Effects of dietary pectin and cellulose on hepatic and intestinal mixed-function oxidations and hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase in the rat

The aim of this study was to determine if feeding dietary fiber (cellulose or pectin) to male rats could influence hepatic and intestinal mixed-function oxidation. We simultaneously compared hepatic drug-oxidizing activity with the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for cholesterol biosynthesis. Three groups of six animals were fed a purified diet containing by weight either 10.4% cellulose or 10.4% pectin, or a standard cereal-based diet containing 4.5% crude fiber; the caloric contributions by carbohydrate, protein and fat in the three diets were similar. In the cellulose-fed rats, the hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were significantly lower when compared with those of rats fed pectin or the cereal-based diet. The hepatic microsomal cytochrome P-450 content and the activities of ethylmorphine N-demethylase and aniline hydroxylase were similar in the pectin-fed and cereal diet-fed rats. Hepatic HMG-CoA reductase activity, hepatic microsomal cytochrome b₅ content, and intestinal benzo[a]pyrene hydroxylase activity were comparably lower in rats fed the purified diet with either dietary fiber when compared to those fed the cereal diet. It is concluded that dietary pectin and cellulose exert distinctly different influences on the hepatic microsomal mixed-function oxidase system for drug metabolism, but not on liver cholesterol synthesis or intestinal benzo[a]pyrene hydroxylation, suggesting that different physiological mechanisms control these enzyme systems.     

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.     

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.     

Differential effects of diabetes on microsomal metabolism of various substrates: Comparison of streptozotocin and spontaneously diabetic wistar rats

We have examined the effect of recent onset diabetes on several aspects of hepatic microsomal metabolism in both streptozotocin (STZ)-induced and spontaneously diabetic BioBreeding (BB) male and female Wistar rats. Differential alterations of the diabetic state on hepatic microsomal enzyme activities were observed. Female diabetic rats exhibited no change in benzo[a]pyrene (BP) hydroxylase activity, a decrease in testosterone Δ⁴-hydrogenase, and an increase in aniline hydroxylase. On the other hand, male diabetic rats demonstrated a decrease in hepatic BP hydroxylase activity, no change in testosterone Δ⁴-hydrogenase, and an increase in aniline hydroxylase. Insulin treatment corrected these effects. No change in kidney BP hydroxylase activity was apparent in either female or male diabetics. There were no marked differences between the chemically induced and genetic models of diabetes with respect to the metabolism studies. Serum testosterone levels were significantly lower than control in male BP diabetics, whereas no change was apparent in female diabetics. Light microscopy and serum insulin determinations indicated that the spontaneously diabetic animals we examined were not severely diabetic. From electrophoresis of hepatic microsomal proteins we determined that spontaneous diabetes of short duration does alter the protein distribution in the cytochrome P-450 region. We conclide that the acute effects of STZ-induced and spontaneous diabetes on hepatic microsomal metabolism are quantitatively and qualitatively similar, despite probable differences in etiology of the diabetic state.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) effects on hepatic microsomal cytochrome P-448-mediated enzyme activities.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal mixed function oxidase (MFO) enzyme systems were examined in female rats. Although TCDD had little effect on NADPH-cytochrome c reductase activity and cytochrome P-450 content, the activities of the cytochrome P-448-mediated enzymes benzo[α]pyrene hydroxylase, ethoxyresorufin O-deethylase, and biphenyl 2-hydroxylase were greatly increased. Three months after a single oral dose of 2 μg/kg TCDD, the cytochrome P-450 content and benzo[α]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were still significantly increased. In addition, the microsomal metabolism of the novel substrate 4,4′-dimethylbiphenyl was greatly increased by TCDD pretreatment. Low dose studies revealed that the ED50 of TCDD induction of benzo[α]pyrene hydroxylase was 0.63 μg/kg and the lowest dose of TCDD which caused a significant increase in enzyme activity was 0.002 μg/kg. Studies in which [1,6-³H]TCDD was used to determine the extent of hepatic uptake of orally administered TCDD at the lowest effective dose of 0.002 μg/kg lead to the estimate that only 65 molecules of TCDD per hepatocyte were required to produce a measurable increase in benzo[α]pyrene hydroxylation. These results attest to the specificity and persistence of TCDD in the induction of cytochrome P-448-mediated enzyme activities in rat liver. The small number of molecules required to induce benzo[α]pyrene hydroxylase suggests that TCDD is among the most potent MFO-inducing agents yet demonstrated in mammalian liver.     

Quantitative and qualitative changes in the metabolism of benzo(a)pyrene in rat tissues after intragastric administration of TCDD

Intragastric treatment of rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) dramatically increased the metabolism of benzo(a)pyrene in the liver of Wistar and Gunn rats. The formation of both ethyl acetate and water-soluble metabolites increased several fold in liver homogenate and microsomal preparations. TCDD treatment increased the rate of formation of unidentified polar ethyl acetate-soluble metabolites 300-fold in hepatic microsomes of Wistar rats and 410-fold in those of Gunn rats. The corresponding increases were about 80- and 30-fold for both 9, 10- and 7,8-dihydrodiols. The rate of formation of 4,5-dihydrodiol increased 18-fold in hepatic microsomes from both Wistar and Gunn rats. The amounts of phenols and quinones increased relatively less. TCDD pretreatment also increased the metabolism of benzo(a)pyrene in the microsomes of the lung and kidney, but the increase was relatively smaller than in the liver. Benzo(a)pyrene was metabolized faster by the microsomes of the kidney from Gunn rats than by those from Wistar rats. The amount of covalently bound metabolites of benzo(a)pyrene in the microsomal protein of the liver increased about 20-fold after TCDD treatment, but no significant changes could be detected in the lung or kidney.     

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.     

Stimulation of drug and carcinogen metabolism by prolonged oral tobacco consumption

Oral administration of tobacco to rats for 21 days caused remarkable stimulation of the metabolism of phenacetin, aniline and benzo[a]pyrene, a carcinogen, by hepatic microsomal mixed function oxidases (MFO). Such treatment for 6 days resulted in a small increase in the activities of phenacetin O-dealkylase and aromatic hydrocarbon hydroxylase (AHH) without affecting aniline hydroxylase activity. Nicotine given orally was found to be a relatively weak inducer of phenacetin O-dealkylase and aniline hydroxylase, and elicited a maximum increase in their activities within 6 days which remained unchanged even after 21 days of continuous administration. However, these two enzyme systems were not affected following only one or two doses of tobacco and nicotine. Both tobacco and nicotine inhibited these biotransformations in vitro.     

The effect of cimetidine on in vitro and in vivo microsomal drug metabolism in the rat

The effect of cimetidine on rat liver microsomal drug metabolism in vitro and in vivo was studied. Cimetidine inhibits aminopyrine N-demethylation and benzo[a]pyrene hydroxylation in a noncompetitive manner with inhibition constants between 1 and 10 mM. Benzo[a]pyrene hydroxylation in liver microsomes from 3-methylcholanthrene-pretreated rats is not appreciably inhibited by cimetidine indicating some specificity in terms of different cytochrome P-450 forms. Cimetidine gives rise to a type II spectral change with a spectral dissociation constant of about 0.1 mM. The prolonged administration of cimetidine does not result in the induction of hepatic drug metabolism. Pretreatment of rats with cimetidine prolongs aminopyrine half-life and hexobarbital sleeping time. These results demonstrate that cimetidine is an in vitro inhibitor of microsomal drug metabolism in the rat and this inhibition leads to pharmacokinetic drug-drug interactions in vivo.     

Dose-dependent actions of thyroxine on hepatic drug metabolism in male and female rats

Studies were carried out to compare the effects of various doses of thyroxine (T₄) on hepatic drug metabolism in male and female rats and to evaluate the role of the pituitary gland in the modulation of T₄ action. Administration of small amounts of T₄ (2.5 to 5 μg/100g body wt/day) to hypophysectomized rats of either sex increased hepatic ethylmorphine demethylase, benzo(a)pyrene hydroxylase and aniline hydroxylase activities. Larger amounts of T₄ (12.5 to 50 μg) reversed the stimulatory effects of the smaller doses. T₄ treatment produced dose-dependent decreases in hepatic cytochrome P-450 content and increases in NADPH-cytochrome c reductase activity in hypophysectomized rats of both sexes. Qualitatively similar effects were produced by T₄ administration to thyroidectomized male and female rats. However, larger doses of T₄ were required for maximum stimulation of drug metabolism in thyroidectomized than in hypophysectomized animals. The results indicate that physiological amounts of T₄ Uniformly stimulate hepatic drug metabolism in both male and female rats. Supraphysiological amounts, however, inhibit metabolism of some substrates and produce sex differences in T₄ actions. The effects of T₄ are demonstrable in the absence of the pituitary gland but pituitary-dependent factors appear to modulate the magnitude of the response to T₄.     

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.     

Effect of cadmium on phenobarbital and polycyclic hydrocarbon-induced alterations in hepatic microsomal monooxygenase activity in the rat

The heavy metal, cadmium, is a potent inhibitor of the hepatic microsomal monooxygenase enzyme system in male, but not female, rats. The selectivity of this inhibitory effect of Cd was further examined by utilizing rats treated with inducers of this drug metabolizing enzyme system. Animals received phenobarbital (PB) sodium (100 mg/kg, ip, 72, 48, and 24 hr before sacrifice), or 3-methylcholanthrene or benzo[a]pyrene (20 mg/kg, ip, 72 and 48 hr prior to sacrifice) as inducers. Designated groups of these animals also received cadmium (1.0 mg Cd²⁺/kg), ip) either 120 or 72 hr before sacrifice. Noninduced male rats exhibited significant decreases in cytochrome P-450 content and drug-metabolizing enzyme activity following Cd treatment. The magnitude of the reductions in drug-metabolizing enzyme activity produced by Cd paralleled the magnitude of the sex difference in biotransformation of the substrate examined. Phenobarbital-treated male rats receiving a simultaneous Cd injection (72 hr prior to sacrifice) were also sensitive to Cd-induced inhibitions in cytochrome P-450 content and monooxygenase activity, although the extent of the reductions produced by Cd in PB-treated animals was less than that observed in noninduced male rats. In PB-treated male rats receiving a prior dose of Cd (120 hr before sacrifice), only the metabolism of the highly sex-dependent substrate, ethylmorphine, was significantly reduced. Cytochrome P-448 levels, and cytochrome P-448-mediated biotransformations which are elevated following hydrocarbon treatment, were not decreased by either simultaneous or prior Cd administration to male rats. Control, PB-treated, and hydrocarbon-treated female rats were not susceptible to Cd-induced reduction in hemoprotein content or inhibition of drug-metabolizing enzyme activity with the exception of those animals receiving both benzo[a]pyrene and Cd, which displayed slight but significant reductions in the oxidation of sex-dependent substrates. These results demonstrate the selective nature of the inhibitory effects of Cd upon drug metabolism in the rat.     

Comparative effects of ethanol administration on hepatic monooxygenases in rats and mice

The inducing effects of chronic ethanol ingestion on hepatic monooxygenases in Sprague-Dawley and Long-Evans rats, and A/J and C57BL/6J mice, were studied. Cytochrome P-450 content was significantly increased in livers of all animals receiving the experimental ethanol-containing liquid diet. The CO-difference spectra of microsomes from ethanol-treated animals showed a shift in the absorbance maximum to 451–452 nm, compared to the absorbance maximum of 450 nm observed with microsomes from control animals. Ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities in livers of ethanol-treated animals were minimally affected. The shift in the absorbance maxima to longer wavelengths in the CO-difference spectrum and the minimal effects on the metabolism of ethylmorphine and benzo[a]pyrene demonstrate that ethanol differs in its inducing properties, when compared to the properties of the two widely used hepatic microsomal enzyme inducers, phenobarbital and 3-methylcholantrhene. In contrast to the minimal effects observed on the metabolism of ethylmorphine and benzo[a]pyrene, several fold increases were observed in hepatic 7-ethoxycoumarin 0-deethylase and aniline hydroxylase activities in the treated animals. Polyacrylamide gel electrophoresis of hepatic microsomes from those animals receiving ethanol revealed protein band(s) in the cytochrome P-450 molecular weight region, the intensities of which were markedly increased relative to that from control animals. The heme-associated peroxidase activity was also increased in the same molecular weight region. The results of the present spectral, catalytic, and electrophoretic studies demonstrate that in mice, as in rats, chronic ethanol treatment causes the induction of specific cytochrome(s) P-450 with preferential activity toward aniline and 7-ethoxycoumarin.     

Lithium-induced increase in the metabolism of benzo[a]pyrene and drugs in rat liver.

Administration of lithium chloride (2.5 mEq/kg/day) to rats for 4 or 12 days increased the rates of hepatic hydroxylation of benzo[a] pyrene, antipyrine and zoxazolamine by 100, 114 and 56 per cent, respectively, and the rate of hepatic conjugation of 4-methylumbelliferone with glucuronic acid by 19 per cent. Liver lithium levels were highly correlated with plasma lithium levels but not with hepatic benzo[a] pyrene hydroxylase activities. In vitro addition of lithium to liver 10,000 g supernatants did not affect the hydroxylation of benzolalpyrene, suggesting that the lithium-induced increase in hepatic drug hydroxylation is not due to enzyme activation and is probably a result of lithium-mediated enzyme induction.     

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.     

Effect of some 6-substituted benzo(a)pyrene derivatives upon liver enzymes and ascorbic acid excretion in mice

The effects of benzo[a]pyrene, 6-hydroxymethylbenzo[a]pyrene, benzo[a]-pyrene-6-carboxaldehyde, 6-methylbenzo[a]pyrene, benzo[a]pyrene-6-carbonitrile and benzo[a]pyrene-6-carbonamide on urinary ascorbic acid excretion and hepatic tyrosine transaminase, aniline hydroxylase, aminopyrine N-demethylase and hexobarbital metabolizing activity in mice were examined. The effect of 6-bromobenzo[a]pyrene on the hepatic enzymes was examined. None of the compounds altered aminopyrine N-demethylase or hexobarbital metabolizing activity whilst all produced a statistically significant rise in aniline hydroxylase activity. The tyrosine transaminase activity was increased by 6-hydroxymethylbenzo[a]pyrene but was unaffected by the other compounds. All the compounds raised urinary ascorbic acid excretion after an initial 3 day lag. The level of excretion remained elevated for about a week or more except for 6-hydroxy-methylbenzo [a]pyrene-treated animals which showed a high raised level of excretion for 1 day followed by a drop to a lower than normal level of excretion. The significance of these observations and their relation to other work was discussed.     

Induction of benzo[a]pyrene hydroxylase in skin and liver by cutaneous application of jute batching oil

Skin paintin with 30 μl of jute barching oil (JBO) for 8 days resulted in increased gross liver weight, microsomal protein content and benzo[α]pyrene hydroxylase activity of liver. Skin benzo[a]pyrene hydroxylase activity at the treated site increased by 10-fold. An investigation of cytochrome pigment status in liver and skin of treated animals showed a specific increase in P-448 level in both tissues. Single skin applications of JBO elevated the level of skin and liver benzo[a]pyrene hydroxylase activity to its maximum after 1 day and 2 days, respectively, which, in absence of further treatment with mineral oil, declined gradually to normal levels in due course. The results suggest that single or multiple cutaneous exposure(s) with JBO can increase carcinogen metabolising status of skin and liver which maybe one of the causative factors for the tumorigenic effects of JBO in skin.     

Xenobiotic biotransformation in wild birds: Activity,induction, characterization and comparison with rat and mouse microsomal enzymes

1. Benzo[a]pyrene hydroxylase, aminopyrine demethylase and glutathione S-trans-ferase activities in hepatic and extrahepatic tissues of five wild birds, rat and mouse were compared.

2. Hepatic benzo[a]pyrene hydroxylase of wild pigeon was at least three times higher than that of rat or mouse. Hepatic aminopyrine demethylase and GSH S-transferase activities of wild birds were lower than those of rodents.

3. Renal GSH S-transferase of wild birds was 2-3 times more than in rat and mouse.

4. Avian benzo[a]pyrene hydroxylase activity of hepatic and renal tissue was linear up to 3.0?mg enzyme protein with pH optima of 7.4 and 7.2, at 42°C Apparent K_m values were 11.76 and 3.33 μM respectively.

5. Hepatic enzyme activity was induced four-fold by 3-methylcholanthrene and about two-fold by phenobarbitone administration.     

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.     

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.