* Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina; and
Department of Biology, North Carolina Central University, Durham, North Carolina
Received February 16, 2000; accepted April 11, 2000
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ABSTRACT |
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Key Words: disinfection by-products; intestinal microflora; bioactivation; nitroreductase; azoreductase; dechlorinase; intestinal metabolism; dichloroacetic acid; dibromoacetic acid; bromochloroacetic acid; bromochloroacetic acid.
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INTRODUCTION |
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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., 1995).
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., 1992). 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, 1980
; 1984
). Treatment of rats with oligofructose or inulin, in combination with bifidobacteria, reduced colon aberrant crypt formation (Gallaher and Khil, 1999
; Rowland et al., 1998
). Inulin and Bifidobacterium longum treatment was associated with decreased ß-glucuronidase activity (Rowland et al., 1998
). The lactic acid bacteria have been shown to be protective against genotoxic damage in vitro and in vivo (Pool-Zobel et al., 1993a
,b
).
Epidemiology studies have linked human consumption of chlorinated drinking water to bladder, kidney, and gastrointestinal cancers (Koivusalo et al., 1994; Morris et al., 1992
). Drinking water disinfection by-products (DBPs) have been associated with cancer in laboratory rodents and humans (Bull et al., 1990
; DeAngelo et al., 1991
, 1996
). 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.
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MATERIALS AND METHODS |
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Animal Treatment
Twenty-one-dayold 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, 1990) 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 70130%. 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., 1977), weighed, homogenized under CO2, and stored on ice. Next, the homogenized tissues (0.10.2 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., 1993
). 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, 1995; 1998
). 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.
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RESULTS |
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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.
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DISCUSSION |
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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., 1996). 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., 1997
). 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., 1993
; Chang et al., 1994
). 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., 1994). 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., 1994) 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, 1998) and catabolite repression has been described in many bacterial species (Saier, 1998
). 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, 1997
).
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, 1989). 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., 1971, 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., 1996
; Wadstrom and Ljungh, 1991
). 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., 1997
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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.
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