Pharmacologic effects of 4-chlorophenol in rats: comparison to clofibrate

4-Chlorophenol (4-CP) is an identified trace contaminant in commercial clofibrate preparations and the pharmacologic effects of 4-CP have not yet been widely established. We have examined the dose-dependent effects of oral 4-CP and clofibrate administration on selected hepatic parameters and on serum glucose, cholesterol, and triglyceride concentrations in male rats. 4-CP treatment (0.00125-0.08 mmol/kg, twice a day) of rats for 2 weeks increased hepatic microsomal protein (20-30%) and cytochrome P-450 (20-190%) contents without changing liver/body weight ratios. Both 4-CP (0.0025 mmol/kg body wt, twice a day) and CPIB (0.4 mmol/kg body wt, twice a day) treatment to rats for 2 weeks caused significant elevations in microsomal cytochrome P-450 content and in the maximal activities of ethylmorphine, aminopyrine, and benzphetamine N-demethylase, but not in the activity of zoxazolamine 6-hydroxylase. With the same dose of 4-CP, time-dependent increases in hepatic microsomal protein, cytochrome P-450, and the activity of benzphetamine N-demethylase were observed for a 4-week period, and the induction of hepatic microsomal benzphetamine N-demethylase activity by 4-CP was associated with an increased enzyme synthesis. 4-CP treatment produced a marked morphologic change in liver cell ultrastructure, including a proliferation of mitochondria and endoplasmic reticulum at lower 4-CP doses. A clustering of intracellular organelles (mitochondria and endoplasmic reticulum) and a foamy cytoplasm were seen at doses greater than 0.01 mmol/kg, twice a day for 2 weeks, and at 0.0025 mmol/kg, twice a day for greater than 4 weeks. The effects of 4-CP and clofibrate on fasting blood glucose and fasting serum lipid levels were also monitored throughout an 8-week period. Both 4-CP (0.005 mmol/kg body wt, twice a day) and clofibrate (0.2 mmol/kg body wt, twice a day) produced significant elevations in fasting serum glucose levels, but this dosage of 4-CP did not alter serum lipid and lipoprotein parameters, whereas clofibrate significantly reduced serum total cholesterol and high density lipoprotein cholesterol levels. These results lead us to conclude that 4-CP does not contribute to the antilipidemic effects of clofibrate.     

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

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

Topically applied nitropyrenes are potent inducers of cutaneous and hepatic monooxygenases

The inducibility of skin and liver microsomal cytochrome P-450 dependent aryl hydrocarbon hydroxylase and other monooxygenases by a mixture of nitropyrenes was assessed and compared with the parent non-nitrated compound, pyrene. A single topical application of nitropyrenes to neonatal rats resulted in highly significant induction of aryl hydrocarbon hydroxylase, ethoxycoumarin O-de-ethylase, and ethoxyresorufin O-de-ethylase activities in skin and liver after 24 hours. Inducibility of the skin and liver enzymes was 3.9-5.7 fold and 1.8-10.3 fold respectively. On the other hand, aminopyrine N-demethylase, benzphetamine N-demethylase and epoxide hydrolase activities in the liver were unaffected by topically applied nitropyrenes. Furthermore, treatment with nitropyrenes produced a 1 nm shift to the blue region in the wavelength maximum of hepatic microsomal cytochrome P-450. Topically applied pyrene produced only marginal or no effects on cutaneous and hepatic enzyme activities. Our results suggest that nitration of pyrene, a relatively ineffective enzyme inducer, produces nitropyrenes which are potent inducers of hepatic and cutaneous monooxygenases and they resemble 3-methylcholanthrene in this inducing effect.     

Altered sexual differentiation of hepatic uridine diphosphate glucuronyltransferase by neonatal hormone treatment in rats

The hepatic microsomal enzyme UDP-glucuronyltransferase undergoes a complex developmental pattern in which enzyme activity is first detectable on the 18th day of gestation in rats. Prepubertal activities are similar for males and females. However, postpubertal sexual differentiation of enzyme activity occurs in which male activities are twice those of females. Neonatal administration of testosterone propionate or diethylstilboestrol to intact animals resulted in lowered UDP-glucuronyltransferase activity in liver microsomal fractions of adult male rats, whereas no changes were observed in the adult females and prepubertal male and female animals. Neonatal administration of testosterone propionate and diethylstilboestrol adversely affected male reproductive-tract development as evidenced by decreased weights of testes, seminal vesicles and ventral prostate. Diethylstilboestrol also markedly decreased spermatogenesis. Hypophysectomy of adult male rats resulted in negative modulation of microsomal UDP-glucuronyltransferase and prevented the sexual differentiation of enzyme activity. In contrast hypophysectomy had no effect on female UDP-glucuronyltransferase activity. A pituitary transplant under the kidney capsule was not capable of reversing the enzyme effects of hypophysectomy, therefore suggesting that the male pituitary factor(s) responsible for positive modulation of UDP-glucuronyltransferase might be under hypothalamic control in the form of a releasing factor. Neonatal testosterone propionate and diethylstilboestrol administration apparently interfered with the normal sequence of postpubertal UDP-glucuronyltransferase sexual differentiation.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Characteristics of renal UDP-glucuronyltransferase

Rat renal microsomes catalyzed the glucuronidation of l-naphthol, 4-methylumbelliferone and p-nitrophenol, whereas morphine and testosterone conjugation were not detected. In contrast, all five substrates were conjugated by hepatic microsomes; the activity was typically 5-10 times greater than with renal microsomes. Renal microsomal UDP-glucuronyltransferase toward l-naphthol was fully activated (six-fold) by 0.03% deoxycholate while the hepatic enzyme was fully activated (eight-fold) by 0.05% deoxycholate. Full activation of hepatic UDP-glucuronyltransferase occurred when microsomes had been preincubated at 0 C with deoxycholate for 20 min. This effect of preincubation was not observed with renal microsomes. The presence of 0.25M sucrose in the buffers during renal microsomal preparation resulted in a two-fold greater rate of l-naphthol conjugation in both unactivated and activated microsomes than renal microsomes prepared in phosphate buffers alone. Preparation of hepatic microsomes with or without 0.25M sucrose had no effect on UDP-glucuronyltransferase activity. Unactivated (-deoxycholate) renal enzyme was activated when incubations were done at a low pH (5.7), whereas fully activated (0.03% deoxycholate) renal microsomal UDP-glucuronyltransferase displayed a pH optimum at 6.5. Renal microsomal UDP-glucuronyltransferase activity toward l-naphthol, p-nitrophenol and 4-methylumbelliferone was induced by pretreatment of rats with beta-naphthoflavone and trans-stilbene oxide but not by phenobarbital or 3-methylcholanthrene. These data demonstrate that renal UDP-glucuronyltransferases are different from the hepatic enzymes with regard to biochemical properties, substrate specificity and in response to chemical inducers of xenobiotic metabolism.     

Role of the kallikrein-kinin system in glandular secretion

Microsomes were prepared from liver, lung and kidney of rats and rabbits using a Ca⁺² aggregation method. Microsomal protein yield from the lung of both species was higher by this method of preparation as compared with ultracentrifuged samples. Specific activities of rat and rabbit pulmonary p-chloro-N-methylaniline (CMA) demethylase, biphenyl 4-hydroxylase and rat pulmonary TPNH cytochrome c reductase also were decreased. Specific activities of rabbit hepatic TPNH cytochrome c reductase, CMA N-demethylase, UDP-glucuronyltransferase and biphenyl hydroxylase were decreased by calcium aggregation Renal enzyme activities were unchanged by this method of preparation. These data indicate an apparent species and organ difference in microsomal enzyme response to calcium aggregation.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Purification and characterization of two forms of chicken liver cytochrome P-450 induced by 3,4,5,3',4'-pentachlorobiphenyl

3,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16 alpha-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16 alpha-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.     

Biphasic effect of methadone on hepatic drug metabolism

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Identification of increased amounts of UDP-glucuronyltransferase protein in phenobarbital-treated chick-embryo liver cells.

UDP-glucuronyltransferase activity of neonatal-chick liver or phenobarbital-treated chick-embryo liver catalysed the glucuronidation of 1-naphthol, 4-nitrophenol and 2-aminophenol. Only low transferase activity towards testosterone was detected, and activity towards bilirubin was not detectable. Liver microsomal transferase activity towards the three phenols was increased approx. 20-50-fold by phenobarbital treatment of chick embryos or by transfer of liver cells into tissue culture. A single form of UDP-glucuronyltransferase, which appears to catalyse the glucuronidation of these three phenols, was purified to near homogeneity from phenobarbital-treated chick-embryo liver microsomal fraction for the first time. The use of this purified enzyme as a standard protein facilitated the identification of this protein in chick-embryo liver microsomal fraction. Further, the accumulation of this microsomal protein was observed following phenobarbital treatment of chick embryos and during tissue culture of chick-embryo liver cells. The value of this model system for the study of the induction of UDP-glucuronyltransferase by drugs and hormones is discussed.     

Steroid glucuronyltransferases of rat liver. Properties of oestrone and testosterone glucuronyltransferases and the effect of ovariectomy, castration and administration of steroids on the enzymes.

1. Microsomal preparations from rat liver, kidney and intestine were tested for UDP-glucuronyltransferase activity by using oestrone, oestradiol-17 beta, oestriol, testosterone, cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone as substrates. The microsomal preparation from the liver glucuronidated oestrone, oestradiol-17 beta and testosterone. 2. The specific activity of the enzyme was significantly higher in livers from female rats than in those from male rats. 3. Testosterone was actively glucuronidated by both sexes. Cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone were not glucuronidated by any of the three tissues. 4. The non-ionic detergent Lubrol WX activates liver microsomal UDP-glucuronyltransferase 2-3-fold with oestrone and testosterone as substrates. 5. Oestrone glucuronyltransferase was inhibited by oestradiol-17 beta, predominantly competitively and by testosterone non-competitively. Bilirubin was a non-competitive inhibitor of oestrone glucuronidation. p-Nitrophenol had no effect. 6. Oestrone glucuronyltransferase could not be stimulated by either acute or prolonged treatment of animals with phenobarbital, whereas a single dose of 3-methylcholanthrene led to a moderate stimulation. 7. Ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity; administration of oestradiol-17 beta induces the enzyme to normal activity after 12 days, and after 15 days the activity is twice the control value. Actinomycin D and cycloheximide block the oestradiol-17 beta-induced increase in enzyme activity. 8. Castration has no effect on the activity of testosterone glucuronyltransferase, nor does administration of testosterone influence enzyme activity. The results provide strong evidence for the existence of multiple steroid glucuronyltransferases in the liver of the rat.     

In vitro drug metabolism in male and female athymic,nude mice

Drug metabolism was studied in hepatic microsomal and post microsomal supernatant fractions from male and female athymic nude mice (nu/nu) and heterozygous (+/nu) and homozygous (+/+) wild-type controls. In males, the following enzyme activities were higher in athymic mice than in the wild-type: NADPH cytochrome c reductase, ethylmorphine and aminopyrine N-demethylases, native UDP glucuronyltransferase, and glutathione (GSH) S-aryltransferase. No differences were observed between groups in UDPNAG-activated UDP-glucuronyltransferase, N-acetyltransferase, or aniline hydroxylase activities or in amounts of cytochrome P-450. In female athymic mice, only ethylmorphine and aminopyrine N-demethylase activities were significantly higher than in female wild-type controls (+/+). The female athymic mice had mixed function oxidase activities that were less than the male athymic mice. There were no sex or strain differences in response to treatment with phenobarbital or 3-methylcholanthrene.     

Strain differences in rat liver (UDP-glucuronyltransferase activity towards androsterone.

Male Donryu, Wistar King rats showed discontinuous variations in hepatic microsomal UDP-glucuronyltransferase activities towards androsterone, but not towards testosterone, bilirubin, phenolphthalein and 4-nitrophenol. Fresh microsomal fraction with a low transferase activity towards androsterone formed 0.049--0.080 nmole of glucuronide/min per mg of protein, whereas fresh microsomal fraction with a high transferase activity towards androsterone formed 0.335--0.557 nmol of glucuronide/min per mg of protein. The microsomal fraction with low enzyme activity towards androsterone was not stimulated by treatment with Triton X-100 or freezing and thawing. In contrast, male Long Evans and Sprague-Dawley rats did not exhibit such diversity.     

Induction and suppression of hepatic and extrahepatic microsomal foreign-compound-metabolizing enzyme systems by 2,3,7,8-tetrachlorodibenzo-p-dioxin

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on a number of hepatic and extrahepatic foreign-compound-metabolizing enzyme systems in microsomes from rats, rabbits and guinea pigs were investigated.Following TCDD treatment, the N-demethylation of benzphetamine, aminopyrine and ethylmorphine was suppressed in hepatic microsomes from male but not from female rats. However, both cytochrome P-450 and benzpyrene hydroxylase were significantly stimulated in hepatic microsomes from both male and female rate at doses as small as 1 μg TCDD/kg body weight. The inductive effect on rat hepatic microsomal enzymes was considerably more persistent than the suppressive effect. Following a single oral dose of 25 μg TCDD/kg body weight, benzpyrene hydroxylase of male rat liver microsomes remained significantly elevated for 73 days but the suppression of benzphetamine N-demethylase had gone after 35 days.The induction of benzpyrene hydroxylase in male rat liver microsomes by TCDD was independent of the age of the rat and the levels to which this enzyme was increased was similar in male rats of all ages. However, the suppression of benzphetamine N-demethylase in male rat liver microsomes was age related: the suppression was seen only in adult animals and in the very young (10 days old) the enzyme was actually induced by TCDD. Inductive effects appeared in both smooth and rough-surfaced hepatic microsomes from male rats but the suppression of N-demethylidon occurred perhaps the derepression arises through the interaction of TCDD or metabolite of TCDD, with the operator gene itself.     

Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7 alpha-hydroxylation.

Rat hepatic cytochrome P-450 form 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) and P-450 form RLM2 (testosterone 15 alpha-hydroxylase; P-450 gene IIA2) are 88% identical in primary structure, yet they hydroxylate testosterone with distinct and apparently unrelated regioselectivities. In this study, androstenedione and progesterone were used to assess the regioselectivity and stereospecificity of these two P-450 enzymes towards other steroid substrates. Although P-450 RLM2 exhibited low 7 alpha-hydroxylase activity with testosterone or progesterone as substrate (turnover number less than or equal to 1-2 nmol of metabolite/min per nmol of P-450), it did catalyse androstenedione 7 alpha-hydroxylation at a high rate (21 min-1) which exceeded that of P-450 3 (7 min-1). However, whereas P-450 3 exhibited a high specificity for hydroxylation of these steroids at the 7 alpha position (95-97% of total activity), P-450 RLM2 actively metabolized these compounds at four or more major sites including the nearby C-15 position, which dominated in the case of testosterone and progesterone. The observation that androstenedione is actively 7 alpha-hydroxylated by purified P-450 RLM2 suggested that this P-450 enzyme might make significant contributions to microsomal androstenedione 7 alpha-hydroxylation, an activity that was previously reported to be associated with immunoreactive P-450 3. Antibody inhibition experiments were therefore carried out in liver microsomes using polyclonal anti-(P-450 3) antibodies which cross-react with P-450 RLM2, and using a monoclonal antibody that is reactive with and inhibitory towards P-450 3 but not P-450 RLM2. P-450 3 was thus shown to catalyse only around 35% of the total androstenedione 7 alpha-hydroxylase activity in uninduced adult male rat liver microsomes, with the balance attributed to P-450 RLM2. The P-450-3-dependent 7 alpha-hydroxylase activity was increased to approximately 65% of the total in phenobarbital-induced adult male microsomes, and to greater than 90% of the total in untreated adult female rat liver microsomes. These observations are consistent with the inducibility of P-450 3 by phenobarbital and with the absence of P-450 RLM2 from adult female rat liver respectively. These findings establish that P-450 RLM2 and P-450 3 can both contribute significantly to microsomal androstenedione 7 alpha-hydroxylation, thus demonstrating that the 7 alpha-hydroxylation of this androgen does not serve as a specific catalytic monitor for microsomal P-450 3.     

Mouse liver testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha). Purification, regioselectivity, stereospecificity, and immunochemical characterization

Microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) was purified from the livers of male 129/J mice based on enzyme activity in the eluates from columns of DEAE Bio-Gel A, hydroxylapatite, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified P-450(16) alpha fraction was 9.5 nmol/mg of protein. The specific testosterone 16 alpha-hydroxylation activity of the purified P-450(16) alpha fraction was 80 nmol/min/nmol of cytochrome P-450 or 764 nmol/min/mg of protein, and these values were about 40- and 400-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(16) alpha showed extremely high regioselectivity and stereospecificity for testosterone hydroxylation; more than 90% of the testosterone metabolites formed by the purified P-450(16) alpha fraction was 16 alpha-hydroxytestosterone. The purified anti-P-450(16) alpha antibody exhibited absolute specificity for inhibition of testosterone 16 alpha-hydroxytestosterone was inhibited by the anti-P-450(16) alpha. Anti-P-450(16) alpha inhibited the 16 alpha-hydroxylation activity of intact microsomes prepared from livers of male or female 129/J mice more than 90%, indicating that P-450(16) alpha is the major cytochrome P-450 isozyme catalyzing 16 alpha-hydroxylation activity of testosterone in these microsomal preparations. The purified P-450(16) alpha fraction also possessed high benzphetamine N-demethylation activity relative to the rates found with other xenobiotic substrates tested in this report.     

Inhibition of rat hepatic mixed function oxidases by antimalarial drugs: selectivity for cytochromes P-450 and P-448

Intraperitoneal administration of chloroquine, primaquine and quinacrine to rats resulted in inhibition of the hepatic microsomal mixed-function oxidases. The N-demethylation of benzphetamine (cytochrome P-450) was inhibited by chloroquine only while the O-deethylation of ethoxyresorufin (cytochrome P-448) was inhibited by primaquine and quinacrine. When incubated with hepatic microsomes from phenobarbital-pretreated rats, chloroquine and primaquine, but not quinacrine, caused a concentration-dependent inhibition of benzphetamine N-demethylase activity. Incubation of hepatic microsomes from beta-naphthoflavone rats with primaquine and quinacrine, but not chloroquine, resulted in a concentration-dependent inhibition of the O-deethylation of ethoxyresorufin. These observations demonstrate that chloroquine and quinacrine are specific inhibitors of cytochromes P-450 and P-448, respectively.     

Effect of DDT on hepatic mixed-function oxidase activity in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks resulted in significant decrease in the body weight and marked reduction in the hepatic vitamin A content. The levels of hepatic phase I microsomal enzymes cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase were found to be substantially reduced by vitamin A-deficiency. Also, the activity of phase II microsomal UDP - glucuronyl transferase enzyme was significantly decreased in deficient animals. Following repeated oral administration of DDT (15 mg/kg/body wt/day) for 21 days, the phase I microsomal enzymes were induced to a greater extent in controls as compared to deficient animals. UDP - glucuronyltransferase remained insensitive to DDT induction. The results imply that the capacity for induction of the hepatic mixed-function oxidase enzyme system is impaired in deficient animals concurrently exposed to DDT.