Elizabeth W. Carter

Measures of genotoxicity in Gulf war I veterans exposed to depleted uranium

Exposure to depleted uranium (DU), an alpha‐emitting heavy metal, has prompted the inclusion of markers of genotoxicity in the long‐term medical surveillance of a cohort of DU‐exposed Gulf War veterans followed since 1994. Using urine U (uU) concentration as the measure of U body burden, the cohort has been stratified into low‐u (<0.10 μg U/g creatinine) and high‐u groups (≥0.10 μg U/g creatinine). Surveillance outcomes for this cohort have historically included markers of mutagenicity and clastogenicity, with past results showing generally nonsignificant differences between low‐ vs. high‐U groups. However, mean hypoxanthine‐guanine phosphoribosyl transferase (HPRT) mutant frequencies (MFs) have been almost 50% higher in the high‐U group. We report here results of a more comprehensive protocol performed in a 2009 evaluation of a subgroup (N = 35) of this cohort. Four biomarkers of genotoxicity [micronuclei (MN), chromosome aberrations, and MFs of HPRT and PIGA] were examined. There were no statistically significant differences in any outcome measure when results were compared between the low‐ vs. high‐U groups. However, modeling of the HPRT MF results suggests a possible threshold effect for MFs occurring in the highest U exposed cohort members. Mutational spectral analysis of HPRT mutations is underway to clarify a potential clonal vs. a threshold uU effect to explain this observation. This study provides a comprehensive evaluation of a human population chronically exposed to DU and demonstrates a relatively weak genotoxic effect of the DU exposure. These results may explain the lack of clear epidemiologic evidence for U carcinogenicity in humans. Environ. Mol. Mutagen., 2011.

Both PIGA and PIGL mutations cause GPI‐a deficient isolates in the Tk6 cell line

Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI‐a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X‐linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI‐a deficient isolates, real‐time analysis for all the 25 autosomal GPI‐a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5′ region of PIGL (17p12‐p22) extending 5′ (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000 bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12‐p22 (275,712 bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI‐a deficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI‐a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously. Environ. Mol. Mutagen. 56:663–673, 2015.

Multi-source development of an integrated model for family health history

OBJECTIVE To integrate data elements from multiple sources for informing comprehensive and standardized collection of family health history (FHH). MATERIALS AND METHODS Three types of sources were analyzed to identify data elements associated with the collection of FHH. First, clinical notes from multiple resources were annotated for FHH information. Second, questions and responses for family members in patient-facing FHH tools were examined. Lastly, elements defined in FHH-related specifications were extracted for several standards development and related organizations. Data elements identified from the notes, tools, and specifications were subsequently combined and compared. RESULTS In total, 891 notes from three resources, eight tools, and seven specifications associated with four organizations were analyzed. The resulting Integrated FHH Model consisted of 44 data elements for describing source of information, family members, observations, and general statements about family history. Of these elements, 16 were common to all three source types, 17 were common to two, and 11 were unique. Intra-source comparisons also revealed common and unique elements across the different notes, tools, and specifications. DISCUSSION Through examination of multiple sources, a representative and complementary set of FHH data elements was identified. Further work is needed to create formal representations of the Integrated FHH Model, standardize values associated with each element, and inform context-specific implementations. CONCLUSIONS There has been increased emphasis on the importance of FHH for supporting personalized medicine, biomedical research, and population health. Multi-source development of an integrated model could contribute to improving the standardized collection and use of FHH information in disparate systems.

1,3-Butadiene, CML and the t(9:22) translocation: A reality check

Epidemiological studies of 1,3-butadiene have suggest that exposures to humans are associated with chronic myeloid leukemia (CML). CML has a well-documented association with ionizing radiation, but reports of associations with chemical exposures have been questioned. Ionizing radiation is capable of inducing the requisite CML-associated t(9:22) translocation (Philadelphia chromosome) in appropriate cells in vitro but, thus far, chemicals have not shown this capacity. We have proposed that 1,3-butadiene metabolites be so tested as a reality check on the epidemiological reports. In order to conduct reliable testing in this regard, it is essential that a positive control for induction be available. We have used ionizing radiation to develop such a control. Results described here demonstrate that this agent does in fact induce pathogenic t(9:22) translocations in a human myeloid cell line in vitro, but does so at low frequencies. Conditions that will be required for studies of 1,3-butadiene are discussed.

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. Environ. Mol. Mutagen. 56:581–593, 2015.

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. Environ. Mol. Mutagen. 56:594–608, 2015.