Mixed function oxidases in kidney and duodenum of camel,guinea pig and rat

The activities of the drug-metabolizing enzymes, aniline 4-hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase have been measured in vitro in kidneys and duodenum of camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus). In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P(450), cytochrome b(5) and NADPH-cytochrome c reductase have also been determined. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. Rats showed the highest activity in NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethoxycoumarin O-deethylase among these species. However, guinea pigs showed the highest enzyme activity in cytochrome P(450), cytochrome b(5) and benzphetamine N-demethylase.     

Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream (Sparus aurata)

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2–3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M_r of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 × 10⁻⁶ M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 7-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.     

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.     

Effects of methylbenzenes on microsomal enzymes in rat liver,kidney and lung

The effects of pretreatment with toluene, o-, m-, p-xylene and mesitylene were investigated on the microsomal enzymes of liver, kidney and lung in rats. The activities of aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase, NADPH-cytochrome c reductase, as well as the concentrations of cytochrome P-450 and cytochrome b₅ were determined. The effects were most marked in the liver, where toluene caused increase in aniline hydroxylase and cytochrome P-450; o-xylene in aminopyrine N-demethylase and cytochrome b₅; m-xylene and mesitylene in all the enzymes investigated. In kidneys, all the compounds increased the activity of aniline hydroxylase; m-xylene induced cytochrome P-450 and b₅ as well as NADPH-cytochrome c reductase; p-xylene induced cytochrome P-450, and mesitylene cytochrome P-450 and b₅. Aminopyrine N-demethylase activity was decreased by toluene. In lungs, only mesitylene caused any significant differences from the controls: increase in aminopyrine N-demethylase and aryl hydrocarbon hydroxylase, decrease in aniline hydroxylase. The methylbenzenes tested induced the microsomal enzymes in a rough correlation to the number of their methyl groups and their hydrophobic properties.     

Genetic expression of aryl hydrocarbon hydroxylase induction. VI. Control of other aromatic hydrocarbon-inducible mono-oxygenase activities at or near the same genetic locus

When mice are administered aromatic hydrocarbons, the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase, p-nitroanisole O-demethylase, 7-ethoxycoumarin O-deethylase, and 3-methyl-4-methylaminoazobenzene N-demethylase activities—all membrane-bound mono-oxygenases having cytochrome P₄₅₀ associated with their active sites—is associated with the same genetic locus or with closely linked loci; we have previously proposed that this genetic region be designated the Ah locus for aromatic hydrocarbon responsiveness. Expression of these four inducible enzyme activities occurs as a single autosomal dominant trait in offspring from a genetic cross between inbred C57BL/6N and DBA/2N mice and from the appropriate backcrosses and intercross. There are no striking differences in relative thermolability or ontogenetic expression among these four closely linked aromatic hydrocarbon-induced mono-oxygenase activities. All four of these microsomal enzyme activities exist in two forms—one predominantly present in control or aromatic hydrocarbon-treated genetically nonresponsive mice and the other predominantly present in aromatic hydrocarbon-treated genetically responsive mice; the latter form is preferentially inhibited in vitro by such compounds as α-naphthoflavone. Whether a single induction-specific protein or a group of induction-specific proteins is associated with the Ah locus remains uncertain. The expression of aminopyrine N-demethylase, d-benzphetamine N-demethylase, NADPH-cytochrome c reductase, and NADPH-cytochrome P₄₅₀ reductase activities in aromatic hydrocarbon-treated genetically responsive and nonresponsive mice is not correlated with the Ah locus.     

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel

When Cd (3.58 mg CdCl₂·H₂O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphoneN-demethylase (EMND) (26%), and aminopyrineN-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b₅ (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomalp-nitroanisoleO-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl₂·6H₂O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%),p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b₅ (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b₅ (26%), and microsomal heme (30%) compared to those of controls. In the case ofp-NAOD activity, the influence was in favor of Ni, i.e, the inhibition was about 61% by the combined treatment. These results reveal that:

Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Xenobiotic metabolizing enzymes in rainbow trout exposed to river water

• 1.1. Juvenile rainbow trout were exposed to river water in a flow-through system. After 15 days of exposure, hepatic biotransformation activities and related parameters were measured and compared to those of the control group organisms that were maintained in tap water under identical experimental conditions.
• 2.2. Liver somatic index (LSI), microsomal protein and cytochrome P-450 contents, benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD) and UDP glucuronyl transferase activities were not significantly affected.
• 3.3. Aminopyrine-N-demethylase (APD) activity showed a slight yet significant increase in exposed trout.

     

Differential control of adrenal drug and steroid metabolism in the guinea pig.

Studies were carried out to compare the effects of several physiological variables on adrenal microsomal drug (ethylmorphine demethylation) and steroid (21-hydroxylation) metabolism in guinea pigs. The rate of adrenal ethylmorphine (EM) metabolism increased with maturation in males but not females, resulting in a sex difference (M > F) in adrenal enzyme activity in adult guinea pigs. Twenty-one hydroxylase activity, in contrast, was similar in adrenals from males and females. The concentration of adrenal microsomal cytochrome P-450 was unaffected by age or sex. ACTH administration decreased adrenal EM demethylase activity but did not affect 21-hydroxylation. Testosterone, when given to female guinea pigs, increased the rate of EM metabolism and decreased 21-hydroxylase activity. Various compounds known to interact with adrenal microsomal cytochrome P-450 had divergent effects on EM metabolism and 21-hydroxylation $\text{in}$$\text{vitro}$. Prostaglandins E₁ and F_2α, spironolactone, and canrenone inhibited EM demethylation but not 21-hydroxylation. Simple aromatic hydrocarbons (benzene, toluene), in contrast, inhibited 21-hydroxylation but did not affect EM metabolism. The results indicate that adrenal drug and steroid metabolism are independently regulated and that different terminal oxidases (cytochrome P-450) are probably involved in adrenal 21-hydroxylation and EM demethylation.     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Microsomal marker enzymes of Manduca sexta (L) midgut

The subcellular distribution of four enzymes (glucose-6-phosphatase, phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase) in the midgut of “wandering” fifth-instar larvae of the tobacco hornworm, Manduca sexta (L), was determined and the composition of mitochondrial and microsomal pellets was examined by electron microscopy. Most of the glucose-6-phosphatase activity and one-third of the phosphodiesterase I activity were found in the high-speed supernatant. NADPH-cytochrome c reductase activity was marginal and O-demethylase activity was undetectable in the supernatant. The highest specific activities for phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase were measured in microsomes, but the relative specific activity of phosphodiesterase I was only half that obtained with the latter two enzymes. In all subcellular preparations the relative specific activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase were closely correlated. It is concluded that glucose-6-phosphatase and phosphodiesterase I are not microsomal marker enzymes in the midgut, but the activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase are quantitative measures of microsomal content.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.     

Drug-metabolizing enzyme activities in freshly isolated oval cells and in an established oval cell line from carcinogen-fed rats

The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and alsoin vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrineN-demethylase and ethoxyresorufinO-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal expoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication ofDL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.Abbreviations CDE choline-deficient/DL-ethionine-supplemented diet - GST glutathione transferase - mEH microsomal epoxide hydrolase - UGT UDP-glucuronosyltransferase     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37°C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Cryopreservation and long-term storage of primary rat hepatocytes: Effects on substrate-specific cytochrome P450-dependent activities and unscheduled DNA synthesis

The effects of cryopreservation and long-term storage on substrate-specific cytochrome P45O-dependent activities and unscheduled DNA synthesis were studied in freshly isolated and cryopreserved hepatocytes derived from adult male Fischer 344 and Sprague-Dawley rats. Primary rat hepatocytes were isolated via an in situ collagenase perfusion technique, cryopreserved at –196°C, and thawed at 5 weeks and 104 and 156 weeks post-freezing. In Fischer 344 and Sprague-Dawley rats, cryopreserved hepatocytes were equivalent or similar to freshly isolated hepatocytes in substrate-specific activities for 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase and unscheduled DNA synthesis responses. No significant differences in activities toward 7-ethoxyresorufin-0-deethylase and dimethylnitrosamine-N-demethylase, the substrate-specific activities for cytochromes P4501A1 and P4501A2 and cytochrome P4502E1, respectively, were observed between freshly isolated and cryopreserved hepatocytes. Similar unscheduled DNA synthesis responses, a measure of DNA damage and repair, were observed after exposure to the genotoxic carcinogens 2-acetylaminofluorene, 7,12-dimethyEbenz[a]anthracene, and dimethylnitrosamine; although some decreases were also observed in Fischer 344 hepatocytes after 104 weeks and Sprague-Dawley hepatocytes after 156 weeks in the highest concentrations tested. These results suggest that cryopreserved hepatocytes, stored for extended periods of time in liquid nitrogen, are metabolically equivalent to freshly isolated hepatocytes in their ability to activate precarcinogens.Abbreviations 2-AAF 2-acetylaminofluorene - DDH₂O distilled deionized water - DMBA 7,12-dimethyIbenz[a]anthracene - DMN dimethylnitrosamine - DMNA dimethylnitrosamine-N-demethylase - DMSO dimethyl sulfoxide - EROD 7-ethoxyresorufin-O-deethylase - F344 Fischer 344 - FBS fetal bovine serum - %IR percentage of cells in repair - LN₂ liquid nitrogen - LSD least significant difference - CG cytoplasmic grains - NNG net nuclear grains - SD Sprague-Dawley - UDS unscheduled DNA synthesis - WE Williams' Medium E     

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b₅ from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol (α,α-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H₂¹⁸O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

The drug and carcinogen metabolism system of rat colon microsomes

The hydroxylation of N- and O-methyl drugs and polycyclic hydrocarbons has been demonstrated in microsomes prepared from colon mucosal cells. The hydroxylation of the drugs benzphetamine, ethylmorphine, p-nitroanisole, and p-nitrophenetole by colon microsomes is inducible two- to fourfold by pretreatment with phenobarbital/hydrocortisone. Colon microsomal benzo[α]pyrene hydroxylation is inducible 35-fold by pretreatment with β-naphthoflavone. Phenobarbital/hydrocortisone pretreatment also induces a fourfold increase in the specific content of colon microsomal cytochrome P-450, while β-naphthoflavone pretreatment causes a shift in the reduced CO difference spectrum peak to 448 nm and an eightfold increase in the specific content of this cytochrome. SKF 525-A inhibits the hydroxylation of the drug benzphetamine by colon microsomes or liver microsomes by 77% at a concentration of 2.0 mm. 7,8-Benzoflavone, on the other hand, inhibits the hydroxylation of the polycyclic hydrocarbon benzo[α]pyrene by colon microsomes by 76% and by liver microsomes by 44% at a concentration of 10 μm. Carbon monoxide, an inhibitor of oxygen interaction with cytochromes P-450 and P-448, inhibits benzphetamine hydroxylation and benzpyrene hydroxylation by colon microsomes 30 and 51%, respectively, at an oxygen to carbon monoxide ratio of 1:10. The K_m values of colon microsomal cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, dichloroindophenol, and ferricyanide (10–77 μm) are in agreement with those for purified rat liver cytochrome P-450 reductase. These data support the conclusions that hydroxylation of drugs and polycyclic hydrocarbons is catalyzed by colon mucosal microsomes and that the hydroxylation activity is attributable to a cytochrome P-450-dependent drug metabolism system similar to that found in liver microsomes.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.