aqueous vehicle and further suggest that the kidney may be a more

Transcription

1 Acute Renal And Hepatic Toxicity Of Bromodichloromethane (BDCM) In F-344 Rats Following Oral Administration In Different Vehicles. By Patrick David Lilly (Under the direction of Dr. R.A. Pegram and Dr. A. Gold) ABSTRACT BDCM, a water disinfection byproduct, was administered by gavage at 0, 200, and 400 mg/kg in either corn oil or 10% Emulphor to 90- day- old F-344 male rats. Urine was collected from 0-6, 6-12, 12-24, 24-36, and hours. Animals were killed at 24 or 48 hr, and serum collected. In the high dose groups for both vehicles after 48 hours, significant increases were observed in serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), cholesterol and bile acids. At the apparent time of peak hepatotoxicity (48 hours), 400 mg BDCM/kg caused significantly greater elevations in AST, creatinine, and bile acids when administered in corn oil than in 10% Emulphor. Significant interactions between dose and vehicle of administration were noted for serum enzymes AST, ALK, and LDH, indicating that vehicle differences observed in BDCM hepatotoxicity may be dosedependent. At 200 mg/kg, the only significant response of a serum enzyme was a slight increase in LDH following aqueous administration of BDCM. In contrast to the virtual lack of effects on liver function enzymes at 200 mg/kg, urinary enzymes were dramatically elevated at the low dose in both vehicles. At 24 hours, BDCM increased urine ALT (33%), AST (390%), LDH (300%), and alkaline phosphatase (ALK) (230%) in the low dose corn oil group with respective increases of 75%, 590%, 540%, and 380% in the 10% Emulphor group. Significant vehicle differences were noted for urinary AST and total protein at the 200 mg/kg dose after 36 hours and glucose at the high dose level at 48 hours. Significant interactions between BDCM dose and dosing vehicle were observed for urinary AST, LDH, and total protein at 36 hours post-gavage and total protein at 24 hours, suggesting again that vehicle effects noted in BDCM nephrotoxicity may be dependent on dose. At 400 mg/kg, the time to peak nephrotoxicity appeared greater with corn oil than with the aqueous vehicle. These data suggest that BDCM is more acutely hepatotoxic when administered in corn oil than in an aqueous vehicle and further suggest that the kidney may be a more sensitive indicator of BDCM toxicity than the liver. «

2 Table of Contents page List of Tables...i List of Figures...ii I. Literature Review...2 A.Trihalomethanes (THMs) Formation and Chemical Properties Prevelance and Occurrence Human Exposure and Risk...3 B. Bromodichloromethane (BDCM) Systemic Toxicity...5 a. Acute and Short Term Exposure...5 b. Subchronic Toxicity...7 c. Carcinogenicity and Chronic Toxicity Toxicokinetics and Metabolism...10 C, Effects of Different Dosing Vehicles Effects on Acute and Subacute Toxicity Effects on Subchronic Toxicity Effects on Pharmacokinetics...15 II. Introduction...17 III. Materials and Methods...19 a. Animals and Husbandry...19 b. Chemicals...19 c. Study Design...20 d. Safety Precautions...20 e. Clinical Chemistry...20 f. Necropsy and Histopathology...21 g. Preliminary Pharmacokinetics Study...22 h. Statistical Analysis...23 IV. Results...23 a. Effects of Dosing Vehicle on Acute Nephrotoxicity b. Effects of Dosing Vehicle on Acute Hepatotoxicity V. Discussion...31 VI. Conclusions...40 VII. References...65

3 List of Tables Tables Page 1. Trihalomethane risk estimates Effect of BDCM on terminal kidney and relative kidney weights of male F-344 rats Urine ph and osmolarity over time following administration of BDCM in different dosing vehicles Urinary activities of renl damage indicators over time following administration of BDCM in different dosing vehicles Urinary concentrations of glucose and total protein over time following administration of BDCM in different dosing vehicles Levels of serum indicators of renal damage 24 and 48 hours after oral administration of bromodichloromethane (BDCM) in different dosing vehicles Kidney histopathology 24 and 48 hours following exposure to BDCM in corn oil or an aqueous vehicle Body and liver weights of male F-344 rats 24 and 48 hours following administration of BDCM in corn oil or aqueous (10% Emulphor) dosing solution Serum enzyme levels 24 hours after oral administration of bromodichloromethane (BDCM) in different dosing vehicles Levels of serum hepatic damage indicators 24 hours after oral administration of bromodichloromethane (BDCM) in different dosing vehicles Levels of serum hepatic damage indicators 48 hours after oral administration of bromodichloromethane (BDCM) in different dosing vehicles Levels of serum hepatic damage indicators 48 hours after oral administration of bromodichloromethane (BDCM) in different dosing vehicles Liver histopathology 24 and 48 hours following administration of BDCM in different dosing vehicles... 54

4 List of Figures Figure Page 1. Structures of the four most widely studied trihalomethanes (THMs) Oxidative metabolism of BDCM Reductive metabolism of BDCM Time course of nephrotoxicity following oral administration of 200 mg BDCM/kg in corn oil or aqueous vehicle Time course of nephrotoxicity following oral administration of 400 mg BDCM/kg in different vehicles Response of renal indicators of BDCM nephrotoxicity 36 hours post-exposure Response of renal indicators of BDCM nephrotoxicity 48 hours post-exposure Concentration-time profile following administration of 400 mg BDCM/kg in corn oil or 10% Emulphor Increases in hepatic toxicity indicators compared to increases in renal toxicity indicators at apparent times of peak toxicity following challenge with 200 mg BDCM/kg Increases in hepatic toxicity indicators compared to increases in renal toxicity indicators at apparent times of peak toxicity following challenge with 200 mg BDCM/kg

5 Literature Review I. Trihalomethanes A. Formation and Chemical Properties Trihalomethanes (THMs) are formed when surface waters containing organic substances are disinfected via chlorination. Hypochlorous acid (HOCl) reacts with endogenous organic molecules, such as humic and fulvic acids, to form chloroform and many other halogenated by-products. HOCl can also oxidize bromide ion to form hypobromous acid (HOBr) which reacts with organic acids to form reactive brominated compounds (Jolley et al., 1978). Some of these reactions are shown below: (1) Fulvic acid + HOCl > CHCI3 + acid residual (2a) Br~ + HOCl > HOBr + Cl~ (2b) Fulvic acid + HOBr > CHBr3 + acid residual The four most commonly studied trihalomethanes are chloroform (CHCI3), bromodichloromethane (BDCM), chlorodibromomethane (CHBr2Cl) and bromoform (CHBr3). The structures of these compounds are illustrated in Figure 1. THMs are lipophilic, volatile compounds which are colorless and have a slight sweet non-irritating odor associated with them. B. Prevalence and Occurrence A number of surveys of THM prevalence in the U.S. have been conducted in the past decade. These studies have found that, in finished drinking water, chloroform levels range from 0.7 to 540 ug/l, BDCM from 1.9 to 183 ug/l, CHBr2Cl from 0.4 to 280 ug/l

6 (U.S. EPA, 1990) and bromoform from 0.1 to 2.7 ug/l (National Organics Reconnaissance Survey, 1975). Higher levels of THM's have been reported in drinking waters which have surface waters as a primary source rather than groundwater. This may be due to lower concentrations of organic precursors and smaller disinfection requirements in groundwater compared to surface waters (Jolley et al., 1978). Increased use of ozonation by municipalities as an alternative to chlorination may result in formation of higher concentrations of brominated THMs (Jacangelo et al., 1989). In addition to being found in chlorinated drinking water, THMs are extensively used in industry and are commonly found in consumer products. Chloroform is used as a solvent and in the production of plastics, refrigerants and other solvents (U.S. EPA, 1980). Chloroform was widely used as an anesthetic but is no longer utilized in this capacity. Brominated THMs have been widely used in chemical and pharmaceutical manufacturing and as solvents (U.S. EPA, 1975). C. Human Exp>osure and Risk Recently, chlorination by-products have been implicated in increased risks of bladder and rectal cancer in humans (Morris et al., 1992). Cantor et al. (1978) also reported a correlation between human bladder cancer and THM exposure in drinking water. In addition to exposure to THMs via drinking water and industrial settings, humans can be exposed through a number of other media.

7 Some foods and beverages, such as soft drinks (Abdel-Rahmen, 1982) which are made with chlorinated water, can be media for exposure. Human exposure can also occur in swimming pools (Beech et al., 1980) and showers (Jo et al., 1990) via dermal absorption and inhalation of vapors. However, it is generally accepted that consumption of drinking water is the primary route of human exposure to THMs, although the study conducted by Jo et al. (1990) introduces inhalation and dermal absorption as significant exposure mechanisms. Due to the presence of these compounds in most drinking water supplies and discovery of their carcinogenic and toxic potential, regulation of THMs has become neccessary. Acceptable human exposure levels and health advisories have been determined for the four THMs with the highest mean concentrations in municipal water supplies. Levels of risk, maximum likelihood estimates (MLE's) and upper 95% confidence limits for the four primary THM's are compiled in Table 1. Levels were determined from data from animal studies and human epidemiological studies. Both carcinogenic and noncarcinogenic endpoints were used in these determinations. BDCM has been assigned the lowest MLE of the four primary THMs suggesting that BDCM poses the greatest risk of increasing cancer rates in humans.

8 II. Bromodichloromethane (BDCM) A. Health effects 1. Systemic Toxicity a. Acute and shoirt term exposure The most extensive study of acute BDCM toxicity was performed by the National Toxicology Program (NTP, 1987) at the National Institute of Environmental Health Sciences (NIEHS). Male and female F-344 rats and male and female B6C3F1 mice were exposed to single doses of 150, 300, 600, 1,250 or 2500 mg BDCM/kg via corn oil gavage. All animals dosed with 2,500 or 1,250 mg BDCM/kg died while 2 of 5 male rats, 1 of 5 female rats, and 2 of 5 female mice survived gavage with 600 mg BDCM/kg. All animals receiving lower doses of BDCM survived. The LD50 values (with 95% confidence interval in parentheses) were calculated to be 651 mg/kg ( mg/kg) and 751 mg/kg ( mg/kg) in male and female F-344 rats, respectively. For female B6C3F2 mice, an LD50 value of 651 mg/kg was reported with a 95% confidence interval of mg/kg. Values for male mice were not reported. In a fourteen-day study conducted as part of the NTP evaluation of BDCM, male and female F-344 rats were administered BDCM in corn oil at 0, 38, 75, 150, 300, and 600 mg/kg. Male and female B6C3F2 mice received the same dosages with the exception of the 600 mg/kg dosage. All male rats survived at all dosage levels while one female rat died at 38 mg/kg and one at the highest dosage. All other female rats survived. Reddened renal

9 medullae were noted in 100% of the male rats and 1 of 5 of the female rats in the high dosage groups. A decrease in body weight with BDCM was also noted in both sexes. Following administration of 150 and 300 mg/kg, no male mice survived while only one female died at the highest dosage. At 150 mg BDCM/kg, renal medullae appeared reddened in 90% of the male mice and 1 of 5 of the female mice with 100% of the males exhibiting the same effect at the 300 mg BDCM/kg dose. Chu et al. (1980,1982a) reported lethality when dosages of BDCM ranging from 54 6 mg/kg to 1500 mg/kg were administered in corn oil to both male and female Sprague-Dawley rats with the latter dose killing 100% of the male rats and 90% of the female rats. Values for the LD50 were reported to be 916 mg/kg with a 95% confidence interval of mg/kg in male rats and 969 mg/kg with a 95% confidence interval of mg/kg in female rats. The values determined in this study were somewhat higher than those reported in the NTP study, perhaps due to different dosing volumes (5 ml/kg in NTP study versus 2 ml/kg in Chu et al. study) or strain differences. In a subacute {28-day) study, male and female Sprague-Dawley rats received 0.14, 1.4 or 11 mg/rat/day of BDCM in drinking water (Chu et al., 1982). No pathological or biochemical changes were noted following administration of the compound. However, slight increases in relative kidney weights were observed in the 1.4 mg/rat/day group (statistical significance not reported).

10 Fourteen-day exposure to 0, 37, 74 or 148 mg BDCM/kg/day in corn oil to male CD-I mice resulted in decreased uptake of p- aminohippurate (PAH) into renal cortical slices at the two highest dose levels (Condie et al., 1983), suggesting damage to the organic anion uptake function of the proximal tubule. A dosage of 148 mg BDCM/kg caused significant increases in serum ALT (alanine aminotransferase) levels as well as increasing histopathological lesions in both the kidney and liver implicating these organs as primary targets of BDCM. Munson et al. (1982) gavaged male and female CD-I mice with 0, 50, 125 and 250 mg BDCM/kg in 10% Emulphor solution and noted signifcant increases in serum ALT, AST (aspartate aminotransferase) and BUN (blood urea nitrogen) while blood glucose levels and body weights decreased significantly. This study is consistent with the previous reports noting the liver and kidney to be target sites of BDCM toxicity. b. Subchronic toxicity A 13-week NTP study resulted in death of 5/10 male and 2/10 female rats following administration of 300 mg BDCM/kg. Both male and female rats survived delivery of 0, 19, 38, 75, and 150 mg/kg doses while both sexes of mice survived doses ranging from 6.25 to 400 mg BDCM/kg. Histopathological findings revealed increases in necrosis of proximal tubule epithelium cells in the kidney and enlarged, vacuolated hepatocytes in the centrilobular region of the liver in mice. Similar histological changes were

11 noted in rats with cellular alterations also occurring in the spleen, thymus, and lymph nodes. After administration of 2500 mg BDCM/liter of drinking water to male and female Sprague-Dawley rats for 90 days and allowing a 90-day recovery period, Chu et al. (1982b) noted no significant effects of BDCM on serum biochemical indicators. However, decreases in water and feed consumption were noted after dosing with BDCM for 90 days while thyroid and liver lesions (vacuolation, increased cytoplasmic volume, homogeneity and density of hepatocytes with vesiculation of biliary epithelial cells and increase in thyroid epithelial height with reduction of follicular size and colloid density) were observed after the 90- day recovery period. Histopathological changes in the thyroid suggest a delayed effect of BDCM, indicating a longer latency period may be required for development of thyroid pathology. 2. Carcinogenicity and chronic toxicity. Administration of 0, 50, and 100 mg BDCM/kg five days a week in corn oil to male and female F-344 rats for 104 weeks resulted in increased incidences of neoplastic changes compared to controls (NTP, 1987; Dunnick et al., 1987) Large increases (90% and 26% in male and female rats, respectively) in incidences of adenomatous polyps or adenocarcinomas in the large intestine were noted in both sexes at the 100 mg BDCM/kg dosage. Overall rates of tubular cell adenomas or adenocarcinomas in the kidney increased by 26% in male rats and 30% in female rats following

12 administration of 100 mg BDCM/kg. Increases in cellular changes were also noted in rat liver, adrenal gland, lung, and mammary gland. Fifty mg BDCM/kg dosages increased overall incidences of renal tubular cell adenoma or adenocarcinomas in male mice by 18% with increases of 58% in hepatocellular tumors in female mice following chronic challenge with 150 mg BDCM/kg (NTP, 1987; Dunnick et al., 1987). Additional pathological changes were also noted in mouse thyroid and anterior pituitary glands, as well as lesions in the testis and ovaries. Tumasonis et al. (1985) administered BDCM to male and female Wistar rats at 2.4 grams of BDCM per liter of drinking water for the lifetime of the animal (approximately 180 weeks). Incidence rates of hepatic adenofibrosis, lymphosarcomas, and pituitary gland tumors increased 2%, 19% and 21%, respectively, in male rats while female rats exhibited increases of 23%, 17% and 9%, respectively, with a 6% increase in tumor incidence in the mammary gland. In contrast to the NTP study, no renal lesions or large intestinal tumors were noted suggesting the dosing vehicle may influence the site of tumor formation. However, animals in the Tumasonis et al. study limited their water intake due to palatability problems and therefore, were likely diet-restricted as well, which could have resulted in significantly lessened expression of neoplasms (Pollard et al., 1985). Chronic administration of microencapsulated BDCM in feed for two years to male and female Wistar rats resulted in increases in relative liver and kidney weights of both sexes with significant

13 10 increases in biochemical indices of toxicity (ALT and AST) after 12 months in groups administered 138 mg/kg/day (Aida et al., 1992). One hundred percent of female rats examined at 24 months exhibited histopathological changes in the liver (fatty degeneration, granulomas, altered cell foci, bile duct proliferation or cholangiofibrosis) after ingesting high dosages of BDCM. Although nonneoplastic responses were noted in other organs in both sexes, the authors concluded that these alterations could not be attributed to BDCM exposure. 3. Toxicokinetics and Metabolism. The proposed metabolic pathways of BDCM are illustrated in Figures 2 and 3. The first step in the oxidative metabolism is mediated by NADPH-dependent cytochrome P-450, converting BDCM into the unstable intermediate alcohol, bromodichloromethanol. This alcohol rapidly decomposes to form phosgene (Pohl et al., 1978), which may undergo further reactions with water to form CO2 and HCl, glutathione to form CO and corresponding conjugates (Anders et al., 1978), cysteine to form cysteine conjugates (Stevens and Anders, 1979), or may bind directly to macromolecules (DNA, RNA, proteins, etc.). Gao and Pegram (1992) reported BDCM bound more extensively to protein and lipid than CHCI3, and CHCI3 has also been demonstrated to bind to DNA (Colacci et al., 1991). Therefore, due to the higher reactivity of BDCM compared to CHCI3, DNA binding may play an important role

14 11 in BDCM toxicity and carcinogenicity and BDCM may be a more important compound for study. Reductive metabolism of BDCM begins with addition of an electron via the cytochrome P-450 mixed function oxidase system and formation of the unstable bromodichloromethane radical anion (Testai and Vittozzi, 1986). This intermediate spontaneously decomposes to form the dichloromethyl free-radical (Tomasi et al., 1985) which may covalently bind to tissue or initiate lipid peroxidation. The distribution of a single administration of '-^C-labeled bromodichloromethane (BDCM) to male F-344 rats at 0, 10, 32 or 100 mg/kg for one day and 10 or 100 mg/kg/day for 10 days in corn oil was investigated by Mathews et al. (1990). The highest dose level of BDCM appeared to be extensively metabolized to -'-^C02 (71% of dose) and partially metabolized to ' ^CO (5% of dose) after 24 hours. Small amounts of radioactivity were found in the urine or feces, suggesting little conjugation of intermediates or, perhaps, excretion of negligible amounts of parent compound. Although total tissue amounts of radiolabeled BDCM never exceeded 4.4% of the total dose, greatest levels of radioactivity were reported in the liver (up to 3.06%) and kidney (up to 0.15%) with liver tissue to blood ratios (TBR) decreasing and kidney TBRs increasing with dose. TBRs in the liver and kidney were greater and tissue levels of -^^C-labeled BDCM were less in the liver and kidney after administration of BDCM for 10 days. Consequently, the authors suggested that BDCM may induce its own metabolism.

15 12 Mink et_al. (1986) reported 81.2% of a 150 mg/kg dose of ^^Clabeled BDCM was expired as CO2 when administered in corn oil to B6C3Fi mice, with 7% being exhaled unmetabolized, 2% found in the urine, and 3% remaining in the tissue eight hours post-gavage. Sprague-Dawley rats receiving 100 mg/kg of radiolabeled compound expired 41.7% of the total dose as parent compound, 14.2% as ' ^C02, 1.4% in the urine, and 3.3% remained in the tissue. Absorption from the gastrointestinal tract appeared to be relatively rapid with 92.7% and 62.7% of the total dose being eliminated from mice and rats, respectively, after 8 hours. Notable dissimilarities of amounts of the total dose expired as parent and as ' ^002 between mice and rats may indicate species differences in rates of metabolism of BDCM. The differences between this study and the investigation by Mathews et_al. also suggests possible strain differences in the Sprague-Dawley and F- 344 rats and their strain-specific ability to metabolize BDCM. III. Effects of different dosing vehicles. A. Effects on acute and subacute toxicity. A number of studies have been performed to determine the effect of vehicle of administration on the toxicity of VOCs. 1,1-dichloroethylene (1,1-DCE) toxicity was reported to change markedly with dosing vehicle 6 hours after male Sprague-Dawley rats were given 200 mg/kg dosages (Chieco et_al., 1981). Levels of serum AST and ALT increased 100-fold after administration of 1,1-DCE in mineral or corn oil while aqueous (0.5% Tween-80

16 13 solution) delivery of the compound resulted in only a 15-fold increase. Histological findings included massive centrilobular and mid-zonal necrosis in the liver following a 1,1-DCE dose in the oil vehicle while 0.5% Tween-80 administration caused only slight necrotic responses. The results of this study suggest that corn oil administration can greatly increase the hepatotoxicity of 1,1-DCE. < Acute CHCI3 administration in corn oil resulted in greater hepatic and renal toxicity in male F-344 rats compared to delivery in 10% Emulphor (M. Lilly, 1992; personal communication). Increases in serum and urinary enzyme activities were greater following delivery of CHCI3 in corn oil, suggesting vehicle of administration can influence the acute toxicity of CHCI3. The subacute toxicity of trichloroethylene (TCE) in different dosing vehicles has also been examined. Merrick et al. (1989) noted greater elevations in serum enzymes ALT, AST, and LDH in male B6C3F]^ mice following administration of TCE for four weeks in corn oil than the aqueous vehicle (20% Emulphor). A similar trend was observed in liver histopathology with higher incidences of necrosis in mice receiving 600 or 1200 mg TCE/kg in corn oil compared to an aqueous solution. These data are consistent with results reported by Chieco et al., suggesting corn oil can influence the toxicological responses of VOCs in experimental animals.

17 14 Kim et al. (1990a) investigated the effect of dosing vehicle on the acute hepatotoxicity of carbon tetrachloride (CCI4) in male Sprague-Dawley rats. In contrast to the previously described studies, aqueous (0.25% Emulphor) delivery of dosages of CCI4 ranging from mg/kg caused significantly greater elevations in serum SDH (sorbital dehydrogenase), ALT and AST than corn oil adminstration. Histological examination of the liver lobule revealed significantly greater hepatocellular injury in the centrilobular region following administration of CCI4 in the aqueous vehicle compared to corn oil. CCl4-induced hepatotoxicity appears to be attenuated by corn oil while aqueous delivery increases CCI4 toxicity. This phenomenon may be due to more rapid uptake rates of CCI4 from the GI tract when it is administered in an aqueous vehicle while corn oil may be acting as a reservoir for the compound, thus slowing absorption. A. Effects on siibchronic toxicity. In contrast to the results from the acute study conducted by Kim et al. (1990), subchronic studies of effects of dosing vehicles on VOC toxicity suggest corn oil administration enhances toxicity. Ninety-day administration of relatively low doses (12 and 120 mg/kg/day) of CCI4 in corn oil to male and female CD-I mice resulted in markedly greater increases in serum ALT, AST, and LDH compared to delivery in 1% Tween-60 solution (Condie et al., 1986). Following administration of 120 mg CCl4/kg/day in

18 15 corn oil to both sexes, a higher incidence of hepatocellular necrosis was noted than in the aqueous vehicle. Bull et al (1986) reported greater hepatotoxicity resulting from 90-day CHCI3 administration in corn oil than in an aqueous vehicle. Male and female B6C3F;2^ mice given 270 mg/kg/day in corn oil exhibited significantly higher levels of serum AST and triglycerides. At the same dose level in corn oil, histopathological examination revealed enlarged, vacuolated hepatocytes and altered hepatic structure in 50% of the males and 70% of the females. However, only minimal necrosis was noted in two of ten females and one of ten males following administration of an equal dose in 2% Emulphor. These studies suggest that results from subchronic administration of VOC's in corn oil can be substantially different from aqueous delivery of the compounds. C. Influence on pharmacokinetics. In addition to influencing the acute, subacute and subchronic toxicity of VOC's, vehicle of chemical administration can markedly affect the pharmacokinetics of compounds. Withey et al. (1983) reported greater areas under the blood-concentration curves (AUC) when four halogenated hydrocarbons (methylene chloride, dichloroethane, TCE, and CHCI3) were administered to male Wistar rats in an aqueous dosing vehicle compared to corn oil. Peak blood concentrations (Cjjj^j^) were markedly greater when the compounds were administered in an aqueous solution with less

19 16 time required to reach maximum concentratons in the blood (Tjjia^) compared to corn oil. The authors also reported corn oil delivery of VOCs resulted in complex or "pulsed" uptake of the compounds in the GI tract. Consistent with the results previously described, CCI4 pharmacokinetics were markedly influenced by dosing vehicle. Kim et al. (1990b) noted a ten-fold increase in Cmax when 25 mg CCl4/kg was administered to male Sprague-Dawley rats in water or aqueous emulsion (0.25% Emulphor) compared to corn oil. Although AUCs were not significantly different between dosing vehicles, ''^max values were 30 times greater following corn oil delivery of CCI4 compared to water or aqueous emulsion administration. These data and results from the investigation by Withey et al. (1983) suggest that the rates of VOC uptake from the GI tract are markedly affected by dosing vehicle and differences in toxicity may be related to these dissimilarities.

20 17 Introduction Bromodichloromethane (BDCM) is a common contaminant of finished drinking water produced when surface waters containing organic substances are disinfected via chlorination. Found in many municipal drinking water supplies in the U.S. (U.S. EPA, 1990), trihalomethanes (THMs) have been measured in concentrations ranging from 0.1 to 540 ug/1 (U.S. EPA, 1990). THMs have been linked by epidemiological data with human bladder and rectal cancer (Morris et al., 1992; Cantor et al., 1978). In chronic studies with experimental animals, BDCM caused increased incidences of neoplasms in the kidney and large intestine of male and female rats, in the liver of female mice, and in the kidney of male mice (Dunnick, 1987; NTP, 1987). In comparison with chloroform (CHCI3), the most prevalent and widely studied THM, BDCM appears to be more carcinogenic (Dunnick et al., 1987) and more acutely toxic (Chu et al., 1982), further indicating the importance of studying this brominated THM. The bromine moiety of BDCM may cause these differences by increasing the reactivity of the compound compared to chloroform. Due to the volatile and lipophilic nature of VOCs (volatile organic compounds), many investigators have administered these compounds in a corn oil vehicle. However, corn oil can influence the biological activity of chemicals. Chronic administration of BDCM to male and female rats in drinking water increased hepatic neoplastic nodules (Tumasonis et al., 1985); in contrast, only renal and large intestinal tumors were observed in rats when BDCM

21 18 was delivered in corn oil (NTP, 1987). CHCI3 hepatotoxicity in mice was enhanced following administration in corn oil for 90 days when compared to dosing in an aqueous vehicle (Bull et al., 1986). Condie et al. (1986) noted the same trend when carbon tetrachloride (CCI4) was delivered subchronically by corn oil gavage compared to an aqueous vehicle in CD-I mice. However, when vehicle effects on the acute toxicity of CCI4 were studied, aqueous administration led to greater hepatotoxicity than dosing in corn oil (Kim et al., 1990a). Several investigators have noted that the pharmacokinetics of certain VOCs are markedly changed when different dosing vehicles are used. Withey et al. (1983) noted greater peak blood concentrations (Cjy^^) and total area under blood concentration-time curves (AUC) and shorter times to peak blood concentration (Tjyj;^) for four halogenated hydrocarbons when administered in an aqueous solution compared to a corn oil vehicle. CCI4 delivery in an aqueous solution resulted in a greater Cj.^^^ and AUC and shorter Tjyjj^ than in corn oil (Kim et al., 1990b). Because of the variability in responses with different dosing vehicles and the scarcity of data concerning BDCM administration in water, comparisons of BDCM toxicity in different dosing vehicles are needed. Results from such comparisons should be useful in the interpretation of corn oil studies for drinking water risk assessment while also providing data which more closely simulates human exposure to THMs via drinking water.

22 19 Materials and Methods Animals and husbandry. Male Fischer-344 rats were obtained from Charles River Breeding Laboratories (Raleigh, N.C.)/ at 90 days of age, housed 2 per cage and were acclimated for 3 days to a 12-hr light/dark cycle with light from 0600 to 1800, The animal room was maintained at C with 40 to 60% relative humidity. Rats were provided Purina Rodent Chow 5001 (Ralston Purina Co., St. Louis, MO) and tap water water ad libitum during the acclimation period. Animals were then assigned to groups based on body weight and housed individually in Nalgene plastic metabolism cages (Nalgene Corp., Rochester, NY). Additional dose groups were placed individually in polyethylene shoebox cages with heat-treated pine-shavings as bedding. Rats were acclimated to the metabolism cages for 3 days prior to dosing and were provided with Bio-Serv 45 mg pelleted Rodent Chow (Bio-Serv, Frenchtown, N.) and tap water ad libitum. Animals were not fasted prior to dosing. Chemicals. BDCM (Lot no DX; purity=98.09%) was obtained from Aldrich Chemical Co. (Milwaukee, WI) and was dissolved in either 10% Emulphor EL-620 solution (GAF Chemical Corp., Wayne, NJ) or corn oil (Sigma Chemical Co.). BDCM dosages were administered in a constant volume of 5 ml/kg by oral intubation with 20-gauge, 2.5 inch ball-tipped gavage needles attached to a 3.0 ml disposable syringe. Control animals received vehicles at the same volume.

23 20 Study Design. The rats in metabolism cages were assigned to six treatment groups consisting of 6 animals each. The doses were 0, 200, and 400 mg BDCM/kg body weight for each vehicle, and the rats were killed 48 hours post-dosing. Additional groups (6 rats per group) were given 0 or 400 mg BDCM/kg body weight and were killed at 24 hours. The rats were dosed between 0900 and Body weights were measured daily. Safety precautions. Personnel were required to wear respirators with cartridges for removing organic vapors and protective lab coats and gloves. Dosing was performed in an animal room under negative pressure ventilation and restricted for VOC study. Clinical chemistry. Urine samples were collected for 12 hours prior to dosing, then from 0 to 6 hours, 6 to 12 hours, 12 to 24 hours, 24 to 36 hours, and 36 to 48 hours. Urine was cooled by Uotek refrigerant packs (Polyform Packer Corp., Wheeler, IL). Urine samples were centrifuged at 800Xg for 10 minutes, and volume, ph and osmolality were measured. After 24 or 48 hours, rats were anesthetized with carbon dioxide, and blood was drawn for clinical chemistry measurements from the dorsal aorta with a 21-gauge, 1.5-inch sterile syringe and a Vacutainer serum-separation tube (Becton Dickinson Vacutainer Systems, Rutherford, NY). Blood samples were held on ice for 30 minutes and allowed to clot. Samples were then centrifuged at 1400Xg for 30 minutes at A'^C. Aliquots of serum were taken for clinical chemistry analysis. Serum samples were stored at 4 C

24 21 for 12 to 24 hours. Various analyses were conducted on sera and urine, which included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), blood urea nitrogen (BUN), creatinine (CRE), gamma glutamyl transferase (GGT), glucose (GLU), lactate dehydrogenase (LDH), and total protein (TPR). Additional analyses were carried out on sera to determine levels of sorbitol dehydrogenase (SDH), cholesterol (CHOL), total bilirubin (TOT BIL), triglycerides (TRIG), albumin (ALB), 5' nucleotidase, and bile acids. Urine and sera analyses were conducted with a CentrifiChem-500 centrifugal analyzer (Baker Instruments Co., Allentown, PA) and appropriate reagent kits. Necropsy and histopathology. After blood collection, the livers were excised, weighed, and sections taken for histopathology. Slices of the left lobe were triimned to 2 to 3 mm in thickness, placed in tissue cassettes, and fixed in 10% phosphate-buffered Formalin solution. Kidneys were also removed and sliced, either laterally (right kidney) or longitudinally (left kidney), and placed in 10% phosphate-buffered Formalin solution. Liver and kidney slices were subsequently prepared for histological evaluation using hematoxylin and eosin (H&E) staining. Prepared liver and kidney sections were evaluated by a certified veterinary pathologist from Pathco, Inc. (Research Triangle Park, NO). Hepatocellular vacuolar degeneration and necrosis were graded separately for location in the liver lobule (centrilobular, periportal, mid-zonal) and for severity, as

25 22 described previously (Simmons et al., 1988). Lesions were evaluated according to the following criteria: none, minimal (one to several hepatocytes affected) ; mild (no more than 25% of the hepatocytes in the affected zone involved); moderate (up to 50% of hepatotcytes in affected zone appeared damaged); marked (over 50% of cells in affected zone damaged). Histopathological evaluations of kidney slices were graded on a scale of increasing severity as none, minimal (up to 25% of cells in affected area damaged); mild (25-50% of cells in damaged area affected); moderate (51-75% of cells affected); and marked (greater than 75% of the area damaged and may contribute to death). Preliminary pharmacokinetic study. Three rats per vehicle per time point were dosed with 400 mg BDCM/kg in corn oil or 10% Emulphor in a constant dosing volume of 5 ml/kg. Animals were killed 4, 16 or 64 minutes post-exposure. Blood was collected via the dorsal aorta with a 20-gauge heparin-coated syringe and EDTA-treated Vacutainer tube to prevent clotting. Blood (0.5 ml) was analyzed with a headspace method on a Hewlett-Packard 5890A gas chromatograph (GC) and headspace sampler with a 6 meter, 1/8- inch diameter SP-1000 stainless steel column (Supelco, Bellafonte, PA). GC parameters for the method were as follows: initial oven temperature of 100 C for 2.00 minutes increasing to 140 C at a rate of 15.0 C/minute and maintaining that temperature for 5.0 minutes. Flame Ionization Detector (FID) temperature was 250 C and injection port temperature was 225 C. Total carrier gas (He) flow rate was set at 30 ml/minute (18 ml/minute from

26 23 headspace and 12 ml/minute from GC). Standards were made from heparinized blood samples of control animals. Statistical analysis. Data were subjected to Bartlett's test for homogeneity of variances (Sokal and Rohlf, 1981) with p<0.001 as the level of significance. Upon failure of the homogeneity test, data were transformed to corresponding logarithms. Analyses of sera and urine data were performed at individual time points. Due to variability in urine volume, values obtained for urinary indicators of toxicity were normalized to creatinine prior to analysis. A two-way analysis of variance (ANOVA) was performed to determine effects of BDCM concentration, dosing vehicle, and to test for significant interactions between vehicle and BDCM dose (PROC GLM, SAS, 1989). Differences were determined to be significant at the p<0.05 level. When appropriate, a student Newman-Keul's range test was used to determine if differences either between doses or between vehicles were significant. Analysis of data from a preliminary pharmacokinetic study was performed with a one-way ANOVA to determine if significant vehicle differences were present at individual time points. Results Effects of dosing vehicle on acute nephrotoxicity. Kidneys appeared lighter in color following administration of BDCM in either corn oil or aqueous vehicle. Kidney weight and relative

27 24 kidney weight {% body weight) are compiled in Table 2. Kidney and relative kidney weights were increased significantly by 400 mg BDCM/kg in both dosing vehicles. Corn oil delivery of 400 mg BDCM/kg significantly changed kidney (18% greater than aqueous dose) and relative kidney weights (12% greater than aqueous dose) compared to dosing with the aqueous vehicle. Mean urine volumes were approximately 80% greater in groups administered BDCM, but these increases were not statistically significant (data not shown). No vehicle differences were noted in urine volume. Urine osmolality and ph decreased significantly following administration of BDCM in both vehicles (Table 3). Forty-eight hours after administration of low doses of BDCM, urine ph and osmolality appeared to begin to return to normal levels. Urine ph was decreased to a significantly greater extent following delivery of 200 mg BDCM/kg in corn oil than in a aqueous vehicle 24 hours post-exposure. Corn oil administration of 400 mg/kg of BDCM resulted in a significantly greater reduction of osmolality 48 hours post-gavage when compared to the aqueous vehicle. Urinary levels of renal damage indicators AST, ALT, LDH, ALK, glucose and total protein are compiled in Tables 4 and 5. Significant interactions between BDCM dose and vehicle of administration were noted for the nephrotoxicity indicators, LDH, AST and total protein at 36 hours and in total protein at 12 and 24 hours, respectively, suggesting that the effect of vehicle on acute BDCM nephrotoxicity changes with dose.

28 25 Examination of the time course of nephrotoxicity of BDCM reveals that the time to apparent peak toxicity was both dose and vehicle dependent. Highest mean levels of the renal damage indicators LDH and AST (Figure 4) were noted 24 hours following administration of 200 mg BDCM/kg in both vehicles. ALT activity was greatest at 24 and 36 hours after delivery of 200 mg BDCM/kg in both vehicles. In contrast, 400 mg BDCM/kg dosages in corn oil caused highest levels of ALT and AST 48 hours post-exposure while aqueous delivery of the compound produced greatest activity after 24 and 36 hours, respectively (Figure 5). LDH levels peaked 36 hours after administration of 400 mg BDCM/kg in either dosing vehicle. Delivery of 200 and 400 mg BDCM/kg in both dosing vehicles resulted in peak ALK activities at 24 hours. In addition, urinary ALK levels in control animals for both dosing vehicles shifted temporally in a cyclical fashion. Total protein levels were greatest 36 hours post-gavage with 200 mg BDCM/kg in both vehicles while peak levels following delivery of 400 mg BDCM/kg were noted at 36 hours in corn oil and 48 hours in the aqueous vehicle. Glucose levels were highest 48 hours after administration of 400 mg BDCM/kg in either dosing vehicle. Exposure of groups to 400 mg BDCM/kg in corn oil caused significantly greater increases in BUN 48 hours post-gavage compared to the same aqueous dosage (Table 6) with corresponding decreases (50% in corn oil and 40% in the aqueous vehicle) in urine urea nitrogen at the same time point (data not shown). Creatinine levels were significantly increased 24 hours following

29 26 challenge with 400 mg BDCM/kg in both dosing vehicles with the same dosage in corn oil resulting in significantly greater increases in creatinine at 48 hours compared to the aqueous vehicle (Table 6). Changes in urinary indicators of nephrotoxicity were noted as early as 12 hours post administration of BDCM. Significant increases in LDH activity followed administration of 200 and 400 mg/kg in corn oil while significant increases were noted strictly in the high dose group when BDCM was delivered in 10% Emulphor. Glucose levels were increased only following exposure to 400 mg BDCM/kg in corn oil 12 hours post-gavage. Activities of renal indicators AST and ALK markedly increased 24 hours post-exposure with significant differences noted when control groups at 24 hours were compared to low and high dose groups in both vehicles. Groups given 400 mg BDCM/kg in either dosing vehicle exhibited significant increases in LDH 24 hour post-gavage when compared to the low dose and control groups. The same trend was noted in total protein levels after administration of 400 mg/kg in an aqueous vehicle. Delivery of 200 and 400 mg BDCM/kg in both dosing vehicles caused significant dose-dependent increases in AST and total protein levels 36 hours post-dosing while aqueous administration of the low dose of BDCM caused significantly greater increases in AST and total protein than corn oil (Figure 6). The activity of urinary LDH following exposure to 400 mg BDCM/kg in both vehicles was significantly greater than control and low dose groups.

30 27 Groups given 200 or 400 mg BDCM/kg in corn oil exhibited significantly greater levels of urinary ALT when compared to corn oil controls. Forty-eight hours following administration of 400 mg BDCM/kg in both dosing vehicles, serum BUN and urinary LDH, glucose, and total protein levels were significantly different than control and low dose groups, and delivery of 400 mg BDCM/kg in corn oil resulted in significantly greater increases in glucose than the aqueous vehicle (Tables 4,5,6; Figure 7). The activity of AST increased in a significant dose-dependent manner in both dosing vehicles. Elevations of ALT persisted at 48 hours after delivery of 400 mg/kg of BDCM in corn oil, but not 10% Emulphor, while ALK activity was increased significantly at 48 hours post-exposure to 200 and 400 mg BDCM/kg in corn oil, but not the aqueous vehicle. Therefore, at specific time points, the nephrotoxicity of low doses of BDCM was greater when the compound was administered in an aqueous vehicle while 400 mg BDCM/kg doses appeared more acutely toxic when delivered in corn oil. Results of histopathological examination of kidney slices of dosing groups killed 24 and 48 hours post exposure are shown in Table 7. BDCM caused a dose-dependent increase in the incidence of renal tubule necrosis 48 hours post-exposure. Corn oil delivery of 400 mg BDCM/kg caused greater damage to the kidney with a higher number of moderate necrotic events than the aqueous vehicle. Renal tubular degeneration increased in a dosedependent manner 48 hours after administration of BDCM in both

31 28 vehicles. Groups administered 400 mg BDCM/kg in corn oil exhibited a higher incidence of marked degeneration of the renal tubule when compared to the aqueous vehicle. Effect of dosing vehicle on acute hepatotoxicity. Effects of acute BDCM exposure on body weight, liver weight and relative liver weight (% body weight) are presented in Table 8. BDCM caused a significant loss of body weight at 400 mg/kg in the aqueous vehicle at 48 hours. No significant vehicle differences were noted. BDCM administered in an aqueous vehicle significantly decreased liver weights at 48 hours post-dosing but not at 24 hours. Relative liver weights (% body weight) were generally lowered slightly by BDCM administration, but were only significantly decreased by an aqueous dose of 400 mg/kg at 24 hours. Serum levels of hepatic damage indicators 24 hours postdosing are shown in Tables 9 and 10. Activities of serum enzymes, ALT, AST, LDH and SDH were significantly increased following administration in both vehicles, with higher levels in rats administered 400 mg BDCM/kg dosage in corn oil than the aqueous vehicle. ALK activity was significantly increased only following delivery of the high dose of BDCM in corn oil. Although no statistically significant vehicle differences were noted, aqueous administration of 400 mg BDCM/kg resulted in relative increases in serum enzymes AST, ALT, ALK, SDH and LDH of 350%, 630%, 6%, 620%, and 140%, respectively, while corn oil delivery caused 430%, 750%, 27%, 500%, and 110% increases.

32 29 Groups exposed to 400 mg/kg of BDCM in both dosing solutions exhibited significant decreases (p<0.05) in triglycerides (TRIG), glucose, and cholesterol and significantly increased bile acid levels. Total bilirubin levels did not change. The activity of 5' nucleotidase was significantly greater 24 hours following administration of 400 mg BDCM/kg in corn oil compared with corn oil controls and the aqueous 400 mg/kg dose group. Activities of the serum enzymatic hepatotoxicity indicators, AST, ALT, LDH, SDH, and ALK 48 hours after dosing, are compiled in Table 11. Significant interactions between dose and vehicle were noted for the hepatic damage indicators, LDH, ALK and AST (p<0.05). While significant differences were noted only in the aqueous vehicle at the 200 mg/kg dosage, four serum enzymes were significantly increased at 400 mg BDCM/kg in both vehicles. Administration of 400 mg/kg of BDCM in corn oil resulted in significantly greater AST and ALK activities than in the aqueous vehicle (p<0.05). In addition to the statistically significant difference between dosing vehicles noted with AST and ALK, BDCM administration in corn oil caused greater relative elevations in mean enzyme activity than in the aqueous vehicle. Percent increases in serum activities of ALT, AST, LDH and SDH were approximately 390%, 650%, 390%, and 510%, respectively, in corn oil, and 240%, 300%, 300%, and 470% in 10% Emulphor. Levels of hepatic damage indicators triglycerides, total bilirubin, bile acids, cholesterol, glucose, and 5' nucleotidase are compiled in Table 12. Decreases in serum triglycerides

33 30 (TRIG) were noted following dosing with 200 and 400 mg BDCM/kg in both the aqueous and corn oil vehicles; glucose was decreased only by corn oil BDCM administration; and decreases in total bilirubin were noted only in rats dosed with the aqueous vehicle. Administration of 400 mg BDCM/kg in corn oil, but not 10% Emulphor, caused significant increases in bile acids. Administration of BDCM in corn oil caused significantly greater increases in bile acid levels at the high dose level than in the aqueous vehicle. There were no significant changes in 5'nucleotidase 48 hours post-administration in either dosing vehicle. Histopathological examination of liver slices sampled at 24 hours post-dosing with 400 mg BDCM/kg revealed centrilobular vacuolar degeneration and hepatocellular necrosis in 100% of the rats in both vehicle groups (Table 13). At 48 hours, centrilobular cellular necrosis was still present in the 400 mg/kg dose groups but at lower frequency in the aqueous vehicle, while mid-zonal vacuolar degeneration was noted in both 400 mg/kg dose groups. Necrosis was evident in 83% of the rats administered the high dose of BDCM in corn oil but only in 33% of the aqueous group. The only histopathological observation at 200 mg/kg was a finding of midzonal vacuolar degeneration in one rat in the corn oil group. On a five-level severity scale (none, minimal, mild, moderate and marked), hepatocellular necrosis was characterized as minimal and vacuolar degeneration as mild with

34 31 the exception of 2 rats in the 400 mg/kg aqueous vehicle group which exhibited minimal vacuolar degeneration. Results of a preliminary pharmacokinetics study of the effects of dosing vehicle on blood concentrations of BDCM absorbed from the GI tract following oral gavage are presented in Figure 8. This study revealed significantly greater levels (92% and 100%) of BDCM in the blood 4 and 16 minutes after administration of 400 mg BDCM/kg in an aqueous vehicle compared to corn oil delivery. Discussion Although the effects of oil and aqueous dosing vehicles on CHCl3-induced chronic and subchronic hepatotoxicity have been studied, vehicle differences in the acute toxicity of BDCM had not been determined previously. The characterization of the acute toxicity of BDCM in aqueous media is vital for assessing risks associated with drinking water exposures to trihalomethanes since BDCM produces a wider spectrum of carcinogenic responses (Dunnick et al., 1987; NTP, 1987) and is more acutely toxic (Chu et al., 1980) than CHCI3 in experimental animals. Unfortunately, nearly all of the acute and chronic studies conducted to examine the toxicity or carcinogenic potential of BDCM have employed administration of the compound to experimental animals in corn oil (Chu et al., 1982; Condie, et al., 1983; NTP, 1987) which may confound interpretation of results for drinking water risk assessments. This study was designed to determine if aqueous