The Disinfection By-Products Dichloro-, Dibromo-, and Bromochloroacetic Acid Impact Intestinal Microflora and Metabolism in Fischer 344 Rats upon Exposure in Drinking Water

S. E. George*,1, G. M. Nelson*, A. E. Swank*, L. R. Brooks*, K. Bailey{dagger}, M. George* and A. DeAngelo*

* Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; and {dagger} Department of Biology, North Carolina Central University, Durham, North Carolina

Received February 16, 2000; accepted April 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human consumption of chlorinated drinking water has been linked epidemiologically to bladder, kidney, and rectal cancers. The disinfection by-product (DBP) dichloroacetic acid is a hepatocarcinogen in Fischer 344 rats and B6C3F1 mice. The objective of this study is to determine the effect of the DBPs dichloro-, bromochloro-, and dibromoacetic acids (DCA, BCA, DBA) on intestinal microbial populations and their metabolism, with emphasis on enzymes involved in the bioactivation of procarcinogens and promutagens. One-month-old male Fischer 344 rats were provided water ad libitum containing 1 g/l DCA, BCA, or DBA for up to 5 weeks. At 1, 3, and 5 weeks of treatment, ß-glucuronidase (GLR), ß-galactosidase (GAL), ß-glucosidase (GLU), nitroreductase (NR), azoreductase (AR), and dechlorinase (DC) activities were determined in cecal and small and large intestinal homogenates. After 5 weeks of treatment, intestinal populations were enumerated on selective media. Cecal GAL (DCA, BCA, DBA) and GLR (DCA, DBA) activities were reduced after 1 and 3 weeks of treatment and GAL activity was elevated at 5 weeks (BCA). Large intestinal GAL (DCA, BCA) and GLU (DCA, BCA, DBA) activities were elevated after 5 weeks of treatment. Week 5 cecal AR (DCA, BCA, DBA), NR (DCA), and DC (DCA, DBA) activities were reduced. Even though some significant changes in intestinal populations were observed, use of selective media was not sensitive enough to explain fluctuations in enzyme activity. Haloacetic acids in the drinking water alter intestinal metabolism, which could influence bioactivation of promutagens and procarcinogens in the drinking water.

Key Words: disinfection by-products; intestinal microflora; bioactivation; nitroreductase; azoreductase; dechlorinase; intestinal metabolism; dichloroacetic acid; dibromoacetic acid; bromochloroacetic acid; bromochloroacetic acid.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme activities of the normal mammalian intestinal microbial population are involved in the bioactivation of many procarcinogens and promutagens (Chadwick et al., 1992Go). For example, both nitroreductase and ß-glucuronidase activity are required for 2,6-dinitrotoluene genotoxicity, shown by the use of conventional and germ-free animals (George et al., 1994Go; Mirsalis et al., 1982Go). The intestinal flora (and specifically, ß-glucosidase activity) are required for the formation of quercetin, a mutagenic compound, from rutin, a nonmutagenic glycoside found in many plants (Brown and Dietrich, 1979Go). Another nongenotoxic plant glycoside, cycasin, is hydrolyzed to a genotoxic product, methylazoxymethanol (MAM), by the intestinal flora (Goldin, 1986Go). Cycasin causes tumors in rats with normal intestinal flora, but not in germ-free rats (Laquer et al., 1967Go). Microbial azoreductase can reduce environmental azo-compounds to genotoxic metabolites (Chung et al., 1992Go). Moghaddam et al. (1996) have suggested that microbial dechlorination may be involved in the metabolism of trichloroacetic and dichloroacetic acids in the intestines.

Intestinal metabolism can be affected by chemical treatment. Chadwick et al. (1991) reported that pentachlorophenol treatment of Fischer 344 rats resulted in elevation of nitroreductase activity and reduction in ß-glucuronidase and ultimately impacted the genotoxicity of 2,6-dinitrotoluene. Five weeks of atrazine treatment produced elevated large intestinal nitroreductase and ß-glucuronidase activities and potentiated urine mutagenicity in rats co-treated with 2,6-dinitrotoluene (George et al., 1995Go).

Chemical and biological treatments can alter the composition of the intestinal microbiota which impacts intestinal metabolism. 2,4,5-trichlorophenoxyacetic acid eliminated Lactobacillus fermentum and elevated levels of oxygen-tolerant lactobacilli and unidentified aerobes from the intestinal tract of rats after 1 week of treatment. This change in microbiota was accompanied by a decrease in 2,6-dinitrotoluene-related urine mutagenicity (George et al., 1992Go). L. acidophilus treatment has been associated with reduction of fecal ß-glucuronidase, azoreductase, and nitroreductase activities and protection against 1,2-dimethylhydrazine colon carcinoma (Goldin and Gorbach, 1980Go; 1984Go). Treatment of rats with oligofructose or inulin, in combination with bifidobacteria, reduced colon aberrant crypt formation (Gallaher and Khil, 1999Go; Rowland et al., 1998Go). Inulin and Bifidobacterium longum treatment was associated with decreased ß-glucuronidase activity (Rowland et al., 1998Go). The lactic acid bacteria have been shown to be protective against genotoxic damage in vitro and in vivo (Pool-Zobel et al., 1993aGo,bGo).

Epidemiology studies have linked human consumption of chlorinated drinking water to bladder, kidney, and gastrointestinal cancers (Koivusalo et al., 1994Go; Morris et al., 1992Go). Drinking water disinfection by-products (DBPs) have been associated with cancer in laboratory rodents and humans (Bull et al., 1990Go; DeAngelo et al., 1991Go, 1996Go). The DBP dichloroacetic acid (DCA) is a hepatocarcinogen in B6C3F1 mice and Fischer 344 rats. The carcinogenic activity of dibromoacetic (DBA) and bromochloroacetic (BCA) acids has not been evaluated.

The purpose of this study is to determine if a carcinogenic dose of dichloroacetic acid (DCA) and relative concentrations of bromochloro- and dibromoacetic acids (BCA, and DBA) alter the intestinal microbial populations and their metabolism, with emphasis on the enzymes often involved in the bioactivation of procarcinogens and promutagens. If the DBPs impact intestinal metabolism, they could alter the bioactivation of promutagens and procarcinogens present in the environment.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Dichloroacetic acid (DCA, CAS#79–43–6, 99+%), dibromoacetic acid (DBA, CAS#631–64–1, 97%), and bromochloroacetic acid (BCA, CAS#5589–96–8, 97%) were purchased from Aldrich Chemical Company (Milwaukee, WI). The enzyme substrates p-nitrophenyl-ß-D-glucuronide (CAS#137629–36–8), p-nitrophenyl-ß-D-glucopyranoside (CAS#2492–87–7), and p-nitrophenyl-ß-D-galactopyranoside (CAS#3150–24–1) were obtained from Sigma Chemical Company (St. Louis, MO) and 3,4-dichloronitrobenzene(CAS#99–54–7, 99%), 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane (p,p`-DDT,CAS#5029–3, 98% ), and methyl orange (CAS#547–58–0, 85+%) were from Aldrich Chemical Co. Unless indicated below, all other chemicals were of reagent grade and obtained commercially.

Animal Treatment
Twenty-one-day–old male Fischer 344 (CDF) rats were obtained from Charles River Laboratory (Raleigh, NC) and acclimated for 12 days. Animals were housed 3 per cage and provided food (Purina 5001, Purina Mills, St. Louis, MO) and water with or without 1 g/l haloacetic acid ad libitum. Rats (24/treatment) were assigned to DCA, DBA, BCA, and control treatment groups. Animals were weighed at 1, 3, and 5 weeks of treatment.

Drinking Water Analyses
Water consumption per cage and treatment was measured over a 4-day period prior to weeks 1, 3, and 5 of treatment, and were averaged. Final water intake was determined by averaging the values obtained at 1, 3, and 5 weeks and calculated per kg body weight. Theoretical treatment, based on the theoretical concentration in the bottle as 1 g/l, was determined.

Actual chemical consumption was determined after chemical analysis of DCA-, DBA-, or BCA-water samples. Aqueous DCA dosing samples were analyzed by a reverse phase direct injection of 25 µl of the sample on a Waters Corporation (Milford, MA) HPLC-PDA. Samples were eluted isocratically at 0.6 ml/min with a 0.5 % phosphoric acid solvent from a 300 mm x 7.8 mm anion exclusion column manufactured by Alltech Associates, Inc. (Deerfield, IL) stabilized at 25°C. The instrument was calibrated with aqueous DCA. The PDA software (Millinium32, Waters Corp., Deerfield, IL ) was set to derive a maximum absorbance plot between 190 and 240 nm. BCA and DBA were analyzed by EPA method 552 (U.S. EPA, 1990Go) using a Hewlet Packard 5890 gas chromatography system equipped with an ECD detector and an RTX-1701 column (Restek Corporation, Bellefonte, PA). An instrument performance check (IPC) was done daily and a laboratory fortified sample matrix (LFSM) was run every 5 samples after the initial 10 samples. The IPC was used to ensure the calibration was still within +/– 15%. For this analysis, the IPCs were at 102 ± 5% for DBA and 101 ± 5% for BCA throughout the analyses. The LFSM was used to determine whether the sample matrix contributed bias to the analytical results. The acceptable recovery of this method was 70–130%. For this analysis, the LFMs were at 85 ± 15% for DBA and 90 ± 7% for BCA.

Enzyme Assays
Azoreductase (AR), nitroreductase (NR), dechlorinase (DC), ß-glucuronidase (GLR), ß-galactosidase (GAL), ß-glucosidase (GLU) activities were determined in control and treated rats after 1, 3, and 5 weeks of treatment. The animals were euthanized by CO2 asphyxiation and the intestinal tract removed aseptically. The small intestine, cecum, and large intestine (tissue and contents) from each animal were placed individually into preweighed tubes containing 20 ml (small intestine and cecum) or 10 ml (large intestine) of pre-reduced buffer (2.0 g gelatin, 0.5 g cysteine, 500 ml deionized water, and 500 ml salts solution [0.1 g anhydrous CaCl2, 0.1 g anhydrous MgSO4, 0.5 g K2HPO4, 5.0 g NaHCO3, and 1.0 g NaCl]) (Holdeman et al., 1977Go), weighed, homogenized under CO2, and stored on ice. Next, the homogenized tissues (0.1–0.2 g tissue/ml) were placed into an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI) with an atmosphere of 85% N2, 5% CO2, and 10% H2. For AR, NR, and DC, a 1-ml aliquot of the tissue homogenate was pipetted into a 16 x 125 mm tube containing 5 ml of the pre-reduced buffer described above. The screw top tubes, each fitted with a butyl rubber septum (Bellco Glass, Inc., Vineland NJ), were removed from the chamber, placed on ice, and 25 µl of the enzyme substrate mix (25 mg/ml dichloronitrobenzene, 12.5 mg/ml p,p`-DDT, and 23 mg/ml methyl orange) was introduced into each tube, using a syringe (Chadwick et al., 1993Go). The enzyme reactions were initiated by incubation in a 37°C water bath with slow mixing. After 1 h of incubation, the reactions were terminated by placement on ice. The resulting metabolites were extracted, derivatized and analyzed by gas chromatography according to the method of Chadwick et al. (1993).

For determination of GLR, GAL, and GLU activities, 100 µl of small intestinal or 20 µl cecal or large intestinal tissue homogenate was injected into 16 x 125 mm screw-top tubes with a butyl rubber septum containing 9.9 or 10.0 ml of reduced buffer. A 37.5 µl aliquot of substrate mix containing 170 mg/ml of the NR competitor 3,4-dichloronitrobenzene and 130 mg/ml of the specific substrate (GLR, p-nitrophenyl-ß-D-glucuronide; GAL, p-nitrophenyl-ß-D-galactopyranoside; GLU, p-nitrophenyl-ß-D-glucopyranoside, 3,4-dichloronitrobenzene) was injected into each tube and incubated at 37°C for 1 h. Reactions were terminated by placing the tubes on ice. Next, the particulate was sedimented by centrifugation at 3000 x g and absorbancy at 405 nm was read in a Spec20+ (Spectronic Instruments, Inc., Rochester, NY). For each enzyme activity, results from 6 animals per time x treatment combination were averaged. Activity for enzymes is reported as µg metabolite/g tissue/h and metabolites are as follows: NR, 3,4-dichloroaniline; AR, N,N-dimethyl-p-phenylenediamine; DC, p,p`-DDE, and GLR, GAL, GLU, p-nitrophenol.

Enumeration of Intestinal Microflora
Tissue homogenates (3 rats per treatment group) described in the previous section were maintained in the anaerobic chamber. Dilutions (-2, –4, –6) were prepared in reduced buffer and the appropriate dilution (determined in a preliminary experiment) was duplicate-plated onto selective media (Nelson and George, 1995Go; 1998Go). MacConkey plates (Mac; Difco, Detroit, MI), selective for lactose fermenting and non-fermenting enterobacteria, were counted after a 24-h aerobic incubation at 37°C. KF Streptococcus agar (KF; Unipath Co., Oxoid Division, Ogdensburg, NY) was incubated aerobically at 37°C for 48 h. Blood Agar (total anaerobic count; BA), BA supplemented with vancomycin and kanamycin (selective for obligately anaerobic gram negative rods; VK), and Rogosa plates (selective for lactobacilli, Difco) were counted after a 72-h anaerobic incubation at 37°C.

Bacterial counts/g tissue were determined and values reported represent an average of 3 rats/time/treatment unless otherwise indicated.

Statistical Methods
A 2-way analysis of variance (Sigma Stat Version 2.0, San Rafael, CA) was used to determine if there was a treatment or time effect on body weight, tissue weight (tissue weight/body weight x 100), and intestinal enzymes. A one-way analysis of variance was used to determine treatment effects on microflora counts. To isolate groups that were statistically different from the others, a multiple comparison procedure (Student-Newman-Keuls method, Sigma Stat Version 2.0) was used. In all cases, treatments were considered significantly different if p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drinking Water Consumption and DBA, DCA, or BCA Dose
The treated-rat groups had greater water intake than the control-group water consumption (DBA > BCA > DCA; Table 1Go). Therefore, increased consumption was directly related to an increase in the mean daily dose. Even though the target concentration of each chemical was 1 g/l, actual daily doses were 87%–96% of the target value. When the actual doses were converted to molar equivalents, rats ingested approximately 20% less BCA and DBA compared to those that received DCA.


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TABLE 1 Dosing and Survival Data for Fisher 344 Rats Exposed to DCA, BCA, and DBA
 
Effect of DBA, DCA, and BCA on Animal Body and Tissue Weights
All 3 of the haloacetic acids studied significantly depressed body weight by week 3 of treatment (Table 2Go). However, by week 5, only DBA-treated animals remained affected. Cecal weights (analyzed as percent body weight) were significantly elevated throughout the experiment for all 3 treatment groups (Table 3Go). Small intestine weights were significantly higher throughout the experiment in DBA-treated animals, and at week 1 in BCA-treated animals. Large intestine weights were unaffected by treatment with haloacetic acids.


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TABLE 2 Effect of DCA, DBA, and BCA Treatment on Male Fischer 344 Rat Body Weights
 

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TABLE 3 DBA, DCA, and BCA Treatment Impact on Fischer 344 Rat Intestinal Weight
 
Impact of DCA, BCA, and DBA on Intestinal Microflora
In rats treated with DCA for 5 weeks, significant reductions were seen in the nonlactose-fermenting enterobacteria and enterococci populations of the small intestine, accompanied by a significant increase in the total anaerobic count in the large intestine. No alterations of the microflora were seen in BCA-treated rats. DBA-treated rats had higher counts of obligately anaerobic Gram-negative bacilli in the small intestine and lower counts of enterococci in the large intestine. No alterations in the cecal microflora were seen for any of the treatments. Results are sumarized in Table 4Go.


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TABLE 4 Effect of 5 Week Haloacetic Acid Treatment on the Intestinal Microflora
 
Fluctuations in Intestinal Enzymes following Haloacetic Acid Administration
In the cecum, GLR activity was significantly reduced at weeks 1 and 3 by BCA and DBA treatment (Fig. 1Go). Cecal GAL activity was significantly depressed at weeks 1 and 3 by all three haloacetic acids but a 5-week elevated activity was observed in the ceca of BCA-treated rats. Significant reductions in cecal AR activity were detected at week 5 for all 3 treatment groups. Cecal NR activity was depressed at weeks 1, 3, and 5 by DCA treatment, and at week 1 by BCA and DBA treatment. Cecal DC activity was depressed at weeks 3 and 5 by DCA treatment, and at week 5 by DBA treatment.




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FIG. 1. Impact of DCA, BCA, and DBA treatment on intestinal enzymes. Fischer 344 rats were treated in their drinking water with DCA, BCA, or DBA at a concentration of 1 g/l. Control animals received water only. Animals were sacrificed by CO2 asphyxiation at 1, 3, and 5 weeks of treatment. Tissues were prepared and assayed for enzymes as described by Chadwick et al. (1993). Activities are expressed as follows: Nitroreductase, µg 3,4-dichloroaniline/g tissue/h; azo reductase, N,N-dimethyl-p-phenylenediamine/g tissue/h; ß-glucuronidase, ß-glucosidase, and ß-galactosidase, µg p-nitrophenol/g tissue/h; dechlorinase, µg 1,1`-(2,2-dichloroethylidene)bis[4-chlorobenzene]/g tissue/h. *Significantly different from control values (p < 0.05).

 
In the large intestine, GAL activity was depressed at week 1 by DBA treatment, but elevated at week 5 by DCA and BCA treatment. GLU activity of the large intestine was significantly elevated at week 5 by all three haloacetic acids.

Small intestinal GAL activity was lower at week 1 in BCA- and DBA-treated animals, but was elevated at week 3 by DCA treatment. NR activity in the small intestine was depressed at week 1 by BCA and DBA treatment, but elevated by DCA treatment. A decline in DC activity was detected following 1 week of DBA treatment.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Haloacetic acid concentrations used in this study (1 g/l) were significantly higher that those found in finished drinking water. Actual concentrations of haloacetic acids in finished water vary and are dependent on the source water and disinfection method. For example, Coleman et al. (1991) reported an average of 0.24 ppb BCA in finished (Cl2 treatment) Mississippi River water. Average DBA and DCA were 0.05 ppb and 0.46 ppb. Ozone treatment followed by Cl2 treatment resulted in average concentrations of 0.13 ppb, 0.11 ppb, and 0.30 ppb for BCA, DBA, and DCA. Uden and Miller (1983) reported a range of 34–160 ppb of DCA while Krasner (1989) surveyed 35 water utilities and reported an average of 19 ppb of DCA. Fair (1996) detected concentrations of DCA up to 74 ppb with an average concentration of 15 ppb. The U.S. Environmental Protection Agency regulates DBPs in drinking water and has published the final rule for dichloroacetic acid with a maximum contaminant level goal (MCLG) of 0 ppb for DCA and 300 ppb for TCA (U.S. EPA, 1998). The maximum contaminant level (MCL) for total haloactic acids (MCA, MBA, DBA, DCA, and TCA) is 60 ppb.

Intestinal microflora metabolize halogenated acetic acids. In in vitro cultures inoculated with mouse or rat cecal contents, trichloroacetic acid was dehalogenated to dichloroacetic acid following anaerobic incubation (Moghaddam et al., 1996Go). However, no dichloroacetic acid was detected upon aerobic incubation, suggesting that the strict anaerobes play a role in the dehalogenation process. In vivo studies where the rat intestine was "sterilized" with Colyte® or antibiotic treatment, resulted in the detection of a significantly reduced concentration of DCA compared to that observed from conventional control animals (Moghaddam et al., 1997Go). Furthermore, DeMarini et al. (1994) have suggested a potential model that involves reduction and dechlorination of DCA, which leads to the formation of the reactive chloroacetaldehyde intermediate, which ultimately can form an etheno DNA adduct and a mutational event. Dechlorinase activity has been associated with the intestinal microflora (Chadwick et al., 1993Go; Chang et al., 1994Go). In the current study, dechlorinase activity is observed in all 3 regions of the intestinal tract at all time points, but it seems to reach a plateau by 3 weeks in the young rats. Surprisingly, where a treatment effect is observed, activity is reduced. An induction might have been expected in the treatment groups due to increased concentrations of haloacetic acids available for metabolism. In the current study, dechlorinase activity does not appear to be impacted by changes in intestinal microflora.

In general, elevated enzyme activities are indicative of increased bioactivation of co-exposed promutagens and procarcinogens. However, a decrease in enzymatic activity does not always reflect less bioactivation. Chadwick et al. (1991) reported a decline in small intestinal nitroreductase activity in Fischer 344 rats orally exposed to pentachlorophenol. They proposed that due to decreased nitroreductase activity in the small intestine, less co-administered 2,6-DNT was reduced, leading to an increase in small-intestinal absorption and liver metabolism. The liver metabolites were either excreted or transported by bile to the GI tract where elevated ß-glurucronidase deconjugated the metabolites, and, following enterohepatic circulation, led to an increase in hepatic DNA adducts compared to controls.

General cecal microflora populations were not impacted by 5 weeks of DCA, BCA, or DBA treatment, even though significant reductions in 5-week azoreductase activity were observed for all treatments. In addition, DCA treatment resulted in reduced nitroreductase and dechlorinase activities and a DBA treatment effect was observed for dechlorinase activity. In other studies, changes in microflora have been linked to enzyme production. In the developing rat, nitroreductase and azoreductase activities increase as anaerobes colonize the large intestine (Chang et al., 1994Go). In the small intestines, nitroreductase and azoreductase activities remain constant. In the small and large intestine, elevated levels of ß-glucuronidase activity occur concurrently with the appearance of large numbers of coliforms.

The changes in the intestinal enzymes may be explained by several mechanisms. The haloacetic acids may induce or repress mucosal enzymes. This explanation is less likely because studies in germ-free rats (George et al., 1994Go) revealed that nitroreductase and ß-glucuronidase were negligible and Chadwick et al. (1990) reported similar findings for dechlorinase activity in antibiotic-treated rats. Cecal and large intestinal azoreductase activity was minimal in germ-free rats; however, small intestinal azoreductase activity was equivalent in germ-free and conventional animals. Because microbial activities in the small intestine are typically lower, alterations in mucosal enzymes may have a larger impact, but reduction in cecal and large-intestinal mucosal enzymes most likely would be masked by the microbial activity. In a previous study, Chadwick et al. (1991) suggested a role for reduced small-intestinal mucosal nitroreductase activity in the potentiation of 2,6-dinitrotoluene genotoxicity, because the small intestinal microorganism contribution to the activity was minimal.

Another possible explanation for modulation of intestinal enzyme activity is that the haloacetic acids may alter the activities in specific populations without changing the actual number of microorganisms through enzyme repression or induction. ß-Glucuronidase activity is inducible in Escherichia coli and Bacillus spp (Tryland and Fiksdal, 1998Go) and catabolite repression has been described in many bacterial species (Saier, 1998Go). Germ-free rats associated with human fecal flora, fed a diet of alpha-gluco-oligosaccharides, demonstrated cecal microflora induction of ß-galactosidase and ß-glucosidase activities and repression of ß-glucuronidase activity (Djouzi and Andrieux, 1997Go).

DCA was toxic to non-lactose-fermenting enterobacteria and enterococci in the small intestine. Typically, the duodenum and proximal jejunum are colonized primarily by the lactobacilli. In the distal jejunum and ileum, the lactobacilli remain but increasing numbers of Bifidobacterium spp, Bacteroides spp, and Enteroccus spp. are detected (Drasar, 1989Go). Both DCA and DBA tended to be toxic to the small and large intestinal enterococci. However, cecal populations remained constant. In the small intestine (most likely in the extreme distal region), the elevation in DBA-treated obligately anaerobic gram-negative rods (e.g., Bacteroides spp.) may indicate that DBA treatment is creating a more favorable environment for anaerobes, possibly by decreasing colonization by enterococci.

Disturbing the microbial balance in the intestinal tract can lead to reduced colonization resistance (CR; also known as competitive exclusion, CE), which results in increased colonization by invading pathogens (van der Waaij et al., 1971Go, 1989). Intestinal pathogens such as Aeromonas hydrophila, Shigella sonnei, S. flexneri, and Escherichia coli O157:H7 have been associated with untreated water, thus the need for chlorination (Kramer et al., 1996Go; Wadstrom and Ljungh, 1991Go). Opportunistic pathogens, such as Pseudomonas aeruginosa, Acinetobacter spp., and Xanthomonas maltophilia, may be able to colonize the intestinal tract and cause disease, especially in immunocompromised hosts (Rusin et al., 1997Go). If these microbes escape the disinfection process and CR/CE is reduced due to disinfection or other factors, exposed individuals may be at an elevated risk for infection.

Even though the concentrations of DCA, DBA, and BCA used in this study are considerably higher than those detected in finished drinking water, at 1 g/l the impacts of these haloacetic acids on the small and large intestinal microflora may result in an elevated risk to invasion by pathogenic microorganisms that escape the water disinfection process. No direct correlation was made between changes in specific populations and metabolism in this 5-week study; however, longer exposure to equivalent or more environmentally relevant doses may prove otherwise. Synergistic effects due to contributions from other disinfection by-products in finished drinking water also may impact the intestinal metabolism and microorganisms. The changes in intestinal metabolism suggest that 5 weeks of ingestion of DCA, DBA, and DCA increases the potential for altered bioactivation of co-occuring promutagens and procarcinogens. In future studies, Fischer 344 rats will be treated subchronically with the haloacetic acids and promutagens or procarcinogens. The findings from these investigations will provide insight on the ability of the haloacetic acids to alter the production of bioactive metabolites.


    ACKNOWLEDGMENTS
 
The authors are appreciative of Ms. Gloria Huggins-Clark`s and Ms. Peggy Matthews` valuable technical assistance. The authors would like to thank Drs. Douglas Wolf and Hugh Barton for their critical review of this manuscript and Dr. Marc Mass for his constructive comments.


    NOTES
 
This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at the Environmental Carcinogenesis Division, Mail Drop 68, U.S. EPA, Research Triangle Park, NC 27711. Fax: (919) 541-0694. E-mail: george.elizabeth{at}epa.gov. Back


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 RESULTS
 DISCUSSION
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