Grantee Research Project Results
Final Report: Metabolic Fate of Halogenated Disinfection By-Products In Vivo, and Relation to Biological Activity
EPA Grant Number: R825957Title: Metabolic Fate of Halogenated Disinfection By-Products In Vivo, and Relation to Biological Activity
Investigators: Ball, Louise M.
Institution: University of North Carolina at Chapel Hill
EPA Project Officer: Hahn, Intaek
Project Period: January 23, 1998 through January 22, 2001
Project Amount: $460,848
RFA: Drinking Water (1997) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objectives of this research project were focused on: (1) identification and quantitation of macromolecular adducts formed by haloacetic acids; and (2) investigation of the metabolism and disposition of MX. Halogenated by-products of drinking water disinfection are of concern because of their widespread human consumption and the current uncertainty over their health effects. Haloacids as a class are the most abundant of the halogenated disinfection by-products. Many of these compounds are suspect carcinogens, and in addition may form macromolecular adducts that could be useful as biomarkers of exposure to disinfectants. This project attempts to elucidate mechanisms of biological activity and disposition of haloacids.
Summary/Accomplishments (Outputs/Outcomes):
Our initial hypothesis was that the aliphatic two-carbon dihaloacetic acids (DCA) could be metabolically activated to genotoxic intermediates by a glutathione-dependent enzyme pathway, analogous to that involving nucleophilic halide displacement reactions catalyzed by glutathione S-transferase theta and its bacterial analogues, to form thioether conjugates. Such a pathway previously was reported for methylene chloride. Identification of the relevant metabolic pathway could thus guide our selection of appropriate synthetic targets for macromolecular adducts. We used the Ames plate incorporation assay to measure the production of genotoxic species in the form of mutations, and we manipulated the metabolic activation conditions to favor glutathione conjugation, cytochrome P450-linked oxidation, or NAD+-dependent oxidation, by addition of the appropriate co-factors, alone or in combination, corresponding to three possible pathways for generation of species capable of binding to DNA and causing mutations. Bacterial strains were maintained, and mutagenicity assays were carried out, according to the standard protocol (Maron and Ames, 1983). The Salmonella typhimurium variant TA100 was selected for detection of mutations because DCA had previously been reported to be weakly mutagenic in that strain (DeMarini, et al., 1997). As TA100 posesses low endogenous levels of the enzymes responsible for catalyzing the reactions that we postulated would lead to formation of mutagenic species, we supplemented the bacterial culture with:
(a) Aroclor-treated rat liver S9 protein, containing cytochrome P450 isoforms (S9) plus NADPH-generating co-factor mix, to ensure oxidizing activity.
(b) S9 plus NADPH-generating co-factor mix plus glutathione (GSH), to test whether P450 catalyzed oxidation produced species that required glutathione for activation.
(c) S9 plus glutathione.
(d) S9 plus NAD+, to test whether an NAD+-dependent pathway could generate mutagens.
(e) S9 plus NAD+ plus glutathione, to test whether an NAD+-dependent pathway could generate species that required glutathione for activation.
(f) Glutathione alone.
(g) NAD+ alone.
(h) Glutathione plus NAD+, to test whether the pathway running horizontally along the top would generate active intermediates.
Sodium azide was used as a positive control, and gave plate counts within the range of historical values. The haloacetic acids were assayed at doses of 0.05, 0.1, 0.5, and 1 mg/plate. Preliminary studies showed that the first DCA to be tested was toxic to the bacteria at doses above 1 mg per plate.
DCA alone at the dose levels tested did not result in the doubling of the background mutation rate, and also was toxic at the highest dose tested. Under conditions where cytochrome P450-catalyzed oxidation could be expected to occur (a), an increase in mutagenicity was seen at the lowest doses, but this was not consistent across the dose range. When glutathione was added to the active P450 (b), plate counts increased more consistently with increasing dose, but not to the point of doubling the background rate. Where glutathione was added with S9 protein, but without the NADPH that is essential for cytochrome P450 activity (c), again an increase was seen at the lowest dose, but plate counts declined at higher doses, suggestive of toxicity to the bacteria. Conditions (b) and (c) would allow for activity of glutathione transferases present in the S9 fraction.
In the presence of glutathione alone (f), no DCA-induced mutagenicity was seen, only toxicity, suggesting that putative direct glutathione conjugates of DCA would be of interest for their toxicity rather than their capacity to bind to DNA. Hence, thioether adducts can be assigned a low priority for synthesis.
Where NAD+ was included in the bacterial culture along with either S9 protein (d), or with glutathione (h), a consistent dose-related increase in mutagenicity was observed that exceeded double the background rate at the highest dose of DCA. NAD+ alone (g) also increased plate counts but only at the highest dose used, with decreases below background at intermediate dose levels. One possible explanation for this pattern is that NAD+ acts as a co-factor for a bacterial enzyme that activates DCA to both toxic intermediates (inferred from the lowered plate counts seen in [g]) and to genotoxic intermediates that cause mutations and increase plate count. When NAD+ was added in the presence of both S9 and glutathione (e), a trend was seen towards a dose-related increase, but this was not sufficiently marked to be meaningful. Although no overt toxicity or genotoxicity was seen, the higher standard deviations in plate count at the higher doses suggest some DCA-related destabilization of the bacterial growth.
Further investigations directed towards determining optimal levels of NAD+ for generation of mutagenicity and of glutathione for induction of toxicity were carried out by including those agents at higher concentrations in the assay system. Varying the amounts of NAD+ or of glutathione by two-, three-, and four-fold did not affect the plate counts produced by DCA.
We extended these investigations to the brominated analogues: our goal at this stage was to prioritize the different haloacetic acids for in vivo metabolic studies; preference would be given to the most mutagenic compounds, for investigation of potential in vivo DNA adduct formation.
The monohaloacetic acids chloroacetic acid (MCA) and bromoacetic acid (MBA), and the DCAs dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) were assayed over a dose range from 0.05 to 1 mg/plate. These chemicals were obtained as stock items from a commercial source, Aldrich or Fluka, and used as received. Our previous studies has indicated that NAD+ enhanced the mutagenicity of DCA, hypothetically by acting as a co-factor for oxidation of an intermediate alcohol to a carboxyl group. Hence, we supplemented the bacterial culture with NAD+ (8 mM), with glutathione (2 mg/plate), or with both, as indicated. The levels of supplementation were selected based on the results of previous experiments with DCA, in which similar plate counts were obtained from supplementation with 4 to 12 mM NAD+ and from 1 to 5 mg GSH per plate.
MCA did not increase plate counts above background, and was slightly toxic at the highest dose tested. The brominated haloacids each exhibited some interesting features. Monobromoacetic acid clearly was toxic to the bacteria, because few or no colonies (either revertant or background) were able to grow at the two highest doses, and this toxicity was mitigated when glutathione was included in the assay mix. NAD+ also did not appear to have any effect on plate counts with or without glutathione. With DBA, BCA plate counts increased at low doses, just barely reached a doubling of the background rate, and fell off at the highest dose. A similar pattern was seen when glutathione was added alone. In the presence of NAD+, either alone or in combination with glutathione, no increase in plate counts was seen, nor was toxicity especially marked. The only compound of those tested here to exhibit a clear mutagenic response was the mixed haloacid BCA. BCA more than doubled the background rate, consistently, either alone or in the presence of NAD+. This increase in plate counts was not observed when GSH was added, either alone or in combination with NAD+.
In a further series of experiments originally designed to ensure that the co-factor NAD+ was available to the bacteria by pre-incubating them in its presence for 30 minutes, a more consistently positive dose-response was seen with DBA, almost doubling the background at the highest dose, with no overt signs of toxicity. Virtually identical results were obtained with and without pre-incubation with NAD+, hence increased availability of NAD+ was unlikely to contribute to the effect. Our next hypothesis that could explain these findings is that proteins or other co-factors leaked from less-viable cells during the pre-incubation somehow mitigated the toxicity of DBA. We tested this hypothesis by carrying out experiments in which the bacterial cell walls were permeabilized by treatment with CaCl2 prior to exposure to DBA test compounds. Again, virtually identical results were obtained, whether or not the incubations were supplemented with NAD+ or with GSH, which also might be expected to exert a protective effect.
Synthesis of putative DNA adducts has been unproductive, as have experiments with MX; hence, our explicit original specific aims have not been met.
Conclusions:
Originally, we had developed the hypothesis that a glutathione-dependent pathway could contribute to formation of genotoxic metabolites from DCAs. We showed that the presence of glutathione does not enhance the mutagenicity of haloacetic acids in the Ames plate incorporation assay. However, while although glutathione appeared to increase the toxicity of DCA, for the brominated species MBA and BCA, the effects of glutathione amounted to detoxication, in the form of a decrease in toxicity for MBA and of mutagenicity for BCA. Thus, interaction with glutathione is biologically important for this class of compounds; the outcome of that interaction would appear to differ depending on the nature of the halogen substituent. Thioether conjugates derived from haloacetic acids are unlikely to contribute greatly to the genotoxicity of these compounds. Nevertheless, sufficient evidence has been developed to show that glutathione-dependent pathways play important, and perhaps different, roles in the overall biological activity of the haloacetic acids. Overall BCA, though not the most abundant DCA produced from drinking water disinfection, would appear to be the most mutagenic HAA.
References:
Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutation Research 1983;113:173-215.
DeMarini DM, Abu-Shakra A, Felton CF, Patterson KS, Shelton ML. Mutation spectra in Salmonella of chlorinated, chloraminated, or ozonated drinking water extracts: comparison to MX. Environmental and Molecular Mutagenesis 1995;26:270-285.
Supplemental Keywords:
drinking water, exposure, effects, metabolism, dose-response, carcinogen, mutagen, mammalian, animal, organism, enzymes, chemicals, environmental chemistry, environmental biology, analytical., RFA, Scientific Discipline, Water, Chemical Engineering, Environmental Chemistry, Chemistry, Analytical Chemistry, Biochemistry, Drinking Water, Biology, halogenated disinfection by-products, biomarkers, bacterial mutagen, exposure and effects, animal model, chemical byproducts, disinfection byproducts (DPBs), exposure, community water system, treatment, carcinogenicity, genotoxicity, haloacetic acids, macromolecular adducts, metabolism, drinking water contaminants, drinking water system, ratProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.