Acrylonitrile's genotoxicity profile: mutagenicity in search of an underlying molecular mechanism.

Acrylonitrile (ACN) is a known rodent and possible human carcinogen. There have also been concerns as to it causing adverse reproductive health effects. Numerous genotoxicity studies at the somatic level in a variety of test systems have demonstrated ACN's mutagenicity; its potential to induce mutations in germ cells has also been evaluated. ACN is metabolized to reactive intermediates capable of forming adducts with macromolecules including DNA, a necessary first step in establishing a direct mutagenic mode of action (MOA) for its carcinogenicity. The mutagenicity of ACN has been well demonstrated, however, numerous studies have found no evidence for the capacity of ACN to induce direct DNA lesions that initiate the mutagenic process. Although ACN and its oxidative metabolite (2-cyanoethylene oxide or CNEO) have been shown to bind in vitro with isolated DNA and associated proteins, usually under non-physiological conditions, studies in mammalian cells or in vivo have provided little specification as to an ACN-DNA reaction. Only one early study in rats has shown an ACN/CNEO DNA adduct in liver, a non-target tissue for its carcinogenicity in the rat. By contrast, numerous studies have shown that ACN can act indirectly to induce at least one DNA adduct by forming reactive oxygen species (ROS) in vivo, but it has not been definitively shown that the resulting DNA damage is causative for the induction of mutations. Genotoxicity studies for ACN in somatic and germinal cells are summarized and critically reviewed. Significant data gaps have been identified for bringing together the massive data base that provides the basis of ACN's current genotoxicity profile.

Using mechanistic information to support evidence integration and synthesis: a case study with inhaled formaldehyde and leukemia.

Formaldehyde is one of the most comprehensively studied chemicals, with over 30 years of research focused on understanding the development of cancer following inhalation. The causal conclusions regarding the potential for leukemia are largely based on the epidemiological literature, with little consideration of cancer bioassays, dosimetry studies, and mechanistic research, which challenge the biological plausibility of the disease. Recent reanalyzes of the epidemiological literature have also raised significant questions related to the purported associations between formaldehyde and leukemia. Because of this, considerable scientific debate and uncertainty remain on whether there is a causal association between formaldehyde inhalation exposure and leukemia. Further complexity in evaluating this association is related to the endogenous production of formaldehyde. Multiple modes of action (MOA) have been postulated for the development of leukemia following formaldehyde inhalation that includes unsupported hypotheses of direct or indirect toxicity to the target cell population. Herein, the available evidence relevant to evaluating the postulated MOAs for leukemia following formaldehyde inhalation exposure is organized in the IPCS MOA Framework. The integration of all the available evidence clearly highlights the limited amount of data that support any of the postulated MOAs and demonstrates a significant amount of research supporting the null hypothesis that there is no causal association between formaldehyde inhalation exposure and leukemia. These analyses result in a lack of confidence in any of the postulated MOAs, increasing confidence in the conclusion that there is a lack of biological plausibility for a causal association between formaldehyde inhalation exposure and leukemia.

Do chromosome changes in blood cells implicate formaldehyde as a leukemogen?

Formaldehyde (FA) is a mutagenic chemical - a property mitigated in vivo by rapid detoxification and limited tissue distribution following inhalation of the free agent. Endogenously produced FA is necessary for life and required for one-carbon transfer reactions; however, FA derived from external sources (exogenous FA), which may be in the form of methanol, may increase in vivo concentrations above naturally occurring physiological levels. Both endogenous and exogenous FA produce DNA monoadducts, DNA-DNA and DNA-protein cross-links (DDX and DPX) but, when exposed to exogenously-derived free FA, DNA monoadducts, DDX, and DPX are only produced at initial sites of contact. In contrast, methanol may systemically induce DNA adducts distally. FA also induces oxidative stress/lipid peroxidation with some individuals suggesting the resulting reactive aldehydes may have the potential to induce distal site DNA damage with the resulting reactive aldehydes having the potential to induce distal site DNA damage. Chromosome changes in the form of aberrations or micronuclei in blood cells have been studied in FA-exposed animals and humans, with most of the former being negative. Human occupational studies have given mixed results for such changes in peripheral blood lymphocytes (PBLs) which circulate widely but do not reflect recent bone marrow (BM) events. Recent studies reporting changes in human BM or hematopoietic precursor cells (HPCs) either have had confounding exposures or could not distinguish in vivo from in vitro occurrences. The reported genetic changes in circulating blood cells do not provide convincing support for FA's classification as a human leukemogen.

The lower alkyl methacrylates: Genotoxic profile of non-carcinogenic compounds.

All of the lower alkyl methacrylates are high production chemicals with potential for human exposure. The genotoxicity of seven mono-functional alkyl esters of methacrylic acid, i.e. methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, n-, i- and t-butyl methacrylate and 2 ethyl hexyl methacrylate, as well as methacrylic acid itself, the acyl component common to all, is reviewed and compared with the lack of carcinogenicity of methyl methacrylate, the representative member of the series so evaluated. Also reviewed are the similarity of structure, chemical and biological reactivity, metabolism and common metabolic products of this group of compounds which allows a category approach for assessing genotoxicity. As a class, the lower alkyl methacrylates are universally negative for gene mutations in prokaryotes but do exhibit high dose clastogenicity in mammalian cells in vitro. There is no convincing evidence that these compounds induce genotoxic effects in vivo in either sub-mammalian or mammalian species. This dichotomy of effects can be explained by the potential genotoxic intermediates generated in vitro. This genotoxic profile of the lower alkyl methacrylates is consistent with the lack of carcinogenicity of methyl methacrylate.

Vinyl acetate monomer (VAM) genotoxicity profile: relevance for carcinogenicity.

Vinyl acetate monomer (VAM) is a site-of-contact carcinogen in rodents. It is also DNA reactive and mutagenic, but only after its carboxylesterase mediated conversion to acetaldehyde (AA), a metabolic reaction that also produces acetic acid and protons. As VAM's mutagenic metabolite, AA is normally produced endogenously; detoxification by aldehyde dehydrogenase (ALDH) is required to maintain intra-cellular AA homeostasis. This review examines VAM's overall genotoxicity, which is due to and limited by AA, and the processes leading to mutation induction. VAM and AA have both been universally negative in mutation studies in bacteria but both have tested positive in several in vitro studies in higher organisms that usually employed high concentrations of test agents. Recently however, in vitro studies evaluating submillimolar concentrations of VAM or AA have shown threshold dose-responses for mutagenicity in human cultured cells. Neither VAM nor AA induced systemic mutagenicity in in vivo studies in metabolically competent mice when tested at non-lethal doses while treatments of animals deficient in aldehyde dehydrogenase (Aldh in animals) did induce both gene and chromosome level mutations. The results of several studies have reinforced the critical role for aldehyde dehydrogenase 2 (ALDH2 in humans) in limiting AA's (and therefore VAM's) mutagenicity. The overall aim of this review of VAM's mutagenic potential through its AA metabolite is to propose a mode of action (MOA) for VAM's site-of-contact carcinogenesis that incorporates the overall process of mutation induction that includes both background mutations due to endogenous AA and those resulting from exogenous exposures.

Formaldehyde exposure and leukemia: critical review and reevaluation of the results from a study that is the focus for evidence of biological plausibility.

A recent study (Zhang et al., 2010) has provided results attributed to aneuploidy in circulating stem cells that has been characterized as providing potential support for proposed mechanisms for formaldehyde to impact bone marrow. A critical review of the study, as well as a reanalysis of the underlying data, was performed and the results of this reanalysis suggested factors other than formaldehyde exposure may have contributed to the effects reported. In addition, although the authors stated in their paper that "all scorable metaphase spreads on each slide were analyzed, and a minimum of 150 cells per subject was scored," this protocol was not followed. In fact, the protocol to evaluate the presence of monosomy 7 or trisomy 8 was followed for three or less samples in exposed workers and six or less samples in non-exposed workers. In addition, the assays used (CFU-GM) do not actually measure the proposed events in primitive cells involved in the development of acute myeloid leukemia. Evaluation of these data indicates that the aneuploidy measured could not have arisen in vivo, but rather arose during in vitro culture. The results of our critical review and reanalysis of the data, in combination with recent toxicological and mechanistic studies, do not support a mechanism for a causal association between formaldehyde exposure and myeloid or lymphoid malignancies.

SURROGATE SELECTION OVERSAMPLES EXPANDED T CELL CLONOTYPES.

Inference from immunological data on cells in the adaptive immune system may benefit from modeling specifications that describe variation in the sizes of various clonal sub-populations. We develop one such specification in order to quantify the effects of surrogate selection assays, which we confirm may lead to an enrichment for amplified, potentially disease-relevant T cell clones. Our specification couples within-clonotype birth-death processes with an exchangeable model across clonotypes. Beyond enrichment questions about the surrogate selection design, our framework enables a study of sampling properties of elementary sample diversity statistics; it also points to new statistics that may usefully measure the burden of somatic genomic alterations associated with clonal expansion. We examine statistical properties of immunological samples governed by the coupled model specification, and we illustrate calculations in surrogate selection studies of melanoma and in single-cell genomic studies of T cell repertoires.

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

Molecular characterization of hypoxanthine guanine phosphoribosyltransferase mutant T cells in human blood: The concept of surrogate selection for immunologically relevant cells.

Somatic cell gene mutations arise in vivo due to replication errors during DNA synthesis occurring spontaneously during normal DNA synthesis or as a result of replication on a DNA template damaged by endogenous or exogenous mutagens. In principle, changes in the frequencies of mutant cells in vivo in humans reflect changes in exposures to exogenous or endogenous DNA damaging insults, other factors being equal. It is becoming increasingly evident however, that somatic mutations in humans have a far greater range of interpretations. For example, mutations in lymphocytes provide invaluable probes for in vivo cellular and molecular processes, providing identification of clonal amplifications of these cells in autoimmune and infectious diseases, transplantation recipients, paroxysmal nocturnal hemoglobinuria (PNH), and cancer. The assay for mutations of the X-chromosomal hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has gained popular acceptance for this purpose since viable mutant cells can be recovered for molecular and other analyses. Although the major application of the HPRT T cell assay remains human population monitoring, the enrichment of activated T cells in the mutant fraction in individuals with ongoing immunological processes has demonstrated the utility of surrogate selection, a method that uses somatic mutation as a surrogate marker for the in vivo T cell proliferation that underlies immunological processes to investigate clinical disorders with immunological features. Studies encompassing a wide range of clinical conditions are reviewed. Despite the historical importance of the HPRT mutation system in validating surrogate selection, there are now additional mutational and other methods for identifying immunologically active T cells. These methods are reviewed and provide insights for strategies to extend surrogate selection in future studies.

1,3-Butadiene: III. Assessing carcinogenic modes of action.

1,3-Butadiene (BD) is a multisite carcinogen in laboratory rodents following lifetime exposure, with greater potency in the mouse than the rat, and is associated with an increase in leukemia mortality in highly exposed workers. Species differences in the formation of reactive metabolites underlie observed species differences in sensitivity to the carcinogenic effects of BD. The modes of action (MOAs) for human leukemia and rodent tumors are both likely related to mutagenic potencies of one or more of these metabolites. However, differences in the nature of genotoxic lesions associated with human leukemia and rodent tumors, along with their implications for risk assessment, require that they be discussed separately. The MOAs for BD are assessed in this review using the modified Hill criteria and human relevance framework. Key events in MOAs for human and rodent cancers are identified, along with important species differences and sources of nonlinearity for each event that can affect extrapolations made from high- to low-dose exposures. Because occupational exposures to BD have also included co-exposures to styrene and dimethyldithiocarbamide (DMDTC), potential interactions with BD carcinogenicity are also discussed. The MOAs for BD carcinogenesis will be used to guide key decisions made in the quantitative cancer dose-response assessment.

1,3-Butadiene: I. Review of metabolism and the implications to human health risk assessment.

1,3-Butadiene (BD) is a multisite carcinogen in laboratory rodents following lifetime exposure, with mice demonstrating greater sensitivity than rats. In epidemiology studies of men in the styrene-butadiene rubber industry, leukemia mortality is associated with butadiene exposure, and this association is most pronounced for high-intensity BD exposures. Metabolism is an important determinant of BD carcinogenicity. BD is metabolized to several electrophilic intermediates, including epoxybutene (EB), diepoxybutane (DEB), and epoxybutane diol (EBD), which differ considerably in their genotoxic potency (DEB >> EB > EBD). Important species differences exist with respect to the formation of reactive metabolites and their subsequent detoxification, which underlie observed species differences in sensitivity to the carcinogenic effects of BD. The modes of action for human leukemia and for the observed solid tumors in rodents are both likely related to the genotoxic potencies for one or more of these metabolites. A number of factors related to metabolism can also contribute to nonlinearity in the dose-response relationship, including enzyme induction and inhibition, depletion of tissue glutathione, and saturation of oxidative metabolism. A quantitative risk assessment of BD needs to reflect these species differences and sources of nonlinearity if it is to reflect the current understanding of the disposition of BD.

1,3-Butadiene: II. Genotoxicity profile.

1,3-Butadiene’s (BD’s) major electrophilic metabolites 1,2-epoxy-3-butene (EB), 1,2-dihydroxy-3,4-epoxybutane (EBD), and 1,2,3,4-diepoxybutane (DEB) are responsible for both its mutagenicity and carcinogenicity. EB, EBD, and DEB are DNA reactive, forming a variety of adducts. All three metabolites are genotoxic in vitro and in vivo, with relative mutagenic potencies of DEB >> EB > EBD. DEB also effectively produces gene deletions and chromosome aberrations. BD’s greater mutagenicity and carcinogenicity in mice over rats as well as its failure to induce chromosome-level mutations in vivo in rats appear to be due to greater production of DEB in mice. Concentrations of EB and DEB in vivo in humans are even lower than in rats. Although most studies of BD-exposed humans have failed to find increases in gene mutations, one group has reported positive findings. Reasons for these discordant results are examined. BD-related chromosome aberrations have never been demonstrated in humans except for the possible production of micronuclei in lymphocytes of workers exposed to extremely high levels of BD in the workplace. The relative potencies of the BD metabolites, their relative abundance in the different species, and the kinds of mutations they can induce are major considerations in BD’s overall genotoxicity profile.

Mutagenicity monitoring in humans: Global versus specific origin of mutations.

An underappreciated aspect of human mutagenicity biomonitoring is tissue specificity reflected in different assays, especially those that measure events that can only occur in developing bone marrow (BM) cells. Reviewed here are 9 currently-employed human mutagenicity biomonitoring assays. Several assays measure chromosome-level events in circulating T-lymphocytes (T-cells), i.e., traditional analyses of aberrations, translocation studies involving chromosome painting and fluorescence in situ hybridization (FISH) and determinations of micronuclei (MN). Other T-cell assays measure gene mutations. i.e., hypoxanthine-guanine phosphoriboslytransferase (HPRT) and phosphoribosylinositol glycan class A (PIGA). In addition to the T-cell assays, also reviewed are those assays that measure events in peripheral blood cells that necessarily arose in BM cells, i.e., MN in reticulocytes; glycophorin A (GPA) gene mutations in red blood cells (RBCs), and PIGA gene mutations in RBC or granulocytes. This review considers only cell culture- or cytometry-based assays to describe endpoints measured, methods, optimal sampling times, and sample summaries of typical quantitative and qualitative results. However, to achieve its intended focus on the target cells where events occur, kinetics of the cells of peripheral blood that derive at some point from precursor cells are reviewed to identify body sites and tissues where the genotoxic events originate. Kinetics indicate that in normal adults, measured events in T-cells afford global assessments of in vivo mutagenicity but are not specific for BM effects. Therefore, an agent's capacity for inducing mutations in BM cells cannot be reliably inferred from T-cell assays as the magnitude of effect in BM, if any, is unknown. By contrast, chromosome or gene level mutations measured in RBCs/reticulocytes or granulocytes must originate in BM cells, i.e. in RBC or granulocyte precursors, thereby making them specific indicators for effects in BM. Assays of mutations arising directly in BM cells may quantitatively reflect the mutagenicity of potential leukemogenic agents.

Genotoxicity as a toxicologically relevant endpoint to inform risk assessment: A case study with ethylene oxide.

The purpose of the present investigation is to analyze the in vivo genotoxicity dose-response data of ethylene oxide (EO) and the applicability of the derived point-of-departure (PoD) values when estimating permitted daily exposure (PDE) values. A total of 40 data sets were identified from the literature, and benchmark dose analyses were conducted using PROAST software to identify a PoD value. Studies employing the inhalation route of exposure and assessing gene or chromosomal mutations and chromosomal damage in various tissues were considered the most relevant for assessing risk from EO, since these effects are likely to contribute to adverse health consequences in exposed individuals. The PoD estimates were screened for precision and the values were divided by data-derived adjustment factors. For gene mutations, the lowest PDE was 285 parts per trillion (ppt) based on the induction of lacI mutations in the testes of mice following 48 weeks of exposure to EO. The corresponding lowest PDE value for chromosomal mutations was 1,175 ppt for heritable translocations in mice following 8.5 weeks of EO exposure. The lowest PDE for chromosomal aberrations was 238 ppt in the mouse peripheral blood lymphocytes following 48 weeks of inhalation exposure. The diverse dose-response data for EO-induced genotoxicity enabled the derivation of PoDs for various endpoints, tissues, and species and identified 238 ppt as the lowest PDE in this retrospective analysis.

Derivation of inhalation toxicity reference values for propylene oxide using mode of action analysis: example of a threshold carcinogen.

Propylene oxide (PO) is an important industrial chemical used primarily in the synthesis of other compounds. Inhalation carcinogenesis studies in rodents, with no-observed-adverse-effect levels (NOAELs) of 100 and 200 ppm, have revealed that chronic, high exposure to PO can induce tumors at the site of contact. Despite these characteristics, there is no evidence that typical environmental or occupational exposures to PO constitute a health risk for humans. The nongenotoxic effects of PO (glutathione depletion and cell proliferation) that augment its DNA-reactive and non-DNA-reactive genotoxicity are expected to be similar in humans and rodents. Available evidence on mode-of-action suggests that cancer induction by PO at the site of contact in rodents is characterized by a practical threshold. Human toxicity reference values for potential carcinogenic effects of PO were derived based on nasal tumors identified in rodent studies and specified uncertainty factors. The 95% lower confidence limit on the dose producing a 10% increase in additional tumor risk (LED10) was calculated using the rat and mouse data sets. The human reference values derived from the rat and mouse LED10 values were 0.7 and 0.5 ppm PO, respectively. A similar noncancer reference value, 0.4 ppm, was derived on the basis of non-neoplastic nasal effects in rats.

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.

1,3-Butadiene metabolite 1,2,3,4 diepoxybutane induces DNA adducts and micronuclei but not t(9;22) translocations in human cells.

Epidemiological studies of 1,3-butadiene (BD) exposures have reported a possible association with chronic myelogenous leukemia (CML), which is defined by the presence of the t(9;22) translocation (Philadelphia chromosome) creating an oncogenic BCR-ABL fusion gene. Butadiene diepoxide (DEB), the most mutagenic of three epoxides resulting from BD, forms DNA-DNA crosslink adducts that can lead to DNA double-strand breaks (DSBs). Thus, a study was designed to determine if (±)-DEB exposure of HL60 cells, a promyelocytic leukemia cell line lacking the Philadelphia chromosome, can produce t(9;22) translocations. In HL60 cells exposed for 3 h to 0-10 µM DEB, overlapping dose-response curves suggested a direct relationship between 1,4-bis-(guan-7-yl)-2,3-butanediol crosslink adduct formation (R = 0.977, P = 0.03) and cytotoxicity (R = 0.961, P = 0.002). Experiments to define the relationships between cytotoxicity and the induction of micronuclei (MN), a dosimeter of DNA DSBs, showed that 24 h exposures of HL60 cells to 0-5.0 µM DEB caused significant positive correlations between the concentration and (i) the degree of cytotoxicity (R = 0.998, p = 0.002) and (ii) the frequency of MN (R = 0.984, p = 0.016) at 48 h post exposure. To determine the relative induction of MN and t(9;22) translocations following exposures to DEB, or x-rays as a positive control for formation of t(9;22) translocations, HL60 cells were exposed for 24 h to 0, 1, 2.5, or 5 µM DEB or to 0, 2.0, 3.5, or 5.0 Gy x-rays, or treatments demonstrated to yield 0, 20%, 50%, or 80% cytotoxicity. Treatments between 0 and 3.5 Gy x-rays caused significant dose-related increases in both MN (p < 0.001) and t(9;22) translocations (p = 0.01), whereas DEB exposures causing similar cytotoxicity levels did not increase translocations over background. These data indicate that, while DEB induces DNA DSBs required for formation of MN and translocations, acute DEB exposures of HL60 cells did not produce the Philadelphia chromosome obligatory for CML.

Effect of chronic azathioprine treatment on germ-line transmission of Hprt mutation in mice.

Azathioprine (Aza), a prodrug of 6-mercaptopurine, is used in human medicine to prevent transplant rejection and for the treatment of autoimmune diseases. Extremely high HPRT lymphocyte mutant frequencies (MFs) are found in humans and mice chronically treated with Aza, and these elevated MFs appear to be caused by selection and amplification of pre-existing HPRT mutant lymphocytes. In the present study, we investigated if in vivo selection by Aza also promotes the germ-line transmission of Hprt mutants. Fifty-five male C57BL/6 mice were treated with 10 mg/kg Aza three times/week for 24 weeks; 10 control mice were treated with the vehicle. Each of these males then was bred to unexposed females for a total of 8 weeks. Analysis of the Aza-treated males after the breeding period indicated that 12 had highly elevated Hprt lymphocyte MFs (1 x 10(-4)-2.5 x 10(-1) vs. normal MFs of <1 x 10(-5)), indicating that the Aza treatment successfully selected somatic cell mutants. The female offspring from the breeding were sacrificed at 28 days of age and Hprt MFs were measured in spleen lymphocytes. Most of the 364 female offspring (332 from Aza-treated fathers) had Hprt MFs of 0-6 x 10(-6), but seven of the offspring had moderately elevated MFs of 16 x 10(-6)-55 x 10(-6). Since one of these mice was fathered by a control male, these relatively high MFs appear to be part of the normal variation in lymphocyte Hprt MF. The present results provide no evidence that long-term Aza treatment promotes high levels of germ-line Hprt mutation transmission in mice.

International Symposium on the Evaluation of Butadiene and Chloroprene Health Risks.

These proceedings represent nearly all the platform and poster presentations given during the International Symposium on Evaluation of Butadiene and Chloroprene Health Risks, held in Charleston, South Carolina, USA, on September 20-22, 2005. The Symposium was attended by 78 participants representing private industry (37), academia (21), government (11), not-for-profit organizations (5), and consulting (4). The program followed the format of previous symposia on butadiene, chloroprene, and isoprene in London UK (2000) and butadiene and isoprene in Blaine, Washington USA (1995). This format enabled the exchange of significant new scientific results and discussion of future research needs. Isoprene was not evaluated during the 2005 Symposium because of lack of new data. For background information, the reader is referred to the proceedings of the London 2000 meeting for a thorough historical perspective and overview of scientific and regulatory issues concerning butadiene, chloroprene, and isoprene [Chem.-Biol. Interact. (2001) 135-136:1-7]. The Symposium consisted of seven sessions: (1) Introduction and Opening Remarks, (2) Butadiene/styrene-butadiene rubber (SBR)--Process Overview, Exposure and Health Effects/Human Studies; (3) Chloroprene--Process Overview, Exposure and Health Effects/Human Studies; (4) Mode of Action/Key Events; (5) Risk Assessment; (6) Poster Presentations; and (7) Panel Discussion and Future Directions. The Symposium concluded with a discussion by all participants of issues that arose throughout the course of the Symposium. The Proceedings of the Symposium published in this Special Issue are organized according to the Sessions outlined above. The purpose of this foreword is to summarize the presentations and their key findings and recommend future research directions for each chemical.