Carbon Disulphide Induced Activation of Liver UDP glucuronosyltransferase in Rats Pretreated with Phenobarbitone

Abstract Carbon disulphide (CS₂) exposure has been shown to activate the UDPglucuronosyltransferase of liver microsomes in rats pretreated with phenobarbitone. Now the nature of CS₂ induced activation of the enzymes has been studied further. Phenobarbitone pretreated rats were exposed to 0.15% CS₂ for 2 hrs on two successive days. The activity of UDPglucuronosyltransferase was measured from the liver microsomes after the enzymes was activated by incubation of the microsomes with various concentrations of the detergents Triton X-100, digitonin and cetylpyridinium chloride. The exposed animals showed an increased enzyme activity at all applied concentrations of the detergents; therefore in addition to membrane destruction by CS₂ exposure, some other mechanism must also be involved in the CS₂ induced activation of liver microsomal UDPglucuronosyltransferase. The changes in membrane lipid-protein interactions with 1-anilino-8-naphthalene sulphonate (ANS) were also probed. The CS₂ exposed animals had more high-affinity binding sites for ANS in their liver microsomal membranes, and in addition the quantum yield of ANS fluorescence was enhanced by CS₂. The changes differed from those found after carbon tetrachloride exposure and suggest that, even if the two drugs have some common effects on microsomes, e.g. UDPglucuronosyltransferase activation, P-450 destruction and lipid peroxidation induction, the changes they cause in the microsomal micro-environment differ.     

A Comparative Study on Drug Hydroxylation and Glucuronidation in Liver Microsomes of Phenobarbital and 3-Methylcholanthrene Treated Rats

The treatment of hepatic microsomes with phospholipase A or phospholipase C decreased the drug hydroxylase activity. The exception was the aryl hydrocarbon hydroxylase in hepatic microsomes from 3-methylcholanthrene pre-treated rats. Its specific activity remained unchanged after phospholipase A digestion. In hepatic microsomes from phenobarbital pretreated rats the aryl hydrocarbon hydroxylase was not significantly increased and after phospholipase A or phospholipase C digestion, it decreased in both treated and control rats. The phenobarbital or 3-methylcholanthrene induced p-nitroanisole 0-demethylase activity in liver microsomes decreased in a manner corresponding to the decrease in cytochrome P-450 (P-448) concentrations. The treatment of hepatic microsomes with phospholipase A or phospholipase C activated the membrane-bound UDP glucuronosyltransferase both in control and drug-treated rats. Phospholipase A increased the measurable UDP glucuronosyltransferase activity particularly in hepatic microsomes from 3-methylcholanthrene pretreated rats. However, digestion with trypsin was necessary in order to detect the induction in UDP glucuronosyltransferase after phenobarbital pretreatment of rats. Other agents studied, such as phospholipase A or C, digitonin, cetylpyridinium chloride, or NaSCN, were unable to do this. Digestion with trypsin released considerable amounts of UDP glucuronosyltransferase activity into 27,000 g supernatant, especially from phenobarbital microsomes, whereas phospholipase A was more active in releasing UDP glucuronosyltransferase into 27,000 g supernatant from hepatic microsomes of 3-methylcholanthrene pretreated rats. Membrane perturbating agents seem to differ in their action towards the hepatic microsomal membranes obtained from rats treated with phenobarbital or 3-methylcholanthrene.     

The in vivo toxicity of CS2 to liver microsomes: binding of labelled CS2 and changes of the microsomal enzyme activities.

The binding of 35S and 14C labelled CS2 to liver microsomes was studied in control and phenobarbitone pretreated rats 3 and 6 hrs after an intraperitoneal injection. The level of hepatic cytochrome P-450, the activities of epoxide hydratase and UDP-glucuronosyltransferase were analyzed in the same animals. The binding of the sulphur label was considerably higher than that of carbon 3 hrs after the injection, the difference being less evident at 6 hrs.T he measurable P-450 declined after the CS2 injection. It was approximately 40% in the phenobarbitone pretreated rats and 60% in control rats of the values of animals which were not treated with CS2. CS2 did not affect microsomal epoxide hydratase activity, while it increased the measurable activity of UDP-glucuronosyltransferase. The increase was evident 3 hrs after the injection of CS2 in the phenobarbitone pretreated rats. It could also be detected in the control animals 6 hrs after the injection. The present data suggest that the change in the measureble P-450 results from the binding of the metabolite(s) of CS2 to the cytochrome, and its subsequent degradation. The increase in measurable UDP-glycuronosyltransferase activity results probably from the activated perturbation of the structure of microsomal membrane by the metabolites of CS2 in vivo.     

Action of styrene and its metabolites styrene oxide and styrene glycol on activities of xenobiotic biotransformation enzymes in rat liver in vivo

The effects of the administration of styrene and its metabolites, styrene oxide and styrene glycol, on drug metabolizing enzymes of rat liver were studied. Styrene (three or six daily doses of 500 g/kg) doubled the activities of microsomal p-nitroanisole O-demethylase, epoxide hydrase (styrene oxide as substrate), and UDP glucuronosyltransferase (p-nitrophenol) when measured in microsomes pretreated in vitro with digitonin or trypsin. On the other hand, glycine conjugation, the cytochrome P-450 content, and activities of NADPH cytochrome c reductase and benzpyrene hydroxylase, were less affected by styrene. Styrene oxide was more toxic to rats than styrene or styrene glycol and it also caused (one dose of 375 mg/kg) a significant decrease in the activities of benzpyrene hydroxylase and p-nitroanisole O-demethylase and in the cytochrome P-450 content. Epoxide hydratase NADPH cytochrome c reductase, and UDP glucuronosyltransferase were more resistant towards styrene oxide. Styrene glycol did not significantly alter the activities of drug metabolizing enzymes. The current data show that styrene causes an increase in the activities of some enzymes involved in its own metabolism.     

Drug metabolism in Gunn rats: inability to increase bilirubin glucuronidation by phenobarbital treatment

The components of the microsomal mixed-function oxidase system in Wistar and Gunn rats were the same, as were the overall hydroxylation reactions (p-nitroanisole O-demethylase, aryl hydrocarbon hydroxylase). The Gunn rat was, however, almost totally devoid of bilirubin glucuronidation activity. The UDP glucuronosyltransferase activity towards p-nitrophenol in the Gunn rat was in liver about 60 per cent and in the small intestinal mucosa about 10 per cent of that in the Wistar rat. The mono-oxygenation step in hepatic microsomal drug metabolism was induced in a similar way by phenobarbital treatment in both Wistar and Gunn strain rats, but no significant enhancement of the mono-oxygenase activity could be observed in the gut. The trypsin digestion of hepatic microsomes increased the specific UDPglucuronosyltransferase activity. Phenobarbital treatment enhanced the p-nitrophenol glucuronidation in livers of both Wistar (3-fold) and Gunn strain (2-fold) rats, but the bilirubin glucuronidation was enhanced (2-fold) only in Wistar rats. Phenobarbital-enhanced transferase activities could be detected only if the microsomes had been digested with trypsin before determination of the enzyme activity. The UDPglucuronosyltransferase activity in the small intestinal mucosa was not enhanced by phenobarbital. The results suggest that bilirubin and p-nitrophenol are conjugated by distinct enzymes and that the enzyme activities for mono-oxygenation and glucuronidation are controlled by different mechanisms. The microenvironment of the UDP glucuronosyltransferase is most probably similar in Gunn and Wistar rats.     

Aryl hydrocarbon hydroxylase activity induced by injected diesel particulate extract vs inhalation of diluted diesel exhaust

The effects of long-term inhalation of diluted diesel exhaust on aryl hydrocarbon hydroxylase activity (AHH) and cytochrome P-450 content in lung and liver microsomes were investigated in male Fischer-344 rats and compared with repeated parenteral administration of organic solvent extracts of hydrocarbons adsorbed on the diesel particulate surface during the combustion process. The animals were exposed to concentrations of 750 micrograms m-3 or 1500 micrograms m-3 of diesel particulates from a 5.7L GM diesel engine 20 h per day, 5 1/2 days per week for up to 9 months or treated by repeated IP injections of diesel particulate extract (dissolved in corn oil) from the same engine at several dose levels for 4 days. No significant effects of long-term inhalation exposure were observed in liver microsomal AHH activity. A slight decrease in lung microsomal AHH activity was found in rats following 6 months of exposure to diesel exhaust at the particulate concentration of 1500 micrograms m-3. The total mass of particles deposited in the lung during the inhalation exposure was estimated and an equivalent dose of extractable hydrocarbons was administered intraperitoneally; no increase in AHH activity was observed in the lung or liver microsomes. In contrast, 1.4- to 9-fold increases in AHH activity were observed in liver and lung microsomes of rats pretreated by intraperitoneal doses 10-50 times larger than the most conservative estimate of the deposited lung burden. No changes in cytochrome P-450 content were observed in the microsomes of rat liver after inhalation or injection treatment studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals

The mutagenic activation activity of hepatic microsomes from three meat-producing animals (cattle, deer and horses) was compared with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using α-naphthoflavone, anti-rat CYP1A1, CYP3A2 and CYP2E1 antibodies suggests that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucoronosyl transferase and cytosolic glutathione-S-transferase, reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. This report is the first to investigate the mutagenic activation activity of the hepatic microsomes and the role of phase II enzymes against this activity in meat-producing animals.     

Combined effect of ethanol and carbon disulphide on cytochrome P-450 mono-oxygenase, lipid peroxidation and ultrastructure of the liver in chronically exposed rats

A 5-month treatment of rats with ethanol (10% solution in drinking water) stimulated aniline p-hydroxylase and the microsomal ethanol oxidizing system (MEOS) by 140 and 70%, respectively, cytochrome P-450 by 22% and accompanied by lipid peroxidation by 40% in microsomes. It also caused smooth endoplasmic reticulum (SER) proliferation and rough endoplasmic reticulum (RER) degranulation in hepatocytes. Repeated inhalatory exposure of rats to 1.5 g/m3 of CS2, 5 h daily, 5 days a week for 5 months decreased aniline p-hydroxylase and MEOS by 70 and 55% respectively, doubled hexobarbital sleeping time and depressed cytochrome P-450 by 30% and its conversion to cytochrome P-420; these effects were accompanied by the appearance of cytochrome P-420, the intensification of lipid peroxidation in microsomes and some degranulation of RER in hepatocytes. Combined exposure of rats to ethanol and CS2 resulted in a significant potentiation of the inhibitory effects of CS2 on cytochrome P-450 mono-oxygenase and MEOS but with enhancement of CS2 effects on the liver microsomal mono-oxygenase, but CS2 decreased the effect of ethanol on SER proliferation. The interaction both on the biochemical and the morphological level can be explained with the ethanol-stimulated biotransformation of CS2 to reactive electrophilic derivative(s), the subsequent destruction of cytochrome P-450 to cytochrome P-420 and the intensification of lipid peroxidation.     

Mixed-function oxidase system induction and propylene hepatotoxicity

Propylene is hepatotoxic to male Charles River COBS Sprague-Dawley rats pretreated with polychlorinated biphenyls (PCB: Aroclor 1254). Four-hour inhalation exposure to 50,000 ppm propylene increased liver weight/body weight ratios and elevated serum enzyme activities in PCB-pretreated animals. Hepatic microsomal cytochrome P-450 content of PCB-pretreated rats dropped profoundly during propylene exposure and remained depressed for at least 24 h. In addition, PCB-pretreated, propylene-exposed rats exhibited a decrease in the specific activity of hepatic microsomal aniline hydroxylase. However, there was no change in activities of either hepatic microsomal aminopyrine demethylase or glucose-6-phosphatase. Propylene exposure of rats pretreated with beta-naphthoflavone (BNF), phenobarbital (PB), or a mixture of BNF and PB was not hepatotoxic. However, there was, in these animals, a substantial decline in hepatic microsomal cytochrome P-450 levels 24 h after the start of propylene exposure. Hence, the propylene-dependent process resulting in hepatic cytochrome P-450 destruction is qualitatively or quantitatively different from the process that causes acute hepatotoxicity. Preexposure fasting had no effect on the hepatotoxicity resulting from a 4-h exposure of PCB-pretreated rats to 50,000 ppm propylene. Administration of SKF-525A to PCB-pretreated rats immediately prior to propylene exposure completely prevented elevations in serum enzyme activities and liver weight/body weight ratios. In vitro incubation of hepatic microsomes prepared from either BNF-, PB-, or PCB-pretreated rats with an atmosphere of 20% propylene/80% air produced in NADPH-dependent decrease in cytochrome P-450 content. These results suggest that PCB pretreatment is a prerequisite for propylene hepatotoxicity in the rat. Cytochrome P-450-dependent bioactivation of propylene is associated with this hepatotoxicity, but further studies are needed to characterize the mechanism of the PCB-propylene interaction.     

In vitro inhibition of oxidative N-demethylation with carbon disulfide]

Earlier findings have shown that in experimental animals (rat) and in man inhaled carbon disulphide (CS2) reversibly inhibits the non-specific oxidative drug metabolism caused by hepatic microsomal enzymes. Very little is known concerning the underlying mechanism. The present investigations were undertaken to throw light on this question. After addition of an NADPH-regenerating system to liver microsomes isolated from adult female Wistar rats, the oxidative N-demethylation of aminopyrine was measured under simultaneous exposure to CS2 by quantitatively determining the formaldehyde obtained; the resulting data were evaluated using enzyme-kinetic parameters according to Lineweaver-Burk: 1. Following acute exposure to low and medium-grade CS2 concentrations (20-400 ppm/8 h), the pattern of inhibition in rat liver microsomes is identical to that obtained in normal liver microsomes to which CS2 had been added. This finding seems to suggest that the inhibitory process under in vivo and in vitro conditions is based on one and the same molecular mechanism. 2. Upon addition of CS2 the in vitro pattern of inhibition corresponds to a strong mixed-type inhibition. 3. It is concluded from the enzyme-kinetic behaviour that CS2 attacks at two different sites of the enzyme molecule: Binding to the first site is followed by inhibition, as evidenced by the rise of Km; after saturation of this site, a second site is occupied resulting in a reactivation and, beyond this, an activation of enzyme output, as shown by the decrease of Km. CS2 exhibits a high affinity for the first site, and a low affinity for the second site. Binding at the first site is reversible. The possibility that the active centre in the enzyme molecule is the site where binding of the inhibitor occurs is ruled out. 4. A continuous decrease of Vmax at increasing inhibitor concentration is causally related with the formation of an enzyme/substrate inhibitor complex. 5. The CS2 added to the microsomes can be eliminated by helium gas; this is followed by the return of the original enzyme activity. It is concluded from this behaviour that under in vitro conditions CS2 itself (rather than its metabolites) acts as the inhibitor. 6. Oxygen treatment of the microsome-containing reaction mixture enhances the inhibition. In substrate-free control mixtures, addition of CS2 was followed by the dose-dependent formation of formaldehyde; a causal explanation is not readily available at this time.     

Phthalate esters

Male Sprague Dawley rats were exposed to dibutylphthalate (DBP) by inhalation with concentrations of 0.5, 2.5, and 7.0 ppm in the air for 5 days. The concentrations were considered relevant to human exposure. No quantitative changes were observed in liver microsomal cytochrome P-450 related enzymes, but significant increase was observed in the liver microsomal metabolism of benzo(a)pyrene and n-hexane, in the 2.5 ppm and 0.5 ppm groups, respectively. Inhaled DBF decreased in a dose-dependent way the lung microsomal concentration of cytochrome P-450 by as much as 63%, which was reflected in a significant reduction of the microsomal metabolism of n-hexane and benzo(a)pyrene in the 7.0 ppm group. It is concluded that DBP in doses relevant to human air exposure influences the cytochrome P-450 enzyme system in both liver and lung, with lung as the main target organ. The observed effects in lung microsomes were similar to those earlier reported after IP administration of high doses of DBP.     

The in Vivo Toxicity of CS2 to Liver Microsomes: Binding of Labelled CS2 and Changes of the Microsomal Enzyme Activities

Abstract The binding of ³⁵S and ¹⁴C labelled CS₂ to liver microsomes was studied in control and phenobarbitone pretreated rats 3 and 6 hrs after an intraperitoneal injection. The level of hepatic cytochrome P-450, the activities of epoxide hydratase and UDP-glucuronosyltransferase were analyzed in the same animals. The binding of the sulphur label was considerably higher than that of carbon 3 hrs after the injection, the difference being less evident at 6 hrs. The measurable P-450 declined after the CS₂ injection. It was approximately 40 % in the phenobarbitone pretreated rats and 60 % in control rats of the values of animals which were not treated with CS₂. CS₂ did not affect microsomal epoxide hydratase activity, while it increased the measurable activity of UDP-glucuronosyltransferase. The increase was evident 3 hrs after the injection of CS, in the phenobarbitone pretreated rats. It could also be detected in the control animals 6 hrs after the injection. The present data suggest that the change in the measurable P-450 results from the binding of the metabolite(s) of CS₂ to the cytochrome, and its subsequent degradation. The increase in measurable UDP-glucuronosyltransferase activity results probably from the activated perturbation of the structure of microsomal membrane by the metabolites of CS₂ in vivo.     

Organ specific induction of drug metabolizing enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied on the activities of arylhydrocarbon hydroxylase, ethoxycoumarin deethylase, cytochrome c reductase, epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase in the liver, kidney, lung, small intestinal mucosa, and testis of male Wistar rats. There was a severalfold increase in the activity of monooxygenase in the liver, kidney, and lung, whereas virtually no effect could be detected in the intestine or testes. The proportion of 3- and 9-hydroxylation of the total hydroxylation of benzo(a)pyrene decreased in the liver, but increased in the kidney. TCDD had no significant effect on epoxide hydratase or glutathione S-transferase activities in any tissues. UDP glucuronosyltransferase exhibited a sevenfold increase in the liver, less than twofold in the kidney, and none in other tissues. Treatment of the microsomes with digitonin, trypsin, or phospholipase A did not reveal additional induction UDP glucurnosyltransferase, although all were able to increase measurable enzymatic activity in control and TCDD-treated animals. TCDD seems to be different from phenobarbital and polycyclic hydrocarbons as an effector of not only monooxygenase but also epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase.     

Induction of cytochrome P-450 in the rat liver after exposure to xylenes, dose-response relationship and dependence on endocrine factors

Male rats were exposed to different concentrations of xylenes for 3 days. Small but statistically significant increases in cytochrome P-450 content, NADPH-cytochrome c reductase activity and O-deethylation of 7-ethoxyresorufin in liver microsomes were detected already at an exposure level of 75 ppm. Morphological studies of livers from rats exposed to relatively high concentrations of xylenes for 5 days showed marked proliferation of smooth ER with little changes of the rough ER. No pathological alterations were observed. Castration of male rats influenced the response to xylene exposure only to a minor extent. Hypophysectomy alone was shown to cause significant increases in cytochrome P-450 and cytochrome b5 content and epoxide hydrolase activity. Induction of cytochrome P-450 dependent enzymatic activities after exposure to xylenes was reduced but qualitatively similar to that obtained with normal male rats whereas the induction of epoxide hydrolase activity was prevented. The difference in response to exposure to xylenes between male and female rats was mainly quantitative in nature. Polyacrylamide gel electrophoresis of liver microsomes from animals exposed to xylenes in the presence of SDS showed increases in protein bands comigrating with cytochrome P-450 PB-B2 and epoxide hydrolase purified from phenobarbital treated rats. It is concluded that in the rat exposure to xylenes mimics the effects of phenobarbital treatment as judged by both biochemical and morphological criteria and that the pituitary seems to have a regulatory function with regard to the induction of several drug-metabolizing enzymes.     

Activation of 6-aminochrysene to genotoxic products by different forms of rat liver cytochrome P450 in an O-acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

Metabolic activation of a potent mutagen, 6-aminochrysene, to genotoxic products in a newly developed tester strain, Salmonella typhimurium NM2009, was studied in a rat liver microsomal monooxygenase system containing cytochrome P450 (P450). Since the tester strain was constructed by introducing an O-acetyltransferase gene into the original strain S. typhimurium TA1535/pSK1002, it is highly sensitive toward the reactive metabolites of carcinogenic arylamines. DNA-damaging activities of 6-aminochrysene were detected at very low concentrations of substrate (between 0.01 and 0.2 microM) and liver microsomes (from 0.2 to 2 micrograms protein/mL) in the S. typhimurium NM2009 strain. Thus, the potency of genotoxic activities induced by 6-aminochrysene was about 10- to 20-times greater than those induced by the well-known mutagens 2-aminoanthracene and 2-amino-3,5-dimethylimidazo[4,5-f]quinoline. Liver microsomes isolated from rats treated with phenobarbital (PB) and a polychlorinated biphenyl mixture, Kanechlor 500, catalyzed very efficiently the activation of 6-aminochrysene to genotoxic metabolites. Treatment of rats with beta-naphthoflavone (BNF) and with dexamethasone also caused moderate induction of the microsomal activation of 6-aminochrysene. Studies employing immunoinhibition of microsomal catalytic activities and reconstitution with purified P450 enzymes suggested that the most important enzymes involved in the activation of 6-aminochrysene were P450 2B1 and 2B2; other enzymes including P450 1A1 and 1A2 participated to some extent. We also found that the microsomal activation of 6-aminochrysene was catalyzed more effectively in an acetyltransferase-overexpressing strain (NM2009) than in the original TA1535/pSK1002 strain and that these activities could be inhibited by an acetyltransferase inhibitor, pentachlorophenol, in liver microsomes from PB-treated rats, but not in those from BNF-treated rats. These results suggest that the P450/acetyltransferase system is one of the most important catalysts for the activation of 6-aminochrysene in liver microsomes of PB-treated rats, and that activation by BNF-induced P450 enzymes occurs by different mechanisms, probably through the ring oxidation pathway.     

The hepatotoxicity of 3-amino-1,2,4-triazole and carbon disulphide in phenobarbitone-treated starved rats

In phenobarbitone-treated starved male rats 1 g/kg 3-amino-1,2,4-triazole produced moderate liver necorsis and increased the serum glutamic-pyruvic transaminase activity. If half an hour after the administration of aminotriazole animals were exposed for 4 h to 2.0 mg/l CS2, the necrotic damage in the liver was larger and the serum glutamic-pyruvic transaminase activity higher than in rats not exposed to CS2. Carbon-disulphide in phenobarbitone-treated starved male rats caused only a very slight increase in the serum transminase activity in spite of the widespread hydropic degeneration in the liver. These experiments indicated that increase in serum transaminase activity is the consequence of necrosis and not hydropic degeneration; aminotriazole is hepatotoxic in rats when microsomal enzymes are induced and the hepatotoxicity of aminotriazole and carbon disulphide is potentiated by the administration of the other compound.     

Effects of alpha-carbon substituents on the N-demethylation of N-methyl-2-phenethylamines by rat liver microsomes

The effects of methyl, ethyl, isopropyl, isobutyl, and benzyl substituents at the alpha-carbon of N-methyl-2-phenethylamine on the kinetics of its N-demethylation in liver microsomes from both control and phenobarbital pretreated rats were studied. In control microsomes, the kinetic studies indicated that more than one enzyme was active for N-demethylation of N-methyl-alpha-methylphenethylamine (methamphetamine) while the other N-methyl-2-phenethylamines appeared to be demethylated by a single enzyme. In microsomes from phenobarbital pretreated animals, there appeared to be more than one enzyme system which was active for N-demethylation of all compounds except N-methyl-alpha-benzylphenethylamine. One of these had a much higher affinity for alpha-ethyl, isopropyl, and isobutyl N-methylphenethylamines while another exhibited affinities for substrates similar to the constitutive enzyme in control microsomes. A correlation was observed between the octanol-buffer or heptane-buffer distribution ratios of the compounds and the negative logarithm of the Michaelis constant (pKm) for the enzyme in control microsomes and for each of the enzyme systems in microsomes from phenobarbital-pretreated animals. Therefore, it is indicated that the concentration of a substrate at the active site of these microsomal enzymes is a function of its lipid solubility.     

The effects of cigarette smoke compared to 3-methylcholanthrene and phenobarbitone on alkoxyresorufin metabolism by lung and liver microsomes from rats

The rates of metabolism of phenoxazone and a homologous series of its ethers (alkoxyresorufins) by liver and lung microsomes of rats exposed to cigarette smoke were compared with the metabolism in rats pretreated with 3-methylcholanthrene (3MC) or phenobarbitone (PB). The rate of resorufin production was dependent on the length of the ether side chain. Liver and lung microsomes from control rats differed in their activity profiles (rate of resorufin production plotted against side-chain length), showing highest activity with ethoxy- and benzyloxyresorufin respectively. 3MC and PB selectively induced hepatic microsomal resorufin production with only certain of the substrates and the two agents differed in their selectivity, inducing most greatly with ethoxy- and benzyloxyresorufin respectively. Pulmonary microsomal resorufin production was induced by 3MC with a substrate selectivity similar to that shown for liver, but PB suppressed pulmonary metabolism with all the substrates. A single, short exposure to cigarette smoke induced ethoxyresorufin O-deethylase activity transiently in liver and lung microsomes. Three consecutive daily short exposures to cigarette smoke caused a weak 3MC-like induction of liver microsomal alkoxyresorufin metabolism, but the effect on lung microsomes was like weak 3MC and PB inductions combined. It is concluded that cigarette smoke induces selected cytochrome P-450-linked alkoxyresorufin O-dealkylase activities to a similar extent in both lung and liver and that the effects of cigarette smoke are characteristic of both 3MC-type and non-3MC-type inducers.     

Oxidation of aldose reductase inhibitors ALO-4114 and ALO-3152 catalyzed by liver microsomes.

Rat and Cynomolgus monkey liver microsomes catalyze the oxidation of 2.7-difluoro-4.5-dimethoxyspiro (9H-fluorene-9,4'-imidazolidine)-2',5'-dione (ALO-4114) to its monomethoxymetabolite (ALO-4417). Formation of this product by O-demethylation of ALO-4114 is catalyzed by NADPH and oxygen-dependent microsomal enzymes with the properties of P-450 monooxygenases. The reaction is blocked by inhibitors selective for these enzymes and activity increases about 2-fold in rats pretreated with phenobarbital or methylcholanthrene. The increase in the O-demethylation of ALO-4114 was, however, considerably less than the increase in benzphetamine N-demethylation or nitrophenetole O-deethylation activities in liver microsomes from rats pretreated with either phenobarbital or methylcholanthrene. Rats pretreated with 20 or 40 mg/kg of ALO-4114 for 3-4 days failed to change significantly the rate of ALO-4114 O-demethylase activity of liver microsomes. O-Demethylation of the achiral ALO-4114 yields the chiral ALO-4417. The enantiomers separated on a Daicel Chiracel AS column by HPLC indicated that O-demethylation of ALO-4114 by microsomes from untreated rats was only slightly stereoselective. However, rats pretreated with methylcholanthrene not only enhanced activity, but also increased the formation of one enantiomer. Further oxidative metabolism of the enantiomers was slow and barely detectable in vitro. Studies conducted with Cynomolgus monkey liver microsomes from one male and one female per experimental group were generally consistent with those from the rat, but some differences were noted. Whether the differences are real or only reflect individual variations caused by the small sample size is not known at present.     

The mechanism of the suicidal, reductive inactivation of microsomal cytochrome P-450 by carbon tetrachloride

1. Stoichiometric losses of microsomal haem and cytochrome P-450 were observed when carbon tetrachloride (CCl4) was incubated anaerobically with rat liver microsomes using NADPH or sodium dithionite as a reducing agent. A rapid destruction of haem was also observed during the non-enzymatic reductive incubation of CCl4 with soluble haem preparations (methaemalbumin) in presence of sodium dithionite. The results indicate that haem is both the site and the target of the suicidal activation of CCl4 by cytochrome P-450. 2. When an additional, fluorimetric assay for haem determination was used, an equimolar loss of protoporphyrin IX fluorescence was also observed in both the enzymatic and non-enzymatic system, indicating that the haem moiety of cytochrome P-450 has undergone a structural change, involving either loss or labilization of the porphyrin tetrapyrrolic structure. In both systems the loss of porphyrin was prevented by carbon monoxide (CO). 3. A dichlorocarbene-cytochrome P-450 ligand complex is partially responsible for the difference spectrum obtained on addition of CCl4 to anaerobically reduced rat liver microsomes. A molar extinction coefficient for this complex has been calculated. The carbene trapping agent 2,3-dimethyl-2-butene (DMB) strongly inhibited (greater than 95%) the formation of this spectrum but did not modify the loss of haem in reduced CCl4-supplemented microsomal incubations. The results suggest that dichlorocarbene (:CCl2) is not significantly involved in CCl4-dependent haem destruction. 4. Pretreatment of rats with different microsomal enzyme inducers was responsible for similar but not identical patterns of :CCl2 and CO formation and haem loss during incubation of CCl4 with reduced microsomes. This indicates a critical role of CCl4 metabolism in the suicidal destruction of cytochrome P-450 haem and suggests that the apoprotein of cytochrome P-450 is capable of modulating not only the metabolism of CCl4 to :CCl2 but also the hydrolysis of :CCl2 to CO. 5. Inactivation of cytochrome P-450 by CCl4 with reduced microsomes from Aroclor-pretreated rats was saturable and followed pseudo first-order kinetics. This provides further evidence to conclude that CCl4 activation is a suicidal process where the reactive metabolite(s) formed bind to haem, we predict, in a one to one stoichiometry. 6. The partition ratio between loss of cytochrome P-450 haem and CCl4 metabolism by liver microsomes from Aroclor pretreated rats has been investigated using limiting concentrations of CCl4. It was calculated that approximately 26 molecules of CCl4 had to be metabolised to achieve the loss of one molecule of haem.