Acute, Short-Term, and Subchronic Oral Toxicity of 1,1,1-Trichloroethane in Rats

James V. Bruckner*,1, Glenna M. Kyle{dagger}, Raja Luthra{ddagger}, Daniel Acosta§, Sanjay M. Mehta*, Sankar Sethuraman and Srinivasa Muralidhara*

* Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602-2352; {dagger} Exxon Company, USA, P.O. Box 2180, Houston, Texas 77252; {ddagger} Department of Pathology, M. D. Anderson Cancer Center, Houston, Texas 77030; § College of Pharmacy, University of Cincinnati, Cincinnati, Ohio 45267-0004; and Department of Mathematics and Computer Science, Augusta State University, Augusta, Georgia 30904

Received September 19, 2000; accepted December 18, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,1,1-Trichloroethane (TRI) is a widely used solvent that has become a frequent contaminant of drinking water supplies in the U.S. There is very little information available on the potential for oral TRI to damage the liver or to alter its P450 metabolic capacity. Thus, a major objective of this investigation was to assess the acute, short-term, and subchronic hepatotoxicity of oral TRI. In the acute study, male Sprague-Dawley (S-D) rats were gavaged with 0, 0.5, 1, 2, or 4 g TRI/kg bw and killed 24 h later. No acute effects were apparent other than CNS depression. Other male S-D rats received 0, 0.5, 5, or 10 g TRI/kg po once daily for 5 consecutive days, rested for 2 days, and were dosed for 4 additional days. Groups of the animals were sacrificed for evaluation of hepatotoxicity 1, 5, and 12 days after initiation of the short-term experiment. This dosage regimen caused numerous fatalities at 5 and 10 g/kg, but no increases in serum enzymes or histopathological changes in the liver. For the subchronic study, male S-D rats were gavaged 5 times weekly with 0, 0.5, 2.5, or 5.0 g TRI/kg for 50 days. The 0 and 0.5 g/kg groups were dosed for 13 weeks. A substantial number of rats receiving 2.5 and 5.0 g/kg died, apparently due to effects of repeated, protracted CNS depression. There was evidence of slight hepatocytotoxicity at 10 g/kg, but no progression of injury nor appearance of adverse effects were seen during acute or short-term exposure. Ingestion of 0.5 g/kg over 13 weeks did not cause apparent CNS depression, body or organ weight changes, clinical chemistry abnormalities, histopathological changes in the liver, or fatalities. Additional experiments did reveal that 0.5 g/kg and higher doses induced hepatic microsomal cytochrome P450IIE1 (CYP2E1) in a dose- and time-dependent manner. Induction of CYP2E1 activity occurred sooner, but was of shorter duration than CYP2B1/2 induction. CYP1A1 activity was not enhanced. In summary, 0.5 g/kg po was the acute, short-term, and subchronic NOAEL for TRI, for effects other than transient CYP2E1 induction, under the conditions of this investigation. Oral TRI appears to have very limited capacity to induce P450s or to cause liver injury in male S-D rats, even when administered repeatedly by gavage in near-lethal or lethal dosages under conditions intended to maximize hepatic effects.

Key Words: 1,1,1-trichloroethane; methylchloroform; liver toxicity; cytochrome P450 induction; CYP2E1; CYP2B1/2; noncancer risk assessment.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1,1,1-Trichloroethane (TRI) is a short-chain, aliphatic chlorinated hydrocarbon (halocarbon) that has a myriad of uses in contemporary society. TRI is used as a degreaser; as a solvent for adhesives, oils, and inks; and as a component of aerosols, lubricants, cleaners, and a variety of other industrial and household products (ATSDR, 1995Go). Release into the environment occurs primarily by volatilization during TRI's manufacture and formulation, as well as during normal usage of the products containing it. Contamination of surface and groundwater frequently occurs in the proximity of production facilities, wastewater plants, and hazardous waste disposal sites. TRI was found at 696 of 1408 hazardous waste sites included in the U.S. National Priorities List (ATSDR, 1994Go). TRI has been identified as a groundwater contaminant at many of the sites, often in combination with trichloroethylene and/or perchloroethylene (Fay and Mumtaz, 1996Go).

TRI is currently assigned the classification of D (not classifiable as to carcinogenicity in humans) by the U.S. EPA (1998). The NCI (1977) conducted a 78-week study in which B6C3F1 mice and F344 rats of both sexes received high doses of TRI daily by gavage. There was no increase in cancers attributable to TRI. In a screening study, Maltoni et al. (1986) observed an apparent increase in leukemias in male and female S-D rats gavaged with 500 mg TRI/kg/day for 104 weeks. Statistical analyses were not presented, and the authors stated that definite conclusions could not be drawn from their work due to limitations in the design and size of the experiment. A 2-year inhalation study in B6C3F1 mice and F344 rats of both sexes revealed no evidence of tumorigenicity due to TRI (Quast et al., 1988Go). Thus at present, assessments of potential health risks posed by TRI are based upon noncancer endpoints.

As inhalation is the primary route of exposure to TRI in industrial and occupational settings, much of the existing toxicology database is comprised of results of inhalation studies. One of the first such studies in laboratory animals was conducted by Adams et al. (1950). They observed central nervous system (CNS) depression in the 4 species they examined. The only organ found to exhibit histological change was the liver of the rats and guinea pigs. Long-term inhalation of high concentrations of TRI has been demonstrated by several research groups to produce CNS depression and modest steatosis in the centrilobular region of the liver of rats, guinea pigs and mice (McNutt et al., 1975Go; Quast et al., 1988Go; Torkelson et al., 1958Go). No organ/system, other than the CNS and liver, appeared to be consistently affected. Although overexposure to TRI vapors has caused fatalities in humans, liver injury was usually absent or quite modest (ATSDR, 1995Go). Hodgson et al. (1989) described 4 men with occupational exposures to TRI who developed liver injury characterized by elevated serum alanine aminotransferase (ALT) activity and fatty vacuolation of hepatocytes. Other instances of liver effects in persons who inhaled TRI, sometimes in combination with trichloroethylene, have been reported by Stahl et al. (1969), Halevy et al. (1980), Thiele et al. (1982), Hodgson et al. (1989), and Cohen and Frank (1994).

Data of the toxic potential of oral TRI in humans and laboratory animals are quite limited. Stewart and Andrews (1966) described a 47-year-old man who accidently drank ~1 ounce (~600 mg/kg) of TRI. Evaluation during a 2-week hospital stay revealed only slight, transient elevation of urinary protein and serum bilirubin. Other clinical chemistry indices were normal, as were EKGs and neurological examinations. Male rats given a single oral dose of ~2500 mg TRI/kg bw in corn oil exhibited a transient increase in serum aspartate aminotransferase (AST) activity, but no increase in ALT (Tyson et al., 1983Go). Klaassen and Plaa (1966) reported the ED50 for a small, but statistically significant elevation of ALT in mice to be 3350 mg TRI/kg ip. Nonneoplastic histological changes have not been observed in the liver or other organs of mice or rats given oral doses of up to 1500 mg/kg in chronic bioassays (NCI, 1977Go; NTP, 1983Go). In the absence of oral dose-response data, the U.S. EPA (1999) recently considered deriving provisional oral reference doses (RfDs) for TRI by extrapolating from CNS effects seen in gerbils in an inhalation study.

In light of the foregoing, one of the objectives of the present investigation was to evaluate dose-response relationships for liver injury in rats from acute, short-term, and subchronic ingestion of TRI. As CNS depression is rapidly reversible, the liver was selected as the target organ of interest.

In anticipation of a low order of hepatotoxic potency for TRI, an effort was made to employ a sensitive animal model. Rats were chosen for the studies, since they have been found to be more sensitive than mice to TRI hepatotoxicity and lethality in cancer bioassays (NCI, 1977Go; NTP, 1983Go; Quast et al., 1988Go). Many halocarbons, including TRI, are metabolically activated by P450s, notably CYP2E1 (Guengerich et al., 1991Go; Raucy et al., 1995). Although hepatic microsomal CYP2E1 and CYP2B1/2 do not appear to be sex-specific in rats, males were selected, since CYP2E1 is more inducible in males (Correia, 1995Go; Morel et al., 1999Go). Our rats were acclimated to a reverse light/dark cycle, so they could be conveniently dosed near the beginning of their active/dark cycle. Bruckner et al. (manuscript submitted) recently observed that hepatic CYP2E1 activity is highest, and that carbon tetrachloride (CCl4) bioactivation and hepatotoxicity are greatest, when rats are exposed to CCl4 during their initial waking hours. Halocarbons such as CCl4 are also more hepatotoxic when given as an oral bolus rather than in divided doses (Sanzgiri et al.,1995).

Although the metabolism of TRI is limited, it may be able to induce the P450s that catalyze its biotransformation. Pretreatment of rats with phenobarbital enhances TRI metabolism (Ivanetich and Ven Den Honert, 1981). Fasting and ethanol pretreatment, conditions which induce CYP2E1, also enhance TRI metabolism in rats (Kaneko et al., 1994Go; Nakajima and Sato, 1979Go). Guengerich et al. (1991) report that human CYP2E1 is a major catalyst of TRI oxidation. Since TRI appears to be a substrate for CYP2E1 and CYP2B1/2, it was hypothesized that TRI is capable of inducing both isoforms in rat liver. CYP2E1 seems to be unique, in that its induction is frequently attributable to stabilization by certain of its substrates (Lieber, 1997Go). If TRI acts in this manner, the time course of CYP2E1 induction should differ significantly from that of CYP2B1/2. The second objective of this project was to test this assumption and the hypothesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Male Sprague-Dawley (S-D) rats for toxicity and for cytochrome P450 experiments were obtained from TIMCO Breeding Laboratories (Houston, TX) and from Charles River (Raleigh, NC). The rats for the toxicity studies were acclimated to reverse light-dark conditions for 2 weeks prior to use, with light from 2100 to 0900 h. Subjects of the P450 experiments were acclimated for at least 1 week to a normal light cycle (light from 0700 to 1900 h). The animals were randomly assigned to dosage groups. Each group of 4 to 6 rats was housed together in stainless steel cages in a light- and humidity-controlled biohazard suite. Ralston Purina Chow 5001® and tap water were available ad libitum throughout the acclimation and dosing periods.

Chemicals and dosing.
Analytical grade 1,1,1-trichloroethane (TRI) of 99%+ purity was obtained from Aldrich Chemical Co. (Milwaukee, WI). The proclaimed purity was assessed by gas chromatography and found to be accurate. Amersham (Piscataway, NJ) supplied 14C-Citrulline. All other chemicals and biologicals were purchased from Sigma Chemical Co. (St. Louis, MO). Mazola® corn oil, from Best Foods/CPC International, Inc. (Englewoods Cliffs, NJ), was used as a diluent. Appropriate quantities of TRI for each dosage level were incorporated into corn oil the day of dosing. Controls received corn oil only. The solutions were administered as a bolus by gavage, using a curved, ball-tipped intubation needle affixed to a glass syringe. Dosings for the toxicity studies were conducted between 1000 and 1100 h, or 1 to 2 h into the reverse cycle rats' dark/active period. Animals in the P450 studies were dosed between 0900 and 1000 h, or 1 to 2 h into the normal cycle rats' light/inactive cycle.

Acute toxicity study protocol.
Five doses of TRI were administered orally to rats of 200–220 g: 0, 0.5, 1.0, 2.0, and 4.0 g/kg bw. The appropriate amounts of TRI were diluted in corn oil and given in a total volume of 1 ml as an oral bolus. The animals were weighed and euthanized with ether 24 h postdosing, so that blood and liver samples could be taken for evaluation.

Short-term toxicity study protocol.
Four dosage levels of TRI were given orally to rats of 230–260 g: 0, 0.5, 5.0, and 10.0 g/kg bw. The appropriate amounts of TRI for the 0.5 and 5.0 g/kg doses were diluted with corn oil, such that a total volume of 1 ml was given as a bolus by gavage. The 10.0 g/kg dose was similarly diluted and given by gavage in a total volume of 2 ml. Controls received 1 ml of corn oil. The rats were dosed once daily for 5 consecutive days, allowed 2 days without dosing, and then dosed for 4 additional days. Moribund animals were removed and examined for gross morphological changes. Groups of up to 5 animals per dosage level were etherized 1, 5, and 12 days after initiation of dosing for evaluation of clinical chemistry parameters and liver histopathology.

Subchronic toxicity study protocol.
Four dosage levels of TRI were given orally to groups of 15–20 rats of 200–260 g: 0, 0.5, 2.5, or 5.0 g/kg bw. The appropriate amounts of TRI for the 0.5 and 2.5 g/kg doses were diluted with corn oil, such that a total volume of 0.5 ml was given as a bolus by gavage. Similarly, the 5.0 g/kg dose was diluted and given in a total volume of 1.0 ml/kg. The rats were dosed on a daily basis 5 times weekly for up to 13 weeks. Blood samples were taken at the following intervals during the regimen: 2, 4, 6, 8,10, 12, and 13 weeks and 1 week posttreatment. Five rats at each dosage level were randomly selected to serve as blood donors. These animals were lightly etherized and 2.5 ml of blood taken by needle puncture of the caudal artery for measurement of serum enzymes. Five different rats from each group were utilized for blood sampling at each of the next 2 sampling intervals, so that each animal served once as a blood donor during the first half of the study. On day 51 of the regimen, 1,1-dichloroethylene was mistakenly given in place of TRI to each member of the 2.5 and 5.0 g/kg groups. All of these animals died within 24 h of massive liver damage. Fortunately, they were housed in a different isolation module from the 0.5 g/kg and control groups. The 0.5 g/kg and control rats were dosed properly on day 51 and thereafter. Blood samples were again taken for serum enzyme analysis from alternate animals in these 2 groups every 2 weeks for the reminder of the study. Moribund rats were removed, killed, and examined for gross morphological changes. The terminal sacrifice was performed on day 92, after 13 weeks of the dosing regimen. Five additional control and five 0.5 g/kg animals were killed after 1 week of recovery.

Clinical chemistry and histopathology evaluations for toxicity studies.
At sacrifice, each rat in the toxicity studies was anesthetized with ether and blood taken by closed-chest cardiac puncture. Serum was prepared and frozen at –20°C prior to analysis. Alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities were measured with a Gemini centrifugal analyzer. Serum ornithine carbamoyl-transferase (OCT) activity was determined by the 14CO2 trapping technique of Drotman (1975), as modified by Kyle et al. (1983).

After taking blood, the abdominal cavity of each animal was opened and the liver excised and weighed. A 1 cm-wide strip of the left median lobe of the liver was removed and placed into buffered formalin. The tissue specimens were routinely processed into paraffin, cut at 2 µ and stained with hematoxylin and eosin. Slides were coded and examined in a single-blind fashion by a veterinary pathologist.

The nonprotein sulfhydryl (NPSH) content of 300-mg portions of the liver was measured by the technique of Richardson and Murphy (1975). A 5-g portion of the central hepatic lobe was removed and microsomes prepared by differential centrifugation. Microsomal protein content was assayed according to Lowry et al. (1951). An aliquot of the microsomal suspension was incubated with histidine, EDTA and glucose-6-phosphate. Liberation of inorganic phosphate was the measure of glucose-6-phosphatase (G-6-Pase) activity (Reynolds and Yee, 1968Go). G-6-Pase activity was monitored as an index of liver microsomal injury.

Hepatic P450 assays and experiments.
The liver was perfused in situ with cold normal saline in order to remove as much blood as possible. A 5-g portion of the central lobe of the liver was then taken for preparation of microsomes by differential centrifugation. The total cytochrome P450 content of hepatic microsomes was determined by the standard spectrophotometric method of Omura and Sato (1964). The microsomal protein concentration was measured by the procedure of Lowry et al. (1951). CYP2E1 activity was estimated by measuring p-nitrophenol hydroxylation (PNP-OH). The hydroxylation of PNP to 4-nitrocatechol (4-NC) was determined according to Koop (1990). A substrate concentration of 100 µM PNP was used in this assay. The activities of pentoxyresorufin-O-dealkylase (PROD) and ethoxyresorufin-O-deethylase (EROD) were measured as indices of the activities of CYP2B1/2 and CYP1A1, respectively. PROD was assayed by the fluorimetric method of Lubet et al. (1985). The fluorimetric technique of Burke and Mayer (1974), as modified by Lubet et al. (1985), was used to measure EROD activity.

The objective of the initial experiment was to determine whether TRI could induce total hepatic microsomal cytochrome P450, CYP2E1, or CYP2B1/2 and to establish the time course(s) of that induction. Rats of 250–280 g were given a single oral bolus dose of 5.0 g TRI/kg bw in corn oil and killed by cervical dislocation at the following times postdosing: 1, 6, 12, 24, 36, 48, and 72 h. Each liver was prepared as described before for measurement of microsomal P450, protein content, and activities of CYP2E1 and CYP2B1/2. An additional experiment was conducted to determine whether the time course of CYP2E1 induction varied with dose. Rats were gavaged with 0.5 and 5.0 g TRI/kg and sacrificed at the same time intervals, so that CYP2E1 activity could be monitored.

The objective of the second series of experiments was to assess the dose dependency of induction of total hepatic microsomal P450, CYP2E1, CYP2B1/2 and CYP1A1. Six dosage levels of TRI were given orally to male S-D rats of 250 g: 0, 0.1, 0.5, 2.5, 5.0 and 10.0 g/kg bw. Appropriate amounts of TRI were diluted in corn oil, as described for the short-term study protocol. The rats were killed at 24 h post dosing by cervical dislocation, in order to isolate the liver microsomes rapidly and without the use of ether or other anesthetics. Each microsomal parameter was measured as described above. Another experiment was carried out to assess the influence of the short-term exposure regimen on hepatic P450 levels and CYP2E1 activity. Rats were dosed once daily for 5 consecutive days, allowed 2 days of rest, and dosed for 4 additional days. Groups of 6 animals per dosage level were sacrificed 1, 5, and 12 days after initiation of the regimen, for analyses of total P450 levels and CYP2E1 activity.

Statistical analyses.
The statistical significance of changes in toxicity and P450 indices as a function of dose was assessed by one-way analysis of variance. The significance of apparent differences over time between values for adjacent time-points was evaluated by Student-Newman-Keul's test. The significance of differences between 0-time and late time-points was assesssed by Duncan's multiple range test. The minimum level of statistical significance selected for these tests was p <= 0.05. Data for each index of liver injury in the short-term and subchronic studies were analyzed using a Minitab statistical package Version 12.0. A response surface model was used to fit each data set (Kuehl, 1998Go). Both linear and quadratic equations were derived for the data with respect to effects of TRI dosage and days of dosing. Regression analysis was utilized to assess the linearity of changes in indices as a function of dose.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute Study
There was little evidence of toxicity 24 h after a single oral dose of 0, 0.5, 1, 2, or 4 g TRI/kg. There was no mortality from TRI exposure. Histopathological examination and measurements of liver:body weight, serum enzymes, hepatic NPSH levels, and G-6-Pase activity did not reveal hepatocellular damage at any dosage level (data not shown).

Short-Term Study
The decision was made to give even higher doses of TRI in the short-term study, in an effort to produce liver injury. The acute oral LD50 for male rats was reported to be 12.3 g/kg by Torkelson et al. (1958). Thus, 10 g/kg was selected as the highest dose to administer in the current study. Since no rat given 4 g/kg died or exhibited hepatotoxicity in our acute experiment, 5 g/kg seemed to be a reasonable choice for the intermediate dosage.

The high, repetitive oral doses of TRI caused multiple fatalities. The 10 g/kg dose proved to be excessive, in that 5 of 10 rats remaining after the 1-day sacrifice died by the third day of the 11-day dosing regimen. Only one 10 g/kg animal survived to the terminal sacrifice. There were 3 deaths at 5 g/kg, 2 of which occurred within the first 24 h (data not shown). Members of the 5 and 10 g/kg groups exhibited hyperexcitability for 20 to 30 min after each dosing. This stage was followed by a protracted period of narcosis. Neither CNS effects nor fatalities were observed in the 0.5 g/kg animals.

Despite daily gavage dosing with lethal or near-lethal amounts of TRI, there were relatively few manifestations of toxicity in the surviving rats. Body weight gain was comparable in the 0 and 0.5 g/kg groups, but the 5 and 10 g/kg subjects did not gain weight during the first 5 days of the regimen (Fig. 1Go). The weight of the 5 g/kg animals remained significantly lower than that of controls throughout the study. The one 10 g/kg survivor at the terminal sacrifice weighed only 205 g and had a liver weighing 11.7 g versus 14.3 ± 1.5 SD for controls.



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FIG. 1. Body weight gain/loss of rats gavaged with 0, 0.5, 5.0, or 10.0 g TRI/kg for 1, 5, or 12 days of a short-term dosage regimen (see Materials and Methods for details). Each point represents the mean ± SD for a group of ~15 rats at each monitoring period. *Value significantly different from control at p <= 0.05.

 
Liver injury was not apparent at any TRI dosage level during the short-term study. Serum enzyme (OCT, SDH, and ALT) activities were not elevated at any time. Hepatic G-6-Pase activity and NPSH levels were not significantly different from controls (data not shown). No treatment-related lesions were found upon microscopic examination of the liver.

Subchronic Study
There was a conscious effort to administer maximally tolerated doses in the 13-week study, in order to elicit hepatotoxicity. The NTP (1983) previously found that daily gavage doses of up to 1.5 g TRI/kg/day for 105 weeks failed to produce histopathological changes in the liver or in any other organ of F344 rats. Although 3 of 15 S-D rats given 5 g/kg died during our short-term study, 5 g/kg was chosen as the highest dose, with 1/2 and 1/10 this amount selected as the medium and low doses, respectively.

A substantial number of fatalities occurred during the first half of the subchronic study (Fig. 2Go); 33% and 47% of the rats receiving 2.5 and 5.0 g/kg, respectively, died within the first 50 days of the regimen. It can be seen in Figure 2Go that the deaths occurred sooner at the highest dose. Pulmonary congestion was the only anomaly apparent upon gross examination of tissues of moribund and dead animals. One death occurred in the 0.5 g/kg group due to accidental instillation of the test solution into a subject's trachea. Rats given 2.5 or 5.0 g TRI/kg exhibited initial hyperexcitability followed by hours of narcosis, after each day's dose of TRI.



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FIG. 2. Survival of rats receiving 0, 0.5, 2.5, or 5.0 g/kg of TRI by gavage 5 times weekly for up to 91 days. Surviving members of the 2.5 and 5.0 g/kg groups were mistakenly killed on day 51. Each point represents mean percentage survival in groups originally numbering 15–20 animals.

 
Long-term intake of TRI resulted in some depression in body weight gain (Fig. 3Go). The (mean ± SD) bw of the 0.5 g/kg group (218 ± 14 g) at the initiation of the study appeared to be slightly lower than that of the controls (226 ± 19 g). Seemingly lower bws of the 0.5 g/kg group members were manifest throughout the study. At no time-point, however, were the apparent differences statistically significant. Body weight gains in the 2.5 and 5.0 g/kg groups were significantly lower than in the controls.



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FIG. 3. Body weight gain of rats receiving 0, 0.5, 2.5, or 5.0 g/kg of TRI by gavage 5 times weekly for up to 91 days. Brackets encase the mean ± SD for groups of 15–20 rats at the beginning of the study. Asterisks indicate statistically significant difference from the 0 g/kg group. The weight of the 0.5 g/kg animals did not differ significantly from the controls at any time point during the 91-day study period.

 
TRI had very limited hepatotoxicity potential under the conditions of the subchronic study. Small but statistically significant increases of serum OCT and ALT levels were manifest after 2 and 4 weeks at the highest dosage level (5 g/kg) (Table 1Go). ALT activity was not different from controls at 6 weeks at this dosage. OCT looked to remain elevated, but substantial intersubject variability in this high-dose group precluded a statistically significant difference from the control. SDH activity was slightly higher than controls at 6 weeks for all three dosages of TRI, likely due to a relatively low control value.


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TABLE 1 Serum Enzyme Levels for the First Half of the Subchronic Study
 
The 0.5 g/kg po dose of TRI did not produce recognizable injury during the latter half of the subchronic study. There were no significant alterations from controls in serum OCT, SDH, or ALT (data not shown). No histopathological changes were seen in the livers of the 0.5 g/kg animals at sacrifice on day 92 of the dosage regimen. Absolute and relative liver weights were not different from controls. As would be anticipated, there were no differences from controls in any of these indices after the 1-week recovery period (data not shown).

P450 Induction Experiments
A relatively high oral dose (5 g/kg) of TRI produced substantial increases in hepatic microsomal CYP2E1 and CYP2B1/2 activities but no change in total P450 levels (Fig. 4Go). The P450 values are not pictured. The time courses of induction of the 2 isozymes differed. CYP2E1 activity was significantly increased over the preexposure level within 6 h postdosing, and maximally induced by 12 h. A significant increase in CYP2B1/2 activity was first seen 12 h after TRI. CYP2B1/2 activity was maximal at 24 h. The maximum elevations in CYP2E1 and CYP2B1/2 were approximately 4x and 5x times controls, respectively. CYP2B1/2 steadily decreased from its peak, returning to preexposure levels within 48 h. In contrast, CYP2E1 activity diminished more rapidly, reaching its preexposure range by 36 h.



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FIG. 4. Relative time courses of induction of CYP2E1 and CYP2B1/2 activities in liver microsomes of rats give a single oral dose of 5.0 g TRI/kg bw. CYP2E1 activity was estimated by measuring hydroxylation of p-nitrophenol to 4-nitrocatechol (4-NC). CYP2B1/2 activity was assessed by measurement of O-dealkylation of pentoxyresorufin to resorufin. Values are expressed as mean ± SD for groups of 5–6 rats. SD bars are omitted from some data points for clarity. Values for each isozyme, which are significantly different at different sampling times, are designated by a different letter.

 
The magnitude and the time course of induction of CYP2E1 by TRI were dose-dependent. As illustrated in Figure 5Go, a maximal increase of ~50% over the preexposure level was manifest at 6 and 12 h in animals receiving 0.5 g TRI/kg. CYP2E1 activity was significantly elevated at 6 h, but did not reach its peak (an ~380% increase) until 12 h after 5.0 g TRI/kg. Although there appeared to be some decrease between 12 and 24 h in the high-dose group, the 12- and 24-h values were not significantly different. During this time frame, CYP2E1 activity in the low-dose group dropped below the preexposure level.



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FIG. 5. Time course of induction of CYP2E1 activity in liver microsomes of rats given a single oral dose of 0.5 or 5.0 g TRI/kg bw. CYP2E1 activity was estimated by measuring hydroxylation of p-nitrophenol to 4-nitrocatechol (4-NC). Values are expressed as mean ± SD for groups of 5–6 rats. Values for each dosage level, which are significantly different at different sampling times, are designated by a different letter.

 
Orally administered TRI produced dose-dependent elevations in 2 of the 3 P450 isozymes assayed (Fig. 6Go). PNP-OH, PROD, and EROD activities were measured in liver microsomes prepared 24 h postdosing, as indices of CYP2E1, CYP2B1/2, and CYP1A1 activities, respectively. CYP1A1 activity was not enhanced at any dosage level (data not shown). The lowest dose to significantly induce CYP2E1 and CYP2B1/2 was 2.5 g/kg. The activity of each isozyme was maximally induced by the 2 highest doses of TRI.



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FIG. 6. Dose dependency of induction of hepatic microsomal CYP2E1 and CYP2B1/2 activities in male S-D rats. CYP2E1 activity was estimated by measuring p-nitrophenol hydroxylation to 4-nitrocatechol (4-NC). O-dealkylation of pentoxyresorufin to resorufin was used as an index of CYP2B1/2. Measurements were made 24 h after administration of a single oral dose of 0, 0.1, 0.5, 2.5, 5.0, or 10.0 g TRI/kg bw. Values are expressed as the mean ± SD for groups of 4–6 animals. Values for each isozyme that are significantly different are designated by a different letter.

 
Repetitive oral exposure to TRI did not result in progressive increases in hepatic microsomal P450 levels or in CYP2E1 activity. The only statistically significant increase in total P450 was manifest on day 1 at the highest dosage level (10 g/kg) (Fig. 7Go). Small, but statistically significant reductions in total P450 levels were apparent on days 5 and 12 in the 0.5 g/kg subjects. Dose-dependent increases in CYP2E1 activity were manifest at 5 and 10 g/kg on days 1 and 5 of the short-term exposure regimen (Fig. 8Go). The magnitude of CYP2E1 induction in response to each of these 2 highest doses did not vary substantially during the 12-day exposure period. This isozyme's activity, like P450 levels, was lower than controls in the 0.5 g/kg animals on days 5 and 12.



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FIG. 7. Influence of short-term oral dosing with TRI on total P450 levels in liver microsomes of male S-D rats. Rats were given 0, 0.1, 0.5, 5.0, or 10.0 g TRI/kg po for 1, 5, or 12 days of a repetitive dosage regimen (see Materials and Methods for details). Values are expressed as mean ± SD for groups of 4–6 rats. Values that are significantly different at each sampling period are designated by a different letter.

 


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FIG. 8. Influence of short-term exposure to TRI on hepatic microsomal CYP2E1 activity. Male S-D rats received 0, 0.1, 0.5, 5.0, or 10.0 g TRI/kg po for 1, 5, or 12 days of a repetitive dosage regimen (see Materials and Methods for details). CYP2E1 activity was estimated by measuring hydroxylation of p-nitrophenol to 4-nitrocatechol (4-NC). Values are expressed as mean ± SD for groups of 4–6 rats. Values that are significantly different at each sampling period are designated by a different letter.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Findings in the present report may be useful in assessing noncarcinogenic risks of short- and long-term ingestion of TRI. A single oral bolus dose of 4 g/kg was not lethal to male S-D rats, but 2 of 15 rats given 5 g/kg succumbed within 24 h of their initial dose. Most animals receiving 10 g/kg po died. Moser and Balster (1985) observed a very steep concentration-lethality relationship for CD-1 mice exposed to a series of vapor concentrations of TRI. Their animals, like those in the current study, exhibited a characteristic progression of signs ranging from hyperactivity to lethargy, unconsciousness, shallow rapid breathing, and death. The apparent cause of death in their investigation and in our own was CNS/respiratory depression. Cardiac arrhythmias and decreased peripheral vascular resistance could be contributory, since TRI can induce cardiac arrhythmias and exert autonomic effects when present systemically in very high concentrations (Aoki et al., 1997; Clark and Tinston, 1973Go; Kobayashi et al., 1987Go). Hepatotoxicity does not appear to play a role in the subjects' demise, since little or no evidence of acute liver injury has been found in rodents given near-lethal to lethal doses of TRI by ip injection (Klaassen and Plaa, 1966Go), inhalation (Carlson et al., 1973) or gavage (present study).

CNS depression was obvious in rats given >= 2 g/kg po in the current investigation. CNS effects would likely have been detected at lower doses, had sensitive, objective measures of animal behavior or performance been employed. A battery of tests by P. J. Spencer et al. (1990, unpublished) failed to show behavioral changes in rats given a single dose of 705 mg TRI/kg po. After several such daily doses, alterations in flash- and somatosensory-evoked potentials, as well as changes in EEG recordings, were described in these investigators' unpublished report. Despite TRI's ability to produce CNS depression, the most comprehensive neurotoxicological evaluation published to date failed to reveal evidence of neurotoxicity in F344 rats exposed to <= 2000 ppm TRI vapor for 6 h/day, 5 days/week for 13 weeks (Mattsson et al., 1993Go). Thus, reversible CNS depression may be the most sensitive measure of acute biological effect by TRI.

Short-term administration of extremely high doses of TRI by gavage caused pronounced CNS depression and ensuing deaths of rats in the current study, but no evidence of hepatotoxicity. The 2 highest dosage levels, 5 and 10 g/kg, caused multiple fatalities. No signs of organ damage were apparent upon gross examination. Body and liver weights were relatively low in survivors, likely the result of reduced food and water intake due to the prolonged periods of narcosis and recovery. Consumption of water and food were not monitored. Light microscopic examination of the liver and serum enzyme measurements failed to reveal hepatic anomalies at any dosage level. Maurissen et al. (1994) gavaged pregnant rats with up to 0.75 g/kg/day on gestation day 6 through lactation day 10, but saw no maternal toxicity or neuropathology in the offspring. No other TRI ingestion or inhalation study of comparable length was found in the literature. Thus, 0.5 g/kg was the short-term oral NOAEL, for effects other than temporary alteration of hepatic CYP2E1, under the conditions of the present study. Three deaths occurred at 5 g/kg, the short-term LOAEL here.

TRI does not appear to be more toxic on subchronic than on acute or short-term exposure. Oral administration of 2.5 and 5.0 g/kg over 7 weeks produced a substantial number of fatalities. The only histopathological changes found in these rats were manifestations of chronic murine pneumonia, which may have contributed to their deaths. The 5.0 g/kg dose produced slight elevations in serum enzyme activities. There was no apparent progression or regression of hepatocellular damage with repeated TRI exposure, since comparable increases in serum enzymes were present at the 2-, 4-, and 6-week monitoring periods. It is unlikely that more severe liver injury would have occurred had the 2.5 or 5.0 g/kg animals been dosed for the intended 13 weeks. Nonneoplastic changes were not found in the liver or in any other organ of male or female rats gavaged with TRI doses as high as 1.5 g/kg for up to 2 years (NCI, 1977Go; NTP, 1983Go). Both sexes, however, exhibited decreased survival rates. It is possible that deaths would have occurred in the present study at doses below 2.5 g/kg, the lowest dosage employed. We saw no adverse effects, however, in male rats given 0.5 g/kg po for 13 weeks. Maltoni et al. (1986) found no reduction in bw gain or survival of S-D rats gavaged with 0.5 g TRI/kg in olive oil 4–5 times weekly for 104 weeks. The NOAEL in the current subchronic study was 0.5 g/kg, but the LOAEL of 2.5 g/kg resulted in multiple fatalities.

A finding in this investigation demonstrates that TRI can induce CYP2E1 and CYP2B1/2 activities in rat liver, but has little effect on CYP1A1 activity or total P450 levels. These results might be anticipated, in that substrates for a particular isozyme are often capable of inducing that isozyme. Phenobarbital pretreatment enhances TRI metabolism in rats, but ß-naphthoflavone pretreatment does not (Ivanetich and Van Den Honert, 1981Go; Koizumi et al., 1983Go). Previous studies have yielded conflicting findings on effects of TRI on P450-catalyzed metabolism of xenobiotics in rats. Toftgard et al. (1981) found no change in hepatic P450 levels in S-D rats inhaling 800 ppm TRI intermittently for 4 weeks. Vainio et al. (1976), and Savolainen et al. (1977) observed significant decreases in hepatic microsomal P450 in rats administered 1.37 g/kg orally or 500 ppm 6 h daily for 4 days (total absorbed dose ~150 mg/kg) by inhalation, respectively. We did see small decreases in total P450 and CYP2E1 at 0.5 g/kg after 5 and 12 days of our short-term oral dosing regimen. Wang et al. (1996) reported that inhalation of 4000 ppm for 6 h (total absorbed dose ~1.86 g/kg) did not affect P450 levels or CYP2E1, CYP2B1/2, or CYP1A1 activities or apoenzyme levels in the livers of male Wistar rats. The lack of induction in these studies may have been due to the prolonged administration of low TRI doses, relative to the bolus doses that were effective in the current study. Fuller et al. (1970) did report increases in P450 and in metabolism of several drugs in liver microsomes of rats that inhaled 2500–3000 ppm TRI for 24 h (total absorbed dose ~4.64–5.56 g/kg). We measured a small increase in total P450 24 h after giving 10 g/kg by gavage. CYP2B1/2 is not a constitutive isoform, while CYP2E1 normally comprises only ~10% of P450s in rat liver (Johansson et al., 1988Go). Thus 4- to 5-fold increases in these isozymes had relatively little influence on total P450 levels.

The time courses of TRI induction of P450s are dissimilar in the present study. CYP2B1/2 activity increases steadily to a maximum 24 h post-TRI, and then progressively declines to preexposure values by 48 h. Induction of CYP2B1/2 by phenobarbital occurs within a similar time frame in rat liver. Induction by PB requires sequential increases in gene transcription, mRNA levels, and isozyme synthesis (Ronis et al., 1991Go; Waxman and Azaroff, 1992Go). CYP2E1 activity was induced more rapidly by TRI (Fig. 4Go). CYP2E1 may be unique among P450 isozymes, in that a number of its substrates (e.g., acetone, ethanol, trichloroethylene) act as inducers by binding to it and thereby delaying its normal degradation by cAMP-dependent proteolysis (Lee et al., 2000Go; Roberts et al., 1995Go; Ronis et al., 1991Go; Song et al., 1989Go). The reversibility of CYP2E1 induction by TRI is apparently dependent upon metabolism of TRI and release of the isozyme for degradation. The relatively small amount of TRI received by the 0.5 g/kg rats induces CYP2E1 somewhat, but is apparently soon metabolized and exhaled. As more TRI is present for a longer time in the 5.0 g/kg animals, there is longer for CYP2E1 to be synthesized, to be bound/stabilized by TRI, and to accumulate to higher levels (Fig. 5Go).

Induction of hepatic P450 isozymes by TRI would appear to be of limited toxicological significance in humans. CYP2B1/2 is apparently not expressed to a significant extent in man (Rendic and DiCarlo, 1997Go), but rat and human CYP2E1s are very similar (Wrighton and Stevens, 1992Go). Human CYP2E1 is responsible for the metabolic activation of a variety of toxic chemicals, including many halocarbons (Guengerich et al., 1991Go; Raucy et al., 1995). Its induction by doses of TRI, which may be encountered occupationally, should be modest and transitory if it occurs at all, judging from our observations in rats. The high doses that were effective here in enhancing CYP2E1 and CYP2B1/2 activities would be life-threatening in man. Effects on total P450 and CYP2E1 did not become more pronounced with repeated TRI exposure. Koizumi et al. (1983) did find that continuous inhalation of 200 ppm TRI for 10 days enhanced the hepatic metabolism of TRI to trichloroethanol in rats. Schumann et al. (1982a), however, reported that 16 months of inhalation of 1500 ppm for 6 h daily (absorbed dose each day ~700 mg/kg) had no effect on the metabolism or disposition of 14C-TRI absorbed by rats during a subsequent 6-h exposure. A variety of other xenobiotics are more potent inducers than TRI and appear more likely to cause clinically significant chemical/drug interactions.

TRI has a very limited cytotoxic potential in the livers of S-D rats, even when administered repeatedly in lethal or near-lethal doses. It should be recognized that we utilized experimental conditions anticipated to maximize TRI's hepatotoxic potential, although the chemical's toxic moiety(ies) and mechanism(s) of action have not been clearly identified. Takano et al. (1985) observed that TRI was bound more tightly to rat liver P450 than 1,1,2-trichloroethane, but was metabolized more slowly. Rodents and humans normally metabolize <= 5–6% of systemically absorbed TRI and rapidly exhale the rest (Schumann et al., 1982bGo; Nolan et al., 1984Go). TRI is apparently exhaled so quickly that there is relatively little time for its metabolic activation. Mitoma et al. (1985) observed a modest degree of covalent binding to liver proteins of mice and rats given an oral dose of TRI as high as 3 to 4 g/kg. Results of experiments by Durk et al. (1992) indicated that TRI can also be reduced to a limited extent by male S-D rats to radical intermediates and trace amounts of acetylene. Durk et al. (1992) presented limited evidence that TRI may cause lipid peroxidation in rats. Tomasi et al. (1984) reported that substantial amounts of free radicals were formed from TRI by freshly isolated rat hepatocytes under hypoxic conditions, but found no association between the rate of free radical formation and covalent binding or carcinogenicity for eight halocarbons. Xia and Yu (1992) were unable to detect free radicals in the liver of rats dosed in vivo with TRI. Pretreatment of mice or rats with ethanol (Klaassen and Plaa, 1966Go) or phenobarbital (Carlson, 1973Go) does potentiate the hepatotoxicity of TRI, but not to the extent of its more hepatotoxic congeners.


    ACKNOWLEDGMENTS
 
This study was supported in part by the U.S. Environmental Protection Agency (EPA) Cooperative Agreement CR-811215 to JVB. The authors would like to express their appreciation to Dr. Rex Pegram of the Experimental Toxicology Division of NHEERL, U.S. EPA, Research Triangle Park, NC, for his assistance with the PROD and EROD assays. The authors are grateful to Mary Eubanks for her assistance in preparation of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (706) 542-3398. E-mail: bruckner{at}rx.uga.edu. Back

The work presented in this paper has not been reviewed by the EPA. It does not necessarily reflect the views of the EPA, and no official endorsement should be inferred.


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 RESULTS
 DISCUSSION
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