2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and the most potent agonist of the aryl hydrocarbon receptor (AhR). Persistent activation of the AhR has been shown to be responsible for most TCDD-mediated toxic responses, including liver tumour promotion. However, the mechanisms responsible for these complex toxic reactions are still unknown. TCDD (1 nM) has previously been shown to reduce DNA synthesis of primary hepatocyte cultures and cell contact inhibition of confluent WB-F344 cells. The latter model was used to study early effects of TCDD on protein tyrosine kinase c-Src in confluent WB-F344 cells. It was found that TCDD decreased cytosolic c-Src (protein and tyrosine kinase activity) after 20-60 min, and increased c-Src in the membrane fraction. Membrane translocation of c-Src occurred in the presence of 100 microM cycloheximide and was observed after treatment with 1 nM TCDD or 50 nM 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Under these conditions epidermal growth factor (EGF) receptor tyrosine phosphorylation was also studied. As expected, its phosphorylation was low in confluent cells but was significantly enhanced by TCDD treatment. Pretreatment of WB-F344 cells for 1 h with 1 microM geldanamycin, which disrupts cytosolic heat shock protein Hsp90 complexes with AhR and Src, abolished TCDD-mediated Src translocation and TCDD-mediated reduction of cell contact inhibition. The WB-F344 cell model appears to be very useful to study TCDD effects on protein tyrosine kinases and of signaling pathways responsible for modulation of the cell cycle by TCDD.

Animals↗

PubMedDate not supplied

Influence of P-4502E1 induction on benzene metabolism in rat hepatocytes and on biliary metabolite excretion.

The influence of P-4502E1 induction on the metabolite pattern of benzene was studied in hepatocytes in vitro and in bile in vivo, and compared with that obtained with phenol (the major benzene metabolite). Eight metabolites from benzene and four from phenol (including conjugates) represented over 90% of total metabolites. Benzene metabolism (0.1 mM) in hepatocytes from isopropanol-treated rats (2.5 ml/kg, orally) was 3-fold higher than in corresponding cells from control rats, primarily because of increased formation of hydroquinone and phenylglutathione. Immunoblotting of microsomes revealed a parallel induction of P-4502E1 in hepatocytes from isopropanol-treated rats. In contrast, treatment with 3-methylcholanthrene or phenobarbital caused a decrease of P-4502E1, together with reduced benzene metabolism at 0.01 mM benzene. Addition of isoniazid (5 mM) resulted in a strong inhibition of benzene and phenol metabolism. Benzene metabolites were determined in bile following intraperitoneal administration of benzene (2.5 and 150 mg/kg). Biliary benzene metabolites were increased 2- to 3-fold after isopropanol treatment. Hydroquinone sulfate was identified as a major biliary metabolite of phenol. The results suggest that treatment with inducers of P-4502E1 leads, even at low benzene exposure, to an increased release of potentially myelotoxic metabolites from liver into the systemic circulation.

1-Propanol↗

PubMedDate not supplied

Stereoselective (S)- and (R)-naproxen glucuronosyl transferases of rat liver.

Stereoselective glucuronidation of naproxen, one of the 2-arylpropionic acids that are widely used as anti-inflammatory drugs, was investigated in chromatofocusing fractions of solubilized liver microsomes from 3-methylcholanthrene- (MC) and phenobarbital- (PB) treated rats. On chromatofocusing of solubilized microsomes of PB-treated rats, two naproxen glucuronosyltransferase (GT) fractions were separated. The fraction eluting at pH 8.7 preferentially conjugated (S)-naproxen (S/R ratio = 1.6) and the fraction eluting at pH 7.8 mostly conjugated (R)-naproxen (S/R ratio = 0.7). Chromatofocusing of solubilized microsomes from MC-treated rats also resulted in the separation of two naproxen GT fractions, eluting at pH 9.4 (S/R ratio 0.2) and at pH 8.7 (S/R ratio 0.8). These two fractions coincided with the elution of known MC-inducible GT activities assigned to a GT isozyme variously termed 4-nitrophenol GT or GT-I. Interestingly, kidney microsomes, known to contain a high constitutive expression of GT-I, preferentially glucuronidated (R)-naproxen (S/R ratio 0.2). The S/R ratio of 0.8, observed with the pI 8.7 fraction of MC-treated rat liver, may be explained by the presence of a mixture of naproxen GTs, consisting of (R)-naproxen GT (S/R ratio 0.2) and of (S)-naproxen GT (S/R ratio 1.6). The results suggest that naproxen is conjugated by at least 3 GT isozymes in rat liver; these have been operationally designated (S)-naproxen GTPB (S/R ratio 1.6), (R)-naproxen GTPB (S/R ratio 0.7), and (R)-naproxen GTMC (S/R ratio 0.2). The latter isozyme is probably identical to the previously characterized MC-inducible GT-I. Thus, (S)- and (R)-naproxen represent useful substrates to distinguish different GT isozymes.

Adult↗

PubMedDate not supplied

Metabolism of benzene in rat hepatocytes. Influence of inducers on phenol glucuronidation.

Alterations of benzene metabolism in liver markedly influence benzene toxicity at extrahepatic target tissues. Therefore, generation of 11 phase I and II metabolites of benzene (including phenol, hydroquinone, catechol, benzene-1,2-dihydrodiol, their sulfates and glucuronides, and phenylglutathione) was compared in hepatocytes from 3-methylcholanthrene (MC)- or phenobarbital-treated rats and from untreated controls. At 0.1 mM benzene, total metabolism appeared to be unchanged by treatment with inducers. Phenylsulfate (35%), phenylglucuronide (15%), and phenylglutathione (12%) represented the major metabolites in hepatocytes from untreated controls. With hepatocytes from MC-treated rats, a pronounced shift from phenylsulfate to phenylglucuronide (increase to 34%) was observed, while the formation of unconjugated phenol, hydroquinone, and catechol was decreased (from 16 to 10%). A similar shift from sulfation to glucuronidation was seen in similar studies with phenol. Lineweaver-Burk analysis of microsomal phenol UDP-glucuronosyltransferase activity suggested that MC-treatment induced a high affinity isozyme (KM = 0.14 mM), in addition to the low affinity isozyme (KM = 3.1 mM) present in liver microsomes from untreated and phenobarbital-treated rats. It is concluded that induction by MC of a high affinity hepatic phenol UDP-glucuronosyltransferase effectively shifts benzene metabolism toward formation of less toxic metabolites. This shift may reduce toxic risks at extrahepatic target tissues.

Animals↗

PubMedDate not supplied

Studies of UDP-glucuronyltransferase activities in human liver microsomes.

Human liver samples from a "liver bank" which have been previously characterized by the ability to catalyze cytochrome P-450 dependent reactions were analyzed for various UDP-glucuronyltransferase activities. When stored at -80 degrees C, UDP-glucuronyltransferase activities and latency characteristics of the enzyme appeared to be stable for up to 2 years. The correlation of UDP-glucuronyltransferase activity (4-methylumbelliferone as substrate) with enzyme activities towards 4-nitrophenol and 1-naphthol was much higher (r greater than 0.8) than that (r less than 0.3) observed with other enzyme activities (4-hydroxybiphenyl, morphine, and chloramphenicol as substrates), suggesting the presence of multiple enzyme forms in human liver. Livers of three patients treated with phenytoin or pentobarbital showed significantly higher UDP-glucuronyltransferase activities towards 1-naphthol, 4-methylumbelliferone, and bilirubin than those from five patients who did not receive these inducing agents.

Enzyme Induction↗