Porphyrinogenic activity and ferrochelatase-inhibitory activity of sydnones in chick embryo liver cells

3-[2-(2,4,6-Trimethylphenyl)thioethyl]-4-methylsydnone was shown to be a potent porphyrinogenic agent in chick embryo liver cells. The accumulation of protoporphyrin IX was consistent with the finding that ferrochelatase activity was inhibited. 3-Benzyl-4-phenylsydnone did not inhibit ferrochelatase activity and protoporphyrin IX was found to constitute only a minor fraction of the prophyrins. These results support the idea that the porphyrinogenicity of 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone is due to its catalytic activation by cytochrome P-450 leading to heme alkylation and formation of N-vinylprotoprophyrin IX which inhibits ferrochelatase.     

Ferrochelatase and N-alkylated porphyrins

Summary The final step in heme synthesis is catalyzed by the mitochondrial enzyme, ferrochelatase. Characterization of this enzyme has been complicated by a number of factors including the dependence of enzyme activity on lipids. Purification of ferrochelatase from rat and bovine sources has been achieved only relatively recently using blue Sepharose CL-6B chromatography. When 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) is given to animals, it produces a hepatic porphyria resembling human variegate porphyria thus providing an experimental system in which to study this disease. DDC has been found to cause the accumulation of a green pigment, identified as N-methyl protoporphyrin IX (N-MePP), which is a potent inhibitor of ferrochelatase. The source of the N-methyl substituent of N-MePP was found to be the 4-methyl group of DDC. Considerable evidence indicates that the protoporphyrin IX moiety of N-MePP originates from the heme moiety of cytochrome P-450 and that DDC is a suicide substrate for this hemoprotein. Some studies suggest that cytochrome P-450 isozymes differ in their susceptibility to destruction by DDC and its 4-alkyl analogues. Griseofulvin has also been reported to inhibit hepatic ferrochelatase in rodents but not in the 17-day old chick embryo nor in hepatocyte culture systems. Thus, the mechanism by which griseofulvin produces an experimental porphyria in chick embryo liver cell culture is different from that for rodents.     

Inactivation of cytochrome P450 and inhibition of ferrochelatase by analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine with 4-nonyl and 4-dodecyl substituents.

Cytochrome P450- and heme-destructive effects of the 4-nonyl and 4-dodecyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were determined using hepatic microsomal preparations obtained from untreated, beta-naphthoflavone-treated, and phenobarbital-treated chick embryos. The 4-nonyl analogue of DDC was less efficacious than 4-ethyl DDC and 4-hexyl DDC, but more efficacious than 4-dodecyl DDC with respect to cytochrome P450-destructive activity. In all hepatic microsomal preparations, cytochrome P450 destruction by 4-nonyl DDC was accompanied by loss of microsomal heme. In contrast, 4-dodecyl DDC caused loss of heme only in hepatic microsomal preparations obtained from phenobarbital-treated chick embryos. The ability of 4-nonyl DDC and 4-dodecyl DDC to lower ferrochelatase activity was compared with that of 4-ethyl DDC and 4-hexyl DDC in cultured chick embryo hepatocytes. As the length of the 4-alkyl group was increased, the ferrochelatase-lowering efficacy and potency of the DDC analogue decreased. The 4-dodecyl DDC analogue was unable to lower ferrochelatase activity, which accorded with the finding that the administration of 4-dodecyl DDC to phenobarbital-treated rats did not lead to the accumulation of an N-alkylprotoporphyrin. The ability of 4-nonyl DDC to lower ferrochelatase activity was attributed to the formation of N-nonylprotoporphyrin IX following the administration of 4-nonyl DDC to phenobarbital-treated rats.     

Comparison of the effects of 3-ethoxycarbonyl-1,4-dihydro-2,4-dimethylpyridine and 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on ferrochelatase activity and heme biosynthesis in chick embryo liver cells in culture

3-Ethoxycarbonyl-1,4-dihydro-2,4-dimethylpyridine (EDP) was shown to lack the ferrochelatase-lowering activity of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) in chick embryo liver cells in culture. This was attributed to the inability of EDP to cause destruction of the heme moiety of cytochrome P-450 with concomitant formation of N-methylprotoporphyrin IX. EDP was less potent as a porphyrinogenic agent than DDC and caused the accumulation of uroporphyrin, heptacarboxylic porphyrin, and coproporphyrin in contrast with DDC which caused primarily protoporphyrin to accumulate. The inactivity of EDP as a ferrochelatase-lowering agent and its low porphyrinogenic potency was explained, at least in part, by its rapid transformation in aqueous solution to other nondihydropyridine products. The two ethoxycarbonyl substituents of DDC are therefore essential for N-methylprotoporphyrin formation, ferrochelatase-lowering activity, and optimal porphyrin-inducing activity.     

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 microM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.     

Inhibition of ferrochelatase by N-methylprotoporphyrin IX is not accompanied by delta-aminolevulinic acid synthetase induction in chick embryo liver cell culture

N-Methylprotoporphyrin has been shown to markedly inhibit ferrochelatase activity in chick embryo liver cell culture without inducing delta-aminolevulinic acid (ALA) synthetase activity. This result supports the idea that the effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) on ALA synthetase activity and ferrochelatase activity are dissociated and that inhibition of ferrochelatase alone is not sufficient to cause induction of ALA synthetase. We conclude that the porphyrinogenic activity of DDC can be explained only in part by the actions of N-methylprotoporphyrin.     

Isolation of regioisomers of N-alkylprotoporphyrin IX from chick embryo liver after treatment with porphyrinogenic xenobiotics

Several porphyrinogenic xenobiotics cause mechanism-based inactivation of cytochrome P450 (P450) isozymes with concomitant formation of a mixture of four N-alkylprotoporphyrin IX (N-alkylPP) regioisomers, which have ferrochelatase inhibitory properties. To isolate the four regioisomers of N-methylprotoporphyrin IX (N-methylPP), 3,5-diethoxycarbonyl, 1-4-dihydro-2,4,6-trimethylpyridine (DDC) was administered to untreated, beta-naphthoflavone-, phenobarbital-, and glutethimide-pretreated 18-day-old chick embryos. Separation of the N-methylPP regioisomers by high pressure liquid chromatography (HPLC) revealed no marked difference in the regioisomer pattern among the different treatments. After administration of griseofulvin, allylisopropylacetamide (AIA), or 1-[4-(3-acetyl-2,4,6-triemethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime (ATMP) to untreated and glutethimide-pretreated 18-day-old chick embryos, an N-alkylPP was isolated after AIA administration only. This finding strengthened previous reports of the species specificity of N-alkylPP formation with griseofulvin and ATMP. A series of dihydropyridines, namely 4-ethylDDC, 4-hexylDDC, and 4-isobutylDDC were administered to untreated and glutethimide-pretreated 18-day-old chick embryos and hepatic N-alkylPPs were isolated and separated by HPLC into regioisomers. The regioisomer patterns obtained did not support a previous proposal of masked regions above both rings B and C in the heme moieties of the P450 isozymes responsible for N-alkylPP formation. However, the data support the hypothesis of a partially masked region above ring B alone. The regioisomer patterns were in agreement with results previously obtained in rats showing that the percentage of Nc and (or) ND regioisomers in the regioisomer mixture increases as the length and bulk of the 4-alkyl substituent of a DDC analogue increase. Differences in the regioselectivity of heme N-alkylation may be due to intrinsic chemical features of DDC analogues themselves or to differences in the P450 isozymes inactivated.     

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

Inactivation of rat liver microsomal steroid hydroxylations by 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine: evidence for selectivity among steroid-inducible cytochrome P450IIIA forms

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6- trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) via destruction of the heme prosthetic group. This is an important component of these compounds' porphyrinogenic mechanism. In an attempt to map the P-450 isozyme selectivities of DDC analogues, we have examined the effects of these compounds on the regioselective and stereoselective hydroxylation of androstenedione (AD) and progesterone (PG) in rat liver microsomal systems. In microsomes from phenobarbital-treated male rats, DDC analogues did not cause time-dependent inactivation of AD 7 alpha-hydroxylase, AD 16 beta-hydroxylase, and PG 21-hydroxylase, selective markers for P450IIA 1/2, IIB1, and IIC6, respectively. In contrast, DDC analogues were effective inactivators of PG 2 alpha-hydroxylase and steroid 6 beta-hydroxylases, selective markers for P450IIC11 and IIIA forms, respectively. We conclude that differences in porphyrinogenicity observed with various DDC analogues are not likely to be due to the selective destruction of different P-450 isozymes by different analogues, but rather to properties of the DDC analogues themselves. 4-Ethyl DDC was found to be capable of discriminating between P450IIIA subfamily forms. In microsomes from untreated male rats, which express P450IIIA2 but not IIIA1, 4-ethyl DDC inactivated both AD and PG 6 beta-hydroxylases. However, in microsomes from dexamethasone-treated female rats, which express P450IIIA1 but not IIIA2, no inactivation of the steroid 6 beta-hydroxylases was observed. Thus, 4-ethyl DDC appears to be a potentially valuable tool for differentiating between P450IIIA forms.     

Role of inducer binding in cytochrome P-450 IA2-mediated uroporphyrinogen oxidation

The oxidation of uroporphyrinogen, an intermediate of the heme biosynthetic pathway, by methylcholanthrene-inducible isozymes(s) of cytochrome P-450 has been proposed to play a role in the development of chemically induced uroporphyria. Prior work from this laboratory indicated that although addition of 3,4,3',4'-tetrachlorobiphenyl is required for uroporphyrinogen oxidation by methylcholanthrene-induced chick embryo liver microsomes, this biphenyl is not required for the oxidation catalyzed by hepatic microsomes from methylcholanthrene-induced rodents. Here we investigated whether rodent microsomes catalyze uroporphyrinogen oxidation without addition of 3,4,3',4'-tetrachlorobiphenyl because the chemical used as an inducer remains bound to cytochrome P-450. Hepatic microsomes containing almost no residual inducer were isolated from rats treated with a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These microsomes oxidized uroporphyrinogen at high rates without addition of 3,4,3',4'-tetrachlorobiphenyl. Inducer-free microsomal cytochrome P-450 was also obtained by inducing cytochrome P-450 in rats and mice with isosafrole, which was then removed from the isolated microsomes by butanol treatment. This procedure resulted in microsomes with high activity for uroporphyrinogen oxidation. Furthermore, addition of chlorobiphenyl to these inducer-free microsomes was inhibitory. Hepatic microsomes from isosafrole-induced C57BL/6 and DBA mice, rendered inducer-free by butanol treatment, oxidized uroporphyrinogen at the same rate even though these two strains differ markedly in their susceptibility to chemically induced uroporphyria. We conclude that uroporphyrinogen oxidation is catalyzed by cytochrome P-450 that is free of inducer.     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats

Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes delta-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, delta-aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors.     

Drug-induced protoporphyria in the olfactory mucosa of the hamster

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Uroporphyrin formation induced by chlorinated hydrocarbons (lindane, polychlorinated biphenyls, tetrachlorodibenzo-p-dioxin). Requirements for endogenous iron, protein synthesis and drug-metabolizing activity

The chlorinated hydrocarbons, lindane (gamma-hexachlorocyclohexane), polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin, induce chick embryo liver cells in culture to rapidly accumulate uroporphyrin III. This is in marked contrast to the delayed effect in animals. The induction is dependent on protein synthesis (cycloheximide inhibited), endogenous iron (chelators inhibited) and perhaps cytochrome P-450 (SKF-525A inhibited). Evidence was obtained for an unstable intermediate that is generated in the liver from chlorinated hydrocarbons which inhibits uroporphyrinogen decarboxylase thus causing uroporphyrin to accumulate. The data suggest that the intermediate may be a hydroxylated derivative of the chlorinated hydrocarbon.     

Inhibition of uroporphyrinogen decarboxylase by halogenated biphenyls in chick hepatocyte cultures. Essential role for induction of cytochrome P-448.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was assayed in cultures of chick-embryo hepatocytes by the changes in composition of porphyrins accumulated after addition of excess 5-aminolaevulinate. Control cells accumulated mainly protoporphyrin, whereas cells treated with 3,4,3',4'-tetrachlorobiphenyl or 2,4,5,3',4'-pentabromobiphenyl accumulated mainly uroporphyrin, indicating decreased activity of the decarboxylase. 3-Methylcholanthrene and other polycyclic-hydrocarbon inducers of the P-448 isoenzyme of cytochrome P-450, did not affect the decarboxylase in the absence of the biphenyls. Induction of P-448 was detected as an increase in ethoxyresorufin de-ethylase activity. Pretreatment of cells with methylcholanthrene decreased the time required for the halogenated biphenyls to inhibit the decarboxylase. The dose response of methylcholanthrene showed that less than 40% of the maximal induction of cytochrome P-448 was needed to produce the maximum biphenyl-mediated inhibition of the decarboxylase. In contrast, induction of the cytochrome P-450 isoenzyme by propylisopropylacetamide had no effect on the biphenyl-mediated decrease in decarboxylase activity. Use of inhibitors of the P-450 and P-448 isoenzymes (SKF-525A, piperonyl butoxide and ellipticine) supported the concept that only the P-448 isoenzyme is involved in the inhibition of the decarboxylase by the halogenated biphenyls. The effect of preinduction with methylcholanthrene to enhance inhibition of the decarboxylase was also shown by the increased rate at which porphyrin accumulated from endogenously synthesized 5-aminolaevulinate after treatment of cells with the combination of propylisopropylacetamide and the biphenyls. Antioxidants, chelators of iron, and chromate affected the decrease in decarboxylase activity only if they prevented the induced increase in cytochrome P-448. We conclude that the P-448 and not the P-450 isoenzyme of cytochrome P-450 plays an obligatory role in the inhibition of uroporphyrinogen decarboxylase caused by halogenated biphenyls.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10,000g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein.min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3'4,4'-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This results accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.     

Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.     

Inhibition of testicular cytochrome P-450-dependent steroid biosynthesis by cis-platinum. Reversal by human chorionic gonadotropin

The treatment of rats with cis-platinum for 7 days caused a profound, and seemingly selective, decrease (70-80%) in the microsomal cytochrome P-450 levels in the testis. This decrease was accompanied by marked reductions (70-80%) in steroid 17 alpha-hydroxylase activity and in plasma testosterone concentration. The treatment of rats with human chorionic gonadotropin partially restored the cytochrome P-450 concentration and 17 alpha-hydroxylase activity and permitted the plasma testosterone level to approach control values. The effect of cis-platinum on the testicular cytochrome P-450 appeared unrelated to deficiencies in heme metabolic processes, in so far that neither was the activity of delta-aminolevulinate synthetase decreased, nor was that of heme oxygenase increased. These enzymes are rate-limiting in heme biosynthesis and degradation pathways, respectively. Also, the activities of uroporphyrinogen I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the concentration of total porphyrins in the testis remained unchanged. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation did not reveal a diminished level of apocytochrome; however, in this preparation, heme could not be detected in molecular weight regions corresponding to cytochrome P-450. The microsomal cytochrome b5 and the mitochondrial heme concentrations were not decreased in cis-platinum-treated rats. It is suggested that the mechanism of depletive action of cis-platinum on microsomal cytochrome P-450 involves an impairment of the effective assembly of heme and apoprotein moieties. It is further suggested that the anterior pituitary hormones control the factor(s) involved in this assembly, a process which is interrupted by cis-platinum.     

3,5-Diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine inactivates rat liver cytochrome P-450c, but not its orthologue, rabbit lung form 6

Various rat liver cytochrome P-450 (P-450) isozymes are targets for mechanism-based inactivation by 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4- dihydropyridine (4-ethyl DDC). Unlike rat liver, which contains multiple P-450 isozymes, rabbit lung contains only three major isozymes referred to as forms 2, 5, and 6. We have examined the ability of 4-ethyl DDC to destroy P-450 heme in hepatic and pulmonary microsomes from untreated and beta-naphthoflavone (beta NF)-treated rabbits. This compound destroyed 31% of the P-450 in either hepatic microsomal preparation, but was ineffective at lowering P-450 and heme levels in pulmonary microsomes when examined at a range of concentrations (0.45-5.0 mM). These data suggest that rabbit pulmonary P-450 forms 2, 5, and 6 are not targets for destruction by 4-ethyl DDC, despite the ability of this compound to inactivate rat liver P-450c, the orthologue of rabbit lung form 6.