No evidence for transgenerational genomic instability in the F1 or F2 descendants of Muta™Mouse males exposed to N-ethyl-N-nitrosourea.

Exposure of male mice to genotoxic agents can increase mutation frequencies in their unexposed descendants. This phenomenon, known as transgenerational genomic instability (TGI), can persist for several generations. However, little is known about the underlying mechanisms. Chemically-induced TGI has been demonstrated in non-coding unstable tandem repeat DNA regions, but it is unclear whether it extends to other genetic endpoints. We investigated whether exposure of Muta™Mouse males to a single dose of 75mg/kg N-ethyl-N-nitrosourea (ENU) increased the spontaneous frequency of gene mutations or chromosome damage in their offspring. Treated males were mated with untreated females 3 days, 6 weeks or 10 weeks post-exposure to produce the F1 generation. Offspring were thus conceived from germ cells exposed to ENU as mature spermatozoa, dividing spermatogonia, or spermatogonial stem cells, respectively. F2 mice were generated by mating F1 descendants with untreated partners. Mutations in the lacZ transgene were quantified in bone marrow and micronucleus frequencies were evaluated in red blood cells by flow-cytometry for all F0 and their descendants. LacZ mutant frequencies were also determined in sperm for all exposed males and their male descendants. In F0 males, lacZ mutant frequencies were significantly increased in bone marrow at least 10-fold at all three time points investigated. In sperm, lacZ mutant frequency was significantly increased 7-11-fold after exposure of dividing and stem cell spermatogonia, but not in replication-deficient haploid sperm. Micronucleus frequencies assessed two days after ENU treatment were increased 5-fold in F0 males, but returned to control levels after 10 weeks. Despite the strong mutagenic response in F0 males, pre- and post-meiotic ENU exposure did not significantly increase lacZ mutant or micronucleus frequencies in F1 or F2 offspring. These findings suggest that TGI may not extend to all genetic endpoints and that further investigation of this phenomenon and its health relevance will require multiple measures of genomic damage.

Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

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

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

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.

Meiotic interstrand DNA damage escapes paternal repair and causes chromosomal aberrations in the zygote by maternal misrepair.

De novo point mutations and chromosomal structural aberrations (CSA) detected in offspring of unaffected parents show a preferential paternal origin with higher risk for older fathers. Studies in rodents suggest that heritable mutations transmitted from the father can arise from either paternal or maternal misrepair of damaged paternal DNA, and that the entire spermatogenic cycle can be at risk after mutagenic exposure. Understanding the susceptibility and mechanisms of transmission of paternal mutations is important in family planning after chemotherapy and donor selection for assisted reproduction. We report that treatment of male mice with melphalan (MLP), a bifunctional alkylating agent widely used in chemotherapy, induces DNA lesions during male mouse meiosis that persist unrepaired as germ cells progress through DNA repair-competent phases of spermatogenic development. After fertilization, unrepaired sperm DNA lesions are mis-repaired into CSA by the egg's DNA repair machinery producing chromosomally abnormal offspring. These findings highlight the importance of both pre- and post-fertilization DNA repair in assuring the genomic integrity of the conceptus.

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

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.

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 assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

Mutation spectrum in FE1-MUTA(TM) Mouse lung epithelial cells exposed to nanoparticulate carbon black.

It has been shown previously that carbon black (CB), Printex 90 exposure induces cII and lacZ mutants in the FE1-Muta(TM) Mouse lung epithelial cell line and causes oxidatively damaged DNA and the production of reactive oxygen species (ROS). The purpose of this study was to determine the mutation spectrum in the cII gene of Printex 90 exposed cells. Cells exposed to CB have a substantially different mutation spectrum in the cII gene compared with vehicle exposed controls. The mutation spectra differ both in the positions (P < 0.0001) and types of the mutations (P < 0.0001). Exposure to Printex 90 increased the number of single base deletions by 2.3-fold and larger deletions by 1.9-fold. Most single base deletions were within two repetitive sequences in cII, but the large deletions were not. The mechanism behind the large deletions is not yet known. The largest increases in base substitutions were observed in G:C→T:A, G:C→C:G, and A:T→T:A transversion mutations; this is in keeping with a genetic finger print of ROS and is further substantiated by the observations that Printex 90 generates ROS and oxidatively damaged DNA.

Mutagenicity of carbon nanomaterials.

Carbon nanomaterials such carbon nanotubes, graphene and fullerenes are some the most promising nanomaterials. Although carbon nanomaterials have been reported to possess genotoxic potential, it is imperitive to analyse the data on the genotoxicity of carbon nanomaterials in vivo and in vitro and check the validity and predictability of different assays.

Simultaneous measurement of benzo[a]pyrene-induced Pig-a and lacZ mutations, micronuclei and DNA adducts in Muta™ Mouse.

In this study we compared the response of the Pig-a gene mutation assay to that of the lacZ transgenic rodent mutation assay, and demonstrated that multiple endpoints can be measured in a 28-day repeat dose study. Muta™Mouse were dosed daily for 28 days with benzo[a]pyrene (BaP; 0, 25, 50 and 75 mg/kg body weight/day) by oral gavage. Micronucleus (MN) frequency was determined in reticulocytes (RETs) 48 hr following the last dose. 72 h following the last dose, mice were euthanized, and tissues (glandular stomach, small intestine, bone marrow and liver) were collected for lacZ mutation and DNA adduct analysis, and blood was evaluated for Pig-a mutants. BaP-derived DNA adducts were detected in all tissues examined and significant dose-dependent increases in mutant Pig-a phenotypes (i.e., RET(CD24-) and RBC (CD24-)) and lacZ mutants were observed. We estimate that mutagenic efficiency (i.e., rate of conversion of adducts into mutations) was much lower for Pig-a compared to lacZ, and speculate that this difference is likely explained by differences in repair capacity between the gene targets, and differences in the cell populations sampled for Pig-a versus lacZ. The BaP doubling doses for both gene targets, however, were comparable, suggesting that similar mechanisms are involved in the accumulation of gene mutations. Significant dose-related increases in % MN were also observed; however, the doubling dose was considerably higher for this endpoint. The similarity in dose response kinetics of Pig-a and lacZ provides further evidence for the mutational origin of glycosylphosphatidylinositol (GPI)-anchor deficiencies detected in the Pig-a assay.

Induction of lacZ mutations in MutaMouse primary hepatocytes.

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.

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

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.