Vinylidene chloride: Its metabolism by hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of vinylidene chloride by hepatic microsomal cytochrome P-450 are reported. Hanes plots for the Type I binding of vinylidene chloride to cytochrome P-450 were biphasic with hepatic microsomes from untreated and β-naphthoflavone- or phenobarbital-treated male rats. Neither pretreatment affected the value of the K_s (ca. 0.22 mM) for the high-affinity binding site for vinylidene chloride, while phenobarbital induction, but not β-naphthoflavone treatment, decreased the value of the K_s for the low-affinity site by 3-fold to ca. 1.6 mM. The maximum extents of binding (ΔA_max or ΔA_max/nmole cytochrome P-450) of vinylidene chloride were decreased or not affected by β-naphthoflavone induction, while ΔA_max but not ΔA_max/ nmole cytochrome P-450 was elevated following phenobarbital induction. The rate of vinylidene chloride stimulated CO-inhibitable hepatic microsomal NADPH oxidation was not affected by β-naphthoflavone induction, but was increased significantly following phenobarbital induction. Vinylidene chloride was converted to monochloroacetate and to the previously unreported metabolite, dichloroacetaldehyde, by hepatic microsomes plus NADPH-generating system. Measurable levels of 2-mono- and 2,2-dichloroethanol, and of chloroacetaldehyde and dichloroacetic acid, were not produced from vinylidene chloride under these conditions. SKF-525A and CO:O₂ (80:20, v/v) inhibited the conversion of vinylidene chloride to monochloroacetate and dichloroacetaldehyde by approximately 60%. The rates of production of monochloroacetate and dichloroacetaldehyde in the presence of NADH were ca. 15% of the rates seen with NADPH-generating system. The rate of monochloroacetate production per mg microsomal protein was not affected by β-naphthoflavone induction but was increased slightly following phenobarbital induction. In contrast, the V_max values per mg microsomal protein for the metabolism of vinylidene chloride to dichloroacetaldehyde were not elevated by either pretreatment. Incubation of vinylidene chloride, NADPH-generating system, EDTA and hepatic microsomes from untreated and β-naphthoflavone- or phenobarbital-treated rats did not result in any significant alterations in the levels of microsomal cytochrome P-450 and heme or in the covalent binding of the mono- or dichloroacetyl moieties to microsomal or buffer constituents, but it did result in significant production of H₂O₂. It is concluded that multiple forms of cytochrome P-450 bind and metabolize vinylidene chloride. However, the form of the enzyme elevated by phenobarbital plays, at most, a minor role in these processes, while the form induced by β-naphthoflavone is not involved in either process. The effect of metabolism of vinylidene chloride by cytochrome P-450 on the relationship between the metabolism and toxicity of vinylidene chloride in vivo and its mutagenicity in vitro is considered.     

Enflurane and methoxyflurane: Their interaction with hepatic cytochrome P-450 in vitro

The binding and metabolism of enflurane (CClFHCF₂OCF₂H) and of methoxyflurane (CCl₂HCF₂OCH₃) were investigated in vitro with hepatic microsomes from male rats. The metabolism of these anesthetic agents was monitored by NADPH consumption and fluoride ion production. In addition, from methoxyflurane, the production of acid-labile fluoride was monitored. The effects of inducing agents for different forms of cytochrome P-450 on the binding (K_s, ΔA_max) and metabolism (K_M, V_max) of both anesthetic agents are reported. The effects of CO, SKF-525A and metyrapone on the production of fluoride from enflurane and on fluoride and acid-labile fluoride from methoxyflurane are compared. The results of these studies indicate that only a form of cytochrome P-450 induced by phenobarbital binds and metabolizes enflurane in vitro. In contrast, at least two forms of cytochrome P-450 are involved in the binding and metabolism of methoxyflurane in vitro. Methoxyflurane interacts with a form of cytochrome P-450 induced by phenobarbital and at least one other form of cytochrome P-450, but not with cytochrome P-448. The relative amounts of free and acid-labile fluoride produced from methoxyflurane in vitro are altered after induction with 3-methylcholanthrene but not with phenobarbital. Although K_s, ΔA_max, K_M and V_max (NADPH consumption) are similar in magnitude for enflurane and methoxyflurane, the rate of production of fluoride from enflurane is approximately 8-fold less than from methoxyflurane. The observed stoichiometry of 140:1 for NADPH consumption to fluoride production for enflurane suggests that enflurane is enhancing NADPH oxidation far in excess of its metabolism. The relevance of these results to the proposed pathways for the metabolism of enflurane and methoxyflurane is discussed. The results are compared with reports of the metabolism and toxicity of enflurane and methoxyflurane in vivo.     

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.     

Metabolism of monochlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of the monochlorobiphenyls (MCBs) 2-, 3- and 4-chlorobiphenyl, has been investigated as a model for the metabolism of polychlorinated biphenyl pollutants. MCB metabolism was catalyzed by cytochrome P-450, as indicated by a dependence on NADPH and O₂, inhibition by 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF 525-A), metyrapone and CO, and the formation of type I difference spectra, on the addition of MCBs to microsomes. All MCBs yielded a 4'-monohydroxy MCB as the major metabolite, as determined by mass and nuclear magnetic resonance spectroscopy, dechlorination to 4-hydroxybiphenyl, and high-pressure liquid chromatography retention times. Minor monohydroxy and dihydroxy metabolites were also produced from the MCBs. The regioselectivity of control cytochrome P-450 for metabolism of MCBs at the 4' position was not altered by preinduction of cytochrome P-450 with 2,4,2',4'-tetrachlorobiphenyl (TCB) or cytochrome P-448 with 3,4,3', 4'-TCB. 2-Chlorobiphenyl was metabolized only by control and induced cytochrome P-450; 3- and 4-chlorobiphenyl were metabolized by control and by induced cytochrome P-450 and P-448. Thus, the regioselectivity of metabolism of MCBs is independent of the chlorine position or the form of the induced cytochrome involved, but the extent of metabolism of polychlorinated biphenyls (PCBs) is determined by induction of the hepatic cytochromes P-450.     

Metabolism of dichlorobiphenyls by hepatic microsomal cytochrome P-450

In vitro rat hepatic microsomal metabolism of ten individual dichlorobiphenyls (DCBs) has been investigated as part of a major study of the role of metabolism in the toxicity of polychlorinated biphenyl (PCB) pollutant mixtures. The DCBs were metabolized to monohydroxy and dihydrodiol metabolites and unstable metabolites of intermediate polarity. DCBs with both chloro substituents on the same ring, one or both of which were ortho substituents, were susceptible to the same regioselectivities for hydroxylation by control, phénobarbital (PB)- or β-naphthoflavone (BNF)-induced cytochromes P-450 (principally in the 4-position), with the greatest rates of hydroxylation arising with PB-induced cytochrome P-450. In contrast, DCBs with no ortho chlorosubstituents had regioselectivities for hydroxylation by control and PB-induced cytochrome P-450 which differed from that of BNF-induced cytochromes P-450; the greatest rates of hydroxylation were with BNF-induced systems. DCBs with one chloro substituent on each ring were metabolized, with the site of hydroxylation being under the electronic influence of the chloro substituent. With 4,4'-DCB, 60 per cent of the hydroxylated DCB metabolite underwent an NIH shift [G. Guroff, J. W. Daly, D. M. Jerina, J. Renson, B. Witkop and S. Udenfriend, Science157, 1524 (1967)]. The BNF-induced system produced the highest rates of dihydrodiol fomation that were eliminated by an epoxide hydratase inhibitor. The results indirectly prove that arene oxides are intermediates in DCB metabolism and are possibly the source of DCB mutagenicity. The PCBs 2,4,2'4'- and 3,4,3',4'-tetrachlorobiphenyl induced the same effects as PB and BNF respectively. Thus, PCBs differentially affect the metabolism of their individual components and are, possibly, responsible for enhancing their own toxicity by inducing enhanced rates of formation of arene oxide intermediates.     

Interaction of nitromethane with reduced hepatic microsomal cytochrome P-450.

Nitromethane interacts with sodium dithionite reduced rabbit liver microsomes to generate a difference spectrum characterized by maxima at 388, 423, 454 and 520 nm and minima at 405, 436, 480 and 550 nm. Spectral binding constants (K_s) of 0.306 ± 0.082 mM (A_max = 0.032 ± 0.004), 0.178 ± 0.029 mM (A_max = 0.040 ± 0.002), and 1.168 ± 0.250 mM (A_max = 0.035 ± 0.006) were calculated for the 388, 423 and 454 nm peaks respectively. These difference spectra are qualitatively different from those previously reported for aromatic nitro compounds [L. A. Sternson and R. E. Gammans, Drug Metab. Dispos.3, 266 (1975)]. Interaction of nitromethane with microsomes from rabbits pretreated with phenobarbital produced absorbance maxima and minima within 2 nm of the controls. Interaction of nitromethane with reduced microsomes from 3-methyl-cholanthrene (3-MC)-pretreated animals produced ferrohemochromes in which maximal absorbance changes were shifted to maxima at 384, 422, 451 and 514 nm and minima at 407, 433, 473 and 552nm. Peak-height ratios derived from difference spectra generated by the addition of 1 mM nitromethane to the sample cuvette were considerably different depending on whether the microsomes were obtained from control, phenobarbital- or 3-MC-pretreated rabbits and may indicate that phenobarbital, like 3-MC, induces qualitative changes in cytochrome P-450. Nitromethane apparently competes with carbon monoxide for a common binding site. Addition of nitromethane to CO-saturated microsomes reduced the magnitude of the 450 nm peak with a concomitant increase in the 423 nm peak of nitromethane. Similarly, addition of CO to reduced microsomes containing nitromethane caused reduction of the 423 nm peak of nitromethane with a corresponding increase of the 450 nm peak of CO. Nitromethane does not generate difference spectra with oxidized microsomes nor does it alter the K_s or A_max of aminopyrine, hexobarbital, aniline or zoxazolamine binding spectra. Nitromethane does inhibit the binding of the type II compound, nicotinamide. Addition of nitromethane to incubation flasks enhanced the metabolism of aniline while tending to inhibit the oxidative demethylation of ethylmorphine.     

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.     

Human hepatic cytochrome P-450 composition as probed by in vitro microsomal metabolism of warfarin

Human liver microsomal fractions from 27 renal donors (tissue obtained post mortem) and from six cancer patients (tissue obtained during surgery) were used to investigate human hepatic cytochrome P-450 isozyme compositions. In vitro microsomal metabolism of the R and S enantiomers of warfarin to dehydrowarfarin and 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin is catalyzed by cytochrome P-450 isozymes and was used as the basis for evaluating similarities and differences between human cytochrome P-450 isozyme compositions. The mean hepatic cytochrome P-450 concentration from postmortem samples was not significantly different from that of surgical patients (0.51 +/- 0.16 vs. 0.35 +/- 0.14 nmol/mg protein), but the NADPH-cytochrome P-450 reductase activity of the former was significantly higher than that of the latter (141 +/- 56 vs. 29 +/- 6 nmol cytochrome c reduced/min/mg protein). In general, the microsomal preparations were overall stereoselective for R warfarin metabolism. The stereoselectivities for formation of the individual metabolites of the R enantiomer were 6-, 8-, and 10-hydroxywarfarin and the S enantiomer were 4'- and 7-hydroxywarfarin. Of the 33 microsomal preparations, 21 exhibited qualitatively similar warfarin metabolite profiles with 6R- and 7S-hydroxywarfarin having the highest formation rates. Some of the preparations exhibited markedly different metabolite profiles, the most notable having 10R-hydroxywarfarin as the major metabolite. Based on the known warfarin metabolite profiles of five purified cytochrome P-450 isozymes, the isozyme composition of the microsomes can be estimated. The majority of the microsomal preparations apparently had similar isozyme compositions but some preparations were markedly different.     

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.     

Induction of hepatic microsomal cytochrome P-450 by mirex and kepone.

The chlorinated insecticides, mirex and Kepone, pose a threat to human health as a consequence of their pollution of the environment. We investigated their potential to affect synergistically the toxicity of other xenobiotics and the pharmacological function of drugs by induction of hepatic microsomal enzymes. Male rats were induced by ip injection of mirex (50 or 5 mg/kg/day for 5 days) or Kepone (10 or 1 mg/kg/day for 5 days). Metabolic activity was tested with warfarin and biphenyl using high-performance liquid chromatographic assays. The high doses of both compounds induced cytochrome P-450 with absorbance bands (reduced, CO complex) at 449 nm. Cytochrome concentrations were enhanced twofold relative to controls. Mirex resembled 3-methylcholanthrene and benzo[a]pyrene by inducing formation of 6-hydroxywarfarin but differed in not inducing 8-hydroxywarfarin. Kepone resembled phenobarbital in inducing 7-hydroxywarfarin but differed in its effects on the other metabolites. The low dose of mirex induced higher amounts of 4′-hydroxywarfarin than did the high dose. The metabolite profiles with high and low doses of Kepone also showed marked variations from one another. Mirex and Kepone are carcinogenic in rats and mice but, in contrast to the polycyclic aromatic carcinogens, do not markedly enhance the activity of microsomal biphenyl 2-hydroxylase relative to biphenyl 4-hydroxylase. We conclude that mirex and Kepone induce hepatic mixed-function oxidase profiles which differ from one another and from the classical inducers, phenobarbital and 3-methylcholanthrene. Mirex apparently only induces one of the enzymes induced by 3-methylcholanthrene. The enzyme profiles arising from the insecticides are dose dependent and will thus potentiate qualitatively differing effects depending on the level of ingestion.     

Relation between hepatic microsomal metabolism of N-nitrosamines and cytochrome P-450 species

Effects of SKF 525A (0.1 mM), metyrapone (0.1 mM), alpha-naphthoflavone (ANF) (0.5 mM) and pyrazole (1.0 mM) on N-nitrosodimethylamine (NDMA), N-nitrosomethylbutylamine (NMBuA) and N-nitrosomethylbenzylamine (NMBeA) metabolism by hepatic microsomes from rats pretreated with inducers were investigated. NDMA demethylation was weakly increased by phenobarbital (PB) treatment. The demethylation was inhibited by SKF 525A and enhanced by metyrapone in non-treated and PB-treated microsomes, and weakly inhibited by ANF in 3-methylcholanthrene(MC)-treated microsomes. NMBuA demethylation was increased by PB treatment and inhibited by SKF 525A in all microsomes. Metyrapone inhibited the demethylation in PB-treated microsomes. NMBuA debutylation was increased by PB and MC treatments, and inhibited by metyrapone in all microsomes. The strongest inhibition by metyrapone was observed in PB-treated microsomes. The debutylation was inhibited by SKF 525A in non-treated and PB-treated microsomes and by ANF in MC-treated microsomes. NMBeA demethylation was decreased by MC treatment and weakly inhibited by SKF 525A in all microsomes. The effects of the inducers and inhibitors on NMBeA debenzylation were almost the same as those on NMBuA debutylation except that the increasing effect of MC was small. Pyrazole was a relatively selective inhibitor of NDMA demethylation. These results suggest the following: NDMA demethylation is catalyzed by PB-induced cytochrome P-450 species (P450-PB) and MC-induced cytochrome P-450 species (P448-MC). But their specific activity is low and the other cytochrome P-450 species demethylate NDMA. NMBuA demethylation is catalyzed by P450-PB. But the specific activity is not high and the other cytochrome P-450 species also demethylate NMBuA. NMBuA debutylation is catalyzed by P450-PB and P448-MC. Almost all of NMBeA demethylation is catalyzed by cytochrome P-450 species other than P450-PB and P448-MC. NMBeA debenzylation is catalyzed by P450-PB and P448-MC, but the specific activity of P448-MC is not high.     

Impaired in vitro metabolism of the flukicidal agent nitroxynil by hepatic microsomal cytochrome P-450 in bovine fascioliasis

In vitro metabolism by liver tissue of the flukicidal agent nitroxynil has been studied in cattle naturally infected with Fasciola hepatica. A dramatic impairment of the cytochrome P-450-dependent nitroxynil metabolism both in the acute and in the milder stage of the disease has been observed and this is due to a loss in the integrity and functionality of the cytochrome P-450 enzyme system. These results suggest that in bovine fascioliasis the in vivo metabolism of nitroxynil will be decreased with consequent increase of nitroxynil retention in the animal's body.     

Role of hepatic microsomal cytochrome P-450 in the toxicity of fluorinated ether anesthetics

To evaluate the role of cytochrome P-450 in anesthetic toxicity, we investigated the effects of hepatic microsomal cytochrome P-450 inducers [phenobarbital (PB), 3-methylcholanthrene (3-MC) and pregnenolone-16 α-carbonitrile (PCN)] and inhibitors [SKF 525-A, metyrapone, and 2allyl2isopropylacetamide (ALA)] on the potentiation of lethal effects to rats of i.p. administered 2,2,2-trifluoroethyl vinyl ether (TFVE), ethyl 2,2,2-trifluoroethyl ether (TFEE), allyl 2,2,2-trifluoroethyl ether (TFAE) and 2,3-epoxypropyl 2,2,2-trifluoroethyl ether (EPTFE). The time courses of tail-vein blood anesthetic concentrations and quantities of exhaled anesthetics together with the in vitro metabolism of the anesthetics and their binding to microsomal cytochromes P-450 were also determined. The results indicate that (1) the majority of the administered anesthetics make a single pass through the liver prior to exhalation and apparently are metabolized to toxic products, (2) the epoxide (EPTFE) exerts its lethal effects independently of cytochrome P-450 catalyzed metabolism and does not lie on the major path of TFAE metabolism, (3) all the anesthetics yield 2,2,2-trinuoroethanol (TFE) on metabolism in vitro but lethality does not always correlate with the rates of TFE formation, (4) PB induced cytochromes P-450 potentiate lethal effects of TFVE and TFEE but not of TFAE, and inhibitors differentiate mechanisms of TFVE and TFEE lethality, (5) PCN induced cytochromes P-450 potentiate the toxicity of TFVE, TFAE, and TFEE in a similar manner, and (6) 3-MC induction potentiates TFEE and TFAE lethality apparently independently of cytochrome P-450 catalyzed metabolism.     

Induction of the hepatic microsomal cytochrome P-450 system by trialkyl phosphorothioates in rats

Single i.p. doses of O,O,O-triethyl phosphorothioate [OOO-Et(S)], one of the suicide substrates for cytochrome P-450, caused a rapid increase of NADPH-cytochrome c reductase activity in rat liver microsomes. The increase was dose dependent but did not coincide with the recovery from the inhibition of drug-metabolizing activities. There was no change of Km value of the reductase in the induced state. The co-administration of cycloheximide repressed the stimulatory effect of OOO-Et(S), suggesting that a de novo synthesis of enzyme protein may be responsible for the increase in activity. Of four homologous tri-n-alkyl esters tested, the triethyl compound was the most effective at 24 and 48 hr after administration. Triethyl phosphate, the oxygen analog of OOO-Et(S), also caused an increase of the reductase activity, but carbon disulfide had no influence on this activity. Although O,O,S-triethyl phosphorodithioate [OOS-Et(S)] and its n-alkyl homologs also caused the inhibition of drug-metabolizing activities and the increase of the reductase activity, the recovery and the stimulation of enzyme activity were different from that of O,O,O-tri-n-aklyl phosphorothioates.     

The 1,2-dichloroethylenes: Their metabolism by hepatic cytochrome P-450 in vitro

Cis- and trans-1,1-dichloroethylene bound to the active site of hepatic microsomal cytochrome P-450 with the production of a Type I difference spectrum and stimulated CO-inhibitable hepatic microsomal NADPH oxidation. Incubation of cis- and trans-1,2-dichloroethylene plus hepatic microsomes, NADPH-generating system-EDTA resulted in the production of measurable levels of 2,2-dichloroethanol and dichloroacetaldehyde but not of 2-chloroethanol, chloroacetaldehyde or chloroacetic acid and, also, resulted in decreased levels of hepatic microsomal cytochrome P-450 and heme. In addition, dichloroacetic acid was produced from trans-dichloroethylene under these experimental conditions. The omission of any component of the incubation mixture eliminated the above effects, while the inclusion of SKF-525A, metyrapone or CO: O₂ (80, v/v) diminished these effects. The effects of β-naphthoflavone and phenobarbital pretreatment on the values of K_s, ΔA_max, K_m and V_mam for the binding and metabolism of the 1,2-dichloroethylenes are reported. The binding and metabolism of the 1,2-dichloroethylenes and the 1,2-dichloroethylene-mediated inactivation of cytochrome P-450 were enhanced per mg of microsomal protein, but generally not per nmole of cytochrome P-450 by prior induction with β-naphthoflavone or phenobarbital. It is concluded that multiple forms of hepatic microsomal cytochrome P-450 bind and metabolize the 1,2-dichloroethylenes. The role of cytochrome P-450 in the metabolic activation of the dichloroethylenes is considered.