Health surveillance of Gulf War I veterans exposed to depleted uranium: updating the cohort.

A cohort of seventy-four 1991 Gulf War soldiers with known exposure to depleted uranium (DU) resulting from their involvement in friendly-fire incidents with DU munitions is being followed by the Baltimore Veterans Affairs Medical Center. Biennial medical surveillance visits designed to identify uranium-related changes in health have been conducted since 1993. On-going systemic exposure to DU in veterans with embedded metal fragments is indicated by elevated urine uranium (U) excretion at concentrations up to 1,000-fold higher than that seen in the normal population. Health outcome results from the subcohort of this group of veterans attending the 2005 surveillance visit were examined based on two measures of U exposure. As in previous years, current U exposure is measured by determining urine U concentration at the time of their surveillance visit. A cumulative measure of U exposure was also calculated based on each veteran's past urine U concentrations since first exposure in 1991. Using either exposure metric, results continued to show no evidence of clinically significant DU-related health effects. Urine concentrations of retinol binding protein (RBP), a biomarker of renal proximal tubule function, were not significantly different between the low vs. high U groups based on either the current or cumulative exposure metric. Continued evidence of a weak genotoxic effect from the on-going DU exposure as measured at the HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus and suggested by the fluorescent in-situ hybridization (FISH) results in peripheral blood recommends the need for continued surveillance of this population.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

Analysis for loss of heterozygosity (LOH) of p53 allele in tumors derived from p53+/- and CD-1 mice following repeated subcutaneous injections of solutions containing antioxidants.

Genomic DNA was isolated from subcutaneous masses observed in CD-1 and p53+/- heterozygous mice during the course of carcinogenicity studies in the vehicle control groups. These masses resulted after daily subcutaneous injection of an antioxidant vehicle with a pH adjusted to 3-4. The vehicle was 1.0% ascorbic acid plus 0.05% sodium metabisulfite in 0.75% saline in a dosing volume of 10 ml/kg/day. These masses were first palpable after 13 and 37 weeks of dosing among p53+/- and CD-1 mice, respectively. By week 26, the incidence of these masses was 89% and 80% in male and female p53+/- mice, respectively (n = 15 mice/sex) and was 0% in both male and female CD-1 mice (n = 60 mice/sex). These masses originated from panniculus carnosus muscle. Histopathological examination of the p53+/- mouse masses indicated the tumors to be sarcomas of spindle-cell origin. The histopathological examination of the masses in the CD-1 mice revealed fibrosarcomas. Five mice/sex/strain were randomly selected from a pool of mice that developed these masses in the course of the two studies. Frozen tissues from these masses were used to examine the DNA for loss of the functional p53 allele in the p53+/- mice (i.e., loss of heterozygosity, or LOH) or for loss of one of the alleles in the wild type (p53+/+) CD-1 mice by the polymerase chain reaction (PCR) technique. Loss of the functional allele was observed only in the tumor from one p53+/- male mouse. These results support a nongenotoxic mechanism for these injection site masses.

Surveillance of depleted uranium exposed Gulf War veterans: health effects observed in an enlarged "friendly fire" cohort.

To determine clinical health effects in a small group of US Gulf War veterans (n = 50) who were victims of depleted uranium (DU) "friendly fire," we performed periodic medical surveillance examinations. We obtained urine uranium determinations, clinical laboratory values, reproductive health measures, neurocognitive assessments, and genotoxicity measures. DU-exposed Gulf War veterans with retained metal shrapnel fragments were excreting elevated levels of urine uranium 8 years after their first exposure (range, 0.018 to 39.1 micrograms/g creatinine for DU-exposed Gulf War veterans with retained fragments vs 0.002 to 0.231 microgram/g creatinine in DU exposed but without fragments). The persistence of the elevated urine uranium suggests ongoing mobilization from the DU fragments and results in chronic systemic exposure. Clinical laboratory outcomes, including renal functioning, were essentially normal. Neurocognitive measures showing subtle differences between high and low uranium exposure groups, seen previously, have since diminished. Sister chromatid exchange frequency, a measure of mutation in peripheral lymphocytes, was related to urine uranium level (6.35 sister chromatid exchanges/cell in the high uranium exposure group vs 5.52 sister chromatid exchanges/cell in the low uranium exposure group; P = 0.03). Observed health effects were related to subtle but biologically plausible perturbations in central nervous system function and a general measure of mutagen exposure. The findings related to uranium's chemical rather than radiologic toxicity. Observations in this group of veterans prompt speculation about the health effects of DU in other exposure scenarios.

Lack of mutagenic activity of 1,6-hexamethylene diisocyanate.

1,6-Hexamethylene diisocyanate (HDI) is an aliphatic diisocyanate used in the manufacture of higher molecular weight biuret and trimer polyisocyanate resins. These resins are commonly used in polyurethane paints, resulting in potential occupational, and to a lesser extent consumer exposures. Because some isocyanates have been reported to be mutagenic, HDI was tested in the bacterial reverse mutation assay (Ames test), CHO/HGPRT gene mutation assay, and in the mouse micronucleus test, using vapor-phase exposures. Although indicators of toxicity were observed in each test, HDI did not induce mutagenic or clastogenic effects in any of the three assays.

Use of transgenic animals for carcinogenicity testing: considerations and implications for risk assessment.

Advances in genetic engineering have created opportunities for improved understanding of the molecular basis of carcinogenesis. Through selective introduction, activation, and inactivation of specific genes, investigators can produce mice of unique genotypes and phenotypes that afford insights into the events and mechanisms responsible for tumor formation. It has been suggested that such animals might be used for routine testing of chemicals to determine their carcinogenic potential because the animals may be mechanistically relevant for understanding and predicting the human response to exposure to the chemical being tested. Before transgenic and knockout mice can be used as an adjunct or alternative to the conventional 2-year rodent bioassay, information related to the animal line to be used, study design, and data analysis and interpretation must be carefully considered. Here, we identify and review such information relative to Tg.AC and rasH2 transgenic mice and p53+/- and XPA-/- knockout mice, all of which have been proposed for use in chemical carcinogenicity testing. In addition, the implications of findings of tumors in transgenic and knockout animals when exposed to chemicals is discussed in the context of human health risk assessment.

Health effects of depleted uranium on exposed Gulf War veterans.

A small group of Gulf War veterans possess retained fragments of depleted uranium (DU) shrapnel, the long-term health consequences of which are undetermined. We evaluated the clinical health effects of DU exposure in Gulf War veterans compared with nonexposed Gulf War veterans. History and follow-up medical examination were performed on 29 exposed veterans and 38 nonexposed veterans. Outcome measures employed were urinary uranium determinations, clinical laboratory values, and psychiatric and neurocognitive assessment. DU-exposed Gulf War veterans with retained metal shrapnel fragments are excreting elevated levels of urinary uranium 7 years after first exposure (range 0.01-30.7 microg/g creatinine vs 0.01- 0.05 microg/g creatinine in the nonexposed). The persistence of the elevated urine uranium suggests on-going mobilization from a storage depot which results in a chronic systemic exposure. Adverse effects in the kidney, a presumed target organ, are not present at this time, though other effects are observed. Neurocognitive examinations demonstrated a statistical relationship between urine uranium levels and lowered performance on computerized tests assessing performance efficiency. Elevated urinary uranium was statistically related to a high prolactin level (>1.6 ng/ml; P=0.04). More than 7 years after first exposure, DU-exposed Gulf War veterans with retained metal fragments continue to excrete elevated concentrations of urinary uranium. Effects related to this are subtle perturbations in the reproductive and central nervous systems.

Biomonitoring of United States Army soldiers serving in Kuwait in 1991.

Biomarkers of polycyclic aromatic hydrocarbon (PAH) exposure and genetic biomarkers of potential cancer susceptibility were determined in a group of United States Army soldiers who were deployed to Kuwait and Saudi Arabia in 1991 in the aftermath of the Persian Gulf War. Because hundreds of oil well fires were still burning, there was concern that ground troops stationed in Kuwait might be exposed to high levels of PAHs and other toxicants. The United States Army Environmental Hygiene Agency monitored air and soil for ambient PAHs. In addition, a group of 61 soldiers was involved in the biomonitoring study reported here. These soldiers kept diaries of daily activities and provided blood and urine samples in Germany (June) before deployment to Kuwait, after 8 weeks in Kuwait (August), and 1 month after the return to Germany (October). Here we present data for PAH-DNA adducts measured by immunoassay in blood cell DNA samples obtained at all three sampling times from 22 soldiers and bulky aromatic adducts measured by 32P-postlabeling in blood cell DNA samples from 20 of the same soldiers. Urinary 1-hydroxypyrene-glucuronide levels were determined by synchronous fluorescence spectrometry in a matched set of samples from 33 soldiers. Contrary to expectations, environmental monitoring showed low ambient PAH levels in the areas where these soldiers were working in Kuwait. For both DNA adduct assays, levels were the lowest in Kuwait in August and increased significantly after the soldiers returned to Germany (October). Urinary 1-hydroxypyrene-glucuronide levels were also lowest in Kuwait and highest in Germany, but the differences were not statistically significant. The PAH-exposure biomarker levels were not significantly influenced by polymorphic variations of CYP1A1 (MspI) and glutathione S-transferases M1 and T1. Overall, the data suggest that this group of soldiers was not exposed to elevated levels of PAHs while deployed in Kuwait.

Evaluation of potential genotoxicity of pulsed electric and electromagnetic fields used for bone growth stimulation.

Medical devices emitting pulsed electric and electromagnetic fields have been found to be effective for a number of clinical applications including stimulation of bone and tissue growth. To determine whether pulsed fields of the type used in these clinical applications present a mutagenic hazard, electric and electromagnetic fields at two exposure levels were tested in the Ames test, CHO cell chromosomal aberration assay, BALB/3T3 cell transformation assay and unscheduled DNA synthesis assay in primary rat hepatocytes. For both field types, initial and independent repeat studies were performed for each assay at both clinical and supra clinical doses. In all assays, the results show a lack of cytotoxic, transforming and mutagenic activity. The data suggest that pulsed electric and electromagnetic fields of the type and dose levels used in bone growth stimulation lack mutagenic and transforming activity.

Genetic toxicology assessment of HI-6 dichloride.

The oxime HI-6 dichloride [1-(2 hydroxyiminomethyl -1-pyridino)-3-(4-carbamoyl-1-pyridino)-2-oxapropane dichloride monohydrate] has shown to be a potent reactivator of cholinesterase activity and may have efficacy for the treatment of organophosphate intoxication [SIPRI, 1976; Schenk et al.; Arch Toxicol 36:71-81, 1976]. As part of a preclinical safety assessment program, the genetic toxicology of HI-6 dichloride was evaluated in a series of assays designed to measure induction of gene mutations and chromosomal aberrations. HI-6 dichloride gave negative responses in the Salmonella mutagenicity assay and in the CHO/HGPRT gene mutation assay. Dose-dependent increases in the frequency of chromosomal aberrations were noted when HI-6 dichloride was tested in cultured CHO cells and in cultured human peripheral blood lymphocytes. The mouse lymphoma gene mutation assay, reputed to measure both gene mutations and chromosomal deletions, was negative in the absence of metabolic activation. Depending on the criteria employed, a negative or equivocal response was seen in the presence of rat liver-derived S-9 mix. An in vivo rat bone marrow metaphase assay performed to further investigate the in vitro clastogenic responses was negative. The results from these studies indicate that HI-6 dichloride does not induce gene mutations in vitro; however, it is clastogenic in vitro but does not appear to be clastogenic in vivo.

Molecular and genetic toxicology of 1,3-butadiene.

During the last 9 years, there have been many studies published concerning the mutagenic potential of butadiene in mammalian systems, including alterations at the molecular level. Butadiene has tested positive in several mouse in vivo and in vitro assays, but has generally tested negative in rat studies. Most of these studies are cytogenetic and include positive data in mice for chromosomal aberrations, micronucleus formation, and sister chromatid exchanges. Butadiene also induces mutations in lung, spleen, and bone marrow of transgenic mice. The positive bone marrow cytogenetic and transgenic data may be significant in view of the increased lymphohematopoietic malignancies observed in mice and probably in humans. In addition, butadiene causes mutations in the K-ras protooncogene and in the p53 tumor suppressor gene in mouse studies. Mutations in these genes are associated with oncogenesis in humans as well as in rodents. Also, positive mutagenicity data have been obtained in a pilot study of workers exposed to butadiene. Positive dominant lethal studies in rodents suggest that exposure to butadiene can result in germ cell mutation and heritable risk. These mutagenicity and molecular data suggest that butadiene is both a somatic and germ cell mutagen in mammals, possibly including humans.

Increased frequencies of sister chromatid exchange in soldiers deployed to Kuwait.

Frequencies of sister chromatid exchange (SCE), a measure of genotoxic exposure, were assessed in military troops deployed to Kuwait in 1991. Soldiers completed health questionnaires and had blood collected prior to, during and following deployment to Kuwait. Frequency of spontaneous SCE was determined on blood samples as a measure of mutagenic exposure. Compared to pre-deployment baseline SCE frequency means, levels obtained 2 months into the Kuwaiti deployment were significantly increased (P < 0.001) and persisted for at least 1 month after return to Germany. Outcome was unaffected by known personal SCE effect modifiers including smoking, age and diet. Potential sources of the apparent mutagenic exposure are discussed.

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Sister-chromatid exchange: second report of the Gene-Tox Program.

This paper reviews the ability of a number of chemicals to induce sister-chromatid exchanges (SCEs). The SCE data for animal cells in vivo and in vitro, and human cells in vitro are presented in 6 tables according to their relative effectiveness. A seventh table summarizes what is known about the effects of specific chemicals on SCEs for humans exposed in vivo. The data support the concept that SCEs provide a useful indication of exposure, although the mechanism and biological significance of SCE formation still remain to be elucidated.