Jean C. Parker

Identification of S-(1,2-dichlorovinyl)glutathione in the blood of human volunteers exposed to trichloroethylene

Healthy male and female human volunteers were exposed to 50 ppm or 100 ppm trichloroethylene (Tri) by inhalation for 4 h. Blood and urine samples were taken at various times before, during, and after the exposure period for analysis of glutathione (GSH), related thiols and disulfides, and GSH-derived metabolites of Tri. The GSH conjugate of Tri, S-(1,2-dichlorovinyl)glutathione (DCVG), was found in the blood of all subjects from 30 min after the start of the 4-h exposure to Tri to 1 to 8 h after the end of the exposure period, depending on the dose of Tri and the sex of the subject. Male subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 2 h after the start of the exposure of 46.1 +/- 14.2 nmol/ml (n = 8), whereas female subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 4 h after the start of the exposure of only 13.4 /- 6.6 nmol/ml (n = 8). Pharmacokinetic analysis of blood DCVG concentrations showed that the area under the curve value was 3.4-fold greater in males than in females, while the t1/2 values for systemic clearance of DCVG were similar in the two sexes. Analysis of the distribution of individual values indicated a possible sorting, irrespective of gender, into a high- and a low-activity population, which suggests the possibility of a polymorphism. The mercapturates N-acetyl-1,2-DCVC and N-acetyl-2,2-DCVC were only observed in the urine of 1 male subject exposed to 100 ppm Tri. Higher contents of glutamate were generally found in the blood of females, but no marked differences between sexes were observed in contents of cyst(e)ine or GSH or in GSH redox status in the blood. Urinary GSH output exhibited a diurnal variation with no apparent sex- or Tri exposure-dependent differences. These results provide direct, in vivo evidence of GSH conjugation of Tri in humans exposed to Tri and demonstrate markedly higher amounts of DCVG formation in males, suggesting that their potential risk to Tri-induced renal toxicity may be greater than that of females.

Role of cytochrome P450 and glutathione S-transferase α in the metabolism and cytotoxicity of trichloroethylene in rat kidney

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent Vmax values of 0.65 and 0.47 nmol/min per mg protein and Km values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) alpha (GSTalpha) but not GSTpi. Low levels of GSTmu were detected in cortical and DT cells. Purified rat GSTalpha2-2 exhibited markedly higher affinity for TRI than did GSTalpha1-1 or GSTalpha1-2, but each isoform exhibited similar VmaX values. Triethyltinbromide (TETB) (9 microM) inhibited DCVG formation by purified GSTalpha-1 and GSTalpha2-2, but not GSTalpha1-2. Bromosulfophthalein (BSP) (4 microM) only inhibited DCVG formation by GSTalpha2-2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTalpha1-1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.

Metabolism and Tissue Distribution of Orally Administered Trichloroethylene in Male and Female Rats: Identification of Glutathione- and Cytochrome P-450-Derived Metabolites in Liver, Kidney, Blood, and Urine

Male and female Fischer 344 rats were administered trichloroethylene (TRI) (2, 5, or 15 mmol/kg body weight) in corn oil by oral gavage, and TRI and its metabolites were measured at times up to 48 h in liver, kidneys, blood, and urine. Studies tested the hypothesis that gender-dependent differences in distribution and metabolism of TRI could help explain differences in toxicity. Higher levels of TRI were generally observed in tissues of males at lower doses. Complex patterns of TRI concentration, sometimes with multiple peaks, were observed in liver, kidneys, and blood of both males and females, consistent with enterohepatic recirculation. Higher concentrations of cytochrome P-450 (P450)-derived metabolites were observed in livers of males than in females, whereas the opposite pattern was observed in kidneys. Trichloroacetate was the primary P450-derived metabolite in blood and urine, although it generally appeared at later times than chloral hydrate. Trichloroethanol was also a significant metabolite in urine. S-(1,2-Dichlorovinyl)glutathione (DCVG) was recovered in liver and kidneys of female rats only and in blood of both males and females, with generally higher amounts found in females. S-(1,2-Dichlorovinyl)-l-cysteine (DCVC), the penultimate nephrotoxic metabolite, was recovered in male and female liver, female kidneys, male blood, and in urine of both males and females. The relationship between gender-dependent differences in distribution and metabolism of TRI and susceptibility to TRI-induced toxicity is discussed.

Kinetics of chloral hydrate and its metabolites in male human volunteers.

Chloral hydrate (CH) is a short-lived intermediate in the metabolism of trichloroethylene (TRI). TRI, CH, and two common metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA) have been shown to be hepatocarcinogenic in mice. To better understand the pharmacokinetics of these metabolites of TRI in humans, eight male volunteers, aged 24-39, were administered single doses of 500 or 1,500 mg or a series of three doses of 500 mg given at 48 h intervals, in three separate experiments. Blood and urine were collected over a 7-day period and CH, DCA, TCA, free trichloroethanol (f-TCE), and total trichloroethanol (T-TCE=trichloroethanol and trichloroethanol-glucuronide [TCE-G]) were measured. DCA was detected in blood and urine only in trace quantities (<2 microM). TCA, on the other hand, had the highest plasma concentration and the largest AUC of any metabolite. The TCA elimination curve displayed an unusual concentration-time profile that contained three distinct compartments within the 7-day follow-up period. Previous work in rats has shown that the complex elimination curve for TCA results largely from the enterohepatic circulation of TCE-G and its subsequent conversion to TCA. As a result TCA had a very long residence time and this, in turn, led to a substantial enhancement of peak concentrations following the third dose in the multiple dose experiment. Approximately 59% of the AUC of plasma TCA following CH administration is produced via the enterohepatic circulation of TCE-G. The AUC for f-TCE was found to be positively correlated with serum bilirubin concentrations. This effect was greatest in one subject that was found to have serum bilirubin concentrations at the upper limit of the normal range in all three experiments. The AUC of f-TCE in the plasma of this individual was consistently about twice that of the other seven subjects. The kinetics of the other metabolites of CH was not significantly modified in this individual. These data indicate that individuals with a more impaired capacity for glucuronidation may be very sensitive to the central nervous system depressant effects of high doses of CH, which are commonly attributed to plasma levels of f-TCE.

Renal activation of trichloroethene and S-(1,2-dichlorovinyl)-L-cysteine and cell proliferative responses in the kidneys of F344 rats and B6C3F1 mice

Covalent binding of reactive intermediates formed by renal beta-lyase activation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) has been suggested to be responsible for the greater renal sensitivity of rats than mice to the carcinogenic effects of chronic treatment with trichloroethene (TRI). Previous work demonstrated that the activation of DCVC results in acid-labile adducts to protein that can be distinguished from adducts formed by other pathways of TRI metabolism. By analyzing acid-labile adduct formation, the relationship between DCVC formation and activation from TRI and increases in rates of cell division in the kidneys of male F344 rats and B6C3F1 mice could be investigated. The delivered dose of DCVC from an oral dose of 1000 mg/kg TRI was approximately six times greater in rats than mice. However, renal activation of DCVC in mice was approximately 12 times greater than in rats. Therefore, the overall activation of TRI was about two times greater in mice than rats. Induction of cell replication in liver and kidney following doses of 1, 5, or 25 mg/kg DCVC or 1000 mg/kg TRI was also measured through the use of miniosmotic pumps that delivered BrdU subcutaneously for 3 d. Acid-labile adduct formation from DCVC and TRI displayed a consistent relationship with increased cell replication in mice and between mice and rats. Both cell replication and acid-labile adduct formation in rats given 25 mg/kg DCVC were approximately equal to that observed in mice given 1 mg/kg. Increased cell replication was not observed in rats receiving 1 or 5 mg/kg DCVC or 1000 mg/kg TRI, nor were there histological signs of nephrotoxicity. Thus, net activation of TRI by the cysteine S-conjugate pathway was found to be greater in mice than rats and these findings appeared related to differences in cell proliferative responses of the kidneys of the two species. Based on these data, it would appear that other factors must contribute to the greater sensitivity of the rat to the induction of renal carcinogenesis by TRI.

Acid‐labile adducts to protein can be used as indicators of the cysteine S‐conjugate pathway of trichloroethene metabolism

Covalent binding of radiolabel to tissue proteins following [14C]trichloroethene (TRI) exposure has been used as a measure of TRI activation. Gross binding of 14C label does not differentiate between alternate routes of metabolism and can be confounded when there is significant metabolic incorporation of radiolabel. We examined the covalent association of 14C label to hepatic and renal proteins in male F344 rats and B6C3F1 mice following oral treatment with [14C]TRI and three metabolites of TRI: [14C]trichloroacetate (TCA), [14C]dichloroacetate (DCA), and [14C]dichlorovinylcysteine (DCVC) in vivo. Association of radiolabel from [14C]TRI with hepatic proteins reached a maximum at 2 and 4 h in mouse and rat hepatic proteins, respectively. Association of radiolabel with renal proteins reached a maximum at 8 h in both species. An approach was developed based upon formation of protein adducts that release acetate and monochloroacetate (MCA) on acid hydrolysis. These adducts were found to be specifically associated with the activation of DCVC to reactive intermediates. Acetate and MCA were identified by using two different conditions of high-performance liquid chromatography (HPLC) separation with differing selectivity. Diethylmaleate and aminooxyacetic acid pretreatment inhibited the formation of these adducts from TRI, consistent with requirements for glutathione and beta-lyase. No evidence of these adducts was detected following [14C]TCA and [14C]DCA treatment. Renal acid-labile adduct formation from 25 mg/kg DCVC was approximately 12-fold greater in male B6C3F1 mice than in male F344 rats. They accounted for 7.8 and 4.6% of the total adducts to renal protein in rats and mice, respectively. Acid-labile adducts formed from 1000 mg/kg TRI were approximately two times greater in mice than rats. In this case, they accounted for 1.4 and 3.3% of the total adduct formed in renal proteins from TRI (corrected for metabolic incorporation), respectively. This greater dilution of adducts associated with DCVC in renal proteins of the rat suggests that covalent binding of TRI has less specificity for the DCVC pathway in rats than in mice.

Glutathione conjugation of perchloroethene in subcellular fractions from rodent and human liver and kidney.

Perchloroethene (Per) is a widely used industrial solvent and common environmental contaminant. In rats, long-term inhalation of Per is known to cause a small increase in the incidence of renal tubule cell tumors in males only; renal toxicity is seen in female rats and in both sexes of mice after prolonged Per exposure. The renal toxicity of Per is likely mediated by a glutathione-dependent bioactivation reaction. Glutathione S-transferase mediated formation of S-(1,2,2-trichlorovinyl)glutathione is the first step in a sequence of reactions finally resulting in the formation of reactive intermediates in the kidney. In this study, we compared the enzymatic rates of formation of S-(1,2,2-trichlorovinyl)glutathione in liver and kidney subcellular fractions from rats, mice, and from both sexes of humans (n = 11). In microsomal fractions from the liver and kidney of all three species, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione from Per could not be observed. S-(1,2,2-Trichlorovinyl)glutathione formation (the structure was confirmed by electrospray mass spectrometry) was observed in liver cytosol from both male and female rats and mice. However, the rates of S-(1,2,2-trichlorovinyl)glutathione formation in liver cytosol from male rats (84.5+/-12 pmol/mg per min) were approximately four times higher than from female rats (19.5+/-8 pmol/mg per min) and from both sexes of mice (27.9+/-6 and 26.0+/-4 pmol/mg per min). Low rates of S-(1,2,2-trichlorovinyl)glutathione formation were also seen in kidney cytosol from mice (12+/-6 pmol/mg per min), but not from rats. In human liver subcellular fractions, enzymatic formation of S-(1,2,2-trichlorovinyl)glutathione could not be detected. The human liver cytosolic fractions, however, exhibited glutathione S-transferase activity (as determined using 1-chloro-2,4-dinitrobenzene and hexachlorobutadiene as marker substrates) in the same order of magnitude as rat and mouse liver cytosol. In contrast to other marker activities for glutathione S-transferases, the ability of all human liver cytosol samples to catalyze the glutathione conjugation of 1,2-dichloro-4-nitrobenzene was three orders of magnitude lower compared to rat and mouse liver cytosol. 1,2-Dichloro-4-nitrobenzene conjugation was also four times higher in liver cytosol from male rats compared to female rats. The results suggest that the ability of the human liver to catalyze the formation of S-(1,2,2-trichlorovinyl)glutathione from Per is at least two orders of magnitude lower than that of rat liver, and that sex-specific differences in the extent of hepatic conjugation of Per with glutathione, which may be relevant for nephrotoxicity, occur in rats.

Effect of enterohepatic circulation on the pharmacokinetics of chloral hydrate and its metabolites in F344 rats.

Chloral hydrate (CH) is a commonly found disinfection by-product in water purification, a metabolite of trichloroethylene, and a sedative/hypnotic drug. CH and two of its reported metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), are hepatocarcinogenic in mice. Another metabolite of CH, trichloroethanol (TCE), is also metabolized into TCA, and the enterohepatic circulation (EHC) of TCE maintains a pool of metabolite for the eventual production of TCA. To gain insight on the effects of EHC on the kinetics of CH and on the formation of TCA and DCA, dual cannulated F344 rats were infused with 12, 48, or 192 mg/kg of CH and the blood, bile, urine, and feces were collected over a 48-h period. CH was cleared rapidly (>3000 ml/h/kg) and displayed biphasic elimination kinetics, with the first phase being elimination of the dose and the second phase exhibiting formation rate-limited kinetics relative to its TCE metabolite. The effects of EHC on metabolite kinetics were only significant at the highest dose, resulting in a 44% and 17% decrease in the area under the curve (AUC) of TCA and TCE, respectively. The renal clearance of CH, free TCE (f-TCE), and TCA of 2, 2.7, and 38 ml/h/kg, respectively, indicates an efficient reabsorption mechanism for all of these small chlorinated compounds. DCA was detected at only trace levels (<2 microM) as a metabolite of CH, TCA, or TCE.

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.