Diethylnitrosamine genotoxicity evaluated in sprague dawley rats using Pig‐a mutation and reticulocyte micronucleus assays

Diethylnitrosamine (DEN) is a genotoxic carcinogen, but in vivo DNA‐damaging activities are not usually evident in hematopoietic cells because the short‐lived active metabolite is formed mainly in the liver. DEN therefore represented an interesting case for evaluating the performance characteristics of blood‐based endpoints of genotoxicity that have been automated using flow cytometric analysis—frequency of micronucleated reticulocytes and Pig‐a mutant phenotype reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?). Male Sprague Dawley rats were treated for 28 consecutive days with DEN at levels up to 12.5 mg/kg/day. Serial blood samples were collected and micronucleus frequencies were determined on Days 4 and 29, while RET^CD59? and RBC^CD59? frequencies were determined on Days 15, 29, and 42. The Pig‐a analyses were conducted with an enrichment step based on immunomagnetic column separation to increase the statistical power of the assay. Modest but significant reductions to reticulocyte frequencies demonstrated that bone marrow was exposed to reactive intermediates. Even so, DEN did not affect micronucleus frequencies at any dose level tested. However, RET^CD59? frequencies were significantly elevated in the high dose group on Day 29, and RBC^CD59? were increased at this same dose level on Days 29 and 42. These results demonstrate that the Pig‐a assay is sufficiently sensitive to evaluate chemicals for genotoxic potential, even in the case of a promutagen that has traditionally required direct assessment(s) of liver tissue for detection of DNA‐damage. Environ. Mol. Mutagen. 55:400–406, 2014. © 2014 Wiley Periodicals, Inc.     

Mouse Pig‐a and micronucleus assays respond to N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and ethyl carbamate,but not pyrene or methyl carbamate

This laboratory previously described a method for scoring the incidence of peripheral blood Pig‐a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24‐negative reticulocytes (RET^CD24−) and erythrocytes (RBC^CD24−) as phenotypic reporters of Pig‐a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N‐ethyl‐N‐nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD‐1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RET^CD24− and RBC^CD24− frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose‐related increases in the frequencies of Pig‐a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N‐ethyl‐N‐nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig‐a mutant cells (e.g., Day 15 mean no. RET^CD24− per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10⁻⁶, respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10⁻⁶). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results demonstrate the utility of the cross‐species Pig‐a and micronucleated reticulocyte assays, and add further support to the value of studying both endpoints in order to cover two distinct genotoxic modes of action. Environ. Mol. Mutagen. 57:28–40, 2016. © 2015 Wiley Periodicals, Inc.     

Pig‐a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil,thiotepa, melphalan,and 1,3‐propane sultone

To evaluate whether blood‐based genotoxicity endpoints can provide temporal and dose‐response data within the low‐dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry‐based micronucleus and Pig‐a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN‐RET) was used to evaluate chromosomal damage, and the frequency of CD59‐negative reticulocytes (RET^CD59?) and erythrocytes (RBC^CD59?) served as phenotypic reporters of mutation at the X‐linked Pig‐a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3‐propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN‐RET were determined at Days 4 and 29, and RET^CD59? and RBC^CD59? data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose‐related increases were observed for each endpoint, and time to maximal effect was consistently: MN‐RET < RET^CD59? < RBC^CD59?. For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig‐a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood‐based analyses to provide dose‐response and temporality information that relates genetic damage to cancer induction. Environ. Mol. Mutagen. 55:299–308, 2014. © 2014 Wiley Periodicals, Inc.     

Flow cytometric analysis of Pig‐a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD‐1 mice

Procarbazine is a genotoxic carcinogen whose DNA‐damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD‐1 mice using a multi‐endpoint study design that scored micronucleated reticulocyte (MN‐RET) frequency and gene mutation at the Pig‐a locus. CD‐1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN‐RETs, with the high dose group exhibiting a mean 29‐fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig‐a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Environ. Mol. Mutagen. 54:294–298, 2013. © 2013 Wiley Periodicals, Inc.     

In vivo pig‐a and micronucleus study of the prototypical aneugen vinblastine sulfate

The Pig‐a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig‐a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty‐eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days ?4, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well‐established aneugenic effect on hematopoietic cells, the lack of a Pig‐a response indicates that this tubulin‐binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30–37, 2018. © 2017 Wiley Periodicals, Inc.     

Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5‐fluorouracil (5‐FU) and 4‐nitroquinoline‐1–oxide (4NQO) as part of an international validation trial of the Pig‐a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5‐FU for three consecutive days (Days 1–3); blood was sampled on Days ?1, 4, 15, 29, and 45. Pig‐a mutant phenotype reticulocyte (RET^CD59?) and mutant phenotype erythrocyte (RBC^CD59?) frequencies were determined on Days ?1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN‐RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC^CD59? and RET^CD59? frequencies were determined on Days ?1, 15, and 29, and MN‐RET were quantified on Day 29. Whereas 5‐FU was found to increase %MN‐RET, no significant increases were observed for RBC^CD59? or RET^CD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC^CD59? and RET^CD59? frequencies, and this same dose level caused a weak but significantly elevated increase in MN‐RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig‐a mutation and MN‐RET into acute and 28‐day repeat‐dose studies. Environ. Mol. Mutagen. 55:735–740, 2014. © 2014 Wiley Periodicals, Inc.     

Interlaboratory Pig-a gene mutation assay trial: Studies of 1,3-propane sultone with immunomagnetic enrichment of mutant erythrocytes

An international collaborative trial was established to systematically investigate the merits and limitations of a rat in vivo Pig-a gene mutation assay. The product of this gene is essential for anchoring CD59 to the plasma membrane, and mutations in this gene are identified by flow cytometric quantification of circulating erythrocytes without cell surface CD59 expression. Initial interlaboratory data from rats treated with several potent mutagens have been informative, but the time required for those flow cytometric analyses (～20 min per sample) limited the number of cells that could be interrogated for the mutant phenotype. Thus, it was desirable to establish a new higher throughput scoring approach before expanding the trial to include weak mutagens or nongenotoxicants. An immunomagnetic column separation method that dramatically increases analysis rates was therefore developed (Dertinger et al. [2011]: Mutat Res 721:163-170). To evaluate this new method for use in the international collaborative trial, studies were conducted to determine the mutagenic response of male Sprague Dawley rats treated for 3 or 28 consecutive days with several doses of 1,3-propane sultone (1,3-PS). Pig-a mutant frequencies were measured over a period of several weeks and were supplemented with another indicator of genetic toxicity, peripheral blood micronucleated reticulocyte (MN-RET) counts. 1,3-PS was found to increase Pig-a mutation and MN-RET frequencies in both 3- and 28-day study designs. While the greatest induction of MN-RETs was observed in the 3-day study, the highest Pig-a responses were found with 28-days of treatment. Pig-a measurements were acquired in approximately one-third the time required in the original method, while the number of erythrocyte and reticulocyte equivalents analyzed per sample were increased by factors of 100 and 10, respectively. The data strongly support the value of using the immunomagnetic separation technique for enumerating Pig-a mutation frequencies. These results also demonstrate that the ongoing international trial will benefit from the inclusion of studies that are based on both acute and protracted repeat dosing schedules in conjunction with the acquisition of longitudinal data, at least until more data have been accumulated.     

In vivo mutagenesis induced by benzo[a]pyrene instilled into the lung of gpt delta transgenic mice

Benzo[a]pyrene (B[a]P) is a ubiquitous airborne pollutant whose mutagenicity has been evaluated previously by oral and intraperitoneal administration to experimental animals. In this study, mutagenesis in the lungs, the target organ of air pollutants, was examined after a single intratracheal instillation of 0-2 mg B[a]P into gpt delta transgenic mice. Intratracheal injection of B[a]P resulted in a statistically significant and dose-dependent increase in gpt mutant frequency as measured by 6-thioguanine selection. The mutant frequencies at B[a]P doses of 0.5, 1, and 2 mg were 2.8, 4.2, and 6.8 times higher than the frequency seen in nontreated mice (0.60 +/- 0.13 x 10(-5)). The most frequent mutations induced by B[a]P treatment were G:C-->T:A transversions, which are characteristic of B[a]P mutagenesis in other models, and single-base deletions of G:C base pairs. To characterize the hotspots of B[a]P-induced mutations in the gpt gene, we analyzed sequences adjacent to the mutated G:C base pairs. Guanine bases centered in the nucleotide sequences CGT, CGA, and CGG were the most frequent targets of B[a]P. Our results indicate that intratracheal instillation of B[a]P into gpt delta mice causes a dose-dependent increase in gpt mutant frequency in the lung, and that the predominant mutation induced is G:C-->T:A transversion.     

Absence of formation of benzo[a]pyrene/DNA adducts in the cuttlefish (sepia officinalis,mollusca: Cephalopoda)

Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, the site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection of B[a]P. The dose of the carcinogen injected into the cuttlefish was 2—4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in the vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods. © 1994 Wiley-Liss, Inc.     

Human erythrocyte PIG‐A assay: An easily monitored index of gene mutation requiring low volume blood samples

This laboratory has previously described a method for scoring the incidence of rodent blood Pig‐a mutant phenotype erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59‐ and CD55‐negative reticulocytes (RET^CD59?/CD55?) and erythrocytes (RBC^CD59?/CD55?) serve as phenotypic reporters of PIG‐A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythrocytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulocyte, RET^CD59?/CD55? or RBC^CD59?/CD55? frequencies. Specimens from a total of 52 nonsmoking, self‐reported healthy adult subjects were evaluated. The mean frequency of RET^CD59?/CD55? and RBC^CD59?/CD55? were 6.0 × 10^?6 and 2.9 × 10^?6, respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra‐subject variability was low, inter‐subject variability was relatively high, with RET^CD59?/CD55? frequencies differing by more than 30‐fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG‐A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent‐based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non‐invasive nature of the assay should benefit myriad research areas involving DNA damage, including studies of environmental factors that modify “spontaneous” mutation frequencies. Environ. Mol. Mutagen. 56:366–377, 2015. © 2014 Wiley Periodicals, Inc.     

Evaluation of in vivo genotoxicity induced by N‐ethyl‐N‐nitrosourea,benzo[a]pyrene,and 4‐nitroquinoline‐1‐oxide in the Pig‐a and gpt assays

The recently developed Pig‐a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor‐deficient red blood cells caused by a forward mutation in the Pig‐a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig‐a assay could be integrated into repeat‐dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig‐a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N‐ethyl‐N‐nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4‐nitroquinoline‐1‐oxide (4NQO, 50 mg/kg) in the Pig‐a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig‐a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7‐week terminal sacrifice. ENU increased both Pig‐a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig‐a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two‐ or threefold above control) were detected in the bone marrow in both the Pig‐a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig‐a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen. 54:747–754, 2013. © 2013 Wiley Periodicals, Inc.     

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene‐induced genotoxicity in vivo

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock‐in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP‐induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven‐week‐old inactivated Polk KI and wild‐type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP‐induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644–653, 2017. © 2017 Wiley Periodicals, Inc.     

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.     

K‐Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

K‐Ras mutant fraction (MF) was measured to examine the default assumption of low‐dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of 10 male A/J mice (7‐ to 9‐weeks old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P and were sacrificed 28 days after treatment. K‐Ras codon 12 TGT and GAT MFs in lung DNAs were measured using Allele‐specific Competitive Blocker‐PCR (ACB‐PCR). The K‐Ras codon 12 TGT geometric mean MF was 3.88 × 10^?4 in controls, indicating an average of 1 mutation in every ～1,288 lung cells. The K‐Ras codon 12 TGT geometric mean MFs were as follows: 3.56 × 10^?4; 6.19 × 10^?4; 2.02 × 10^?3, and 3.50 × 10^?3 for the 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The 5 and 50 mg/kg dose groups had TGT MFs significantly higher than did controls. Although 10^?5 is considered as the limit of accurate ACB‐PCR quantitation, K‐Ras codon 12 GAT geometric mean MFs were as follows: 8.38 × 10^?7, 1.47 × 10^?6, 2.19 × 10^?6, 5.71 × 10^?6, and 8.99 × 10^?6 for the 0, 0.05, 0.5, 5, and 50 mg/kg B[a]P treatment groups, respectively. The K‐Ras TGT and GAT MFs increased in a B[a]P‐dose‐dependent manner, with response approximately linear over the 0.05 to 5 mg/kg dose range. K‐Ras MF increased with B[a]P adduct burden measured for identical doses in a separate study. Thus, ACB‐PCR may be useful in characterizing the shape of a dose‐response curve at low doses and establishing relationships between DNA adducts and tumor‐associated mutations. Environ. Mol. Mutagen. 2010. Published 2009 Wiley‐Liss, Inc.     

Induction of centrosome amplification and chromosome instability in p53-deficient lung cancer cells exposed to benzo[a]pyrene diol epoxide (B[a]PDE)

Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking-associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53-deficient H1299 lung cancer cells to B[a]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence-tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE-guanine lesions in an error-free manner, suppressed the B[a]PDE-induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non-functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE-DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity.     

Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig‐a gene Mutation: Support from an in vitro assay based on L5178Y/Tk+/− cells and the CD90.2 antigen

Lack of cell surface glycosylphosphatidylinositol (GPI)‐anchored protein(s) has been used as a reporter of Pig‐a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk^+/?. Cells were exposed to mutagenic and nonmutagenic compounds for 24 hr followed by washout and incubation for an additional 7 days. Following this mutant manifestation time, cells were labeled with fluorescent antibodies against CD90.2 and CD45 antigens. These reagents indicated the presence of GPI‐anchored proteins and general cell surface membrane receptor integrity, respectively. Instrument set‐up was aided by parallel processing of a GPI anchor‐deficient subclone. Results show that the mutagens reproducibly caused increased frequencies of mutant phenotype cells, while the nonmutagens did not. Further modifications to the method, including application of a viability dye and an isotype control for instrument set‐up, were investigated. As a means to verify that the GPI‐anchored protein‐negative phenotype reflects bona fide Pig‐a gene mutation, sequencing was performed on 38 CD90.2‐negative L5178Y/Tk^+/? clones derived from cultures treated with ethyl methanesulfonate. All clones were found to have mutation(s) within the Pig‐a gene. The continued investigation of L5178Y/Tk^+/? cells, CD90.2 labeling, and flow cytometric analysis as the basis of an in vitro mutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor‐deficiency as a valid reporter of Pig‐a gene mutation. Environ. Mol. Mutagen. 59:18–29, 2018. © 2017 Wiley Periodicals, Inc.