Influence of exposure route and oral dosage regimen on 1, 1-dichloroethylene toxicokinetics and target organ toxicity.

The objective of this investigation was to elucidate the effects of route of exposure and oral dosage regimen on the toxicokinetics (TK) of 1,1-dichloroethylene (DCE). Fasted male Sprague-Dawley rats that inhaled 100 or 300 ppm for 2 h absorbed total systemic doses of (10 or 30 mg/kg DCE, respectively. Other groups of rats received 10 or 30 mg/kg DCE by intravenous injection, bolus gavage (by mouth), or gastric infusion (g.i.) over a 2-h period. Serial microblood samples were taken from the cannulated, unanesthetized animals and analyzed for DCE content by gas chromatography to obtain concentration versus time profiles. Inhalation resulted in substantially higher peak blood concentrations and area under blood-concentration time curves (AUC(0)(2)) than did gastric infusion of the same dose over the same time frame at each dosage level, although inhalation (AUC(0)(infinity)) values were only modestly higher. Urinary N-acetyl-beta-D-glucosaminidase (NAG) and gamma-glutamyltranspeptidase (GGT) activities were monitored as indices of kidney injury in the high-dose groups. NAG and GGT excretion were much more pronounced after inhalation than gastric infusion. Administration of DCE by gavage also produced much higher Cmax and AUC(0)(2) values than did 2-h g.i., although AUC(0)(infinity) values were not very different. The 30 mg/kg bolus dose produced marked elevation in serum sorbitol dehydrogenase, an index of hepatocellular injury. Administration of this dose by inhalation and gastric infusion was only marginally hepatotoxic. These findings demonstrate the TK and target organ toxicity of DCE vary substantially between different exposure routes, as well as dosage regimens, making direct extrapolations untenable in health risk assessments.

Presystemic elimination of trichloroethylene in rats following environmentally relevant oral exposures.

1,1,2-Trichloroethylene (TCE), a volatile organic contaminant (VOC) of drinking water in the Unites States, is frequently present in trace amounts. TCE is currently classified by the International Agency for Research on Cancer and the U.S. Environmental Protection Agency as a probable human carcinogen, because it produces tumors in some organs of certain strains of mice or rats in chronic, high-dose bioassays. Previous studies (Toxicol Appl Pharmacol 60:509-526, 1981; Regul Toxicol Pharmacol 8:447-466, 1988) used physiological modeling principles to reason that the liver should remove virtually all of a well metabolized VOC, such as TCE, as long as concentrations in the portal blood were not high enough to saturate metabolism. To test this hypothesis, groups of unanesthetized male Sprague-Dawley rats received intravenous injections of 0.1, 1.0, or 2.5 mg TCE/kg as an aqueous emulsion. Other rats were gavaged with 0.0001, 0.001, 0.01, 0.1, 1, 2.5, 5, or 10 mg TCE/kg b.wt. Serial microblood samples were taken via an indwelling carotid artery cannula, to generate blood TCE versus time profiles. Headspace solid-phase microextraction gas chromatography with negative chemical ionization mass spectrometry (limit of quantitation = 25 pg/ml) was used to quantify TCE. TCE was undetectable in rats given 0.0001 mg/kg, but it exhibited linear kinetics from 0.1 to 5.0 mg/kg. Bioavailability was consistent over this dosage range, ranging from 12.5 to 16.4%. The presence of these limited amounts of TCE in the arterial blood disprove the aforementioned hypothesis, yet demonstrate that first-pass hepatic and pulmonary elimination in the rat afford its extrahepatic organs protection from potential adverse effects by the majority of the low levels of TCE absorbed from drinking water.

Acute, subacute, and subchronic oral toxicity studies of 1,1-dichloroethane in rats: application to risk evaluation.

1,1-Dichloroethane (DCE) is a solvent that is often found as a contaminant of drinking water and a pollutant at hazardous waste sites. Information on its short- and long-term toxicity is so limited that the U.S. EPA and ATSDR have not established oral reference doses or minimal risk levels for the volatile organic chemical (VOC). The acute oral LD(50) in male Sprague-Dawley (S-D) rats was estimated in the present study to be 8.2 g/kg of body weight (bw). Deaths appeared to be due to CNS depression and respiratory failure. In an acute/subacute experiment, male S-D rats were given 0, 1, 2, 4, or 8 g DCE/kg in corn oil by gavage for 1, 5, or 10 consecutive days. The animals were housed in metabolism cages for collection of urine and sacrificed for blood and tissue sampling 24 h after their last dose. There were decreases in body weight gain and relative liver weight at all dosage levels, as well as increased renal nonprotein sulfhydryl levels at 2 and 4 g/kg after 5 and 10 days. Elevated serum enzyme levels, histopathological changes, and abnormal urinalyses were not manifest. For the subchronic study, adult male S-D rats were gavaged with 0.5, 1, 2, or 4 g DCE/kg 5 times weekly for up to 13 weeks. Animals receiving 4 g/kg exhibited pronounced CNS depression, with more than one-half dying by week 11. The 2-g/kg rats exhibited moderate CNS depression. One 2-g/kg rat died during week 6. There were very few manifestations of organ damage in animals that succumbed or in survivors at any dosage level. Decreases in bw gain and transient increases in enzymuria were noted at 2 and 4 g/kg. Serum enzyme levels and blood urea nitrogen were not elevated, nor were glycosuria or proteinuria present. Chemically induced histological changes were not seen in the liver, kidney, lung, brain, adrenal, spleen, stomach, epididymis, or testis. Hepatic microsomal cytochrome P450 experiments revealed that single, high oral doses of DCE did not alter total P450 levels, but did induce CYP2E1 levels and activity and inhibit CYP1A1 activity. These effects were reversible and regressed with repeated DCE exposure. There was no apparent progression of organ damage during the 13-week subchronic study, nor appearance of adverse effects not seen in the short-term exposures. One g/kg orally (po) was found to be the acute, subacute, and subchronic LOAEL for DCE, under the conditions of this investigation. In each instance, 0.5 g/kg was the NOAEL.

Acute, short-term, and subchronic oral toxicity of 1,1,1-trichloroethane in rats.

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.

Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes.

A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of acetaminophen and the toxicological significance of glutathione (GSH) depletion. The relative time of onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time- and dose-dependent depletion of cellular GSH. The acetaminophen-induced GSH depletion and ensuing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset, in contrast to that caused by iodoacetamide (IAA) and by diethylmaleate (DEM), 2 well-known depletors of GSH. There was comparable LDH leakage, irrespective of drug treatment, when GSH levels decreased to about 20% of normal. Reduction of GSH levels below the 20% threshold by IAA treatment resulted in marked LDH leakage and loss of viability. Maximal LDH leakage in response to IAA and acetaminophen preceded maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence of cell damage as well as GSH depletion. IAA and DEM produced a comparable, modest accumulation of MDA, yet IAA was much more cytotoxic. These findings indicate that lipid peroxidation does not play a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in vivo, the system can be a useful and valid model with which to study mechanisms of chemical toxicity.

Development of an animal model of solvent abuse for use in evaluation of extreme trichloroethylene inhalation.

Self-intoxication by inhalation of vapors of trichloroethylene (TCE) and other solvents is widespread. In order to develop exposure protocols which typify episodes of TCE "sniffing", male Wistar-Munich rats were exposed to TCE vapor levels ranging from 9000 to 16 000 ppm. TCE in concentrations of 14 000 ppm and greater quickly produced loss of righting reflex. Recovery from the narcosis was very rapid. Central nervous system (CNS) depression was found to be cumulative in rats subjected for 5 h to alternating periods of 5 min of 15 000 ppm TCE and 15 min of fresh air. Ethanol markedly potentiated depression in these subjects. No evidence of liver or kidney damage was seen in rats subjected to the repetitive 5-h TCE inhalation regimen, nor in rats fasted for 16 h before the TCE-exposure session. Oral administration of 5 ml/kg body wt of ethanol 1 h, 16 h, or once daily for 3 days before the TCE-exposure regimen had little if any potentiating effect on hepatorenal toxicity potential. Animals that received ethanol and were fasted before TCE exposure exhibited slight elevations in SGOT and SGPT levels.

Effects of long-term exposure to environmental levels of polychlorinated biphenyls on pharmacokinetics of pentobarbital in rats.

The pharmacokinetics of pentobarbital, 30mg/kg iv, were studied in untreated rats and rats pretreated with 1,5, and 25 ppm of polychlorinated biphenyls in food for up to 140 days. Environmental contaminants may contribute to variations in metabolic rates of drugs by causing enzyme induction. The objective of this work was to quantitate the effects of environmental levels of the contaminant and enzyme inducer, a polychlorinated biphenyl, on the pharmacokinetics of pentobarbital, a drug whose primary elimination route is liver metabolism. The pharmacokinetics of pentobarbital in rats could be fit to a biexponential equation of the type Cp = Ae-alpha t+ Be-beta t. After 35 days of pretreatment, only the 25-ppm-treated rats showed any significant acceleration of pentobarbital elimination. At the 70- and 140-day samplings, both the 5- and 25-ppm pretreatments showed significant acceleration of pentobarbital elimination. There were no significant effects on A, alpha, B, and Vd for any pretreatment. The beta-values for the 25-ppm-pretreated rats reached a constant value from the 35-day pretreatment period onward. A calculation of total body clearance suggested that pentobarbital elimination in those rats had approached portal blood flow rate-limited metabolism.

Cephaloridine toxicity in primary cultures of rat renal cortical epithelial cells.

Primary cultures of rat renal cortical epithelial cells were used to assess the in vitro nephrotoxicity of cephaloridine (Cph). Several different indices were used to follow the course of Cph-induced nephrotoxicity in the cultures. Plasma membrane integrity was determined by the effect of Cph on lactate dehydrogenase (LDH) leakage, cellular K(+) content and plasma membrane Na(+)/K(+) ATPase activity. Significant LDH leakage and decreased cellular K(+) content occurred after 8 hr of exposure to Cph. Na(+)/K(+) ATPase activity was depressed as early as 4 hr after Cph treatment. Brush border integrity was assessed by the effect of Cph on the activity of the brush border enzyme alkaline phosphatase, which was significantly decreased following 12 hr of exposure to Cph. Treatment with Cph resulted in an initial elevation of cellular glutathione (GSH)-as indicated by cellular non-protein sulphydryl content-followed by a decrease in GSH content 16 hr later. The mitochondrial response to Cph was assessed by determining mitochondrial succinate dehydrogenase (SDH) activity and cellular ATP content. SDH activity was significantly depressed after 4 hr of Cph exposure; ATP content was not significantly depressed until 12 hr after treatment. The time course of Cph-induced injury in our culture system suggests that early injury involves alterations at the level of the mitochondrial membrane and the plasma membrane. The most sensitive indicators of Cph-induced toxicity in this system were mitochondrial SDH activity and plasma membrane Na(+)/K(+) ATPase activity.

Development of a primary culture system of rat kidney cortical cells to evaluate the nephrotoxicity of xenobiotics.

A method has been developed for preparing primary monolayer cultures of postnatal rat kidney cortical epithelial cells. These cultures maintained differentiated cell functions and epithelial-like morphology for several days in culture. The presence of alkaline phosphatase and maltase was used to confirm the presence of cells from the renal cortex. The concentrations of these enzymes were maintained in culture until day 3, but had declined significantly by day 5. Similar patterns were observed with cytochrome P-450 and glutathione content, although their concentrations remained stable from day 3 to day 5. Mercuric chloride, cadmium chloride and acetaminophen were evaluated for nephrotoxicity in this culture system. Treatment with these compounds resulted in dose-dependent changes in cell morphology and in biochemical parameters such as lactate dehydrogenase leakage, alkaline phosphatase activity and cellular glutathione content. With this culture system, it was possible to detect the acute toxicities of compounds that produce varying degrees of renal injury. Further development of this kidney culture system may have value in detecting potential nephrotoxins and in studying their mechanisms of toxicity.

Organ weights and fat volume in rats as a function of strain and age.

The Fischer 344 (F344) rat and the Sprague-Dawley (SD) rat are used commonly to evaluate potential adverse health effects resulting from environmental exposure to chemicals. They are also the most common rat strain/stock used in physiologically based pharmacokinetic (PBPK) modeling. Accurate characterization of model input parameters will improve the usefulness of PBPK model predictions. Thus, organ (i.e., liver, kidneys, spleen, stomach, small intestine, large intestine, heart, lungs, brain) weights and body fat were measured in male SD rats of different ages (4 to 40 wk) and in young (9 to 10 wk) and old (22 to 23 mo) male F344 rats. Comparison of age-matched (9 to 10 wk) F344 and SD rats revealed that the SD rats weighed significantly more and had significantly higher absolute organ weights. These significant differences usually disappeared when organ weights were expressed as a percentage of body weight (relative organ weight). Percent body fat was significantly lower in the age-matched SD rats (6.48%) than in their F344 counterparts (8.67%). As expected, both body weight and absolute organ weights were significantly higher in old than in young F344 rats. However, these differences were largely reversed when relative organ weights were considered, with most relative organ weights significantly lower in the old F344 rats. Body fat as a percentage of body weight was 14.02% in the old F344 rats. When SD rats of various ages were examined, relative organ weights declined between the ages of 4 and 14 wk. In contrast, significant differences in percent body fat were not detected among the SD rats of different ages and weights examined in this study (4 to 40 wk, approximately 75 to approximately 450 g). In summary, values for physiological input parameters are provided that should prove useful in development and implementation of more accurate PBPK models.

PBPK modeling of the metabolic interactions of carbon tetrachloride and tetrachloroethylene in B6C3F1 mice.

Potential exists for widespread human exposure to low levels of carbon tetrachloride (CT) and tetrachloroethylene (TET). These halocarbons are metabolized by the cytochrome P450 system. CT is known to inhibit its own metabolism (suicide inhibition) and to cause liver injury by generation of metabolically derived free radicals. The objective of this research was to use develop a physiologically based pharmacokinetic (PBPK) model to forcast the metabolic interactions between orally administered CT and TET in male B6C3F1 mice. Trichloroacetic acid (TCA), a stable metabolite of TET, was used as a biomarker to assess inhibition of the cytochrome P450 system by CT. Metabolic constants utilized for CT were 1.0mg/kg/h for Vmaxc_CT and 0.3 for Km_CT (mg/l). Values for TET (based in TCA production), were 6.0mg/kg/h for Vmaxc_TET was 3.0mg/l for Km_TET. The rate of loss of metabolic capacity for CT (suicide inhibition) was describe as: Vmaxloss ( mg / h )=- Kd ( RAM × RAM ) , where Kd (h/kg) is a second-order rate constant, and RAM (mg/h) is the Michaelis-Menten description of the rate of metabolism of CT. For model simplicity, CT was assumed to damage the primary enzymes responsible for metabolism of CT (CYP2E1) and TET (CYP2B2) in an equal fashion. Thus, the calculated fractional loss of TET metabolic capacity was assumed to be equivalent to the calculated loss in metabolic capacity of CT. Use of a Kd value of 400h/kg successfully described serum TCA levels in mice dosed orally with 5-100mg/kg of CT. We report, for the first time, suicide inhibition at a very low dose of CT (1mg/kg). The PBPK model under-predicted the degree of metabolic inhibition in mice administered 1mg/kg of CT. This PBPK model is one of only a few physiological models available to predict the metabolic interactions of chemical mixtures involving suicide inhibition. The success of this PBPK model demonstrates that PBPK models are useful tools for examining the nature of metabolic interactions of chemical mixtures, including suicide inhibition. Further research is required to compare the inhibitory effects of inhaled CT vapors with CT administered by oral bolus dosing and determine the interaction threshold for CT-induced metabolic inhibition.

Differences in sensitivity of children and adults to chemical toxicity: the NAS panel report.

The National Academy of Sciences (NAS) Committee on Pesticides in the Diets of Infants and Children worked for some 4 years to evaluate the extent and the health-related consequences of exposure of infants and children to pesticides. The focus of this paper is on deliberations and recommendations of the committee relevant to protection of infants and children from toxic effects of pesticides. The most comprehensive data available for contrasting the toxicity of chemicals in the young and adults were compilations of rodent mortality studies. Age-dependent differences in chemical lethality were less than 1 order of magnitude and usually varied no more than 2- to 3-fold. Findings in studies of pesticides and other chemicals revealed that toxicity was age- and compound-dependent. The younger and more immature the subject, the more different its response from that of an adult. Substantial anatomical, biochemical, and physiological changes occur during infancy, childhood, and adolescence. These maturational changes can substantially affect the absorption, distribution, metabolism, and elimination of chemicals. The net effect of immaturity on pharmacokinetics and pharmacodynamics is difficult to predict. Measurements of physiological functions in different age groups can be made and input into physiologically based pharmacokinetic (PBPK) models. The committee felt that PBPK models could be effectively utilized for different exposure scenarios, to predict the time course of potentially toxic chemicals and metabolites in different organs of children. The committee recognized that maturing organ systems of infants and children may be susceptible to injury by chemicals. There may be developmental periods (i.e., windows of vulnerability) when the endocrine, reproductive, immune, visual, or nervous systems are particularly sensitive to certain chemicals. The committee recommended early assessments using sensitive indices of injury to these organ systems of test animals. Only limited information was available on the therapeutic efficacy and toxicity of drugs in pediatric populations. The most definitive data were maximally tolerated doses (MTDs) of chemotherapeutic agents. MTDs were frequently higher for children than adults, though the differences between age groups were usually < or =2. It was concluded by the NAS committee that immaturity does not necessarily entail greater sensitivity to chemical toxicity; age-dependent toxicity is chemical-dependent; and the existing 10-fold interspecies uncertainty factor provides adequate protection of infants and children, based on current knowledge.

A rapid analytical method for measuring toluene in biological specimens.

A rapid and accurate method for the direct extraction and quantitation of toluene in blood and tissues has been developed. The technique involved extraction with methanol, selective adsorption onto Tenax, and desorption from the Tenax with heat and injection into a gas chromatograph. Measurement of standards indicated values were linear over a range of tissue concentrations of 1-1,734 microgram per gram of sample. The procedure described in this paper is a rapid, accurate method for measuring volatile hydrocarbon solvent in both blood and solid tissues of experimental animals. This technique should be applicable to basic research including animal studies, as well as to clinical cases involving abuse of or industrial exposure to solvents.

Simple method for rapid measurement of trichloroethylene and its major metabolites in biological samples.

A simple and rapid, yet sensitive technique was developed for concurrent measurement of trichloroethylene (TCE) and its major metabolites (i.e., trichloroacetic acid, trichloroethanol and dichloroacetic acid) in blood and in solid tissues. The method involves addition of an esterizer (water, sulfuric acid, methanol; 6:5:1; v/v/v) to blood or tissue homogenate in sealed vials, and subsequent gas chromatographic headspace analysis. The procedure should be useful in medical monitoring of TCE exposure as well as in experimental work, notably pharmacokinetic and pharmacodynamic studies pertaining to TCE carcinogenesis.

Biological factors which may influence an older child's or adolescent's responses to toxic chemicals.

Currently, there is considerable interest in scientific and regulatory issues relating to protection of children's health. Attention to date has largely been focused on establishing the efficacy and safety of drugs in children and on assessing potential risks of pesticides and similar agents to infants and young children. Older children and adolescents, however, have received little attention as special subgroups at risk from exposure to toxic and carcinogenic chemicals. Adolescence is the second most rapid period of growth and development, after infancy. Several organ systems experience substantial structural and functional changes during puberty. Attention is focused in this review on the more important organ systems that are undergoing maturation and therefore may be the most likely to exhibit aberrant responses to toxicants. Attention is also paid to age-related changes in processes which govern the disposition and metabolism of chemicals in the body.