Vasily N. Dobrovolsky

Development of an in vivo gene mutation assay using the endogenous Pig‐A gene: I. Flow cytometric detection of CD59‐negative peripheral red blood cells and CD48‐negative spleen T‐cells from the rat

The product of the phosphatidylinositol glycan complementation group A gene (Pig‐A) is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link various protein markers to the surface of several types of mammalian cells, including hematopoietic cells. Previous observations indicate that Pig‐A mutation results in the lack of GPI synthesis and the absence of GPI‐anchored proteins on the cell surface. As a first step in designing a rapid assay for measuring Pig‐A mutation in the rat, we developed flow cytometry (FCM) strategies for detecting GPI‐negative cells in rat peripheral blood and spleen. Anti‐CD59 was used to detect GPI‐anchored proteins on red blood cells (RBCs), and anti‐CD48 was used to detect GPI‐anchored proteins on spleen T‐cells. The spontaneous frequency of CD59‐negative RBCs in five male F344 rats ranged from 1 × 10−6 to 27 × 10−6. In contrast, treatment of five rats with three doses of 40 mg/kg N‐ethyl‐N‐nitrosourea (ENU) increased the frequency of CD59‐negative RBCs to 183 × 10−6 to 249 × 10−6 at 2 weeks and to 329 × 10−6 to 413 × 10−6 at 4 weeks after dosing. In the same 4‐week posttreatment rats, the frequency of CD48‐negative T‐cells was 11 × 10−6 to 16 × 10−6 in control rats and 194 × 10−6 to 473 × 10−6 in ENU‐treated rats. The frequencies of GPI‐deficient cells were similar for RBCs and spleen T‐cells. These results indicate that FCM detection of GPI‐linked markers may form the basis for a rapid in vivo mutation assay. Although RBCs may be useful for a minimally invasive assay, T‐cells are a promising tissue for both detecting GPI‐deficient cells and confirming that Pig‐A gene mutation is the cause of the phenotype. Environ. Mol. Mutagen., Published 2008 Wiley‐Liss, Inc.

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The Pig‐a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N‐acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig‐a is the only gene involved in GPI anchor synthesis that is on the X‐chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI‐anchored cell surface proteins, almost always have mutations in the Pig‐a gene. These properties of the Pig‐a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig‐a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10−6) and rats treated with multiple doses of the potent mutagen, N‐ethyl‐N‐nitrosourea, display Pig‐a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat‐dose toxicology studies. Environ. Mol. Mutagen., 2010. Published 2010 Wiley‐Liss, Inc.

Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea.

We previously reported the development of an in vivo gene mutation assay using the phosphatidylinositol glycan complementation group A gene (Pig-A) as an endogenous reporter. The assay quantifies mutation in rat peripheral red blood cells (RBCs) by flow cytometric detection of cells negative for glycosylphosphatidyl inositol (GPI)-anchored protein surface markers. In this study, we examined the accumulation and persistence of Pig-A mutant RBCs in rats treated with N-ethyl-N-nitrosourea (ENU) using two dosing schedules. Male F344 rats were given single i.p. injections of 8.9, 35.6, or 142.4 mg/kg ENU or four equal weekly doses totaling 35.6 or 142.4 mg/kg ENU (8.9 mg/kgx4 or 35.6 mg/kgx4; split-dose groups). Before the treatment and through 26 weeks after the single dose or beginning the split-dose regimen, peripheral RBCs were collected and Pig-A mutant frequencies measured as RBCs negative for the GPI-anchored protein, CD59. Mean CD59-negative RBC frequencies in negative control rats ranged from 3.9 x 10(-6) to 28.7 x 10(-6) and displayed no time-related trend. With single ENU doses, CD59-negative RBC frequencies increased in a time- and dose-related manner. Maximum responses were observed beginning at 6 weeks post-treatment (57.3 x 10(-6) in the 8.9 mg/kg group; 186.9 x 10(-6) in the 35.6 mg/kg group; 759.2 x 10(-6) in the 142.4 mg/kg group), and these elevated mutant frequencies persisted to the last sampling time. In addition, splitting the dose of ENU into four weekly doses produced nearly the same mutant frequency as when given as a single dose: the maximum responses after four weekly doses of 8.9 or 35.6 mg/kg were 176.8 x 10(-6) and 683.3 x 10(-6), respectively. These results indicate that ENU-induced Pig-A mutant RBCs accumulate in a near additive fashion in rats, and once present in the peripheral blood, persist for at least 6 months. These characteristics of Pig-A mutation could be important for detecting weak mutagens by repeated or subchronic/chronic dosing protocols.

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup.

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Although the assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans, currently it is optimized only for measuring CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. An expert workgroup formed by the International Workshop on Genotoxicity Testing considered the state of assay development and the potential of the assay for regulatory use. Consensus was reached on what is known about the Pig-a assay and how it should be conducted, and recommendations were made on additional data and refinements that would help to further enhance the assay for use in hazard identification and risk assessment.

Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants†‡

We previously reported that rat spleen T‐cells and peripheral red blood cells that are deficient in glycosylphosphatidylinositol (GPI) synthesis [presumed mutants for the phosphatidylinositol glycan complementation group A gene (Pig‐A)] could be detected by flow cytometry (FCM) as cells negative for GPI‐linked markers (CD48 and CD59, respectively). To establish this procedure as a rapid in vivo gene mutation assay, we have examined the Pig‐A gene of GPI‐deficient rat spleen T‐cells for DNA sequence alterations. Splenocytes were isolated from male F344 rats, primed with ionomycin and phorbol‐12‐myristate‐13‐acetate, and seeded at limiting‐dilution into 96‐well plates. To select for GPI‐deficient T‐cells, the cells were cultured for 10 days in a medium containing rat T‐STIM® and 2 nM proaerolysin (ProAER). The frequency of ProAER‐resistant (ProAERr) spleen T‐cells from control rats ranged from 1.3 × 10−6 to 4.8 × 10−6, while administration of three doses of 40 mg/kg N‐ethyl‐N‐nitrosourea increased the frequency of ProAERr T‐cells 100‐fold at 4 weeks after dosing. FCM analysis of the cells in ProAERr clones revealed that they were CD48‐negative, and thus presumably GPI‐deficient. Sequencing of Pig‐A cDNA from six ProAERr clones indicated that they all contained alterations in the Pig‐A protein coding sequence; five had base pair substitutions and one had multiple exons deleted. These results indicate that GPI‐deficient spleen T‐cells are Pig‐A gene mutants and support the use of FCM analysis of GPI‐deficient cells as a rapid assay for measuring in vivo gene mutation. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.

The genotoxicity of acrylamide and glycidamide in big blue rats.

Acrylamide (AA), a mutagen and rodent carcinogen, recently has been detected in fried and baked starchy foods, a finding that has prompted renewed interest in its potential for toxicity in humans. In the present study, we exposed Big Blue rats to the equivalent of approximately 5 and 10 mg/kg body weight/day of AA or its epoxide metabolite glycidamide (GA) via the drinking water, an AA treatment regimen comparable to those used to produce cancer in rats. After 2 months of dosing, the rats were euthanized and blood was taken for the micronucleus assay; spleens for the lymphocyte Hprt mutant assay; and liver, thyroid, bone marrow, testis (from males), and mammary gland (females) for the cII mutant assay. Neither AA nor GA increased the frequency of micronucleated reticulocytes. In contrast, both compounds produced small (approximately twofold to threefold above background) but significant increases in lymphocyte Hprt mutant frequency (MF, p < 0.05), with the increases having dose-related linear trends (p < 0.05 to p < 0.001). Neither compound increased the cII MF in testis, mammary gland (tumor target tissues), or liver (nontarget tissue), while both compounds induced weak positive increases in bone marrow (nontarget tissue) and thyroid (target tissue). Although the genotoxicity in tumor target tissue was weak, in combination with the responses in surrogate tissues, the results are consistent with AA being a gene mutagen in the rat via metabolism to GA.

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes.

Recent studies indicate that the Pig‐a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig‐a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid‐specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 × 106 CD71‐positive RETs could be evaluated by flow cytometry for Pig‐a mutant frequency within 5 to 8 min. CD59‐deficient RET and BME frequencies of more than 100 × 10−6 and 80 × 10−6 were detected 1 week after treating rats with 40 mg/kg N‐ethyl‐N‐nitrosourea; by comparison, the frequency of CD59‐deficient total RBCs in these rats was 13.2 × 10−6. The frequency of spontaneous Pig‐a mutant RETs and BMEs was less than 5 × 10−6 and 15 × 10−6, respectively. Since ∼98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59‐deficient Pig‐a mutations in cells of erythroid lineage. Conducting concurrent Pig‐a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.

Mice deficient for cytosolic thymidine kinase gene develop fatal kidney disease

The thymidine kinase (Tk) gene codes for a cytosolic protein involved in the pyrimidine nucleotide salvage pathway. A functional Tk gene is not necessary for cells in culture, and a naturally occurring Tk deficient phenotype has not been described in humans or animal models. In order to determine the biological significance of the Tk gene, we created Tk(-/-) knockout (KO) mice through homologous recombination in mouse embryonic stem cells. Tk KO mice have shortened life spans compared with their wild-type or Tk heterozygous (HET) siblings. All Tk KO mice develop sclerosis of kidney glomeruli and die before one year of age of kidney failure. Among other changes in KO animals, the most consistent is a switch from exclusively mucous secretion to predominantly serous secretion in the sublingual salivary gland. HET parents can produce KO mice at a frequency approaching Mendelian inheritance. Other observations in KO animals include an elevated level of serum thymidine, a significant decrease in the cloning efficiency of splenic lymphocytes, an increase in the frequency of hypoxanthine guanine phosphoribosyl transferase gene mutant lymphocytes, and histological alteration in the lymphoid structure of the spleen. In addition, KO animals sporadically exhibit inflammation of the arteries, which taken together with the lymphocyte and spleen abnormalities, suggest an abnormal immune system. Alterations in Tk KO mice indicate that the pyrimidine nucleotide salvage pathway is indispensable in vivo.

Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression.

Our previous rat studies indicate that the endogenous Pig-a gene is a promising reporter of in vivo mutation and potentially useful as the basis for an in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100mg/kgN-ethyl-N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM Pig-a mutants transit from the BM and accumulate in PB.

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

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