Erythrocyte PIG-A mutant frequencies in cancer patients receiving cisplatin.

BACKGROUND: Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin-induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies. In the current study, we have used the erythrocyte PIG-A assay to evaluate mutagenesis in non-tumor hematopoietic tissue of cancer patients receiving cisplatin chemotherapy. METHODS: Twenty-one head and neck cancer patients undergoing treatment with cisplatin were monitored for the presence of PIG-A mutant total erythrocytes and the young erythrocytes, reticulocytes (RETs), in peripheral blood for up to five and a half months from the initiation of the anti-neoplastic chemotherapy. RESULTS: PIG-A mutant frequency (MF) in RETs increased at least two-fold in 15 patients at some point of the monitoring, while the frequency of total mutant RBCs increased at least two-fold in 6 patients. A general trend for an increase in the frequency of mutant RETs and total mutant RBCs was observed in 19 and 18 patients, respectively. Only in one patient did both RET and total RBC PIG-A MFs did not increase at any time-point over the monitoring period. CONCLUSION: Cisplatin chemotherapy induces moderate increases in the frequency of PIG-A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG-A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

Effect of life stage and target tissue on dose-response assessment of ethyl methane sulfonate-induced genotoxicity.

In order to investigate the possibility that treatment age affects the genotoxic response to ethyl methane sulfonate (EMS) exposure, we dosed gpt-delta neonatal mice on postnatal days 1-28 with 5-100 mg/kg/day of EMS and measured micronucleus (MN) induction in peripheral blood and gpt gene mutation in liver, lung, bone marrow, small intestine, spleen, and kidney. The data were compared to measurements from similarly exposed adult gpt-delta mice. Our results indicate that the peripheral blood MN frequencies in mice treated as neonates are not substantially different from those measured in mice treated as adults. There were, however, differences in tissue-specific gpt mutation responses in mice treated with EMS as neonates and adults. Greater mutant frequencies were seen in DNA isolated from kidney of mice treated as neonates, whereas the mutant frequencies in bone marrow, liver, and spleen were greater in the animals treated as adults. Benchmark dose potency ranking indicated that the differences for kidney were significant. Our data indicate that there are differences in EMS-induced genotoxicity between mice treated as adults and neonates; the differences, however, are relatively small.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.

Pig-a mutations in bone marrow erythroblasts of rats treated with 7,12-dimethyl-benz[a]anthracene.

Flow cytometry-based phenotypic detection of red blood cells (RBCs) deficient in surface markers anchored by glycosylphosphatidylinositol (GPI) is an efficient tool for monitoring somatic mutation in mammalian species. Biochemical considerations suggest that GPI-anchored marker-deficient RBCs found in peripheral blood are due to mutations in the endogenous X-linked phosphatidylinositolglycan, class A gene (Pig-a gene). Yet the linkage between the detected mutant phenotype and the actual mutation in the Pig-a gene is difficult to establish directly in mammalian RBCs that are naturally free of genomic DNA and may have only traces of heavily degraded mRNA. We have traced the origin of the marker-deficient RBC phenotype in the precursors of peripheral RBCs, bone marrow erythroid cells (BMEs, also known as erythroblasts), in rats treated by gavage with 75 mg/kg of the potent mutagen, 7,12-dimethyl-benz[a]anthracene (DMBA). The frequencies of marker-deficient BMEs were significantly increased in DMBA-treated rats. We identified Pig-a mutations in sorted mutant phenotype BMEs. The spectrum of DMBA-induced Pig-a mutations in erythroid lineage cells was identical to the spectra of mutations previously determined for the Pig-a and for another X-linked reporter gene, hypoxanthine-guanine phosphoribosyltransferase gene, in cells of lymphoid lineage, spleen T-lymphocytes. Our observations lend additional support to the hypothesis that GPI-anchored marker-deficient RBCs are true Pig-a mutants.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/- cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/- cells, but only at concentrations of 100 µg/ml and greater, and only in the presence of S9 activation. Pig-a mutations in L5178Y/Tk+/- cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G→A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

Lifespan Kras mutation levels in lung and liver of B6C3F1 mice.

Somatic mutations accumulate in the human genome and are correlated with increased cancer incidence as humans age. The standard model for studying the carcinogenic effects of exposures for human risk assessment is the rodent 2-year carcinogenicity assay. However, there is little information regarding the effect of age on cancer-driver gene mutations in these models. The mutant fraction (MF) of Kras codon 12 GGT to GAT and GGT to GTT mutations, oncogenic mutations orthologous between humans and rodents, was quantified over the lifespan of B6C3F1 mice. MFs were measured in lung and liver tissue, organs that frequently develop tumors following carcinogenic exposures. The MFs were evaluated at 4, 6, 8, 12, 21, and 85 weeks, with the 12-week and 21-week time points being coincident with the conclusion of 28-day and 90-day exposure durations used in short-term toxicity testing. The highly sensitive and quantitative Allele-specific Competitive Blocker PCR (ACB-PCR) assay was used to quantify the number of mutant Kras codon 12 alleles. The mouse lung showed a slight, but significant trend increase in the Kras codon 12 GAT mutation over the 85-week period. The trend with age can be equally well-fit by several non-linear functions, but not by a linear function. In contrast, the liver GAT mutation did not increase, and the GTT mutation did not increase for either organ. Even with the slight increase in the lung GAT MFs, our results indicate that the future use of Kras mutation as a biomarker of carcinogenic effect will not be confounded by animal age. Environ. Mol. Mutagen. 59:715-721, 2018. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Analysis of mutation in the rat Pig-a assay: II. Studies with bone marrow granulocytes.

The in vivo erythrocyte Pig-a gene mutation assay measures the phenotypic loss of GPI-anchored surface markers. Molecular analysis of the marker-deficient erythrocytes cannot provide direct proof that the mutant phenotype is due to mutation in the Pig-a gene because mammalian erythrocytes lack genomic DNA. Granulocytes are nucleated cells that originate from myeloid progenitor cells in bone marrow as is the case for erythrocytes, and thus analysis of Pig-a mutation in bone marrow granulocytes can provide information about the source of mutations detected in the erythrocyte Pig-a assay. We developed a flow cytometric Pig-a assay for bone marrow granulocytes and evaluated granulocyte Pig-a mutant frequencies in bone marrow from male rats treated acutely with N-ethyl-N-nitrosourea (ENU). Bone marrow cells from these rats were stained with anti-CD11b for identifying granulocytes and anti-CD48 for detecting the Pig-a mutant phenotype. The average Pig-a mutant frequency in granulocyte precursors of control rats was 8.42 × 10-6 , whereas in ENU-treated rats it was 567.13 × 10-6 . CD11b-positive/CD48-deficient mutant cells were enriched using magnetic separation and sorted into small pools for sequencing. While there were no Pig-a mutations found in sorted CD48-positive wild-type cells, Pig-a mutations were detected in mutant granulocyte precursors. The most frequent mutation observed was T→A transversion, followed by T→C transition and T→G transversion, with the mutated T on the nontranscribed DNA strand. While the spectrum of mutations in bone marrow granulocytes was similar to that of erythroid cells, different Pig-a mutations were found in mutant-phenotype granulocytes and erythroids from the same bone marrow samples, suggesting that most Pig-a mutations were induced in bone marrow cells after commitment to either the granulocyte or erythroid developmental pathway. Environ. Mol. Mutagen. 59:733-741, 2018. © 2018 Wiley Periodicals, Inc.

Analysis of mutation in the rat Pig-a assay: I) studies with bone marrow erythroid cells.

We have established a flow cytometry-based Pig-a assay for rat bone marrow erythroid cells (BMEs). The BME Pig-a assay uses a DNA-specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI-anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N-ethyl-N-nitrosourea (ENU) the frequency of CD59-deficient phenotypically mutant BMEs increased approximately 24-fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59-deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X-linked Pig-a gene of CD59-deficient BMEs revealed that they are Pig-a mutants. The spectrum of ENU-induced Pig-a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59-deficient erythrocytes measured in the flow cytometric erythrocyte Pig-a assay develop from BMEs containing mutations in the Pig-a gene. Thus, the erythrocyte Pig-a assay detects mutation in the Pig-a gene. Environ. Mol. Mutagen. 59:722-732, 2018. © 2018 Wiley Periodicals, Inc.

Establishing a novel Pig-a gene mutation assay in L5178YTk+/- mouse lymphoma cells.

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk+/- mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk+/- cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC. Also it is known as an in vitro and in vivo mutagen and genotoxicant. However, the molecular mechanism by which procarbazine induces mutations is not thoroughly understood and the spectrum of procarbazine-induced in vivo mutations is described insufficiently. We employed flow cytometry-based erythrocyte and T lymphocyte assays in order to quantify the frequencies of cells deficient in glycosylphosphatidyl inositol-anchored surface markers CD59 and CD48 (presumed mutants in the endogenous X-linked Pig-a gene) in rats. The rats were treated once daily with 100 mg/kg procarbazine HCl for 3 days. In addition, we sorted mutant-phenotype spleen T cells and immediately analysed their Pig-a gene using next generation sequencing of dual-indexed multiplex libraries and error-correcting data filtering. More than 100-fold increase in the frequencies of CD59-deficient RBCs was observed at Day 29 after the last administration, and a 10-fold increase in the frequency of CD48-deficient T cells was observed at Days 45 to 50. Sequencing revealed that, in T cells from procarbazine-treated rats, mutations in the Pig-a gene occurred predominantly at A:T basepairs when A was located on the non-transcribed DNA strand. A→T transversion was the most common mutation. Our results suggest that, at least for the transcribed X-linked Pig-a gene, in vivo methyl guanine adducts are not the major contributors to mutations induced by procarbazine.

In vivo genotoxicity of estragole in male F344 rats.

Estragole, a naturally occurring constituent of various herbs and spices, is a rodent liver carcinogen which requires bio-activation. To further understand the mechanisms underlying its carcinogenicity, genotoxicity was assessed in F344 rats using the comet, micronucleus (MN), and DNA adduct assays together with histopathological analysis. Oxidative damage was measured using human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified comet assays. Results with estragole were compared with the structurally related genotoxic carcinogen, safrole. Groups of seven-week-old male F344 rats received corn oil or corn oil containing 300, 600, or 1,000 mg/kg bw estragole and 125, 250, or 450 mg/kg bw safrole by gavage at 0, 24, and 45 hr and terminated at 48 hr. Estragole-induced dose-dependent increases in DNA damage following EndoIII or hOGG1 digestion and without enzyme treatment in liver, the cancer target organ. No DNA damage was detected in stomach, the non-target tissue for cancer. No elevation of MN was observed in reticulocytes sampled from peripheral blood. Comet assays, both without digestion or with either EndoIII or hOGG1 digestion, also detected DNA damage in the liver of safrole-dosed rats. No DNA damage was detected in stomach, nor was MN elevated in peripheral blood following dosing with safrole suggesting that, as far both safrole and estragole, oxidative damage may contribute to genotoxicity. Taken together, these results implicate multiple mechanisms of estragole genotoxicity. DNA damage arises from chemical-specific interaction and is also mediated by oxidative species.

Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats.

Cyproterone acetate (CPA), a synthetic hormonal drug, induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females. In order to evaluate the potential of the in vivo alkaline Comet assay to predict the sex-specific carcinogenicity of CPA, CPA-induced direct DNA damage (DNA strand breaks and alkali-labile sites) were evaluated in the livers of both male and female F344 rats. In addition, secondary oxidative DNA damage was measured concurrently utilizing the human 8-oxoguanine-DNA-N-glycosylase (hOGG1) and EndonucleaseIII (EndoIII)-modified in vivo alkaline Comet assays and the reticulocyte micronucleus (MN) frequency was analyzed in peripheral blood. Groups of 5 seven-week-old male and female F344 rats received olive oil or 10, 25, 50 or 100 mg/kg bw CPA in olive oil by gavage at 0, 24, and 45 h and were sacrificed at 48 h. CPA-induced direct DNA damage in rat liver showed the same sex-specific pattern as its hepatotumorigenicity: a five-fold-higher dose of CPA was needed to induce a statistically significant increase in direct DNA damage in livers of males compared to females. However, peripheral blood MN frequency was weak in both sexes and CPA-induced oxidative DNA damage was generally greater in male than female rat livers. Taken together, our results demonstrate concordance in the sex-specificity of CPA in the in vivo alkaline Comet assay and cancer bioassay, while the induction of oxidative DNA damage by CPA was not directly correlated with its tumorigenicity.

Quantitative dose-response analysis of ethyl methanesulfonate genotoxicity in adult gpt-delta transgenic mice.

The assumption that mutagens have linear dose-responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA-reactive mutagen and carcinogen, exhibited sublinear or thresholded dose-responses for LacZ mutation in transgenic Muta™Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101-107). In order to explore variables in establishing genotoxicity dose-responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta™Mouse. Male gpt-delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no-observed-genotoxic-effect-levels (NOGELs), lower confidence limits of threshold effect levels (Td-LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10 s). Similar PoDs were calculated from the published EMS dose-responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig-a MFs were 13-40-fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose-responses in gpt-delta mice had lower PoDs than those calculated from the Muta™Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds.

Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.

Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens.

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay.

Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, aristolochic acids.

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.75, 5.5, and 11mg/kg AAs for 28 days or with 0, 11, 22, and 30mg/kg AAs for 3 days. Day 1 being the first day of treatment, Pig-a mutant frequencies (MFs) were assayed in peripheral blood erythrocytes up to Day 56 for the 28-day treatment or Day 42 for the 3-day treatment; micronuclei were assayed in peripheral blood reticulocytes on Day 4 (both treatment protocols) and on Day 29 of the 28-day treatment protocol; and at the final sampling times (Day 59 or Day 42), the animals were sacrificed and Hprt mutant lymphocytes were measured. In a separate study, the Comet assay was performed on liver, kidney, and bone marrow of animals gavaged with 0, 11, 22, and 30mg/kg AAs for 4 days and sacrificed 3h after the last treatment. While only weak increases in micronucleated reticulocyte frequency were observed in treated animals, Pig-a MFs increased in a dose- and time-dependent manner with both treatment schedules. Lymphocyte Hprt mutant frequencies also increased dose dependently in treated animals, and the Comet assay detected elevated levels of DNA damage in all the tissues evaluated. These findings indicate that the DNA damage produced by AAs in rat bone marrow is a weak inducer of micronuclei but a relatively strong inducer of gene mutation.