Aniline Hydroxylation Research Articles

Ketone potentiation of haloalkane-induced hepato- and nephrotoxicity. II. Implication of monooxygenases.

Previous results in Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatment (3 d, po) at dosages of 6.8 and 13.6 mmol/kg potentiate CCl4 hepatotoxicity and CHCl3 nephrotoxicity, respectively. The potentiation potency profile observed was MiBK > A > MEK for liver and A > MEK > or = MiBK for kidney toxicity (Raymond & Plaa, 1995). In the present study, hepatic and renal microsomes from A-, MEK-, and MiBK-pretreated rats (6.8 or 13.6 mmol/kg) were examined for cytochrome P-450 content, substrate-specific monooxygenase activity (aminopyrine and benzphetamine N-demethylase, aniline hydroxylase) and in vitro covalent binding of 14CHCl3 and 14CCl4. Of the three ketones, only MiBK significantly increased P-450 content of liver and renal cortical microsomes. Similarly, 14CCl4 covalent binding under aerobic and anaerobic conditions was significantly increased by MiBK pretreatment only. 14CHCl3 covalent binding by renal cortical microsomes was significantly increased only under aerobic conditions by MiBK pretreatment. MiBK (13.6 mmol/kg) increased (threefold) aminopyrine N-demethylation in both liver and kidney, but only benzphetamine N-demethylation (two-fold, at 6.8 and 13.6 mmol/kg) in liver; A and MEK had no effect on either monooxygenase. All ketones at dosages of 6.8 and 13.6 mmol/kg increased aniline hydroxylation in liver (two-fold) and kidney (fivefold). Comparable profiles for P-450 induction, haloalkane covalent binding, and aminopyrine or benzphetamine N-demethylase activity were observed in liver and kidney microsomes. This profile was consistent with the ketone potentiation potency ranking profile observed in vivo for liver but not kidney injury. These findings affirm the importance of ketone-enhanced bioactivation for potentiation of CCl4 hepatotoxicity but suggest an alternative mechanism for CHCl3 nephrotoxicity.

Difference between in vivo and in vitro effects of propofol on defluorination and metabolic activities of hamster hepatic cytochrome P450-dependent mono-oxygenases

We have compared the in vivo and in vitro effects of propofol on cytochrome P450-dependent monooxygenase activities in hamster liver microsomes. Propofol (Diprivan) 10 mg/100 g body weight was injected i.p. twice a day for 2 weeks to induce cytochrome P450 enzymes. Liver microsomes were prepared by differential centrifugation. Metabolism of the cytochrome P450-dependent mono-oxygenase system was evaluated by measuring aniline hydroxylation, benzphetamine demethylation and benzo(a)pyrene hydroxylation. Defluorination of enflurane was assayed by detecting free fluoride metabolites. At similar concentrations as in the in vivo group, propofol in vitro exhibited concentration-dependent inhibition of metabolism of benzphetamine and benzo(a)pyrene. Aniline hydroxylation and defluorination of enflurane were inhibited to 78% of control with propofol 0.25 mmol litre-1. In propofol-treated hamsters, there was only minimal inhibitory or inductive effects on either mono-oxygenase activities or capacity for defluorination. This difference between the in vitro and in vivo effects of propofol on cytochrome P450 mono-oxygenase activities emphasizes the need for care when comparing in vitro and clinical data.

Effects of flavonoids on cytochrome P‐450 from rat liver microsomes: Inhibition of enzyme activities and protection against peroxidative damage

AbstractA series of flavonoids, able to inhibit non‐enzymic and enzymic lipid peroxidation, were investigated as inhibitors of cytochrome P‐450 mediated reactions (aminopyrine N‐demethylation and aniline hydroxylation) in rat liver microsomes. The most potent compounds were chrysin, luteolin, amentoflavone and eriodictyol (aminopyrine N‐demethylation) and apigenin, kaempferol and quercetin (aniline hydroxylation). Some structure‐activity relationships were established for inhibition of aminopyrine N‐demethylation and it was observed that the resorcinol configuration of the ring A is a determinant feature, whereas O‐glycosylation abolished the activity. Most of the compounds showed lower activity on aniline hydroxylation. The presence of these flavonoids during incubation of microsomes in a medium containing NADPH did not modify the cytochrome P‐450 concentration and protected against lipid peroxidation induced degradation of this cytochrome.

Human cytochrome P450 mono-oxygenase system is suppressed by propofol

We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward specific cytochrome P450 isoform test substrates, aniline, 7-ethoxycoumarin, benzphetamine and benzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylation to a greater extent than the oxidative metabolism of the other test substrates, even at 0.05 mmol litre-1. The degrees of inhibition of benzphetamine N-demethylation and 7-ethoxy-coumarin O-de-ethylation were similar, while aniline hydroxylation was least affected by propofol. Spectral analysis showed that propofol competed with carbon monoxide for binding to the haem moiety of haemoprotein in the P450 enzyme. The variable inhibition observed may be caused by the differential binding of propofol to P450 isoforms. Propofol 0.05-1.0 mmol litre-1 exhibited a concentration-dependent inhibitory effect on human cytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions of propofol on human liver microsomal enzymes in vitro suggest that potential drug interactions may exist between propofol and other drugs administered clinically.

Characterization of the independent and combined effects of two inhibitors on oxidative drug metabolism in rat liver microsomes

To evaluate how two inhibitors influence oxidative drug metabolism, this study investigated the inhibitory effects of mexiletine with cimetidine and mexiletine with lidocaine, both individually and in combination, on the oxidative metabolism of two probe substrates, aminopyrine and aniline in rat liver microsomes. Mexiletine was a competitive inhibitor of aminopyrine N-demethylation, whereas cimetidine was a mixed type of inhibitor ( K i = 2.00 ± 0.04 and 0.20 ± 0.02 mM, respectively). For aniline hydroxylation, mexiletine exhibited a mixed type of inhibition, whereas lidocaine was a noncompetitive inhibitor ( K i = 0.60 ± 0.07 and 8.50 ± 0.12 mM, respectively). The combined inhibition of either mexiletine with cimetidine or mexiletine with lidocaine on aminopyrine and aniline metabolism was close to the fully additive effects of the individual compounds when their individual concentrations were below a 2-fold K i concentration, regardless of the apparent kinetic inhibition type. The combined inhibition was less than fully additive when the individual concentrations were twice the K i or above. These results demonstrate that, when two inhibitors of oxidative drug metabolism are combined, both the k i values and the concentrations of inhibitors play important roles in determining the extent of additive inhibition of enzyme activity.

Effects of intra- and intermolecular crosslinking on the free radical reactions of bovine hemoglobins

Chemical modifications of human or bovine hemoglobins are designed to produce proteins that can act as oxygen-carrying blood substitutes. Concerns about the redox reactivity of cell-free hemoglobin and its contribution to tissue-damaging oxygen free radicals has not been fully established. We determined that bovine hemoglobins intra- or intermolecularly crosslinked differ in their ability to generate or interact with reactive oxygen species. These differences do not correlate with their oxygen affinities. We compared HbBv-FMDA, produced by the reaction of bovine hemoglobin with fumaryl-monodiaspirin and Poly HbBv, a glutaraldehyde polymerized bovine hemoglobin, with unmodified bovine hemoglobin (HbBv). Superoxide radicals are produced during the spontaneous oxidation of hemoglobin. Relative to the other two proteins, Poly HbBv was found to be more susceptible to autoxidation. Spectral changes indicative of protein modification and ferrylhemoglobin formation during the enzymatic peroxidation of these hemoglobins differ qualitatively and occur at an increasing order, poly HbBv > HbBv > HbBv-FMDA. The proteins also differ in the rate of hemoglobin catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. Taken together, our results and those reported previously on modified human hemoglobins, suggest that redox and oxygen-carrying functions of hemoglobin can be experimentally manipulated as independently selectable parameters that may ultimately aid in the design of a safer reperfusion agent.

Protective Effect of Rifampicin against Acute Liver Injury Induced by Carbon Tetrachloride in Mice

Rifampicin conferred significant protection against carbon tetrachloride (CCl4)-induced liver injury. Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were not markedly altered and only hepatocellular fatty degeneration was found in mice pretreated with rifampicin (200 mg/kg), whereas severe centrilobular necrosis was observed and serum ALT and AST activities were as high as 281 and 271 I.U./l, respectively, in the control group following administration of CCl4, (400 μl/kg). The contents and activities of microsomal drug-metabolizing enzymes in rifampicin-pretreated animals were also much higher than those of the controls. CCl4-mediated malondialdehyde (MDA) formation was increased in rifampicin-treated liver microsomes, demonstrating that rifampicin was capable of increasing the NADPH-dependent metabolism of CCl4 catalyzed by P-450 2E1 to produce free radicals. However, MDA formation was obviously depressed by rifampicin at varying concentrations from 2 to 32 × 10-6 M in an in vitro cytochrome P-450 (P-450) enzyme system. On the other hand, NADPH oxidation in the metabolism of CCl4, and aniline hydroxylation were not suppressed in the presence of rifampicin in this systems, suggesting that rifampicin did not influence the biotransformation of CCl4, by P-450 2E1 in vitro. Therefore, the protective effect of rifampicin against CCl4, hepatotoxicity appeared to result from the direct inhibition of lipid peroxidation generated by CCl4,-derived free radicals.

Effects of Apple Leaf Allelochemistry on Tufted Apple Bud Moth (Lepidoptera: Tortricidae) Resistance to Azinphosmethyl

The dihydrochalcone glycoside phloridzin is a dominant allelochemical in foliage of apple. This allelochemical varies within and among apple cultivars and can influence distribution and abundance of tufted apple bud moth, Platynota idaeusalis (Walker), in orchards. Laboratory bioassays were used to investigate the influence of phloridzin on tolerance of both susceptible and resistant colonies of P. idaeusalis to the organophosphate insecticide azinphosmethyI. Addition of phloridzin to diet did not influence tolerance of first ins tars of the susceptible strain to azinphosmethyI. Third instars of the susceptible strain exhibited reduced tolerance to azinphosmethyl in the presence of phloridzin, and this response was associated with inhibition of the activities of glutathione transferase, esterase, and aniline hydroxylation enzymes. Neonate larvae of the resistant strain fed on diet with phloridzin were more tolerant to azinphosmethyl at high insecticide concentrations only. Third instars of the resistant strain were equally tolerant to azinphosmethyl in the presence and absence of phloridzin. Although phloridzin inhibited glutathione transferase activity in resistant larvae, as in the susceptible strain, it increased activity of esterases. Our data provide further evidence that both host-plant allelochemistry and insect selection history influence results of pesticide bioassays.

Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO2. The distribution of radioactivity obtained after transmethylation of the adducts of 14CHCl3 intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to .CHCl2 radicals) increased with decreasing pO2. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl3 in the liver of Osborne-Mendel rats were not influenced by ethanol consumption.

Molecular orbital-based quantitative structure-activity relationship for the cytochrome P450-catalyzed 4-hydroxylation of halogenated anilines.

The cytochrome P450 (P450) catalyzed 4-hydroxylation of halogenated anilines was investigated with special emphasis on possible relationships between kinetic parameters and physicochemical and electronic characteristics of the substrates. The most important observation of the present study was a correlation (r = 0.96) between the natural logarithm of the apparent maximum reaction rate kcats for 4-hydroxylation of the aniline substrates in a iodosobenzene-supported microsomal cytochrome P450-catalyzed reaction and the energy of the highest molecular orbital [E(HOMO)] of the anilines. This result is in accordance with a mechanism that proceeds by an initial electrophilic attack of the P450 (FeO)3+ intermediate on the frontier pi electrons of the aniline substrates. In the iodosobenzene-supported aniline 4-hydroxylation this electrophilic attack is the rate-limiting step. In the NADPH/oxygen-supported cytochrome P450-catalyzed 4-hydroxylation of the anilines a correlation of the natural logarithm of kcats with E(HOMO) was not observed and the kcats values were lower than observed in the iodosobenzene-supported reaction. From this result it is concluded that, although the NADPH/oxygen-supported microsomal 4-hydroxylation of the halogenated anilines proceeds by the same cytochrome P450 (FeO)3+ intermediate and, thus, by a similar electrophilic attack of the (FeO)3+ on the pi electrons of the substrate, this attack is no longer the rate-limiting step of the reaction. Additional results of the present study demonstrate that the apparent Michaelis constant Kms of the NADPH/oxygen-supported 4-hydroxylation of the anilines decreases with increasing hydrophobicity of the aniline derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal changes in P-4502E1 expression with continued ethylbenzene exposure

The goal of this study was to examine the effect of duration of ethylbenzene exposure on cytochrome P-450-dependent activities. Male rats were treated with ethylbenzene by intraperitoneal injection for either 1 or 3 days, and microsomal preparations were examined for changes in the microsomal proteins and activities as well as the expression of specific P-450 isozymes. Two general pattems of induction were evident when different P-450-dependent activities were examined. (i) Cytochrome P-450 2B-dependent activities (e.g., p-nitroanisole demethylation, benzphetamine demethylation, and aromatic toluene hydroxylations) were induced both after 1 and 3 days of ethylbenzene exposure. (ii) Cytochrome P-450 2El-dependent activities (e.g., N, N-dimethylnitrosamine demethylation and aniline hydroxylation) were induced after treatment with ethylbenzene for one day; however, after 3 days of ethylbenzene treatment these activities returned to control levels. Changes in these activities were consistent with changes in the levels of specific P-450 isozymes as determined by immunoblotting. Cytochrome P-450 2B levels were increased and P-450 2Cll levels were suppressed at both 1 and 3 days of ethylbenzene exposure. A temporal response in P-450 2E1 expression was observed, with P-450 2E1 levels increasing after a single ethylbenzene injection and returning to controls after administration of the hydrocarbon for 3 days. Rats were also subjected to a pair-feeding regimen to determine whether these effects were related to altered dietary status in ethylbenzene-treated rats. Neither P-450-dependent activities nor immunoreactive protein levels were altered in pair-fed rats. These results demonstrate that prolonging the duration of hydrocarbon exposure can produce differential effects on the expression of P-450 2E1, with levels being elevated after acute hydrocarbon administration, but not after more prolonged hydrocarbon exposure.

Tiamulin selectively inhibits oxidative hepatic steroid and drug metabolism in vitro in the pig.

The simultaneous use of the antibiotic tiamulin with certain ionophoric antibiotics (monensin, salinomycin) may give rise to a toxic interaction in pigs and poultry. In the present study, effects of tiamulin on hepatic cytochrome P450 activities in vitro were studied using pig liver microsomes. When tiamulin was added to the incubation medium the N-demethylation rate of ethylmorphine and the hydroxylation of testosterone at the 6 beta- and 11 alpha-positions was strongly inhibited. Tiamulin inhibited these activities more than SKF525A or cimetidine, but less than ketoconazole. The microsomal N-demethylation rate of erythromycin and the hydroxylation of testosterone at the 2 beta-position were inhibited to a lesser degree, whereas the ethoxyresorufin-O-deethylation, aniline hydroxylation and testosterone hydroxylations at the 15 alpha- and 15 beta-positions were not affected by tiamulin. No in vitro complexation by tiamulin of cytochrome P450 resulting in a loss of CO-binding capacity could be demonstrated. Results from the present study suggest a selective inhibition of cytochrome P450 enzymes in pigs, probably belonging to the P4503A subfamily. The mechanism of this interaction is still unclear. However, interactions between tiamulin and those veterinary drugs or endogenous compounds which undergo oxidative metabolism by P450 enzymes must be considered. More research is needed to reveal which of the P450 enzymes are affected by tiamulin in order to improve the understanding and probably the predictability of this interaction.

Comparison of aniline hydroxylation by hemoglobin and microsomal cytochrome P450 using stable isotopes

Hemoglobin (Hb) and cytochrome P450 carry out aromatic ring hydroxylation of aniline. In the presence of reductants and Hb, para- and ortho-aminophenol were formed. Under [ 18O]O 2, 100% of product was labeled; no incorporation occurred with [ 18O]H 2O. Deuterium (1.9%) was detectable in p-aminophenol formed from p-[ 2 H] aniline by Hb, as compared with 6% retention observed with cytochrome P450. These observations are consistent with a mechanism for Hb-dependent reaction involving formation of an ironoxo complex competent to hydroxylate substrate. Hb-mediated reactions may represent a source of extrahepatic metabolism since Hb is a major carrier for small organic molecules. The similarities of P450- and Hb-mediated aniline hydroxylation using stable isotopes preclude their use as in vivo probes.

The role of radiation produced peroxides and superoxide in the radiation response of A549 cells

Rates of ethanol oxidation by perfused livers from fasted female rats were decreased from 82 ± 8 to 11 ± 7 μmol/g/hr by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. The subsequent addition of fatty acids of various chain lengths in the presence of 4-methylpyrazole increased rates of ethanol uptake markedly. Palmitate (1 mM) increased rates of ethanol oxidation to 95 ± 8 μmol/g/hr, while octanoate and oleate increased rates to 58 ± 11 and 68 ± 15 μmol/g/hr, respectively. Hexanoate, a short-chain fatty acid oxidized predominantly in the mitochondria, had no effect. Addition of oleate also increased the steady-state level of catalase-H2O2. Pretreatment of rats for 1.5 hours with 3-amino-1,2,4-triazole (1.0 g/kg), an inhibitor of catalase, prevented the ethanol-dependent decrease in the steady-state level of catalase-H2O2 completely. Under these conditions, aminotriazole decreased rates of ethanol oxidation by about 50% and blocked the stimulation of ethanol oxidation by fatty acids. Oleate decreased rates of aniline hydroxylation by about 50%, indicating that cytochrome P450 is not involved in the stimulation of ethanol uptake by fatty acids. Furthermore, oleate stimulated ethanol uptake in livers from ADH-negative deermice indicating that fatty acids do not simply displace 4-methylpyrazole from alcohol dehydrogenase. It is concluded that the stimulation of ethanol oxidation by fatty acids is due to increased H2O2 supplied by the peroxisomal β-oxidation of fatty acids for the catalase-H2O2 peroxidation pathway.

Changes in amounts of cytochrome P450 isozymes and levels of catalytic activities in hepatic and renal microsomes of rats with streptozocin-induced diabetes

Hepatic microsomal cytochrome P450s, which are involved in the metabolism of drugs, hormones, prostaglandins and fatty acids, change when animals develop diabetes. We studied changes in cytochrome P450 isozymes in both hepatic and renal microsomes of rats with diabetes caused by streptozocin, and compared the results with changes in catalytic activities in the microsomes. In hepatic microsomes of diabetic rats, the amount of cytochrome P450 2E1, an acetone-inducible isozyme, was two and a half times that of control rats, and that of P450 4A2, a major renal isozyme, was three times that in the controls. The amounts of cytochrome P450s 2A1, 2C6, 2C7, 3A2, and 4A3 increased in hepatic microsomes of diabetic rats, and P450 2C11 decreased. Treatment with insulin restored these to the levels in the controls. The catalytic activities of aniline hydroxylation, 7-ethoxycoumarin O-dealkylation, testosterone 2β, 6β, 7α, and 16β-hydroxylation, and ω-, (ω − 1)-hydroxylation of lauric acid were low. In renal microsomes of diabetic rats, and testosterone 2α and 16α-hydroxylation activities were low. In renal microsomes of diabetic rats, cytochrome P450s 2E1, 4A2 and K-4 were induced, and ω- and (ω − 1)-hydroxylation activities were high. These changes were reversed by insulin treatment. The induction and suppression of cytochrome P450 isozymes in diabetic rats were consistent with changes in the catalytic activities. In both hepatic and renal microsomes, P450s 2E1 and 4A2 were induced, altered metabolism of ketones and fatty acids in diabetes may contribute to these changes.

Induction and inhibition of cytochrome P-450-dependent monooxygenases in hamster tissues by ethanol

The effects of ethanol on hamster hepatic and extrahepatic monooxygenases were determined in the present study. Chronic ethanol administration increased cytochrome P-450 ( P-450) content and monooxygenase activities towards aniline, N-nitrosodimethylamine, and 7-ethoxyresorufin. In contrast, benzphetamine and benzo( a)pyrene oxidation rates were decreased 21–24% by ethanol. In kidney, ethanol pretreatment increased P-450 content, aniline and N-nitrosodimethylamine oxidation activities. In lung, ethanol ingestion selectively increased aniline hydroxylation without affecting other monooxygenase activities. Intestinal monooxygenase activity was refractory to ethanol induction. Immunoblotting of the microsomal proteins showed that ethanol induced a protein cross-reactive with rabbit antibody raised against human P-450 2E1 in hamster liver, kidney, and lung. Immunoblotting analysis using mouse monoclonal antibody 1-12-3 raised against scup P-450 1A1 revealed that ethanol induced an immunorelated protein in hamster liver, kidney, and lung. Induction of P-450 2E1 and 1A was not observed with intestinal protein blots. Immunoblotting analysis using mouse monoclonal antibody 2-66-3 against rat P-450 2B1 showed inhibition of an immunorelated protein in ethanol-treated hamster liver. The inhibitory effect on P-450 2B was not observed with extrahepatic tissues. These results suggest that ethanol has the ability to induce P-450s 2E1 and 1A and to inhibit P-450 2B in hamster tissues.

Selective para hydroxylation of phenol and aniline by singlet molecular oxygen.

Phenol reacts with singlet oxygen (1O2) generated in aqueous solution (H2O or D2O) by (a) the exposure of methylene blue to light or (b) the thermal dissociation of the endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate to lead selectively to hydroquinone as the primary product. The other isomers of phenol hydroxylation, catechol and resorcinol, were not observed. In agreement with the involvement of 1O2 as the reactive species in the hydroxylation, in D2O the yield of hydroquinone is 7 times that in H2O, and the 1O2 quenchers azide and the thiols, glutathione and dithiothreitol, suppress the production of hydroquinone. In contrast, the hydroxyl radical scavengers, tert-butyl alcohol, propanol, or sodium formate, are without effect. In a follow-up reaction, hydroquinone is converted into benzoquinone. Reaction of 1O2 with aniline leads to the selective formation of 4-hydroxyaniline as the initial product. This is further converted to hydroquinone with formation of ammonia (deamination), and then to benzoquinone. On the basis of these results, the selective para hydroxylation of phenol or aniline may be used as an indicator for the involvement of singlet oxygen as compared to .OH radical- or cytochrome P450-mediated reactions.

Picrotoxin as a potent inducer of rat hepatic cytochrome P450, CYP2b1 and CYP2b2

The induction by the central stimulant picrotoxin of hepatic drug-metabolizing enzymes was studied in rats. The hepatic content of P450 and the activity of benzphetamine N-demethylation increased gradually after administration of picrotoxin dissolved in drinking water (2 mg/mL), to three-times higher levels than the initial values at the third day of treatment. The increase in benzphetamine N-demethylase activity by picrotoxin was somewhat higher than the increase produced by phenobarbital. Supporting these results, immunoblot analysis showed that CYP2B1 and 2B2 proteins in the liver microsomes were increased by picrotoxin. Picrotoxinin and picrotin, which are components of the picrotoxin molecule, had the same ability to induce the hepatic activity of benzphetamine N-demethylation. The liver microsomal activities of testosterone 16β- and 16β-hydroxylation were enhanced significantly after treatment with picrotoxinin and picrotin. However, benzo[a]pyrene 3-hydroxylation, aniline 4-hydroxylation, and testosterone hydroxylations at the 2α- and 7α-positions were not increased by picrotoxinin and picrotin treatment. In addition to monooxygenase, significant induction of glutathione S-transferase activity for 1-chloro-2,4-dinitrobenzene and UDP-glucuronyltransferase activity for 4-hydroxybiphenyl and 4-nitrophenol was also observed by pretreatment of picrotoxin. These results clearly indicate that picrotoxin is an inducer of phenobarbital-inducible liver enzymes.

Induction of Cytochrome P450-Dependent Monooxygenases in Hamster Tissues by Fasting

The effects of fasting on liver, kidney, and lung monooxygenases were studied using hamsters starved for 4 days. Fasting treatment increased microsomal cytochrome P450 content and NADPH-cytochrome P450 reductase activity in kidney and lung. The treatment caused significant increases of aniline hydroxylation, N-nitrosodimethylamine demethylation, and 7-ethoxycoumarin 0-deethylation activities in the liver, kidney, and lung. Fasting caused a threefold increase of benzphetamine N-demethylation activity in lung and a 25% increase in liver and had no effect in kidney. Benzo[a]pyrene hydroxylation activities in the fasted hamster liver, kidney, and lung were higher, lower, and similar to the controls, respectively. Gel electrophoresis of tissue microsomes from control and fasted hamsters revealed that fasting enhanced the intensity of protein band(s) in the P450 molecular weight region. Immunoblotting of the microsomal proteins showed that fasting induced a protein cross-reactive with rabbit antibody raised against human P450 2E1 in hamster liver, kidney, and lung. Immunoblotting analysis using mouse monoclonal antibody 2-66-3 raised against rat P450 2B1 revealed that fasting induced an immunorelated protein preferentially in hamster lung, with minimal effects on liver and kidney. Protein blots probed with mouse monoclonal antibody 1-12-3 indicated that fasting induced a protein related to P450 1A1 in hamster liver, kidney, and lung. These results demonstrate that fasting causes a variety of inductive effects on the enzyme components and catalytic activities of monooxygenase systems as well as on the P450s 2E, 2B, and 1A in the hamster tissues.

Characterization of cytochrome P450 dependent mixed-function oxidase system of gilthead seabream ( sparus aurata; sparidae) liver

1. 1. The average cytochrome P450 content of gilthead seabeam liver microsomes was found to be 0.078 ± 0.012 ( n = 4) nmol/mg protein, which was about 50%, 24% and 13% of that measured in trout, sturgeon and scup liver microsomes, respectively. 2. 2. Gilthead seabream liver microsomes contained a relatively higher amount of cytochrome b5, as 0.32 ± 0.05 ( n = 4) nmol/mg protein. 3. 3. Effects of microsomal protein amount and temperature ranging from 15 to 30°C on aniline 4-hydroxylation and ethylmorphine N-demethylation activities were examined. No significant differences were observed between the enzyme activities at these temperatures. 4. 4. The liver microsomal aniline 4-hydroxylase activity of gilthead seabream in the 1990 fall (0.016–0.021 nmol p-aminophenol/min/mg protein) was 4.5–6 times higher than that in the 1991 spring (0.003–0.004 nmol p-aminophenol/min/mg protein). 5. 5. Liver microsomal ethylmorphine N-demethylase activity measured in 1991 spring (0/371) nm ol formaldehyde/min/mig protein) was 41% higher than that in 1990 fall (0.263 nmol formaldehyde/min/mg protein).