Ethanol-induced enhancement of trichloroethylene metabolism and hepatotoxicity: difference from the effect of phenobarbital

Male Wistar rats pretreated with ethanol (2.0 g in 80 ml liquid diet/day for 3 weeks) or phenobarbital (PB, 80 mg/kg/day ip for 4 days) were exposed by inhalation to 500, 1000, 2000, 4000, or 8000 ppm trichloroethylene (TRI) for 2 or 8 hr, and the blood concentration of TRI and the urinary concentration of TRI metabolites (trichloroethanol (TCE) and trichloroacetic acid (TCA] were determined at various times. Plasma glutamic-pyruvic transaminase (GPT) activity was measured 22 hr after the end of exposure as an indicator of hepatic damage. Both ethanol and PB enhanced TRI metabolism as evidenced by accelerated disappearance of TRI from the blood and increased excretion of total trichloro compounds (TCE + TCA) in the urine. However, the effects of ethanol and PB were different from each other: ethanol markedly enhanced the metabolism particularly at TRI concentration of 2000 ppm or lower, whereas PB enhanced it only at 4000 ppm or higher. This difference was also reflected in the effect of TRI on liver: ethanol potentiated TRI hepatotoxicity more markedly than did PB when TRI concentration remained 2000 ppm or lower, whereas PB potentiated the toxicity more markedly than ethanol when the concentration was 4000 ppm or higher. It is noteworthy that ethanol potentiated TRI hepatotoxicity at a TRI concentration as low as 500 ppm. The severity of hepatic damage expressed by plasma GPT activity essentially paralleled the urinary excretion rate of total trichloro compounds during and 4 hr after exposure (r = 0.87 to 0.93). Compared between the contribution of concentration and duration of exposure to the toxicity, a higher concentration of TRI tended to cause more severe liver damage to PB-treated rats than did a prolonged period of exposure, whereas the toxicity in ethanol-treated rats was generally more marked in rats exposed to TRI for a longer period than in rats exposed to a higher concentration.     

Three forms of trichloroethylene-metabolizing enzymes in rat liver induced by ethanol, phenobarbital, and 3-methylcholanthrene

In vitro metabolism of trichloroethylene (TRI) and trichloroethanol (TCE) was investigated using liver microsomes from control and ethanol-, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated rats. At least three forms of enzymes were involved in TRI metabolism. One was a low-Km type normally existing in microsomes from control rats. The ethanol-inducible enzyme was found to be catalytically identical to this low-Km isozyme. Another was a high-Km type which was induced exclusively by PB, and a third was an MC-inducible isozyme with a Km value between those of ethanol- and PB-inducible isozymes. Although MC treatment did not affect the rate of TRI metabolism in vitro, both ethanol and PB treatment markedly enhanced the metabolism. Ethanol-induced enhancement was different from PB-induced enhancement in that ethanol enhanced the metabolism predominantly at low substrate concentrations, whereas PB did so at high concentrations. In addition, TRI metabolism with enzymes from ethanol-treated rats was inhibited by the substrate itself at high concentrations. MC treatment of rats had little or no influence on the rate of TCE metabolism in vitro, whereas both ethanol and PB enhanced the microsomal conversion of TCE to chloral hydrate. As in the case of TRI metabolism, ethanol induced a microsomal TCE-metabolizing enzyme of low Km, whereas PB preferentially induced an enzyme of high Km.     

The metabolite ratio as a function of chloral hydrate dose and intracellular redox state in the perfused rat liver

Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase, and is also oxidized to trichloroacetic acid (TCA) by the nicotinamide adenine dinucleotide (NAD)-dependent enzyme, CH dehydrogenase. Alcohol dehydrogenase requires reduced NAD (NADH), aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and CH dehydrogenase requires NAD to complete the reaction. It is unclear which reaction is predominant at the physiological redox level in intact liver cells. To study this question, we perfused the livers of well-fed rats with Krebs-Ringer buffer solution containing 0.1 mM pyruvate/1.0 mM lactate. The levels of TCE and TCA in the effluent were measured by gas chromatography, and the fluorescence of reduced pyridine nucleotides was measured with a surface fluorometer. When a low concentration (below 0.25 mM) of CH was administered, more TCA than TCE was produced. When a high concentration of CH was administered (over 0.5 mM), TCE production was greater. Reduced pyridine nucleotides decreased inversely with the CH concentration. Even at low CH concentrations, pyridine nucleotides were not reduced. When 10 mM lactate was added to the perfusate in order to reduce the pyridine nucleotides in the liver cells, the TCE/TCA ratio increased. On the other hand, the TCE/TCA ratio tended to fall following the addition of 5.0 mM pyruvate. In conclusion, the TCE/TCA ratio was altered according to the concentration of CH, and to the redox level of pyridine nucleotides in the liver.     

The metabolism of trichloroethylene and its metabolites in the perfused liver

The metabolism of trichloroethylene (TRI) and its metabolites, chloral hydrate (CH), trichloroethanol (free-TCE) and trichloroacetic acid (TCA), were examined in the isolated perfused rat liver, to clarify the role of the liver in the metabolism of TRI. TRI was rapidly converted to TCE and TCA by the perfused liver. TCA was produced from TRI about 2.5 times greater than was total-TCE. CH was metabolized to TCE and TCA immediately. TCA was also a dominant metabolite of CH over total-TCE. TCE(free type) was speedily conjugated by the liver. A portion of TCE was converted to TCA. Less than 10% of these metabolites produced by the liver were excreted into the bile. Most of them appeared in the perfusate.     

Effect of ethanol on the metabolism of trichloroethylene

Trichloroethylene (TCE) is metabolized to chloral hydrate (CH) by the cytochrome P-450 monooxygenase system. CH can either be oxidized by chloral hydrate dehydrogenase to trichloroacetic acid (TCA) or reduced by alcohol dehydrogenase to trichloroethanol (TCEtOH). The oxidation reaction requires NAD+, while the reduction reaction requires NADH. Since ethanol (EtOH) is known to alter the NAD+/NADH ratio in the hepatocyte, it was coadministered with TCE in an attempt to alter the metabolism of TCE. This would provide a means for predicting interactions of ethanol on the hepatotoxicity and carcinogenicity of TCE. Male Sprague-Dawley rats were administered oral doses of either 1.52, 4.56, or 22.8 mmol/kg TCE, with the treatment group receiving an additional 1.52, 4.56, or 22.8 mmol/kg EtOH, respectively. Blood and urine samples were collected over 72 h. The clearance of TCE appeared to be saturated at the 4.56 mmol/kg dose, as evidenced by prolonged residence times for TCE in the body. Consistent with this result, there was an attenuation of the increases in the levels of TCEtOH and TCA in blood. However, the time to peak concentration of these metabolites was delayed with increasing doses and their residence time in the body was prolonged. Therefore, the area under the curve (AUC) for TCEtOH and TCA continued to increase with the higher doses of TCE. Measurement of the net output of these metabolites in urine confirmed that, although metabolism was saturated, the net metabolic conversion of TCE increased. As predicted, EtOH decreased blood levels of TCA, but only at early times at the high dose. EtOH did increase the urinary TCEtOH/TCA ratio at all dose levels. These results are consistent with the hypothesis of a more reduced state in the hepatocyte caused by the generation of excessive reducing equivalents by EtOH metabolism. The metabolism of TCE is shifted toward reduction to TCEtOH, away from oxidation to TCA. However, the effect was prominent only at extremely high doses of TCE and EtOH.     

Trichloroacetic acid in urine as biological exposure equivalent for low exposure concentrations of trichloroethene

A urinary trichloroacetic acid (TCA) concentration of 100 mg/l at the end of the last work shift (8 h/day, 5 days/week) of the week has been established in workers as exposure equivalent for the carcinogenic substance trichloroethene (EKA for TRI) at an exposure concentration of 50 ppm TRI. Due to the continuous reduction of atmospheric TRI concentrations during the last years, the quantitative relation given by the EKA for TRI is revised for exposures to low TRI concentrations. A physiological two-compartment model is presented by which the urinary TCA concentrations are calculated that result from inhaled TRI in humans. The model contains one compartment for trichloroethanol (TCE) and one for TCA. Inhaled TRI is metabolized to TCA and to TCE. The latter is in part further oxidized to TCA. Urinary elimination of TCA is modeled to obey first order kinetics. All required model parameters were taken form the literature. In order to evaluate the model performance on the urinary TCA excretion at low exposure concentrations, predicted urinary TCA concentrations were compared with data obtained in two volunteer studies and in one field study. The model was evaluated at exposure concentrations as low as 12.5 ppm TRI. It is demonstrated that the correlation described by the hitherto used EKA for TRI is also valid at low TRI concentrations. For TRI exposure concentrations of 0.6 and 6 ppm, the resulting urinary TCA concentrations at the end of the last work shift of a week are predicted to be 1.2 and 12 mg/l, respectively.     

Metabolism of chloral hydrate in the anoxic perfused liver

The metabolism of chloral hydrate (CH) under anoxic conditions was investigated in the non-recirculating, hemoglobin-free liver perfusion system. CH uptake in the anoxic liver decreased to about 80% of that in the oxygen-supplied liver. The reduction of CH to trichloroethanol (TCE) increased and the oxidation of CH to trichloroacetic acid (TCA) decreased. The TCE/TCA ratio increased; however, the total trichloro compounds, that is TCE and TCA, were not significantly altered by anoxia. Though approximate 14% of the CH infused into the oxygen-supplied liver was changed to substances other than TCE or TCA, the unknown part was a very small portion in the anoxic liver. The decrease in CH uptake, by the anoxic liver, is thought to be equivalent to the decrease of the unknown metabolites. The TCE/TCA ratio under anoxia was also altered by pyruvate or lactate infusion.     

Effect of ethanol on uptake of trichloroethylene in isolated perfused rat liver

The effect of ethanol on uptake of trichloroethylene in isolated perfused rat liver was investigated. The uptake of trichloroethylene was measured in both phenobarbital (PB) non-treated and PB treated rat livers. Furthermore, for PB treated rat livers, the fluorescence of reduced pyridine nucleotides, oxygen consumption, and scanning reflectance spectrum were measured in the liver, perfused with Krebs-Henseleit buffer saturated with a 92%O2-5%CO2-3%CO gas mixture. The uptake of trichloroethylene was decreased by 6.0% in the PB non-treated rat liver and 10.6% in the PB treated rat liver following the addition of ethanol. This uptake decrease was thought to arise mainly from the inhibitory effect of ethanol on mixed-function oxidation in the liver because of the corresponding decrease in oxygen consumption and absorbance difference delta A450-490nm. The inhibition was considered to be due to interference with electron transfer to the complex of substrate and cytochrome P-450. Increase in intracellular NADH might also affect the formation of trichloroacetic acid since the reduction of NADH in the cytosol attained a maximum with 20 mM ethanol.     

Extrahepatic metabolism of chloral hydrate, trichloroethanol and trichloroacetic acid in dogs

To examine the details concerning that part of TRI metabolism which was carried out by the extrahepatic organs, we studied the extrahepatic metabolism of chloral hydrate (CH), free-trichloroethanol (F-TCE) and trichloroacetic acid (TCA) using a method developed in our laboratory. Bypass and non-bypass dogs were given CH, F-TCE and TCA, and we compared the concentrations these substances and their metabolites in the serum and urine of the two groups of animals. In the bypass dogs, F-TCE, TCA and conjugated-trichloroethanol (Conj-TCE) appeared in the blood and urine 30 min. after the CH administration, and TCA and Conj-TCE appeared 30 min. after the F-TCE. All levels of administered substance were higher in bypass dogs than in non-bypass dogs, and the compounds were metabolized in small amounts in the extrahepatic organs compared with the liver. Therefore, administered substances remained at high levels in the serum and were excreted in large amounts in the urine in the form of unchanged substances. The metabolized percentage volumes of CH to TCA in the bypass dogs were 10-20%, and those of F-TCE to TCA were very small, while these percentage values of CH to F-TCE were the same or slightly smaller, respectively. Moreover, trichloroethylene (TRI) acts to decrease the leukocyte count in the blood, but the TRI metabolites described above do not have this function.     

Biotransformation of trichloroethylene in collagen gel sandwich cultures of rat hepatocytes

The collagen gel sandwich culture of hepatocytes has been proposed as one of the most suitable culture models available for biotransformation studies of xenobiotics. It is a complex model which imitates the cascade of enzymatic events of in vivo biotransformation and allows investigation of biological endpoints under realistic conditions. The biotransformation of trichloroethylene (TRI) has been studied in this model using rat hepatocytes. Headspace gas chromatographic measurements revealed that hepatocytes, cultured for 4 days in this in vitro system, metabolised TRI into the major oxidative metabolites trichloroacetic acid (TCA) and trichloroethanol (TCE). Cultured hepatocytes were exposed either to TRI, or to TCA and TCE. Endpoints studied were albumin secretion and the cytochrome P450 (CYP)-dependent enzymatic activities ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD) and N-nitrosodimethylamine demethylase (NDMA). The results show that both the parent compound and its metabolites exert specific effects on different CYP-dependent mono-oxygenase activities, as seen in vivo. It is suggested that collagen gel sandwich cultures represent a useful in vitro model for the investigation of metabolism-linked toxicity studies.     

Physiologically based pharmacokinetic modeling of the lactating rat and nursing pup: a multiroute exposure model for trichloroethylene and its metabolite, trichloroacetic acid

A physiologically based pharmacokinetic (PB-PK) model was developed to describe trichloroethylene (TCE) kinetics in the lactating rat and nursing pup. The lactating dam was exposed to TCE either by inhalation or by ingestion in drinking water. The nursing pup's exposure to TCE was by ingestion of maternal milk containing TCE. The kinetics of trichloroacetic acid (TCA), a metabolite of TCE, were described in the lactating dam and developing pup by a hybrid one-compartment model. The lactating dam's exposure to TCA was from metabolism of TCE to TCA. The pup's exposure to TCA was from metabolism of TCE ingested in suckled milk and from direct ingestion of TCA in maternal milk. For the PB-PK model, partition coefficients (PCs) were determined by vial equilibration, and metabolic constants for TCE oxidation, by gas uptake methods. The blood/air and the fat/blood PCs for the dam were 13.1 and 34.2, and for the pup, 10.6 and 42.3, respectively. The milk/blood PC for the dam was 7.1. In lactating rats and rat pups (19-21 days old) the maximum velocities of oxidative metabolism were 9.26 +/- 0.073 and 12.94 +/- 0.107 mg/kg/hr. The plasma elimination rate constant (K = 0.063 +/- 0.004 hr-1) and apparent volume of distribution (Vd = 0.568 liter/kg) for TCA in the lactating dam were estimated from both intravenous dosing studies and an inhalation study with TCE. For the pup, K (0.014 +/- hr-1) and Vd (0.511 liter/kg) were estimated from a single 4-hr inhalation exposure with TCE. The dose-rate-dependent stoichiometric yield of TCA from oxidative metabolism of TCE in the lactating rat is 0.17 for a low-concentration inhalation exposure (27 ppm TCE) and 0.27 for an exposure above metabolic saturation (about 600 ppm TCE). For the pup, the stoichiometric yield of TCA is 0.12. With changing physiological values during lactation for compartmental volumes, blood flows, and milk yields obtained from the published literature and kinetic parameters and PCs determined by experimentation, a PB-PK model was constructed to predict maternal and pup concentrations of TCE and TCA. To test the fidelity of the PB-PK lactation model, a multiday inhalation exposure study was conducted from Days 3 to 14 of lactation and a drinking water study, from Days 3 to 21 of lactation. The inhalation exposure was 4 hr/day, 5 days/week, at 610 ppm. The TCE concentration in the drinking water was 333 micrograms/ml. Prediction compared favorably with limited data obtained at restricted time points during the period of lactation.     

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)     

Morphological and biochemical analyses of trichloroethylene hepatotoxicity: differences in ethanol- and phenobarbital-pretreated rats

The histological and biochemical differences between ethanol- and phenobarbital (PB)-potentiated hepatotoxicity of trichloroethylene (TRI) in Wistar strain male rats were investigated. Both ethanol (2 g in daily liquid diet for 3 weeks) and PB (80 mg/kg/day for 4 days, ip) pretreatments enhanced TRI (inhalation exposures of 500 ppm for 8 hr, 2000 ppm for 2 or 8 hr, and 8000 ppm for 2 hr)-induced hepatic damage as judged by increases in plasma transaminase activities. Livers from PB-treated rats exposed to TRI displayed centrilobular necrosis, whereas livers from ethanol-treated rats exposed to TRI were characterized by ballooning degeneration mainly in midzonal areas. TRI exposure decreased the in vitro metabolism of TRI, high-Km benzene aromatic hydroxylase (BAH) activity, and cytochrome P450 content in livers of PB-treated rats with severe hepatic damage. In ethanol-treated rats, TRI exposure increased both the in vitro metabolism of TRI and the low-Km BAH activity but did not cause an apparent decrease in cytochrome P450 content even in animals with severe hepatic damage. These results suggest that TRI caused necrosis of centrilobular hepatocytes in PB-pretreated rats, which was accompanied by loss of xenobiotic metabolizing functions, whereas ballooning degeneration of hepatocytes mainly in midzonal areas occurred in ethanol-pretreated rats without loss of xenobiotic metabolizing functions.     

Acute,nonfatal intoxication with trichloroethylene

Nonfatal acute inhalation of trichloroethylene (TRI) at work was described. The subject, male, 54 years old, was drawn unconscious by a metal-degreasing machine and immediately sheltered in intensive care unit. Other than basic life support and common laboratory indices, blood and urine were collected to measure dose and kidney effect parameters such as TRI in blood and urine, trichloroethanol (TCE) and trichloroacetic acid (TCA) in urine, and total urinary proteins (TUP), urinary glutamine synthetase (GS) and urinary N-acetyl-beta-D-glucosaminidase (NAG). Two hours after accident, TRI in blood was 9 mg/l, but after 38 h it was below 1 mg/l. TCE and TCA have a peak 11 and 62 h after poisoning, respectively. Acute renal involvement was revealed by a peak of urinary proteins and enzymes 7 h after exposure with a second peak 74 h after. Seven day after hospitalisation the patient was dismissed with complete recovery. This nonfatal intoxication with TRI shows that the exposure was approximately 150 ppm, three times the ACGIH TLV (50 ppm) and that kidney was the only organ affected. Urinary enzymes, in particular GS, are good indices to monitor transient effects of TRI on the kidney.     

Regulation of NADPH-dependent mixed-function oxidation in perfused livers: Comparative studies with sorbitol and ethanol

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD⁺/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP⁺/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.     

代谢活化在三氯乙烯对小鼠致敏及肝毒性中的作用

目的研究三氯乙烯(TCE)通过什么代谢通路活化后才具有致敏活性。方法以TCE和细胞色素P450(CYP450)诱导剂(乙醇和苯巴比妥)分别及联合给小鼠染毒,测定血清中丙氨酸氨基转移酶(GPT)和天冬氨酸氨基转移酶(GOT)活性;分离脾细胞体外培养,采用噻唑兰(MTT)法测定TCE所致小鼠脾淋巴细胞特异性增殖反应和细胞毒性,并观察体外培养过程中加入代谢酶抑制剂SKF525A和氨氧乙酸(AOAA)后对上述效应的影响。结果TCE致敏组小鼠脾淋巴细胞与TCE共孵育后,细胞相对数(79±10)%明显高于溶剂对照组(63±11)%,差异有统计学显著性(P<0.05),在培养液中加入β裂合酶抑制剂AOAA后,这种抗原特异性淋巴细胞增殖反应消失;而乙醇诱导+TCE组和苯巴比妥诱导+TCE组小鼠脾淋巴细胞未出现TCE特异性增殖反应。溶剂对照组、TCE致敏组、乙醇诱导+TCE组和苯巴比妥诱导+TCE组小鼠脾淋巴细胞与TCE+SKF共培养后,细胞相对数均明显高于TCE单独培养的淋巴细胞,差异有统计学显著性(P<0.05)。乙醇诱导+TCE组血清GPT活性(49.5±8.4)μmol·min-1·L-1和苯巴比妥诱导+TCE组血清GPT,GOT活性(47.3±9.9)和(170±36)μmol·min-1·L-1均明显高于溶剂对照组(34.7±2.1)和(117±34)μmol·min-1·L-1,差异有统计学显著性(P<0.05),而TCE致敏组小鼠血清GPT和GOT活性与溶剂对照组相比均无显著性差异(P>0.05)。结论TCE谷胱甘肽结合通路的代谢产物巯基烯酮类物质具有致敏活性,可能是TCE致敏小鼠的特异性半抗原,而TCE氧化通路的代谢产物是产生细胞毒性和肝损伤的主要物质,乙醇和苯巴比妥能够增加TCE的肝脏毒性。     

Amino acid and protein targets of 1,2-diacetylbenzene,a potent aromatic gamma-diketone that induces proximal neurofilamentous axonopathy

Determining the key events in the induction of liver cancer in mice by trichloroethylene (TRI) is important in the determination of how risks from this chemical should be treated at low doses. At least two metabolites can contribute to liver cancer in mice, dichloroacetate (DCA) and trichloroacetate (TCA). TCA is produced from metabolism of TRI at systemic concentrations that can clearly contribute to this response. As a peroxisome proliferator and a species-specific carcinogen, TCA may not be important in the induction of liver cancer in humans at the low doses of TRI encountered in the environment. Because DCA is metabolized much more rapidly than TCA, it has not been possible to directly determine whether it is produced at carcinogenic levels. Unlike TCA, DCA is active as a carcinogen in both mice and rats. Its low-dose effects are not associated with peroxisome proliferation. The present study examines whether biomarkers for DCA and TCA can be used to determine if the liver tumor response to TRI seen in mice is completely attributable to TCA or if other metabolites, such as DCA, are involved. Previous work had shown that DCA produces tumors in mice that display a diffuse immunoreactivity to a c-Jun antibody (Santa Cruz Biotechnology, SC-45), whereas TCA-induced tumors do not stain with this antibody. In the present study, we compared the c-Jun phenotype of tumors induced by DCA or TCA alone to those induced when they are given together in various combinations and to those induced by TRI given in an aqueous vehicle. When given in various combinations, DCA and TCA produced a few tumors that were c-Jun+, many that were c-Jun-, but a number with a mixed phenotype that increased with the relative dose of DCA. Sixteen TRI-induced tumors were c-Jun+, 13 were c-Jun-, and 9 had a mixed phenotype. Mutations of the H-ras protooncogene were also examined in DCA-, TCA-, and TRI-induced tumors. The mutation frequency detected in tumors induced by TCA was significantly different from that observed in TRI-induced tumors (0.44 vs 0.21, p < 0.05), whereas that observed in DCA-induced tumors (0.33) was intermediate between values obtained with TCA and TRI, but not significantly different from TRI. No significant differences were found in the mutation spectra of tumors produced by the three compounds. The presence of mutations in H-ras codon 61 appeared to be a late event, but ras-dependent signaling pathways were activated in all tumors. These data are not consistent with the hypothesis that all liver tumors induced by TRI were produced by TCA.     

Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate.

Differing rates and extent of trichloroethylene (TCE) metabolism have been implicated as being responsible for varying sensitivities of mice and rats to the hepatocarcinogenic effects of TCE. Recent data indicate that the induction of hepatic tumors in mice may be attributed to the metabolites trichloroacetate (TCA) and/or dichloroacetate (DCA). The present study was directed at determining whether mice and rats varied in (1) the peak blood concentrations, (2) the area under the blood concentration over time curves (AUC) for TCE and metabolites in blood, and (3) the net excretion of TCE to these metabolites in urine in the dose range used in the cancer bioassays of TCE, and to contrast the kinetic parameters observed for TCE-derived TCA and DCA with those obtained following direct administration of TCA and DCA. Blood and urine samples were collected over 72 hr from rats and mice after a single oral dose of TCE of 1.5 to 23 mmol/kg. The AUC values from the blood concentration with time profiles of TCE, TCA, and trichloroethanol (TCOH) were similar for Sprague-Dawley rats and B6C3F1 mice. Likewise, the percentages of initial TCE dose recovered as the urinary metabolites TCA and TCOH were comparable. Nevertheless, the peak blood concentrations of TCE, TCA, and TCOH observed in mice were much greater than those in rats, while the residence time of TCE and metabolites was prolonged in rats relative to that of mice. DCA was detected in the blood of mice but not in rats. The blood concentrations of DCA observed in mice given a carcinogenic dose of TCE (15 mmol/kg) were of the same magnitude as those observed with carcinogenic doses of DCA. In conclusion, the net metabolism of TCE to TCA and TCOH was similar in rats and mice. The initial rates of metabolism of TCE to TCA, however, were much higher in mice, especially as the TCE dose was increased, leading to greater concentrations of TCA and DCA in mice approximated those produced by carcinogenic doses of the chlorinated acetates makes it highly likely that both compounds play a role in the induction of hepatic tumors in mice by TCE.     

The Extent of Dichloroacetate Formation from Trichloroethylene, Chloral Hydrate, Trichloroacetate, and Trichloroethanol in B6C3F1 Mice

Conflicting data have been published related to the formationof dichloroacetate (DCA) from trichloroethylene (TRI), chloralhydrate (CH), or trichloroacetic acid (TCA) in B6C3F1 mice.TCA is usually indicated as the primary metabolic precursorto DCA. Model simulations based on the known pharmacokineticsof TCA and DCA predicted blood concentrations of DCA that were10- to 100-fold lower than previously published reports. BecauseDCA has also been shown to form as an artifact during sampleprocessing, we reevaluated the source of the reported DCA, i.e.,whether it was metabolically derived or formed as an artifact.Male B6C3F1 mice were dosed with TRI, CH, trichloroethanol (TCE),or TCA and metabolic profiles of each were determined. DCA wasnot detected in any of these samples above the assay LOQ of1.9 µM of whole blood. In order to slow the clearanceof DCA, mice were pretreated for 2 weeks with 2 g/liter of DCAin their drinking water. Even under this pretreatment condition,no DCA was detected from a 100 mg/kg iv dose of TCA. Althoughthere is significant uncertainty in the amount of DCA that couldbe generated from TRI or its metabolites, our experimental dataand pharmacokinetic model simulations suggest that DCA is likelyformed as a short-lived intermediate metabolite. However, itsrapid elimination relative to its formation from TCA preventsthe accumulation of measurable amounts of DCA in the blood.     

Interaction of ethanol with codeine metabolism in rat hepatocytes: a multicompartmental model

A multicompartmental pharmacokinetic model is presented, which based upon data from a previous study, describes the effects of ethanol (60 mM) on the metabolism of codeine (10 microM) in isolated rat hepatocytes. According to this model, about one third of codeine metabolized was transformed to morphine (13%) and norcodeine (18%), and two-thirds to unknown metabolites in the absence of ethanol. In the presence of ethanol, the apparent first order fractional rate of total codeine metabolism was reduced by 66% (0.0783 vs 0.0271 min-1). There was no alteration in the portion of codeine metabolized to norcodeine, but there was a 44% decrease in the fraction transformed to unknown metabolites and a tripling in the portion transformed to morphine. The fractional rate of codeine O-demethylation to morphine was apparently not sensitive to ethanol. In the absence of ethanol, about two-thirds of morphine was metabolized to morphine-3-glucuronide and the other third to unidentified metabolites. Only the glucuronidation process seemed to be inhibited by ethanol. The fractional rate of further metabolism of norcodeine to normorphine was similar in the absence or presence of ethanol. In conclusion ethanol co-incubation with codeine resulted in an inhibition of codeine conversion to unknown metabolites and norcodeine, and with morphine to morphine-3-glucuronide, but no inhibition in morphine production.