Comparison of in vivo derived and scaled in vitro metabolic rate constants for several volatile organic compounds (VOCs).

Metabolic rate parameters estimation using in vitro data is necessary due to numbers of chemicals for which data are needed, trend towards minimizing laboratory animal use, and limited opportunity to collect data in human subjects. We evaluated how well metabolic rate parameters derived from in vitro data predict overall in vivo metabolism for a set of environmental chemicals for which well validated and established methods exist. We compared values of VmaxC derived from in vivo vapor uptake studies with estimates of VmaxC scaled up from in vitro hepatic microsomal metabolism studies for VOCs for which data were available in male F344 rats. For 6 of 7 VOCs, differences between the in vivo and scaled up in vitro VmaxC estimates were less than 2.6-fold. For bromodichloromethane (BDCM), the in vivo derived VmaxC was approximately 4.4-fold higher than the in vitro derived and scaled up VmaxC. The more rapid rate of BDCM metabolism estimated based in vivo studies suggests other factors such as extrahepatic metabolism, binding or other non-specific losses making a significant contribution to overall clearance. Systematic and reliable utilization of scaled up in vitro biotransformation rate parameters in PBPK models will require development of methods to predict cases in which extrahepatic metabolism and binding as well as other factors are likely to be significant contributors.

The Impact of Scaling Factor Variability on Risk-Relevant Pharmacokinetic Outcomes in Children: A Case Study Using Bromodichloromethane (BDCM).

Biotransformation rates extrapolated from in vitro data are used increasingly in human physiologically based pharmacokinetic (PBPK) models. This practice requires use of scaling factors, including microsomal content (mg of microsomal protein/g liver, MPPGL), enzyme specific content, and liver mass as a fraction of body weight (FVL). Previous analyses indicated that scaling factor variability impacts pharmacokinetic (PK) outcomes used in adult population dose-response studies. This analysis was extended to pediatric populations because large inter-individual differences in enzyme ontogeny likely would further contribute to scaling factor variability. An adult bromodichloromethane (BDCM) model (Kenyon, E. M., Eklund, C., Leavens, T. L., and Pegram, R. A. (2016a). Development and application of a human PBPK model for bromodichloromethane (BDCM) to investigate impacts of multi-route exposure. J. Appl. Toxicol. 36, 1095-1111) was re-parameterized for neonates, infants, and toddlers. Monte Carlo analysis was used to assess the impact of pediatric scaling factor variation on model-derived PK outcomes compared with adult findings. BDCM dose metrics were estimated following a single 0.05-liter drink of water or a 20-min bath, under typical (5 µg/l) and plausible higher (20 µg/l) BDCM concentrations. MPPGL, CYP2E1, and FVL values reflected the distribution of reported pediatric population values. The impact of scaling factor variability on PK outcome variation was different for each exposure scenario, but similar for each BDCM water concentration. The higher CYP2E1 expression variability during early childhood was reflected in greater variability in predicted PK outcomes in younger age groups, particularly for the oral exposure route. Sensitivity analysis confirmed the most influential parameter for this variability was CYP2E1, particularly in neonates. These findings demonstrate the importance of age-dependent scaling factor variation used for in vitro to in vivo extrapolation of biotransformation rates.

The impact of variation in scaling factors on the estimation of internal dose metrics: a case study using bromodichloromethane (BDCM).

A rate for hepatic metabolism (Vmax) determined in vitro must be scaled for in vivo use in a physiologically based pharmacokinetic (PBPK) model. This requires the use of scaling factors such as mg of microsomal protein per gram of liver (MPPGL) and liver mass (FVL). Variation in MPPGL and FVL impacts variation in Vmax, and hence PBPK model-derived estimates of internal dose used in dose response analysis. The impacts of adult human variation in MPPGL and FVL on estimates of internal dose were assessed using a human PBPK model for bromodichloromethane (BDCM), a water disinfection byproduct, for multiple internal dose metrics for two exposure scenarios (single 0.25 liter drink of water or 10 min shower) under plausible (5 µg/L) and high level (20 µg/L) water concentrations. For both concentrations, all internal dose metrics were changed less than 5% for the showering scenario (combined inhalation and dermal exposure). In contrast, a 27-fold variation in area under the curve (AUC) for BDCM in venous blood was observed at both oral exposure concentrations, whereas total amount of BDCM metabolized in liver was relatively unchanged. This analysis demonstrates that variability in the scaling factors used for in vitro to in vivo extrapolation (IVIVE) for metabolic rate parameters can have a significant route-dependent impact on estimates of internal dose under environmentally relevant exposure scenarios. This indicates the need to evaluate both uncertainty and variability for scaling factors used for IVIVE.

Development of a human physiologically based pharmacokinetic (PBPK) model for dermal permeability for lindane.

Lindane is a neurotoxicant used for the treatment of lice and scabies present on human skin. Due to its pharmaceutical application, an extensive pharmacokinetic database exists in humans. Mathematical diffusion models allow for calculation of lindane skin permeability coefficients using human kinetic data obtained from in vitro and in vivo experimentation as well as a default compound-specific calculation based on physicochemical characteristics used in the absence of kinetic data. A dermal model was developed to describe lindane diffusion into the skin, where the skin compartment consisted of homogeneous dermal tissue. This study utilized Fick's law of diffusion along with chemical binding to protein and lipids to determine appropriate dermal absorption parameters which were then incorporated into a physiologically based pharmacokinetic (PBPK) model to describe in vivo kinetics. The estimation of permeability coefficients using chemical binding in combination with in vivo data demonstrates the advantages of combining physiochemical properties with a PBPK model to predict dermal absorption.

Development and application of a human PBPK model for bromodichloromethane to investigate the impacts of multi-route exposure.

As a result of its presence in water as a volatile disinfection byproduct, bromodichloromethane (BDCM), which is mutagenic, poses a potential health risk from exposure via oral, dermal and inhalation routes. We developed a refined human physiologically based pharmacokinetic (PBPK) model for BDCM (including new chemical-specific human parameters) to evaluate the impact of BDCM exposure during showering and bathing on important measures of internal dose compared with oral exposure. The refined model adequately predicted data from the published literature for oral, dermal and bathing/showering exposures. A liter equivalency approach (L-eq) was used to estimate BDCM concentration in a liter of water consumed by the oral route that would be required to produce the same internal dose of BDCM resulting from a 20-min bath or a 10-min shower in water containing 10 µg l(-1) BDCM. The oral liter equivalent concentrations for the bathing scenario were 605, 803 and 5 µg l(-1) BDCM for maximum venous blood concentration (Cmax), the area under the curve (AUCv) and the amount metabolized in the liver per hour (MBDCM), respectively. For a 10-min showering exposure, the oral L-eq concentrations were 282, 312 and 2.1 µg l(-1) for Cmax, AUC and MBDCM, respectively. These results demonstrate large contributions of dermal and inhalation exposure routes to the internal dose of parent chemical reaching the systemic circulation, which could be transformed to mutagenic metabolites in extrahepatic target tissues. Thus, consideration of the contribution of multiple routes of exposure when evaluating risks from water-borne BDCM is needed, and this refined human model will facilitate improved assessment of internal doses from real-world exposures. Published 2015. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Estimating Potential Increased Bladder Cancer Risk Due to Increased Bromide Concentrations in Sources of Disinfected Drinking Waters.

Public water systems are increasingly facing higher bromide levels in their source waters from anthropogenic contamination through coal-fired power plants, conventional oil and gas extraction, textile mills, and hydraulic fracturing. Climate change is likely to exacerbate this in coming years. We estimate bladder cancer risk from potential increased bromide levels in source waters of disinfecting public drinking water systems in the United States. Bladder cancer is the health end point used by the United States Environmental Protection Agency (EPA) in its benefits analysis for regulating disinfection byproducts in drinking water. We use estimated increases in the mass of the four regulated trihalomethanes (THM4) concentrations (due to increased bromide incorporation) as the surrogate disinfection byproduct (DBP) occurrence metric for informing potential bladder cancer risk. We estimate potential increased excess lifetime bladder cancer risk as a function of increased source water bromide levels. Results based on data from 201 drinking water treatment plants indicate that a bromide increase of 50 µg/L could result in a potential increase of between 10(-3) and 10(-4) excess lifetime bladder cancer risk in populations served by roughly 90% of these plants.

Improving in vitro to in vivo extrapolation by incorporating toxicokinetic measurements: a case study of lindane-induced neurotoxicity.

Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans. Time- and concentration-dependent lindane dosimetry was determined in primary cultures of rat cortical neurons in vitro using "faux" (without electrodes) microelectrode arrays (MEAs). In vivo data were derived from literature values, and physiologically based pharmacokinetic (PBPK) modeling was used to extrapolate from rat to human. The previously determined EC50 for increased firing rates in primary cultures of cortical neurons was 0.6µg/ml. Media and cell lindane concentrations at the EC50 were 0.4µg/ml and 7.1µg/ml, respectively, and cellular lindane accumulation was time- and concentration-dependent. Rat blood and brain lindane levels during seizures were 1.7-1.9µg/ml and 5-11µg/ml, respectively. Brain lindane levels associated with seizures in rats and those predicted for humans (average=7µg/ml) by PBPK modeling were very similar to in vitro concentrations detected in cortical cells at the EC50 dose. PBPK model predictions matched literature data and timing. These findings indicate that in vitro MEA results are predictive of in vivo responses to lindane and demonstrate a successful modeling approach for IVIVE of rat and human neurotoxicity.

Comparison of trihalomethanes in tap water and blood: a case study in the United States.

BACKGROUND: Epidemiological studies have used various measures to characterize trihalomethane (THM) exposures, but the relationship of these indicators to exposure biomarkers remains unclear. OBJECTIVES: We examined temporal and spatial variability in baseline blood THM concentrations and assessed the relationship between these concentrations and several exposure indicators (tap water concentration, water-use activities, multiroute exposure metrics). METHODS: We measured water-use activity and THM concentrations in blood and residential tap water from 150 postpartum women from three U.S. locations. RESULTS: Blood ΣTHM [sum of chloroform (TCM), bromodichloromethane (BDCM), dibromo-chloromethane (DBCM), and bromoform (TBM)] concentrations varied by site and season. As expected based on variable tap water concentrations and toxicokinetic properties, the proportion of brominated species (BDCM, DBCM, and TBM) in blood varied by site (site 1, 24%; site 2, 29%; site 3, 57%) but varied less markedly than in tap water (site 1, 35%; site 2, 75%; site 3, 68%). The blood-water ΣTHM Spearman rank correlation coefficient was 0.36, with correlations higher for individual brominated species (BDCM, 0.62; DBCM, 0.53; TBM, 0.54) than for TCM (0.37). Noningestion water activities contributed more to the total exposure metric than did ingestion, but tap water THM concentrations were more predictive of blood THM levels than were metrics that incorporated water use. CONCLUSIONS: Spatial and temporal variability in THM concentrations was greater in water than in blood. We found consistent blood-water correlations across season and site for BDCM and DBCM, and multivariate regression results suggest that water THM concentrations may be an adequate surro-gate for baseline blood levels.

Development of an inhalation physiologically based pharmacokinetic (PBPK) model for 2,2, 4-trimethylpentane (TMP) in male Long-Evans rats using gas uptake experiments.

2,2,4-Trimethylpentane (TMP) is a volatile colorless liquid used primarily to increase the octane rating of combustible fuels. TMP is released in the environment through the manufacture, use, and disposal of products associated with the gasoline and petroleum industry. Short-term inhalation exposure to TMP (< 4 h; > 1000 ppm) caused sensory and motor irritations in rats and mice. Like many volatile hydrocarbons, acute exposure to TMP may also be expected to alter neurological functions. To estimate in vivo metabolic kinetics of TMP and to predict its target tissue dosimetry during inhalation exposures, a physiologically based pharmacokinetic (PBPK) model was developed for the chemical in Long-Evans male rats using closed-chamber gas-uptake experiments. Gas-uptake experiments were conducted in which rats (80-90 days old) were exposed to targeted initial TMP concentrations of 50, 100, 500, and 1000 ppm. The model consisted of compartments for the closed uptake chamber, lung, fat, kidney, liver, brain, and rapidly and slowly perfused tissues. Physiological parameters were obtained from literature. Partition coefficients for the model were experimentally determined for air/blood, fat, liver, kidney, muscle, and brain using vial equilibration methods. Common to other hydrocarbons, metabolism of TMP via oxidative reactions is assumed to mainly occur in the liver. The PBPK model simulations of the closed chamber data were used to estimate in vivo metabolic parameters for TMP in male Long-Evans rats.

Disposition of bromodichloromethane in humans following oral and dermal exposure.

Exposure to bromodichloromethane (BDCM), one of the most prevalent disinfection byproducts in drinking water, can occur via ingestion of water and by dermal absorption and inhalation during activities such as bathing and showering. The objectives of this research were to assess BDCM pharmacokinetics in human volunteers exposed percutaneously and orally to (13)C-BDCM and to evaluate factors that could affect disposition of BDCM. Among study subjects, CYP2E1 activity varied fourfold; 20% had the glutathione S-transferase theta 1-1 homozygous null genotype; and body fat ranged from 7 to 22%. Subjects were exposed to (13)C-BDCM in water (target concentration of 36 mug/l) via ingestion and by forearm submersion. Blood was collected for up to 24 h and analyzed for (13)C-BDCM by solid-phase microextraction and high-resolution GC-MS. Urine was collected before and after exposure for mutagenicity determinations in Salmonella. After ingestion (mean dose = 146 ng/kg), blood (13)C-BDCM concentrations peaked and declined rapidly, returning to levels near or below the limit of detection (LOD) within 4 h. The T(max) for the oral exposure ranged from 5 to 30 min, and the C(max) ranged from 0.4 to 4.1 ng/l. After the 1 h dermal exposure (estimated mean dose = 155 ng/kg), blood concentrations of (13)C-BDCM ranged from 39 to 170 ng/l and decreased to levels near or below the LOD by 24 h. Peak postdose urine mutagenicity levels that were at least twice that of the predose mean level occurred in 6 of 10 percutaneously exposed subjects and 3 of 8 orally exposed subjects. These results demonstrate a highly significant contribution of dermal absorption to circulating levels of BDCM and confirm the much lower oral contribution, indicating that water uses involving dermal contact can lead to much greater systemic BDCM doses than water ingestion. These data will facilitate development and validation of physiologically based pharmacokinetic models for BDCM in humans.

In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1.

The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2). First, rodent hepatic cytosol incubations containing [(14)C]CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA. The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2). Second, exposure of GSTT1-1-expressing S. typhimurium to [(14)C]CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity. Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis. A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed. To circumvent problems of adduct instability, reactions of [(14)C]CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine. Hydroxyapatite chromatography of [(14)C]-labeled DNA or HPLC chromatography of [(14)C]-labeled deoxyguanosine derivatives demonstrated the covalent binding of [(14)C]CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of [(14)C]CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts). Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue). Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared. In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues. The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats).

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure.

BACKGROUND: Epidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water. METHODS: DNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB. RESULTS: CCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs. CONCLUSION: CCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

Bromodichloromethane inhibits human placental trophoblast differentiation.

Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans.

Effect of bromodichloromethane on chorionic gonadotrophin secretion by human placental trophoblast cultures.

Bromodichloromethane (BDCM) is a trihalomethane found in drinking water as a by-product of disinfection processes. BDCM is hepatotoxic and nephrotoxic in rodents and has been reported to cause strain-specific full-litter resorption in F344 rats during the luteinizing hormone-dependent phase of pregnancy. In humans, epidemiological studies suggest an association between exposure to BDCM in drinking water and increased risk of spontaneous abortion. To begin to address the mechanism(s) of BDCM-induced spontaneous abortion, we hypothesized that BDCM targets the placenta. Primary cultures of human term trophoblast cells were used as an in vitro model to test this hypothesis. Trophoblasts were allowed to differentiate into multinucleated syncytiotrophoblast-like colonies, after which they were incubated for 24 h with different concentrations of BDCM (20 nM to 2 mM). Culture media were collected and assayed for immunoreactive and bioactive chorionic gonadotropin (CG). Cultures exposed to BDCM showed a dose-dependent decrease in the secretion of immunoreactive CG as well as bioactive CG. The lowest effective BDCM concentration was 20 nM, approximately 35-times higher than the maximum concentration reported in human blood (0.57 nM). Trophoblast morphology and viability were similar in controls and cultures exposed to BDCM. We conclude that BDCM perturbs CG secretion by differentiated trophoblasts in vitro. This suggests that the placenta is a likely target of BDCM toxicity in the human and that this could be related to the adverse pregnancy outcomes associated with BDCM.

[35S]-labeling of the Salmonella typhimurium glutathione pool to assess glutathione-mediated DNA binding by 1,2-dibromoethane.

Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation. However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g. conjugates of 1,2-dibromoethane). The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a [35S]-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA. The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates. Bacteria were grown to mid-log phase and then incubated with [35S]-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached. At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium. The [35S]-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify [35S]-binding to DNA. The amount of [35S]-label that was covalently bound to DNA in the GSTT1-1-expressing Salmonella strain (33.2 nmol/mg DNA) was sevenfold greater than that of the control strain that does not express GSTT1-1. Neutral thermal hydrolysis of the DNA yielded a single [35S]-labeled adduct with a similar t(R) as S-[2-(N(7)-guanyl)ethyl]GSH, following HPLC analysis of the hydrolysate. This adduct accounted for 95% of the total [35S]-label bound to DNA. Thus, this [35S]-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels. This is also, to our knowledge, the first demonstration of de novo incorporation of [35S]-L-cysteine into the bacterial GSH pool.

Induction of DNA strand breaks by trihalomethanes in primary human lung epithelial cells.

Trihalomethanes (THMs) are disinfection by-products and suspected human carcinogens present in chlorinated drinking water. Previous studies have shown that many THMs induce sister chromatid exchanges and DNA strand breaks in human peripheral blood lymphocytes in vitro. Exposure to THMs occurs through oral, dermal, or inhalation routes, with the lung being a target of exposure by the latter route, although not a target for rodent carcinogenicity. Thus, to examine the genotoxicity of THMs in this tissue, we used the comet assay to examine the DNA damaging ability of five THMs in primary human lung epithelial cells. Cells were collected by scraping the large airways of four volunteers with a cytology brush and then passaging the cells no more than three times in order to have sufficient numbers for the experiments. Cells were exposed for 3h to 10, 100, or 1000 microM CHCl(3), CHCl(2)Br, CHClBr(2), or CHBr(3); CH(2)Cl(2) was also used as a model dihalomethane for comparison to the THMs. The compounds ranked as follows for DNA damaging ability: CHCl(2)Br>CHBr(3)>CHCl(3) approximately equal CH(2)Cl(2); CHClBr(2) was negative. Considerable inter-individual variation was observed. For example, CHCl(3) was genotoxic in only two subjects, and the interaction between dose and donor was highly significant (P<0.001). The same variation was observed for CHCl(2)Br, which was positive only in the two subjects in which CHCl(3) was negative. This variation was not due to the GSTT1-1 genotype of the subjects. Although two subjects were GSTT1-1(+), and two were GSTT1-1(-), no cultured cells with a GSTT1-1(+) genotype had detectable GSTT1-1 enzymatic activity nor did any frozen epithelial cells that had not been cultured. However, GSTT1-1 enzymatic activity was detected in fresh (neither frozen nor cultured) lung cells. These results show that freezing or culturing causes lung cells to lose GSTT1-1 activity and that factors other than GSTT1-1 activity play a role in the variable responses of these human cells to the genotoxicity of the halomethanes studied here.

Glutathione transferase theta 1-1-dependent metabolism of the water disinfection byproduct bromodichloromethane.

Bromodichloromethane (CHBrCl(2)), a prevalent drinking water disinfection byproduct, was previously shown to be mutagenic in Salmonella that express rat GSH transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the kinetics of CHBrCl(2) reactions mediated by GST in different species as well as the isoform specificity and reaction products of the GST pathway. Conjugation activity of CHBrCl(2) with GSH in mouse liver cytosol was time- and protein-dependent, was not inhibited by the GST alpha, mu and pi inhibitor S-hexyl-GSH, and correlated with GST T1-1 activity toward the substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane. Conjugation activities in hepatic cytosols of different species toward CHBrCl(2) followed the order mouse > rat > human. As compared with CH(2)Cl(2), the catalytic efficiency (k(cat)/K(m)) of conjugation of CHBrCl(2) with GSH by pure recombinant rat GST T1-1 was approximately 3-6-fold less. Taken together, this suggests that GST T1-1 is the primary catalyst for conjugation of CHBrCl(2) with GSH and that flux through this pathway is less than for CH(2)Cl(2). The initial GSCHCl(2) conjugate formed was unstable and degraded to several metabolites, including GSCH(2)OH, S-formyl-GSH, and HCOOH. Addition of NAD(+) to cytosol did not alter the rate of conjugation of CHBrCl(2) with GSH; however, it did increase the amount of [(14)C]HCOOH produced ( approximately 10-fold). A similar result was seen in a reaction containing pure rat GST T1-1 and GSH-dependent formaldehyde dehydrogenase, indicating that GSCH(2)OH was formed as a precursor to S-formyl-GSH. The half-life of synthetic S-formyl-GSH in pH 7.4 buffer was approximately 1 h at ambient temperature and decreased to approximately 7 min in pH 9.0 buffer, and it does not react with deoxyguanosine. In conclusion, GST T1-1 conjugation of CHBrCl(2) has been definitively demonstrated and the kinetics of conjugation of CHBrCl(2) with GSH characterized in mouse, rat, and human hepatic cytosols. The significance of this GST pathway is that reactive GSH conjugates are produced resulting in possible formation of DNA adducts. Comparisons with CH(2)Cl(2) suggest that the reactive intermediates specific to GSH conjugation of CHBrCl(2) are more mutagenic/genotoxic than those derived from CH(2)Cl(2).

Evidence for the involvement of CYP1A2 in the metabolism of bromodichloromethane in rat liver.

Bromodichloromethane (BDCM) is a drinking water disinfectant by-product that has been implicated in liver, kidney and intestinal cancers in rodents and in intestinal tumors and low birth weight effects in humans. BDCM is also hepatotoxic and requires metabolic activation for both toxicity and carcinogenicity. We have recently reported that CYP1A2 may participate in that metabolism and we now report experiments to support that implication. Induction of CYP1A2 in male F344 rats without inducing CYP2E1 or CYP2B1/2, using TCDD, increased the hepatotoxicity of BDCM when compared to earlier work conducted under similar protocols. Inhibition of CYP1A2, with isosafrole, reduced the metabolism and toxicity of BDCM in the previously induced rats. In addition, specific activities and Western blots for these CYP isoenzymes were measured 24 h after exposure. Activity data show that only CYP1A2 was inhibited by isosafrole; isosafrole forms a complex with CYP1A2 that persists for more than 24 h. Western blot results generally agree with the activity data except that isosafrole induced the protein for all isoenzymes measured. A physiologically based pharmacokinetic model, developed previously, estimated that BDCM metabolism was complete about 7 h after gavage dosing. It is noteworthy that the reduction in CYP1A2 activity was still measurable despite the production of additional CYP1A2 protein during the period of approximately 18 h after BDCM metabolism was complete. These results demonstrate that CYP1A2 does metabolize BDCM and does contribute to hepatotoxicity under certain conditions.