Richard J. Bull

Potential carcinogenic hazards of non-regulated disinfection by-products: Haloquinones, halo-cyclopentene and cyclohexene derivatives, N-halamines, halonitriles, and heterocyclic amines

Drinking water disinfectants react with natural organic material (NOM) present in source waters used for drinking water to produce a wide variety of by-products. Several hundred disinfections by-products (DBPs) have been identified, but none have been identified with sufficient carcinogenic potency to account for the cancer risks projected from epidemiological studies. In a search for DBPs that might fill this risk gap, the present study projected reactions of chlorine and chloramine that could occur with substructures present in NOM to produce novel by-products. A review of toxicological data on related compounds, supplemented by use of a quantitative structure toxicity relationship (QSTR) program TOPKAT®) identified chemicals with a high probability of being chronically toxic and/or carcinogenic among 489 established and novel DBPs. Classes of DBPs that were specifically examined were haloquinones (HQs), related halo-cyclopentene and cyclohexene (HCP&H) derivatives, halonitriles (HNs), organic N-chloramines (NCls), haloacetamides (HAMs), and nitrosamines (NAs). A review of toxicological data available for quinones suggested that HQs and HCP&H derivatives appeared likely to be of health concern and were predicted to have chronic lowest observed adverse effect levels (LOAELs) in the low μg/kg day range. Several HQs were predicted to be carcinogenic. Some have now been identified in drinking water. The broader class of HNs was explored by considering current toxicological data on haloacetonitriles and extending this to halopropionitriles. 2,2-dichloropropionitrile has been identified in drinking water at low concentrations, as well as the more widely recognized haloacetonitriles. The occurrence of HAMs has been previously documented. The very limited toxicological data on HAMs suggests that this class would have toxicological potencies similar to the dihaloacetic acids. Organic N-halamines are also known to be produced in drinking water treatment and have biological properties of concern, but no member has ever been characterized toxicologically beyond bacterial or in vitro studies of genotoxicity. The documented formation of several nitrosamines from secondary amines from both natural and industrial sources prompted exploration of the formation of additional nitrosamines. N-diphenylnitrosamine was identified in drinking waters. Of more interest, however, was the formation of phenazine (and subsequently N-chorophenazine) in a competing reaction. These are the first heterocyclic amines that have been identified as chlorination by-products. Consideration of the amounts detected of members of these by-product classes and their probable toxicological potency suggest a prioritization for obtaining more detailed toxicological data of HQs>HCP&H derivatives>NCls>HNs. Based upon a ubiquitous occurrence and virtual lack of in vivo toxicological data, NCls are the most difficult group to assign a priority as potential carcinogenic risks. This analysis indicates that research on the general problem of DBPs requires a more systematic approach than has been pursued in the past. Utilization of predictive chemical tools to guide further research can help bring resolution to the DBP issue by identifying likely DBPs with high toxicological potency.

Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate

Differing rates and extent of trichloroethylene (TCE) metabolism have been implicated as being responsible for varying sensitivities of mice and rats to the hepatocarcinogenic effects of TCE. Recent data indicate that the induction of hepatic tumors in mice may be attributed to the metabolites trichloroacetate (TCA) and/or dichloroacetate (DCA). The present study was directed at determining whether mice and rats varied in (1) the peak blood concentrations, (2) the area under the blood concentration over time curves (AUC) for TCE and metabolites in blood, and (3) the net excretion of TCE to these metabolites in urine in the dose range used in the cancer bioassays of TCE, and to contrast the kinetic parameters observed for TCE-derived TCA and DCA with those obtained following direct administration of TCA and DCA. Blood and urine samples were collected over 72 hr from rats and mice after a single oral dose of TCE of 1.5 to 23 mmol/kg. The AUC values from the blood concentration with time profiles of TCE, TCA, and trichloroethanol (TCOH) were similar for Sprague-Dawley rats and B6C3F1 mice. Likewise, the percentages of initial TCE dose recovered as the urinary metabolites TCA and TCOH were comparable. Nevertheless, the peak blood concentrations of TCE, TCA, and TCOH observed in mice were much greater than those in rats, while the residence time of TCE and metabolites was prolonged in rats relative to that of mice. DCA was detected in the blood of mice but not in rats. The blood concentrations of DCA observed in mice given a carcinogenic dose of TCE (15 mmol/kg) were of the same magnitude as those observed with carcinogenic doses of DCA. In conclusion, the net metabolism of TCE to TCA and TCOH was similar in rats and mice. The initial rates of metabolism of TCE to TCA, however, were much higher in mice, especially as the TCE dose was increased, leading to greater concentrations of TCA and DCA in mice approximated those produced by carcinogenic doses of the chlorinated acetates makes it highly likely that both compounds play a role in the induction of hepatic tumors in mice by TCE.

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice.

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.

Therapeutic dose as the point of departure in assessing potential health hazards from drugs in drinking water and recycled municipal wastewater

The detection of drugs in drinking water sources has raised questions related to safety. In the absence of regulatory or other official guidance, water utilities are faced with a problem of which drugs should be monitored and the detection limits that should be required. The US FDA summarizes data required for drug approval and post marketing adverse reaction reporting. The use of these data as a means of arriving at concentrations in water where adverse health effects are minimal or non-existent was explored. The minimum therapeutic dose was assumed an appropriate point of departure. Appropriate uncertainty factors could be applied depending upon the qualitative and quantitative nature of the data that are available. Assumptions inherent in US FDAs approval of drugs for use in subsets of the population relative to the broader concerns that arise for exposures of the entire population had to be considered. Additional questions are; whether the drug under consideration is carcinogenic, carries pregnancy and lactation warnings, approval for limited vs. chronic use, exposures to multiple compounds that could act in additive or synergistic ways, and the seriousness of toxicities that are observed. Aside from these considerations, a combined uncertainty factor of 1000 appeared adequate.

Early induction of reparative hyperplasia in the liver of B6C3F1 mice treated with dichloroacetate and trichloroacetate

Trichloroacetate (TCA) and dichloroacetate (DCA) were administered at concentrations of 0, 300, 1000 or 2000 mg/l in the drinking water of male B6C3F1 and male and female Swiss-Webster mice for up to 14 days. At 2, 5 or 14 days of treatment, mice were injected with [3H]thymidine 2 h prior to sacrifice. The livers were examined histologically and autoradiographically and DNA was isolated and counted. As observed in chronic studies dichloroacetate induced a marked increase in liver weights, but only after 14 days of treatment and local necrosis in both B6C3F1 and Swiss-Webster mice. A significant increase in the labeling index of hepatocytes was observed in animals treated with DCA, but only at 14 days of treatment. No such increases were observed in animals treated with TCA. In contrast, significant increases in [3H]thymidine were observed in the livers of both DCA- and TCA-treated animals after 5 days of treatment. This effect remained apparent with TCA after 14 days of treatment. These data support the hypothesis that the tumorigenic effect of DCA is strongly influenced by necrosis and reparative hyperplasia. On the other hand, the carcinogenic effects of TCA appear to be more closely associated with [3H]thymidine incorporation that can be separated from cell division, suggesting an elevated rate of repair synthesis of DNA. Thus the carcinogenic effects of TCA (and perhaps lower doses of DCA) may involve damage to DNA.

Cellular and molecular mechanisms of bromate-induced cytotoxicity in human and rat kidney cells

The mechanisms of bromate (BrO(3)(-))-induced toxicity in Normal Rat Kidney (NRK) and human embryonic kidney 293 (HEK293) cells were investigated. BrO(3)(-) (added as KBrO(3)) induced concentration-dependent decreases in 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) staining after 48 h. BrO(3)(-)-induced necrosis based on tandem increases in annexin V and PI staining. Cell cycle analysis demonstrated that BrO(3)(-) also induced G2/M arrest and nuclear fragmentation, prior to alterations in MTT staining or annexin V and PI staining. Immunoblot analysis demonstrated that the G2/M arrest correlated to induction of phosphorylated (p)-p53, p21, cyclin B1 and p-cdc2. Further, BrO(3)(-) induced time-dependent increases in the activity of the mitogen activated protein kinases p38 and ERK1/2. Treatment of cells with the p38 inhibitor SB202190, but not the ERK1/2 inhibitor PD98059, partially reversed BrO(3)(-)-induced G2/M arrest and decreased BrO(3)(-)-induced p-p53, p21 and cyclin B1 expression. In addition, BrO(3)(-) treatment induced reactive oxygen species (ROS) based on increases in CM-H(2)DCFDA fluorescence. The antioxidant ascorbic acid inhibited BrO(3)(-)-induced p38 activation, G2/M arrest, p-p53, p21 and cyclin B1 expression; however, ascorbic acid had no effect on BrO(3)(-)-induced formation of 8-OHdG, a marker of DNA oxidative damage, whose increases preceded cell death by 24h. These data suggest that ROS mediated MAPK activation is involved in the molecular mechanisms of BrO(3)(-)-induced cell cycle arrest, which occurs independently of 8-OH-dG production. The similar mode of action in both NRK and HEK293 cells suggests that the mechanisms of BrO(3)(-)-induced renal cell death are model-independent.

Differences in the Distribution of Iodine and Iodide in the Sprague–Dawley Rat

Use of iodine as a drinking water disinfectant for extended space flight raises concerns about potential chronic effects on health. A key question is whether the chemical form of iodine might play a role. To address this question the influence chemical form has on the uptake and distribution of radioiodine was studied in fed and fasted rats. Following oral administration of 125I2 or 125I-, blood 125I levels were maximal at 2 hr and reached similar concentrations in fed animals receiving 125I- and fasted animals receiving either 125I2 or 125I-. However, when 125I2 was administered to fed animals the initial levels of 125I into blood were significantly lower than after the other treatments. The half-life of elimination of 125I from the blood appeared independent of the form of iodine administered. The initial distribution of 125I to the thyroid depended sharply on chemical form, being greater when iodide rather than iodine was administered, whether animals were fed or fasted. In fed animals administered I2, this may largely be explained by the increased retention of 125I in the stomach contents. In fasted animals, both stomach content and blood levels of 125I were similar whether I2 or I- was administered. Since thyroid uptake of iodine is specific for I-, this suggests that the form of iodine in the blood was different in animals administered I2. This notion was further supported by the finding that pretreatment of animals with varying concentrations of I- in drinking water was four times as effective in suppressing the uptake of a test dose of 125I- than pretreatment with equivalent concentrations of I2.

The role of dichloroacetate in the hepatocarcinogenicity of trichloroethylene

The induction of hepatic tumors in B6C3F1 mice treated with trichloroethylene (TRI) has been attributed to its metabolism to trichloroacetate (TCA). Trichloroacetate is an effective peroxisome proliferator in mice at blood concentrations that are readily achieved with carcinogenic doses of TRI. Recent data has demonstrated that both TCA and dichloroacetate (DCA) are capable of inducing liver tumors in B6C3F1 mice. Although long recognized as a metabolite of TRI, little attention has focussed on the role DCA might play in the hepatocarcinogenic effects of TRI. There are significant differences in the effects of DCA and TCA on the liver of B6C3F1 mice. Trichloroacetate treatment induces peroxisome proliferation, increases lipid deposition, and results in a marked accumulation of lipofuscin in the liver with long-term exposures. Dichloroacetate induces a markedly enlarged liver associated with a cytomegaly and large accumulations of glycogen. The cytomegaly is associated with the development of focal areas of recurrent liver necrosis which in turn lead to high levels of cell proliferation in the area surrounding these lesions. Induction of peroxisomes with DCA is transitory and the accumulation of lipofuscin is much less evident than with TCA treatment. Studies of TRI metabolism demonstrate that blood levels of DCA produced are sufficient to account for the hepatocarcinogenic effects of TRI. The rather low concentrations of DCA found in the urine of mice treated with TRI relative to TCA concentrations are due to the much more rapid and complete metabolism of DCA. These data do not support the conclusion that the hepatocarcinogenic effects of TRI are simply related to peroxisome proliferation.

Modification of lipoperoxidative effects of dichloroacetate and trichloroacetate is associated with peroxisome proliferation

Pretreatment of male B6C3F1 mice with clofibric acid (CFA) or trichloroacetic acid (TCA) in the drinking water results in a marked decrease in the lipoperoxidative response as measured by the production of thiobarbituric acid reactive substances (TBARS) in mouse liver homogenates following acute dosing with TCA or dichloroacetic acid (DCA). Pretreatment with TCA or CFA also increased palmitoyl-CoA oxidase activity, microsomal 12-(omega) hydroxylation of lauric acid and expression of P450 4A isoforms. At the doses utilized, DCA-pretreatment did not increase the level of P450 4A protein, or markers of peroxisome proliferation. However, DCA-pretreatment did result in enhanced levels of TBARS, following acute dosing with DCA, compared to controls. Pretreatment with DCA, TCA, or CFA did not alter p-nitrophenol hydroxylation (an assay specific for P450 2E1), and no increases in immunodetectable P450 2E1, 4A, 1A1/2, 2B1/2 or 3A1 protein were observed. Assays from CFA- and TCA-pretreated mice suggest that the reduction in the TBARS response seen in TCA-pretreated animals results from activities associated with peroxisome proliferation. This might result from the induction of systems efficient in scavenging of peroxide intermediates or detoxification of aldehyde by-products of lipid peroxidation.

Determinants of Whether or Not Mixtures of Disinfection By-Products are Similar

Reactive chemicals have been used to disinfect drinking waters for over a century. In the 1970s, it was first observed that the reaction of these chemicals with the natural organic matter (NOM) in source waters results in the production of variable, complex mixtures of disinfection by-products (DBP). Because limited toxicological and epidemiological data are available to assess potential human health risks from complex DBP mixture exposures, methods are needed to determine when health effects data on a specific DBP mixture may be used as a surrogate for evaluating another environmental DBP mixture of interest. Before risk assessors attempt such efforts, a set of criteria needs to be in place to determine whether two or more DBP mixtures are similar in composition and toxicological potential. This study broadly characterizes the chemical and toxicological measures that may be used to evaluate similarities among DBP mixtures. Variables are discussed that affect qualitative and quantitative shifts in the types of DBP that are formed, including disinfectants used, their reactions with NOM and with bromide/iodide, pH, temperature, time, and changes in the water distribution system. The known toxicological activities of DBP mixtures and important single DBPs are also presented in light of their potential for producing similar toxicity. While DBP exposures are associated with a number of health effects, this study focuses on (1) mutagenic activity of DBP mixtures, (2) DBP cancer epidemiology, and (3) toxicology studies to evaluate similarity among DBP mixtures. Data suggest that further chemical characterization of DBP mixtures and more systematic study of DBP toxicology will improve the quality and usefulness of similarity criteria.