In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase

1. The specific activity of the aniline 4-hydroxylase which catalyses hydroxylation of aniline to p-aminophenol was found to be 0.65 (N = 10) and 0.15 (N = 13) nmol p-aminophenol formed/mg protein/min, in sheep liver and lung microsomes, respectively. 2. The effects of aniline concentration, pH, cofactors, amount of enzyme and incubation period, on enzyme activity were studied, and the optimum conditions for maximum activity of liver and lung microsomes were determined. 3. Liver and lung microsomal aniline 4-hydroxylase activity was found to be completely dependent on the presence of cofactor NADPH. 4. The Lineweaver Burk and Eadie Hofstee plots of the liver enzyme were found to be curvilinear, suggesting that the enzyme did not follow the Michaelis Menten kinetics. From these graphs, two different Km values were calculated for the liver enzyme as 3.21 and 0.072 mM aniline. Km of the lung enzyme was calculated to be 1.43 mM aniline from its Lineweaver Burk graph. 5. The effects of magnesium, nickel and cadmium ions on the liver and lung aniline 4-hydroxylase activity were examined. Magnesium ion was found to have stimulatory effect, whereas nickel and cadmium ions inhibited the activity of the both liver and lung enzyme.     

Effect of cadmium on pulmonary and renal microsomal drug metabolizing enzymes of the guinea-pig

1. The effect of acute cadmium (Cd) treatment on pulmonary and renal microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase enzyme activities of adult male guinea-pigs were assessed 72 hr following a single dose of Cd ion (2 mg Cd2+/kg i.p.). Tissue and microsomal Cd levels were also determined. 2. There were no significant differences between either lung or kidney tissue weights, microsomal protein contents or enzyme activities of Cd treated and control animals. 3. The tissues and microsomes of Cd-treated animals were found to have significantly higher levels of Cd than those of control animals. In Cd treated animals, tissue and microsomal Cd levels of kidney were found to be higher than that of lung. 4. In vitro addition of cadmium chloride (CdCl2) to incubation mixtures produced concentration related inhibitions of microsomal drug metabolizing enzymes in each tissue. However, in vitro effect of CdCl2 was found to be stronger on drug metabolizing enzymes of kidney than those of lung. In addition, while the strength of Cd effect was more pronounced on the activity of ethylmorphine N-demethylase than that of aniline 4-hydroxylase in the lung, the opposite was observed in the kidney.     

Stimulation of microsomal dimethylnitrosamine-N-demethylase by pretreatment of mice with acetone

To further investigate the relationship between in vivo microsomal enzyme modifiers and in vitro dimethylnitrosamine (DMN) metabolism, male C57BL/6J mice were pretreated with acetone or Aroclor 1254, two compounds known to influence DMN-N-demethylase activity. Pretreatment with acetone enhanced the in vitro microsomal activity of DMN-N-demethylase, as measured by formaldehyde production from DMN. Accompanying this acetone-enhanced demethylase activity was an increase in the covalent binding of [14C]DMN to RNA, protein and DNA. Four distinct Km values dependent on the substrate concentration were observed for the N-demethylase present in control microsomes. Only one Km value was observed for the demethylase in microsomes from acetone-treated animals, but it was significantly lower than the lowest Km observed in the control microsomes. At DMN concentrations of 1 and 10 mM, acetone significantly increased N-demethylation of DMN as compared to control, but not at 100 mM DMN. Aroclor 1254 pretreatment repressed DMN-N-demethylase at 1 mM DMN but enhanced it at 100 mM. These results suggest that there may be multiple forms of DMN-N-demethylase which are dependent on DMN concentration and respond differently to modifiers of the microsomal drug-metabolizing enzymes.     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

Effects of cage beddings on microsomal oxidative enzymes in rat liver

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.     

Effect of 2-mercaptopropionylglycine on lipid peroxidation and drug oxidation in rat liver microsomes

2-Mercaptopropionylglycine, a synthetic thiol, significantly stimulated NADPH-dependent lipid peroxidation by rat liver microsomes, while the thiol inhibited the microsomal aminopyrine N-demethylase activity with an increase in lipid peroxidation. But, a strong inhibition of lipid peroxidation by EDTA could not abolish the inhibition of the N-demethylase activity by the thiol. Besides, the thiol markedly increased not only the Km value for aminopyrine N-demethylase but also the apparent Ks value for aminopyrine binding to the microsomal oxidized cytochrome P-450 by interacting with the cytochrome P-450.     

Hepatic effects of ketoconazole in the male Swiss Webster mouse: temporal changes in drug metabolic parameters

There have been conflicting observations regarding the effects of ketoconazole on hepatic metabolism. The objectives of these studies were to determine whether ketoconazole was an enzyme inducer or inhibitor in the mouse and then to establish the time frame of these ketoconazole-induced enzyme changes. Ketoconazole was administered (150 mg/kg p.o. X 4 days) to male Swiss Webster mice. Biochemical observations over a period of 6 days following treatment indicated that ketoconazole had a temporal biphasic effect on the liver. Although liver weight and microsomal protein were elevated, all other parameters monitored were lower at 2 h following ketoconazole treatment. At 24 h after the last dose of ketoconazole, hepatic biochemical parameters (liver wt., % liver wt./body wt., microsomal protein, and cytochrome P-450) were statistically elevated, while enzyme activities (benzphetamine N-demethylation, 6 beta- and 7 alpha-hydroxylation of testosterone, formation of androstenedione and UDP-glucuronyltransferase) were inhibited. At 72 h the ketoconazole-induced changes in the hepatic biochemical parameters were comparable to those observed at 24 h, and enzymatic parameters generally appeared to be induced by ketoconazole, with the exception of benzphetamine N-demethylase and UDP-glucuronyltransferase, which exhibited lower enzyme activities. Ethoxyresorufin O-deethylase, 7 alpha-hydroxylation of testosterone and glutathione S-transferase, on the other hand, were unaltered by ketoconazole treatment. The opposing effects of ketoconazole on benzphetamine N-demethylase and ethylmorphine N-demethylase at 72 h were further examined. Enzyme kinetics studies indicated that ketoconazole did not effect the Michaelis constants (Km) of the two substrates, but the maximum velocity (Vmax) of the reactions was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.     

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.     

Xenobiotic-metabolizing enzyme systems in test fish--II. The ethylmorphine N-demethylase activity of guppy (Poecilia reticulata) liver

The oxidative demethylation of the model substrate ethylmorphine has been characterized for the first time in the liver of a fish (Poecilia reticulata). The enzyme showed maximal activity at 35 degrees C and pH values higher than 8. The values of Km and Vmax for the reaction were 0.83 +/- 0.11 mM and 4.64 +/- 0.81 nmol HCHO/(mg microsomal protein) per min. The activity is attributed to the cytochrome P-450-dependent monoxygenase system, since it is inhibited by CO and requires NADPH; moreover it is inhibited competitively by alpha-naphthoflavone and non-competitively by metyrapone. The enzyme activity is induced by a two-week treatment of fish with phenobarbital and may be associated with a protein band of Mr 54,000.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.     

Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.     

Further studies on microsomal drug metabolizing enzyme activity and protein levels in animal models showing different susceptibilities to aflatoxicosis

1. The total protein content and the activities of aniline hydroxylase and p-aminopyrine N-demethylase were measured in the microsomal fraction of rat, mouse, duck, pigeon and turkey. The relative Km values of the enzymes were also determined. 2. The microsomal protein content and the enzyme activities in the birds were lower than that in the rodents. Km values for aniline hydroxylase and p-aminopyrine N-demethylase were higher in birds than in either rat or mouse. 3. The microsomal protein content of mouse was relatively lower than that in rat, although its aniline hydroxylase and p-aminopyrine N-demethylase activities were higher than those in the rat. Km values for aniline hydroxylase in mouse and rat were 0.98 and 1.25 mM, respectively, while the Km values for p-aminopyrine N-demethylase in mouse and rat were 0.55 and 0.62 mM, respectively. 4. These results are discussed in relation to the species differences in susceptibility of animals to toxic or carcinogenic substances.     

Comparison of hepatic and renal drug-metabolising enzyme activities in sheep given single or two-fold challenge infections with Fasciola hepatica

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases (P<0.05) (17-44%) were observed in the microsomal content of cytochrome P450 and in the two measured P450-dependent monooxygenase activities, benzphetamine and ethylmorphine N-demethylations. Moreover, Western blot analysis of microsomes demonstrated a decrease in the expression of cytochrome P4503A subfamily correlative with that of its presumed corresponding activity ethylmorphine N-demethylase. By contrast, the conjugation of chloro-dinitrobenzene to glutathione remained unchanged in liver cytosolic fractions prepared from all these animals. In kidneys, a significant decrease (P<0.05) (30%) in microsomal cytochrome P450 level of 10-week mono-infected sheep was observed whereas there was no change in the other groups of animals. The inflammatory origin and the consequences in terms of pathology and animal productivity of the fascioliasis-induced decreases in tissue-oxidative drug metabolism are discussed, particularly in the case of adult sheep suffering repetitive infections.     

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.     

Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties

1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.