Kinetic alterations of cytochrome c oxidase in carbon tetrachloride induced cirrhotic rat liver

In a study of the chronic effects of CCl4 on the respiratory activities of rat liver mitochondria, the content of cytochrome c oxidase increased from 0.077 +/- 0.010 (nmol/mg protein) for normal rats to 0.101 +/- 0.009, and its specific activity increased from Vmax = 345 +/- 24 (e-/s/cytochrome aa3) to 431 +/- 19 in mitochondria of CCl4 treated rats. There was a slight increase in Km for cytochrome c from 5.63 +/- 0.08 microM to 7.79 +/- 0.80. These results would strongly suggest that an appreciable decrease in the steady state concentration of ATP in hepatic cells of CCl4 treated rats brought about a compensatory increase in the overall activity of cytochrome c oxidase. However, when the rate of oxygen uptake by mitochondria was measured in the presence of rotenone and tetramethyl-p-phenylene-diamine with NADH as substrate, the specific activity in CCl4 treated rats was lower than that of normal rats (Vmax = 345 +/- 31 (e-/s/cytochrome aa3), as compared to Vmax = 408 +/- 21) in spite of the increased activity of cytochrome c oxidase. This phenomenon was ascribed to a decrease in the activity of NADH cytochrome b5 reductase in the mitochondrial outer membrane due to CCl4 treatment.     

Inhibition of CCl4 metabolism by oxygen varies between isoenzymes of cytochrome P-450

Oxygen inhibition of CCl4 metabolism by different isoenzymes of cytochrome P-450 was assessed by studying liver microsomes isolated from control rats and rats treated with phenobarbital or isoniazid. Rates of CCl4 metabolism were similar for all microsomes under a nitrogen atmosphere. An air atmosphere inhibited metabolism by microsomes from control rats to 12% of the value under nitrogen and metabolism by microsomes from rats treated with phenobarbital to 5%. It inhibited metabolism by microsomes from rats treated with isoniazid only to 32%. Rats treated with phenobarbital, which increases hepatic cytochrome P-450 content, or isoniazid, which does not increase hepatic cytochrome P-450 content, both metabolized more CCl4 than control rats as indicated by exhalation of greater quantities of CCl4 metabolites and by an increase in CCl4 toxicity. These results indicate that some isoenzymes of cytochrome P-450 are more effective than others in metabolizing CCl4 when oxygen is present.     

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent Km values for cytochrome c of 13 microM in the presence of phenazine methosulfate or 19 microM with N,N,N',N'-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O/min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.     

Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus

Cytochrome oxidase from T. thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R. M., Choc, M. G., Tarr, G. E., Findling, K. L., and Fee, J. A. (1983) J. Biol. Chem. 258, 112-123). We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T. thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD). These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions. Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme. The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3. When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized. The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme.     

Kinetic and structural differences between cytochrome c oxidases from beef liver and heart

1. The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eightfold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content. 2. The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c-depleted membranes, deoxycholate-dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low-affinity and for the high-affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes. 3. Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme. 4. By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase isoenzymes in liver and heart is discussed.     

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.     

Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.     

Effect of chronic aurothioglucose treatment of rats on kidney catalase and cytochromes

Catalase activity and cytochrome content were measured in kidneys of Fisher 344 rats injected with aurothioglucose (ATG) either daily for 3 days or 5 days a week for up to 8 wk. Catalase activity was decreased 39%, 59%, and 48% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. Microsomal cytochrome P-450 levels decreased 71%, 86%, and 80% (all p less than 0.001) after 3 days, 2 wk, and 8 wk, respectively. In contrast, cytochrome b5 was significantly increased at 3 days and 2 wk, but not at 8 wk. Microsomal heme contents decreased 44% (p less than 0.001), 34% (p less than 0.001), and 22% (p greater than 0.05) at 3 days, 2 wk, and 8 wk, respectively. The content of mitochondrial cytochromes aa3, b, c1, and c were not affected after 8 wk of ATG treatment. In vitro inhibition of the heme-containing enzyme delta-aminolevulinic acid dehydratase by ATG was reversible in the presence of physiological concentrations of small thiols. Although the activity of this enzyme in kidneys of ATG-treated rats was not measured, its significant inhibition in vivo by ATG appears unlikely. This study demonstrates that there were differential effects of gold on the various cytochromes and that changes in catalase activity paralleled changes in cytochrome P-450 and heme contents in the kidneys of ATG-treated rats. The findings are relevant to nephrotoxicity during chrysotherapy.     

Selective protection of curcumin against carbon tetrachloride-induced inactivation of hepatic cytochrome P450 isozymes in rats

We investigated the effects of curcumin, a major antioxidant constituent of turmeric, on hepatic cytochrome P450 (CYP) activity in rats. Wistar rats received curcumin-containing diets (0.05, 0.5 and 5 g/kg diet) with or without injection of carbon tetrachloride (CCl(4)). The hepatic CYP content and activities of six CYP isozymes remained unchanged by curcumin treatment, except for the group treated with the extremely high dose (5 g/kg). This suggested that daily dose of curcumin does not cause CYP-mediated interaction with co-administered drugs. Chronic CCl(4) injection drastically decreased CYP activity, especially CYP2E1 activity, which is involved in the bioactivation of CCl(4), thereby producing reactive free radicals. Treatment with curcumin at 0.5 g/kg alleviated the CCl(4)-induced inactivation of CYPs 1A, 2B, 2C and 3A isozymes, except for CYP2E1. The lack of effect of curcumin on CYP2E1 damage might be related to suicidal radical production by CYP2E1 on the same enzyme. It is speculated that curcumin inhibited CCl(4)-induced secondary hepatic CYPs damage through its antioxidant properties. Our results demonstrated that CYP isozyme inactivation in rat liver caused by CCl(4) was inhibited by curcumin. Dietary intake of curcumin may protect against CCl(4)-induced hepatic CYP inactivation via its antioxidant properties, without inducing hepatic CYPs.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Kinetic characterization of cytochrome c oxidase from Bacillus subtilis

Bacillus subtilis aa3-type cytochrome c oxidase is capable of oxidizing cytochrome c from different origins. The kinetic properties of the enzyme are influenced by ionic strength. The affinity for Saccharomyces cerevisiae cytochrome c declines with increasing ionic strength whereas the Vmax remains almost constant. An increase of Vmax is observed when the enzyme is incorporated in artificial membranes. Negatively charged phospholipids allow high turnover rates of the aa3-type oxidase. The effect of ionic strength on oxidation of horse heart cytochrome c results in significant changes of both Km and Vmax. These effects can be explained by disturbances of enzyme-substrate interactions and are not related to changes in the aggregation state of the enzyme. The respiration control index of the enzyme reconstituted in artificial membranes appeared to be dependent on phospholipid composition, protein/lipid ratios and also on the external pH. The action of the ionophores nigericin and valinomycin, at various pH values, on the enzyme activity and proton-permeability measurements of the membranes indicate that both components of the proton-motive force, the membrane potential and the pH gradient, can in principle regulate enzyme activity in the reconstituted state.     

Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.     

Protective effect of cornuside against carbon tetrachloride-induced acute hepatic injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl?)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl?. Sixteen h after CCl? treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl?-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl? treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl?-treated rats. Our data indicate that cornuside protects the liver from CCl?-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Seizure-associated pulmonary edema and cerebral oxygenation in the rat

Cerebral partial pressure of O2 (PO2), relative changes in the ratio of reduced/oxidized cytochrome aa3, blood flow, and the arteriovenous difference in O2 content were measured during seizures with and without pulmonary edema. Seizures were induced with bicuculline (0.2-1.2 mg/kg iv) in rats anesthetized with 70% N2O and paralyzed with curare. Briefer seizures were accompanied by increased cerebral PO2 and increased oxidation of cytochrome aa3. Lung water content and arterial O2 partial pressure (PaO2) remained normal. Longer duration seizures were also accompanied initially by increases in cerebral oxygenation. Within minutes, however, PaO2 fell from a mean of 118 to 51 mmHg, and lung water content increased from 76.2 to 83.6%. Cerebral PO2 fell but most often rose back to or above control levels, while cytochrome aa3 became markedly reduced. Simultaneously, cerebral blood flow increased more than 300% above preseizure values and O2 delivery increased more than O2 consumption. The reductive shift of cytochrome aa3 was greater than that produced by lowering PaO2 to equivalent values in seizure-free rats. The reductive shift of cytochrome aa3, despite increased O2 delivery, may be indicative of derangements in cerebral O2 diffusion or energy metabolism.     

Hepatoprotective and antioxidant effect of tender coconut water on carbon tetrachloride induced liver injury in rats

Hepatoprotective and antioxidant effects of tender coconut water (TCW) were investigated in carbon tetrachloride (CCl4)-intoxicated female rats. Liver damage was evidenced by the increased levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and decreased levels of serum proteins and by histopathological studies in CCl4-intoxicated rats. Increased lipid peroxidation was evidenced by elevated levels of thiobarbituric acid reactive substance (TBARS) viz, malondialdehyde (MDA), hydroperoxides (HP) and conjugated dienes (CD), and also by significant decrease in antioxidant enzymes activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and glutathione reductase (GR) and also reduced glutathione (GSH) content in liver. On the other hand, CCl4-intoxicated rats treated with TCW retained almost normal levels of these constituents. Decreased activities of antioxidant enzymes in CCl4-intoxicated rats and their reversal of antioxidant enzyme activities in TCW treated rats, shows the effectiveness of TCW in combating CCl4-induced oxidative stress. Hepatoprotective effect of TCW is also evidenced from the histopathological studies of liver, which did not show any fatty infiltration or necrosis, as observed in CCl4-intoxicated rats.     

Cytochrome aa3 from Nitrosomonas europaea

Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.     

Differential inhibitory effects of carbon tetrachloride on the hepatic plasma membrane, mitochondrial and endoplasmic reticular calcium transport systems: implications to hepatotoxicity

Mitochondrial, endoplasmic reticular and plasma membrane fractions were isolated by a new method from control male Fischer 344 rats and rats given CCl4 by gavage. After 1 h of CCl4 treatment, rats were in glucose and pancreatic hormone balance but plasma levels of T3 and T4 were decreased 29 and 22%, respectively. After 24 hours of CCl4 treatment, rats were: hypoglycaemic and insulin and glucagon levels were increased 33- and 35-fold, respectively; total T4 levels were decreased 62%; while total T3 levels were normalized. In liver fractions from CCl4-treated rats, 1 h after CCl4 administration: (i) calcium binding was decreased 65% in the mitochondrial fraction, 66% in the endoplasmic reticular fraction and 46% in the plasma membrane fraction; (ii) calcium uptake was decreased 59% in the mitochondrial fraction, 46% in the endoplasmic reticular fraction and 37% in the plasma membrane fraction. After 24 h of CCl4 administration: (i) calcium binding was decreased 57% in the mitochondrial fraction, 50% in the endoplasmic reticular fraction and 71% in the plasma membrane fraction; (ii). calcium uptake was decreased 55% in the mitochondrial fraction, 17% in the endoplasmic reticular fraction and 53% in the plasma membrane fraction. In vitro studies indicated the plasma membrane calcium transport system to be rapidly (within a minute) and strongly (>90%) inhibited by CCl4. We conclude that CCl4 produces a differential inhibitory effect on the hepatocyte calcium pumps that are implicated with hepatocellular damage.     

Decrease of oxidative activities in brown adipose tissue mitochondria of cold acclimated rats on short term exposure to heat stress

Exposure of rats to the cold (4-5 degrees C) caused large (2-3-fold) increases in the mass of interscapular brown adipose tissue (BAT), its mitochondrial content and the basal metabolic rate of the animals. The rate of substrate oxidation by BAT mitochondria also increased about 3-fold. When cold-acclimated animals were exposed to heat (37 degrees C), the BMR decreased by half in 3 h, the earliest time interval tested. Mitochondrial substrate oxidation, as well as substrate-dependent H2O2 generation, showed a proportionate decrease in rates. In these mitochondria, activities of cytochrome c reductases, but not dehydrogenases with NADH, alpha-glycerophosphate and succinate as substrates, also showed a significant decrease. The concentration of cytochromes aa3 and b, but not cytochrome c, also decreased in BAT mitochondria from 12-h heat-exposed animals, while the change in concentration of cytochrome b alone was found as early as 3 h of heat exposure. These results identify the change in cytochromes as a mechanism of regulation of oxidative activities in BAT mitochondria under conditions of acute heat stress.     

Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.