene, and chrysene) of the 16 EPA priority polycyclic aromatic hydrocarbons (PAHs) in an extract of carbon black. The detected PAHs are only weakly mutagenic compared with benzo[a]pyrene, and were present in very low amounts. In conclusion, carbon black was weakly mutagenic in vitro at the cII and lacZ loci. It also induced DNA strand breaks and oxidized DNA bases. More studies are essential for understanding the biological significance of these findings, and clearly documenting DNA sequence changes. The results do not necessarily imply that other carbonaceous nano materials are genotoxic.

Increased mutant frequency by carbon black, but not quartz, in the lacZ and cII transgenes of muta mouse lung epithelial cells.

3-Nitrobenzanthrone (3-NBA) is a mutagen and suspected human carcinogen detected in diesel exhaust, airborne particulate matter, and urban soil. We investigated the tissue specific mutagenicity of 3-NBA at the lacZ locus of transgenic MutaMouse following acute single dose or 28-day repeated-dose oral administration. In the acute high dose (50 mg/kg) exposure, increased lacZ mutant frequency was observed in bone marrow and colonic epithelium, but not in liver and bladder. In the repeated-dose study, a dose-dependent increase in lacZ mutant frequency was observed in bone marrow and liver (2- and 4-fold increase above control), but not in lung or intestinal epithelium. In addition, a concentration-dependent increase in mutant frequency (8.5-fold above control) was observed for MutaMouse FE1 lung epithelial cells exposed in vitro. 1-Nitropyrene reductase, 3-NBA reductase, and acetyltransferase activities were measured in a variety of MutaMouse specimens in an effort to link metabolic activation and mutagenicity. High 3-NBA nitroreductase activities were observed in lung, liver, colon and bladder, and detectable N-acetyltransferase activities were found in all tissues except bone marrow. The relatively high 3-NBA nitroreductase activity in MutaMouse tissues, as compared with those in Salmonella TA98 and TA100, suggests that 3-NBA is readily reduced and activated in vivo. High 3-NBA nitroreductase levels in liver and colon are consistent with the elevated lacZ mutant frequency values, and previously noted inductions of hepatic DNA adducts. Despite an absence of induced lacZ mutations, the highest 3-NBA reductase activity was detected in lung. Further studies are warranted, especially following inhalation or intratracheal exposures.

Tissue-specific metabolic activation and mutagenicity of 3-nitrobenzanthrone in MutaMouse.

We have developed an in vitro mutation assay using primary hepatocytes from the transgenic MutaMouse. Primary hepatocytes were isolated using a two-step perfusion method with purification by Percoll, cultured, and treated with benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenyl- imidazo[4,5-b]pyridine (PhIP), 3-nitrobenzoanthrone (3-NBA), and cigarette smoke condensate (CSC). The mean lacZ mutant frequency (MF) for the solvent control was approximately twofold greater than the spontaneous MF observed in liver tissue. A concentration-dependent increase in MF (up to 3.7-fold above control) was observed following exposure to BaP. Fourfold and twofold increases in mutant frequency were observed for 3-NBA and PhIP exposures, respectively, without the addition of any exogenous metabolic activation. A slight but statistically significant increase in lacZ MF was observed for CSC, but only at the lowest concentration. This is the first report demonstrating that mutations can be detected in cultured primary hepatocytes from MutaMouse. The preliminary results presented suggest that the MutaMouse primary hepatocyte mutagenicity assay can be used as a cost-effective tool for screening of environmental mutagens and therapeutic products.

Lynda Soper

Environmental Health Science and Research Bureau,
Health Canada,
Environmental Health Science and Research Bureau, Health Canada

Environmental Health Centre

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

Lynda Soper

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Environmental Health Centre

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

Environmental Health Science and Research Bureau,
Health Canada,
Environmental Health Science and Research Bureau, Health Canada