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.

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.

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.

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.

Contribution of direct solvent injury to the dose-dependent kinetics of trichloroethylene: portal vein administration to rats.

Presystemic elimination of trichloroethylene (TCE), a common contaminant of drinking water, has been shown by Lee et al. (Toxicol. Appl. Pharmacol. 139, 262-271, 1996) to be inversely related to dose. When relatively high doses were administered to rats via the portal vein (PV), first-pass hepatic extraction became negligible. This phenomenon could result not only from metabolic saturation, but from suicidal destruction of cytochromes P450 and hepatocellular injury as well. The objectives of the current investigation were to: (a) clarify the relative roles of P450 depletion and hepatocellular toxicity in the apparent cessation of hepatic elimination of TCE in animals given relatively high doses of TCE via the PV; and (b) investigate mechanism(s) of hepatocellular injury under such exposure conditions. TCE (16 and 64 mg/kg body weight (bw) was incorporated into a 5% aqueous Alkamuls emulsion and injected via an indwelling jugular vein (JV) or PV cannula into male Sprague-Dawley rats. Some animals received 73.5 micromol/kg of p-nitrophenol (PNP), a competitive metabolic inhibitor of TCE, through the PV cannula 3 min before TCE. Administration of TCE via the PV resulted in deposition of relatively high levels of TCE in the liver. PV dosing resulted in lower total hepatic P450 levels than did JV dosing. PV dosing produced marked elevations of cytoplasmic enzymes in serum, but JV dosing did not. Decreases in hepatic P450 were not selective for cytochrome P4502E1. Histological examination of the liver of PV-dosed rats revealed periportal rather than centrilobular necrosis. PNP pretreatment failed to prevent the increase in serum enzymes, decrease in hepatic P450 content, and hepatic necrosis following PV TCE. It is concluded that PV injection of bolus doses of TCE >/= 16 mg/kg causes liver injury within minutes in rats, primarily through direct solvent action on hepatocellular membranes rather than by P450-mediated effects. This liver damage likely plays a modest role in reducing the liver's capacity to metabolize high PV doses of TCE.

Mechanisms of the dose-dependent kinetics of trichloroethylene: oral bolus dosing of rats.

Trichloroethylene (TCE), a common contaminant of drinking water, is oxidized by high-affinity, low-capacity cytochrome P450 isozymes and subsequently converted to metabolites, some of which are carcinogenic in mice and rats. Although the initial oxidation step is known to be rate-limiting and saturable, the oral dosage-range over which saturation materializes is unclear. One objective of this study was to characterize the dose-dependency of gastrointestinal (GI) absorption of TCE and its kinetics over a wide range of oral bolus doses. A related objective was to investigate cause(s) of the apparent saturation kinetics observed. Cannulas were surgically implanted into a carotid artery and the stomach of male Sprague-Dawley rats. TCE was incorporated into a 5% aqueous Alkamuls emulsion and given in doses of 2 to 1200 mg/kg bw via the stomach tube. Serial blood samples were taken from the arterial cannula for up to 14 h postdosing and analyzed for TCE content by headspace gas chromatography. The rate of GI absorption of TCE diminished as the dosage increased. Pharmacokinetic analysis indicated that TCE was eliminated by capacity-limited hepatic metabolism, with incursion into nonlinear kinetics with bolus doses >/=8 to 16 mg/kg. Effects of p-nitrophenol, a competitive metabolic inhibitor, were manifest at a high, but not at a low TCE dose. Gavage bolus doses as high as 1200 mg/kg did not cause rapid elevation of serum enzyme levels, typical of the solvation of hepatocellular membranes observed after portal vein administration of TCE (Lee et al., Toxicol. Appl. Pharmacol. 163, 000-000, 2000). No evidence of cytochrome P4502E1 (CYP2E1) destruction was seen with oral doses up to 1000 mg/kg. Instead, CYP2E1 activity was induced as early as 1 h postdosing. Induction was maximal at 12 h, then returned toward controls during the next 12 h. Pretreatment with cycloheximide did not reduce CYP2E1 activity in rats given 432 or 1000 mg TCE/kg, suggesting that binding of TCE to CYP2E1 may stabilize the isozyme. Metabolic saturation, in concert with relatively slow GI absorption, are responsible for the prolonged elevation of blood TCE levels in rats given high TCE doses, while suicidal inactivation of CYP2E1 and hepatocellular injury apparently play little role.

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.

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.

Effect of Emulphor, an emulsifier, on the pharmacokinetics and hepatotoxicity of oral carbon tetrachloride in the rat.

Emulphor, a polyethoxylated vegetable oil, is now being used widely to incorporate volatile organic compounds (VOCs) and other lipophilic compounds into aqueous solutions for biochemical, pharmacokinetic, and toxicological studies. Previous work in this laboratory demonstrated that 0.25% Emulphor did not alter the kinetics or hepatotoxicity of low doses of CCl4 compared to when the halocarbon was given to rats orally in water. The present study was undertaken as there was concern that higher concentrations of Emulphor (necessary to maintain lipophilic VOCs in stable aqueous emulsions for extended periods) might alter the VOCs' absorption, disposition, and/or toxicity. Dosages of 10 and 180 mg CCl4/kg bw were given, as an aqueous emulsion using 1, 2.5, 5, or 10% Emulphor, by gavage to fasted male Sprague-Dawley rats. Serial microsamples of blood were collected from an indwelling cannula in unanesthetized, freely moving rats at intervals of 2-60 min for up to 12 hr. The samples' CCl4 content was measured by headspace gas chromatography. Thereby, it was possible to obtain blood CCl4 concentration-versus-time profiles. Animals were euthanized 24 hr postdosing and blood was collected for measurement of serum enzymes as indices of hepatotoxicity. No toxicologically significant differences in pharmacokinetic parameters as a function of Emulphor concentration were found. Similarly the hepatotoxic potency of 10 and 180 mg/kg CCl4, as reflected by elevation in serum enzyme activities, did not vary significantly with the concentration of Emulphor utilized. Hence, it can be concluded that Emulphor, in concentrations as high as 10% (equivalent to 260 mg Emulphor/kg bw) in aqueous emulsions, does not significantly affect the absorption, disposition, or acute hepatotoxicity of CCl4 in male Sprague-Dawley rats.

Uptake, distribution, and elimination of carbon tetrachloride in rat tissues following inhalation and ingestion exposures.

Carbon tetrachloride (CCl4) has been studied extensively for its hepatotoxic effects. There is a paucity of information, however, about its tissue deposition following administration by different routes and patterns of exposure. The specific objective of this study was to delineate the uptake, distribution, and elimination of CCl4 in tissues of rats subjected to equivalent oral and inhalation exposures. Male Sprague-Dawley rats (325-375 g) were exposed to 1000 ppm CCl4 for 2 hr. The total absorbed dose (179 mg CCl4/kg bw) was administered to other groups of rats as a single oral bolus or by constant gastric infusion over a period of 2 hr. Animals were terminated at selected time intervals during and postexposure and tissues (liver, kidney, lung, brain, fat, skeletal muscle, spleen, heart, and GI tract) removed for measurement of their CCl4 content by headspace gas chromatography. CCl4 levels in all tissues were much lower in the gastric infusion group than in the oral bolus and inhalation groups. Inhalation resulted in relatively high tissue CCl4 concentrations, because inhaled chemicals enter the arterial circulation and are transported directly to organs throughout the body. It seems logical that the liver should accumulate more CCl4 following ingestion than following inhalation. This did not prove to be the case when comparing liver AUC values for the gastric infusion and inhalation groups. Substantially lower CCl4 concentrations in the liver of animals in the gastric infusion group appeared to be due to very rapid metabolic clearance of the relatively small amounts of CCl4 entering the liver over the 2-hr infusion period. It was hypothesized that the capacity of first-pass hepatic and pulmonary elimination could be exceeded, if CCl4 were given as a single, large oral bolus. Indeed, deposition of CCl4 in all tissues was greater in the oral bolus group than in the gastric infusion group. The time courses of uptake and elimination of CCl4 appeared to be governed largely by a tissue's rate of blood perfusion and lipid content. CCl4 was rapidly taken up, for example, by the brain and liver. These organs' CCl4 content then diminished, as CCl4 was metabolized and redistributed to adipose tissue. CCl4 accumulated slowly, but to very high concentrations, in fat and remained elevated for a prolonged period. Thus, concentrations of CCl4 in some tissues may not be reflective of blood levels. The most appropriate measure of internal dose for CCl4 acute hepatotoxicity appears to be the area under tissue concentrations versus time curve from 0 to 30 min. Tissue time-course data sets are essential for the refinement and validation of physiological models for CCl4 and other volatile organic chemicals.

Lack of volatilization and escape of orally administered trichloroethylene from the gastrointestinal tract of rats.

It is possible that a substantial portion of orally administered volatile organic chemicals (VOCs) may volatilize within the warm environment of the gastrointestinal (GI) tract and escape via the esophagus before being absorbed. The objective of this study was to test this hypothesis with a representative VOC, 1,1,2-trichloroethylene (TCE). Upon hepatic portal vein (PV) injection, complete systemic absorption of TCE was assumed. Thus, exhaled TCE after PV injection should originate only from pulmonary exhalation. In contrast, TCE volatilized in the gut may also contribute to the amounts of TCE exhaled by orally (PO) dosed animals. Male Sprague-Dawley rats (320-380 g) were given 8 or 16 mg TCE/kg bw in an aqueous Alkamuls emulsion (RhonePoulenc, Cranbury, New Jersey). For the PO groups both doses were given by gavage, and for the PV groups the lower dose was injected into the PV as a bolus and the higher dose given as a 20 mm infusion. Serial blood samples were taken from an indwelling carotid arterial cannula and analyzed for their TCE content by headspace gas chromatography (GC), so that the areas-under-blood-concentration-versus-time curves (AUCs) could be determined. Serial exhaled air samples were collected from a sampling port in a miniaturized one-way breathing value and TCE measured by GC analysis to delineate exhaled breath concentration-versus-time-curves (EAUCs). Exhaled breath levels of TCE paralleled blood levels of TCE throughout the monitoring periods. Evidence against the aforementioned hypothesis was provided by comparison of ratios of blood AUCs and exhaled breath EAUCs: values for ratios of AUCPO/AUCPV and EAUCPO/EAUCPV were the same, as were EAUC/AUC ratios for the PO and PV groups. The EAUCs in the PO groups should have been higher, had there been substantial extrusion of volatilized TCE fronm the GI tract.

Use of the vial equilibration technique for determination of metabolic rate constants for dichloromethane.

Metabolism of methylene chloride, or dichloromethane (DCM), plays a key role in determining the kinetics and carcinogenicity of the halocarbon. The objectives of this study were: to evaluate and optimize the vial equilibration technique, originally described by Sato and Nakajima (1979a), in order to characterize the hepatic metabolism of DCM by Sprague-Dawley rats; to employ different hepatic microsomal preparations to examine buffer effects on DCM metabolism; and to assess the relative importance and metabolic constants of the mixed-function oxidase (MFO) and glutathione (GSH) S-transferase (GST) metabolic pathways. A crude liver homogenate (20% W/V) was prepared from perfused livers of male Sprague-Dawley (S-D) rats (275-325 g). A 30% glycerol buffer was found to significantly inhibit DCM metabolism, while 0.25 M sucrose buffer containing 10 mM EDTA and 1.15% KCl did not. DCM was incubated with the liver 10,000 g supernatant or microsomes and cofactors in sealed headspace vials. Disappearance of DCM, as a measure of the chemical's metabolism, was monitored by headspace gas chromatography. Different trials were conducted to elucidate time-, enzyme-, and substrate-activity relationships. The scaled-up K(m) and Vmax values for the microsomal fraction were quite similar to optimized in vivo values reported by other investigators. In the current study, DCM appeared to be metabolized preferentially by cytochrome P450 IIE1, since substrates (e.g., pyrazole, ethanol, and glycerol) for this isozyme completely inhibited DCM metabolism. Thus, glycerol should not be used as a P450 stabilizer for preparation or storage of microsomes. Phorone pretreatment caused marked hepatic GSH depletion, but had little effect on the overall rate of DCM metabolism. Quantitatively, the GST pathway in the cytosol played a very minor role in DCM metabolism. It was not possible to accurately calculate metabolic constants for this pathway in S-D rats. The vial equilibration technique, as described here, is a relatively simple and reliable method, which should be broadly applicable for measuring the microsomal metabolism of DCM and other VOCs.

Characterization of presystemic elimination of trichloroethylene and its nonlinear kinetics in rats.

1,1,2-Trichloroethylene (TCE) is a volatile organic chemical which contaminates drinking water and food supplies and is primarily of concern because of the risk of cancer it may pose. The objectives of the present study were to evaluate the efficiency and dose dependency of presystemic elimination of TCE in rats. Cannulas were surgically implanted into male Sprague-Dawley rats (330-380 g) 24 hr before TCE dosing. TCE (0.17, 0.33, 0.71, 2, 8, 16, and 64 mg/kg) in a 5% aqueous Alkamuls emulsion was administered over 30 sec into the carotid artery, jugular vein (JV), hepatic portal vein, or the stomach. Serial arterial blood samples of 1-500 microliters were collected for up to 12 hr from the unanesthetized animals and analyzed for TCE content by headspace gas chromatography. Pharmacokinetic analyses indicated that TCE was eliminated through dose-dependent nonlinear processes. A three-compartment model with Michaelis-Menten and first-order elimination was derived to fit simultaneously the TCE blood data following JV administration. Total presystemic elimination of TCE was inversely related to dose, ranging from approximately 60 to < 1%. A dose-dependent decrease in hepatic extraction was primarily responsible for the reduction in total first-pass elimination at high doses, whereas pulmonary extraction (i.e., 5-8%) was relatively constant over the dosage range. When metabolic saturation was minimal or absent, hepatic presystemic elimination of TCE accounted for approximately 45-55% of the administered dose. These findings indicate that a substantial proportion of trace amounts of VOCs ingested in environmental media may not enter the systemic circulation nor reach extrahepatic target organs.

Effect of route and pattern of exposure on the pharmacokinetics and acute hepatotoxicity of carbon tetrachloride.

The objectives of this study were to evaluate the influence of both route and pattern of exposure on the pharmacokinetics (PK) and target organ toxicity of a common volatile organic chemical, carbon tetrachloride (CCl4). Male Sprague-Dawley rats, 325-375 g, inhaled 100 or 1000 ppm CCl4 for 2 hr through a one-way breathing valve. The total amount of CCl4 retained by each rat (i.e., the systemically absorbed dose) during the 2-hr period was determined to be 17.5 and 179 mg CCl4/kg body wt, respectively. CCl4, in doses of 17.5 and 179 mg/kg body wt, was administered in an aqueous emulsion by bolus gavage or by constant gastric infusion over 2 hr. Serial micro blood samples from the animals were analyzed for CCl4, in order to delineate blood concentration-versus-time profiles. Serum enzyme activities and total liver microsomal cytochrome P450 level and glucose 6-phosphatase activity were measured 24 hr postdosing as indices of CCl4 hepatotoxicity. The pattern of oral exposure, or dosage regimen, had a significant effect on the PK and acute the hepatotoxicity of CCl4. Arterial blood levels in the gastric infusion group were much lower than in the oral bolus group with both doses. Since CCl4 is quickly and extensively absorbed from the GI tract, large amounts of CCl4 in the portal blood following the oral bolus apparently exceeded the capacity of the liver to metabolize the chemical. Thus, substantially higher Cmax and AUC0 integral of infinity values were manifest. Hepatotoxicity was also significantly greater in these animals. The route of exposure also had a significant effect on the PK of CCl4. Levels of CCl4 in the arterial blood were much higher during inhalation than during gastric infusion. However, AUC0 integral of infinity vales for the two groups were not significantly different, due to relatively slow elimination after gastric infusion. There was little difference between the two groups in hepatotoxicity indices 24 hr postdosing.

Physiologically based pharmacokinetic model useful in prediction of the influence of species, dose, and exposure route on perchloroethylene pharmacokinetics.

The ability of a physiologically based pharmacokinetic (PBPK) model to predict the uptake and elimination of perchloroethylene (PCE) in venous blood was evaluated by comparison of model simulations with experimental data for two species, two routes of exposure, and three dosage levels. Unanesthetized male Sprague-Dawley rats and beagle dogs were administered 1, 3, or 10 mg PCE/kg body weight in polyethylene glycol 400 as a single bolus, either by gavage or by intraarterial (ia) injection. Serial blood samples were obtained from a jugular vein cannula for up to 96 h following dosing. The PCE concentrations were analyzed by headspace gas chromatography. For each dose and route of administration, terminal elimination half-lives in rats were shorter than in dogs, and areas under the blood concentration-time curve were smaller in rats than in dogs. Over a 10-fold range of doses, PCE blood levels in the rat were well predicted by the PBPK model following ia administration, and slightly underpredicted following oral administration. The PCE concentrations in dog blood were generally overpredicted, except for fairly precise predictions for the 3 mg/kg oral dose. These studies provide experimental evidence of the utility of the PBPK model for PCE in interspecies, route-to-route, and dose extrapolations.