Robert P. Beliles

Consideration of the Target Organ Toxicity of Trichloroethylene in Terms of Metabolite Toxicity and Pharmacokinetics

Trichloroethylene (TRI) is readily absorbed into the body through the lungs and gastrointestinal mucosa. Exposure to TRI can occur from contamination of air, water, and food; and this contamination may be sufficient to produce adverse effects in the exposed populations. Elimination of TRI involves two major processes: pulmonary excretion of unchanged TRI and relatively rapid hepatic biotransformation to urinary metabolites. The principal site of metabolism of TRI is the liver, but the lung and possibly other tissues also metabolize TRI, and dichlorovinyl-cysteine (DCVC) is formed in the kidney. Humans appear to metabolize TRI extensively. Both rats and mice also have a considerable capacity to metabolize TRI, and the maximal capacities of the rat versus the mouse appear to be more closely related to relative body surface areas than to body weights. Metabolism is almost linearly related to dose at lower doses, becoming dose dependent at higher doses, and is probably best described overall by Michaelis-Menten kinetics. Major end metabolites are trichloroethanol (TCE), trichloroethanol-glucuronide, and trichloroacetic acid (TCA). Metabolism also produces several possibly reactive intermediate metabolites, including chloral, TRI-epoxide, dichlorovinyl-cysteine (DCVC), dichloroacetyl chloride, dichloroacetic acid (DCA), and chloroform, which is further metabolized to phosgene that may covalently bind extensively to cellular lipids and proteins, and, to a much lesser degree, to DNA. The toxicities associated with TRI exposure are considered to reside in its reactive metabolites. The mutagenic and carcinogenic potential of TRI is also generally thought to be due to reactive intermediate biotransformation products rather than the parent molecule itself, although the biological mechanisms by which specific TRI metabolites exert their toxic activity observed in experimental animals and, in some cases, humans are not known. The binding intensity of TRI metabolites is greater in the liver than in the kidney. Comparative studies of biotransformation of TRI in rats and mice failed to detect any major species or strain differences in metabolism. Quantitative differences in metabolism across species probably result from differences in metabolic rate and enterohepatic recirculation of metabolites. Aging rats have less capacity for microsomal metabolism, as reflected by covalent binding of TRI, than either adult or young rats. This is likely to be the same in other species, including humans. The experimental evidence is consistent with the metabolic pathways for TRI being qualitatively similar in mice, rats, and humans. The formation of the major metabolites--TCE, TCE-glucuronide, and TCA--may be explained by the production of chloral as an intermediate after the initial oxidation of TRI to TRI-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of pregnancy on the acute toxicity of various compounds in mice

Abstract LD50 values were determined in pregnant (day 13) and nonpregnant mice. Iron dextran, iron dextrin, folic acid, colchicine and chlorpromazine hydrochloride were more toxic in the pregnant mouse than in the nonpregnant mouse following iv administration. Aspirin administered po appeared less toxic in the pregnant mouse. No remarkable differences between pregnant and nonpregnant animals in respect to the LD50 values were noted (a) following iv administration of atropine sulfate, caffeine, chlorpheniramine maleate, chloramphenicol, diphenhydramine hydrochloride, ethanol, ergonovine maleate, ferrous sulfate, hexobarbital sodium, isoproterenol hydrochloride, metyrapone ditartrate, pentobarbital sodium and trypan blue, and (b) following po administration of ferrous sulfate, folic acid and naladixic acid.

Effect of mercury vapor exposure on metallothionein and glutathione s-transferase gene expression in the kidney of nonpregnant, pregnant, and neonatal rats.

Elemental mercury (Hg 0 ) is a ubiquitous toxic pollutant. Exposure to Hg 0 vapor typically is by inhalation, and the kidney is the primary target organ. Glutathione (GSH) and metallothionein (MT) appear to mitigate mercury toxicity. However, little is known about GSH or MT regulation after Hg 0 vapor exposure, particularly during pregnancy, a time of high sensitivity to most metals. Thus, this study sought to determine renal mercury accumulation and MT- and GSH-related gene expression following Hg 0 vapor exposure in nonpregnant, pregnant, and neonatal rats exposed in utero. Groups ( n = 5) of pregnant rats (LongEvans) were exposed to Hg 0 vapor (4 mg/m3) or air (control) for 2 h/d from gestational day (GD) 6 to 15, and kidneys from dams and pups were removed at various times during and after the onset of exposure. For comparative purposes, nonpregnant female rats were exposed to Hg 0 for 10 d under the same conditions. Renal mercury, MT protein, and GST activity were assayed by standard analytical techniques. Western blot analysis was also performed using antibodies against MT and GST-pi. GSH-related gene expression was studied by cDNA microarray. Hg 0 vapor exposure produced renal accumulation of mercury in nonpregnant, pregnant, and neonatal rats. However, the transplacentally exposed neonates accumulated approximately 1000-fold less mercury than adults. Hg 0 vapor exposure produced a time-dependent increase in renal MT protein in nonpregnant and pregnant rats, but not in neonatal rats. Maximum MT increases were observed on d 10 (fivefold) in nonpregnant and GD 15 (threefold) in pregnant rats. Activation of the MT gene by Hg 0 was confirmed at the translational level by Western blot analysis and at the transcriptional level by Northern blot analysis. Microarray analysis revealed a significant upregulation in the renal expression of the GST-pi, GST-Ya , and microsomal GST and GST5-5 genes in nonpregnant and pregnant rats. Western blot and enzyme assay confirmed the upregulation of GST genes after Hg 0 exposure. Thus, in response to Hg 0 vapor exposure, the expression of the MT gene and various GST genes is activated in nonpregnant and pregnant rats. Activation of these genes could be part of a defensive response directed at decreasing renal mercury toxicity, and may help divert the metal away from the fetus.

Valproate-induced limb malformations in mice associated with reduction of intracellular pH.

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pHi). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pHi was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pHi of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pHi regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pHi.

Gestational Mercury Vapor Exposure and Diet Contribute to Mercury Accumulation in Neonatal Rats

Exposure of pregnant Long-Evans rats to elemental mercury (Hg0) vapor resulted in a significant accumulation of Hg in tissues of neonates. Because elevated Hg in neonatal tissues may adversely affect growth and development, we were interested in how rapidly Hg was eliminated from neonatal tissues. Pregnant rats were exposed to 1, 2, or 4 mg Hg0 vapor/m3 or air (controls) for 2 hr/day from gestation day 6 (GD6) through GD15. Neonatal brain, liver, and kidney were analyzed for total Hg at various times between birth and postnatal day 90 (PND90). Milk was analyzed for Hg between birth and weaning (PND21). Before weaning, the Hg levels in neonatal tissues were proportional to maternal exposure concentrations and were highest in kidney followed by liver and then brain. There was no elimination of Hg between birth and weaning, indicating that neonates were exposed continuously to elevated levels of Hg during postpartum growth and development. Consumption of milk from exposed dams resulted in a slight increase in kidney Hg concentration during this period. Unexpectedly, neonatal Hg accumulation increased rapidly after weaning. Increased Hg was measured in both control and exposed neonates and was attributed to consumption of NIH-07 diet containing trace levels of Hg. By PND90, tissue Hg levels equilibrated at concentrations similar to those in unexposed adult Long-Evans rats fed the same diet. These data indicate that dietary exposure to trace amounts of Hg can result in a significantly greater accumulation of Hg in neonates than gestational exposure to high concentrations of Hg0 vapor.

Concordance across species in the reproductive and developmental toxicity of tetrachloroethylene.

Reproductive and developmental toxicities resulting from exposure to tetrachloroethylene include delayed or impaired conception, sperm quality, death during development, developmental neurotoxicity, and growth retardation. In most cases there was concordance between rodents and humans. The risk assessments indicated that neurotoxicity was the most sensitive endpoint for inhalation, whereas growth retardation was the most sensitive endpoint when exposure was by the oral route. The reference concentration (RfC) of 0.01 ppm was based on neurotoxicity among human subjects. The reference dose (RfD) of 0.0006 mg/kg per day was based on small for gestation age infants. In both cases, studies in rodents supported the credibility of these assessments. For the RfD, similar findings for other trihalomethanes have been reported. The latter part of pregnancy and early life may constitute a susceptible period for alterations leading to behavioral deficits. During this period, the capacity to metabolize tetrachloroethylene is reduced and may further contribute to the sensitivity during this phase of development. Studies suggest that an effect on dopamine metabolism is a plausible mode of action for some types of neurotoxicity. In addition, it has been suggested that this alteration may affect reproductive integrity by influencing prolactin levels.

Pharmacokinetically based risk assessment of workplace exposure to benzene

Cancer risk from exposure to benzene for a working lifetime was estimated from data obtained in studies with rodents. Cancers of the Zymbal gland and the blood-forming system were selected as endpoints for the assessment because of their consistent occurrence. The combined metabolites were judged from toxicological data to be the best representative of the reactive agent. Because of similarity in the percentages of lifetime exposed in the rodent studies and in the occupational setting, the amount metabolized/day as a result of exposures 5 days a week for a lifetime was judged to be an appropriate dose paradigm for this assessment. Derived Michaelis-Menton constants were used to convert the doses of combined metabolites from the pharmacokinetic studies to the doses used in the bioassays. Scaling across species was based on allometric relationships. Experimental data were used to scale doses across species with body weight ratios raised to the exponents of 0.74 for the inhalation route and 1.0 for the oral route. The occupational lifetime cancer risk estimated from rodent data was 6 to 14 cases/1000 workers, which is consistent with the 9.5 to 174 leukemia cases/1000 estimated by others from epidemiological data. Implications of these estimates and uncertainties associated with making them are discussed.

The subchronic toxicity of 5-benzyl-11-[4-(N-methylpiperidilene)]-5,6-dihydromorphanthridine hydrogen maleate

Abstract The toxicity of 5-benzyl-11-[4-( N -methylpiperidylene)]-5,6-dihydromorphanthridine hydrogen maleate (EX 10-542A), an antitremor and anticholinergic agent, was investigated by oral administration to rats and dogs for 26 weeks. Reduced body weight gain, hyperglycemia, increased relative liver weights, and fibrotic and hydropic degeneration of the pancreatic islets were produced in rats at 100 mg/kg. The changes appeared to be reversible. Secondary myositis was also observed at this dose level. Doses of 25 or 50 mg/kg produced histologic changes in the pancreatic islets, decreased growth and liver weight increases. A dose of 10 mg/kg produced only the pancreatic lesion and the increased liver weights. No such changes were observed in dogs.

Risk Assessment and Oncodynamics of Ethylene Oxide as Related to Occupational Exposure

Two rat inhalation bioassays have been integrated into the risk assessment on the carcinogenicity of ethylene oxide (EO). The car cinogenic findings as well as relevant metabolism and pharmacoki netic data are reviewed. Brain tumors were selected as the endpoint for the assessment of risk because of the indication that adverse effects on the nervous system, related to EO exposure, were con sistent across species. Two methods, time-exposure concentration product and area under the plasma concentration-time curve (A UC) are used as a basis for calculating effective dose. Scaling of the dose to man from both rat and dog is explored based on phar macokinetic studies. Two different mathematical risk extrapolation models, the probit and the multi-stage, are used to estimate the cancer risk for daily exposures to EO of 1.8 μg/liter over a working lifetime. The use of A UC as a basis for dose from a daily exposure of 1.8 μg/liter over a working lifetime gives the higher risk rates (90-142/10,000 workers). The implication of the simulated dose using plasma concentrations versus the time-concentration product approach is discussed in relation to threshold effects.

Interaction of bishydroxycoumarin with chloral hydrate and trichloroethyl phosphate

Abstract Pretreatment by oral administration of trichloroethyl phosphate produced no indication of enzyme induction in rats as revealed by the duration of the effect of the test material or of zoxazolamine. The half-life of bishydroxycoumarin in dogs was reduced by pretreatment with chloral hydrate, but not by trichloroethyl phosphate. Plasma bishydroxycoumarin concentrations and prothrombin times were more affected by concurrent administration of the anticoagulant and chloral hydrate than by the anticoagulant and trichloroethyl phosphate. Chloral hydrate produced significantly higher plasma concentrations of trichloroacetic acid than did trichloroethyl phosphate in the dog. Possible differences in metabolism of trichloroethyl phosphate with respect to species, man and dog, are pointed out.