Günter Blaich

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.

The effects of pretreatment with 7,8-benzoflavone on drug-metabolizing enzymes and diethylstilboestrol metabolism in male hamster liver microsomal preparations

1. Pretreatment of male Syrian golden hamsters with 7,8-benzoflavone (7,8-BF, 0.4% in the diet) for 4 and 20 weeks resulted in a significant increase in the amount of cytochrome P-450 and cytochrome b5 in hepatic microsomes. The activities of microsomal 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) increased by a factor of 2 and 5, respectively, whereas aryl hydrocarbon hydroxylase (AHH) activity was only marginally enhanced. 2. Because the relative increase in cytochrome b5 exceeded that of cytochrome P-450, the ratio of P-450 to b5 decreased from 2.2 in controls to 1.6 in 7,8-BF-treated animals. 3. Hepatic microsomes from untreated and 7,8-BF-pretreated hamsters metabolize E-diethylstilboestrol (E-DES) to its stereoisomer Z-DES and to several oxidative metabolites, of which 3-hydroxy-DES and Z,Z-dienestrol were unambiguously identified by g.l.c.-mass spectrometry. 4. Pretreatment with 7,8-BF led to a marked decrease in the formation of all oxidative DES metabolites but not in the isomerization to Z-DES. The possible implication of these data for the mechanism of liver tumour formation by the combined treatment of hamsters with 7,8-BF and DES is discussed.

Effect of pretreatment of male syrian golden hamsters with 7,8-benzoflavone and with diethylstilbestrol on P-450 isoenzyme activities and on microsomal diethylstilbestrol metabolism

Combined treatment of male Syrian golden hamsters with the synthetic estrogen diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF) gives rise to a high incidence of hepatocellular carcinomas, whereas no such tumors are formed with DES alone nor with 7,8-BF alone. To determine whether alterations in DES metabolism may account for the observed hepatocarcinogenicity, we have studied the effect of pretreatment with 7,8-BF alone, DES alone and 7,8-BF plus DES on the levels of hepatic P-450 and cytochrome b5, on the activities of various P-450 isoenzymes and on microsomal DES metabolism. Hepatic P-450 content was significantly increased after pretreatment with 7,8-BF and decreased after DES, while combined pretreatment led to levels similar to those in untreated control animals. Hepatic cytochrome b5 was also elevated in 7,8-BF-treated hamsters; DES pretreatment had no effect, and combined pretreatment led to a slight increase. Four different substrates were used to probe P-450 isoenzyme activity. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin-O-deethylase (ECOD), 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) were all elevated after 7,8-BF-pretreatment, while DES led to a decrease in these activities with the exception of AHH, where a transient increase which was observed after 8 and 20 weeks of pretreatment was back to control levels after 32 weeks. Combined pretreatment with 7,8-BF and DES led to an intermediate response (slight increase) with AHH, EROD and PROD, but not with ECOD, where a full induction comparable with that observed after 7,8-BF alone was elicited. In spite of the modulation of enzyme levels and activities observed after the various pretreatments, the metabolism of DES in microsomes from pretreated animals was virtually identical with that from controls. Therefore it is concluded that modulation of hepatic DES metabolism is not the reason for the observed hepatotumorigenicity; instead, it is speculated that 7,8-BF is the carcinogenic agent in this tumor model, and DES may act as a promotor.

Effect of pretreatment with 7,8-benzoflavone and diethylstilbestrol on the hepatic metabolism of diethylstilbestrol in the male Syrian golden hamster in vivo

Liver tumors are induced in male Syrian golden hamsters by the combined treatment with diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF), but not with either substance alone. With the aim of clarifying whether metabolic activation of DES is involved in the mechanism of tumorigenesis in this animal model, we have studied the effect of pretreatment with 7,8-BF alone, DES alone, and 7,8-BF plus DES for 2, 8, 20 and 32 weeks on the hepatic in vivo metabolism of DES, using biliary metabolites collected from bile-duct cannulated male hamsters as probe. Formation of glucuronides and sulfates was not affected by treatment with 7,8-BF nor 7,8-BF plus DES. In contrast, animals pretreated with DES alone had a decreased amount of glucuronides and an increased proportion of unconjugated material in the bile. Oxidative metabolism of DES was not significantly altered in hamsters treated with 7,8-BF for up to 20 weeks, whereas pretreatment with DES alone and with 7,8-BF plus DES caused an enhancement of oxidative DES metabolism in vivo, leading mostly to highly polar, as yet unidentified products. From a consideration of various cytochrome P-450-associated enzyme activities, it is concluded that the observed effect on biliary DES metabolites is most likely to be due to an estrogen-induced intrahepatic cholestasis. Taken together, the data do not support a role for the metabolic activation of DES in this tumor model. Alternative mechanisms are proposed.

Effect of 7,8-benzoflavone pretreatment on diethylstilbestrol metabolism, drug-metabolising enzymes and the aromatic hydrocarbon (Ah) receptor in male hamster liver

SummaryPretreatment of male Syrian golden hamsters with 7,8-benzoflavone (7,8-BF) leads to a marked increase of cytochrome P450 and cytochrome b5 levels in the liver, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induce cytochrome P450 but not cytochrome b5 7,8-BF pretreatment has only minor effects on the activities of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin-Odeethylase, but 7-ethoxyresorufin-O-deethylase is increased 3-fold. In contrast to PB, pretreatmant with 7,8-BF or MC reduces the oxidative metabolism of diethylstilbestrol (DES) by hepatic microsomes in vitro. The cytosolic level of the aromatic hydrocarbon (Ah) receptor in hamster liver is decreased by 7,8-BF and slightly enhanced by MC pretreatment. PB increases the receptor level 1.5-fold. The affinity of 7,8-BF to the Ah receptor in vitro is of the same order of magnitude as that of the known ligands 5,6-benzoflavone and 2,3,7,8-tetrachlorodibetizofurane. PB and DES show no binding to the receptor protein.