Effects of polychlorinated terphenyls and paraffins on rat liver microsomal cytochrome P-450 and in vitro metabolic activities

The polychlorinated terphenyl Aroclor 5460 and the polychlorinated paraffins Witaclor 171 P and Witaclor 149 increased to different degrees the total microsomal concentration of cytochrome P-450 in the rat liver after intraperitoneal injection of 0.3, 1.0, and 1.0 g · kg^–1 body weight, respectively, each day for four days. The multiple forms of cytochrome P-450 were affected differently with an induction of RLvMc P-450₅₀ and RLvMc P-450₅₄ by all chemicals, and an additional induction of RLvMc P-450₅₅ by the polychlorinated terphenyl. The rat liver weights were extensively increased after treatment with the polychlorinated paraffins. Alterations were found in the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroid hormones, 4-androstene-3,17-dione and 5-androstane-3,17-diol, after exposure to all chemicals. Changes in the in vitro formation of benzo(a)pyrene metabolites were found to correlate with changes in the multiple forms of cytochrome P-450. The present study demonstrate that only limited information can be obtained from alterations in the total concentration of cytochrome P-450 and show the importance of studying changes in the multiple forms and the metabolism of different substrates. Our results further indicate that exposure to any of the investigated polychlorinated chemicals may alter the biological effects of other environmental contaminants, drugs and endogenous substances which are metabolized by the cytochrome P-450 enzyme system.     

Dual ligand binding of pyridylalkanamides to microsomal cytochrome P-450

Twelve homologous and regioisomeric pyridylalkanamides were examined spectrally for their binding affinity to cytochrome P-450 in phenobarbital- and 3-methylcholanthrene-induced rat liver microsomes. The pKs values were calculated by the Lineweaver-Burk method and by non-linear analysis using both a one ligand-one acceptor and a one ligand-two acceptor model. The latter model best fits most of the data, confirming that two pKs values exist for most derivatives in the 3-pyridyl and 4-pyridyl series. Structure-binding relationships are discussed. The two binding constants are hypothesized to arise from a dual mode of binding to the ferric ion. At low ligand concentrations, binding to hexacoordinated cytochrome P-450 occurs and involves displacement of an endogenous 6th ligand; at higher concentrations, the ligands bind to the pentacoordinated P-450, resulting in a high-to-low spin shift.     

Selective induction of renal microsomal cytochrome P-450-linked monooxygenases by 1,1-dichloroethylene in mice

Intraperitoneal administration of a single dose of 1,1-dichloroethylene (DCE) to C57 B1/6N mice (125 mg/kg) caused a selective 6- to 10-fold increase in renal microsomal 7-ethoxyresorufin O-deethylase ( EROD ) and 7-ethoxycoumarin O-deethylase ( ECOD ), without affecting benzo[a]pyrene hydroxylase activity (AHH) or total microsomal cytochrome P-450 content. The observed increases did not result from in vitro activation of the enzymes or from any analytical artifact. Moreover, studies with actinomycin D and cycloheximide demonstrated that the increases resulted from de novo enzyme synthesis. Maximal enzyme induction was observed after a DCE dose of approximately 125 mg/kg, and the induced enzyme decayed rapidly, returning to control levels in about 3 days. Compared to female mice, male mice had higher basal levels of renal EROD and ECOD and were more responsive to the inductive effects of DCE; this correlated with corresponding differences in microsomal cytochrome P-450 levels. Starvation of mice for 24 or 48 hr increased renal EROD and ECOD activities in both male and female mice, but not the extent observed after DCE. The present results support the view of multiple renal cytochrome P-450 isozymes.     

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.     

Reinduction of the cytochrome P-450 system of the liver in rats exposed to polychlorinated biphenyls during starvation

Contents of cytochrome P-450 and b5, rates of oxidation of aniline, amidopyrine and dimethylaniline as well as activities of NADP-H- and ascorbate-dependent systems of lipid peroxidation (LPO) in rat liver microsomes five months after single administration of the mixture of polychlorinated diphenyls (PCD) significantly exceeded the control level. Starvation of the animals for 120 hours led to an additional increase of cytochrome P-450 content and LPO activation. The rat liver monooxygenase system retained the ability to respond to the inducing action of the mixture of PCD (500 mg/kg) during starvation.     

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.     

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.     

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant (K_s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding (ΔA_max) was increased only following phenobarbital induction. The K_s values (ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant (K_m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V_max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO₂ and H₂O₂, but not NaIO₄, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b₅ or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.     

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.     

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.     

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.     

Interspecies homology of liver microsomal cytochrome P-450. A form of dog cytochrome P-450 (P-450-D1) crossreactive with antibodies to rat P-450-male

P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male.     

Relationship between covalent binding to microsomal protein and the destruction of adrenal cytochrome P-450 by spironolactone

Previous investigations have demonstrated that guinea pig adrenal microsomes catalyze an NADPH-dependent activation of spironolactone (SL) resulting in the degradation of cytochrome(s) P-450 and decreases in steroidogenic enzyme activities. Studies were done to evaluate the relationship between the destruction of cytochrome P-450 and the covalent binding to microsomal protein by SL and by 7 alpha-thiospironolactone (7 alpha-thio-SL), an obligatory intermediate in the activation pathway. NADPH-dependent irreversible binding to guinea pig adrenal microsomal protein was demonstrable with 22-14C- and with 35S-labelled SL or 7 alpha-thio-SL as substrates. In the absence of NADPH, there was relatively little binding. NADPH-dependent covalent binding was not demonstrable with hepatic microsomal preparations. The amount of covalent binding to adrenal microsomes was far greater with 7 alpha-thio-SL than with SL and also greater with 35S-labelled than with 14C-labelled substrates. The latter results suggest the possibility of more than one reactive metabolite. Time-course experiments revealed a good correlation between covalent binding and P-450 destruction by SL and by 7 alpha-thio-SL. In addition, the 17 alpha-hydroxylase inhibitor, SU-10'603, and the 17 alpha-hydroxylase substrate, progesterone, prevented both the degradation of cytochrome P-450 and the NADPH-dependent covalent binding by 7 alpha-thio-SL. Reduced glutathione also decreased covalent binding but did not diminish P-450 destruction. The latter results indicate that some of the covalent binding is unrelated to the degradation of cytochrome P-450. However, all of the data are consistent with the hypothesis that 7 alpha-thio-SL is a suicide inhibitor of adrenal cytochrome P-450 and that covalent binding to protein is involved in the degradation of cytochrome P-450.     

The measurement of FAD-containing mono-oxygenase activity in microsomes containing cytochrome P-450

1. Antibodies to NADPH-cytochrome P-450 reductase have been used to essentially abolish the contribution of cytochrome P-450 to xenobiotic metabolism by mammalian microsomes. This permits the determination of the activity of the FAD-containing monooxygenase and the stoichiometry between substrate, O₂ and NADPH, in the microsomal membrane, and in the absence of cytochrome P-450-dependent activity.

2. FAD-containing mono-oxygenase oxidation rates were determined for sulphur- and nitrogen-containing substrates, including: thiols; sulphides; thioamides; primary, secondary and tertiary amines; hydrazines.

3. Although the enzyme in mouse, rabbit, rat and pig microsomes displays similar substrate specificity, some catalytic characteristics are different between species and tissues.     

The covalent binding of cyclosporin A to rat liver macromolecules in vivo and in vitro: the role of cytochrome P-450

Incubation of rat liver S9 with [3H]cyclosporin A ([3H]CSA) resulted in covalent binding of CSA to macromolecules. Binding was dependent on the presence of NADPH and could be inhibited by SKF-525A. Incubation of isolated rat liver parenchymal cells with CSA resulted in a concentration-dependent binding which increased with incubation time for at least 2 h. Addition of SKF-525 A (0.5 mM) decreased the binding by 83% while viability of the cells was unchanged. Pretreatment of the cells with diethylmaleate doubled the binding of CSA, indicating that glutathione can prevent binding of CSA. When [3H]CSA was injected i.v. radioactivity was covalently bound to liver and kidney macromolecules. Induction of cytochrome P-450 by phenobarbital resulted in enhanced in vivo covalent binding in liver and kidney. Whether this covalent binding of CSA is related to its cytotoxicity is yet unclear.