Yoshinori Kasahara

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.

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.

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.

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.

Analysis of mutations in the Pig‐a gene of spleen T‐cells from N‐ethyl‐N‐nitrosourea‐treated fisher 344 rats

A rapid in vivo somatic cell gene mutation assay is being developed that measures mutation in the endogenous X‐linked phosphatidylinositol glycan, class A gene (Pig‐a). The assay detects Pig‐a mutants by flow cytometric identification of cells deficient in glycosylphosphatidyl inositol (GPI) anchor synthesis. GPI‐deficient, presumed Pig‐a mutant cells also can be detected in a cloning assay that uses proaerolysin (ProAER) selection. Previously, we demonstrated that ProAER‐resistant (ProAERr) rat spleen T‐cells have mutations in the Pig‐a gene. In the present study, we report on a more complete analysis of ProAERr rat spleen T‐cell mutants and describe a mutation spectrum for mutants isolated from rats 4 weeks after treatment with three consecutive doses of 35.6 mg/kg N‐ethyl‐N‐nitrosourea (ENU). We identified a total of 55 independent mutations, with the largest percentage (69%) involving basepair substitution at A:T. The overall spectrum of Pig‐a gene mutations was consistent with the types of DNA adducts formed by ENU and was very similar to what has been described for in vivo ENU‐induced mutation spectra in other rodent reporter genes (e.g., in the endogenous Hprt gene and transgenic shuttle vectors). These data are consistent with the rat Pig‐a assay detecting test‐agent‐induced mutational responses. Environ. Mol. Mutagen., 2011. Published 2011 Wiley‐Liss, Inc.

Decrease in deoxyribonucleotide triphosphate pools and induction of alkaline-labile sites in mouse bone marrow cells by multiple treatments with methotrexate

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei in bone marrow and peripheral blood cells, and this induction was enhanced by multiple treatments with the drug. Furthermore, we have suggested that the multiple-dose effect on the induction of micronuclei by MTX might be explained by intracellular accumulation of the drug, resulting in an enhancement of DHFR inhibition. An imbalance or decrease in the deoxyribonucleotide (dNTP) pool would be generated by this enzyme inhibition. Therefore, we attempted to determine the level of the dNTP pool in mouse bone marrow cells. The levels of three dNTPs (dTTP, dATP, dGTP) as determined by HPLC were only 1/10-1/40 of the levels previously found in mammalian cell lines, but dCTP levels could not be determined precisely because they approached the limits of detectability. The levels of dTTP, dATP and dGTP in mouse bone marrow cells 3 h after four injections of MTX (4 mg/kg/day) decreased to 21.2%, 47.0% and 38.1%, respectively, of those in the control group. The level of dTTP 3 h after four injections of 100 mg/kg of the drug decreased to almost 0%. The results of alkaline elution assays suggested that alkaline-labile sites were generated in mouse bone marrow cells 6 h after four injections of MTX (4 mg/kg). These findings suggest that the multiple-dose effects of MTX on micronucleus induction in mouse bone marrow cells may be explained by the decrease in the dNTP pool and subsequent generation of alkaline-labile sites (possibly apurinic/apyrimidinic sites).

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.

The rat Pig-a assay using an erythroid HIS49 antibody in a single dose study of isopropyl p-toluenesulfonate

As part of a collaborative study in the Mammalian Mutagenicity Study group of the Japanese Environmental Mutagen Society, we evaluated the in vivo mutagenicity of isopropyl p-toluenesulfonate (IPTS) using a peripheral blood Pig-a assay in rats. Pig-a mutant frequency (MF) data was obtained for both red blood cells (RBCs) and reticulocytes (RETs) at 1, 2 and 4 weeks after a single oral administration of IPTS at doses of 125, 250, or 500mg/kg. The results of the RBC Pig-a assay demonstrated that both the 250 and 500mg/kg treatment groups showed significant increases in Pig-a MF only at 4 weeks after IPTS treatment. In comparison, the PIGRET assay showed a clear and dose-related increase in Pig-a MF at 1 week after treatment, with a continuous increase until 4 weeks after treatment observed in the highest dose group. These results indicate that the both the RBC Pig-a assay and PIGRET assay can detect in vivo IPTS mutagenicity under a single dosing protocol. In particular, the PIGRET assay, which uses magnetic enrichment to analyze greater numbers of RETs in a high-throughput manner, showed an increase in Pig-a MF earlier than the RBC Pig-a assay. The PIGRET assay is also considered to be more sensitive than the RBC Pig-a assay because it exhibits a low spontaneous Pig-a MF. For this reason, the PIGRET assay clearly identified small increases in Pig-a MF as significant at the lower doses than in the RBC Pig-a assay under the conditions in this study.

Report on the rat Pig-a assay using an anti-rat erythroid marker HIS49 antibody in a single dose study of 1,2-dimethylhydrazine

As part of a collaborative study in the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society (JEMS), we investigated the in vivo genotoxicity profile of 1,2-dimethylhydrazine (DMH) using a Pig-a assay in total red blood cells (RBC Pig-a assay) or a reticulocyte Pig-a assay (PIGRET assay). We also assessed the genotoxic potential of DMH using both a bone marrow micronucleus test and a liver comet assay as follow-up studies. Single administration of 25, 50, 100mg/kg DMH to male rats did not show time- or dose-related increases in Pig-a mutant frequency (MF) in either the RBC Pig-a or PIGRET assays up to 4 weeks after treatment. The bone marrow micronucleus test under the same dose levels was judged positive, while the liver comet assay was judged inconclusive due to the high number of hedgehogs. Re-evaluation of the rat liver comet assay at lower dose levels (4, 10, and 25mg/kg DMH) showed a dose-related increase in%DNA in tail. Taken together, DMH showed a positive response in both the bone marrow micronucleus test and liver comet assay, while the increases in Pig-a MF in both the RBC Pig-a and PIGRET assays could hardly be detected after single dosing. These results suggest that DMH provides different genotoxicity outcomes depending on the endpoint following acute in vivo dosing.

Prophylactic treatment with sulphonated immunoglobulin G attenuates development of mechanical allodynia-like response in mice with neuropathic pain

Human immunoglobulin G (IgG) concentrates are immune-modulating, anti-inflammatory plasma-derived products. Clinical studies in recent years have suggested that IgG attenuates neuropathic pain. In this study, effects of sulphonated IgG on the development and maintenance of a mechanical allodynia-like response were examined in mice with neuropathic pain induced by a partial sciatic nerve ligation (PSL). When sulphonated IgG (400 or 1,000 mg/kg/day, i.p.) was administered for 5 days, from 1 day before surgery to post-operative day (POD) 3, the development of a mechanical allodynia-like response was attenuated. On the other hand, sulphonated IgG had little effect on the maintenance of a mechanical allodynia-like response when administered for 5 days, from POD 11 to POD 15, at which time a mechanical allodynia-like response had already been developed. To explore the mechanism of sulphonated IgG, the mRNA expression of inflammatory cytokines was evaluated in the injured sciatic nerve. Sulphonated IgG (1,000 mg/kg/day, i.p.) that was administered for 3 days, from 1 day before surgery to POD 1, significantly attenuated the up-regulation of tumor necrosis factor-α and monocyte chemotactic protein-1 mRNAs on POD 1. These results suggest that prophylactic treatment with sulphonated IgG attenuates the development of mechanical allodynia-like response by inhibition of inflammatory cytokine expression in mice with PSL.