Environmental and Molecular Mutagenesis 56:581^593 (2015)

Research Article Mutagenicity Monitoring Following Battlefield Exposures: Longitudinal Study of HPRT Mutations in Gulf War I Veterans Exposed to Depleted Uranium Richard J. Albertini,1 Pamela M.Vacek,2 Elizabeth W. Carter,3 Janice A. Nicklas,4* Katherine S. Squibb,5 Patricia W. Gucer,5 Susan M. Engelhardt,6 and Melissa A. McDiarmid5 1

Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 2 Medical Biostatistics Unit, University of Vermont College of Medicine, Burlington, Vermont 3 Center for Clinical and Translational Science—Biomedical Informatics Unit, University of Vermont, Burlington, Vermont 4 Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont 5 Occupational Health Program, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 6 Department of Veterans Affairs Medical Center, Baltimore, Maryland 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. Fortynine 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 pres-

ent 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, C 2015 Wiley Periodicals, Inc. 2015. V

Key words: depleted uranium; urine uranium; HPRT; biomarkers of genotoxicity; mutation

INTRODUCTION Military service under battlefield conditions presents unique occupational exposures to potentially genotoxic agents. Among these agents are metals, for which sources of exposure may be external via skin contamination or internal through inhalation or in the form of embedded metal fragments, (i.e., shrapnel) which can cause longterm in situ release of metal ions to the circulatory system. We have had the opportunity to longitudinally study such a group of 70 Veterans, most of whom were C 2015 Wiley Periodicals, Inc. V

Grant sponsors: US Department of Veterans Affairs; Vermont Cancer Center; the Lake Champlain Cancer Research Organization; UVM College of Medicine. *Correspondence to: Janice A. Nicklas, PhD, Genetic Toxicology, University of Vermont 665 Spear St., Burlington, VT 05405, USA. E-mail: [email protected] Received 20 January 2015; provisionally accepted 8 April 2015; and in final form 00 Month 2015 DOI 10.1002/em.21955 Published online 13 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

Environmental and Molecular Mutagenesis. DOI 10.1002/em 582

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exposed to depleted uranium (DU) during the first Gulf War (1991), examining somatic mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes (PBL). A much smaller number of Veterans were exposed during Operation Iraqi Freedom in 2003. Our studies extended over a period of 8 years with multiple assays of the same individuals. The initial intent of this research was to assess the mutagenicity of depleted uranium (DU) in exposed soldiers. The exposure scenarios during the first Gulf War occurred during the month of February, 1991, when ;115 US military personnel in Iraq were mistakenly fired upon by U.S. forces using DU penetrators. Exposures of the smaller group occurred in 2003. DU is derived from naturally occurring uranium (U), which contains three radioactive isotopes (U234, U235, and U238). DU is created as a by-product of the U enrichment process, in which the two most radioactive isotopes (U234 and U235) are removed, leaving DU with approximately 60% of the radioactivity of natural U, primarily in the form of alpha particles [AEPI 1995]. DU is used by the military in tank armor and in armor-piercing shells because it is twice as dense as naturally occurring lead. On impact, DU forms DU oxide dust due to its pyrophoric character, as well as metal fragments that may act as shrapnel. In addition to traumatic injuries, these fired-upon personnel were exposed to DU dust by inhalation and/or wound contamination. In some, shrapnel penetrated into soft tissues to remain as embedded DU fragments. Although the DU dust exposures were acute and occurred more than 20 years ago, embedded fragments have served as a constant internal source of DU exposure due to oxidation and release of the metal. In 1993, the US Department of Veterans Affairs, in conjunction with the Department of Defense, initiated a medical surveillance program to assess the health of Gulf War Veterans exposed to DU through friendly fire incidents. This surveillance originally included studies of chromosome aberrations as measures of mutagenicity. In 2001, monitoring for HPRT PBL mutations was added to the test battery. At 2-year intervals for the next 8 years, our laboratory performed HPRT assays on these Veterans as well as the very few Veterans from the more recent Iraq conflict. The early results of the mutagenicity studies suggested that DU was associated with elevated HPRT mutations in a small number of highly exposed individuals, creating some concern in Veterans of this conflict. However, as described in this article, analysis of longitudinal data that allows determination of mutation frequencies (frequencies of initial events that alter the DNA information content) rather than mutant frequencies (frequencies of clonally amplified progeny of the initial mutant cell) through identification of persistent in vivo HPRT mutant T-cell clones

has reversed this impression. The results presented here provide evidence that mutations in humans, at least as measured by HPRT, were not increased by systemic DU exposure, thereby laying that concern to rest. Over time, elevation of other metal concentrations, as reflected by urine metal measurements, has also been determined in this Veteran cohort, allowing their impact on HPRT mutations to be assessed. In addition, the data set developed over the several years of this study has permitted a characterization of HPRT PBL mutation assays in adult men, including the effects of age, smoking and non-selected cloning efficiencies, as well as the inter-individual and intra-individual variability across time points, as encountered by a single laboratory. This is the first such characterization of a large HPRT data set since 1996, when a similar attempt was possible only by pooling the data of several laboratories [Robinson et al., 1994].

MATERIALS AND METHODS Sample Acquisition and Handling Sub-groups of the DU exposed Veteran cohorts have been examined at 2-year intervals at the Baltimore VA Medical Center where surveillance testing was performed and biological samples collected. The protocol used in this work was approved by the Baltimore VAMC’s and University of Maryland School of Medicine’s IRB programs. Each participant completed an informed consent document. The University of Vermont (UVM) IRB reviewed the University of Maryland IRB approval; however, the UVM part of the studies did not require local approval because the UVM researchers had no access to patient identifiers. On the day of sampling, tubes of heparinized venous blood were sent at room temperature to the Genetic Toxicology Laboratory of the University of Vermont by overnight mail. On receipt, peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and cryopreserved in medium containing 90% FCS (HyClone, Logan, UT) and 10% DMSO. PBMCs were then stored in liquid nitrogen until tested. Blood samples were received in Vermont in groups of several each. Individual samples were then assayed randomly on cryopreserved mononuclear cells. Approximate average time from sample receipt to assay varied by year from 11.5 days in 2001, 218 days in 2003, 88 days in 2005, 98 days in 2007 to 20 days in 2009.

Determination of Urine Uranium Concentrations As a measure of DU exposure, total U concentration and the U235/ U238 isotopic ratio of 24-hr urine samples collected during the surveillance visits were measured by the Armed Forces Institute of Pathology’s (AFIP) Department of Environmental Toxicologic Pathology (Washington, DC) (now the Joint Pathology Center) as previously described [McDiarmid et al., 2007] using an Inductively Coupled Plasma-Dynamic Reaction Cell-Mass Spectrometer (ICP-DRC-MS) method developed by Ejnik et al. [2005]. Urine U (uU) concentrations were standardized on the basis of urine creatinine concentrations to account for urine dilution to obtain mg U/g creatinine [Karpas et al., 1998; McDiarmid et al., 2000]. For each participant a mean uU exposure metric was calculated by averaging their standardized uU concentrations from all their surveillance visits up to and including 2007. The distribution of the mean uU values for 77 Veterans for whom values were determined between 1993

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of clonable cells per well. A mean non-selection CE was determined from the non-selection plates; a mean selection CE was determined from the 6TG selection plates. These values were used to calculate the mutant frequency (MF) per 106 clonable cells, defined as: MF 3 1026 ¼ ðCEselection =CEnon2selection Þ 3 106

Molecular Analyses of HPRT Mutations and TCR Gene Rearrangements

Fig. 1. Calculated HPRT mutant frequencies (MF) versus calculated HPRT mutation frequencies (MutF). and 2007 is shown in Figure 1, 68 of whom were involved in this HPRT study.

Determination of Urine Concentrations of Additional Metals The 24-hr urine specimens collected for U concentration analysis were also analyzed by the JPC Biophysical Toxicology Laboratory for 12 additional metals including: aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), nickel (Ni), tungsten (W) and zinc (Zn). These metals were chosen based on their presence in analyzed embedded fragments and their potential toxicity and carcinogenicity [Gaitens et al., 2010]. For quantitative elemental measurements of urine specimens, samples were diluted with 2% nitric acid and analyzed by sector field high resolution ICP-MS. Detection limits (DL) for most of the metals ranged from 0.1 to 1 ng ml21). As with the uU results, concentrations of the addition 12 metals were standardized on the basis of urine creatinine concentrations to account for urine dilution to obtain mg of metal per g creatinine.

HPRT Mutation Assays The method for the T cell cloning assay for HPRT T-cell mutations has been described in detail [O’Neill et al., 1987]. Briefly, the PBMC containing the T-lymphocyte fraction were rapidly thawed, diluted in medium consisting of 80% RPMI-1640, 20%HL-1 and 10% fetal calf serum (FCS) and incubated overnight at 37 8C in a humidified CO2 (6%) incubator. Following this, cells were washed and diluted in RPMI1640 medium supplemented with 10% lymphokine activated killer cell supernatant, 20% HL-1, 10% FCS and 0.25 lg ml21 PHA. PBMCs were then inoculated in limiting dilutions into the wells of 96-well microtiter plates at 1–5 cells/well in growth medium (non-selection plates) or at 2 3 104 cells/well in selection medium (selection plates) consisting of growth medium plus 6-thioguanine (6TG; 1025 M). All wells also received 1 3 104 lethally irradiated (10 3 103 cGy g radiation) accessory feeder cells (TK6 lymphoblastoid cells). Wells were scored for visible colony growth at 11 and 17 days using an inverted phase-contrast microscope.

Calculation of Cloning Efficiencies (CEs) and HPRT Mutant Frequencies (MF) CEs were calculated using the Poisson relationship, CE 5 (2ln P0)/x, in which P0 5 the fraction of negative wells and x 5 the average number

Complete identification of the mutation of the HPRT gene in mutant isolates required several studies. RT-PCR was utilized to amplify cDNA for initial analysis. A 2nd round of PCR was performed if sufficient product was not obtained from the 1st round of amplification. Exon specific genomic PCR, multiplex genomic PCRs, and V(D)J TCR gene rearrangement PCR were performed when necessary to identify a specific mutation. RNA and DNA isolation, specific conditions of PCR and primers used are described in the accompanying paper [Nicklas et al., 2015]. T-cell beta (TRBi) gene rearrangements were determined from cDNA prepared from isolated RNA. TRB transcripts were reverse transcribed using oligo dT or a random primer instead of a specific primer. The specific methods are described in the accompanying paper [Nicklas et al., 2015]. HPRT and TRBi PCR products were ExoSAP-IT (USB Corporation, Cleveland, OH) treated according to manufacturer’s recommendations and DNA sequencing was performed at the University of Vermont, Vermont Cancer Center DNA Analysis Facility utilizing an Applied Biosystems 3130XL.

HPRT Mutant Frequencies (MF) Versus HPRT Mutation Frequencies (MutF) If two isolated T-cell mutants are shown to have the identical HPRT mutation at the genomic level, the question is whether they represent two independent mutations at the same site or whether they arise from in vivo cell division of one original mutation. Pre-T cells travel through the thymus, differentiating and uniquely rearranging their T-cell receptor (TCR) genes. This unique T-cell rearrangement marks a T-cell and all its clonal descendants. Therefore, if two mutants have both the same HPRT mutation and the same TCR gene rearrangement, they can be assumed to be clonal expansion of a single mutation. These “duplicate” mutants arising from clonal expansion rather than new mutation can then be removed from analysis and the mutant frequency (MF) (using all the mutants) corrected to create a true mutation frequency (MutF) (removing the duplicates and leaving only the unique mutants). This adjustment is calculated as follows: Mutant proportion ðMPÞ 5 # unique mutant isolates=ð# 6TG resistant isolatesÞ MutF ¼ MP x HPRT MF

Statistical Analysis HPRT MFs and MutFs are log-normally distributed, so analyses were based on their natural logarithms (lnMF and lnMutF). Values of lnMF at individual time points for all individuals tested were analyzed as hierarchical linear mixed models. Subject identification number was included in the regression models as a random effect to take into account correlations between repeated measurements on the same individual. Age, CE, smoking status, and urine U concentrations were included as time-varying covariates to assess their relationships with lnMF. Results from these analyses were used to adjust MFs for differences in CE by subtracting 1.70 3 (0.24 – CE) from lnMF, where 0.24 is the average CE across all assays. Each person’s overall MF was then

Environmental and Molecular Mutagenesis. DOI 10.1002/em 584

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calculated as the geometric mean of his adjusted MFs from all time points. The overall MutF was obtained by multiplying the overall MF by the mutation proportion. Relationships between average DU exposure (measured by uU concentrations) and overall lnMF and lnMutF were analyzed by linear regression, with exposure as a continuous variable, or by t tests and Kruskal–Wallis tests to determine the difference between low and high U categorical exposure groups defined using a uU cut point of 0.1 ug U/g creatine for placing individuals in the low vs. high groups.

RESULTS Description of Data Set HPRT mutations in peripheral blood T-lymphocytes were determined over an 8-year period (2001–2009) for a total of 70 Veterans evaluated in five separate studies. Each study, spaced at 2-year intervals, included 30–40 individuals. Several Veterans participated in all five studies while others in only one. A total of 186 HPRT cloning assays were performed on these 70 individuals. All study subjects were male. Results of all HPRT assays including non-selected cloning efficiencies (CE) and calculated mutant frequency (MF) values are presented in Table I, together with together with the ages of the study participants at the time of the 2001 study and smoking status across all studies (current smoker at the time of any of their assays or non-smoker at all time points). Associations of Age, Smoking, and CE With lnMF Ages in this data set ranged from 19 to 51 years as of 2001; 25 participants stated that they were current smokers at at least one time point (noted as 1 in Table I), while 43 denied smoking at any of the time points (noted as 0 in Table I), with no information from two. Current smoking at the time an HPRT assay was performed had no significant effect on MF. Non-smokers and current smokers had lnMF values of 2.667 and 2.640 (MF 3 1026 5 14.40 and 14.01), respectively (P 5 0.81). As noted in previous studies ([Robinson et al., 1994] and references within), linear regression of lnMF on CE showed a highly significant inverse association (P < 0.0001). Regression of age on lnMF also showed a highly significant but positive association (P < 0.0001). The magnitude of the age effect on lnMF was an increase of 0.03 per year, corresponding to a 3% increase in MF per year. However, age itself was significantly inversely associated with CE (P < 0.0001) and, when both age and CE are included in the regression analysis, only CE remains significant (P < 0.0001). The associations between CE, age and lnMF allow the following calculation of the expected lnMF given the other two variables. lnMFðexpectedÞ ¼ 2:48 2 1:70 X CE 1 0:015 X age (1) This compares closely to the equation for the expected MF derived from a population of healthy individuals stud-

ied more than 20 years ago [Finette et al., 1994], which was: lnMFðexpectedÞ ¼ 2:26 – 1:68 X CE 1 0:014 X age (2)

Between- and Within-Individual Variability in lnMF The relationship shown in Eq. (1) explained 11.6% of the between-individual variability in lnMF and 19.9% of the within-individual variability across time points. Estimates of between- and within-individual lnMF variances are 0.2421 and 0.1889, respectively, in this population before adjustment for CE and age. Adjustment for both CE and age gives estimates of 0.2140 and 0.1513 for the between and within-individual variances, respectively. HPRT MF Versus HPRT Mutation Frequencies (MutF) in Gulf War I Cohort Our studies of HPRT mutations in the Gulf War I Veterans included molecular analyses of the HPRT gene in 1377 mutant isolates from 62 individuals. Two-hundred eighty-two studies were done at the 2003 visit, 357 at the 2005 visit, 341 at the 2007 visit and 397 at the 2009 visit. The molecular spectrum of the HPRT mutations is described in detail in the accompanying paper [Nicklas et al., 2015]. TCR gene rearrangement analysis was performed on mutant isolates from an individual Veteran that had the same HPRT gene alteration. If the mutants had both the same HPRT alteration and the same TCR gene rearrangement, they were assumed to have originated from clonal expansion of one original founder mutation. Only one mutant isolate of the clonal set was used for MutF calculations. Results of the analyses of HPRT gene mutations in the 6TG resistant T-cell isolates along with their TCR gene rearrangement patterns are shown in Tables II and III. Table II shows the in vivo mutant clones that were discovered for each veteran over the 8-year study. An unexpected finding was that some in vivo mutant clones persisted in some individuals over the several years of study. Definitive mutations in the HPRT gene were found in 1193 mutant isolates (87% of isolates) from the 62 studied individuals, with no mutation being discovered in 184 (13% of isolates) despite extensive study of both cDNA and genomic DNA as described above. Isolates with no discoverable HPRT mutation are discussed below and described in detail in the accompanying paper [Nicklas et al., 2015]. Of the 1193 definitive HPRT mutations, 1050 (88%) were unique within an individual. Table I shows the mutation proportion and MutF calculated as described from the geometric mean of the CE adjusted MF’s for each veteran. It can be seen that the MutF was less than the MF for most individuals, but that

Age in 2001

42 37 43 32 30 32 36 42 30 30 19 30 43 41 30 32 51 33 33 40 36 39 47 33 30 28 30 33 41 32 33 43 34 24 33 32 30 31 34 32 33 35

ID #

1113 1153 1160 1232 1274 1336 1400 1415 1461 1515 1623 1836 1839 1865 2000 2027 2101 2117 2119 2200 2248 2270 2274 2333 2439 2466 2547 2647 2718 2733 2786 2897 2925 2949 3030 3041 3296 3337 3579 3618 3621 3714

1 1 0 1 0 0 1 1 0 0 1 0 1 0 0

0 0 0 0 1 0

0 0 1 1 0 0 0 0 0 1 1 0 0 1 1 1 1 1 1

Smoker (at any visit)

0.016 0.023 0.063 0.026 0.005 0.005 33.389 0.006 0.003 0.008 2.357 16.925 0.130 0.468 0.032 0.005 0.058 0.003 0.006 0.020 0.007 0.015 0.019 0.013 0.006 0.004 0.008 0.008 0.081 0.073 2.685

0.006 4.146 0.008 0.370 0.036 0.072 0.043 0.086 7.852 0.009

Dose (lg g21 creatinine) geometric mean

10.70 18.20 4.50 13.80 5.20

6.00 14.10

15.40 21.60

0.27 0.54 0.25 0.38

0.35 0.20

0.20 0.37

41.40 7.30 14.10

0.31 0.50 0.19

0.45

12.70

69.10 15.90

0.40 0.31 0.33

7.00

0.36

9.90

0.54 5.00 13.00

7.40

0.48

0.40 0.29

15.10 12.60 8.60

MF (31026) 2001

0.34 0.39 0.63

CE 2001

3.90

16.80 7.10

0.31 0.26

0.16

30.60 17.20 8.80

0.17 0.27 0.11

34.30

11.10

0.26

0.16

16.40 161.00

13.30 121.20

7.20

16.00

0.11 0.05

0.19 0.13

0.19

0.40

10.00

9.80

0.25 0.19

16.40 5.90 11.20

MF (31026) 2003

0.13 0.27 0.18

CE 2003

17.30

42.10

0.19 0.42

7.10

4.80 18.80 24.80

15.40 19.30

0.32

0.22 0.27 0.11

0.35 0.24

58.50 8.40 10.60

4.70

0.28

0.16 0.32 0.33

15.60 73.80

11.80

6.10 9.20

9.50 13.20

2.10 8.10 11.50 13.90 5.50 8.50

MF (31026) 2005

0.27 0.33

0.19

0.33 0.45

0.40 0.27

0.14 0.26 0.27 0.28 0.25 0.25

CE 2005

0.19

15.78

30.04 14.81 16.69

23.94 25.65 29.46 9.14

0.11 0.14 0.12 0.09

0.13 0.18 0.12

88.83 11.11 15.50

22.12

45.40 22.89 80.96

24.55

12.74 18.05 11.11 17.25 11.87

14.40 21.14 10.88

MF (31026) 2007

0.14 0.14 0.23

0.11

0.15 0.13 0.27

0.19

0.17 0.11 0.22 0.20 0.21

0.19 0.14 0.26

CE 2007

TABLE I. Overall HPRT Mutant and Mutation Frequencies (MF and MutF) for DU Exposed Veterans: 2003–2009

0.20

0.14 0.13

0.10 0.15 0.27

0.13

0.29

0.11 0.40 0.26 0.18

0.10

0.17 0.22

0.15

20.65

21.94 15.45

22.15 18.63 8.27

12.70

12.97

75.67 10.44 11.98 15.79

22.27

25.33 74.22

40.89

13.19 18.71 5.35

17.32 16.19 17.57 14.61 30.79

0.20 0.21 0.15 0.18 0.09 0.40 0.15 0.23

11.42

MF (31026) 2009

0.32

CE 2009 1.77 12.87 12.15 13.54 9.24 10.89 12.22 13.44 12.22 17.59 13.48 6.26 12.88 34.91 9.65 39.09 17.10 86.23 17.91 11.61 14.80 13.15 66.03 10.91 12.82 14.28 19.33 19.17 21.51 15.46 11.65 9.95 6.84 19.78 20.49 15.92 8.75 13.17 26.80 12.73 18.38 12.62

Mean adjusted MF (31026)

20 10 27 24

8 43 29 42 8 8 33 20 15 11 9 18 10 32 30 38

30

33 30 42 20 31 20 47 20 30 19 15 24 10 4 10 36 43

Total # mutants

14 7 22 20

8 22 27 33 5 5 24 13 10 8 9 14 7 25 19 27

22

19 26 32 16 26 19 40 19 25 17 12 21 8 3 8 27 7

# Unique mutants 1.000 0.576 0.867 0.762 0.800 0.839 0.950 0.894 0.950 0.833 0.895 0.800 0.875 0.800 0.750 0.800 0.750 0.163 1.000 0.733 1.000 1.000 0.512 0.931 0.786 0.625 0.625 0.727 0.650 0.667 0.727 1.000 0.778 0.700 0.781 0.633 0.711 1.000 0.700 0.700 0.815 0.833

Proportion unique

1.77 7.41 10.53 10.32 7.39 9.13 11.61 12.01 11.61 14.66 12.06 5.01 11.27 27.93 7.23 31.27 12.82 14.04 17.91 8.51 14.80 13.15 33.79 10.16 10.07 8.93 12.08 13.94 13.98 10.31 8.47 9.95 5.32 13.85 16.01 10.08 6.22 13.17 18.76 8.91 14.98 10.52

MutF using adjusted mean MF (31026)

3795 3934 3958 4102 4246 4432 4504 4716 4840 4848 4893 4960 4998 5229 5392 5494 5504 5579 5590 5648 5689 5701 5758 5768 5779 5848 5859 5860 Total#

ID #

36 33 34 38 31 37 36 44 34 37 30 48 37 35 33 43 29 35 33 35 31 32 31 35 30 34 35 32

Age in 2001

1 0 0 0 0 0 1 1 1 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1

Smoker (at any visit)

TABLE I. (continued).

1.341 0.003 0.086 37.980 0.010 2.780 0.003 0.014 2.315 0.004 0.004 0.237 0.003 0.006 0.007 0.009

2.017 0.017 0.006 0.005 0.006 0.445 0.007 0.006 0.014 0.190 0.006

Dose (lg g21 creatinine) geometric mean

14.70 9.70 27.10 7.30 9.50 10.80 10.40

4.40 9.50 39

0.30 0.18 0.39 0.38 0.29

0.38 0.29

8.90 21.70 12.20 12.10

0.30 0.28 0.31 0.51

0.46 0.32

6.90 22.00 7.40

MF (31026) 2001

0.52 0.34 0.37

CE 2001

10.50 17.60

0.10 0.07 32

3.90

2.30 27.30

0.28 0.11 0.09

19.50

0.10

33.00 11.20

19.60

0.21 0.17 0.17

26.30

16.70 5.80

0.16 0.18

0.07

11.20

MF (31026) 2003

0.22

CE 2003

5.50 37

19.20 11.20 7.20

0.28 0.14 0.47 0.25

13.20

4.50 27.60

15.40

23.80

9.50

6.20

5.90

MF (31026) 2005

0.27

0.35 0.37

0.20

0.28

0.32

0.17

0.34

CE 2005

0.24 0.08

38

8.00 16.70

19.66 18.61 10.07 10.86 18.91

16.73 23.72

0.20 0.15

0.14 0.14 0.17 0.19 0.15

18.50 19.19

33.62

34.50

8.86

MF (31026) 2007

0.12 0.17

0.08

0.07

0.23

CE 2007

0.19

0.19

0.24 0.19 0.12 0.30

0.23 0.10

0.12

0.20

0.25

0.18 0.26

0.16

0.26

CE 2009

40

9.06

24.15

10.66 15.77 27.12 9.65

12.59 24.92

19.51

26.58

13.44

12.59 13.54

25.07

7.27

MF (31026) 2009 8.88 26.08 14.33 5.37 17.14 14.03 9.86 17.70 13.74 19.15 21.43 14.96 21.23 10.51 13.87 19.23 6.59 6.46 18.96 18.65 8.54 9.92 16.20 21.39 9.45 8.75 9.78 7.61 70

Mean adjusted MF (31026)

16 42 18 23 3 10 23 1 40 12 4 1377

19 7 40 10 18 50

41

21 9 18 10

43

Total # mutants

16 35 15 18 3 9 21 1 29 10 3 1050

12 7 21 9 16 39

33

20 5 13 8

41

# Unique mutants

0.953 1.000 0.952 0.556 0.722 0.800 1.000 0.805 1.000 1.000 0.632 1.000 0.525 0.900 0.889 0.780 1.000 1.000 0.833 0.833 0.783 1.000 0.900 0.913 1.000 0.725 0.833 0.750

Proportion unique

8.47 26.08 13.65 2.98 12.38 11.23 9.86 14.25 13.74 19.15 13.54 14.96 11.15 9.46 12.33 15.00 6.59 6.46 15.80 15.54 6.68 9.92 14.58 19.53 9.45 6.34 8.15 5.70

MutF using adjusted mean MF (31026)

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TABLE II. HPRT and TCR Rearrangement Defined In Vivo Mutant Clones Number of mutants in the clone in year

Number of mutants in the clone in year

Veteran—clone

2003

2005

2007

2009

Total all years

1136 1153 1232 1232 1232 1274 1415 1515 1515 1623 1839 2027 2101 2101 2117 2117 2200 2274 2274 2274 2274 2274 2333 2439 2439 2439 2466 2547

1 5 2 0 0 ND 1 1 0 ND 0 ND 1 0 3 3 ND 0 3 1 0 1 ND 0 1 1 ND ND

1 0 0 0 0 2 0 ND ND ND 0 ND 1 0 8 4 0 2 1 0 2 2 0 2 1 0 ND 0

ND 1 0 1 2 ND 0 1 1 0 2 2 0 1 2 1 2 0 2 1 1 0 0 0 0 1 ND 3

0 6 0 1 0 1 2 0 1 2 ND ND 0 2 1 7 1 1 3 0 1 1 2 0 1 0 2 ND

2 12 2 2 2 3 3 2 2 2 2 2 2 3 14 15 3 3 9 2 4 4 2 2 3 2 2 3

clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone clone

A A A B C A A A B A A A A B A B A A B C D E A A B C A A

Veteran—clone 2647 2647 2647 2718 2718 2733 2733 3030 3030 3041 3041 3296 3579 3795 4893 4998 5392 5494 5494 5590 5590 5648 5648 5689 5689 5689 5768 5848 5848

clone A clone B clone C clone A clone B clone A clone B clone A clone B clone A clone B clone A clone A clone A clone A clone A clone A clone A clone B clone A clone B clone A clone B clone A clone A clone B clone A clone A clone B

2003

2005

2007

2009

Total all years

0 2 1 ND ND 4 1 ND ND 2 0 2 ND 1 3 6 ND 2 1 0 0 ND ND ND ND ND ND 0 0

1 0 0 2 2 ND ND 1 1 ND ND 0 2 0 ND 0 ND 1 0 2 0 ND ND 2 1 1 1 2 0

3 0 0 1 2 0 1 2 1 1 3 0 ND 0 ND 4 1 0 1 0 2 2 0 0 1 0 ND 0 1

1 0 2 ND ND ND ND 0 1 0 0 0 0 1 5 1 1 0 0 0 0 1 2 0 0 1 1 0 1

5 2 3 3 4 4 2 3 3 3 3 2 2 2 8 11 2 3 2 2 2 3 2 2 2 2 2 2 2

ND—veteran not studied in that year.

this difference was substantial for only a few. The relationship between MFs and MutFs is shown graphically in Figure 2. Impact of DU Exposure In this surveillance program, systemic DU levels were estimated by uU excretion for each Veteran at each follow-up visit as calculated using each individual’s uU values collected from the surveillance visits in years from 1993 to 2007. The numbers of individual estimates therefore, varied from five to one. Figure 1 portrays the distribution of mean excretion values for 77 Veterans as determined between 1993 and 2007 visits, 68 of whom were included in the HPRT studies described here. Of the 77 Veterans with values depicted in Figure 1, 23% (n 5 18) had shown evidence of retained embedded fragments when evaluated by an X-ray plain film skeletal survey [Hooper et al., 1999; Squibb and McDiarmid, 2006]. To assess effects of low versus high U exposure, a uU value of 0.10 mg U/g creatinine was established as a cut off level to separate participants into two groups; this

cut-off level is slightly greater than twofold the 95th percentile uU concentration for adults (0.043 mg U/g creatinine) as determined during the 2001–2002 NHANES survey [NHANES, 2003]. Veterans in the high uU group had a mean uU value of 7.694 6 3.707 mg U/g creatinine, two orders of magnitude higher than the mean uU value of 0.012 6 0.003 mg U/g creatinine in the low group. As shown in Figure 1, all Veterans except for one with uU values above this cut off level showed retained shrapnel fragments. By contrast, only two Veterans with uU values below this cut off level showed retained shrapnel fragments. As excretion values over multiple visits were remarkably similar in Veterans when repeat measurements were available, the mean excretion value was calculated for each Veteran and used for statistical analyses of associations of exposure with overall MF and MutF. Association of HPRT MFs and MutFs With Exposure to DU Comparison of the overall lnMF (logarithm of a person’s average MF 3 1026 over all available time points) in subjects with low and high average DU

Environmental and Molecular Mutagenesis. DOI 10.1002/em 588

Albertini et al.

TABLE III. HPRT Mutant Isolates Per Veteran Studied Between 2003 and 2009 No. of mutants analyzed

Veteran 1153 1160 1232 1274 1336 1400 1415 1461 1515 1623 1836 1839 1865 2000 2027 2101 2117 2200 2270 2274 2333 2439 2466 2547 2647 2718 2733 2786 2897 2925 2949 3030 3041 3296 3579 3618 3621 3714 3795 3958 4102 4246 4432 4716 4893 4960 4998 5229 5392 5494 5579 5590 5648 5689 5701 5758

Mutants without determined mutation

2003

2005

2007

2009

Total

2003

2005

2007

2009

Total

Total # with discoverable mutation

11 11 11 2 11 2 11 2 10 2 2 6 2 2 2 9 9 2 8 13 2 12 2 2 10 2 10 11 9 2 2 2 10 9 2 2 2 4 10 11 6 2 2 6 9 2 10 10 2 10 3 12 2 2 2 2

2 10 10 10 10 2 16 10 2 2 5 10 2 4 2 11 14 9 2 10 9 10 2 2 5 10 2 2 2 8 10 10 2 10 10 2 27 13 3 2 2 15 2 2 10 2 2 20 3 10 2 10 2 2

10 9 11 2 2 10 10 10 10 9 2 8 2 2 10 6 10 10 2 10 10 10 2 8 8 10 5 2 2 2 2 10 10 9 2 2 2 10 10 2 2 9 2 10 2 7 10 2 10 10 2 10 8 4 3 10

10 2 10 10 10 10 10 2 10 10 10 2 10 2 2 10 10 11 2 10 10 10 8 2 10 2 2 2 2 10 2 12 10 10 10 10 2 10 10 10 2 9 10 10 10 2 10 2 8 10 10 10 10 9 2 2

33 30 42 20 31 20 47 20 30 19 15 24 10 4 10 36 43 30 8 43 29 42 8 8 33 20 15 11 9 18 10 32 30 38 20 10 27 24 43 21 9 18 10 41 19 7 40 10 18 50 16 42 18 23 3 10

2 4 2 2 3 2 1 2 0 2 2 2 2 2 2 4 1

0 0 0 0 1 2 3 1 2 2 1 0 2 1 2 1 1 1 2 0 0 0 2 2 1 1 2 2 2 1 3 0 2 2 1 2 5 2 0 2 0 2 2 4 2 0 v 2 2 0 0 2 0 2 0

0 0 1 2 2 1 0 2 1 1 2 0 2 2 1 0 7 0 2 2 0 1 2 1 0 1 0 2 2 2 2 1 1 1 2 2 2 0 0 2 2 2 2 1 2 0 2 2 1 1 2 0 0 0 0 1

1 2 4 2 0 0 1 0 2 0 2 2 2 2 2 1 0 5 2 0 1 2 2 2 0 2 2 0 2 3 2 2 2 4 4 3 2 0 0 0 2 3 2 1 0 2 4 2 0 2 0 2 0 2 2 2

3 4 7 2 4 1 5 1 3 1 3 2 2 1 1 6 9 6 0 4 1 5 2 1 2 2 0 3 0 4 3 3 7 10 5 3 5 4 1 1 4 5 2 8 0 0 9 1 1 8 0 3 0 2 0 1

30 26 35 18 27 19 42 19 27 18 12 22 8 3 9 30 34 24 8 39 28 37 6 7 31 18 15 8 9 14 7 29 23 28 15 7 22 20 42 20 5 13 8 33 19 7 31 9 17 42 16 39 18 21 3 9

0 2 2 2 2 2 1 2 0 3 0 2 2 2 4 3 2 2 2 4 1 1 4 2 2 2 0 2 3 1 2 3 0 1 2 2 2

Clones with the same HPRT mutation and same TCR rearrangement (size of clone) 12 2,2,2 3 2 3 2,2 2 2

2 2,3 14,15 3 3,9,2,4,4 2 2,3,2 2 3 5,2,3 3,4 4,2,2

3,3 3,3 2 2

2

8 11 2 3,2 3,2,2 3,2 2,2,2

Total with different HPRT mutations or different TCR rearrangement 19 26 32 16 26 19 40 19 25 17 12 21 8 3 8 27 7 22 8 22 27 33 5 5 24 13 10 8 9 14 7 25 19 27 14 7 22 20 41 20 5 13 8 33 12 7 21 9 16 39 16 35 15 18 3 9

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT MF in DU Exposed Veterans

589

TABLE III. (continued). No. of mutants analyzed

Mutants without determined mutation

Veteran

2003

2005

2007

2009

Total

2003

2005

2007

2009

Total

Total # with discoverable mutation

5768 5779 5848 5859 5860 Total

2 2 5 5 2 282

13 1 15 2 4 357

2 2 10 7 2 341

10 2 10 2 2 397

23 1 40 12 4 1377

2 2 3 2 2 59

1 0 0 2 1 32

2 2 2 0 2 30

0 2 4 2 2 63

1 0 9 2 1 184

22 1 31 10 3 1193

Clones with the same HPRT mutation and same TCR rearrangement (size of clone) 2 2,2

Total with different HPRT mutations or different TCR rearrangement 21 1 29 10 3 1050

Fig. 2. Mean urine uranium values used to estimate systemic exposure to DU were calculated using each veterans’ urine U values collected from earlier surveillance visits between 2001 and 2007. Symbols show which veterans have embedded DU fragments and/or isotopic ratios that are consistent with depleted U in urine samples. Lines on the graph show for comparative purposes urine uranium concentrations observed in other studies [ICRP, 1974; Thun et al., 1985; USDHHS-CDC, 2012].

exposures (< 0.10 lg g21 creatinine versus $ 0.10 lg g21 creatinine) showed no significant difference (P 5 0.28). The mean overall lnMF values were 2.585 (MF 3 1026 5 13.3) for individuals with low average exposures and 2.750 (MF 3 1026 5 15.6) for those with high exposures. The corresponding lnMutF values were 2.376 (MutF 3 1026 5 10.8) and 2.440 (MutF 3 1026 5 11.5) for the low and high exposure groups, respectively, and also did not differ significantly (P 5 0.64). However, when average DU exposure was analyzed as a continuous variable in regression analyses, overall lnMF increased by 0.032 (a 3.26% increase in MF 3 1026) per each 1 unit increase in average DU exposure (P < 0.001) while lnMutF’s association with DU exposure remained not significant (P 5 0.18). As the distribution of DU exposure doses was highly skewed, it is most appropriate to also express doses as the natural logarithm of dose for these regressions [Draper and Smith, 1998]. When dose was expressed as ln dose, the associations between DU exposure (uU) and

Fig. 3. HPRT MF or MutF as a function of ln mean urine uranium excretion as a measure of mean DU exposure. (a) portrays geometric mean MF as a function of exposure; (b) shows MutF as a function of exposure.

lnMF was not as highly significant (P 5 0.043) (Fig. 3A). Importantly, there was no significant association between DU exposure and lnMutF (Fig. 3B). As our earlier papers only dealt with HPRT MF values, the relationship of dose and this endpoint was examined further. The significant association between DU exposure and lnMF (P 5 0.043; Fig. 3A) was attributable to three individuals with average DU exposures over 10 lg g21 creatinine. To demonstrate the influence of these three

Environmental and Molecular Mutagenesis. DOI 10.1002/em 590

Albertini et al.

individuals on this exposure-response relationship, they were excluded from the regression analyses. When this was done, there was no significant relationship between overall lnMF and DU exposure regardless of how exposure was expressed. For lnMF similar results were obtained when mutant frequencies and DU exposures at differing time points were analyzed as repeated measures. A significant association was observed only when the three individuals with very high DU exposures were included in the analysis and DU dose was not ln transformed. This confirms our contention that our original suggestions of a DU effect on HPRT MFs was due entirely to these three individuals. The association between HPRT MF and uU concentration was also tested by repeat measures analysis of lnMF data from all Veterans from all time points using Eqs. (1) and (2) above to calculate residual MF values (observed– expected). When high and low exposure groups were compared, there was no significant difference between the high and low groups using either equation. However, residual ln MFs were significantly greater than zero in both the high and low exposure groups when using the older Eq. (2) derived from populations not exposed to Iraq battlefield conditions. However, the more important point shown by this current analysis is that MFs do not adequately reflect HPRT MutFs, which are the relevant units of mutagenic effect. For overall lnMutF, there was no significant relationship to DU exposure over the entire Veteran cohort with no exclusions, regardless of how DU exposure is expressed.

reported by clinical laboratories, were used [McDiarmid et al., 2013]. Values reported as mg per 24 hr and mg per L were converted to mg per g creatinine, assuming an average urine volume of 1.2 L per 24 hr and an average creatinine output of 1.2 g creatinine per 24 hr [ACGIH, 1991]. Of the 12 additional metals measured, only cobalt showed a possible association with mutant frequency. The overall lnMF for the six individuals with elevated cobalt excretion values was 3.394 (MF 3 1026 5 29.8) while the lnMF for the 30 individuals not showing elevated excretion values was 2.595 (MF 3 1026 5 13.4). Converting to MutF revealed that the ln MutF for the six individuals with elevated excretion values was 2.839 (MF 3 1026 5 17.1) while the lnMutF for the 30 individuals not showing elevated excretion values was 2.359 (MF 3 1026 5 10.6 3 1026). These difference between the two groups were statistically significant by Kruskal–Wallace test (P 5 0.020 for lnMF and P 5 0.024 for ln MutF). Statistical Power Based on the variance in lnMF in the low and high dose groups after adjustment for age and CE observed in this study, the repeated measures analysis of individual MF values had 86% power to detect a 10% difference in lnMF (1.11 fold increase in MF). For the overall MF and MutF analyses, the study had 88% power to detect differences of 15% (1.16 fold increase in MF or MutF). DISCUSSION

Association of HPRT MFs and MutFs With Exposures to Other Metals The urine samples taken for uU determinations during a 2011 visit were analyzed for concentrations of other metals (aluminum (Al), arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), nickel (Ni), tungsten (W) and zinc (Zn), in addition to U (HPRT mutations were not studied in this visit). However, 36 of the Veterans studied for additional metals had been studied for HPRT mutations on one or more visits between 2001 and 2009. Assuming that the metal measurements would be stable over the earlier time points (as were the DU measurements) allowed an analysis of possible associations between these metal exposures and the HPRT mutations determined during the earlier visits. Urine concentrations of these additional metals, as for U, were determined to be either above or below an upper limit reference value established for each metal from unexposed populations (data not shown). When available, the 2003–2004 NHANES 95th percentile creatinine adjusted value was used as the reference value [USDHHS-CDC, 2012]; otherwise, other reported reference values, such as the upper values

Our purpose in initiating these studies of DU exposed Veterans was to assess the mutagenicity of this radioactive, heavy metal exposure, as measured by somatic gene mutations in vivo. Previous, contemporaneous and later studies had either failed to find or inconsistently found cytogenetic changes associated with DU exposures [McDiarmid et al., 2001, 2004, 2006, 2007, 2009, 2011]. Our initial findings came as a surprise then when we measured clearly elevated HPRT MF values in the three individuals with the highest measured exposures, as reflected by uU excretion levels (Veterans 2117, 2274, 5494) [McDiarmid et al., 2004]. Although wary of conclusions based on three individuals, it was possible to construct dose-response scenarios that assumed a threshold, i.e. non-linear dose–response relationship between DU and gene mutations. Although these initial results clearly demanded repetition, we continued to find increased HPRT MFs in these three individuals on repeat studies over years, indicating at least that the elevated MF values were real [McDiarmid et al., 2004, 2006, 2007, 2009]. These three values influenced all subsequent dose-response analyses. Unfortunately, our results raised some concern among

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT MF in DU Exposed Veterans

Gulf War Veterans [Evans, 2004]. Our results came at a time when other illnesses of uncertain cause were being found in armed forces personnel who served during the first Gulf War. It was only after we had accumulated sufficient mutant isolates from repeat HPRT cloning assays performed over an extended period of time that we were able to do complete molecular analyses of large numbers of recovered mutant colonies. These analyses included extensive workup of the mutations themselves as well as determinations of TCR gene rearrangements in the isolates. As described by Albertini and coworkers, combined analyses of HPRT mutations and TCR gene rearrangements provides an unambiguous definition of in vivo clonality of mutant cells ([Albertini, 2001] and references therein). As presented here, such analyses showed that the elevated HPRT MF values in the three individuals in question were due to in vivo clonal amplifications of mutant cells and not to an increase in mutations. While the vast majority of T-cells are quiescent in the G0 stage of the cell cycle at any given time, T-cells are stimulated to divide and proliferate by antigenic or other stimuli. It appears that somatic mutations arise preferentially in the dividing rather than in the quiescent fraction of the in vivo T-cell population ([Albertini, 2001] and references therein). Thus, a number of the mutants observed are the result of cell division, not newly induced mutations, whether spontaneous resulting from background endogenous DNA damage or from exposure to exogenous mutagens, and can be a considerable proportion of the mutants in some individuals. In T-lymphocytes in vivo therefore, it will be the MutF and not the MF that reflects the frequency of mutations. The former is the actual event induced by background DNA damage or by a mutagenic exposure. The MF, to the extent that it differs from the MutF, is a reflection of in vivo mutant cell proliferation after the mutational event. The results presented here show that a continuous DU exposure, such as that resulting from embedded DU containing shrapnel, does not induce HPRT mutations in individuals so exposed. The length of time since initial exposures to DU is not a factor in these veterans as the embedded DU fragment provides a constant source of exposure. Mention should be made of the very short range of alpha emissions from radioactive isotopes such as uranium and how this must influence interpretation of mutagenicity data in T-lymphocytes. It is expected that only cells in almost immediate contact with the retained radioactive particles in these Veterans will receive radiation exposure. However, we can expect that the T-cells obtained from venous blood will contain some that have been in contact with the tissues containing these particles for two reasons: (i) T-cells circulate widely throughout the body, re-entering the circulation after having been in the tissues and, (ii) any inflammation in the shrapnel con-

591

taining tissues will be enriched for T-cells, which then recirculate. Furthermore, many of these T-cells are long lived shown by several-year persistence of some mutant clones, so there has been ample time for cells to circulate to the affected tissues. These are our assumptions. We can only state that the circulating T-cells we studied manifested no increases in HPRT mutations. The caveat must be that the dose of radiation may have been too low for detection with the dilution effect of unexposed T-cells that have never been in close proximity with the embedded particles accentuating this. Although these results resolve the question of detectable induced mutations at these levels and forms of DU exposure, they raise another question. The persistence of in vivo mutant clones over several years at levels that dominate mutant isolate recovery in some individuals was unexpected. This phenomenon was seen in the three individuals (Veterans 2117, 2274, 5494) with elevated MF and uU excretion values but also in others (Tables II and III). Although this clearly shows that mutant cells do proliferate in vivo¸ it begs the question of why. Although the HPRT mutations in these cells may not have hindered their replication, there is presumably some stimulus of these particular clones to proliferate—immunological or other. Our only answers at present are in the negative. Several veterans have been tested by skin tests for allergic response to metals included in the shrapnel that they carry (data not shown). There is no relationship between clonality and allergic responses. There is no association between clonality and DU or the other metal exposures measured. One hypothesis was that these clonal expansions reflected inflammation around the site of shrapnel, which may have also induced the HPRT mutations in these clones, but analysis of PET-CT findings in a sample of the veterans did not support this (data not shown). At present we must entertain the possibility that this is a general phenomenon among HPRT mutant T-cells not recognized earlier because extensive longitudinal studies such as the one reported here had not been performed. What are the implications of mutant T-cell clonality for interpreting HPRT MF data in human population studies? The relationship between MFs and MutFs (Fig. 2) shows severe dissociations only for MF values above ;40 3 1026 and, of course, then not always. This is the same conclusion that we came to several years ago using an entirely different data set [O’Neill et al., 1994]. Certainly, elevated MF may well indicate elevated MutFs, which is the basic assumption underlying mutagenicity monitoring. Furthermore, it is expected that clonality among T-cells will occur equally in test (putatively mutagen exposed) and control populations. However, that was not the case here. As in our study, a small number of outliers should be a signal for caution. In such cases, outliers may require analyses at the molecular level before conclusions are reached.

Environmental and Molecular Mutagenesis. DOI 10.1002/em 592

Albertini et al.

While requiring some increase in effort for studies aimed at mutagenicity monitoring, clonality among mutant T-cells and its persistence over time present a unique population for using HPRT mutations in these cells as mechanistic probes. This area has been discussed in detail in an earlier publication [Albertini, 2001]. Our finding that no changes in the HPRT gene were detected in 14% of the mutant isolates despite an extensive search in each requires some comment. Amplification of HPRT from material remaining from feeder cells, though dead, is ruled out by the fact that our feeder TK6 cells have a complete deletion of the HPRT gene although some HPRT mRNA may have remained from the wild-type T-cells that were killed by 6TK in the cloning assay. Mutants without RNA messages as reflected by absence of RT-PCR products but with wild-type HPRT as reflected in genomic DNA might have resulted from translocations within that separate parts of the HPRT gene or from methylation or other forms of gene silencing. Methylation of HPRT with resultant acquisition of 6TG resistance in proliferating T-cells in vitro has been reported [Gabdoulkhakova et al., 2007]. Operator or other DNA changes in portions of the regulator apparatus are also possibilities. The HPRT mutational spectrum reflected in this large collection of mutant isolates is described and discussed in detail in the accompanying paper [Nicklas et al., 2015]. Our addition of isolates lacking demonstrable mutations of the gene in the total TGresistant isolates is justified because many of these isolates probably have real although undefined HPRT mutations and, in any event, all are included in conventional MF calculations. It was gratifying to see that the influence of nonselective CEs and age in predicting MFs in the Veteran population is approximately the same as it was for an entirely different population comprised of several age groups determined previously [Finette et al., 1994]. The observation of elevations in residual ln MFs in both the low and high uU veterans (greater than zero) when using the previously determined equation could suggest some mutagenic exposures under the conditions experienced by these soldiers but not by earlier population. However, as these are two very different populations, speculation as to the reasons for higher mutant frequencies in the soldiers is probably not warranted. Similarly, the hint of a marginal significance for the elevation of HPRT avg MFs in the group of soldiers more heavily exposed to cobalt compared to those with lesser exposure is again a finding based on small numbers of individuals. Furthermore, as for the analyses of DU exposure-HPRT association, conversion to avg MutFs reveals no significant association between the mutations and the exposures. In summary, the primary purpose of this report is to demonstrate unequivocally that DU in the form and

exposure levels experienced by these Veterans of the first Gulf War did not induce HPRT mutations in Tcells in vivo at levels above background. This demonstration refutes our earlier suggestion that such exposures may have been mutagenic at the gene level. This earlier suggestion was due to the influence of outlier subjects in whom there was in vivo amplifications of in vivo mutant clones that lead to greatly overestimated underlying mutation frequencies (MutFs). Our error therefore, was in basing our association with DU exposure on HPRT Mfs rather than on HPRT MutFs, which are the relevant units of mutation. The secondary purpose of this report is the characterization of the HPRT assay performance in a human population studied over time. A companion paper describes the molecular mutational spectrum of the mutant colonies recovered from the mutation assays [Nicklas et al., 2015]. An unexpected finding reported here is the persistence of mutant clones in vivo in apparently normal individuals, spanning in some cases the several years of study. Although this indicates caution in interpreting purely quantitative studies for exposures at a certain moment in time, especially results dependent on small numbers of outliers, it does highlight a phenomenon that may provide useful material for exploiting HPRT mutations in vivo for studies of T-cell kinetics. ACKNOWLEDGMENTS The authors thank the technical support from the clinical laboratory and patient care personnel at the Veterans Affairs (VA) Medical Center in Baltimore, the staff and administration of the General Clinical Research Center at the University of Maryland Medical Systems Hospital, and the Depleted Uranium Follow-up Program administrative staff for their invaluable assistance. The automated DNA sequencing was performed in the VT Cancer Center DNA Analysis Facility (special thanks to Mary Lou Shane and Jessica Hoffman). AUTHOR CONTRIBUTIONS RJA designed the studies, interpreted the data and created the figures. JAN performed the molecular analysis of the HPRT mutations and the TCR analyses. PMV performed all of the statistical analysis. EWC performed the HPRT cloning assays and collated the MF data. MAM, as Director of the VA DU Follow Up Program, designed the basic DU Health Surveillance Program that served as the basis for this study. SME organized and handled day-today logistics and data collection for the Health Surveillance visits. KSS worked with staff at the Joint Pathology Center (JPC) on the analysis of urine DU and other metal urine concentrations. PWG conducted data analysis for the Health Surveillance visits. All authors provided critical review and approved the final manuscript. None of

Environmental and Molecular Mutagenesis. DOI 10.1002/em HPRT MF in DU Exposed Veterans

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Accepted by— J. Fuscoe