Effect of the structural isomer N-3-fluorenylacetamide on microsomal binding and hydroxylation of the carcinogen N-2-fluorenylacetamide

N-2-Fluorenylacetamide (2-FAA), a carcinogen, or N-3-fluorenylacetamide (3-FAA), the noncarcinogenic isomer, when added to hepatic microsomes from 3-methylcholanthrene (3-MC)-treated rats, in phosphate buffer-l,2-propanediol, exhibited equal affinity for cytochrome P₁-450 as indicated by equal binding constants (K_s). Both isomers were bound to the same site on cytochrome P₁-450 and displaced each other from the common binding site, as shown by competitive inhibition. Microsomal hydroxylation of 2-FAA to N-OH-2-FAA and to 7-OH-2-FAA was also inhibited by 3-FAA. The inhibitory effect was enhanced by omission of 1,2-propanediol from the incubation system. In contrast to the inhibition of the binding of 2-FAA by 3-FAA, the inhibition of hydroxylation of 2-FAA by 3-FAA was noncompetitive or uncompetitive. The contrasting patterns of inhibition of hydroxylation of 2-FAA by 3-FAA and of binding of 2-FAA by the isomer indicated that the binding site of 2-FAA is separate from the site of hydroxylation. This conclusion was supported by (1) a 10³-fold difference in the values of K_m, and K_m of 2-FAA, and (2) opposite effects of 1,2-propanediol on binding and hydroxylation of 2-FAA. Whereas binding of 2-FAA to cytochrome P₁-450 was increased with increasing concentrations of 1,2-propanediol, the microsomal formation of N-OH-2-FAA and of 7-OH-2-FAA was markedly diminished under the same conditions.     

Microsomal metabolism of arylamides by the rat and guinea pig--II. Oxidation of 3-fluorenylacetamide at carbon atom 9. Formation of 3-acetamido-9-fluorenone.

Previous work on the oxidation of N-3-fluorenyl acetamide at carbon atom 9 yielded two products, 9-hydroxy-3-fluorenylacetamide and 3-acetamido-9-fluorenone. The formation of the alcohol, 9-hydroxy-3-fluorenylacetamide, was catalyzed by a microsomal heme protein. The present study deals with the formation of the ketone, 3-acetamido-9-fluorenone, and with the question of whether the alcohol-forming and the ketone-forming enzymes are identical. 3-Acetamido-9-fluorenone was formed from 9-hydroxy-3-fluorenylacetamide, suggesting that 9-hydroxy-3-fluorenylacetamide is an intermediate in the formation of 3-acetamido-9-fluorenone. Cofactor requirements, pH optimum, effect of carbon monoxide and intracellular distribution indicated that the alcohol-forming and the ketone-forming activities are not identical. Unlike the alcohol-forming enzyme, the ketone-forming utilized NADP⁺ and NAD⁺ and does not appear to be a heme protein.     

Characterization of the activation of hepatic microsomal hydroxylation by betamethasone and α naphthoflavone

Biphenyl 2-hydroxylation is selectively activated in vitro by incubation of betamethasone or α naphthoflavone with control male rat liver microsomes. Biphenyl 3- and 4-hydroxylation activities are unchanged or marginally inhibited. The nature of the enzymes involved in the activation has been investigated. Metyrapone (1 mM) completely inhibited the expression of the activation but had a lesser effect on the basal 2-, 3- and 4-hydroxylation activities. SKF525A (1 mM) 2 inhibited both basal and betamethasone-activated enzyme activities by 25–35 per cent. Of other drug metabolizing enzymes investigated, only benzo[a]pyrene hydroxylation activity was increased by betamethasone and α naphthoflavone. Acetone (0.6M) caused a small activation (40 per cent) of biphenyl 2-hydroxylation but inhibited 4-hydroxylation. The non-ionic detergent Brij 35 inhibited biphenyl 2-, 3- and 4-hydroxylation. It was concluded that activation of biphenyl 2-hydroxylation differs from activation of aromatic amine hydroxylation and glucuronyl transferase but may be related to activation of benzo[a]pyrene hydroxylation by naphthoflavones.     

Microsomal metabolism of arylamides by the rat and guinea pig—I.: Oxidation of N-3-fluorenylacetamide at carbon-atom-9—A major metabolic reaction

The N-hydroxylation of N-3-fluorenylacetamide (3-FAA), an isomer of the carcinogen, N-2-fluorenylacetamide (2-FAA), by hepatic microsomes of untreated and 3-methylcholanthrene (3-MC)-treated guinea pigs was found to be of a similar low order as that previously observed in the rat. Hepatic microsomes of the guinea pig and of the rat converted 3-FAA to N-(9-hydroxy)-3-FAA and to N-(9-oxo)-3-FAA. These new metabolites were separated and identified by high-pressure liquid chromatography (h.p.l.c.). N-(9-hydroxy)-3-FAA was the major product of the hydroxylation of 3-FAA by hepatic microsomes of the rat or guinea pig exceeding the formation of N-(7-hydroxy)-3-FAA, the principal phenolic metabolite of 3-FAA. The amounts of N-(9-oxo)-3-FAA formed were about one-third of the amounts of N-(9-hydroxy)-3-FAA produced. In contrast to the formation of phenolic metabolites, the hydroxylation of 3-FAA to N-(9-hydroxy)-3-FAA was not increased by pretreatment of guinea pigs or rats with 3-MC. Similarly, pretreatment of rats with PB did not enhance the yield of N-(9-hydroxy)-3-FAA. Co inhibited the formation of N-(9-hydroxy)-3-FAA by 80 per cent. These data lead us to conclude that the formation of N-(9-hydroxy)-3-FAA is catalyzed by a microsomal hemoprotein not identical with cytochrome P₁-450 or P-450. In contrast to 3-FAA, 2-FAA appeared to be a poor substrate for hydroxylation to the N-(9-hydroxy)-2-FAA. The susceptibility of 3-FAA to hydroxylation at carbon-atom 9 of the fluorene moiety may be rationalized by resonance structures in which carbon-atom 9 is positively charged and acts as a electrophilic center. Similar resonance structures cannot be written for 2-FAA.     

In vivo and in vitro effects of 3-methylcholanthrene on the microsome-mediated in vitro mutagenicity of 2-acetylaminofluorene

Pretreatment of rat, hamster or mouse by 3-methylcholanthrene (3-MC) largely induces the liver microsomal N-hydroxylase activity. The same pretreatment given simultaneously with 2-acetylaminofluorene (2-AAF) inhibits the hepatocarcinogenicity in the rat but not in the hamster.The present report compares the in vivo and in vitro effects of 3-MC on liver microsomal N-hydroxylation and liver microsome-mediated mutagenicity of 2-AAF in hamster, rat and mouse. The induction of hamster or mouse liver microsomal N-hydroxylase activity correlates well with the increase in the microsome-mediated mutagenicity of 2-AAF. With rat, however, even though the N-hydroxylase activity is largely enhanced, microsome-mediated mutagenicity is significantly reduced after pretreatment with 3-MC. Such a reduction parallels a decrease in enzyme affinity.Added in vitro to the incubation medium, 3-MC (μM concentration) inhibits both the N-hydroxylase activity and the microsome-mediated mutagenicity of 2-AAF. Those data are discussed in relationship with the biological interactions between 3-MC and 2-AAF.     

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism of the β-carbolines harmine and harmol

The metabolism of the β-carbolines harmine and harmol by C57/BL10 mouse liver microsomes is reported. Marked changes in apparent Michaelis-Menten kinetics and metabolite profiles were induced differentially by phenobarbitone (PB) or 3-methylcholanthrene (MC). With control or PB-induced microsomes harmine was metabolised by both high- and low-affinity reactions (app. K_m = 1 and 29 μM for controls; app. K_m = 1.9 and 21 μM for PB-induced). Only the high-affinity reaction occurred following MC pretreatment, induced 31-fold to V_max = 22 nmoles·min^?1·mg protein^?1 (app. K_m = 0.4 μM). With control or PB-induced microsomes harmine was metabolised almost exclusively by O-demethylation to harmol (3-fold induction to V_max = 9.8 nmoles·min^?1·mg protein^?1, app. K_m = 73 and 14 μM respectively), which was probably the low-affinity reaction of harmine metabolism. With MC-induced microsomes harmine was metabolised mainly to two unidentified yellow products, with harmol as a minor metabolite formed by a high-affinity reaction (app. K_m = 0.6 μM). Control, PB- and MC-induced microsomes each metabolised harmol by a high-affinity reaction (app. K_m = 0.6–1.9 μM) to unidentified metabolites. Harmol metabolism was not induced by PB, but was induced by MC (22-fold to Vmax = 11 nmoles·min^?1·g protein^?1): this partly explains the relative lack of net harmol production after MC induction. The unidentified MC-induced yellow metabolites of harmine were not formed via harmol. Harmine and harmol metabolism showed the characteristics of cytochrome P-450 catalysed reactions. Harmine gave Type II cytochrome P-450 binding spectra with control and PB-induced microsomes, but a high-affinity Type I spectrum (K_s = 6.5 μM) with MC-induced microsomes. Harmol gave modified Type II spectra in all cases, with again higher-affinity binding to MC-induced microsomes (K_s = 62 μM) than to control (K_s ? 500 μM) or PB-induced microsomes (K_s = 500 μM).     

The effects of harman and norharman on the metabolism of benzo(a)pyrene in isolated perfused rat lung and in rat lung microsomes.

The effects of harman and norharman, nitrogen-containing pyrolysis products of amino acids present in cigarette smoke, on the metabolism of benzo(a)pyrene in rat lung microsomes in vitro and in isolated perfused rat lung were studied. In rat lung microsomes, both harman and norharman inhibited the metabolism of benzo(a)pyrene (BP) to dihydrodiols, phenols and quinones at concentrations over approximately 0.05 mM. The formation of BP-7, 8-dihydrodiol and BP-9, 10-dihydrodiol was inhibited more than that of BP-4, 5-dihydrodiol. No appreciable differences in inhibition were seen between microsomes from control or 3-methylcholanthrene-pretreated rats. In isolated perfused rat lung, 1 mM of harman in the perfusion fluid inhibited the formation of ethyl acetate-soluble metabolites of benzo(a)pyrene except BP-9, 10-dihydrodiol, and inhibited the total covalent binding of benzo(a)pyrene metabolites to lung tissue macromolecules. 0.03 mM of harman seemed to increase other metabolites than BP-7,8-dihydrodiol without changing the total covalent binding. These results suggest that at most concentrations both β-carboline derivatives, harman and norharman, inhibit benzo(a)pyrene metabolism and covalent binding both in lung microsomes in vitro and in isolated perfused rat lung.     

Biotransformation of 4-dimethylaminophenol in the isolated perfused rat liver and in the rat

Isolated rat livers were perfused with various concentrations of 4-dimethylaminophenol-[¹⁴C] (DMAP). During single pass perfusion with modified protein-free Krebs-Henseleit solution up to 70μM prehepatic 4-dimethylaminophenol (DMAP) were metabolized by the liver. The main route of biotransformation was conjugation. At steady state conditions glucuronide formation showed an apparent V_max of 8.5 μmoles × min^?1 × g protein^?1, and K_m of 562 μM, whereas sulfate formation had an apparent V_max of 1.2 and a K_m of 35. Thus, at low substrate concentration the sulfate conjugation outweighed glucuronidation whereas at high substrate concentration the ratio of conjugates was reversed. In contrast to DMAP-sulfate, some DMAP-glucuronide was stored by the liver and was released with a half life of about 15 min which showed positive correlation with the dose of DMAP during the washout period. Perfusion with human or rat erythrocytes demonstrated the other important path of biotransformation of DMAP within erythrocytes, namely thioether formation with glutathione and SH-groups of hemoglobin. The pattern of DMAP-conjugation was affected depending on the time of prehepatic exposure to erythrocytes, and the species of red cells. The results obtained from the isolated metabolic system resemble the hepatic part of the overall metabolism under in vivo conditions.     

Isomerically pure bromobiphenyl congeners and hepatic mono-oxygenase activities in the rat: influence of position and degree of bromination.

Isomerically pure di-, tri-, tetra-, penta-, hexa-, and octabromobiphenyls were injected ip into weanling rats at a dosage of 0.2 mmol/kg/day for 3 consecutive days and the animals were killed 96 hr after the last injection. The influence of position and degree of bromination of the biphenyl nucleus on hepatic function was assessed by measuring changes in activities of hepatic p-nitroanisole O-demethylase (OD) and aniline hydroxylase (AH), by examining morphological changes by electron microscopy, and by comparing the results with those of animals treated with equivalent doses of Firemaster BP6. Bromobiphenyl residues were extracted from liver and analyzed by gas-liquid chromatography. Hepatic O-demethylase activities were increased proportionally as the degree of bromination increased, observations which correlated well with the increased tissue residues and changes in morphology. Marked increases in aniline hydroxylase were observed following treatment with certain di- and tribromobiphenyl congeners but, as the degree of bromination increased above four atoms per molecule, aniline hydroxylase activity was reduced to levels equal to or below those of control, vehicle-treated animals. In contrast, increases in both mono-oxygenases were observed following treatment with Firemaster BP6, fivefold increases for OD and twofold for AH. In vitro experiments with highly brominated congeners suggested that these influenced the AH assay by interacting with microsomal cytochrome P-450.     

Degradation of endomorphin-2 at the supraspinal level in mice is initiated by dipeptidyl peptidase IV: an in vitro and in vivo study

Endomorphin-2 (Tyr-Pro-Phe-PheNH(2)) was discovered as an endogenous ligand for the mu-opioid receptor. The physiological function of endomorphin-2 as a neurotransmitter or neuromodulator may cease through the rapid enzymatic process in the synapse of brain, as for other neuropeptides. The present study was conducted to examine the metabolism of endomorphin-2 by synaptic membranes prepared from mouse brain. Major metabolites were free tyrosine, free phenylalanine, Tyr-Pro and PheNH(2). Both the degradation of endomorphin-2 and the accumulation of major metabolites were inhibited by specific inhibitors of dipeptidyl peptidase IV, such as diprotin A and B. On the other hand, the accumulation of Phe-PheNH(2) and Pro-Phe-PheNH(2) was increased in the presence of bestatin, an aminopeptidase inhibitor, whereas that of free phenylalanine and PheNH(2) was decreased. Furthermore, purified dipeptidyl peptidase IV hydrolyzed endomorphin-2 at the cleavage site, Pro(2)-Phe(3) bond. Thus, degradation of endomorphin-2 by brain synaptic membranes seems to take place mainly through the cleavage of Pro(2)-Phe(3) bond by dipeptidyl peptidase IV, followed by release of free phenylalanine and PheNH(2) from the liberated fragment, Phe-PheNH(2) by aminopeptidase. We have also examined that the effect of diprotin A on the antinociception induced by intracerebroventricularly administered endomorphin-2 in the mouse paw withdrawal test. Diprotin A simultaneously injected with endomorphin-2 enhanced endomorphin-2-induced antinociception. These results indicate that dipeptidyl peptidase IV may be an important peptidase responsible for terminating endomorphin-2-induced antinociception at the supraspinal level in mice.     

Species and sex differences in the metabolism of a chlorinated epoxide by hepatic microsomal enzymes

A dimethylated chlorocyclodiene epoxide (DME), proposed as a suitable substrate for the study of microsomal monooxygenases in rats, yields two major metabolites, M1 and M2, when incubated with NADPH-supplemented hepatic microsomes from adult male rats but only one when incubated with those from adult female rats. Similar microsomes from male and from female mice, pigeons and chickens produce both metabolites. The ratio of the amounts of the two metabolites formed varies with the species but no sex difference within any one of these species has been demonstrated. The rate of metabolism of DME varies both with sex and with species: hepatic microsomes from female mice metabolise it faster than do those from males whereas hepatic microsomes from male rats metabolise it faster than do those from females. Hepatic microsomes from both sexes of the domestic chicken metabolise DME slowly, converting it predominantly to metabolite M2. The use of [¹⁴C]DME has established that the apparent absence of metabolite M2 from the incubates containing liver preparations from female rats is not attributable to low metabolism nor is it caused by metabolite M2 being converted to other metabolites as soon as it is formed. Little radioactivity remains behind in the aqueous phase after hexane extraction of incubation media containing [¹⁴C]DME and female rat liver microsomes. Florisil column separation of the components of the hexane extracts indicates that little radioactivity is present in eluate fractions other than those showing a g.l.c. peak for DME or for metabolite Ml or for metabolite M2. Hepatic microsomes from young rats of both sexes metabolise DME at similar rates until they approach 28 days of age. Thereafter, the rate in those from male rats increases whereas that from those from female rats falls. The hepatic microsomes from male rats castrated when seven days of age metabolise DME in adulthood at the same rate as those from intact female rats. The relevance of these observations to the proposed use of DME is discussed.     

A comparative study on the effects of alpha-hexachlorocyclohexane and its metabolite beta-pentachlorocyclohexene on growth and monooxygenase activities in rat liver

The present study adds support to the hypothesis that β-pentachlorocyclohexene (β-PCH) is a primary intermediate in α-hexachlorocyclohexane (α-HCH)4 metabolism in the rat. Degradation of α-HCH to β-PCH was shown to occur in vitro and in vivo, partially by non-enzymic catalysis. β-PCH accumulated in liver and adipose tissue of α-HCH treated rats, which had received the glutathione-lowering agent diethyl maleate. β-PCH disappears from the body much more rapidly than the parent compound α-HCH: about 50 per cent of a single i.p. dose were degraded within 2.5 hr, while half-life of α-HCH is known to be approximately 130 hr. To maintain equimolar liver concentrations, β-PCH must be given in doses 100-fold higher than α-HCH. β-PCH and α-HCH were fed for a period of ten days at various dose levels to give steady-state liver concentrations. It was found that β-PCH has similar hepatic effects to α-HCH: both agents induced liver growth and a phenobarbital-type pattern of monooxygenase activities, as measured by the following substrates: aminopyrine, ethylmorphine, benzphetamine, 4-nitroanisole, aniline, benzo[α]pyrene, ethoxyresorufin and 2,5-diphenyl-oxazole. Threshold doses for these effects were 30–43 μmoles/kg/day for β-PCH and 1.0–1.7 μmoles/kg/day for α-HCH. However, on the basis of molar hepatic concentrations β-PCH was a more potent inducer than α-HCH (2–10 times). Threshold concentrations ranged from 0.4 to 0.6 nmoles β-PCH/g liver and from 0.7 to 1.5 nmoles α-HCH/g liver. β-PCH concentrations in livers of rats treated even with high doses of α-HCH were below the threshold for induction of liver growth and of monooxygenase increases. It is, therefore, highly unlikely that β-PCH is responsible for the effect of α-HCH on rat livers.     

Structure-activity relationships in the inhibitory effects of ellipticines on benzo(a)pyrene hydroxylase activity and 3-methylcholanthrene mutagenicity

The structural features which determine the ability of ellipticine (5,11dimethyl6Hpyrido[4-3b] carbazole) and its derivatives to interact with cytochrome P-450 and to inhibit rat liver microsomal benzo(a)pyrene hydroxylase as well as to inhibit the mutagenicity of 3-methylcholanthrene have been studied. Spectral interactions studies were carried out with either Aroclor 1254-, 3-methylcholanthrene-or phenobarbital-induced microsomes. Inhibitory activities towards benzo(a)pyrene hydroxylase and 3-methylcholanthrene mutagenicity (Ames test), were determined using Aroclor 1254-induced microsomes. It appears that every ellipticine derivative having significant inhibitory effects on hydroxylation of benzo(a)pyrene or mutagenicity of 3-methylcholanthrene also exhibits a very good affinity for microsomal cytochromes P-450. The accessibility of the pyridinic nitrogen of ellipticine derivatives appears as the most important factor for their binding to cytochromes P-450 and the presence of methyl groups in 5 and 11 positions of ellipticine derivatives is an essential condition for the expression of the inhibitory power. Various substitutions in the A ring of ellipticine appear to be of secondary importance. On the other hand the location of the pyridinic ring and consequently the arrangement of the molecule within the hydrophobic pocket of cytochrome P-450 seems also to play an important role in the inhibitory power since isoellipticines are devoid of such properties. These results should help in the design of particularly efficient inhibitors of drug and carcinogen metabolism.     

Effects of chronic ethanol administration in the rat: Relative dependency on dietary lipids—I.: Induction of hepatic drug-metabolizing enzymes in vitro

Female Sprague-Dawley rats were fed nutritionally adequate liquid diets with or without ethanol, at two ethanol concentrations, 5 and 6% (w/v). In other animals, various degrees of caloric deficiency were obtained by replacing ethanol by water in one animal of a pair. Ethanol given as a 5% (w/v) solution with high amounts of dietary fat increased cytochrome P-450, the activities of NADPH-cytochrome P-450 reductase, benzphetamine demethylation, aniline hydroxylation and microsomal ethanol-oxidizing system (MEOS). When ethanol was given with a low fat diet as a 5% (w/v) solution, the increase in cytochrome P-450 and P-450 reductase was much less than with a high fat diet; the other enzyme activities, however, were enhanced to a level comparable to that achieved with the high fat diet. When ethanol was administered as a 6% (w/v) solution in presence of a low fat diet, caloric deficiency was observed and no significant induction of any parameter except aniline hydroxylation could be found. When it was given with a high fat diet, in spite of caloric deficiency and lower ethanol ingestion, cytochrome P-450 and P-450 reductase activities were enhanced while that of MEOS was not. Ingestion of ethanol as a 6% (w/v) solution with a high fat diet resulted in a negligible weight gain. Higher basal levels of cytochrome P-450, P-450 reductase and benzphetamine demethylation activities were found in animals rendered caloric-deficient. Ethanol is associated with a greater induction of drug-metabolizing enzyme activities in the high fat model compared to the low fat model. Induction of drug-metabolizing enzymes by ethanol is partly dependent on dietary lipids as well as on the amounts of ethanol ingested and on the caloric status of the animal.     

Mutagenic activation of 2,4-diaminoanisole and 2-aminofluorene in vitro by liver and kidney fractions from aromatic hydrocarbon responsive and nonresponsive mice.

The mutagenicity of the two carcinogenic arylamines 2,4-diaminoanisole (2,4-DAA) and 2-aminofluorene (AF) was compared using liver and kidney fractions from two aromatic hydrocarbon (3-methylcholanthrene, MC) responsive and two nonresponsive mouse strains. MC pretreatment of mice caused an increase in 2,4-DAA mutagenicity with liver fractions from all four strains; however, much higher increases were seen in the two responsive than in the two nonresponsive strains. Kidney fractions had very low basal 2,4-DAA mutagenic activity. MC treatment led to 14–27-fold increase in 2,4-DAA mutagenicity in the responsive C57BL/6/BOM (B6) strain, but not in any of the other strains. AF mutagenicity was increased with liver fractions from all four mouse strains, but to the greatest extent in the B6 mice. AF showed high basal mutagenic activity with kidney fractions from all four strains, but MC treatment did not cause any increase in AF mutagenicity in any of the strains. Thus, there was a clear difference in the pattern of metabolic activation of the two arylamines 2,4-DAA and AF by liver and kidney fractions in mice, both with respect to constitutive activities and to the response to aromatic hydrocarbons.     

Enzymatic sulfation of steroids--XX. Effects of ten drugs on the hepatic glucocorticoid sulfotransferase activity of rats in vitro and in vivo

The effects of ten drugs on hepatic glucocorticoid sulfotransferase activity (HGSTA) were examined in male rats. The enzyme activity per 100 g body weight was elevated 152, 94.9, 140, 140, 73.1, 63.9, 76.9, and 140% after administration of daily i.p. doses of 111 mg spironolactone/kg (6-10 days), 66.7 mg WIN-24540/kg (6-10 days), 150 mg metyrapone/kg (19-31 days), 33.3 mg pentachlorophenol/kg (9-16 days), 16.5 mg aspirin/kg (10-16 days), 90.5 mg alloxan/kg (23.27 days), 104 mg aminoglutethimide/kg (12-20 days), and 16.8 mg propranolol/kg (21-27 days). Shorter experimental periods or lower drug doses caused smaller effects on HGSTA. Most notably, spironolactone (111 mg/kg) and WIN-24540 (66.7 mg/kg) caused 50-75% elevation of HGSTA in 2 days. Effects of WIN-24540, aspirin and pentachlorophenol were due mostly to elevation of hepatic levels of sulfotransferase III (STIII), the glucocorticoid-preferring sulfotransferase of rat liver. Effects of the other test drugs were due to elevation of hepatic levels of sulfotransferases I and II (STI and STII), which much prefer dehydroepiandrosterone as substrate, but also catalyze glucocorticoid sulfation. Enzyme inhibition studies showed that the test drugs interacted with the HGSTA in vitro in a fashion that appeared to be related to the in vivo effects already described. None of the drugs interacted exclusively with STI, STII or STIII in vitro. However, some differences of the strengths of individual drug-sulfotransferase interactions were observed. The drug effects are discussed in relation to drug and glucocorticoid actions.     

Effects of microsomal enzyme inducers in vivo and inhibitors in vitro on the covalent binding of benzo[a]pyrene metabolites to DNA catalyzed by liver microsomes from genetically responsive and nonresponsive mice.

The in vitro DNA binding of benzo[a]pyrene metabolites generated by mouse liver microsomes can be resolved into at least nine distinct peaks by elution of a Sephadex LH20 column with a water-methanol gradient. These peaks, representing metabolite-nucleoside complexes, are named A (most polar) through I (least polar). 3-Methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital, Aroclor 1254, pregnenolone-16α-carbonitrile, or ethanol was administered in vivo to genetically “responsive” C57BL/6N or “nonresponsive” DBA/2N mice, in an attempt to understand and identify increases or decreases in reactive BP intermediates that bind to DNA. Rises or falls in these peaks are also noted when liver microsomes from control or 3-methylcholanthrene-treated C57BL/6N or DBA/2N mice were incubated in vitro with [³H]benzo[a]pyrene and microsomal enzyme inhibitors such as α-naphthoflavone, metyrapone or cyclohexene oxide. All of our interpretations concerning the binding of metabolites to DNA are consistent with non-K-region oxygenation of benzo[a]pyrene being mediated predominantly by cytochrome(s) P₁-450 and K-region oxygenation of benzo[a]pyrene being catalysed predominantly by form(s) of P-450 other than P₁-450. All of the biological perturbations are consistent with the following assignments. The major reactive intermediate of benzo[a]pyrene contributing to each peak is suggested to be: peaks A and C, an unknown dihydrodiol oxide; peaks B, D, F and I, quinones oxygenated further (or quinone-derived free radicals); peak E, both cis- and tans-7,8-diol-9,10-epoxides; peak F′, the 7.8-oxide; peak G, the 4.5-oxide; and peak H, an unknown phenol oxide. The DNA nucleosides are not identified in this study. Of the ten peaks listed here, it is of interest that the major metabolite(s) contributing to eight of the peaks (all except peaks F' and G) involve(s) more than a single mono-oxygenation by forms of cytochrome P-450. All peaks, with the exception of peak G, appear to be predominantly associated with benzo[a]pyrene metabolism mediated by P₁-450 and, therefore, controlled by the Ah locus. The use of these microsomal enzyme inducers or inhibitors—combined with the underlying genetic predisposition of the individual, tissue, or cell culture system under study—demonstrates that the balance between P-450 and epoxide hydrase, and the ratio of each form of P-450 to the other forms of P-450, can influence markedly the quantity and quality of reactive intermediates of benzo[a]pyrene that bind to DNA.     

Induction of hepatic microsomal cytochrome P-448-mediated oxidases by 3,3′-Dichlorobenzidine in the rat

Intraperitoneal administratioin of the hepatocarcinogen 3,3′-dichlorobenzidine (4,4′-diamino, 3,3′-dichlorobiphenyl) to adult male rats caused the induction of hepatic microsomal ethoxycoumarin O-deethylase and p-nitrophenetole O-deethylase activities comparable in magnitudes to those induced by 3-methylcholanthrene; neither anilin hydroxylase nor aminopyrine N-demethylase activity was affected by the pretreatment. The induction was not accompanied by a significant increase in content of hepatic microsomal cytochrome P-450; however, a shift in the absorption maxium of the reduced + CO spectrum of the cytochrome to 448 nm and an increase in the ratio of the 455 nm : 430 nm peaks of the reduced + ethylisocyanide spectrum of the hemoprotein was affected. Arylhydrocarbon hydroxylase activitity was stimulated 5-fold by dichlorobenzidine pretreatment in comparison with a 12-fold stimulation following 3-methylcholanthrene pretreatment. However, enzymatically mediated covalent binding of benzo[a]pyrene to microsomal protein was greater in microsomes from dichlorobenzidine-pretreated rats than in those from methylcholanthrene-pretreated rats. All of the dichlorobenzidine-induced enzymic activities were inhibited by α-naphthoflavone but not by SKF-525A. Hepatic microsomes from dichlorobenzidine than those from untreated animalsl both sets of microsomes elicited the Type II spectral change on combination with the compound, albeit with different binding affinities and capacities. The results show that dichlorobenzidine, although only a dihalogenated biphenyl derivative, is a potent inducer of cytochrome P-448.