Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium.

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.

Longitudinal study of t-cell somatic mutations conferring glycosylphosphatidylinositol-anchor deficiency in gulf war I veterans exposed to depleted uranium.

Fifty Veterans of the first Gulf War in 1991 exposed to depleted uranium (DU) were studied for glycosylphosphatidylinositol-anchor (GPIa) deficient T-cell mutants on three occasions during the years 2009, 2011, and 2013. GPIa deficiency was determined in two ways: cloning assays employing aerolysin selection and cytometry using the FLAER reagent for positive staining of GPIa cell surface proteins. Subsequent molecular analyses of deficient isolates recovered from cloning assays (Nicklas JA et al. [2019]: Environ Mol Mutagen) revealed apparent incomplete selection in some cloning assays, necessitating correction of original data to afford a more realistic estimate of GPIa deficient mutant frequency (MF) values. GPIa deficient variant frequencies (VFs) determined by cytometry were determined in the years 2011 and 2013. A positive but nonsignificant association was observed between MF and VF values determined on the same blood samples during 2013. Exposure to DU had no effect on either GPIa deficient MF or VFs. Environ. Mol. Mutagen. 60:494-504, 2019. © 2019 Wiley Periodicals, Inc.

Mutagenicity monitoring following battlefield exposures: Longitudinal study of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

A total of 70 military Veterans have been monitored for HPRT T-cell mutations in five separate studies at 2-year intervals over an 8-year period. Systemic depleted uranium (DU) levels were measured at the time of each study by determining urinary uranium (uU) excretion. Each HPRT study included 30-40 Veterans, several with retained DU-containing shrapnel. Forty-nine Veterans were evaluated in multiple studies, including 14 who were in all five studies. This permitted a characterization of the HPRT mutation assay over time to assess the effects of age, smoking and non-selected cloning efficiencies, as well as the inter- and intra-individual variability across time points. Molecular analyses identified the HPRT mutation and T-cell receptor (TCR) gene rearrangement in 1,377 mutant isolates. An unexpected finding was that in vivo clones of HPRT mutant T-cells were present in some Veterans, and could persist over several years of the study. The calculated HPRT mutant frequencies (MFs) were repeatedly elevated in replicate studies in three outlier Veterans with elevated urinary uranium excretion levels. However, these three outlier Veterans also harbored large and persistent in vivo HPRT mutant T-cell clones, each of which was represented by a single founder mutation. Correction for in vivo clonality allowed calculation of HPRT T-cell mutation frequencies (MutFs). Despite earlier reports of DU associated increases in HPRT MFs in some Veterans, the results presented here demonstrate that HPRT mutations are not increased by systemic DU exposure. Additional battlefield exposures were also evaluated for associations with HPRT mutations and none were found.

Mutagenicity monitoring following battlefield exposures: Molecular analysis of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures.

The effect of dietary folic acid deficiency on the cytotoxic and mutagenic responses to methyl methanesulfonate in wild-type and in 3-methyladenine DNA glycosylase-deficient Aag null mice.

Folic acid deficiency (FA-) augments DNA damage caused by alkylating agents. The role of DNA repair in modulating this damage was investigated in mice. Weanling wild-type or 3-methyladenine glycosylase (Aag) null mice were maintained on a FA- diet or the same diet supplemented with folic acid (FA+) for 4 weeks. They were then treated with methyl methanesulfonate (MMS), 100mg/kg i.p. Six weeks later, spleen cells were collected for assays of non-selected and 6-thioguanine (TG) selected cloning efficiency to measure the mutant frequency at the Hprt locus. In wild-type mice, there was no significant effect of either MMS treatment or folate dietary content on splenocyte non-selected cloning efficiency. In contrast, non-selected cloning efficiency was significantly higher in MMS-treated Aag null mice than in saline treated controls (diet-gene interaction variable, p=0.04). The non-selected cloning efficiency was significantly higher in the FA+ diet than in the FA- diet group after MMS treatment of Aag null mice. Mutant frequency after MMS treatment was significantly higher in FA- wild-type and Aag null mice and in FA+ Aag null mice, but not in FA+ wild-type mice. For the Aag null mice, mutant frequency was higher in the FA+ mice than in the FA- mice after either saline or MMS treatment. These studies indicate that in wild-type mice treated with MMS, dietary folate content (FA+ or FA-) had no effect on cytotoxicity, but FA- diet increased DNA mutation frequency compared to FA+ diet. In Aag null mice, FA- diet increased the cytotoxic effects of alkylating agents but decreased the risk of DNA mutation.

Nucleic acid deletions and copy number in rats.

Rats fed either a cereal-based or purified diet of variable folate content (deficient, replete, or supplemented) inadvertently were infected with sialodacryoadenitis virus, which resulted in an increased frequency of hepatic mitochondrial DNA (mtDNA) deletions that persisted for three weeks after the period of acute signs of disease. The amount of the "common deletion" (4.8 kb, bases 8103-12937) in liver was measured by quantitative co-amplification of the mitochondrial D-loop and the mitochondrial deletion, using a real-time quantitative polymerase chain reaction assay. The relative abundance of mtDNA was determined by co-amplifying mitochondrial D-loop versus the rat beta-actin gene. Virus-infected rats had more mtDNA deletions (P < 0.0001) and higher copy number (P < 0.0001) than did uninfected animals. There was no effect of diet on frequency of deletions. Diet affected mtDNA relative abundance in the infected, but not the uninfected rats. Relative abundance was higher (P = 0.004) in rats of the high folate group than in rats of the low-folate or folate-replete groups, and was significantly higher in rats of the cereal diet group than that in those of the purified diet group. In conclusion, sialodacryoadenitis virus infection in rats was associated with increased frequency of hepatic mtDNA deletions. Thus, sialodacryoadenitis virus infection mitigated biological processes in the liver of rats, and mtDNA damage was modulated by diet.

Dietary modulation of mitochondrial DNA deletions and copy number after chemotherapy in rats.

Mitochondrial DNA (mtDNA) is particularly susceptible to mutation by alkylating agents, and mitochondrial damage may contribute to the efficacy and toxicity of these agents. We found that folate supplementation decreased the frequency of the "common deletion" (4.8kb, bases 8103-12,936) in liver from untreated rats and from animals treated with cyclophosphamide but not 5-fluorouracil (5-FU). The relative abundance of mitochondrial DNA was greater after chemotherapy but there was no effect of diet. Rats fed with a purified diet had fewer mitochondrial deletions than those maintained on a cereal-based diet after chemotherapy. These results indicate that diet can modulate the extent of mitochondrial damage after cancer chemotherapy, and that folic acid supplementation may be protective against mitochondrial DNA deletions.