Mutation Frequency In Cells Research Articles

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.

Compensatory erythropoiesis has no impact on the outcome of the in vivo Pig-a mutation assay in rats following treatment with the haemolytic agent 2-butoxyethanol.

The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450 mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay.

Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, groups of five male and female Sprague Dawley rats were exposed to the mutagens 1,3-propane sultone (80mg/kg/day), ethyl carbamate (600mg/kg/day), or thiotepa (7.5mg/kg/day) for three consecutive days (study days 1–3). Pig-a mutant phenotype reticulocyte (RETCD59−) and mutant phenotype erythrocyte (RBCCD59−) frequencies were determined on study days −4, 15, 30 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). While the percentage of reticulocytes (%RET) was markedly higher for pre-treatment blood samples from males compared to females (6.6% vs. 3.5%), this sex effect was slight or nonexistent at later time points. Treatment-related effects to %RET were generally modest owing to the 12-day interval between last exposure and blood sampling. Mean RETCD59− and RBCCD59− frequencies were consistently low in vehicle control animals of both sexes, with 77% of samples exhibiting mutant cell frequencies ≤1×10−6 over study days 15–46. Treatment with each mutagen caused significant increases to mean RETCD59− and RBCCD59− frequencies. Whereas genotoxicant-induced RETCD59− values were maximal on day 15, induced RBCCD59− frequencies were highest at the last sampling time. Sex did not affect 1,3-propane sultone- or thiotepa-induced mutant cell frequencies. While ethyl carbamate-exposed females exhibited higher mean mutant cell frequencies compared to like-treated males, statistical significance was achieved only for RBCCD59− at one time point (7.6±1.0×10−6 compared to 4.7±0.6×10−6 on day 30). Thus, while some quantitative differences were evident, there were no qualitative differences in how males and females responded to three diverse mutagens. These data support the use of both sexes for Pig-a gene mutation studies.

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.

Genotoxic effects of two-generational selenium deficiency in mouse somatic and testicular cells.

Many studies have investigated genotoxic effects of high Se diets but very few have addressed the genotoxicity of Se deprivation and its consequences in germ cells and none in somatic cells. To address these data gaps, C57BL/6 male mice were subjected to Se deprivation starting in the parental generation, i.e. before conception. Mice were given a diet of either low (0.01mg Se/kg diet) or normal (0.23mg Se/kg diet) Se content. Ogg1-deficient (Ogg1 (-/-) ) mice were used as a sensitive model towards oxidative stress due to their reduced capacity to repair oxidised purines. Ogg1 (-/-) mice also mimic the repair characteristics of human post-meiotic male germ cells which have a reduced ability to repair such lesions. The genotoxicity of Se deficiency was addressed by measuring DNA lesions with the alkaline single cell gel electrophoresis (+ Fpg to detect oxidised DNA lesions) in somatic cells (nucleated blood cells and lung cells) and male germ cells (testicular cells). Total Se concentration in liver and GPx activity in plasma and testicular cells were measured. Gene mutation was evaluated by an erythrocyte-based Pig-a assay. We found that Se deprivation of F1 from their conception and until early adulthood led to the induction of DNA lesions in testicular and lung cells expressed as significantly increased levels of DNA lesions, irrespective of the mouse genotype. In blood cells, Se levels did not appear to affect DNA lesions or mutant cell frequencies. The results suggest that the testis was the most sensitive tissue. Thus, genotoxicity induced by the low Se diet in the spermatozoal genome has potential implications for the offspring.

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

Transgenic rodent assay for quantifying male germ cell mutant frequency.

De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

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.

Role of the Somatic Mutation Rate in Patients with Classical Myeloproliferative Neoplasms (MPN) with and without Other Neoplasms

AbstractAbstract 5055Classical chronic myeloproliferative neoplasms (MPNs) are amongst the best-characterized neoplasms associated with one of a set of specific somatic mutations, the most common of which is in the JAK2 gene. MPN is a relatively rare condition, and it could be surmised that it develops preferentially in people who have an increased tendency to somatic mutations, i. e. an increased somatic mutation rate (μ). Since measuring μ is rather labor-intensive, in this study we have measured instead the frequency of peripheral blood granulocytes that have inactivating mutations in a reporter gene, namely the gene PIG-A, a gene whose protein product is required for numerous glycosyl-phosphatidylinositol-anchored proteins to become surface bound. Since several such proteins are displayed by normal granulocytes, mutant cells can be numbered accurately by flow cytometry; and the frequency (ƒ) of such mutant cells may be a good surrogate of μ (Peruzzi et al. Mutation Research. 2010, 705:3).We have determined ƒ in 74 patients with MPN (including 29 with polycythemia vera (PV), 17 with essential thrombocythemia (ET), 16 with primary myelofibrosis (PMF) 12 with post-PV/post-ET (PPV/PET) MF. Overall the proportion of those with a JAK2(V617F) mutation was 90%. In a subset of 59 of these patients, who had “uncomplicated” MPNs, the median of ƒ was no different compared to 142 healthy controls (4. 74×10−6vs. 4. 87×10−6: Figure 1). However, in a subset of 15, consisting of patients who in addition to MPN had at least one primary malignancy (2 had lymphomas and 13 had one of several types of solid tumors), the median value of ƒ was 15. 22×10−6, i. e. significantly elevated (Mann Whitney test: MPNs vs. MPNs with a second neoplasm, p<0. 013; Healthy controls vs. MPNs with a second neoplasm, p<0. 005: Figure 1). [Display omitted] Clearly we cannot automatically extrapolate from our reporter gene, PIG-A, to the actual probability that another gene undergoes somatic mutation; besides, the rate of mutation of JAK2 is influenced by the local genomic context. With that proviso, our data suggest that JAK2 mutations are unlikely to result from an overall increased mutation rate. In keeping with this notion, we found no correlation between the value of ƒ and the allelic burden of JAK2(V617F) (R2= 0. 001): i. e., there is no evidence for genetic instability in MPNs (although, once there is a JAK2 mutation, this may produce a mutator phenotype). On the other hand, an intrinsically increased mutation rate does play a significant role in subjects with multiple neoplasms. This is not surprising: but, to the best of our knowledge, for patients who did not otherwise appear to be ‘cancer-prone', direct evidence for this is provided here for the first time. Disclosures:No relevant conflicts of interest to declare.

Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds.

The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.

In vivo flow cytometric Pig‐a and micronucleus assays: Highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated‐dose designs

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig-a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig-a endpoint is suitable for both repeated-dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on-going toxicology studies, and use as a short-term assay that provides efficient screening and mechanistic information in vivo.

Regulation of the Mutation Threshold During Immune Diversification by Activation Induced Cytidine Deaminase,

Abstract 3244Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. AID converts cytosine to uracil and resulting U-G mismatches are subsequently processed by low-fidelity base excision and mismatch repair pathways to yield point mutations (for SHM) and DNA strand breaks (for CSR). Under normal conditions, genotoxic stressors activate DNA damage response pathways that result in DNA repair or cell cycle arrest and apoptosis. However, in normal germinal center B cells, key DNA damage checkpoint factors such as ATR, p53 and p21 are repressed by the germinal center transcriptional repressor BCL6. BCL6 thus creates a permissive environment to allow the accumulation of mutations within the Ig loci. However, in addition to this fundamental role in immune diversification, aberrant targeting of AID contributes to point mutations and translocations of proto-oncogenes associated with B cell malignancy. Indeed, the combined effect of BCL6 and AID poses a direct threat to genomic integrity but the mechanism responsible for regulating the mutation threshold in germinal center B cells is not understood. To determine if B cells have a mechanism for down-regulating AID activity in response to genotoxic stress, we subjected the Ramos-A23 cell line to continuous low-dose exposure to several genotoxic agents. Ramos-A23 is a Burkitt lymphoma line that constitutively expresses AID and mutates the Ig heavy chain variable region with high frequency. Mutation frequency can be monitored by flow cytometry through loss of surface expression of IgM (due to AID-dependent nonsense and missense mutations) and by direct sequencing of the variable region. IgM+ Ramos-A23 cells were maintained in continuous culture in the presence or absence of etoposide (100 nM), cytarabine (5 nM), 5-azacytidine (10 nM), or trichostatin A (15 nM). Drug concentrations were titrated to the highest dose that would minimally affect proliferation or survival when compared with untreated cells. After 4 weeks in culture, untreated clones were 20–22% IgM-, consistent with ongoing SHM. In contrast, clones treated with the DNA damaging agents etoposide or cytarabine were only 4–6% IgM- (p<0.0001). Cells treated with the epigenetic modifiers 5-azacytidine or trichostatin A were 15–19% IgM-. Sequence analysis of the Ig variable region (3 clones from each group) demonstrated 6.2 and 3.8-fold reductions in mutation frequency in cells treated with etoposide and cytarabine, respectively, when compared with untreated cells. The mutation frequency in the cells treated with epigenetic modifiers did not differ significantly from untreated controls. This suggests that while epigenetic stress has little effect on SHM, genotoxic stress leads to a significant reduction in AID activity. It has previously been demonstrated that genotoxic stress leads to an ATM-dependent down-regulation of BCL6, a finding that we also observed in our model. In addition, we found that genotoxic stress causes a dose and time-dependent decrease in AID protein and transcript levels, thus explaining the decrease in SHM in our system. Like BCL6, repression of AID by genotoxic stress was inhibited by pre-treating the cells with the ATM inhibitor KU-55933. Further analysis of markers of B cell differentiation revealed up-regulation of IRF4 and Blimp1 with complete down-regulation of PAX5 (a transcriptional regulator of AID) in response to genotoxic stress. Lentiviral expression of BCL6 shRNA in Ramos cells also lead to down-regulation of AID and SHM, consistent with the observation that BCL6-deficient B cells do not undergo SHM. It has also recently been demonstrated that DNA damage in germinal center B cells leads to ATM-dependent inactivation of the CREB transcriptional co-activator CRTC2 with subsequent down-regulation of CRTC2 target genes, including AID. Altogether, these data suggest that, during the germinal center response, a mutation threshold recognized by ATM leads to repression of the SHM machinery and reprogramming to facilitate B cell maturation. Additional investigation is needed to further define the critical steps in this regulatory pathway and how it might breakdown during the pathogenesis of B cell malignancy. Disclosures:No relevant conflicts of interest to declare.

Frequency of TCR Mutant Human Lymphocytes Depending on Genotypes by Loci of Xenobiotics Detoxication

The results of the associative study of predisposition to an elevated somatic mutagenesis in human lymphocytes determined by the test of TCR mutant cells (CD3-CD4+phenotype) for 251 females are presented. The presence of minor alleles of polymorphic sites of CYP1A1 gene, which increase the enzyme activity, correlated with the increasing spontaneous frequency of TCR mutant cells. The analysis of gaplotypes in CYP1A1 locus (3 sites) showed that a minor gaplotype of CG sites T3801C-T606G, which had a strong effect on the parameters of somatic mutation at the frequency of around 10%, has the maximum prognostic importance relative to the studied effects.

Pig-a Mutation: Kinetics in Rat Erythrocytes Following Exposure to Five Prototypical Mutagens

An in vivo mutation assay has been developed based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient rat erythrocytes. With this method, blood is incubated with anti-CD59-PE and SYTO 13 dye, and flow cytometry is used to score the frequency of CD59-negative erythrocytes. The experiments described herein were designed to define the kinetics of mutant erythrocyte appearance and disappearance from peripheral blood to support appropriate treatment and sampling designs for the assay. Wistar Han rats were treated with one of five prototypical mutagens: N-ethyl-N-nitrosourea (ENU); 7,12-dimethyl-1,2-benz[a]anthracene (DMBA); 4-nitroquinoline-1-oxide; benzo[a]pyrene; and N-methyl-N-nitrosourea. ENU and DMBA were also evaluated in Sprague Dawley rats. Animals were treated on three consecutive days (days 1-3) via oral gavage, and blood specimens were obtained on days -1, 4, 15, 30, 45, and 90 (and day 180 for ENU). A second endpoint of genotoxicity, the frequency of peripheral blood micronucleated reticulocytes, was measured on day 4. Each chemical induced micronuclei and the GPI anchor-deficient phenotype. Increased mutant cell frequencies were evident at day 15. Mutant reticulocyte frequencies remained relatively stable for some chemicals, but others peaked and then dropped significantly. The differences in kinetics observed are presumably related to the degree to which mutation occurs in hematopoietic stem cells versus more committed cells with limited self-renewal capacity. Collectively, the results suggest that enumerating GPI anchor-deficient erythrocytes is an efficient means of evaluating the in vivo mutagenic potential of chemicals. The kinetics and ease of scoring this blood-based endpoint suggest that integration into routine toxicology studies will be feasible.

Endogenous expression of Hras G12V induces developmental defects and neoplasms with copy number imbalances of the oncogene

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

Radiation-quality Dependent Cellular Response in Mutation Induction in Normal Human Cells

We studied cellular responses in normal human fibroblasts induced with low-dose (rate) or low-fluence irradiations of different radiation types, such as gamma rays, neutrons and high linear energy transfer (LET) heavy ions. The cells were pretreated with low-dose (rate) or low-fluence irradiations (approximately 1 mGy/7-8 h) of 137Cs gamma rays, 241Am-Be neutrons, helium, carbon and iron ions before irradiations with an X-ray challenging dose (1.5 Gy). Helium (LET = 2.3 keV/microm), carbon (LET = 13.3 keV/microm) and iron (LET = 200 keV/microm) ions were produced by the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan. No difference in cell-killing effect, measured by a colony forming assay, was observed among the pretreatment with different radiation types. In mutation induction, which was detected in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus to measure 6-thioguanine resistant clones, there was no difference in mutation frequency induced by the X-ray challenging dose between unpretreated and gamma-ray pretreated cells. In the case of the pretreatment of heavy ions, X-ray-induced mutation was around 1.8 times higher in helium-ion pretreated and 4.0 times higher in carbon-ion pretreated cells than in unpretreated cells (X-ray challenging dose alone). However, the mutation frequency in cells pretreated with iron ions was the same level as either unpretreated or gamma-ray pretreated cells. In contrast, it was reduced at 0.15 times in cells pretreated with neutrons when compared to unpretreated cells. The results show that cellular responses caused by the influence of hprt mutation induced in cells pretreated with low-dose-rate or low-fluence irradiations of different radiation types were radiation-quality dependent manner.

The JAK2V617F Mutation in Non-MPD Hematopoiesis Occurs at a Low Frequency and in Differentiating Erythroid Cells.

A somatic point mutation in the JAK2 gene results in a constitutive activation of the JAK2 kinase in the erythropoietin (epo) signal transduction pathway. The mutation was found in Polycythemia Vera (PV) patients (>95%) as well as in other myeloproliferative disorders (MPD). In MPD the mutation was shown to occur in pluripotential hematopoietic stem cells and to be present in all blood cell lineages. We analyzed DNA samples from healthy individuals for JAK2V617F mutation by allele specific Real Time PCR. The mutation was detected in 9% of 114 samples with less than 0.001% of JAK2 mutant/wild-type (wt) DNA allelic ratio. The clonality of the mutation was studied by growing blood mononuclear cells from 8 healthy individuals in semi-solid culture in the presence of epo, allowing for erythroid differentiation. Individual erythroid colonies were picked up after 2 weeks. The mutation was detected in all 8 individuals at 5.6 – 24% (a median of 13.4%) of the colonies. The mutant/wt allelic ratio was 0.02% per single colony. This low frequency of positive mutant cells within a colony suggests that the mutation occurred at late stages of colony development concurrently with cell differentiation. In contrast, positive colonies derived from PV patients showed mutant/wt allelic ratio of either 50% (heterozygote) or 100% (homozygote), indicating the presence of the mutation at the early erythroid progenitor stage (BFUe). To further demonstrate that in cells from normal individuals the mutation occurs during erythroid differentiation, we studied 4 samples of CD34+ cells (purity >95%). Cells cultured in the presence of epo differentiated to erythroid precursors within 3 weeks. We found 2–4×10−5% of the JAK2V617F mutation in the CD34+ cells prior to culture, which increased more than 2 folds following cell differentiation. We hypothesized that maintenance of DNA fidelity is down graded with cell differentiation, rendering the cells susceptible for mutations in general and in JAK2 in particular. To prove this hypothesis we used mouse erythroleukemia cells (MEL) as a model for differentiation. MEL cells were transfected with plasmid containing human JAK2 wt or V617F DNA sequences and stimulated for differentiation by hemamethylene bisacetamide. After 5 days in culture human V617F sequence (~ 0.001%) was detected in cells transfected with JAK2 wt-containing plasmid and vice versa, indicating incorrect replication of the introduced plasmid. The changes occurred at 2 folds higher frequency in differentiated cells compared to undifferentiated cells. We speculated that accelerated erythropoiesis with increased erythroid differentiation will result in higher mutation rate. We therefore studied 2 conditions associated with increased hematopoiesis, Thalassemia major and smoking. We analyzed 54 samples from Thalassemic patients and detected the V617F mutation in 12 (22.2%) patients - a 2.4 -fold higher frequency compared to healthy individuals. We also screened peripheral blood of 79 heavy smokers and found the mutation in 20 (25%) individuals – a 2.8 fold higher than in non-smokers. As expected, the mutation was detected at very low frequency ~ 0.0024% of total DNA. Our results demonstrate that unlike in MPD where the JAK2V617F mutation occurs in pluripotential stem cells and expands extensively, in non-MPD individuals it arises in very low mutant/wt DNA allelic ratio during cell differentiation. These differences determine the lack of clinical significance of the JAK2V617F mutation in non-MPD individuals.

The Emergency Workers Medical Observation at the Long Time after the Chernobyl NPP Accident

The approximately 500 emergency workers (liquidators) have been observed in the Treatment and Diagnostic center (Obninsk, Russia). The majority of them were men worked in Chernobyl NPP area in 1986. The total dose of external irradiation was up to 250 mSv. The basic pathology was cardiovascular diseases: ischemic heart disease, hypertension, multifocal atherosclerosis. Associated diseases were osteochondrous, bronchitis, goitre, obesity, prostatitis. In some cases (N=6) the oncopathology has been estimated (thyroid cancer – 2, prostate cancer – 2, kidney cancer – 1, stomach cancer – 1). The frequency of lymphocytes bearing mutated T-cell receptors was assessed in 184 liquidators at the long time after the Chernobyl accident. The mutant cell frequency was elevated in 31 patients of this group as compared with control group. We have not found any difference in the clinical data and the somatic diseases manifestation among liquidators and other nonexposed patients. The treatment of liquidators undergoes according to the modern approaches. The emergency workers demonstrated the alterations in the psychological status: high level of personal anxiety, hesitation, indecision, tension, pressure, inner conflict, the reducing of social activity. The MMPI profile (multifactor method of personal identification) of liquidators with cerebrovascular diseases was characterized by the mixed response with the hypochondria prevalence. The indices of the hypochondria, depression, hysteria and psychoastenia scales in the liquidators group were significantly higher than in the control group patients with the same diseases but had no contact with ionizing radiation. Cerebrovascular diseases presence was not the main reason of the psychological status abnormalities in liquidators. The prolonged psychological impact of the Chernobyl accident caused somatic complains and disease-orientated behavior of liquidators and resulted in reducing their social activity. Thus the medical screening of emergency workers and the special psychological support of them should be continued. The special medical observation of liquidators with the elevated level of mutant cell frequency should be prolonged because of possible high cancer risk in this group.

Genomic instability in mice long term after radiation exposure at low dozes

In this study we measured frequency of lymphocytes bearing mutation at T-cell receptor locus (TCR) and frequency of micronucleated erythrocytes in peripheral blood of mice. Experiments were conducted with male CBA strain of mice which irradiated at 0,2 Gy of γ-rays using Co 60 . Blood sampling was made 3 and 6 months after the irradiation. As it was shown with Mann-Whitney test the frequencies of TCR-mutant cells in exposed and control unexposed groups were not significantly different (p>0,05). According to results of 3 independent experiments medians of TCR mutant cell frequency were 11,7 x 10 -4 . in the controls and 12,5 x 10 -4 in exposed group 3 months after the irradiation (p>0,05). Medians increased slightly in both groups 6 months after radiation exposure: 13,8 x 10 -4 and 15,5 x 10 -4 in control and exposed groups accordingly (p> 0,05). 95% confidence intervals for TCR mutant cell frequency were calculated in control groups. About 15 % of exposed mice had frequencies of the mutant cells exceeding upper boundary of this interval 3 month after the irradiation. The proportion of exposed mice with high TCR mutant cell frequencies increased up to 21% 6 months after the irradiation (p -3 and (3,2±1,0) x 10 -3 in control and exposed groups accordingly. Thus, genomic instability over 3 and 6 months after the irradiation was observed in proportion of mice on gene level and was not found on chromosomal level. It is arguable that the mechanisms underlying formation of genomic instability after low-LET radiation exposure at low doses in vivo involve, as a rule, only little changes in DNA structure (single-strand break, base substitution, etc.), that lead to formation of mainly gene mutations. It is possible also, that elimination of cells with micronuclei occurs more intensively, than cells with TCR mutations.

Cul4A Is Required for Cell Viability and Its Deficiency in Hematopoietic Cells Causes Apoptosis and Is Fatal.

Critical regulators of hematopoiesis are controlled by ubiquitin-mediated proteolysis. Cul4A encodes a core subunit of one ubiquitin ligase, and previous results with hematopoietic cell lines and with Cul4A haploinsufficient mice indicate that Cul4A is required for hematopoietic stem cell function and to maintain the homeostasis of progenitors, precursors, and mature hematopoietic cells. Because Cul4A-deficiency is embryonic lethal, we generated Cul4A conditional knockout mice to examine the requirement of Cul4A for hematopoiesis in adult mice. A mutant Cul4A allele (Cul4Aflox) was constructed where its first coding exon was flanked by loxP sites. Transgenic mice with this mutant allele and the interferon-inducible Cre transgene, Mx1-Cre, were derived. When deletion of Cul4A was induced in Cul4Aflox/floxMx1-Cre mice, the animals died within 3–10 days of the beginning of induction. Necropsies performed four days after the beginning of induction showed that all of the tissues where Mx1-Cre was reported to be expressed appeared normal, except the bone marrow, spleen, and small intestine. The red pulp in the spleen was diminished, there were many fewer nucleated cells in the bone marrow, and the microvilli of the small intestine (duodenum) were dramatically shortened. The mass and total cellularity of mutant spleens were half of controls (Cul4Aflox/floxmice without Mx1-Cre), and bone marrow total cellularity was one-tenth of controls. The frequency of mutant hematopoietic progenitors was reduced 3800-fold in the bone marrow and 80-fold in the spleen. Peripheral blood counts of mature myeloid and lymphoid cells were also dramatically reduced. To separate the in vivo effects of Cul4A-deficiency in hematopoietic cells from those in other cell types, conditional mutant bone marrow was transplanted into wild type recipients, these cells were allowed to engraft for 2 months, and then Cul4A deletion was induced. Mutant animals died within 9–11 days of the beginning of induction with bone marrow nearly empty of cells, spleens only 29% the mass of controls, myeloid and lymphoid counts in the peripheral blood reduced to nearly zero, hematocrits at only 21% of controls, and platelet counts at only 10% of controls. The small intestine, however appeared normal, indicating that Cul4A-deficiency in hematopoietic cells is sufficient to cause death. To examine the fate of Cul4A-deficient hematopoietic cells, deletion was induced in vivo in Cul4Aflox/floxMx1-Cre and control mice, and then bone marrow was harvested and cultured in vitro. Apoptotic cells were detected (either Annexin V positive, 7-AAD negative or TUNEL positive cells) 2–5 days after induction. At 4 and 5 days after induction, the frequency of apoptotic mutant cells was significantly greater than controls (P=0.01 and 0.03, respectively), and at 5 days the frequency of TUNEL positive cells was 4.5-fold greater in the mutant cells. Together, these results indicate that Cul4A-deficiency in hematopoietic cells results in apoptosis, a failure of the hematopoietic system, and death. Analyses of how the expression levels of Cul4A target proteins are altered by Cul4A-deficiency will be presented.