2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated depression of rat testicular heme synthesis and microsomal cytochrome P-450

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces hirsutism, alopecia, and chlorance, symptoms that suggest a possible alteration of endocrine function. Therefore, the effects of TCDD on rat testicular cytochrome P-450 content were investigated. Forty-eight hours after a single, oral dose of TCDD (25 μg/kg) testicular microsomal cytochrome P-450 levels were depressed by approximately 24%. Microsomal cytochrome P-450 continued to decrease to 62% of control levels at 4 days and remained at approximately the same levels 7 days following treatment. Testicular microsomal heme content exhibited a similar pattern after administration of TCDD. No alterations in testicular δ-aminolevulinic acid (ALA) synthase were detected. The incorporation of [¹⁴C]ALA into microsomal heme was decreased to approximately 36% of control values at 24 hr after TCDD administration. Testicular weights were not altered during the 7-day experimental period. These data suggest that TCDD depresses cytochrome P-450 levels in the rat testis through an inhibition of the synthesis of testicular heme.     

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes

The binding constants of an homologous series of n-alkyl (C₂-C₁₀) carbamates

to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/K_s) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. The major metabolites of C₄-C₈ carbamates were unconjugated ω-1 oxidation products. Below hexyl carbamate only the ω-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the ω-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.     

Cumene hydroperoxide-supported microsomal hydroxylations of warfarin—A probe of cytochrome P-450 multiplicity and specificity

The hepatic microsomal metabolism of R and S warfarin, supported by NADPH or cumene hydroperoxide, has been investigated to probe the multiplicity and specificity of cytochromes P-450. Microsomes were uninduccd, and phenobarbital (PB)-, 3-methylcholanthrene (MC)- or 3β-hydroxy-20-oxopregn-5-ene-16-α-carbonitrile (PCN)-induced from rat liver. Cumene hydroperoxide supported the formation of all the NADPH-supported warfarin metabolites (4′-, 6-, 7- and benzylic hydroxywarfarin and dehydrowarfarin). except 8-hvdroxywarfarin. Comparisons of the rates of formation of the metabolites supported by NADPH or cumene hydroperoxide (with uninduced and induced microsomes) revealed that cumene hydroperoxide had the following effects: (1) rates of hydroxylation of the phenyl substituent of warfarin (4′-hydroxywarfarin) were increased; (2) rates of metabolism of the aliphatic portion of warfarin (benzylic hydroxywarfarin and dehydrowarfarin) were increased, except with S warfarin and uninduced microsomes; and (3) rates of hydroxylation of the phenyl ring of the coumarin group of warfarin were (a) decreased (7-. 8-hydroxywarfarin) or (b) decreased (6-hydroxywarfarin) with MC-induced microsomes and increased or unchanged with uninduced and PB- or PC'N-induced microsomes. We concluded from these studies that multiple cytochromes P-450 are implicated in the metabolism of warfarin: that the cytochromes P-450 catalyzing the formation of 7- and 8-hydroxywarfarin differ from those catalyzing the other metabolites. except foro-hydroxylation by MC-induced microsomes: that the cytochromes catalyzing 7- and 8- hydroxywarfarin formation differ from one another; that for each metabolite of warfarin, the cytochrome P-450 type predominantly responsible for its formation is the same. irrespective of the mode of induction of the microsomes: and that 6-hydroxylase activity is the exception to the previous point, and is predominantly associated with different cytochromes P-450 in differently induced microsomes. The effects of cumene hydroperoxide have been ascribed to differences in cumene hydroperoxide affinities, differences in cumene hydroperoxide-induced destruction, and differences in cumene hydroperoxidc inhibitions of warfarin binding to different cytochromes P-450. together with differences in the situation of cytochromes P-450 in the microsomal membrane.     

Comparison of the effects of methyl-N-butyl ketone and phenobarbital on rat liver cytochromes P-450 and the metabolism of chloroform to phosgene

It was previously shown that treatment of rats with methyl-n-butyl ketone (MBK) produced an increase in the total level of liver microsomal cytochromes P-450 and an increase in the rate of metabolism of chloroform (CHCl3) to phosgene (COCl2). In the present study it was found that MBK also produced qualitative changes in the composition of microsomal cytochromes P-450 in rat liver as determined by anion-exchange chromatography. The degree of the chromatographic changes paralleled the effect of MBK on the rate of metabolism of CHCl3 to COCl2 and CHCl3-induced hepatotoxicity, suggesting that MBK potentiated the hepatotoxicity of CHCl3, at least in part, by inducing the formation of cytochromes P-450 that metabolized CHCl3 to the hepatotoxin COCl2. In this regard, reconstitution studies with a form of cytochrome P-450 isolated from rat liver microsomes from rats treated with MBK or phenobarbital (Pb) showed unequivocally that cytochrome P-450 can metabolize CHCl3 to COCl2. Although analysis of rat liver microsomes by SDS-polyacrylamide electrophoresis and anion-exchange chromatography suggested that MBK and Pb had similar effects on the composition of cytochromes P-450, metabolism studies indicated that differences did exist.     

The interaction of hepatic microsomal cytochrome P-450 with fluroxene (2,2,2-trifluoroethyl vinyl ether) in vitro.

The anaesthetic agent fluroxene (2,2.2-trifluoroethyl vinyl ether) and a closely related compound 2,2,2-trifluoroethyl ethyl ether (TFEE) interact with the cytochromc P-450 component of isolated rat hepatic microsomcs to produce a type I difference spectrum. The extent of the absorbance difference (ΔA) between λ_max (390 nm) and λ_min (420 nm) produced with fluroxene or TFEE is dependent on the concentration of the anaesthetic agent and the extent and type of prior induction of the microsomes. Induction of cytochrome P-448 with 3-methylcholanthrene (MC) or 3.4-benzpyrene (BP) does not affect the magnitude of the maximal absorbance difference spectrum (ΔA_max) relative to uninduced microsomes. In contrast, phenobarbital (PB) induced microsomes exhibit ΔA_max values with either anaesthetic agent which, relative to controls, arc increased approximately in proportion to the increase in the level of total type P-450 cytochromes. The K_s values for the binding of fluroxene and TFFE to all microsomal preparations are 9.3 × 10⁴ M and 1.7 × 10^?3 respectively. Both anaesthetics are metabolized by hepatic microsomal cytochrome P-450 as evidenced by enhanced carbon monoxide-inhibitahic NADPH oxidation in the presence of these compounds. The maximum velocities of NADPH consumption in the presence of cither anaesthetic are unaffected by induction with BP or MC but are increased approximately 3-fold following induction of cytochrome P-450 with PB. For fluroxene metabolism by all microsomes K_m was determined to be 8.4 × 10⁴ M. Determination of K_m values for TFEE metabolism is more complex as biphasic effects are observed with some systems. We conclude that fluroxene and TFEE bind to cytochrome P-450 and are metabolized but that TFEE is a poorer substrate. In contrast cytochrome P-448 neither binds nor metabolizes either anaesthetic. Since K_m and K_s values for fluroxene are the same we conclude that the rate-limiting step of its metabolism occurs at a step after the binding of fluroxene to ferricytochrome P-450.     

Tetrachloroethylene metabolism by the hepatic microsomal cytochrome P-450 system

The interaction of tetrachloroethylene with hepatic microsomal cytochromes P-450 has been investigated using male Long-Evans rats. The spectral binding of tetrachloroethylene to cytochromes P-450 in hepatic microsomes from uninduced rats was characterized by a K_s of 0.4 mM. The K_s was not affected by phenobarbital induction, but was increased following pregnenolone-16α-carbonitrile induction. The K_M of 1.1 mM, calculated for the conversion of tetrachloroethylene to total chlorinated metabolites by the hepatic microsomal cytochrome P-450 system, was decreased by phenobarbital induction and increased by pregnenolone-16α-carbonitrile induction. The maximum extents of binding (ΔA_max) and metabolism (V_max) of tetrachloroethylene were increased by both phenobarbital and pregnenolone-16α-carbonitrile induction. Induction with β-naphthoflavone was without effect on any of the above parameters. The effects of the inducing agents on tetrachloroethylene-stimulated CO-inhibitable hepatic microsomal NADPH oxidation followed the same trend as their effects on V_max for the metabolism of tetrachloroethylene, although in all cases the extent of NADPH oxidation was 5- to 25-fold greater than the extent of metabolite production. The inhibitors of cytochromes P-450, viz. metyrapone, SKF 525-A, and CO, inhibited the hepatic microsomal binding and metabolism of tetrachloroethylene. Free trichloroacetic acid was found to be the major metabolite of tetrachloroethylene from the hepatic microsomal cytochrome P-450 system. Neither 2.2,2-trichloroethanol nor chloral hydrate was produced in measurable amounts from tetrachloroethylene. A minor but significant metabolite of tetrachloroethylene by cytochrome P-450 was the trichloroacetyl moiety covalently bound to components of the hepatic microsomes. Incubation of tetrachloroethylene. an NADPH-generating system. EDTA and hepatic microsomes was without effect on the levels of microsomal cytochromes P-450, cytochrome b₅, beme, and NADPH-cytochrome c reductase. It is concluded that hepatic microsomal cytochromes P-450 bind and metabolize tetrachloroethylene. The major product of this interaction is trichloroacetic acid, which is also the major urinary metabolite of tetrachloroethylene in vivo. The forms of cytochrome P-450 that bind and metabolize tetrachloroethylene include those induced by pregnenolone-16α-carbonitrile and by phenobarbital. Cytochrome P-448. which was induced in rat liver by β-naphthoflavone, does not appear to spectrally bind or metabolize tetrachloroethylene. The metabolism and toxicity of tetrachloroethylene are considered in relation to other chlorinated ethylenes.     

The synergism of n-hexane-induced neurotoxicity by methyl isobutyl ketone following subchronic (90 days) inhalation in hens: induction of hepatic microsomal cytochrome P-450

The effect of methyl isobutyl ketone (MiBK) on n-hexane-induced neurotoxicity was investigated via inhalation in seven groups of five hens each for 90 days followed by a 30-day observation period. One group was exposed to vapors containing 1000 ppm n-hexane and another group to vapors having 1000 ppm MiBK. Four groups were exposed simultaneously to 1000 ppm of n-hexane and 100, 250, 500, or 1000 ppm MiBK. Another group was exposed similarly to ambient air in an exposure chamber and used as a control. Hens continuously exposed to 1000 ppm MiBK developed leg weakness with subsequent recovery, while inhalation of the same concentration of n-hexane produced mild ataxia. Hens exposed to mixtures of n-hexane and MiBK developed clinical signs of neurotoxicity, the severity of which depended on the MiBK concentration. Thus, all hens exposed to 1000 ppm n-hexane in combination with 250, 500, or 1000 ppm MiBK progressed to paralysis. Hens continuously exposed to 1000/100 n-hexane/MiBK showed severe ataxia which did not change during the observation period. The neurologic dysfunction in hens exposed simultaneously to n-hexane and MiBK was accompanied by large swollen axons and degeneration of the axon and myelin of the spinal cord and peripheral nerves. The results indicate that the nonneurotoxic chemical MiBK synergized the neurotoxic action of the weak neurotoxicant n-hexane since the coneurotoxicity coefficient for joint exposure was more than two times the additive effect of each treatment alone. In another experiment, to investigate the mechanism of MiBK synergism of n-hexane neurotoxicity, continuous inhalation for 50 days of 1000 ppm n-hexane had no effect on hen hepatic microsomal enzymes, whereas inhalation of 1000 ppm MiBK for 50 days or a mixture of 1000 ppm of each of n-hexane and MiBK for 30 days significantly induced aniline hydroxylase activity and cytochrome P-450 contents in hen liver microsomes. Liver microsomal proteins from these hens and from hens treated with beta-naphthoflavone (beta-NF) and phenobarbital (PB) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While beta-NF increased the 55-kDa band (1408%), PB, MiBK, and MiBK/n-hexane increased the protein band (49 kDa) (258, 335, and 253%, respectively), indicating that MiBK induces chicken hepatic cytochrome P-450. The results suggest that the synergistic action of MiBK on n-hexane neurotoxicity may be related to its ability to induce liver microsomal cytochrome P-450, resulting in increased metabolic activation of n-hexane to more potent neurotoxic metabolites.     

Sex differences in cytochrome P-450 and mixed-function oxygenase activity in gonadally mature trout

Levels of microsomal cytochrome P-450 and aminopyrine demethylase activity in liver and of cytochrome P-450 in kidney of gonadally mature rainbow and brook trout were markedly greater in males than in females. Similar differences appeared in hepatic microsomal NADH- but not in NADPH-cytochrome c reductase activity or cytochrome b₅ content. When normalized to cytochrome P-450 content, benzo[a]pyrene hydroxylase activity in both liver and kidney was greater in females. In liver, there was a pronounced sex difference in the response of this activity to 7,8-benzoflavone, suggesting cytochromes P-450 of different catalytic function. Electron paramagnetic resonance spectra of hepatic microsomal cytochromes P-450 in mature brook trout were not demonstrably different between males and females, and crystal field parameters indicate that axial ligands to the neme are the same in these as in other cytochromes P-450. Mixed-function oxygenase activities in liver of gonadally immature brook trout differed from those in mature fish, and there was no sex difference. The appearance of seasonally dependent sex differences suggests that fish may provide interesting models for studying regulation of sex-specific forms of cytochromes P-450.     

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.     

Fluroxene (2,2,2-trifluoroethyl vinyl ether) mediated destruction of cytochrome P-450 in vitro.

Incubation of rat hepatic microsomes with an NADPH generating system and the anaesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) at 30° resulted in the destruction of hepatic cytochrome P-450 in excess of that observed in the presence of the NADPH generating system alone. The extent of destruction of cytochrome P-450 was markedly enhanced by pre-induction of cytochrome P-450 with phenobarbital. Induction with 3-methylcholanthrene or 3,4-benzpyrene, which induce cytochrome P-448, enhanced the overall level of cytochrome destruction. No destruction was observed when CO was added to the system prior to the addition of the anaesthetic, or when NADH replaced NADPH or when fluroxene was replaced by its chemically reduced from, 2,2,2-trifluoroethyl ethyl ether. The fluorexene potentiated destruction of cytochrome P-450 was accompanied by the loss of heme from the microsomes but not by the appearance of cytochrome P-420 nor by the loss of cytochrome b₅ or NADPH-cytochrome c reductase. We conclude that cytochrome P-450 is specifically destroyed by fluroxene in a metabolic process involving destruction of its heme group. The vinyl group of the anaesthetic is essential for the destructive process.     

Formation of an inactive cytochrome P-450 Fe(II)-metabolite complex after administration of troleandomycin in humans

In rats, it has been shown that troleandomycin induces its own transformation into a metabolite forming an inactive complex with reduced cytochrome P-450. To determine whether similar effects occur in humans, we studied hepatic microsomes from 6 untreated patients and 6 patients treated with troleandomycin, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased by 48%; total cytochrome P-450 concentration was also increased, but 33% of total cytochrome P-450 was complexed by a troleandomycin metabolite. The cytochrome P-450 Fe(II)-metabolite complex exhibited properties identical to those of the inactive complex formed in rats: it exhibited a Soret peak at 456 nm, was unable to bind CO, and was destroyed by addition of 50 μM potassium ferrciyanide.We also measured the clearance of antipyrine in 6 other subjects. This clearance was decreased by 45% when measured again on the seventh day of the troleandomycin treatment. We conclude that repeated administration of troleandomycin induces microsomal enzymes, produces an inactive cytochrome P-450 Fe(II)-metabolite complex, and decreases the clearance of antipyrine in humans.     

Hepatic microsomal epoxidation of bromobenzene to phenols and its toxicological implication

In vitro microsomal hepatic epoxidation of bromobenzene in rats and mice is presented in this study. Formation of o-bromophenol via bromobenzene-2,3-epoxide and p-bromophenol via bromobenzene-3,4-epoxide was assayed enzymatically and identified by a new, rapid and sensitive gas-liquid chromatography method using electron capture detection. Pretreatment of the animals with phenobarbital caused significant increases in both pathways whereas 3-methylcholanthrene or β-naphthoflavone caused a selective and marked increase of only the 2,3-epoxide pathway. Sodium dodecyl sulfate-gel electrophoresis of microsomal preparations resolved multiple forms of cytochrome P-450 and indicated that different forms of the heme protein were responsible for the formation of o-bromophenol and p-bromophenol. it is of interest that various inducers augment particular pathways for a common substrate especially since bromobenzene-3,4-epoxide and not the bromobenzene-2,3-epoxide has been proposed as the cytotoxic reactive metabolite of bromobenzene.     

Effect of diabetes on rat liver cytochrome P-450: Evidence for a unique diabetes-dependent rat liver cytochrome P-450

Detergent-solubilized hepatic microsomal fractions from alloxan diabetic rats exhibited a 52,000 molecular weight hemeprotein band that was not present in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of identically solubilized hepatic microsomal fractions from normal, 3-methylcholanthrene- or phenobarbital-treated rats. This 52,000 mol. wt hemeprotein band disappeared from the protein profile of insulin-treated diabetic rat liver to yield the SDS-PAGE profile of normal rat liver. When P-450 hemeproteins were purified by lauric acid affinity and hydroxylapatite chromatography from solubilized microsomes, only the diabetic rat had a 52,000 mol. wt P-450. This distinct 52,000 mol. wt diabetes-induced P-450 interacted with type II compounds to yield a 2-fold greater absorbance change than was observed with the purified P-450s from either the normal or the chemically induced rats. The properties of this unique 52,000 mol. wt P-450 suggest that it may be the catalytic component responsible for the increased rate of type II substrate (aniline) metabolism observed in the diabetic rat.     

Some characteristics of hamster liver and lung microsomal aryl hydrocarbon (biphenyl and benzo(a)pyrene) hydroxylation reactions

Aryl hydrocarbon hydroxylation (AHH) reactions were compared using liver and lung microsomes of corn oil- and 3-methylcholanthrene (3-MC)-treated hamsters, employing benzo(a)pyrene (BAP) and biphenyl as substrates. The predominant AHH activity of liver and lung microsomes from corn oil- or 3-MC-treated hamsters was biphenyl 4-hydroxylase. Biphenyl 2-hydroxylase and BAP-hydroxylase activities were approximately 50 per cent as active as biphenyl 4-hydroxylase in liver and approximately 1–3 per cent as active as biphenyl 4-hydroxylase in lung microsomes. Biphenyl 4-hydroxylase activity was 70–80 per cent as active in lung as in liver microsomes. Treatment with 3-MC in vivo induced the biphenyl 4-hydroxylation reaction in liver but not in lung microsomes, the biphenyl 2-hydroxylation reaction both in lung and liver microsomes, and the BAP hydroxylation reaction in lung but not in liver microsomes. Biphenyl 2- and 4-hydroxylase activities of liver microsomes displayed similar sensitivities to inhibition by a number of chemical inhibitors in vitro. Inhibition of biphenyl hydroxylation reactions by metyrapone or carbon monoxide did not distinguish between lung or liver microsomal mono-oxygenases of corn oil- or 3-MC-treated hamsters. While small differences were expressed by inhibition with ethylmorphine, large differences became apparent through inhibition studies with BAP or α-naphthoflavone. It is concluded that the major aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the constitutive (uninduced) AHH of the liver microsomes and that the minor aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the induced AHH of the liver microsomes.     

Effects of short-term inhalation of cadmium oxides on rabbit pulmonary microsomal enzymes

Rabbits, guinea pigs, rats and mice were compared for the activity of benzo[a]pyrene hydroxylase. aminopyrine N-demethylase and aniline hydroxylase of pulmonary microsomes. The activity of the microsomal enzymes was highest in rabbits, followed by guinea pigs and then rats and mice. Effects of the inhalation of cadmium oxides (CdO) were studied on the pulmonary microsomal enzymes in male rabbits. Rabbits were exposed for 15 min to air containing microparticles of CdO at four different concentrations ranging from 6.4 ± 0.5 to 22.4 ± 0.4 mg Cd/m³. The animals were killed 24 hr after the inhalation of CdO. The lung weight of the animals was increased markedly at doses higher than 12.6 mg/m³, the increase attaining 50 per cent increase at the highest dose. The activity of benzo[a]pyrene hydroxylase was reduced significantly at the higher doses, the reduction being dose-dependent reaching 50 per cent reduction. The activity of aminopyrine N-demethylase and aniline hydroxylase was reduced moderately by CdO inhalation but the reduction was not dose-dependent. Time-course effects of CdO inhalation on the pulmonary microsomal enzymes were studied on the rabbits exposed for 15 min to air containing CdO at concentrations of 13.0 ± 0.3 mg Cd/m³. The animals were killed 0.5, 1, 2, 4, and 6 days after the inhalation. The body weight increase was less in the treated groups than in the controls and edematous lesions in the lung were observed in most of the treated animals. The lung weight was increased after the inhalation, reaching its plateau on day 4. The activity of the microsomal enzymes was reduced throughout the experimental period, the maximum reduction being obtained on day 2 after the inhalation except for aniline hydroxylase that was reduced at the same degree throughout the experimental period.     

Spectral interaction of orphenadrine and its metabolites with oxidized and reduced hepatic microsomal cytochrome P-450 in the rat

Product inhibition has been suggested to be a determinant in orphenadrine pharmacokinetics. Two possibilities for the mechanism of product inhibition in orphenadrine metabolism are explored in this study. Orphenadrine and its metabolites may compete for cytochrome P-450 catalytic binding sites. Therefore the interaction of orphenadrine and some of its metabolites with hepatic microsomal ferricytochrome P-450 of the rat was investigated. The spectral dissociation constant for the type I (substrate) interaction of orphenadrine and its metabolites displayed no relationship with the lipophilicity of the compounds. Orphenadrine is only partially displaced from its cytochrome P-450 binding sites by its respective metabolites. For this mechanism to be significant in vivo. the metabolites need to reach concentrations near cytochrome P-450 similar to that of orphenadrine. This is not known yet. The significance of this mechanism for the product inhibition phenomenon is therefore uncertain. In this study it is also established that during both in vitro as well as in vivo metabolism of orphenadrine, a metabolic intermediate is formed, which binds irreversibly to ferrous-cytochrome P-450 (MI complex). In vitro, both the rate and extent of the MI complex formation with orphenadrine and metabolites as precursor, decreased in the order N-hydroxytofenacine >; tofenacine > orphenadrine > bisnororphenadrine. The metabolite orphenadrine-N-oxide did not produce an MI complex, in vitro. Furthermore. in vitro, it was shown that the N-demethylation of tofenacine paralleled the concomitant MI complex formation. Together, the data suggest that the first N-demethylation step of orphenadrine occurs via α-carbon oxidation, whereas the second N-demethylation step mainly comes about via N-oxidation. Both metabolic pathways eventually lead to the MI complex forming species. These two parallel pathways also account for the complicated substrate dependency and concentration dependency in MI complex formation. Finally, the formation of the nitroxide radical (the ultimate ligand for MI complexation) has been shown to be susceptible to inhibition by its precursors.The occurrence of MI complex formation resulting in metabolic inactive cytochrome P-450 is probably the main mechanism for the product inhibition phenomenon in orphenadrine metabolism.