Effects of chloroform and bromodichloromethane on DNA synthesis in male F344 rat kidney.

We have been investigating the actions of chloroform (CHCl3) and bromodichloromethane (BDCM) in rat kidney after different routes of exposure. Male F344 rats were exposed by gavage with corn oil or water as the diluting vehicle. All experiments lasted 30 days with gavage exposures 5 days per week for 4 weeks (20 doses). All animals were injected IP with bromodeoxyuridine (BrdU) 3 times over a 6-day period at 50 mg/kg/injection. Kidney tissue was fixed and slides were stained with hematoxylin and eosin for routine viewing and by the PAP (peroxidase-antiperoxidase) technique using anti-BrdU to label cells in DNA synthesis. There were no significant changes in gross parameters evaluated between the control rats and the rats exposed to CHCl3 or BDCM. Rats exposed via corn oil gavage to CHCl3 displayed a segment-specific epithelial cell necrosis (6/6 high dose, 2/6 low dose). The lesions were primarily localized to the second segment of the proximal tubule, although some spread to cells in the first segment was occasionally observed. No histologic lesions were observed in the kidneys of rats exposed to BDCM. Preliminary results indicate a significant increase in DNA synthesis in the CHCl3-treated rats and a slight increase in DNA synthesis in BDCM-treated rats with corn oil as the diluent. The increase in BrdU labeling was primarily in cells of the S2 segment of the proximal tubule and interstitial cells of CHCl3-exposed animals and in cells of the S3 segment of BDCM-exposed animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Chloroform (CHCl3) and bromodichloromethane (BDCM) are trihalomethanes that are commonly detected at low levels in public water supplies (1)(2)(3). CHCl3 induces hepatic and renal toxicity in mice and renal toxicity in male rats (4)(5)(6)(7)(8)(9). BDCM induces renal toxicity in rats and hepatic toxicity in mice (10)(11)(12). Both chemicals have been shown to be metabolized by cytochrome P450, although the active metabolite has only been identified for CHCl3 (13)(14)(15). BDCM (but not CHCl3) has been demonstrated to be mutagenic (16). CHCl3 produces site-specific degeneration and necrosis of epithelial cells in the proximal tubules of rodent kidneys (17)(18)(19), although high doses may also induce damage in the distal nephron. BDCM administered by intragastric gavage at a dose of 10 mg/kg for 10 days did not induce any histopathologic changes in the kidneys of male F344 rats (10). Both CHCl3 and BDCM were shown to be tumorigenic to male rat kidney (4,11,20,21). CHCl3 was also carcinogenic to male and female mouse liver (20,21), and BDCM was carcinogenic in female rat kidney, male and female rat colon/rectum, male mouse kidney, and female mouse liver (16). Little information is available on the effects of either chemical on DNA synthesis in the kidney. However, because CHCl3 is nonmutagenic and nephrotoxic, one may speculate that compensatory cell replication due to renal cell necrosis may play a role in its carcinogenicity. The effect of BDCM on renal DNA synthesis has not been described. The purpose of the present study was to evaluate the effects of subchronic gavage administration of CHC13 and BDCM on DNA synthesis in male rat kidneys at doses that were carcinogenic in chronic bioassays. We also compared the effects of each chemical when administered by corn oil gavage and water gavage.

Animals
Male F344 rats (150-200) were purchased from Charles River Laboratories (Wilmington, MA). They were singly housed in polycarbonate cages with food and water available ad libitum. Temperature and humidity were within recommended ranges for the species, with 12 air changes/hr and a 12-hr light cycle. The animals were observed once a day for signs of morbidity. Before entry in the study, the animals were quarantined for 2 weeks.
The results reported here are a subset of a larger study. This work was performed as two experiments. For both experiments, four groups, each consisting of six rats, were evaluated for histopathologic alterations. A minimum of three rats from each group were also used to quantify DNA synthesis [as determined by labeling with bromodeoxyuridine (BrdU)] All animals were anesthetized with sodium pentobarbital (50 mg/kg, IP), the kidneys were rapidly removed and sectioned into 1-mm thick sections for fixation. The animals were killed by exsanguination while anesthetized.
Chemicals and Dosing CHC13 and BDCM were purchased from Aldrich Chemical Company, Milwaukee, WI. In the first experiment, CHC13 (180 and 90 mg/kg) or BDCM (100 mg/kg) was administered by gavage in corn oil 5 days/week for 4 weeks. In the second experiment, both chemicals were administered by gavage in water for the same time and at the same doses as in the first experiment. Control animals for each experiment were gavaged with the appropriate solvent (equal volume).
The last dose in each experiment was administered the day before sacrifice. BrdU (50 mg/kg) was administered by IP injection every other day over a 6-day period, the last dose being given the day before sacrifice.

Pathology and BrdU Labeling Data
A limited necropsy was performed on each animal with emphasis on the kidneys and liver. The kidneys were removed, weighed, cut in cross-section into 1to 2-mm thick slices, and fixed in 4% phosphate-buffered formaldehyde. Two sections from the same area of the left and right kidneys from each animal were mounted in the same paraffin block for sectioning. Sequential sections were taken for routine hematoxylin and eosin staining and for immunohistochemical staining using the peroxidase-antiperoxidase method with a monoclonal antibody to BrdU. For each experiment, all sections were stained at the same time for BrdU according to methods described by Reimschuessel et al. (22).
All sections were viewed under high power, and consecutive random fields from the cortex and the outer stripe of the outer medulla were reviewed. Labeled and unlabeled nuclei were counted in the following nephron segments: glomerulus, interstitium, distal tubule, collecting duct, the third segment (S3) of the proximal tubule, and the combined first and second segments (Si, S2) of the proximal tubule. Data are expressed as the percentage of total nuclei that stained for BrdU incorporation in each segment.
Results Pathology Animals exposed to CHCl3 by corn oil gavage displayed acute cell injury and necrosis, primarily in the epithelial cells lining the S2 segment of the proximal tubule. Although the extent of the injury made it difficult to determine, it appeared that some necrosis also occurred in the cells of the S1 segment. The injury was present in six of six animals exposed to the high dose of CHCl3 and in two of six animals exposed to the low dose of CHCl3. The morphological appearance of the S3 segment of the proximal tubule and the other nephron segments was unaffected by CHCl3 exposure. Animals exposed to BDCM by corn oil gavage did not display any significant renal pathology related to chemical treatment.
Animals exposed to CHCl3 with water as the vehicle showed minimal pathologic alterations in the kidneys. There was mild injury and necrosis in cells of the S2 segment in one of six animals in the high-dose group. No lesions were seen in the animals in the low-dose group. Animals exposed to BDCM with water as the vehicle did not display any pathological alterations in the kidneys. Table 1 summarizes the DNA labeling data for animals exposed by corn oil gavage. There was a dosedependent increase in total labeling of nuclei in renal cells of the CHCl3-treated animals compared to the DNA SYNTHESIS IN RAT KIDNEY control animals. The BDCM-treated animals had a slight increase in labeled cells. In the CHC13-treated animals, the largest increase in labeling was in the cells of the S2 segment of the proximal tubule (43.5% and 19.9% compared to 2.5% for the control animals). There was also an increase in labeling in the interstitial cells.

DNA Labeling
The 90 mg/kg CHC13-treated animals also had an increase in labeled cells in the S3 segment of the proximal tubule, but this was not seen in the high-dose animals. The BDCM-treated animals had an increase in labeling primarily in the cells of the S3 segment of the proximal tubule and a mild increase in labeling in the interstitial cells.
In the animals exposed by the water gavage (Table  2), there was little to no change in labeling of renal cells by the BrdU method. Small increases were seen in the labeling of cells in the S3 segment of the proximal tubule, collecting duct, and the interstitial cells in animals exposed to BDCM. In general, the control animals in the water gavage experiment had lower levels of DNA labeling compared to the control animals in the corn oil gavage experiment. Discussion CHC13 and BDCM induced renal adenomas and carcinomas when tested in chronic rodent bioassays (4,16,20,21). Although BDCM is mutagenic (16), CHC13 shows no mutagenic potential. CHC13 is nephrotoxic in rats and mice, although the extent of toxicity varies among different strains and sexes of animals (6-8). CHC13 induces renal neoplasms in male rats; however, it exhibits only hepatocarcinogenicity in mice (4,20,21).
The mechanism of toxic action of CHC13 has been correlated to its metabolism by cytochrome P450dependent mixed-function oxidases (6,8). Inhibitors of mixed-function oxidase activity decreased the nephro- toxicity of CHC13 (7). CHC13 was carcinogenic to rat kidney when administered by corn oil gavage or in the drinking water. In the present experiments, CHC13 displayed potent nephrotoxic effects when given by corn oil gavage, but not when given by water gavage. Only one of six animals displayed any morphological evidence of toxicity. In all cases the cell degeneration and necrosis was focused in the cells of the S2 segment of the proximal tubule, with no toxicity in the S3 segment, distal tubules, or collecting ducts. Increases in DNA synthesis (as determined by immunostaining for BrdU incorporation) closely paralleled the pattern of toxicity, with increased labeling primarily in cells of the S2 segment of the proximal tubule. In addition, increases in DNA synthesis were evident only in animals exposed by corn oil gavage, with only minimal increases in labeling noted in animals exposed by water gavage. In the Fischer rats used in this study, it seems that the vehicle for dilution of CHC13 is directly related to the potential for nephrotoxicity. This was not the case with Osborne-Mendel rats, in which the production of renal neoplasms occurred when CHC13 was administered by corn oil gavage or in the drinking water (4,23). Tumasonis et al. (12), however, also found no significant increase in renal lesions in Wistar rats chronically exposed in the drinking water for up to 180 weeks. Strain differences may play a large role in the sensitivity to the toxic and carcinogenic potential of this trihalomethane.
BDCM is a classic carcinogen with mutagenic potential (16). The effect of corn oil and water gavage exposure on DNA synthesis was similar. A mild increase in DNA synthesis was observed in both experiments. The increase was centered in the S3 segment of the proximal tubule. The increase in DNA labeling in the S3 segment of the proximal tubule by BDCM is consistent with the site of origin of some chemically induced renal carcinomas that we have previously proposed (24,25). It is interesting to note that the level of DNA labeling in the corn oil gavage was generally higher than in the water gavage. This was noted for the BDCM-treated animals as well as for the control animals. The additional studies under way in our laboratory will help to determine if this is, in fact, the case or if it was just an anomaly in these reported experiments.