Clifford R. Elcombe

Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450

The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylcholanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. beta-Napthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16 alpha-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the O-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy- and benzyloxyphenoxazone. Very high degrees of induction were seen. MC and ARO each induced preferentially the O-dealkylation of ethoxyphenoxazone (51- and 61-fold respectively). PB and SKF-525A each induced preferentially the O-dealkylation of pentoxyphenoxazone (283- and 324-fold respectively). ISO induced preferentially the O-dealkylation of benzyloxyphenoxazone (43-fold). For any particular induced type of microsomes the substrate with the fastest metabolism was not necessarily the substrate whose metabolism was induced the most, so that in order to characterize each of the 5 different inducers (PB, MC/BNF, ARO, ISO, SKF) it was necessary to compare both the degrees of induction and the specific activities of the reactions. Experiments with purified cyt. P-450 isozymes showed that ethoxyphenoxazone and pentoxyphenoxazone were highly selective substrates for the major isozymes induced by MC and PB respectively, whilst benzyloxyphenoxazone was a good substrate for both isozymes. Experiments using the organic inhibitors metyrapone and alpha-naphthoflavone and inhibitory antibodies against individual cyt. P-450 isozymes indicated that similar substrate selectivities occurred with the monooxygenase system in the microsomal membrane. It is suggested that the use of some or all of these homologous phenoxazone ethers will provide both a simple routine test for the characterization of several types of inducing agents and a powerful tool for investigating the biochemical basis for cyt. P-450 isozyme substrate selectivity.

Induction and characterization of hemoprotein(s) P-450 and monooxygenation in rainbow trout (Salmo gairdneri)

Hepatic microsomes obtained from control rainbow trout showed relatively low monooxygenase activities toward benzo[a]pyrene, ethoxycoumarin, ethoxyresorufin, and ethylmorphine as substrates. However, benzo[a]pyrene hydroxylation, ethoxycoumarin-O-deethylation, and ethoxyresorufin-O-deethylation were greatly induced by pretreatment of trout with β-naphthoflavone and Aroclor 1242. No increase in ethylmorphine-N-demethylation, liver/body ratio, or yield of microsomal protein were observed. Phenobarbital pretreatment of trout failed to affect any of the parameters studied. The λmax of carboxyferrocytochrome P-450 was at 449 nm in control microsomes, and although a small (40%) increase in total hemoprotein(s) P-450 was seen after β-naphthoflavone pretreatment no hypsochromic shift of the λmax was observed. No significant changes in the 430455 ratio of the EtNC ligand spectra were noted after induction. Type I and Type II binding spectra were observed in all microsomal preparations examined. The use of specific inhibitors of monooxygenation (α-naphthoflavone and metyrapone) indicated that control and induced trout hepatic hemoprotein(s) P-450 were similar to rat cytochrome P1-450. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence of a novel hemoprotein in hepatic microsomes after pretreatment of trout with β-naphthoflavone or Aroclor 1242. This induced protein had a molecular weight of approximately 57,000.

The effect of various types of inducing agents on hepatic microsomal monooxygenase activity in rainbow trout.

The effects of various types of inducing agents on the hepatic microsomal monooxygenase (MO) system of rainbow trout were examined and compared to the induction profiles observed following pretreatment with either 3-methylcholanthrene- or phenobarbital-type inducers. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) elevated ethoxycoumarin- and ethoxyresorufin-O-deethylase activities (ECOD, EROD) as well as the cytochrome(s) P-450 content, but had no effect on benzphetamine-N-demethylation (BeND). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of solubilized microsomes from TCDD-pretreated fish demonstrated the intensification of a protein band with a molecular weight of 57,000. Although the magnitude of induction was not as great, isosafrole pretreatment of rainbow trout resulted in an induction profile similar to that observed following TCDD administration to these animals. Neither Kepone nor mirex had a stimulatory effect on any of the measured parameters and neither caused a change in the protein profile following SDS-PAGE of solubilized microsomes. These results suggest that fish respond differently from mammals to these inducers of the hepatic microsomal MO system.

Studies on the interaction of safrole with rat hepatic microsomes

Abstract (1) Similar to previous results with methylenedioxyphenyl compounds microsomes from safrole pretreated rats showed, on reduction with NADH, NADPH or Na 2 S 2 O 4 , characteristic absorption maxima at 427 and 455 nm. The same spectrum can be obtained after incubation in vitro of control microsomes with safrole, NADPH and oxygen. (2) Subsequent addition of carbon monoxide to microsomes of safrole pretreated rats causes an absorption maximum at 448 nm, characteristic of the 3-methylcholanthrene type of induction of microsomal hydroxylase protein. (3) The suspected cytochrome P-450-safrole metabolite complex, which can be visualized only in the reduced state of cytochrome P-450, is very stable as witnessed by its preservation through the preparation procedure for microsomes or after dialysis or detergent treatment. However, when safrole or ethylbenzene is added, both absorption maxima decrease in a time dependent manner. This can be measured for each time point after complete reduction of the microsomal preparation by adding Na 2 S 2 O 4 . (4) From this it is concluded that the carcinogen safrole leads to the biosynthesis of a 3-methylcholanthrene type cytochrome P-450 and formation of a safrole metabolite-cytochrome P-450 complex which in turn can be cleaved in vitro by safrole or other lipophilic compounds.

Further studies on the dissociation of the isosafrole metabolite-cytochrome P-450 complex.

Abstract The dissociation of an in vivo isosafrole metabolite-cytochrome P-450 complex by a number of exogenous and endogenous compounds is described. The rate of dissociation is temperature-dependent, but the process does not require oxygen. Compounds which produce Type I (substrate) binding spectra with oxidized cytochrome P-450 facilitate displacement of the isosafrole metabolite from cytochrome P-450 whereas Type II (ligand-binding) compounds do not dissociate the complex. The factors necessary for dissociation of the complex are discussed and a model for the isosafrole metabolite-cytochrome P-450 complex is proposed.

Sex steroids and drug metabolism. A sex-related difference in hepatic microsomal ethoxyresorufin-O-deethylation in sprague-dawley rats.

Abstract The effect of sex steroids and pregnenolone-16α-carbonitrile (PCN) on hepatic microsomal monooxygenase activity was examined in male and female Sprague-Dawley rats. Male rats had a greater cytochrome(s) P-450 content and ethoxycoumarin-O-deethylase (ECOD) and NADPH-cytochrome c reductase (CcR) activities than females. Ethoxyresorufin-O-deethylation (EROD) in hepatic microsomes from females, however, was greater than from males, suggesting that the female rats possessed inherently higher cytochrome P-448-mediated monooxygenase activity. Testosterone propionate and PCN administration resulted in stimulation of ECOD and CcR activities in female rats, and PCN also increased the cytochrome(s) P-450 content. Estradiol benzoate decreased ECOD and CcR in male rats but enhanced EROD in both sexes. Neither testosterone nor PCN had an effect on EROD activity. In castrated male rats, testosterone increased, and estradiol decreased, ECOD activity without affecting cytochrome(s) P-450 content. Estradiol pretreatment of castrated males resulted in an increase in EROD activity, with testosterone having no effect. Thus, testosterone and estradiol may be, in part, responsible for the inherently higher and lower cytochrome(s) P-450-related activities in male and female rats, respectively, and estradiol may be responsible for the elevated cytochrome P-448-type activity observed in the females.

The transfer of 2,4,5,2′,4′,5′-hexachlorobiphenyl to fetuses and nursing offspring: II. Induction of hepatic microsomal monooxygenase activity in pregnant and lactating mice and their young

Abstract The effect of 2,4,5,2′,4′,5′-hexachlorobiphenyl (6-CB) on hepatic microsomal monooxygenase activity in virgin or pregnant and lactating mice and their offspring was examined. 6-CB was administered in a single dose of 100 mg/kg 14 days before mating. Prior to, and during early pregnancy, hepatic monooxygenase activity in all 6-CB-pretreated mice was greater than that in corn oil controls. No notable differences were observed between pregnant and virgin animals in either group. Corn oil-pretreated mothers had lower cytochrome P-450 content during mid to late pregnancy and early lactation than that observed in virgins sacrificed at the same time, and benzphetamine-N-demethylation and ethoxycoumarin-O-deethylation were also decreased in lactating animals. Mothers pretreated with 6-CB and sacrificed on the day of birth had lower microsomal monooxygenase activity and cytochrome P-450 content than 6-CB-pretreated virgins sacrificed concurrently, but no differences were noted between these groups of animals during lactation. Hepatic enzyme activities and cytochrome P-450 content were not different between newborns (Day 1) of corn oil- and 6-CB-pretreated mothers. However, these parameters were elevated in 5- to 20-day postpartum nursing offspring from 6-CB-pretreated mothers when compared to those from corn oil-pretreated mothers suggesting transfer of 6-CB through breast milk in quantities sufficient to affect hepatic microsomal monooxygenase activity.

Substrate-elicited dissociation of a complex of cytochrome P-450 with a methylenedioxyphenyl metabolite.

Abstract The 14C-isosafrole metabolite-rat hepatic cytochrome P-450 complex is stable to dialysis but is readily dissociated by cyclohexane (1 mM) to release free cytochrome P-450 and radioactive ligand. The ratio of cytochrome P-450: isosafrole metabolite in the complex is unity.

COMPARATIVE ASPECTS OF HEPATIC MONOOXYGENASE INDUCTION IN RAT AND RAINBOW TROUT BY SEVERAL PCB ISOMERS

Publisher Summary This chapter discusses the comparative aspects of hepatic monooxygenase induction in rat and rainbow trout by several polychlorinated biphenyl (PCB) isomers. Because of the widespread distribution of PCBs in the ecosphere including fresh and marine waters, the study described in the chapter investigated the effects of (1) two commercial mixtures of PCBs, namely, Aroclors 1254 and 1242, and (2) two pure non-coplanar PCB isomers and one pure coplanar isomer on the inducibility of rainbow trout hepatic cytochrome(s) P-450 activity. The hepatic cytochrome P-450 system of this strain of fish has proven refractive to induction by phenobarbitone-like agents. From the study, it was found that rodent and trout hepatic monooxygenation activities were induced more with the commercial PCB mixtures than with the individual isomers studied. The finding of chlorinated dioxins and dibenzofurans in these mixtures has been used to explain their potent inductive effects, especially on the cytochrome P-448 system.