cII Mutant Frequency Research Articles

Hydroxychloroquine induces oxidative DNA damage and mutation in mammalian cells

Since the early stages of the pandemic, hydroxychloroquine (HCQ), a widely used drug with good safety profile in clinic, has come to the forefront of research on drug repurposing for COVID-19 treatment/prevention. Despite the decades-long use of HCQ in the treatment of diseases, such as malaria and autoimmune disorders, the exact mechanisms of action of this drug are only beginning to be understood. To date, no data are available on the genotoxic potential of HCQ in vitro or in vivo. The present study is the first investigation of the DNA damaging- and mutagenic effects of HCQ in mammalian cells in vitro, at concentrations that are comparable to clinically achievable doses in patient populations. We demonstrate significant induction of a representative oxidative DNA damage (8-oxodG) in primary mouse embryonic fibroblasts (MEFs) treated with HCQ at 5 and 25 μM concentrations (P = 0.020 and P = 0.029, respectively), as determined by enzyme-linked immunosorbent assay. Furthermore, we show significant mutagenicity of HCQ, manifest as 2.2- and 1.8-fold increases in relative cII mutant frequency in primary and spontaneously immortalized Big Blue® MEFs, respectively, treated with 25 μM dose of this drug (P = 0.005 and P = 0.012, respectively). The observed genotoxic effects of HCQ in vitro, achievable at clinically relevant doses, are novel and important, and may have significant implications for safety monitoring in patient populations. Given the substantial number of the world’s population receiving HCQ for the treatment of various chronic diseases or in the context of clinical trials for COVID-19, our findings warrant further investigations into the biological consequences of therapeutic/preventive use of this drug.

In vivo mutagenicity evaluation of the soil fumigant 1,3-dichloropropene

1,3-Dichloropropene (1,3-D; CAS No. 542-75-6) is a soil fumigant used for the control of nematodes in agriculture. There is an extensive database on the genotoxicity of 1,3-D and many of the published studies are confounded by the presence of mutagenic stabilisers in the test substance. Mixed results were obtained in the in vitro assays, often due to the purity of the 1,3-D sample tested. In order to get further clarity, the mutagenic potential of 1,3-D was investigated in vivo in the transgenic Big Blue rodent models. Inhalation exposure of 150 ppm 1,3-D (×2.5 tumourigenic dose) to transgenic male B6C3F1 mice did not induce lacI mutations in either the lung (tumour target tissue) or liver. Similarly, dietary administration of 1,3-D up to 50 mg/kg/day to transgenic male Fischer 344 rats did not increase the cII mutant frequency in either the liver (tumour target) or kidney. These results, along with other available in vivo data, including the absence of DNA adducts and clastogenic/aneugenic potential, support the conclusion that 1,3-D is efficiently detoxified in vivo and, as such, does not pose a mutagenic hazard or risk.

The mutant frequencies and types of mutations induced by Comfrey in the lungs of transgenic big blue rats

Comfrey (Symphytum Officinale) has been used as a vegetable and herbal remedy for many years. It, however, is a mutagen and carcinogen in liver and a possible genotoxin in lung. In order to evaluate the mutagenicity of comfrey and the mechanisms of action in rat lung, we examined the mutant frequencies (MF) and mutational types in the lung cII gene from transgenic Big Blue rats treated with 8% comfrey for 12 weeks. The CII MF in the lungs for rats fed with comfrey was 48 ± 9 × 10-6, which was significantly greater than the MF for control rats, 34 ± 10 × 10-6 (p = 0.026). The mutational spectrum in lung from comfrey-fed rats is significantly different from that in the control. G:C → T:A transversion (29%) was the major type of mutation in comfrey-fed rats, whereas G:C → A:T transition (63%) was the predominant mutation in the controls. An unusual type of tandem-base substitution (4%) was observed among the mutations from comfrey-fed rats that were not seen in the control. This type of tandem-base substitution has been suggested as a mutational signature for the genetic damage of pyrrolizidine alkaloids (PAs). The results indicate that comfrey is mutagenic in rat lung and the types of mutations induced by comfrey are similar to those by other PAs; and suggest that the mutagenicity of comfrey in rat lung results from the genotoxicity of PAs in the plant.

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization

To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.

The Lambda Select cII Mutation Detection System.

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Mutation Analysis in Cultured Cells of Transgenic Rodents.

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

Dose and temporal evaluation of ethylene oxide-induced mutagenicity in the lungs of male big blue mice following inhalation exposure to carcinogenic concentrations.

Ethylene oxide (EO) is a direct acting alkylating agent; in vitro and in vivo studies indicate that it is both a mutagen and a carcinogen. However, it remains unclear whether the mode of action (MOA) for cancer for EO is a mutagenic MOA, specifically via point mutation. To investigate the MOA for EO-induced mouse lung tumors, male Big Blue (BB) B6C3F1 mice (10/group) were exposed to EO by inhalation, 6 hr/day, 5 days/week for 4 (0, 10, 50, 100, or 200 ppm EO), 8, or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for cII mutant frequency (MF) at 4, 8 and 12 weeks of exposure; the mutation spectrum was analyzed for mutants from control and 200 ppm EO treatments. Although EO-induced cII MFs were 1.5- to 2.7-fold higher than the concurrent controls at 4 weeks, statistically significant increases in the cII MF were found only after 8 and 12 weeks of exposure and only at 200 ppm EO (P ≤ 0.05), which is twice the highest concentration used in the cancer bioassay. Consistent with the positive response, DNA sequencing of cII mutants showed a significant shift in the mutational spectra between control and 200 ppm EO following 8 and 12 week exposures (P ≤ 0.035), but not at 4 weeks. Thus, EO mutagenic activity in vivo was relatively weak and required higher than tumorigenic concentrations and longer than 4 weeks exposure durations. These data do not follow the classical patterns for a MOA mediated by point mutations. Environ. Mol. Mutagen. 58:122-134, 2017. © 2017 Wiley Periodicals, Inc.

Neonatal exposure of 17β-estradiol has no effects on mutagenicity of 7,12-dimethylbenz [a] anthracene in reproductive tissues of adult mice.

IntroductionBiological studies in animals and epidemiological findings in humans clearly demonstrate that estrogens including 17β-estradiol (E2) are weak carcinogens via both genetic and epigenetic mechanisms. Carcinogenesis analyses have indicated that female mice exposed to E2 as neonates develop more mammary and ovarian tumors when compared to adult exposures. In the present study, Big Blue transgenic mice were used to investigate the effects of E2 on mutagenicity of 7,12-dimethylbenz [a] anthracene (DMBA), a genotoxic carcinogen, in mammary gland and ovary following neonatal exposure.ResultsDMBA treatment resulted in significant increases in cII mutant frequencies (MFs) in both mammary glands and ovaries, with A:T → T:A transversion as the predominant type of mutation. However, co-exposure to E2 daily for the first 5 days after birth and to DMBA at 6 months of age did not significantly increase cII MFs compared to DMBA treatment alone. Further, there were also no significant differences in mutational spectra between DMBA exposure alone and E2 + DMBA treatment.ConclusionThese results suggest that early life exposures of mice to estrogens like E2 do not enhance mutagenicity by subsequent exposure to a chemical like DMBA in later life.Electronic supplementary materialThe online version of this article (doi:10.1186/s41021-015-0011-y) contains supplementary material, which is available to authorized users.

Evaluation of cII mutations in lung of male Big Blue mice exposed by inhalation to vanadium pentoxide for up to 8 weeks

Chronic inhalation of vanadium pentoxide (V2O5) increases the incidence of alveolar/bronchiolar tumors in male and female B6C3F1 mice at 1, 2, or 4mg/m3. The genotoxicity of V2O5 has been extensively investigated in the literature with mixed results. In general, tests for gene mutations have been negative. Both positive and negative results were reported for clastogenicity in vitro with some reports suggesting aneugenic potential. In vivo, V2O5 was negative in the mouse micronucleus test (erythrocyte) and comet assay (lung). Previously, K-ras mutations have been detected in the lung tumors in mice exposed to V2O5. Recently, a short-term inhalation study in B6C3F1 mice reported slight induction of 8-oxodGuo DNA lesions in lungs. Because 8-oxodGuo DNA lesions can lead to gene mutations if not repaired or if misrepaired, we have used groups of transgenic Big Blue (BB) mice (B6C3F1) to test whether V2O5 has mutagenic potential in vivo in the tumor target tissue under the conditions of the bioassay. Groups of six male BB mice were exposed to particulate aerosols containing 0, 0.1, or 1mg/m3 (tumorigenic concentration) V2O5 for 4 or 8 weeks (6h/day, 5 days/week) and cII mutant frequencies (MFs) were evaluated in the right lungs. A significant increase in lung weight was noted in mice exposed to 1mg/m3 V2O5 (P≤0.05) compared to sham control, confirming exposure to an inflammatory level of the test material. The mean MFs (×10−6) of mice in the 4-week exposure groups were 30 (sham control), 39 (0.1mg/m3), and 24 (1mg/m3) while the corresponding values in the 8-week exposure groups were 29, 48, and 17, respectively. None of these cII MFs measured at any time point was significantly higher than the corresponding control MFs (P≥0.1). Overall, these results suggest that mutagenicity is not likely to be an initial key event in the lung tumorigenicity of V2O5.

A robust method for assessing chemically induced mutagenic effects in the oral cavity of transgenic Big Blue® rats.

The Big Blue® (BB) in vivo mutation assay uses transgenic rodents to measure treatment-induced mutations in virtually any tissue. The BB assay can be conducted in rats or mice and is ideal for investigating tissue-specific mutagenic mode of action of tumor induction. Some tissues such as oral mucosa have not been thoroughly studied. Due to the small quantity and cartilaginous nature of oral cavity tissues, development of special prosection and DNA isolation methods was required to permit robust analysis of mutations in these tissues. Improved surgical methods permitted collection of adequate and reproducible quantities of tissue (∼45 mg gingiva/buccal and ∼30 mg gingiva/palate). Optimized DNA isolation methods included use of liquid nitrogen pulverization, homogenization, nuclei pelleting, digestion, and phenol/chloroform extraction, to yield sufficient quantities of DNA from these tissues. In preliminary optimization work, mutant frequency (MF) in tongue and gingiva was increased in rats exposed to the promutagen, benzo[a]pyrene, and the direct mutagen, N-ethyl-N-nitrosourea. The oral cavity carcinogen, 4-nitroquinoline-1-oxide (4-NQO; 10 ppm in drinking water; 28 days), was qualified as a positive control for mutagenesis in oral tissues since it caused significant increases in cII MFs in gingiva/palate (50.2-fold) and gingiva/buccal tissues (21.3-fold), but not in liver or bone marrow (0.9- and 1.4-fold, respectively). These results are consistent with the observation that 4-NQO primarily induces tumors in oral cavity. Results also demonstrate the utility of the BB rat mutation assay and optimized methods for investigation of oral cavity mutagenicity, and by extension, analysis of other small and cartilaginous tissues.

Acrylamide-induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of big blue mice exposed to acrylamide and glycidamide for up to 4 weeks.

Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA- or GA-induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19-111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7-5.6-fold higher than the corresponding controls (P ≤ 0.05; control MFs = 17.2 ± 2.2 and 15.8 ± 3.5 × 10(-6) in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P ≤ 0.01). The predominant types of mutations in the lung cII gene from AA- and GA-treated mice were A:T → T:A, and G:C → C:G transversions, and -1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446-456, 2015. © 2015 Wiley Periodicals, Inc.

Temporal Changes in K-ras Mutant Fraction in Lung Tissue of Big Blue B6C3F1 Mice Exposed to Ethylene Oxide

Ethylene oxide (EO) is a genotoxicant and a mouse lung carcinogen, but whether EO is carcinogenic through a mutagenic mode of action remains unclear. To investigate this question, 8-week-old male Big Blue B6C3F₁ mice (10 mice/group) were exposed to EO by inhalation-6 h/day, 5 days/week for 4 weeks (0, 10, 50, 100, or 200 ppm EO) and 8 or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for levels of 3 K-ras codon 12 mutations (GGT→GAT, GGT→GTT, and GGT→TGT) using ACB-PCR. No measureable level of K-ras codon 12 TGT mutation was detected (ie, all lung mutant fractions [MFs] ≤ 10⁻⁵). Four weeks of inhalation of 100 ppm EO caused a significant increase in K-ras codon 12 GGT→GTT MF relative to controls, whereas 50, 100, and 200 ppm EO caused significant increases in K-ras codon 12 GGT→GAT MF. In addition, significant inverse correlations were observed between K-ras codon 12 GGT→GTT MF and cII mutant frequency in the lungs of the same mice exposed to 50, 100, or 200 ppm EO for 4 weeks. Surprisingly, 8 weeks of exposure to 100 and 200 ppm EO caused significant decreases in K-ras MFs relative to controls. Thus, the changes in K-ras MF as a function of cumulative EO dose were nonmonotonic and were consistent with EO causing early amplification of preexisting K-ras mutations, rather than induction of K-ras mutation through genotoxicity at codon 12. The possibility that these changes reflect K-ras mutant cell selection under varying degrees of oxidative stress is discussed.

Mutagenicity and DNA adduct formation by aristolochic acid in the spleen of Big Blue® rats.

Aristolochic acid (AA) is a potent human nephrotoxin and carcinogen. We previously reported that AA treatment resulted in DNA damage and mutation in the kidney and liver of rats. In this study, we have determined the DNA adducts and mutations induced by AA in rat spleen. Big Blue® transgenic rats were gavaged with 0, 0.1, 1.0, and 10.0 mg AA/kg body weight five-times/week for 3 months. Three DNA adducts, [7-(deoxyadenosin-N(6)-yl)-aristolactam I, 7-(deoxyadenosin-N(6)-yl)-aristolactam II and 7-(deoxyguanosin-N(2)-yl)-aristolactam I], were identified by (32)P-postlabeling. Over the dose range studied, there were strong linear dose-responses for AA-DNA adduct formation in the treated rat spleens, ranging from 4.6 to 217.6 adducts/10(8) nucleotides. Spleen cII mutant frequencies also increased in a dose-dependent manner, ranging from 32.7 to 286.2 × 10(-6) in the treated animals. Mutants isolated from the different treatment groups were sequenced; analysis of the resulting spectra indicated that there was a significant difference between the pattern of mutation in the 10 mg/kg AA-treated and the vehicle control rats. A:T → T:A transversion was the major type of mutation in AA-treated rats, whereas G:C → A:T transition was the main type of mutation in the vehicle controls. These results indicate that AA is genotoxic in the spleen of rats exposed under conditions that result in DNA adduct formation and mutation induction in kidney and liver.

New experimental data linking secondhand smoke exposure to lung cancer in nonsmokers

Secondhand smoke (SHS) exposure is a known risk factor for lung cancer development in lifelong nonsmokers; however, the mechanistic involvement of SHS in the genesis of this malignancy remains elusive. The present study is the first comprehensive investigation of SHS mutagenicity in vivo, in which we have established the mutagenic effects of SHS in transgenic Big Blue mice, and subsequently found correlations between our experimental findings and those obtained from our analysis of the largest database of mutations in human TP53, which is the most frequently mutated gene in human lung cancer. We demonstrate that whole-body SHS exposure of mice for 5 h/d, 5 d/wk for a duration of 2 or 4 mo elicits a significant mutagenic response in the lung, trachea, and bladder of exposed animals, as reflected by the elevation of background cII mutant frequency in the respective organs. The organ-specific mutagenicity of SHS is most pronounced in the lung and remains persistent both in the lung and bladder of SHS-exposed animals after a 1-mo recovery in clean air. The induced cII mutagenesis in the lung of SHS-exposed mice perfectly recapitulates our analysis of the TP53 mutations in human lung cancer in nonsmokers. Remarkably, the relative frequencies of all types of mutations in the TP53 gene of nonsmokers' lung tumors and in the cII transgene of lung cellular DNA from SHS-exposed mice are indistinguishable from one another. We provide the first verification of a mechanistic mode of action for SHS of relevance for carcinogenesis and the first experimental evidence linking SHS exposure to lung cancer in nonsmokers.

Organ Specificity of the Bladder Carcinogen 4-Aminobiphenyl in Inducing DNA Damage and Mutation in Mice

Aromatic amines are a widespread class of environmental contaminants present in various occupational settings and tobacco smoke. Exposure to aromatic amines is a major risk factor for bladder cancer development. The etiologic involvement of aromatic amines in the genesis of bladder cancer is attributable to their ability to form DNA adducts, which upon eluding repair and causing mispairing during replication, may initiate mutagenesis. We have investigated the induction of DNA adducts in relation to mutagenesis in bladder and various nontarget organs of transgenic Big Blue mice treated weekly (i.p.) with a representative aromatic amine compound, 4-aminobiphenyl (4-ABP), for six weeks, followed by a six-week recovery period. We show an organ-specificity of 4-ABP in inducing repair-resistant DNA adducts in bladder, kidney, and liver of carcinogen-treated animals, which accords with the bioactivation pathway of this chemical in the respective organs. In confirmation, we show a predominant and sustained mutagenic effect of 4-ABP in bladder, and much weaker but significant mutagenicity of 4-ABP in the kidney and liver of carcinogen-treated mice, as reflected by the elevation of background cII mutant frequency in the respective organs. The spectrum of mutations produced in bladder of 4-ABP-treated mice matches the known mutagenic properties of 4-ABP-DNA adducts, as verified by the preponderance of induced mutations occurring at G:C base pairs (82.9%), with the vast majority being G:C→T:A transversions (47.1%). Our data support a possible etiologic role of 4-ABP in bladder carcinogenesis and provide a mechanistic view on how DNA adduct-driven mutagenesis, specifically targeted to bladder urothelium, may account for organ-specific tumorigenicity of this chemical.

Aristolochic acid-induced carcinogenesis examined by ACB-PCR quantification of H-Ras and K-Ras mutant fraction

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen associated with the development of urothelial cancer in humans. AA induces forestomach, kidney and urothelial tract tumours in rats and mice. This study was conducted to characterise AA's carcinogenic mechanism of action and compare allele-specific competitive blocker-polymerase chain reaction (ACB-PCR)-based early detection of carcinogenic effect using two different tumour-relevant endpoints. H-Ras codon 61 CAA→CTA mutation was analysed because it is found in rodent forestomach tumours and A:T→T:A transversion is the predominant mutational specificity induced by AA. K-Ras codon 12 GGT→GAT mutation was analysed because it is a common spontaneous mutation present in various rodent tissues and may be a useful generic biomarker for carcinogenic effect. DNA samples from Big Blue rats treated with 0, 0.1, 1.0 or 10.0 mg AA/kg body weight (bw) by gavage, 5 days/week for 12 weeks were used in ACB-PCR in order to examine the induction of the two specific mutations. A significant dose-dependent induction of H-Ras mutant fraction (MF) was observed in liver and kidney. Statistically significant correlations were observed between AA-induced DNA adduct levels or cII mutant frequencies (previously measured in the same rats) and H-Ras MF measurements. No correlation between AA dose and K-Ras MF was found in liver or kidney, although there was a significant induction of K-Ras mutation in kidneys exposed to 0.1 mg/kg bw AA relative to controls. Thus, the data establish a straightforward dose-related increase in H-Ras MF due to fixation of AA-induced DNA adducts, whereas the common spontaneous K-Ras mutation showed a non-monotonic dose-response, consistent with loss of non-targeted mutation at cytotoxic doses.

Mutagenicity of Acrylamide and Glycidamide in the Testes of Big Blue Mice

Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and rodent carcinogen. It can also cause damage during spermatogenesis. In this study, we investigated whether AA and its metabolite glycidamide (GA) induce mutagenic effects in the germ cells of male mice. Male Big Blue transgenic mice were administered 1.4 or 7.0mM of AA or GA in the drinking water for up to 4 weeks. Testicular cII mutant frequency (MF) was determined 3 weeks after the last treatment, and the types of the mutations in the cII gene were analyzed by DNA sequencing. The testes cII MFs in mice treated with either the low or high exposure concentrations of AA and GA were increased significantly. There was no significant difference in the cII MFs between AA and GA at the low exposure concentration. The mutation spectra in mice treated with AA (1.4mM) or GA (both 1.4 and 7.0mM) differed significantly from those of controls, but there were no significant differences in mutation patterns between AA and GA treatments. Comparison of the mutation spectra between testes and livers showed that the spectra differed significantly between the two tissues following treatment with AA or GA, whereas the mutation spectra in the two tissues from control mice were similar. These results suggest that AA possesses mutagenic effects on testes by virtue of its metabolism to GA, possibly targeting spermatogonial stem cells, but possibly via different pathways when compared mutations in liver.

Isolated spermatozoa as indicators of mutations transmitted to progeny

Spermatozoa comprise a large and homogeneous population of cells that may serve as an alternative to resource-intensive assays of transmissible mutations based on progeny. To evaluate mutagenic responses in spermatozoa derived from germ cells exposed to a mutagen at different stages of spermatogenesis, we compared cII mutant frequencies (MFs) in spermatozoa collected from male λ transgenic medaka exposed to ethylnitrosourea (ENU) as either post-meiotic or pre-meiotic germ cells. cII MFs in spermatozoa exposed to ENU as spermatogonial stem cells were induced significantly, 9-fold, compared to controls, whereas, cII MFs in spermatozoa exposed as spermatozoa/late spermatids were not elevated. To directly compare responses in spermatozoa with those in progeny, we analyzed cII MFs directly in spermatozoa and in the offspring produced from identical sperm samples of ENU-exposed males. cII MFs in isolated spermatozoa exposed to ENU as post-meiotic germ cells were not significantly elevated, whereas 11–30% of the progeny derived from the identically exposed germ cells exhibited significantly elevated cII MFs, ∼2-fold to >130-fold, compared to controls. The contradictory responses between spermatozoa and progeny analyses can be attributed to induced pre-mutational lesions that remain intact in spermatozoa but were not detected as mutations. Progeny analyses, by contrast, revealed mutant individuals with elevated cII mutant frequencies because persistent DNA damage in the spermatozoa was fixed as mutations in cells of the early stage embryo. Spermatozoa exposed to a mutagen as spermatogonial stem cells can provide an efficient means to detect the portion of transmissible mutations that were fixed as mutations in spermatozoa. The caveat is that direct analyses of mutations in spermatozoa excludes the contribution of mutations that arise from post-fertilization processes in cells of early stage embryos, and therefore may underestimate the actual frequency of mutant offspring.

Evaluation of mutagenic mode of action in Big Blue mice fed methylphenidate for 24 weeks

Methylphenidate hydrochloride (MPH), a widely prescribed pediatric drug for attention deficit hyperactivity disorder, induced liver adenocarcinomas in B6C3F1 mice exposed to 500 ppm in feed for 2 years (Dunnick and Hailey (1995) [14]). In order to determine if the induction of liver tumors was by a mutagenic mode of action, groups of male Big Blue (BB) mice (B6C3F1 background) were fed diets containing 50–4000 ppm MPH for 4, 12, or 24 weeks. At sacrifice, the livers were removed and the cII mutant frequency (MF) and spectrum of cII mutations were determined. In addition, the frequencies of micronucleated reticulocytes (MN-RETs) and normochromatic erythrocytes (MN-NCEs) were measured in peripheral blood erythrocytes as was the Hprt MF in splenic lymphocytes. Food consumption and body weight gain/loss were recorded weekly for each animal. The levels of MPH and RA were determined immediately before sacrifice in the serum of mice fed MPH for 24 weeks. A significant loss in body weights ( p ≤ 0.01) was found in mice fed the 2000 and 4000 ppm doses of MPH; however, there was no significant difference in the food consumption by any of the MPH-treated groups. The average liver cII MF in control animals was 20 ± 4.5 × 10 −6 where as in MPH-treated animals, the average cII MFs ranged between 20 ± 2.5 and 32 ± 6.7 × 10 −6. None of the cII MFs in livers from any of the MPH treatment was significantly higher than the concurrent controls at either 4, 12 or 24 weeks. Further, there was no significant increase in either the Hprt MF or the micronucleus frequency at any time point in the treated animals. These results suggest that MPH is not mutagenic in mice and that the induction of tumors as previously reported in the liver is probably through a nongenotoxic mode of action.

Transgenic λ medaka as a new model for germ cell mutagenesis

To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated lambda transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 x 10(-5)) in the range of 10(-4) to 10(-3) were mosaic mutant offspring, whereas those with MFs approaching 1 x 10(-2) were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks.