Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins)

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent K_m-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes—the ethyl, propyl and butyl ethers—but the magnitudes of the spectra differed in the order 3MC- > PB- > control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold).The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.     

Interactions of heme with hepatic microsomal mono-oxygenase. Effect on benzpyrene hydroxylation.

The addition of heme (1-10 muM) to liver microsomes from phenobarbital (PB)-treated and 3-methylcholanthrene (MC)-treated male rats increased the rate of benzpyrene (BP) hydroxylation by about 20-40%. On the other hand, protoporphyrin IX caused only inhibition of BP hydroxylation. There was no increase of enzymatic activity by heme when solubilized preparations of liver microsomes were used. This suggested the possibility that an apo-cytochrome P-450 was present in intact microsomes. Higher concentrations of heme inhibited BP hydroxylation by either intact or solubilized microsomes. The inhibition by heme with solubilized microsomal preparations was noncompetitive, "mixed-type". However, with intact microsomes, the lack of linearity, precluded the determination of the type of inhibition. To examine possible effects of heme on the binding of BP to microsomal cytochrome P-450, the spectrum elicited by the addition of BP to microsomes was obtained in the presence or absence of added heme. The addition of heme to liver microsomes produced a marked increase in the trough (419-420 nm) of the difference spectrum formed by the subsequent addition of BP. These findings would suggest that heme increased the binding of BP to microsomes. However, the possibility that BP merely displaces the bound heme of the microsomes to yield, as expected, a trough at 413-416 nm (the addition of heme to microsomes yields a peak of 413-416 nm, unpublished) cannot be ruled out. Nevertheless, independent of our understanding of the mechanism involved in the spectral interactions between heme and BP with liver microsomes it is clear that an effect at their binding site(s) must have been elicited by the presence of both compounds.     

Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on a number of hepatic and extrahepatic foreign-compound-metabolizing enzyme systems in microsomes from rats, rabbits and guinea pigs were investigated.Following TCDD treatment, the N-demethylation of benzphetamine, aminopyrine and ethylmorphine was suppressed in hepatic microsomes from male but not from female rats. However, both cytochrome P-450 and benzpyrene hydroxylase were significantly stimulated in hepatic microsomes from both male and female rate at doses as small as 1 μg TCDD/kg body weight. The inductive effect on rat hepatic microsomal enzymes was considerably more persistent than the suppressive effect. Following a single oral dose of 25 μg TCDD/kg body weight, benzpyrene hydroxylase of male rat liver microsomes remained significantly elevated for 73 days but the suppression of benzphetamine N-demethylase had gone after 35 days.The induction of benzpyrene hydroxylase in male rat liver microsomes by TCDD was independent of the age of the rat and the levels to which this enzyme was increased was similar in male rats of all ages. However, the suppression of benzphetamine N-demethylase in male rat liver microsomes was age related: the suppression was seen only in adult animals and in the very young (10 days old) the enzyme was actually induced by TCDD. Inductive effects appeared in both smooth and rough-surfaced hepatic microsomes from male rats but the suppression of N-demethylidon occurred perhaps the derepression arises through the interaction of TCDD or metabolite of TCDD, with the operator gene itself.     

Induction of cytochrome P-450 in rat liver by the antibiotic troleandomycin: partial purificaton and properties of cytochrome P-450-troleandomycin metabolite complexes

Liver cytochrome P-450 from rats treated intraperitoneally with troleandomycin (TAO) were solubilized and partially purified using DE 52 anion exchange chromatography. The major TAO-induced cytochrome P-450 form appears in fraction A which is not bound on the DE 52 column. It is different from the major form induced in rats by phenobarbital or 3-methylcholanthrene in terms of absolute visible spectroscopy, gel electrophoresis (M 45000) and reactions with antibodies. This TAO-induced form mainly exists $\text{in vivo}$ as an iron-TAO metabolite complex and exhibits a characteristic Soret peak at 456 nm. Reconstitution experiments using this partially purified form, after dissociation of its iron-metabolite bond by ferricyanide treatment, underline its particular ability to demethylate TAO itself. TAO also leads to an important induction of other cytochromes P-450 that are present in fraction B (retained on DE 52 column) like the major phenobarbital-induced form, but are immunologically distinct from it.     

Polychlorinated biphenyl isomers and congeners as inducers of both 3-methylcholanthrene- and phenobarbitone-type microsomal enzyme activity

Highly purified synthetic polychlorinated biphenyls substituted in the meta and para positions of both phenyl rings and at one ortho position were administered to male Wistar rats and the effects of these compounds on the microsomal drug-metabolising enzymes were evaluated. The in vivo effects of these compounds were determined by measuring the microsomal benzo[a]pyrene hydroxylase, dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC), 2,2',4,4'-tetrachlorobiphenyl (TCBP-II) (a PB-type inducer), 3,3',4,4'-tetrachlorobiphenyl (TCBP-I) (an MC-type inducer), PB plus MC (coadministered) and TCBP-II + TCBP-I (coadministered) to the test animals. At dosage levels of 30 and 150 mumol . kg-1, pretreatment with 2,3,3',4,4'-pentachlorobiphenyl (PCBP-II), 2,3',4,4',5-pentachlorobiphenyl (PCBP-I), 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-II) and 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-III) gave hepatic microsomes with enzymic and spectral properties consistent with a mixed pattern of induction. These polychlorinated biphenyl (PCB) isomers and congeners have been identified as either major or minor components of the commercial PCB mixtures and must contribute to their activity as MC-type inducers. The only PCB isomer in this series which was not a mixed type inducer was 2,3',4,4',5,5'-hexachlorobiphenyl (HCBP-I) which appeared to be a PB-type inducer. This contrasted to the mixed-type activity observed for 2,3',4,4',5,5'-hexabromobiphenyl which was isolated from a commercial polybrominated biphenyl (PBB) mixture.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Dimethylnitrosamine-demethylase: absence of increased enzyme catabolism and multiplicity of effector sites in repression. Hemoprotein involvement.

Evidence is presented that the previously observed decrease of the Vmax of hepatic microsomal demethylation of dimethylnitrosamine (DMN), following pretreatment of rats with 3-methylcholanthrene (MC), is not due to increase in the rate of breakdown but to decrease of de novo synthesis. Determinations of Vmax at time intervals in the transition from the high steady-state level induced by a carbohydrate-devoid casein diet, down to the low steady-state level of carbohydrate-containing basal diet, yielded two consecutive slopes; descent from the basal diet level to the lower steady-state level following pretreatment with MC yielded one slope. Plotting these slopes against the initial Vmax values gave a typical exponential curve (or straight line if the logs of slopes are used) indicating that the rate of enzyme decay in the MC-treated animals is not greater than that expected from normal enzyme catabolism. A multiplicity of effector sites appears to be involved in the repressor action of different structural types; for example, repression by MC (46.6%) and by phenobarbital (23.9%) in combination are approximately additive (62.0%), rather than competitive, indicating that the two agents act at different sites. A P-450 type cytochrome is involved in the demethylation of DMN. DMN-demethylase is inhibited by carbon monoxide, but the susceptibility to CO is far greater than that observed previously with 3,4-benzopyrene hydroxylation; inhibition of DMN-demethylase as a function of CO concentration follows typical enzyme kinetics. However, while both phenobarbital and MC powerfully repress the DMN-demethylase, we have confirmed that they are strong inducers of the synthesis of P-450 and P-448, respectively, as estimated from the difference spectra.     

Strain differences in the induction of soluble and microsomal enzymes by phenobarbital and 3-methylcholanthrene

The ability of phenobarbital and 3-methylcholanthrene (3MC) to induce liver microsomal and soluble enzymes was compared in Sprague-Dawley and Long-Evans rats. 3MC increased the V for the aniline hydroxylase and stimulated the formation of the hemoprotein P₄₄₈ to a similar extent in the 2 strains of rats. On the other hand phenobarbital increased the V for the microsomal enzyme aniline hydroxylase and aminopyrine demethylase and enhanced the activity of the soluble enzyme aldehyde dehydrogenase only in Sprague-Dawley rats. It induced a more marked increase of cytochrome P₄₅₀ in the Sprague-Dawley than in the Long-Evans strain.     

Cytochrome P-450 and halothane metabolism. Decrease in rat liver microsomal P-450 in vitro

Anaerobic in vitro incubation of microsomes from phenobarbital(PB)-induced rats with halothane results in an irreversible decrease of measurable cytochrome P-450. There is a parallel decrease in heme content under the same incubation conditions. However, microsomes from 3-methylcholanthrene(3-MC)-induced or untreated animals do not show a reduction in cytochrome P-450 content. Aerobic incubation with halothane results in a decrease of cytochrome P-450 which can be completely reversed by dialysis or the addition of potassium ferricyanide. These latter treatments only partially restore the cytochrome P-450 levels following anaerobic incubations. The decrease in cytochrome caused by halothane is not associated with measureable heme N-alkyl adduct formation; lipid peroxidation does not play a role as indicated by the lack of effect of 1 mM EDTA or a decrease in glucose-6-phosphatase activity. Halothane metabolites are bound irreversibly to microsomal protein as determined by gel electrophoresis only when the oxygen concentration is very low. The mechanism of cytochrome P-450 decrease is consistent with the formation of a reactive metabolite which binds to the protein portion and also destroys heme.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: effects of di-ortho substitution

All of the 13 possible polychlorinated biphenyl (PCB) isomers and congeners substituted at both para positions, at least two meta positions (but not necessarily on the same ring) and at two ortho positions have been synthesized and tested as rat hepatic microsomal enzyme inducers. The effects of these compounds were evaluated by measuring microsomal benzo-[a]pyrene (B[a]P) hydroxylase, 4-chlorobiphenyl (4-CBP) hydroxylase, 4-dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the carbon monoxide(CO)- and ethylisocyanide(EIC)-difference spectra of ferrocytochrome P-450. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered). At dose levels of 150 mumol . kg-1, all of the PCB congeners, except 2,3',4,4',5',6-hexachlorobiphenyl, significantly enhanced hepatic microsomal cytochrome P-450 content, B[a] P hydroxylase and/or DMAP N-demethylase activities compared to the control (corn oil-treated) animals. Only 5 of these compounds, namely 2,3,4,4',5,6-hexa-, 2,2',3,3',4,4'-hexa-, 2,2',3',4,4',5-hexa-, 2,3,3',4,4',6-hexa-and 2,2',3,3',4,4',5-heptachlorobiphenyl, enhanced microsomal B[a]P hydroxylase, 4-CBP hydroxylase, NADPH-cytochrome c reductase and DMAP N-demethylase activities in a manner consistent with a mixed pattern of induction. The results suggest that PCB isomers and congeners substituted at both para positions, at least two meta positions, at two ortho positions and containing a 2,3-4-trichloro substitution pattern on one ring are mixed-type inducers; in addition the effects of 2,3,4,4',5,6-hexachlorobiphenyl were also consistent with a mixed pattern of induction.     

Michaelis--Menten kinetic analysis of the hepatic microsomal benzpyrene hydroxylase from control, phenobarbital- and methyl-3-cholanthrene-treated rats.

The sensitive fluorimetric assay for hydroxy-3-benzpyrene (3-OH-BP) described by Dehnen et al., was used to study the effect of microsomal membrane concentration of the benzpyrene hydroxylase activity. Microsomes from phenobarbital (PB) and methyl-3-cholanthrene (3-MC)-treated rats were used in comparison with the microsomal fraction from control animals. At very low protein concentration, benzpyrene hydroxylase follows as Michaelis--Menten type kinetics. When the concentration of microsomal membrane is higher than a minimal value (+/- 6 mug protein/ml) the Km increases with increasing concentration of protein due to competitive inhibition by reversible and non-specific binding of the substrate. The Ki's for such a binding have been calculated. Pretreatment of rats with 3-MC selectively shortens the time linearity, decreases the Ks value, and has no effect on Vmax, while the administration of PB prolongs the time linearity, decreases Vmax and does not modify the Ks. 3-MC and PB specifically act on cytochrome P-450 and do not modify the physico-chemical properties of the microsomal membrane as measured by the non-specific binding of benzpyrene (BP).     

Binding of benzo(a)pyrene to hepatic cytosolic protein enhances its microsomal oxidation

Sephadex G-100 gel permeation chromatography of rat liver cytosol saturated with ¹⁴C-benzo(a)pyrene (BP) resulted in two peaks of protein bound radioactivity. Glutathione-S-transferase (GST) activity (towards 1-chloro 2,4-dinitrobenzene as substrate) was eluted as a single major peak which coincided with one peak of protein bound BP. Oxidation of protein bound BP (GST rich fractions) by microsomes from control or 3-methylcholanthrene treated rats was significantly enhanced as compared to ethanol suspended BP. The formation of oxidized products from the protein-bound BP was dependent on incubation time and microsomal protein concentration, required NADPH and was inhibited by monooxygenase inhibitors α-napthoflavone, 1-benzylimidazole, metyrapone and SKF 525A. Coemergence of BP binding-protein with GST suggests that the soluble protein could be one of the glutathione-S-transferases.     

Metabolic activation of dimethylnitrosamine and diethyl-nitrosamine to mutagens

Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad-3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G₄6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G₄6 are more sensitive than the ad-3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.     

Strain-differences and inducibility of microsomal oxidative enzymes in Drosophila melanogaster flies

Some basic characteristics of the enzyme system involved in the oxidative metabolism of xenobiotic compounds were investigated in Drosophila melanogaster flies. Attention was focussed on (1) the normal levels of these enzymes and their activities in whole flies, in different parts of the fly's body and in different sexes, (2) the changes in levels and activities of the enzymes elicited by pretreatment of the flies with known enzyme inducers and (3) differences between strains.Four commonly used wild-type (WT) strains, three insecticide resistant strains (IR) and one white-eyed mutant strain were employed. Except in those experiments on sex differences and in spatial distribution in the fly's body of the enzymatic activities, microsomes were isolated from whole-body homogenates of mixtures of female and male flies. Microsomal cytochrome P-450, benzo[a]pyrene (BP) hydroxylation, p-nitroanisole (pNA) demethylation and aminopyrine (AP) demethylation were measured in control flies and in flies pretreated with Aroclor 1254 (AC), phenobarbital (PB) or butylated hydroxytoluene (BHT).In flies of the WT strain Berlin-K, there were no significant differences in BP hydroxylation activity and its inducibility between the two sexes. In males, inducibility of BP hydroxylation activity was similar in the head, thorax and abdomen, but significantly lower in testis. Considerable differences in some enzyme activities were found between the strains. pNA demethylation and AP demethylation were substantially higher in all IR strains, while no correlation could be found between their increased insecticide resistance and BP hydroxylating capacity or cytochrome P-450 content of the microsomes.Response to enzyme inducing compounds was found to be strain-dependent. PB proved to be a more efficient inducer of BP hydroxylation than AC, which does induce pNA demethylation. BHT has inducing properties that are intermediate between PB and AC. IR strain Hikone-R turned out to be an exception, possessing very low BP hydroxylating capacity and a low degree of inducibility of mixed-function oxidase activities. Differential temperature dependence was found for BP hydroxylation as compared with pNA demethylation. While BP hydroxylation was doubled when raising the temperature from 25°C to 35°C, pNA demethylation was reduced by 50%.     

Mouse liver and lung glutathione S-epoxide transferase: Effects of phenobarbital and 3-methylcholanthrene administration

The effects of 3-methylcholanthrene (3MC) and phenobarbital (PB) administration on the levels of glutathione-S-epoxide transferase activity in supernatant preparations of liver and lung were studied in a number of different strains of mice, C57Bl/6, C3H, C3H_f^?, Balb/c^?, A⁺ and DBA/2⁺. Three epoxide substrates, 3MC-11,12-oxide, styrene oxide (SO) and 3,3,3-trichloro-1,2-epoxypropane (TCPO), were employed in this investigation. PB administration (75 mg/kg body weight for 3 days) resulted in 13–57% increases in enzyme activity in the liver supernatant but was ineffective in inducing activity in lung. 3MC administration (40 mg/kg body weight for 2 days) on the other hand was without any effect on glutathione-S-epoxide transferase activity in both liver and lung.     

The induction of cytochrome P-450 and monooxygenase activities in the chick embryo by 2-acetylaminofluorene

Administration of 2-acetylaminofluorence to chick embryos increases the cytochrome P-450 level 3.4 fold but causes no increase in total epoxide hydrase activity or other microsomal electron transport enzymes. The induction response shows some similarity to that elicited by phenabarbitone both in terms of the monooxygenase activities induced and their inhibition characteristics. Induction of a specific cytochrome P-450 subform by this agent may increase its detoxification and in part account for the resistance of avian species to its hepatocarcinogenic effect.     

HgCl2 increases the methemoglobin prooxidant activity. Possible mechanism of Hg2+-induced lipid peroxidation in erythrocytes

In an attempt to elucidate the mechanism of initiation of peroxidation in HgCl2-treated erythrocytes, the effect of HgCl2 on methemoglobin-catalyzed lipid peroxidation was studied. It was found that HgCl2 reinforces the prooxidant action of methemoglobin. This effect seems not to be due to dissociation or degradation of the hemoglobin molecule to heme-containing fragments or iron-containing products of low molecular weight. The results obtained indicate that Hg2+ increases the binding of oxy- and methemoglobin to liposomes. A suggestion is made that the acceleration of methemoglobin-catalyzed peroxidation by HgCl2 is mainly due to increased binding of methemoglobin to liposomes. On the basis of these results and the results obtained previously the possible mechanism of initiation of peroxidation in Hg2+-treated erythrocytes is discussed.