Papers by Mugimane Manjanatha
Environmental and Molecular Mutagenesis, 2013
ALTEX
Recently, the human hepatoma-derived HepaRG cell line, which expresses various levels of phase I ... more Recently, the human hepatoma-derived HepaRG cell line, which expresses various levels of phase I and phase II enzymes, transporters, and nuclear receptors, has been cited as a promising cell model to be used as a surrogate for primary human hepatocytes (PHHs) for in vitro genotoxicity assessments (Guillouzo et al., 2007; Pfuhler et al., 2011). Our previous studies demonstrated that when cultured in 2D format, metabolically competent HepaRG cells expressed significantly higher levels of cytochrome P450 (CYP450) enzyme activities and showed higher sensitivity in detecting DNA damage and the micronuclei (MN) formation induced by a number of genotoxicants or carcinogens 1 Introduction Improving current in vitro genotoxicity tests to closely mimic human responses is an ongoing task to improve hazard identification and risk assessment (Pfuhler et al., 2011). To increase the ability of in vitro mammalian cell genotoxicity tests to reliably predict genotoxicity in humans, recommendations have been made to use human-derived, tumor protein p53-proficient, and metabolically competent cells within the context of appropriately set limits of concentration and cytotoxicity (Kirkland, 2011; Kirkland et al., 2007).
Frontiers in Toxicology
The in vivo Comet assay measures the generation of DNA strand breaks under conditions in which th... more The in vivo Comet assay measures the generation of DNA strand breaks under conditions in which the DNA will unwind and migrate to the anode in an electrophoresis assay, producing comet-like figures. Measurements are on single cells, which allows the sampling of a diversity of cells and tissues for DNA damaging effects. The Comet assay is the most common in vivo method for genotoxicity assessment of nanomaterials (NM). The Method outlined here includes a recommended step-by-step approach, consistent with OECD 489, taking into consideration the issues impacting assessment of NM, including choice of cells or systems, handling of NM test articles, dose determination, assay methods and data assessment. This method is designed to be used along with the accompanying “Common Considerations” paper, which discusses issues common to any genotoxicity assay using NM as a test article.
Toxicological Sciences - TOXICOL SCI, 2010
Acrylamide (AA) is an industrial chemical, a by-product of fried starchy foods, and a mutagen and... more 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.
Comparative Medicine, Jul 1, 2007
Daidzein (4&a... more Daidzein (4',7-dihydroxyisoflavone), a soy phytoestrogen, is a weakly estrogenic compound that may have potential health benefits. Biotransformation of daidzein by the human gut microflora after ingestion converts it to either the highly estrogenic metabolite equol or to nonestrogenic metabolites. We investigated the metabolism of daidzein by colonic microflora of rats. Fecal samples, obtained before and after rats were exposed to daidzein at 250 or 1000 parts per million, were incubated in brain-heart infusion (BHI) broth with daidzein under anaerobic conditions. Samples were removed from the cultures daily and analyzed by high-performance liquid chromatography (HPLC) and mass spectrometry. The fecal bacteria of all rats, regardless of prior daidzein exposure, metabolized the added daidzein to dihydrodaidzein. Both compounds disappeared rapidly from BHI cultures incubated for more than 24 h, but no other daidzein metabolites were detected. Only daidzein and dihydrodaidzein were found in a direct analysis of the feces of rats that had consumed daidzein in their diets. Unlike the fecal bacteria of humans and monkeys, the rat flora rapidly metabolized daidzein to aliphatic compounds that could not be detected by HPLC or mass spectral analysis.
Mutat Res Fundam Mol Mech Mut, 1997
J Toxicol Environ Health Pt a, 1996
Liver tumors from mice treated with genotoxic carcinogens often possess mutations in ras protoonc... more Liver tumors from mice treated with genotoxic carcinogens often possess mutations in ras protooncogenes, and these sequence alterations in ras frequently reflect the mutational specificity of the carcinogen. Previous studies suggest that the mouse model used for tumor induction may affect ras mutational patterns. In order to explore this possibility, H- and K-ras mutational profiles were established in liver tumors from male B6C3f1 and CD-1 mice administered benzo[a]pyrene (BaP), 6-nitrochrysene (6-NC), and 4-aminobiphenyl (4-ABP). With the exception of 6-NC-induced tumors in B6C3F1 mice, a high proportion of the tumors induced in both types of mice contained ras mutations. In CD-1 mice, 6-NC predominantly induced C-->A mutations in H-ras codon 61 (90% of tumors analyzed), whereas 4-ABP mainly induced A-->T mutations in H-ras codon 61 (50%) and BaP induced both A-->T (27%) and G--> (50%) mutations in H-ras codon 61 and K-ras codon 13, respectively. In B6C3F1 mice, 85% of BaP tumors had G-->C mutations in K-ras codon 13 and 85% of 4-ABP tumors had C-->A mutations in H-ras codon 61, while among 6-NC tumors, only 4% had G-->C mutations in K-ras codon 13 and none had H-ras mutations. Statistical analysis of these results indicates that the patterns of tumor ras mutations induced by BaP in CD-1 and B6C3F1 mice were indistinguishable, while 6-NC and 4-ABP produced different tumor ras profiles in the two mouse models. Published mutational profiles for active metabolites of BaP and 6-NC from in vitro reporter gene systems were inconsistent with both the CD-1 and B6C3F1 tumor ras mutational responses.
Page 1. REGULATORY RESEARCH PERSPECTIVES Volume 7, Issue 1 July 2007 DHHS/FDA/Jefferson Labs Nati... more Page 1. REGULATORY RESEARCH PERSPECTIVES Volume 7, Issue 1 July 2007 DHHS/FDA/Jefferson Labs National Center for Toxicological Research Impact on Public Health Studies on the Influence of Dietary Isoflavones, Daidzein ...
Carcinogenesis, 2000
ABSTRACT Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations i... more ABSTRACT Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue((R)) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18. 3 +/- 4 x 10(-6) p.f.u.). (32)P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.
The initiating mutations of a tumor are present in each of the cancerous cells comprising the tum... more The initiating mutations of a tumor are present in each of the cancerous cells comprising the tumor. Identification and measurement of the subsequent mutations that occur during tumor progression, however, requires mutation detection in a smaller subset of the tumor cells. In this study, allele-specific competitive blocker PCR (ACB-PCR), a genotypic selection method with the sensitivity to detect a specific
Genes and Environment, 2015
IARC scientific publications
Carcinogenic arylamines and nitroaromatic hydrocarbons are chemicals that present occupational he... more Carcinogenic arylamines and nitroaromatic hydrocarbons are chemicals that present occupational health hazards and share pathways of metabolic activation. The 32P-postlabelled DNA adducts formed in Chinese hamster ovary (CHO) cells treated with metabolites from two pathways that are common to the activation of the nitroaromatic hydrocarbon 6-nitrochrysene (6-NC) and the arylamine 6-aminochrysene (6-AC) compared with the spectra of mutations induced at the CHO hprt locus by these were metabolites. 6-Nitrosochrysene (6-NOC), which is reduced by the cells to N-hydroxy-6-AC, formed adducts mainly with deoxyguanosine, but induced mutations primarily through base-pair substitution involving deoxyadenosine. In contrast, 6-AC 1,2-dihydrodiol produced DNA adducts and mutations that mainly involved deoxyguanosine residues. The two major activation pathways for 6-NC and 6-AC thus produce distinct adduct and mutation spectra in CHO cells, and these adduct and mutational spectra are different from those of other arylamines and nitroaromatic hydrocarbons that have been studied.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2015
Chronic inhalation of vanadium pentoxide (V2O5) increases the incidence of alveolar/bronchiolar t... more Chronic inhalation of vanadium pentoxide (V2O5) increases the incidence of alveolar/bronchiolar tumors in male and female B6C3F1 mice at 1, 2, or 4mg/m(3). 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/m(3) (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/m(3) 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/m(3)), and 24 (1mg/m(3)) 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.
Mutation research, Jan 31, 1998
The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a bio... more 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 de...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997
Direct pulmonary instillation of 1,6-dinitropyrene (DNP) into male Fischer 344 rats results in a ... more Direct pulmonary instillation of 1,6-dinitropyrene (DNP) into male Fischer 344 rats results in a dose-dependent induction of lung tumors and 6-thioguanine-resistant (TGr) T-lymphocytes. The treatment also results in DNP binding to dG in the lung and in T-lymphocytes. In the present study, we have examined the types of mutations associated with these responses to DNP. Sequencing of DNA amplification products from 20 DNP-induced lung tumors identified 5 mutations in K-ras codon 12, 4 GGT-->TGT transversions and one GGT-->GAT transition. No mutations were found in K-ras codons 13 or 61. Single-strand conformation polymorphism analysis of p53 exons 5-8 revealed mobility shifts indicative of mutation in 9 of the 20 tumor samples. Eight of the mutations were substitutions at G:C base pairs, and one was a deletion of a single G:C base pair. DNA from 161 TGr lymphocyte colonies cultured from DNP-treated rats was examined for point mutations by amplification of hprt exons 2, 3, and 8, and screening the products for mutant: wild-type heteroduplex formation by denaturing gradient-gel electrophoresis. Only three mutations were found, a G-->T transversion in exon 3, a G-->A transition in exon 8, and a complex mutation consisting of a tandem G-->T transversion and a one base deletion in exon 3. The mutations identified in the DNP-induced lung tumors and TGr T-lymphocytes are consistent with the formation of dG-DNA adducts by DNP. The extremely low recovery of point mutations from TGr lymphocytes suggests that DNP induces a substantial number of mutations by other mechanisms.
Vegetables, Whole Grains, and Their Derivatives in Cancer Prevention, 2010
Epidemiological studies provide evidence for the possibility of preventing cancer and/or forestal... more Epidemiological studies provide evidence for the possibility of preventing cancer and/or forestalling the complications of menopause through the consumption of foods containing phytoestrogens such as soy isoflavones. Lifestyle changes in technologically advanced societies, however, limit the consumption of adequate foods to meet health needs; thus supplements of phytoestrogen including soy isoflavones daidzein (DZ) and genistein (GE), considered to be compounds
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2001
Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals prov... more Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2001
The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spo... more The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.
Mutation Research/Environmental Mutagenesis and Related Subjects, 1996
Uploads
Papers by Mugimane Manjanatha