Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

Studies of the binding of sulfur released in the mixed-function oxidase-catalyzed metabolism of diethyl p-nitrophenyl phosphorothionate(parathion) to diethyl p-nitrophenyl phosphate (paraoxon)

Incubation of [³⁵S]labeled parathion with rat liver microsomes in the presence of an NADPH-generating system leads to the covalent binding of sulfur to the macromolecules of the microsomal membrane. The maximal binding of sulfur to microsomes requires the presence of NADPH, is increased using microsomes from phenobarbital and 3-methyl-cholanthrene-treated animals, and is inhibited by carbon monoxide. The majority, if not all, of the sulfur bound is in a form free of the remainder of the parathion molecule. These findings, coupled with the fact that the apparent K_m and V_max for sulfur binding are not statistically different from the apparent K_m and V_max for metabolism of parathion to paraoxon, indicate the sulfur bound is that released in the mixed-function oxidase-catalyzed metabolism of parathion to paraoxon. Under the conditions of the experiments described in this report, the binding of sulfur to microsomes decreases the concentration of cytochrome P-450 in the microsomes and inhibits slightly the rate of the mixed-function oxidase-catalyzed metabolism of benzphetamine.     

Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase,from Saccharomyces cerevisiae

1. The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88–97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE.

2. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5–6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE.

3. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H₂O₂ generated in situfrom a glucose oxidase system; in each case V_max is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased K_m, is lowered.

4. The spin state of purified yeast cytochrome P-448 was 94% low spin (22°C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spinat 22°C).

5. Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from β-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites.

6. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine.

7. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.

8. A more specific and efficient form of benzo[a]pyrene hydroxylase is induced by the addition of benzo[a]pyrene to the yeast growth medium at zero time. The efficiency of the enzyme, as indicated by the V_max / K_m ratio, increases progressively with concentration of benzo[a]pyrene. This indicates that multiple forms of yeast cytochrome P-448 occur. Induction of more efficient forms occurs at the expense of less efficient forms as little increase in total enzyme concn. is observed.     

Further studies on the increase in drug-metabolizing capacity adjacent to intrahepatic Morris hepatomas

Microsomal cytochrome P-450 content was higher in histologically non-tumorous liver adjacent to intrahepatically implanted Morris hepatomas 5123D or 7795 than in histologically normal liver far removed from each tumor. V_max values for microsomal benzo[a]pyrene monooxygenase activity and cyclophosphamide activation were also significantly higher in tumor-adjacent liver than in normal liver far removed from tumor. K_m values of these reactions were unchanged. After intrahepatic implantation, inert spheres of several different materials produced no regional differences in hepatic microsomal cytochrome P-450 content. Both intrahepatic Morris hepatomas exhibited markedly reduced cytochrome P-450 content and benzo[a]pyrene monooxygenase activity. Cyclophosphamide biotransformation could not be detected in microsomes from either Morris hepatoma. Similar recoveries from microsomes of far-removed and tumor-adjacent liver indicated that differences between these regions in drug-metabolizing activity could not be attributed to different stabilities or sedimenting properties of their microsomes. Although microsomal recovery was significantly less from hepatomas than from far-removed or tumor-adjacent liver, this loss of tumor microsomes accounted for only a small part of the reductions in cytochrome P-450-mediated monooxygenases observed within tumors. Compared to control rats. tumor-bearing rats exhibited no change in hepatic drug-metabolizing capacity measured in vivo by hexobarbital sleeping times and antipyrine elimination rates. Phenobarbital (PB) pretreatment of tumor-bearing rats induced cytochrome P-450 to different extents within far-removed liver, tumoradjacent liver, and both hepatomas. The same differential inducibility occurred with PB pretreatment for cyclophosphamide activation. After PB induction, differences in drug-metabolizing activity between far-removed and tumor-adjacent liver disappeared; though induced, these activities remained lower in the hepatomas than in other regions. These changes in drug-metabolizing activity in both basal and PB-induced states of various hepatic regions were related to changes in cellularity of tumor-adjacent tissue. Hepatocellular nuclei prepared from tumor-containing liver were separated into diploid and tetraploid classes by sucrose density gradient centrifugation. Compared to far-removed liver, tumoradjacent liver contained significantly more diploid nuclei and less tetraploid nuclei.     

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.     

Hydroxylation of benzo[a]pyrene and binding of (−)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450: Effect of 7,8-benzoflavone,butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsornes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated adult rabbits with respect to benzo[a]pyrene hydroxylation and the activation of (?)(rflw-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene[(?)trans-7,8-diol] to DNA-binding metabolites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phnobarbital led to induction of P-450LM₂ and a concomitant 3-fold enhancement in microsomal benzo[a]pyrene hydroxylase activity, whereas the conversion of (?)trans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM₂ exhibited the highest activity and specificity toward benzo[a]pyrene and the lowest activity toward (?)trans-7,8-diol. Conversely, P-450LM₄ was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or β-naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (?)trans-7, 8-diol but not of benzo[a]pyrene. Homogeneous P-450LM₄ preferentially Catalyzed the oxygénation of (?)trans-7,8-diol, but was largely ineffective with benzo[a]pyrene. Partially purified P-450LM₇ lacked substrate specificity, for it metabolized both benzo[a]pyrene and (?)trans-7, S-diol at comparable rates. Additionally, 7,8-benzoflavone strongly inhibited benzo[a]pyrene hydroxylation by P-450LM₄ and phenobarbital-induced microsomes, as well as (?)trans-7,8-diol metabolism by P-450LM₄ and 3-methyl-cholanthrene-induced microsomes; in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM₄ and LM₂ with benzo[a]pyrene and (?)trans-7 ,8-diol, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8-benzoflavone, butylated hydroxytoluene inhibited benzo[a]pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo[a]pyrene and its 7,8-dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver microsomes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LM₂ and LM₄, respectively.     

Immunological and enzymatic comparison of hepatic cytochrome P-450 fractions from phenobarbital-, 3-methylcholanthrene-, β-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats

A comparison of the cytochrome P-450 forms induced in rat liver microsomes by phenobarbital on the one hand, and 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin on the other hand, was performed using specific antibodies: anti-P-450 B₂ PB IG (against the phenobarbital-induced cytochrome P-450) and anti-P-450 B₂ BNF IG (against the β-naphtoflavone-induced cytochrome P-450). On DEAE-cellulose chromatography, four cytochrome P-450 fractions were separated, called P-450 A (non-adsorbed), P-450 Ba, P-450 Bb and P-450 Bc, from control, phenobarbital-, 3-methylcholanthrene, /gb-naphtoflavone- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Cytochrome P-450 A fractions appeared to be unmodified by the inducers, whereas the specifically induced cytochrome P-450 forms were always recovered in Bb fractions. The P-450 Bb fractions induced by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibited common antigenic determinants, comparable catalytic activities (benzphetamine, N-demethylase, benzo[a]pyrene hydroxylase) and similar mol. wts. Moreover, the inhibition patterns by the two antibodies of benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities catalysed by 3-methylcholanthrene, β-naphtoflavone and 2,3,7,8-tetrachlorodibenzo-p-dioxin microsomes or by the corresponding P-450 Bb fractions in a reconstituted system were quite identical. By these different criteria, β-naphtoflavone, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin seem to induce a common cytochrome P-450 species in rat liver.     

Acute interaction of drugs. I. The effect of volatile anesthetics on the kinetics of aniline hydroxylase and aminopyrine demethylase in rat hepatic microsomes

The in vitro effect of halothane, methoxyflurane, diethyl ether, and chloroform on the Michaelis constant (K_m) and maximal velocity (V_max) of microsomal aniline hydroxylase and aminopyrine demethylase was determined. The microsomes were obtained from rats pretreated with phenobarbital or 3-methylcholanthrene as well as from untreated rats. The halogenated anesthetics increased both the K_m and V_max of aniline hydroxylase in microsomes from untreated rats and this effect was magnified in microsomes from phenobarbital-induced animals. The K_m and V_max of aniline hydroxylase was not stimulated above control levels by halogenated anesthetics in microsomes from methylcholanthrene-induced rats. These anesthetics tended to inhibit the aminopyrine demethylase by lowering the V_max. Enzyme induction did not alter this inhibition. Diethyl ether inhibited aniline hydroxylase and aminopyrine demethylase by lowering the V_max.     

Differential effects of disulfiram and diethyldithiocarbamate on small intestinal and liver microsomal benzo[a]pyrene metabolism

Microsomes isolated from rat small intestinal mucosa and liver were used to study the effects of disulfiram and diethyldithiocarbamate on benzo[a]pyrene monooxygenase activity. This activity was decreased in the intestinal microsomes to 25 per cent of control 24 hr after a single oral dose of disulfiram. In contrast, daily administration of disulfiram for 5 days produced a dose related increase of benzo[a]pyrene monooxygenase activity, above control level. The elevated activities were accompanied by a concomitant increase in the concentration of cytochrome P-450. This benzo[a]pyrene monooxygenase activity was further stimulated by addition of α-naphthoflavone to the incubation medium. Furthermore, the absorption maximum of this cytochrome was at 450 nm in the CO bound reduced difference spectrum. These observations indicate that the disulfiram induced cytochrome P-450 was of the control type. Daily pretreatment with diethyldithiocarbamate impaired both intestinal and liver microsomes at benzo[a]pyrene monooxygenase activities. Pretreatment with a single dose of 3-methylcholanthrene resulted in a more than 10-fold increase of intestinal benzo[a]pyrene monooxygenase activity after 24 hr. Administration of disulfiram 24 hr before treatment appeared to potentiate the 3-methylcholanthrene induced increase of intestinal benzo[a]pyrene monooxygenase activity. In vitro addition of disulfiram and diethyldithiocarbamate to incubates of intestinal or liver microsomes inhibited benzo[a]pyrene metabolism to various extents; the liver being more sensitive. Disulfiram was approximately 50-fold more potent as an inhibitor than diethyldithiocarbamate. The in vitro inhibition of intestinal benzo[a]pyrene monooxygenase activity obtained with disulfiram appeared to be caused both by direct interaction with the monooxygenase system and through NADPH dependent metabolic activation of disulfiram, while the inhibition of diethyldithiocarbamate may be a result of the latter process only.     

Adenosine 3',5'-monophosphate phosphodiesterase activity in experimental animal tumours which are either sensitive or resistant to bifunctional alkylating agents.

The adenosine 3′,5′-monophosphate phosphodiesterase of Walker rat carcinoma 256, ADJ/ PC6 plasma cell tumour, NK lymphoma. Sarcoma 180 and TLX5 lymphoma behaves kinetically as if two separate activities exist, one with a low affinity for the substrate and the other with a high affinity. The high K_m values are 82·5, 566, 590, 1975 and 1075 μM for the enzyme from each tumour respectively, and the low K_m values are 1·1, 17·7, 5·75, 7·1 and 4·4 μM. In the Walker carcinoma and PC6 plasma cytoma, tumours which are sensitive to alkylating agents, the apparent V_max of the low K_m forms are respectively 38 and 25 per cent of the total activity. In those tumours which are naturally resistant to the growth inhibitory properties of the alkylating agents, the apparent V_max of the low K_m form is less than 10 per cent of the total activity. Walker carcinoma showing a 20-fold resistance to chlorambucil[p-(di-2-chloroethylamino) phenylbutyric acid] has the V_max of the high affinity form decreased to 15 per cent of the total, while a 70-fold resistant line has the V_max of this form of the enzyme decreased to 9 per cent of the total. This decrease in the activity of the high affinity form of the enzyme in the resistant tumours does not appear to be due to the presence of an endogenous inhibitor. While the high K_m form of the enzyme in the Walker carcinoma is mainly confined to the cytosol, about half of the activity of the low K_m form appears to be associated with particulate fractions. This subcellular distribution does not differ appreciably in the sensitive and resistant tumours. The possible reasons for such an isoenzyme shift are discussed.     

Tissue and sex differences in the activation of aromatic hydrocarbon hydroxylases in rats

Betamethasone and α-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas α-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas α-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes from female rats betamethasone activated whereas α-naphthoflavone had no effect.Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one ‘latent’ aromatic hydrocarbon hydroxylase in rodents.     

Effects of steroid hormones in vitro on adrenal xenobiotic metabolism in the guinea pig

Studies were carried out to determine the effects of steroid hormones in vitro on adrenal and hepatic microsomal benzphetamine demethylation and benzo[a] pyrene hydroxylation. Testosterone inhibited adrenal drug metabolism but had no effect on hepatic enzymes, whereas 6β-hydroxytestosterone had no effect in either tissue. All of the corticosteroids tested (cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone) produced a concentration-dependent inhibition of adrenal drug metabolism, but had little or no effect on hepatic metabolism. The 17-deoxy-steroids were more potent inhibitors of adrenal metabolism than were their 17-hydroxylated counterparts. Cortisol was a potent inhibitor of adrenal benzphetamine and benzo[a]pyrene metabolism, produced a type I difference spectrum in adrenal microsomes, and diminished the magnitude of the benzphetamine-induced spectrum; 6 β-hydroxycortisol had none of these effects. Prior addition of benzphetamine to adrenal microsomes reduced the size of cortisol-induced spectral change. The results demonstrate that the effects of corticosteroids in vitro are relatively specific for adrenal enzymes and established a close association between the 6 β-hydroxylase and some drug-metabolizing enzymes. Adrenal steroids may have an important role in the regulation of adrenal xenobiotic metabolism.     

Vitamin K epoxide reductase activity in the metabolism of epoxides

The importance of vitamine K epoxide reductase for the metabolism of a range of structurally diverse epoxides has been investigated. Vitamin K₁ epoxide is reduced by rat liver microsomes at a rate of 0.47 nmoles/g liver/min. The rate of menadione oxide reduction is not significantly higher than the non-enzymatic reduction rate. No measurable reduction of benzo[a]pyrene 4,5-oxide, benzo[a]pyrene 7,8-oxide, phenanthrene 9,10-oxide, styrene 7,8-oxide, and dieldrin has been detected, nor could trichothecene T-2 toxin inhibit reduction of vitamin K₁ epoxide. Thus, vitamin K epoxide reductase is very specific for vitamin K₁ epoxide. Taking into account the range of structurally diverse epoxides investigated and the high specific activities of microsomal epoxide hydrolase and cytosolic glutathione transferase for these epoxides it may be concluded that vitamin K epoxide reductase, in all likelihood, generally does not significantly contribute to the control of epoxides metabolically formed from xenobiotics.     

Effect of protein deficiency on the inducibility of the hepatic microsomal drug-metabolizing enzyme system—II: Effect on enzyme kinetics and electron transport system

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed ad lib. (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed ad lib. (group 3). After each group was further subdivided, animals were injected i.p. on days 11–14 with either 0.9% saline or phenobarbital (80 mg/kg) in 0.9 % saline. Twenty-four hr after the last injection, animals were decapitated and liver microsomes were prepared. Apparent V_max and apparent K_m kinetic constants were determined for ethylmorphine and aniline. The V_max per milligram of microsomal protein was 64–66 per cent lower in the protein-deficient group. Equivalent reductions of the content of cytochrome P-450 and activities of cytochrome P-450 and c reductases were also observed. Phenobarbital induction increased specific enzyme activities (V_max per milligram of microsomal protein) in all groups with slightly greater percentage increases seen in the protein-deficient animals. Increases were also noted for the cytochrome P-450 content and cytochromes P-450 and c reductase activities. It was suggested that phosphatidylcholine and cytochrome P-450 both play important roles in the kinetics of metabolism determined after protein deficiency or phenobarbital induction, or both.     

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans,monkeys, rats,and minipigs

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (V_max) and high catalytic efficiency (V_max/K_m, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent K_m values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10–100-fold those of rat FMO1, although these enzymes had similar V_max values. The mean catalytic efficiencies (V_max/K_m, 1.4 and 0.4 min⁻¹ μM⁻¹, respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas V_max/K_m values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.     

A comparison of the effects of benzypyrene administration on some hepatic microsomal mixed-function oxidases of rats and mice

There were marked differences in the responses of the hepatic microsomal enzymes of rats and mice after treatment of these animals with benzpyrene. Benzpyrene treatment caused qualitative as well as quantitative changes in the hepatic microsomal mixed-function oxidases of rats. Qualitative changes included: a decrease in the ratio of the peaks at 430 and 455 mμ in the pH-dependent ethyl isocyanide difference spectrum; an increase in the pH optimum of aniline p-hydroxylation; and changes in the apparent Michaelis-Menten kinetics of aniline, (+)-benzphetamine and benzpyrene metabolism. In addition, there were marked alterations in the substrate concentration-dependent “kinetics” of the difference spectra produced by the interaction of aniline and (+)-benzphetamine with rat liver microsomal P-450. Treatment of rats with benzpyrene increased both the apparent V_max and apparent K_m of aniline p-hydroxylation, and the degree of change was dependent upon substrate concentration and pH of the incubation systems. Benzpyrene treatment also increased the apparent ΔA_max of the aniline-induced microsomal difference spectrum, although the apparent spectral dissociation constant (K_s) was decreased. Both the rate of (+)-benzphetamine metabolism and the magnitude of the (+)-benzphetamine-induced microsomal difference spectrum were decreased after benzpyrene treatment. Treatment of rats with benzpyrene also increased the apparent V_max and decreased the apparent K_m of benzpyrene hydroxylation several-fold. In identical experiments performed concomitantly with mice, benzpyrene treatment produced no measurable effect on any of these characteristics of the mouse hepatic microsomal mixed-function oxidases, except for a decrease in the magnitude of the (+)-benzphetamine-induced microsomal difference spectrum. Although the administration of benzpyrene produced no stimulatory effects on the drug-metabolizing enzymes of the mouse, treatment with 3-methylcholanthrene did enhance the benzpyrene hydroxylase activity of mouse liver microsomes. Studies showed that the apparent V_max of this reaction was increased more than 2-fold.     

Characterization of 1-Hydroxypyrene as a Novel Marker Substrate of 3-Methylcholanthrene-Inducible Phenol UDP-Glucuronosyltransferase(s)

Abstract: Rats were treated with acetone, pyrazole, phenobarbital, 4,4′-methylenebis-(2-chloroaniline) (MOCA), 3-methylcholanthrene, creosote oil, or a mixture of polychlorinated biphenyls (Aroclor 1254) to study the inducibility and enzyme kinetics of UDP-glucuronosyltransferases towards 1-hydroxypyrene, which is a human metabolite and a urinary biomarker of exposure to pyrene. The rate of 1-hydroxypyrene glucuronidation was analysed in rat liver microsomes by a fluorometric HPLC assay of the formed glucuronide. The apparent K_m and V_max values in untreated controls (K_m = 0.27 mM; V_max = 31 nmol/min./mg protein) did not differ markedly from those in rats treated with acetone, pyrazole or phenobarbital, whereas the significantly decreased K_m and increased V_max values of the rats treated with the carcinogenic chemicals, MOCA (0.11; 51), creosote (0.06; 137), 3-methylcholanthrene (0.07; 141) or the Aroclor-1254 polychlorinated biphenyl (PCB) mixture (0.08; 226), implicated major changes in the hepatic expression of UDP-glucuronosyltransferases. 1-Hydroxypyrene proved to be a high affinity substrate and a sensitive marker of the polycyclic aromatic hydrocarbon (PAH) metabolizing UDP-glucuronosyltransferase(s). Catalytically, the most efficient isoforms were induced in creosote, 3-methylcholanthrene and PCB-treated rats showing V_max/K_m ratios which were 22–27 times greater than in untreated controls. Our findings suggest the existence of a 3-methylcholanthrene type inducible and a functionally efficient low-K_m/high-V_max form(s) of UDP-glucuronosyltransferase(s) that detoxify 1-hydroxypyrene and probably other polycyclic aromatic hydrocarbons as well.     

The hydroxylation of the antitumor agent, ellipticine, by liver microsomes from differently pretreated rats

The antitumor agent, ellipticine (5,11-dimethyl-6H-pyrido[4-3,b]carbazole) is mainly hydroxylated in position 9 by liver microsomes of differently pretreated rats, this result being in agreement with that obtained previously in vivo. A quick and reliable fluorometric assay, based on the differential fluorescent properties of ellipticine and 9-hydroxyellipticine, is described for the measurement of the 9-hydroxylase activity of different microsomes. This activity exhibits the usual features of the cytochrome-P450-dependent monooxygenases. Control rat liver microsomes exhibit a good affinity for ellipticine (K_m = 3 × 10^?5 M) but a low specific activity (0.1 nmole min^?1 mg protein ^?1), perhaps related with an excess substrate inhibition. Pretreatment of rats with benzo[a]pyrene or ellipticine enhances the rate of 9-hydroxylation: pretreatment with phenobarbital does not. Metyrapone and 7,8 benzofiavone are poor inhibitors of ellipticine hydroxylation particularly in microsomes from benzo[a]pyreneor ellipticine-pretreated rats.     

Styrene induced modifications of some rat liver enzymes involved in the activation and inactivation of xenobiotics.

Rats were injected intraperitoneally with single doses of styrene. Its effects on the kinetic parameters of liver microsomal monooxygenases and epoxide hydratase were investigated. The results were compared with those produced either by ethylbenzene, the vinyl-saturated analog of styrene or by phenobarbital and 3-methylcholanthrene, the classical inducers of those enzymes. The biochemical modifications were correlated with the altered ability of homogenates obtained from similarly pretreated rats to activate benzo(a)pyrene into intermediates mutagenic towards Salmonella typhimurium. Administration of styrene or 3-methylcholanthrene decreased the K_m, of benzo(a)pyrene hydroxylase and aldrin epoxidase; styrene, but not 3-methylcholanthrene, decreased the K_m of styrene oxide hydratase; none of the two compounds modified the K_m of styrene epoxidase.Pretreatment of the rats by styrene or 3-methylcholanthrene enhanced the S₉ mediated mutagenicity of benzo(a)pyrene several-fold, when compared to the mutagenic response mediated by liver preparations from control rats. Phenobarbital and ethylbenzene did not modify either the K_m of the investigated enzymes or the liver-mediated mutagenicity of benzo(a)pyrene.     

Decreased effect of phenobarbital treatment on microsomal drug metabolizing enzyme activity during gestation

Summary Pregnant and nonpregnant rats were treated with 37.5 mg phenobarbital (PB) per kg body weight twice daily for 4 days. Pregnant rats were injected with PB from day 10 to day 13 and from day 17 to day 20 of gestation and were used for the experiments on day 14 or day 21, respectively. This treatment resulted in a 35% increase in liver weight in nonpregnant rats. The livers of pregnant rats weighed the same as the livers from PB-treated nonpregnant rats, and PB-treatment during gestation had only a small stimulatory effect on liver weight.—The duration of sleeping time after an i.p. injection of 125 mg hexobarbital per kg maternal body weight was identical in control nonpregnant and control pregnant rats. PB-treatment significantly reduced the sleeping time but PB-treated pregnant rats had a longer sleeping time than PB-treated nonpregnant rats.—In liver microsomes, the K _m and V _max values were measured for the ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase. For the two substrates the K _m values were identical in all groups. The V _max values for the two enzyme activities were not different in control pregnant and control nonpregnant rats when calculated per total liver. Treatment of the rats with PB increased the V _max values for the two enzyme activities, but these values were significantly lower in the microsomes obtained from PB-treated pregnant rats.—The content of cytochrome P-450 was higher in PB-treated nonpregnant rats than in control nonpregnant rats, but no increase in the amount of cytochrome P-450 was measured after treating pregnant rats with PB.—The electron microscopic examination of the livers of pregnant rats revealed that the smooth elements of the endoplasmic reticulum were predominant in the cytoplasm, and after PB-treatment the typical proliferation of the smooth ER was not detectable.The abbreviations used are PB Phenobarbital - BP Benzo(a)pyrene - ER endoplasmic reticulum; day 14 or day 21 refer to day 14 of gestation or to day 21 of gestation, respectively - N.P. nonpregnant - CO control This work was supported by a grant from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 29, Embryonale Entwicklung und Differenzierung (Embryonal-Pharmakologie).Parts of this paper were presented at the Spring Meeting of the German Pharmacological Society held in Mainz, March 1973.