Rex A. Pegram

Water Chlorination: Essential Process or Cancer Hazard?

Chlorine has been successfully used for the control of waterborne infectious disease for nearly a century. In the 1970s it was found that chlorine reacted with natural organic matter present in surface waters to produce disinfection by-products (DBP). Concern focused initially on the trihalomethanes (THM), but a wide variety of DBPs are now known to result from chlorination. Chlorination of drinking water has been one of the most effective public health measures ever undertaken. There are a number of alternatives to chlorination that are in active use in many parts of the world, but the risks associated with their by-products are even less well established than for chlorination. Moreover, the use of these alternatives vary in their effectiveness and some require greater sophistication in their application. This can mean less protection to public health as a result of inappropriate application and control. Therefore, hazards associated with the use of such a clearly beneficial process as chlorination must be carefully considered not only in an absolute sense, but also in the context of alternative approaches for producing a safe drinking water. The key question is whether the hazards associated with by-products have been sufficiently well established to warrant regulations that will undoubtedly have both positive and negative impacts on the public health. This symposium examined the toxicological and epidemiological data on chemical hazards associated with chlorination and attempted to measure this hazard against competing microbial risks. The first presentation discussed the available analytical epidemiological studies. A second presentation dealt with the importance of chlorination to the prevention of waterborne infectious disease. Pharmacokinetic, mechanistic, and modeling information on the prototypical DBP, chloroform, were discussed and contrasted with data on brominated THMs to determine if it was scientifically appropriate to regulate THMs as a single toxicological class. The fifth presentation dealt with the carcinogenic properties of a potent mutagen that is produced by chlorination. The final presentation discussed the haloacetates, carcinogenic DBPs whose concentrations approach and occasionally exceed those of the THMs. Clearly, there is a need to carefully weigh these different types and sometimes competing risks when considering the delivery of drinking water to ever-increasing populations for which there are finite sources of fresh water.

Glutathione S-transferase-mediated induction of GC AT transitions by halomethanes in salmonella

Halomethanes are among the most common mutagenic and carcinogenic disinfection by‐products present in the volatile/semivolatile fraction of chlorinated drinking water. Recent studies have demonstrated that the mutagenicity of dichloromethane (CH2Cl2) and bromodichloromethane (BrCHCl2) can be mediated by a theta‐class glutathione S‐transferase (GSTT1‐1). These studies used strain RSJ100 of Salmonella, which is a derivative of the base‐substitution strain TA1535 (hisG46, rfa, δuvrB), into which has been cloned the GSTT1‐1gene from rat. In the present report, we have ex tended these studies by demonstrating that the mutagenicity of two additional brominated trihalomethanes, bromoform (CHBr3) and chlorodibromomethane (ClCHBr2), are also mediated by GSTT1‐1 in RSJ100. Using a Tedlar bag vaporization technique, the mutagenic potencies (revertants/ppm) for these two compounds as well as the compounds tested previously rank as follows: CHBr3 ≈ ClCHBr2 > BrCHCl2 ≈ CH2Cl2. To explore the mutational mechanism, we determined the mutation spectra of all four halomethanes at the hisG46 allele by per forming colony probe hybridizations of ∼100 revertants induced by each compound. The majority (96–100%) of the mutations were GC → AT transitions, and 87–100% of these were at the second position of the CCC/GGG target. In contrast, only15% of mutants induced by CH2Cl2 were GC → AT transitions in the absence of the GSTT1‐1 gene in strain TA100 (a homologue of TA1535 containing the plasmid pKM101). The ability of GSTT1‐1 to mediate the mutagenicity of these di‐ and trihalomethanes and the induction of almost exclusively GC → AT transitions by these compounds suggest that these halomethanes are activated by similar pathways in RSJ100, possibly through similar reactive intermediates. The implications of these findings are discussed in relation to previous experimental work on the GST‐mediated bioactivation of dihalomethanes, which includes the possible formation of GSH intermediates and/or GSH‐DNAadducts. Environ. Mol. Mutagen. 30:440–447, 1997 Published 1997 Wiley‐Liss, Inc. This article is a US Government work and as such, is in the public domain in the United States of America.

Effect of dosing vehicle on the developmental toxicity of bromodichloromethane and carbon tetrachloride in rats.

Several halocarbons have been shown to cause full-litter resorption (FLR) in Fischer-344 rats when administered orally in corn oil. Since halocarbons often occur as contaminants of drinking water, we sought to determine the influence of the vehicle, aqueous versus lipid, on the developmental toxicity of two of these agents. In separate assays, bromodichloromethane (BDCM) and carbon tetrachloride (CCl4) were administered by gavage to Fischer-344 rats on gestation days (GD) 6-15 at 0, 25, 50, or 75 mg/kg/day in either corn oil or an aqueous vehicle containing 10% Emulphor EL-620. Dams were allowed to deliver and the litters were examined postnatally. Uteri of females that did not deliver were stained with 10% ammonium sulfide to detect FLR. Effects of both agents on maternal weight gain were slightly more pronounced in the aqueous vehicle at lower doses, but at the highest dose, CCl4 was more maternally toxic in corn oil. Developmentally, both agents caused FLR at 50 and 75 mg/kg in both vehicles. At 75 mg/kg, dams receiving corn oil had significantly higher rates of FLR (83% for BDCM, 67% for CCl4) compared to their aqueous-vehicle counterparts (21% for BDCM, 8% for CCl4). Blood concentrations of BDCM following GD-6 gavage revealed a shorter elimination half-life in the aqueous dosing vehicle (2.7 h) compared to the oil vehicle (3.6 h). Benchmark doses of CCl4 were similar for the oil (18.9 mg/kg) and aqueous (14.0 mg/kg) vehicles. For BDCM, the corn oil vehicle yielded a less conservative (i.e., higher) value (39.3 mg/kg) than the aqueous vehicle (11.3 mg/kg), reflecting different confidence intervals around the estimated 5%-effect dose levels.

Physiologically based estimation of in vivo rates of bromodichloromethane metabolism

Bromodichloromethane (BDCM) is a rodent carcinogen formed by chlorination of drinking water containing bromide and organic precursors. BDCM is a member of the class of disinfection by-products known as trihalomethanes (THMs), compounds that have been shown to be carcinogenic in rodents. A physiologically-based pharmacokinetic (PBPK) model has been developed and applied to provide estimates of the rates of metabolism of BDCM in vivo in rats. The model consists of five compartments (liver, kidney, fat and slowly and rapidly perfused tissues). Tissue partition coefficients were determined using a modified vial equilibration technique and rates of metabolism were estimated by fitting data obtained from stable metabolite (bromide ion, (Br-)) analysis following 4 h constant concentration BDCM inhalation exposure (50-3200 ppm) and closed chamber gas uptake experiments. Metabolism was described using a single saturable pathway representing a high capacity, high affinity process (Vmaxc = 12.8 mg/h/kg; Km = 0.5 mg/l). Rate constants obtained from Br- data adequately described data from gas uptake experiments and literature data on exhalation of 14CO and 14CO2 produced following oral gavage with 14C-BDCM. Pretreatment with trans-dichloroethylene (t-DCE), an inhibitor of CYP2E1, increased the apparent Km from 0.5 to 225 mg/l indicating that CYP2E1 is the major P450 isoform involved in the bioactivation of BDCM to reactive intermediates.

Integrated Disinfection By-Products Research: Salmonella Mutagenicity of Water Concentrates Disinfected by Chlorination and Ozonation/Postchlorination

Although chemical disinfection of drinking water is a highly protective public health practice, the disinfection process is known to produce toxic contaminants. Epidemiological studies associate chlorinated drinking water with quantitatively increased risks of rectal, kidney, and bladder cancer. One study found a significant exposure-response association between water mutagenicity and relative risk for bladder and kidney cancer. A number of studies found that several types of disinfection processes increase the level of mutagens detected by the Salmonella assay. As part of a comprehensive study to examine chlorinated and ozonated/postchlorinated drinking water for toxicological contaminants, the Salmonella mutagenicity assay was used to screen both volatile and nonvolatile organic components. The assay also compared the use of reverse osmosis and XAD resin procedures for concentrating the nonvolatile components. Companion papers provide the results from other toxicological assays and chemical analysis of the drinking water samples. The volatile components of the ozonated/postchlorinated and chlorinated water samples and a trihalomethane mixture were mutagenic to a Salmonella tester strain transfected with a rat theta-class glutathione S-transferase and predominantly nonmutagenic in the control strain. In this study, the nonvolatile XAD concentrate of the untreated water possessed a low level of mutagenic activity. However, compared to the levels of mutagenicity in the finished water XAD concentrates, the contribution from the settled source water was minimal. The mutagenicity seen in the reverse osmosis concentrates was < 50% of that seen in the XAD concentrates. Overall, mutagenic responses were similar to those observed in other North American studies and provide evidence that the pilot plant produced disinfection by-products similar to that seen in other studies.

Toxicity of bromodichloromethane in female rats and mice after repeated oral dosing.

The carcinogenic water disinfection byproduct, bromodichloromethane (BDCM), produces renal and hepatic toxicity in rodents in acute and subchronic studies. In the present investigation, female rats and mice (n = 6) were dosed daily for 5 consecutive days with BDCM (dissolved in an aqueous, 10% Emulphor solution) by gavage. Rats received 75, 150 and 300 mg BDCM/kg body weight/day and mice received 75 and 150 mg BDCM/kg body weight/day. Two rats in the 300 mg/kg/day treatment group died on day 5. On day 6, the animals were sacrificed and serum samples were taken for analysis of indicators of hepatic and renal toxicity. Livers and kidneys were excised and samples taken for histopathological evaluation. Portions of the livers were also utilized to produce microsomes for analysis of cytochrome P450 enzyme activities and total P450 content. Total hepatic cytochrome P450 was decreased in rats dosed with 150 and 300 mg BDCM/kg body weight/day, but was not significantly affected in BDCM-treated mice. Serum lactate (LDH) and sorbitol (SDH) dehydrogenase, aspartate aminotransferase (AST), creatinine and blood urea nitrogen were increased above those of controls in rats dosed with 300 mg BDCM/kg/day. These data suggested that hepatic and renal damage had occurred in this treatment group. This was confirmed by histopathological analyses which revealed that lesions occurred in both hepatic and renal tissues from rats dosed with 150 and 300 mg BDCM/kg/day. The hepatic lesions were centrilobular and primarily consisted of vacuolar degeneration. The hepatotoxicity indicators alanine aminotransferase (ALT) and SDH were increased in mice dosed with 150 mg BDCM/kg/day. However, no histopathological lesions were observed in these animals. This study shows that BDCM is both hepatotoxic and nephrotoxic to female rats after repeated dosing, but is only weakly hepatotoxic to female mice at the administered doses. Also, reduced activities of hepatic cytochrome P450 were observed in rats, but not mice. These species differences in toxicity and xenobiotic metabolizing enzyme inhibition caused by BDCM suggest that an understanding of the mechanism of toxicity of this compound will be critical when extrapolating rodent toxicity data to humans for this environmental pollutant.

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.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) distribution and cytochrome P4501A induction in young adult and senescent male mice

While the developmental toxicology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its congeners has received considerable attention, the impact of advanced age on the biochemical effects and the pharmacokinetics of dioxins remains largely undetermined. In the present investigation, TCDD tissue distribution and cytochrome P4501A (CYP1A) induction were characterized in male C57BL/6N mice aged 10 weeks and 28 months at 7 days after administration of single oral [3H]TCDD doses ranging from 0.015 to 15 microgram/kg body wt. Determinations of hepatic marker enzyme activities for CYP1A1 (ethoxyresorufin O-deethylation, EROD) and 1A2 (acetanilide-4-hydroxylation, ACOH) indicated that the dose response curves for EROD induction by TCDD were nearly identical for the 2 age groups, but the ACOH induction response was greater in old mice. After receiving the 15 micrograms/kg dose, an increase (approximately 35%) in relative liver weight was observed 7 days after dosing in the 10-week mice, but not in the aged mice, and the hepatic concentration of TCDD was approximately 25% greater in young than old mice. No age difference was found in hepatic nuclear concentrations of TCDD. A dose-dependent increase in liver:fat tissue concentration ratios was noted at both ages, and adipose tissue and blood concentrations of TCDD did not vary significantly with age. In old mice however, TCDD concentrations in skin, kidney and muscle were all approximately twice those of young mice at the 15 micrograms/kg dose. These results suggest that advanced age may have differential effects on Ah receptor-mediated enzyme induction, while increased TCDD concentrations in certain tissues may have toxicological implications for older animals.

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.

The effects of inhalation exposure to bromo-dichloromethane on specific rat CYP isoenzymes

Several cytochrome P450 (CYP) isoenzymes may be involved in the metabolism of bromo-dichloromethane (BDCM), a drinking water disinfection byproduct. After 4-h inhalation exposures of male F344 rats to BDCM between 100 and 3200 p.p.m., hepatic microsomal methoxyresorufin demethylase (MROD), ethoxyresorufin de-ethylease (EROD) and pentoxyresorufin dealkylase (PROD) activities showed modest increases at low exposure levels and larger decreases at high exposure levels, compared with controls. Western blots for CYP1A2 and CYP2B1 showed similar trends. In addition, p-nitrophenol hydroxylase (PNP) activity was measured and Western blots for CYP2E1 were performed. CYP2E1 and CYP2B1 isoenzymes are known to metabolize BDCM (Thornton-Manning, J.R., Gao, P., Lilly, P.D., Pegram, R.A., 1993. Acute bromodichloromethane toxicity in rats pretreated with cytochrome P450 inducers and inhibitors. The Toxicologist 13: 361). When compared with a multiple gavage study of BDCM in female F344 rats (Thornton-Manning, J.R., et al., 1994. Toxicology 94, 3-18), the results of the two studies for EROD, PROD, and PNP activities were qualitatively the same; PNP activity did not change, while both PROD and EROD activities decreased at high exposures. In the current work, Western blots for CYP2E1, CYP2B1 and CYP1A2 supported the results from the PNP, PROD and MROD activities, respectively. The decreases in MROD and PROD activities and in Western blots for CYP1A2 and CYP2B1 at high exposures suggest that BDCM may be a suicide substrate for these CYP isoenzymes. Other important conclusions that can be drawn from the comparison between the current and prior work are that the liver response is similar for both sexes, and it is also similar for inhalation and gavage exposures under these conditions. Finally, the decrease in EROD activity at high doses, found in both studies, may be a further reflection of CYP1A2 activity, since little or no CYP1A1 activity is normally found in uninduced rat liver and CYP1A2 is known to metabolize ethoxyresorufin, although much more slowly than CYP1A1.