Mutant Erythrocytes Research Articles

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

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

The PIG-A gene mutation assay in human biomonitoring and disease.

The blood cell phosphatidylinositol glycan class A (PIG-A) gene mutation assay has been extensively researched in rodents for in vivo mutagenicity testing and is now being investigated in humans. The PIG-A gene is involved in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis. A single mutation in this X-linked gene can lead to loss of membrane-bound GPI anchors, which can be enumerated via corresponding GPI-anchored proteins (e.g., CD55) using flow cytometry. The studies published to date by different research groups demonstrate a remarkable consistency in PIG-A mutant frequencies. Moreover, with the low background level of mutant erythrocytes in healthy subjects (2.9-5.56 × 10-6 mutants), induction of mutation post genotoxic exposure can be detected. Cigarette smoking, radiotherapy, and occupational exposures, including lead, have been shown to increase mutant levels. Future applications of this test include identifying new harmful agents and establishing new exposure limits. This mutational monitoring approach may also identify individuals at higher risk of cancer development. In addition, identifying protective agents that could mitigate these effects may reduce baseline somatic mutation levels and such behaviors can be encouraged. Further technological progress is required including establishing underlying mechanisms of GPI anchor loss, protocol standardization, and the development of cryopreservation methods to improve GPI-anchor stability over time. If successful, this assay has the potential be widely employed, for example, in rural and low-income countries. Here, we review the current literature on PIG-A mutation in humans and discuss the potential role of this assay in human biomonitoring and disease detection.

X-linked C1GALT1C1 mutation causes atypical hemolytic uremic syndrome.

Hemolytic-uremic syndrome (HUS), mostly secondary to infectious diseases, is a common cause of acute kidney injury in children. It is characterized by progressive acute kidney failure due to severe thrombotic microangiopathy, associated with nonimmune, Coombs-negative hemolytic anemia and thrombocytopenia. HUS is caused mostly by Shiga toxin-producing E. Coli, and to a lesser extent by Streptococcus pneumonia. In Streptococcus pneumonia HUS (pHUS), bacterial neuraminidase A exposes masked O-glycan sugar residues on erythrocytes, known as the T antigen, triggering a complement cascade causing thrombotic microangiopathy. Atypical HUS (aHUS) is a life-threatening genetic form of the disease, whose molecular mechanism is only partly understood. Through genetic studies, we demonstrate a novel X-linked form of aHUS that is caused by a de-novo missense mutation in C1GALT1C1:c.266 C > T,p.(T89I), encoding a T-synthase chaperone essential for the proper formation and incorporation of the T antigen on erythrocytes. We demonstrate the presence of exposed T antigen on the surface of mutant erythrocytes, causing aHUS in a mechanism similar to that suggested in pHUS. Our findings suggest that both aHUS caused by mutated C1GALT1C1 and pHUS are mediated by the lectin-complement-pathway, not comprehensively studied in aHUS. We thus delineate a shared molecular basis of aHUS and pHUS, highlighting possible therapeutic opportunities.

Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis.We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage.Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation.Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis.Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR.Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. DisclosuresNo relevant conflicts of interest to declare.

Dietary and lifestyle factors effect erythrocyte PIG-A mutant frequency in humans.

It is well understood that poor diet and lifestyle choices can increase the risk of cancer. It is also well documented that cancer is a disease of DNA mutations, with mutations in key genes driving carcinogenesis. Measuring these mutations in a minimally invasive way may be informative as to which exposures are harmful and thus allow us to introduce primary preventative measures, in a bid to reduce cancer incidences. Here, we have measured mutations in the phosphatidylinositol glycan class A (PIG-A) gene in erythrocytes from healthy volunteers (n = 156) and from non-cancer patients attending the local endoscopy department (n = 144). The X-linked PIG-A gene encodes an enzyme involved in glycosylphosphatidylinositol (GPI) anchor synthesis. A silencing mutation in which leads to the absence of GPI anchors on the extracellular surface which can be rapidly assessed using flow cytometry. The background level of PIG-A mutant erythrocytes was 2.95 (95% CI: 2.59-3.67) mutant cells (10-6). Older age increased mutant cell frequency (P < 0.001). There was no difference in mutant cell levels between males and females (P = 0.463) or smokers and non-smokers (P = 0.186). In the endoscopy group, aspirin users had lower mutant frequencies (P = 0.001). Further information on diet and exercise was available for the endoscopy patient group alone, where those with a higher health promotion index score had lower mutant frequencies (P = 0.011). Higher dietary intake of vegetables reduced mutant cell levels (P = 0.022). Participants who exercised for at least 1 h a week appeared to have reduced mutant frequencies than those who did not exercise, although this was not statistically significant (P = 0.099). This low background level of mutant erythrocytes in a population makes this assay an attractive tool to monitor exposures such as those associated with lifestyles and diet, as demonstrated here.

Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20–50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

Intra- and inter-laboratory reproducibility of the rat blood Pig-a gene mutation assay.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the requirements for OECD approval are demonstrations of assay reliability, including reproducibility within and among laboratories. Experiments reported herein address the reproducibility of the rat blood Pig-a assay using the reference mutagens chlorambucil and melphalan. These agents were evaluated for their ability to induce Pig-a mutant erythrocytes in three separate studies conducted across two laboratories. Each of the studies utilized a common treatment schedule: 28 consecutive days of exposure via oral gavage. Whereas one laboratory studied Crl:CD(SD) rats, the other laboratory used Wistar Han rats. One or two days after cessation of treatment blood samples were collected for mutant reticulocyte and mutant erythrocyte measurements that were accomplished with the same analytical technique whereby samples were depleted of wildtype erythrocytes via immunomagnetic separation followed by flow cytometric enumeration of mutant phenotype cells (MutaFlow®). Dunnett's test results showed similar qualitative outcomes within and between laboratories, that is, each chemical and each study demonstrated statistically significant, dose-related increases in mutant reticulocyte and erythrocyte frequencies. Benchmark dose analysis (PROAST software) provided a means to quantitatively analyze the results, and the relatively tight, overlapping benchmark dose confidence intervals observed for each of the two chemicals indicate that within and between laboratory reproducibility of the Pig-a assay are high, adding further support for the development of an OECD test guideline.

Detection of Pig-a Mutant Erythrocytes in the Peripheral Blood of Rats and Mice.

The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because: (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection. Two endpoints are determined in the assay: the frequency of mutant total RBCs and the frequency of mutant reticulocytes. When Pig-a mutation is used to assess the in vivo mutagenic potential of suspect hazards, the frequency of mutant reticulocytes is an early indicator of mutagenic potential, while the mutant frequency in total RBCs can be measured more rapidly and with greater precision.

The splicing factor Sf3b1 regulates erythroid maturation and proliferation via TGFβ signaling in zebrafish

AbstractThe spliceosomal component Splicing Factor 3B, subunit 1 (SF3B1) is one of the most prevalently mutated factors in the bone marrow failure disorder myelodysplastic syndrome. There is a strong clinical correlation between SF3B1 mutations and erythroid defects, such as refractory anemia with ringed sideroblasts, but the role of SF3B1 in normal erythroid development is largely unknown. Loss-of-function zebrafish mutants for sf3b1 develop a macrocytic anemia. Here, we explore the underlying mechanism for anemia associated with sf3b1 deficiency in vivo. We found that sf3b1 mutant erythroid progenitors display a G0/G1 cell-cycle arrest with mutant erythrocytes showing signs of immaturity. RNA-sequencing analysis of sf3b1 mutant erythroid progenitors revealed normal expression of red blood cell regulators such as gata1, globin genes, and heme biosynthetic factors, but upregulation of genes in the transforming growth factor β (TGFβ) pathway. As TGFβ signaling is a known inducer of quiescence, the data suggest that activation of the pathway could trigger sf3b1 deficiency–induced anemia via cell-cycle arrest. Indeed, we found that inhibition of TGFβ signaling released the G0/G1 block in erythroid progenitors. Surprisingly, removal of this checkpoint enhanced rather than suppressed the anemia, indicating that the TGFβ-mediated cell-cycle arrest is protective for sf3b1-mutant erythrocytes. Together, these data suggest that macrocytic anemia arising from Sf3b1 deficiency is likely due to pleiotropic and distinct effects on cell-cycle progression and maturation.

Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59−) or mutant reticulocyte frequencies (RETCD59−) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Erythrocyte β spectrin can be genetically targeted to protect mice from malaria.

The malaria parasite hijacks host erythrocytes to shield itself from the immune system and proliferate. Red blood cell abnormalities can provide protection from malaria by impeding parasite invasion and growth within the cell or by compromising the ability of parasites to avoid host clearance. Here, we describe 2 N-ethyl-N-nitrosourea-induced mouse lines, SptbMRI26194 and SptbMRI53426 , containing single-point mutations in the erythrocyte membrane skeleton gene, β spectrin (Sptb), which exhibit microcytosis but retain a relatively normal ratio of erythrocyte surface area to volume and are highly resistant to rodent malaria. We propose the major factor responsible for malaria protection is the specific clearance of mutant erythrocytes, although an enhanced clearance of uninfected mutant erythrocytes was also observed (ie, the bystander effect). Using an in vivo erythrocyte tracking assay, we established that this phenomenon occurs irrespective of host environment, precluding the involvement of nonerythrocytic cells in the resistance mechanism. Furthermore, we recapitulated this phenotype by disrupting the interaction between ankyrin-1 and β spectrin in vivo using CRISPR/Cas9 genome editing technology, thereby genetically validating a potential antimalarial target. This study sheds new light on the role of β spectrin during Plasmodium infection and highlights how changes in the erythrocyte cytoskeleton can substantially influence malaria susceptibility with minimal adverse consequences for the host.

Applying the erythrocyte Pig-a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation.

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc.

Diet-induced obesity increases the frequency of Pig-a mutant erythrocytes in male C57BL/6J mice.

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30-week old male mice reared on either a high-fat diet (60% calories from fat) that exhibit an obese phenotype or a normal-fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N-ethyl-N-nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig-a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non-obese mice with respect to Pig-a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig-a mutant frequencies (increased 2.5-3.7-fold, p < 0.02) in erythrocytes as compared to non-obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668-677, 2016. © 2016 Wiley Periodicals, Inc.

Results of rat Pig-a/PIGRET assay with a single dose regimen of 1,3-propane sultone and 2-acetyl aminofluorene

The Pig-a assay is a useful in vivo mutation detecting test and is easier to perform than the in vivo transgenic mutation assay. This assay is now recognized to be able to detect a number of mutagenic chemicals administered to rats in sub-acute or sub-chronic dose studies. The present investigation was conducted to evaluate the usefulness of peripheral blood Pig-a assays with total red blood cells (RBC Pig-a assay) and with reticulocytes (PIGRET assay) using two genotoxic rodent carcinogens, 1,3-propane sultone (1,3-PS) and 2-acetylaminofluorene (2-AAF). Male rats were orally administered a single dose of each test compound, and both the RBC Pig-a and PIGRET assays were performed using flow cytometry to measure the Pig-a mutant frequency (MF) before and after dosing on Days 8, 15 and 29. In the experiment with 1,3-PS, significant increases in Pig-a MF were observed from Day 15 and Day 8 in the RBC Pig-a and PIGRET assays, respectively. The results of both assays demonstrated that the increases in Pig-a MF were detectable after a single treatment with 1,3-PS. Furthermore, the difference in the kinetics of the increase in Pig-a MF between the RBC Pig-a and PIGRET assays with 1,3-PS suggests that the PIGRET assay has an advantage in detecting the mutant erythrocytes earlier than the RBC Pig-a assay. In contrast, no significant increases were observed in the Pig-a assays using either RBC or reticulocytes with 2-AAF. The negative results in both assays with 2-AAF may indicate the limitation of the single dose method; however, further investigation at higher doses is necessary to determine limitation of the single dose method.

Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1.

The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology; however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, Tfrc(MRI24910), identified during an N-ethyl-N-nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami, mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria.

Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate.

Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500 mg/kg/day) or EC (250 mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10(-6) on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10(-6) on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

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.