Journal of Toxicology and Environmental Health

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Metabolism and distribution of bromodichloromethane in rats after single and multiple oral doses J. M. Mathews , P. S. Troxler & A. R. Jeffcoat To cite this article: J. M. Mathews , P. S. Troxler & A. R. Jeffcoat (1990) Metabolism and distribution of bromodichloromethane in rats after single and multiple oral doses, Journal of Toxicology and Environmental Health, 30:1, 15-22, DOI: 10.1080/15287399009531406 To link to this article: http://dx.doi.org/10.1080/15287399009531406

Published online: 15 Oct 2009.

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Date: 12 November 2015, At: 21:05

METABOLISM AND DISTRIBUTION OF BROMODICHLOROMETHANE IN RATS AFTER SINGLE AND MULTIPLE ORAL DOSES J. M. Mathews, P. S. Troxler, A. R. Jeffcoat

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Center for Bioorganic Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina The disposition of [14C]bromodichloromethane (BDCM) was studied in male Fischer rats after single oral doses of 1, 10, 32, or 100 mg/kg and 10-d repeat oral dosing of 10 or 100 mg/kg/d. Methods were developed to quantitate exhaled 14CO and 14CO2. Bromodichloromethane was extensively (~80-90%) metabolized within 24 h postdosing with ~70-80% of the administered dose appearing as 14CO2 and ~3-5% as 14CO. Urinary and fecal elimination were low, accounting for 4-5% and 1-3% of the dose, respectively. Oral administration of BDCM at a level of 10 mg/kg/d for 10 d did not result in the bioaccumulation or altered disposition of the test chemical, but during the course of the repeat 100 mg/kg/d dosing the rate of production of 14CO2 increased, suggesting that this dose of BDCM induced its own metabolism. Persistence of radiolabeled residues in tissues collected 24 h after single-dose administration was low (3-4% of dose), with the most marked accumulation (1-3% of dose) in liver. Kidney tissue, particularly the cortical region, also contained significant concentrations of residues.

INTRODUCTION Bromodichloromethane (BDCM) and other trihalomethanes form from the reaction of chlorine and bromine with natural organic compounds in water (Rook, 1980) and are widespread contaminants of municipal drinking water supplies. The Environmental Protection Agency (EPA) reports that the mean concentration of BDCM in chlorinated water supplies in the United States is 0.017 mg/l with a range of 0-0.125 mg/l (U.S. EPA, 1979). Balster and Borzelleca (1982) have calculated that for an average 70-kg adult male drinking 2 l/d, intake of BDCM could reach 4 /¿g/kg/d. Over 20 yr this would result in a total dose of 30 mg/kg. Two-year gavage studies conducted by the National Toxicology Program (1987) given clear evidence that 50 and 100 mg/kg/d BDCM is carcinogenic in rats and mice. Significantly increased incidences of adenocarcinoma in large intestine and tubular cell adenocarcinoma in kidney The authors are indebted to Dr. H. B. Matthews for his helpful advice in the conduct of these studies and to Pamela Parker for the preparation of this manuscript. This work was performed under National Institute of Environmental Health Sciences contract NO1-ES-65137. Requests for reprints should be sent to James M. Mathews, Center for Bioorganic Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709-2194. 15 Journal of Toxicology and Environmental Health, 30:15-22, 1990 Copyright © 1990 by Hemisphere Publishing Corporation

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J. M. MATHEWS ET AL.

were observed in both male and female Fischer 344 rats. There were also compound-related increases in the incidence of neoplasms in the liver of female mice and in the kidney of male mice. BDCM has not been the subject of extensive metabolism studies, but oral doses of BDCM have been shown to be metabolized to CO2 in rats and mice (Mink et al., 1986) and to CO and CO2 in rats (Anders et al., 1978). In studies of a closely related compound, chloroform, Mansuy et al. (1977) and shortly thereafter Pohl et al. (1977) demonstrated that CO2 formation from chloroform, catalyzed by hepatic microsomal cytochrome P-450, proceeds with the formation of phosgene as an intermediate. Chloroform hepatotoxicity has been associated with the covalent binding of reactive metabolites to tissue macromolecules. The present study was conducted to determine if formation and accumulation of phosgene during the oxidative metabolism of BDCM could lead to the toxicity observed in the chronic toxicity studies of BDCM. This study also provides the first thorough study of the disposition of BDCM performed to date. MATERIALS AND METHODS Bromodichloromethane, 98 + %, was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). [14C]Bromodich!oromethane was prepared by Amersham (Buckinghamshire, England). Dose formulations were made so as to contain ~10 /¿Ci of radiolabel, an appropriate amount of unlabeled BDCM, and sufficient corn oil in a single dose volume of 5 ml/kg to rats. The radiochemical purity of the [14C]BDCM in dose formulations was determined by reverse-phase HPLC to be 98% on the day used. Adult male Fischer 344 rats (225-325 g) were used in all studies and were fed Certified Purina Rat Chow 5002 and water ad libitum. Doses were administered by intragastric gavage. Rats were housed individually in glass, Roth-type metabolism chambers designed for the separate collection of urine, feces, and expired air. Radiolabeled metabolites in breath were collected separately by drawing 200-500 ml/min of room air by vacuum through the chambers and then through a series of traps. The first two traps in the series each contained 50-80 ml of ethanol. The first trap was maintained at 0°C, and the second at — 60°C. The next two traps each contained 500 ml of 1 N NaOH. Next, the air was dried by drawing it through a tube containing a glass wool matrix cooled to — 60°C, then through a tube containing Drierite desiccant. The dried air was drawn through a tube (1 cm ID x 12 cm) containing ~ 5 g Hopcalite catalyst (Mine Safety Appliances Co., Pittsburgh, Pa.), which converts CO to CO2. The CO2 formed was recovered in the last set of two traps each containing 500 ml of 1 N NaOH solution. Hopcalite was reactivated by heating overnight in an oven at 120°C after a day's use and was stored in a dessicator. Prior to sacrifice, animals were anesthetized with ketamine/xylazine, and blood was removed by cardiac puncture. Rats were sacrificed

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DISPOSITION OF BROMODICHLOROMETHANE IN RATS

17

with an overdose of sodium pentobarbital, and tissue samples (0.1-0.3 g) of adipose, muscle, and skin, and entire kidneys, liver, large and small intestine, and stomach were excised and weighed directly into scintillation vials containing 2 ml Soluene-350 tissue solubilizer (Packard Instrument Co., Inc., Downers Grove, III.). After digestion (1-2 d), samples requiring bleaching were decolorized with 135 /d of 70% perchloric acid and 300 /xl of 30% hydrogen peroxide prior to addition of scintillation cocktail (Scintiverse E, Fisher Chemical Co.). Aliquots of ethanol from breath traps were added directly to vials containing scintillation cocktail. Aliquots of urine, plasma, and NaOH trapping solution were added to empty vials and counted after the addition of scintillation cocktail. Vials containing scintillator and either Soluene-350 or NaOH were stored in the dark overnight before assaying by scintillation counting. RESULTS Despite the volatility of BDCM, material balances in these studies were excellent, and recoveries of radiolabel were >90%. In a check of the methodology for trapping/quantitating carbon monoxide, H14COOH was decomposed with concentrated sulfuric acid in a reaction vessel open to the series of breath traps described in the Materials and Methods section. The 14CO formed was efficiently converted to CO2 and recovered ( — 90%) in the last series of NaOH-containing traps. The effect of dose on the profile of excretion of radioactivity is shown in Table 1. Oral doses of BDCM were extensively (-80-90%) metabolized within 24 h postdosing, with -70-80% of the administered dose appearing as CO2 and - 3 - 5 % as CO. There were no significant differences in the profiles of excretion of radioactivity between the dose levels of 1 and 10 (and 32, data not shown) mg/kg. There was indication of saturation of metabolism at a dose of 100 mg/kg, as evidenced by slower CO2 production. This difference was apparent at 1 h postdosing and particularly striking at 8 h postdosing, where CO2 production accounted for 62 and 66% of the 1 and 10 mg/kg doses, respectively, but only for 33% of the 100 mg/kg dose. At all dose levels urinary and fecal elimination were low, accounting for 4% and 1-3% of the administered doses, respectively. Persistence of BDCM equivalents in tissues collected 24 h after single doses was low (3-4% of dose), with the most marked accumulation (1-3% of dose) in liver (Table 2). Kidney, a target tissue for BDCM, was the next highest in radiochemical concentration of the tissues removed, exhibiting elevated tissue/blood ratios (TBR) of 5-8. Kidneys from animals administered a single oral dose of 32 mg BDCM/kg, 10 mg BDCM/kg/d for 10 d or 100 mg BDCM/kg/d for 10 d, were further dissected to separate cortex from medulla prior to determination of carbon-14 content. The ratios of carbon-14 concentration (cortex:medulla) were 7.9 ± 2.3, 6.4 ± 0.7, and 6.1 ± 1 . 1 , respectively. Renal cortex is the site of neopla-

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J. M. MATHEWS ET A L

TABLE 1. Cumulative Excretion of Radioactivity for 24 Hours after Oral Administration of [14C] BDCM to Male F-344 Rats (n - 4) a Percent of dose

End of interval (h)

Volatiles

CO 2

1 4 8 16 24

2.1 ± 1.5 2.7 ± 1.8

9.5

1 4 8 16 24

2.0 ± 0.8 2.7 ± 1.1

CO

Urine

Feces

Total excreta

2.7 ± 1 . 5

11.6 41.1 68.3 81.8 90.7

0.7 ± 0.2

10.0 44.5 72.1 87.4 94.2

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1 mg/kg

3.0 ± 1.6

2.8 ±1.1

0.5 1 1.5 2 6 8 24

1.5 2.6 3.6 4.2

± 1.2 ±1.7 ±1.8 ±1.9

± 37.0 ± 62.9 ± 76.4 ± 77.5 ± 8.0

39.9 66.0 81.3 82.1 0.4 1.9 3.8 5.5

1.1 3.2 2.2 3.2 3.3

± ± ± ± ±

2.0 3.2 4.0 1.7 1.8

±

0.2 0.9 0.9 1.8

± ±

1.5 ± 0.7 2.7 ± 1.1 3.3 ±1.5 10 mg/kg

4.1 ± 0.2

1.9 ± 0.4 3.4 ± 0.9 4.3 ± 0.2 4.3 ± 1.0 100 mg/kg ND

33.4 ± 7.4 71.0 ± 1.7

2.3 ± 0.7 5.2 ± 0.3

±2.8 ± 1.7 ± 2.9 ± 1.8

± 1.5 ± 3.0 ± 3.9 ± 1.5

± 1.6

1.8 ± 1.2 4.6 ± 1.8 7.6 ± 2.0

0.1 ± 0 0.2 ± 0.1 0.3 ± 0.1 0.6 ± 0.4

5.7 ± 2.1 6.3 ± 2.1

± 1.3

4.1 ±0.2

0.7 ± 0.3

10.0 10.6 42.0 87.3

± ± ± ±

2.9 3.0 8.3 1.6

a

Values are means ± SD.

sias in rats and mice treated with BDCM. In intestine, another major target tissue, the TBR ranged from 2 to 4. There were no statistically significant differences in radiochemical concentration between duodenum, jejunum, and ileum at 1,10, and 100 mg/kg (data not shown). The effects of repeated oral administration of BDCM at a level of 10 and 100 mg/kg/d were also studied. The daily excretion of radiolabel was unchanged during the 10 d of the 10 mg/kg/d dosing. An examination of the production of radiolabeled volatiles, CO2, and CO during the course of d 1, 3, and 10 (Fig. 1) demonstrated that the rate of formation of these metabolites was unchanged. Only about 1% of the administered doses was recovered in the tissues (Table 3). Again, only liver and kidney demonstrated markedly high TBR (14.3 and 6.5, respectively). The ratio of concentration of kidney residues, cortexrmedulla, found in animals administered 10 mg BDCM/kg/d for 10 d was 6.4 ± 0.70. As was the case with the 10 mg/kg/d doses, daily 100 mg/kg oral doses of BDCM were extensively (—75%) metabolized in the 24-h period following each dose. There were, however, significant changes in the initial rate of production of 14CO2 going from d 1 to subsequent days (Fig. 2). The rate of CO2 production was —30% of dose during the 8 h following dosing on d 1, and increased to —60% of dose during that interval for

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TABLE 2. Tissue Distribution of Radioactivity 24 Hours after Oral Administration of [14C]BDCM to Male F-344 Rats (n - Af 1 mg/kg

10 mg/kg

100 mg/kg

Tissue

Tissue/blood ratio

Percent dose in total tissue

Tissue/blood ratio

Percent dose in total tissue

Tissue/blood ratio

Percent dose in total tissue

Adipose Blood Large intestine Small intestine Kidney Liver Muscle Plasma Skin Stomach

0.83 unity 3.33 3.71 4.93 44.46 0.52 2.38 1.28 4.21

0.15 0.12 0.02 0.09 0.07 3.06 0.47 0.14 0.35 0.03

0.42 unity 2.30 2.91 5.98 20.05 0.39 1.99 0.94 3.31

0.09 0.13 0.02 0.07 0.09 1.61 0.41 0.14 0.29 0.02

0.68 unity 2.89 2.45

0.19 0.17 0.02 0.08 0.15 1.20 0.56 0.14 0.37 0.09

Total a

Values are means ± SD.

± ± ± ± ± ± ± ± ± ±

0.04 0.02 0.00 0.02 0.01 0.15 0.07 0.01 0.07 0.01

4.36 ± 0.22

± ± ± ± ± ± ± ± ± +

0.04 0.01 0.00 0.02 0.01 0.09 0.10 0.01 0.09 0.00

2.74 ± 0.14

8.2

11.41 0.42 1.56 0.90 8.33

± ± ± ± ± ± ± ± ± ±

0.06 0.04 0.01 0.03 0.04 0.10 0.07 0.02 0.09 0.05

2.87 ± 0.33

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J. M. MATHEWS ET AL.

O — O Day I A — A Day 3 D ODay 10

o Q

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'(O Q

CO

~T—i—i—n—i—i—r" —I—i—1—i—I—1—i—r

16

24 2

8

16

24

Time at End of Interval (h) FIGURE 1. Comparison of daily excretion of radioactive metabolites in breath. Dose regimen: [14C]BDCM, 10 mg/kg/d orally for 10 days.

the remainder of the 10-d experiment. No other marked changes in disposition were apparent. Only about 1% of the administered radioactivity was recovered in the tissues sampled, with liver and kidney demonstrating high TBR, 14.7 and 13.6, respectively (Table 3). The ratio of carbon-14 concentration in the cortex and medulla was 6.1 ± 1.1 (data not shown). TABLE 3. Tissue Distribution of Radioactivity 10 Days after Daily Oral Administration of [14C]BDCM to Male F-344 Rats3 1 mg/kg/d

100 mg/kg/d

Tissue

Tissue/blood ratio

Percent dose in total tissue

Tissue/blood ratio

Percent dose in total tissue

Adipose Blood Large intestine Small intestine Kidney ' Liver Muscle Plasma Skin Stomach

1.01 unity 1.74 1.91 6.51 14.30 0.59 1.32 1.23 2.01

0.09 0.06 0.01 0.02 0.04 0.46 0.26 0.04 0.16 0.01

1.99 unity 3.03 3.18 13.64 14.72 1.14 2.49 2.21 2.99

0.09 0.03 0.01 0.02 0.04 0.27 0.27 0.04 0.16 0.01

Total a

± ± ± ± ± ± ± ± ± ±

0.01 0.00 0.00 0.00 0.00 0.01 0.02 0.00 0.02 0.00

1.11 ± 0.04

± ± ± ± ± ± ± ± ± +

0.02 0.00 0.00 0.00 0.00 0.02 0.01 0.00 0.01 0.00

0.90 ± 0.03

Values are means ± SD. N - 4 for the 100 mg/kg/d dose and N - 5 for the 10 mg/kg/d dose.

DISPOSITION OF BROMODICHLOROMETHANE IN RATS

21

35 O — O Day 1 A A Day 3 D — G Day 10

in O Q

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Q

16

24 2

Time at End of Interval (h) FIGURE 2. Comparison of daily excretion of radioactive metabolites in breath. Dose regimen: [14C]BDCM, 100 mg/kg/d orally for 10 days.

DISCUSSION AND CONCLUSIONS The data presented describe the disposition of >90% of orally administered doses of bromodichloromethane as exhaled volatiles, CO2, and CO, as well as into urine, feces, and tissue residues. Previous studies did not report or quantitate total CO production and did not achieve recoveries of radiolabel adequate for the delineation of the disposition of this haloform (Mink et al., 1986; Anders et al., 1978). Described here is a convenient room-temperature method for the continuous quantitation of carbon monoxide generated in vivo during multiple-animal, multipleday studies. Other methods of monitoring CO production have relied on measuring carboxyhemoglobin levels in blood, maintaining catalytic columns at temperatures of 160°C or higher, and/or using GC/MS (Kubic et al., 1974). Perhaps the perceived difficulty in trapping/quantifying exhaled carbon monoxide has contributed to the lack of literature citations of CO as a metabolite. Accumulation and changes in disposition of BDCM, an environmental contaminant to which large populations are exposed on a daily basis, were studied in repeat dosing experiments. Oral doses of BDCM were well absorbed and extensively metabolized, mostly to CO2 with a few percent each of the administered dose appearing as carbon monoxide or in urine, feces, and tissues. Following single or repeated dosing, the body burden of BDCM was low, with liver and kidney (particularly the cortical region) exhibiting the highest tissue-

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J. M. MATHEWS ET AL.

to-blood ratios. Like liver, the cortical region of the kidney is enriched in cytochrome P-450, and the known production of phosgene from trihalomethanes by that enzyme may account for those higher levels of tissue residues. Unfortunately, the BDCM-derived residues remaining in the tissues were too low to permit chemical characterization. Oral administration of BDCM at a level of 10 mg/kg/d for 10 d did not result in the bioaccumulation or altered disposition of the test chemical, nor were any histopathological changes in liver or kidney induced by this regimen. Despite the —80% production of CO? from BDCM, presumably through the intermediacy of the reactive intermediate phosgene, and residues in liver and kidney, the metabolic and histological data suggest no immediate toxic consequences of exposure of rats to a total dose of BDCM of 100 mg/kg over a period of 10 d (10 mg/kg/d). At an estimated daily intake of 4 ¿tg/kg/d for a 70-kg adult male it would take 68 yr to ingest this amount of BDCM in drinking water. At a still higher dose of 100 mg/kg/d, BDCM was also extensively metabolized to CO2. The initial rate of CO2 production from daily doses, however, doubled after d 1 and stayed at that rate for the remainder of the 10-d experiment. Therefore, BDCM apparently induces its own metabolism, though no attendant increase in liver weight or toxicity was observed. REFERENCES Anders, M. W., Stevens, J. L, Sprague, R. W, Shaath, Z., and Ahmed, A. E. 1978. Metabolism of haloforms to carbon monoxide II. In vivo studies. Drug Metab. Dispos. 6(5)556-560. Balster, R. L., and Borzelleca, J. F. 1982. Behavioral toxicity of trihalomethane contaminants of drinking water in mice. Environ. Health Perspect. 46:127-136. Kubic, V. L, Anders, M. W., Engel, R. R., Barlow, C. H., and Caughey, W. S. 1974. Metabolism of dihalomethanes to carbon monoxide. Drug. Metabl. Dispos. 2(1):53-57. Mansuy, D., Beaune, P., Cresteil, T., Lange, M., and Leroux, J-P. 1977. Evidence for phosgene formation during liver microsomal oxidation of chloroform. Biochem. Biophys. Res. Commun. 79(2):513-517. Mink, F. L., Brown, T. J., and Rickabaugh, J. 1986. Absorption, distribution and excretion of 14Ctrihalomethanes in mice and rats. Bull. Environ. Contam. Toxicol. 37:752-758. National Toxicology Program. 1987. Toxicology and Carcinogenesis Studies of Bromodichloromethane in F344/N Rats and B6C3F1 Mice. Technical Report No. 321, NIH Publication No. 88-2537. Pohl, L. R., Bhooshan, B., Whittaker, N. F., and Krishna, G. 1977. Phosgene: A metabolite of chloroform. Biochem. Biophys. Res. Commun. 79(3):684-691. Rook, J. J. 1980. Possible pathways for the formation of chlorinated degradation products during chlorination of humic acids and resorcinol. In Wafer Chlorination: Environmental Impact and Health Effects, vol. 3, eds. R. L. Jolly, W. A. Brungs, and R. B. Cumming, pp. 85-98. Ann Arbor, Mich.: Ann Arbor Science. U. S. Environmental Protection Agency. 1979. Part III. Environmental Protection Agency. National Interim Primary Drinking Water Regulations; Control of Trihalomethanes in Drinking Water; Final Rule. Fed. Reg. 44:68624-68707. Received July 25, 1989 Accepted January 6, 1990

Metabolism and distribution of bromodichloromethane in rats after single and multiple oral doses.

The disposition of [14C]bromodichloromethane (BDCM) was studied in male Fischer rats after single oral doses of 1, 10, 32, or 100 mg/kg and 10-d repea...
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