Mugimane G. Manjanatha

Accumulation of point mutations in mitochondrial DNA of aging mice.

Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P<0.001). The estimated mutation frequency in mtDNA from old mice was 11.6+/-2.7 or 25.4+/-7.8 per 10(5) nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Transgenic Animal Models in Toxicology: Historical Perspectives and Future Outlook

Transgenic animal models are powerful tools for developing a more detailed understanding on the roles of specific genes in biological pathways and systems. Applications of these models have been made within the field of toxicology, most notably for the screening of mutagenic and carcinogenic potential and for the characterization of toxic mechanisms of action. It has long been a goal of research toxicologists to use the data from these models to refine hazard identification and characterization to better inform human health risk assessments. This review provides an overview on the applications of transgenic animal models in the assessment of mutagenicity and carcinogenicity, their use as reporter systems, and as tools for understanding the roles of xenobiotic-metabolizing enzymes and biological receptors in the etiology of chemical toxicity. Perspectives are also shared on the future outlook for these models in toxicology and risk assessment and how transgenic technologies are likely to be an integral tool for toxicity testing in the 21st century.

p53, mutations, and apoptosis in genistein-exposed human lymphoblastoid cells.

The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.

Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue® rats treated with 7,12-dimethylbenz[a]anthracene

The lacI transgene of Big Blue(R) (BB) rats was evaluated as a reporter of in vivo mutation by comparing mutant frequencies (MFs) in it and in the endogenous Hprt gene. Seven-week old female BB rats were given single doses of 0, 20, 75 and 130 mg/kg of 7, 12-dimethylbenz(a)anthracene (DMBA) by gavage, and Hprt and lacI MFs in splenic lymphocytes were measured over a period of 18 weeks. The Hprt MFs in treated rats increased for 10 weeks and then declined; 130 mg/kg of DMBA produced a maximum Hprt MF of 168+/-11.4x10-6 clonable lymphocytes, while the MF in control rats was 7.4+/-1. 5x10-6. DMBA exposure of generic F344 rats resulted in a similar time-course of mutant induction but produced about 50% higher Hprt MFs with the 75 and 130 mg/kg doses. In contrast, the lacI MFs increased for 6 weeks and then remained relatively constant; 130 mg/kg of DMBA produced a maximum increase in lacI MF of 341+/-83x10-6 PFU compared with 25+/-5x10-6 PFU in control rats. The Hprt mutant frequencies in DMBA-treated BB and F344 rats were significantly increased over control values for every dose-time combination examined, while only the 130 mg/kg dose consistently produced lacI MFs that were significantly above the controls. In addition, the fold-increase in MF for treated vs. control rats was two times higher for the Hprt gene than the lacI gene due to the higher MFs in the lacI gene of control rats. Differences between the lacI and Hprt genes in the kinetics of mutant induction, in the frequency of induced mutants, and in the sensitivity of mutant detection could be explained at least partially by the properties of these two genes.

Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: Application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats†

A modified flow cytometry assay for Pig‐A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague‐Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 × /day on postnatal days (PNDs) 29–50. Eight additional rats (4/sex) were injected i.p. with N‐ethyl‐N‐nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/μl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614–629), the HIS49 Pig‐A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU‐treated rats, the modification increased the overall sensitivity of the assay 2–3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig‐A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El‐Zein et al. [2005]: Cancer Lett 230: 284–291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans. Environ. Mol. Mutagen. 2010. Published 2009 by Wiley‐Liss, Inc.

Genotoxicity of nanomaterials: Refining strategies and tests for hazard identification

A workshop addressing strategies for the genotoxicity assessment of nanomaterials (NMs) was held on October 23, 2010 in Fort Worth Texas, USA. The workshop was organized by the Environmental Mutagen Society and the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute. The workshop was attended by more than 80 participants from academia, regulatory agencies, and industry from North America, Europe and Japan. A plenary session featured summaries of the current status and issues related to the testing of NMs for genotoxic properties, as well as an update on international activities and regulatory approaches. This was followed by breakout sessions and a plenary session devoted to independent discussions of in vitro assays, in vivo assays, and the need for new assays or new approaches to develop a testing strategy for NMs. Each of the standard assays was critiqued as a resource for evaluation of NMs, and it became apparent that none was appropriate without special considerations or modifications. The need for nanospecific positive controls was questioned, as was the utility of bacterial assays. The latter was thought to increase the importance of including mammalian cell gene mutation assays into the test battery. For in‐vivo testing, to inform the selection of appropriate tests or protocols, it was suggested to run repeated dose studies first to learn about disposition, potential accumulation, and possible tissue damage. It was acknowledged that mechanisms may be at play that a standard genotoxicity battery may not be able to capture. Environ. Mol. Mutagen. 54:229–239, 2013.

Comparison of the types of mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI and hprt genes of Big Blue rats.

An important question regarding the use of transgenic reporter genes to detect mutation in rodents is how the types of mutations recovered in transgenes compare with the types of mutations found in the endogenous genes. In this study, we examined mutations induced by 7,12‐dimethylbenz‐[a]anthracene in the lacI transgene and the endogenous hprt gene of lymphocytes from Big Blue® rats and in the hprt gene of lymphocytes from non‐transgenic Fischer 344 rats. The overall mutation profiles found in these genes were remarkably similar: the majority of mutations were base pair substitutions, with the most common mutation being A:T → T:A transversion. Differences were found for the mutational profiles endogenous gene and transgene with respect to the location of the mutations and the orientation of basepair substitutions in the DNA strands. In most cases, these differences could be explained by the nature of the target genes. The results support the use of the lacI transgene for detecting in vivo mutation. Environ. Mol. Mutagen. 31:149–156, 1998 Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Evaluation of Macaca mulatta as a model for genotoxicity studies.

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.