Microsomal Monooxygenase Research Articles

Hepatic P450 monooxygenase response in rainbow trout (Oncorhynchus mykiss (Walbaum)) administered aquaculture antibiotics

The effects of the 4-quinolones, oxolinic acid and flumequine on hepatic microsomal cytochrome P450 monooxygenases in rainbow trout were examined during this study. Following antibiotic administration in the diet for 10 days at a representative commercial medicated feed concentration, fish were killed for assay at various periods up to 12 days following a return to normal diet. Ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were significantly elevated as a result of antibiotic treatment. The effects of oxolinic acid were delayed and longer-lasting compared with flumequine. The effects of flumequine were detectable 4 days earlier than those of oxolinic acid and lasted for less than ten days after treatment cessation. In contrast, oxolinic acid effects were apparent even on day 12 of the recovery period. The results of O-dealkylation, isoform-selective inhibition, and immunoblotting showed that the effects of both oxolinic acid and flumequine were related to P4501A subfamily. P450-binding spectra and experiments in vitro suggested that both antibiotics are weak dose-independent inhibitors of P450 activity, with flumequine exhibiting slightly higher binding affinity and inhibitory activity.

In vitro metabolism of pyriproxyfen by microsomes from susceptible and resistant housefly larvae.

Levels of cytochrome P450 and b5 were investigated in microsomal enzymes of houseflies from the gut and fat body of the third instar larvae of a pyriproxyfen-resistant strain (YPPF) and two pyriproxyfen-susceptible strains (YS and SRS). In comparison to the YS and SRS strains, YPPF microsomes had higher levels of total cytochrome P450s in both the gut and fat body. Furthermore, microsomes from the gut and fat body of YPPF larvae were found to have a much greater ability to hydroxylate aniline than YS larvae. In vitro metabolism studies of pyriproxyfen indicated that the metabolic rates were much higher in both the gut and fat body of YPPF larvae than of YS and SRS larvae. The major metabolites of pyriproxyfen in houseflies were identified to be 4'-OH-pyriproxyfen and 5"-OH-pyriproxyfen. Cytochrome P450 inhibitors, piperonyl butoxide (PB) and 2-propynyl 2,3,6-trichlorophenyl ether (PTPE), decreased the metabolic rates significantly in all three strains. This study confirmed that microsomal cytochrome P450 monooxygenases play an important role in the pyriproxyfen resistance of the housefly. Furthermore, it suggests that the fat body must be as important as the gut for the metabolism of pyriproxyfen in resistant housefly larvae.

Changes in hepatic cytochrome P450 enzymes by cis- and trans-1,2-dichloroethylenes in rat

1. We examined the effect of cis- and trans-1,2-dichloroethylen es (DCEs) on hepatic microsomalcytochrome P450 (P450) enzymes in the male and female rat. Rats were treated intraperitoneal ly with 7 5 mmol kg cis- or trans-1,2-DCE daily for 4 days. 2. Among the hepatic microsomal P450-dependent monooxygenase activities tested, testosterone 2α-hydroxylase (T2AH) activity in the male rat, which is associated with CYP2C11, was decreased by both cis- and trans-1,2-DCE isomers. The levels to control activities were 53 and 63 % respectively. Furthermore, immunoblottingshowed that both isomers significantly reduced the CYP2C11 6 protein level in liver microsomes from the male. The levels of testosterone 6β-hydroxylase (T6BH) activity and CYP3A2 1 protein in the male rat were reduced by cis-1,2-DCE but not trans-1,2-DCE. 3. cis-1,2-DCE decreased ethoxycoumarin O-deethylase (ECOD), benzyloxyresorufin O-debenzylase (BROD), chlorzoxazone 6-hydroxylase (CZ6H) and testosterone 7α-hydroxylase (T7AH) activities in the male rat by 29, 28, 34 and 27 % respectively. On the other hand, trans-1,2-DCE significantly increased 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) activities in the male rat 1.4- and 1.9-fold respectively. Immunoblottingshowed that cis-1,2-DCE reduced CYP1A1 2 and CYP2B1/2 protein levels, whereas trans-1,2-DCE increased CYP1A1/2 and CYP2B1/2 protein levels in the male rat. 4. The activities of other P450-dependent monooxygenases, namely benzphetamine N-demethylase (BZND), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND) and lauric acid ω-hydroxylase (LAOH), in the male rat were hardly affected by either 1,2-DCE isomer. In the female rat there were no apparent changes in P450-dependent monooxygenase activities upon treatment with the 1,2-DCE isomers. 5. These results suggest that 1,2-DCEs mainly affect male-specific P450 isoforms in the rat liver and that these changes may relate to the toxicity of 1,2-DCEs.

Effect of licorice and glycyrrhizin on murine liver CYP-dependent monooxygenases

This study is aimed to investigate the effect of the prolonged intake of conspicuous amounts of licorice (LE), or its natural constituent glycyrrhizin (G) on murine liver CYP-catalyzed drug metabolism. For this purpose the modulation of the regio- and stereo-selective hydroxylation of testosterone, together with the use of highly specific substrates as probes for different CYP isoforms such as ethoxyresorufm (CYP1A1), methoxyresorufin (1A2), pentoxyresorufin (2B1), p-nitrophenol (2E1) and aminopyrine (3A), were investigated. Daily doses of licorice root extract (3,138 or 6,276 mg kg b.w. Per os), or G (240 or 480 mg kg b.w. Per os), were administered to different groups of Swiss Albino CD1 mice of both sexes for 1, 4 or 10 consecutive days. While a single LE or G dose was unable to affect the multienzymatic CYP-system, using both schedules of repeated treatment, either LE or G were able to significantly induce hepatic CYP3A- and, to a lesser extent, 2B1- and 1A2-dependent microsomal monooxygenase activities, as well as 6β- (mainly associated to CYP3A), 2α-, 6α- (CYP2A1, 2B1), 7α-, 16α- (CYP2B9) and 16β-testosterone hydroxylase (TH) activities in male and female mice. Data on CYP3A modulation, the major isoform present in human liver, was confirmed by using Western immunoblotting with anti-CYP3A1/2 rabbit polyclonal antibodies raised against purified rat CYP3A. Northern blotting analysis using CYP3A cDNA biotinylated probe showed that the expression of such isozyme is regulated at the mRNA level. These results suggest that the induction of cytochrome P450-dependent activities by the prolonged intake of high LE or G doses, may result in accelerated metabolism of coadministered drugs with important implications for their disposition. The adverse effects associated with CYP changes such as toxicity/cotoxicity and comutagenicity may also have clinical consequences.

Expression and Distribution of Cytochrome P450 Enzymes in Male Rat Kidney: Effects of Ethanol, Acetone and Dietary Conditions

Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.

The biochemical toxicology of 1,3-difluoro-2-propanol, the major ingredient of the pesticide gliftor: the potential of 4-methylpyrazole as an antidote.

Administration to rats of 1,3-difluoro-2-propanol (100 mg kg-1 body weight), the major ingredient of the pesticide gliftor, resulted in accumulation of citrate in the kidney after a 3 hour lag phase. 1,3-Difluoro-2-propanol was found to be metabolized to 1,3-difluoroacetone and ultimately to the aconitate hydratase inhibitor (-) erythrofluorocitrate and free fluoride. The conversion of 1,3-difluoro-2-propanol to 1,3-difluoroacetone was found to be catalyzed by an NAD(+)-dependent alcohol dehydrogenase, while the defluorination was attributed to microsomal monooxygenase activity induced by phenobarbitone and inhibited by piperonyl butoxide. 4-Methylpyrazole was found to inhibit both of these processes in vitro and when administered (90 mg kg-1 body weight) to rats, 2 hours prior to 1,3-difluoro-2-propanol, eliminated signs of poisoning, prevented (-) erythrofluorocitrate synthesis, and markedly decreased citrate and fluoride accumulation in vivo. 4-Methylpyrazole also appeared to diminish (-) erythrofluorocitrate synthesis from fluoroacetate in vivo, and this was attributed to its capacity to inhibit malate dehydrogenase activity. The antidotal potential of 4-methylpyrazole and the potential for 1,3-difluoro-2-propanol to replace fluoroacetate (compound 1080) as a vertebrate pesticide is discussed.

Suppression of microsomal cytochrome P450-dependent monooxygenases and mitochondrial oxidative phosphorylation by fullerenol, a polyhydroxylated fullerene C 60

The acute toxicity of fullerenol-1 was determined using mice pretreated intraperitoneally (i.p.) with polyhydroxylated C 60 derivatives. The LD 50 value of fullerenol-1 was estimated to be 1.2 g/kg. Pretreatments with 0.5 and 1.0 g/kg fullerenol-1 decreased cytochromes P450 and b5 contents, and NADPH-cytochrome P450 reductase, benzo[ a]pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. Pretreatments with 0.01 and 0.1 g/kg fullerenol-1 had no effect on these monooxygenases. Additions of fullerenol-1 to mouse liver microsomes suppressed monooxygenases activities toward benzo[ a]pyrene, 7-ethoxycoumarin, aniline, and srythromycin with IC 50 values of 42, 94, 102 and 349 μM, respectively. Fullereno1-1 exhibited noncompetitive and mixed-type of inhibition in benzo[ a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. Additions of fullereno1-1 to rat liver mitochondria resulted in a dose-dependent inhibition of ADP-induced uncoupling and markedly inhibited mitochondrial Mg 2+-ATPase activity with an IC 50 value of 7.1 μM. These results demonstrate that fullerenol-1 can suppress the levels of the microsomal enzymes in vivo and decrease the activities of P450-dependent monooxygenase and mitochondrial oxidative phosphorylation in vitro.

Purification and Characterization of 2-Hydroxybiphenyl 3-Monooxygenase, a Novel NADH-dependent, FAD-containing Aromatic Hydroxylase from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from a recombinant of Escherichia coli JM109, which efficiently expressed the hbpA gene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl, the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparent Km values (30 degrees C, pH 7.5) were 2.8 microM for 2-hydroxybiphenyl, 26.8 microM for NADH, and 29.2 microM for oxygen. The enzyme was inactivated by p-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the betaalphabeta-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.

Serum B esterases as a nondestructive biomarker in the lizard Gallotia galloti experimentally treated with parathion

AbstractLizards (Gallotia galloti) were given either single or consecutive acute oral treatments of the organophosphorus (OP) insecticide parathion in two different experiments. Brain, serum, and liver microsomal esterase activities and liver microsomal monooxygenase activities were measured 6 and 24 h after the single acute treatment at each of four different doses (Experiment 1) or periodically up to 72 d after a number of consecutive acute treatments at two different doses (Experiment 2). Inhibition of serum butyrylcholinesterase (BChE) and carboxylesterase activities was observed in all treatment groups after 24 h and in the groups treated with 2.5, 5, and 7.5 mg/kg of parathion 6 h after treatment. Brain acetylcholinesterase (AChE) was inhibited at all doses after 6 h but only at the highest dose after 24 h. Highly significant nonlinear correlations, based on a piecewise linear regression model, were obtained between brain AChE activity and serum esterase activities at two sampling times after the single acute treatment. Liver microsomal carboxylesterase was found to be induced at the lower doses 6 and 24 h after treatment. Liver microsomal monooxygenase activity was higher 6 h after treatment than at 24 h, but the difference was not statistically significant. In Experiment 2, serum esterase activities recovered exponentially over a period of weeks. An increase in the recovery time to normal esterase activity was observed after each consecutive acute treatment. Brain AChE activity was inhibited at the end of consecutive administrations of parathion at the higher dose, and liver microsomal monooxygenase activity was inhibited at both doses. Symptoms of poisoning were observed in lizards treated with the higher dose of parathion, but no mortality was recorded. Two main conclusions can be drawn: (1) serum esterase activities recovered extremely slowly after acute treatment with parathion and even more slowly after consecutive acute treatments, and (2) there was a nonlinear correlation between the nondestructive biomarkers, serum “B” esterases, and the destructive biomarker, brain AChE, 6 and 24 h after exposure. These results suggest that G. galloti should be an ideal bioindicator organism to assess OP exposure in the Canary Islands instead of the birds commonly used for this purpose.

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.

Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans

Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be ∼5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher K m with recombinant FMO3 than did methimazole (2 mM vs. 35 μM). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high K m substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli

Age-related effects of heat stress on protective enzymes for peroxides and microsomal monooxygenase in rat liver.

To evaluate the age-related response of essential cell functions against peroxidative damage in hyperthermia, we studied the biochemical response to heat stress in both young and aged rats. Passive hyperthermia was immediately observed in rats after exposure to hot environments. In aged rats, the rectal temperature maintained thermal homeostasis and increased to the same degree as in young rats. In these aged animals, the damage from heat stress was more serious than in young animals. In aged rats under normal environmental conditions, hepatic cytosolic glutathione peroxidase (GSH peroxidase) activities were markedly higher than those activities in younger rats. Hepatic cytosolic GSH peroxidase activities were induced by heat stress in young rats but were decreased by hot environments in aged rats. Hepatic catalase activities in young rats were not affected by hot environments, whereas in aged rats, hepatic catalase activities were seriously decreased. Catalase activities in the kidney of aged rats were also reduced by hot environments. Lipid peroxidation in the liver was markedly induced in both young and aged rats. Because the protective enzymes for oxygen radicals in aged rats were decreased by hot environments, lipid peroxidation in the liver was highly induced. In aged rats, lipid peroxidation in intracellular structures such as mitochondria and microsomes was also markedly induced by hot environments. In both young and aged rats, hyperthermia greatly increased the development of hypertrophy and vacuolated degeneration in hepatic cells. In aged rats, both mitochondria and endoplasmic reticulum of the hepatic cells showed serious distortion in shape as a result of exposures to hot environments. Microsomal electron transport systems, such as cytochrome P450 monooxygenase activities, were seriously decreased by heat stress in aged rats but not in young rats. Although the mitochondrial electron transport systems were not affected by acute heat stress in young rats, their activities were simultaneously inhibited after long-lasting heat exposure. In isolated hepatic cells and polymorphonuclear leukocytes in animals, the 70-kDa heat shock-induced proteins were markedly increased by heat stress. In conclusion, the heat stress-inducible oxygen radical damage becomes more severe according to the age of rats. Because aging and hyperthermia have a synergistic effect on lipid peroxidation, protective enzyme activities for oxygen radicals may be essential for surviving and recovering from thermal injury in aged animals and also in humans.

Age-Related Effects of Heat Stress on Protective Enzymes for Peroxides and Microsomal Monooxygenase in Rat Liver

Age-Related Effects of Heat Stress on Protective Enzymes for Peroxides and Microsomal Monooxygenase in Rat Liver

Functional interactions between synthetic alkyl phospholipids and the ABC transporters P-glycoprotein, Ste-6, MRP, and Pgh 1.

The ABC superfamily of transporters includes the mammalian P-glycoprotein family (Class I and Class II P-gps), the multidrug resistance-associated protein (MRP), the Pgh-1 product of Plasmodium falciparum gene pfmdr1, all of which are associated with cellular pleiotropic drug resistance phenomena. STE6, the yeast transporter for the farnesylated peptide pheromone a, is also a member of this family. Structural similarities in this family translate into functional homology as expression of mouse Mdr3S (P-gp), P. falciparum Pgh-1, and human MRP partially restore mating in a sterile yeast mutant lacking a functional STE6 gene. The demonstration that Class II P-gps function as phosphatidylcholine (PC) translocators raise the possibility that other ABC transporters may also interact with physiological lipids. We report the identification of the synthetic lipid and PC analog ET-18-OCH3 (edelfosine) as a substrate for not only Class II P-gp but also for Class I P-gps and surprisingly for the other ABC transporters MRP, Pgh-1, and STE6. Expression of these proteins in the yeast Saccharomyces cerevisiae JPY201 was found to confer cellular resistance to cytotoxic concentrations of this lipid by a factor of 4-20-fold in a growth inhibition assay. The noted activity of ABC transporters toward this synthetic lipid was specific as a mutant variant of Mdr3 (Mdr3F) with reduced activity could not convey cellular resistance to ET-18-OCH3. ET-18-OCH3 was also found capable of blocking a-peptide pheromone transport and STE6 complementation by these ABC proteins. The inhibitory effect of ET-18-OCH3 on cell growth and a-factor transport could be abrogated by incubation with the lipid acceptor protein BSA or by enzymatic cleavage by microsomal alkylglycerol mono-oxygenase (MAMO). MAMO and BSA reversal of the ether lipid effect was only seen in the presence of a functional transporter. These results suggest that the group of cytotoxic synthetic PC analogs studied reveal possible structural and functional aspects common to the ABC transporters tested. Furthermore, the studies with BSA and MAMO suggest that the mechanism of transport of ET-18-OCH3 by these ABC transporters may be related to the flippase mechanism of PC transport by Mdr2.

Status of Insecticide Resistance inSpodoptera liturain Andhra Pradesh, India

Twenty-two strains of the tobacco caterpillar, Spodoptera litura (F.) (Lepidoptera: Noctuidae), collected from groundnut crops of eight locations in Andhra Pradesh, India, between 1991 and 1996 were assayed in the F1 generation for resistance to commonly used insecticides. Resistance levels ranged as follows: cypermethrin, 0·2- to 197-fold; fenvalerate, 8- to 121-fold; endosulfan, 1-to 13-fold; quinalphos, 1- to 29-fold; monocrotophos, 2- to 362-fold and methomyl, 0·7- to 19-fold. In nearly all strains pre-treatment with the metabolic inhibitor, piperonyl butoxide, resulted in complete suppression of cypermethrin resistance (2- to 121-fold synergism), indicating that enhanced detoxification by microsomal P450-dependent monooxygenases was probably the major mechanism of pyrethroid resistance. Pre-treatment with the synergist DEF, an inhibitor of esterases and the glutathione S-transferase system, resulted in a 2- to 3-fold synergism with monocrotophos indicating that esterases and possibly glutathione S-transferases were at least to some extent contributing to organophosphate resistance.

Alteration of Cytochrome P-450 Isozymes by Captopril and Idrapril in Hepatic and Renal Microsomes of Normotensive and Spontaneously Hypertensive Rats

To examine the effects of angiotensin-converting enzyme (ACE)inhibitors such as captopril and idrapril on the P-450 system, these compounds were administered 100 mg/kg i.p. for 4 days to spontaneously hypertensive (SHRs) and normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats; thereafter, the principal hepatic and renal microsomal monooxygenase activities were determined. In all the rat strains used, both captopril and idrapril decreased only the P-450 2C11, (as determined by immunoblotting) and its linked activities such as 16alpha-, 2alpha- and 17-testosterone hydroxylases. These changes were accompanied by a significant decrease of blood testosterone levels both in normotensive and, more markedly, in hypertensive rats and by a reduction of systolic blood pressure, but only in SHRs. Only in SHRs as well, the renal immunodetectable P-450 4A content and the P-450 4A-dependent activities, such as the (omega)-lauric acid hydroxylase, diminished after captopril or idrapril treatment. These data suggest that the decrease of increased blood pressure in hypertensive SHRs by the ACE inhibitors may be linked to the downregulation of the circulating testosterone level, the renal P-450 4A expression, and the related formation of the potent vasoconstrictor (omega)-hydroxy arachidonic acid.

Reduction of oxysterol levels up-regulates HMG-CoA reductase activity in rat liver

Cholesterol regulates hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity by feedback inhibition. It has been suggested that oxidized derivatives of cholesterol (oxysterols) play an important role, as an intracellular mediator, in the feedback inhibition of cholesterol biosynthesis. We, therefore, investigated the role of intracellular oxysterols in the regulation of HMG-CoA reductase activity. Rats were fed with food (control), cholesterol, clofibrate as a potentiator of the microsomal monooxygenase cytochrome P-450 enzyme system, ketoconazole as a strong inhibitor of the system, or butylated hydroxytoluene (BHT) as an antioxidant. We analyzed and compared hepatic microsomal oxysterol levels among the groups. The results of this study indicated that the oxysterol level, especially 7 β-hydroxycholesterol and 7-ketocholestrol, in the liver was lowered by the administration of ketoconazole and BHT, and HMG-CoA reductase activity was increased in response to these agents. However, there was no change in the HMG-CoA reductase activity, after the administration of clofibrate. We conclude that reduced levels of oxysterol may release the inhibitory effect on the HMG-CoA reductase enzyme and lead to up-regulation of the enzyme.

Interaction of tributyl- and triphenyltin with the microsomal monooxygenase system of molluscs and fish from the Western Mediterranean

The in vitro interaction of tributyltin (TBT) and triphenyltin (TPhT) with the microsomal cytochrome P450 monooxygenase system of different marine organisms has been evaluated. The selected organisms were two bivalve species, the mussel Mytilus galloprovincialis and the clam Tapes decussata, and the gastropod Thais haemastoma. The fish Mullus barbatus was also studied and the results compared with those obtained for invertebrates. The microsomal fraction isolated from the hepatopancreas or liver of these organisms was incubated in the presence of different concentrations of TBT and TPhT and the interaction with cytochrome P450, cytochrome b 5, NAD(P)H cytochrome c reductases and 7-ethoxyresorufin O-deethylase (EROD) activity evaluated. Direct effects on spectrally determined P450 and b 5 were only studied in mussels and detected at very high concentrations of TBT or TPhT (1 mM). In general, TBT and TPhT differed in their specificity. TBT led to inhibition of both NADH and NADPH cytochrome c reductase activity in the studied molluscs, whereas TPhT showed little effect. On the contrary, no significant effects on fish hepatic NAD(P)H cytochrome c reductases were detected. EROD activity, however, was significantly inhibited by both TBT and TPhT in Mullus barbatus at a concentration as low as 5–10 μM. The study shows speciesrelated differences and selective effects of TBT and TPhT on different components of the microsomal monooxygenase system.

Plant Monooxygenases: Participation in Xenobiotic Oxidation

The induction of individual components of the plant microsomal monooxygenase system have been studied. Quantitative distribution of NADPH reducible equivalents at monooxygenase and oxidase reactions and rereduction of coenzyme have been exhibited when there is deficiency of oxygen. Xenobiotic oxidation specificity in NADPH and NADH synergism with NADPH have been detected. The dependence of hydroxilation rate on the degree of hydrophobicity of xenobiotic molecules has been established. The possibility of cytochrome P-450 switching over from biosynthetic into detoxication pathway and transformation of monooxigenase mechanism of oxidation into peroxidase according to plant age have been demonstrated.

Effect of gossypol on hepatic and serum gamma-glutamyltransferase activity in rats.

A single intraperitoneal dose (25 mg/kg) of gossypol given to male Sprague-Dawley rats caused marked changes in the activity of the hepatic and serum gamma-glutamyltransferase (GGT) and microsomal monooxygenases. The GGT activity in liver homogenate, S-9 supernatant fraction and microsomes was significantly depressed; however, the level of serum GGT was elevated. While the hepatic glutathione concentration was not greatly changed, the aminopyrine N-demethylase activity and microsomal cytochrome P450 content of the liver were significantly decreased in the treated rats. At necropsy, the livers of the treated rats appeared generally pale with distinct pinpoint foci. Histopathological examination of the liver showed degenerative changes and coagulative necrosis. The results indicate that gossypol is a strong hepatotoxic agent which can produce severe hepatic damage.