Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate).

Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively).

The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.     

Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates

The side-chain cleavage of cholesterol by cytochrome P-450_scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3β-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450_scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450_scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450_scc-20,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20-hydroxycholesterol, 22R-hydroxycholesterol and 20,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450_scc occur at approximately the same rate.     

Effects of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase and glutathione S-transferase activities in rat brain

The effects of acute and therapeutic doses of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase (EC 1.14.14.1) and glutathione S-transferase (EC 2.5.1.18) activities have been studied in rat brain and compared with those of rat liver. P-450 enzymic activity was assayed by N-demethylation of p-chloro-N-methylaniline and 1-chloro-2,4-dinitrobenzene was used as substrate for glutathione S-transferase activity. The acute effects of a single daily dose of phenobarbital (75 mg/kg/day;i.p.) and sodium salicylate (500 mg/kg/day;i.p.) for 3 days increased cytochrome P-450 as well as glutathione S-transferase in rat liver. But the same doses of both drugs decreased glutathione S-transferase levels in rat brain and increased cytochrome P-450 dependent N-demethylation of p-chloro-N-methylaniline. The therapeutic doses of sodium salicylate (50 mg/kg/day;i.p.) and phenobarbital (10 mg/kg/day;i.p.) daily for 21 days increased cytochrome P-450 in rat liver as well as in brain. The increase in brain glutathione S-transferase by prolonged treatment of phenobarbital was significant compared to the control values.     

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O₂ tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O₂ carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Molecular orbital studies of oxygen activation and mechanisms of cytochromes P-450-mediated oxidative metabolism of xenobiotics

Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth², and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth² and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): studies on the effects of monooxygenase inducers and role of cytochromes P-450

The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (>90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography—mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with β-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55–60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.     

A detailed molecular model for human aromatase

Using a variety of techniques, including sequence alignment, secondary struucture prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450_AROM (human aromatase) based on that of P-450_cam, the only cytochrome P-450 enzyme for which crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: effect of Tween20 concentration

Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], [R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Evaluation of immobilized cytochrome P-448 from Saccharomyces cerevisiae using permeabilized whole cell, microsomal fraction and highly purified reconstituted forms, with benzopyrene-3-monooxygenase activity

Cytochrome P-448 from Saccharomyces cerevisiae in permeabilized whole cell, microsomal fraction and in a highly purified reconstituted benzopyrene-3-monooxygenase (EC 1.14.14.1) system have been immobilized on various supports. Calcium alginate was found to be especially useful and the kinetics of hydroxylation were close to that of the free enzyme system with all three forms of enzyme, even with permeabilized whole yeast cells (V _max of 664 pmol 3-hydroxybenzo(a)pyrene produced per h per nmol cytochrome P-448 compared with 1000 for free highly purified reconstituted enzyme system). Only the highly purified reconstituted form was successfully immobilized by BrCN-activated Sepharose-4B or by acrylamide. Both of these supports stabilized the highly purified reconstituted cytochrome P-448 benzopyrene-3-monooxygenase activity in prolonged storage at 4°C. Applications for various immobilized enzymes and cells are assessed.     

Effects of two oral antimycotics, ketoconazole and fluconazole, upon steroidogenesis in rat adrenal cells in vitro

Rat adrenal cells were incubated with various concentrations of two orally active azole antimycotics in order to evaluate the effects on steroidogenesis. The first compound was ketoconazole, a well-known inhibitor not only of fungal cytochrome P-450 but at higher concentrations also of mammalian cytochrome P-450 dependent enzymes. The second was fluconazole, a newly developed oral antimycotic with a triazole structure, which likewise inhibits fungal cytochrome P-450. The influence of both drugs on mammalian cytochrome P-450 dependent enzymes was investigated in this study. Ketoconazole inhibited ACTH-stimulated corticosterone (IC50 = 0.9 microM) and aldosterone secretion (IC50 = 1.4 microM) and enhanced 11-deoxycorticosterone output at low concentrations but reduced it at higher concentrations. Radiotracer experiments with [3H]pregnenolone or [3H]11-deoxycorticosterone as exogenous substrates revealed a 50% inhibition of the oxidative substrate metabolism at about 1 microM ketoconazole. These effects could also be observed with fluconazole but occurred at concentrations approximately two orders of magnitude higher as compared to ketoconazole. We conclude that fluconazole has a much higher selectivity for fungal cytochrome P-450 than ketoconazole. The order of sensitivity of the cytochrome P-450 dependent enzymes of rat adrenal steroidogenesis to ketoconazole was the 11 beta/18-hydroxylase, the cholesterol side chain cleavage enzyme and the 21-hydroxylase with decreasing sensitivities.     

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f.

Incubation of chloroplasts with KCN or HgCl₂ decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.

At low concentrations of salt and sugar the fast phases of P-700⁺ reduction were largely inhibited. Increasing concentrations of MgCl₂, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl₂ and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.

The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.

Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl₂ suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.