Inactivation of chick embryo hepatic cytochrome P450 1A, 2H and 3A following in ovo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine and 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone.

Rat hepatic cytochrome P450 (P450) isozymes 1A1, 2C6, 2C11, 3A1 and 3A2 are targets for mechanism-based inactivation by the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC). It is of interest to determine whether similar P450 isozymes are targets of porphyrinogenic drugs in the chick embryo liver. The chick embryo expresses P450 2H1/2 isozymes, which are similar to the rat P450 2B1/2 isozymes, a polycyclic aromatic hydrocarbon-inducible P450 1A isozyme, and a pregnenolone 16 alpha-carbonitrile-inducible P450 3A isozyme. We have found previously that chick embryo hepatic P450 1A and 3A isozymes are targeted for in vitro mechanism-based inactivation by 4-ethyl DDC and by the sydnone 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS). Marked differences have been observed between the in vitro and in vivo effects of porphyrinogenic drugs on P450 isozymes. Thus, the first objective of this study was to determine whether chick embryo hepatic P450 1A and 3A isozymes are subject to in ovo inactivation by these porphyrinogenic compounds. Our second objective was to determine whether the chick embryo hepatic P450 2H isozyme(s) was subject to in ovo and in vitro inactivation by 4-ethyl DDC and TTMS. Using hepatic microsomes prepared from beta-naphthoflavone-, dexamethasone-, phenobarbital-, and glutethimide-induced 19-day-old chick embryos, we found that total P450 content was decreased significantly in microsomes prepared from all treatment groups following in ovo administration of 4-ethyl DDC and TTMS. Moreover, in ovo administration of both 4-ethyl DDC and TTMS caused a significant decrease of 7-ethoxyresorufin O-deethylase, erythromycin N-demethylase, and benzphetamine N-demethylase activities, which are selective catalytic markers for chick embryo hepatic P450 1A, 3A and 2H isozymes, respectively. In addition, in vitro administration of 4-ethyl DDC and TTMS caused mechanism-based inactivation of benzphetamine N-demethylase activity in microsomes from phenobarbital- and glutethimide-treated chick embryos, showing that the chick embryo hepatic P450 2H isozyme is a target for mechanism-based inactivation. Therefore, it was concluded that the chick embryo hepatic P450 1A, 2H and 3A isozymes serve as targets for both in ovo and in vitro mechanism-based inactivation by 4-ethyl DDC and TTMS.

Evidence for mechanism-based inactivation of rat and chick embryo hepatic cytochrome P4501A and P4503A by dihydropyridines, sydnones, and dihydroquinolines.

Rat hepatic P4501A1 and 3A1/2 have been shown previously to be targets for mechanism-based inactivation by the 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), namely, 4-ethyl DDC and 4-isopropyl DDC. In this study we have shown that rat hepatic P4501A and P4503A are targets for mechanism-based inactivation by the sydnones, 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) and 3-(2-phenylethyl)-4-methylsydnone (PEMS). The dihydroquinoline, 2,4-diethyl-2-methyl-1,2-dihydroquinoline (DMDQ), caused mechanism-based inactivation of rat hepatic P4501A but not of P4503A. The P4501A isozyme(s) of chick embryo liver was found to share the ability of rat liver P4501A to serve as a target for mechanism-based inactivation by the dihydropyridines, 4-ethyl DDC and 4-isopropyl DDC, the sydnones, TTMS and PEMS, and the dihydroquinoline, DMDQ. A P4503A-like isozyme of chick embryo liver shared the ability of the rat liver P4503A isozyme(s) to serve as a target for mechanism-based inactivation by the dihydropyridines, 4-ethyl DDC and 4-isopropyl DDC, and the sydnone, TTMS, but not of the sydnone PEMS. The dihydropyridine, DDC, was found to serve as a mechanism-based inactivator of the chick embryo P4501A isozyme(s), but not of the P4503A isozyme(s), in contrast to its previously reported inactivity with both the rat hepatic P4501A1 and 3A1/2 isozymes.

11 beta-hydroxyandrostenedione: a marker of adrenal function in hirsutism.

To assess the role of the adrenal glands in the development of hirsutism, levels of 11 beta-hydroxyandrostenedione (11 beta-OHA), 17 alpha-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulphate (DHEAS), androstenedione (delta 4A), and free and total testosterone (T) were measured in 63 hirsute females and 30 control patients. Six of the hirsute patients had basal levels of 11 beta-OHA and 17-OHP and responses to adrenocorticotropic hormone that were significantly greater than these values in controls and the other hirsute women. These women were designated as having an adrenal source for their hirsutism. Women with polycystic ovarian syndrome and idiopathic hirsutism had normal values of 11 beta-OHA and 17-OHP. Levels of total and free T, DHEAS and delta 4A were significantly higher than control values in all of the hirsute women. This study demonstrates that 11 beta-OHA can be used as a marker to assess the adrenal contribution to hirsutism.

Thin-layer chromatographic separation of the ferrochelatase-inhibitory ring A and ring B regioisomers of N-ethylprotoporphyrin from a mixture of the four regioisomers.

When N-alkylprotoporphyrins are prepared synthetically or biologically, a mixture of four regioisomers is obtained. For our studies, separation of the potent ferrochelatase-inhibitory ring A (NA) and ring B (NB) regioisomers from the ring C (NC) and ring D (ND) regioisomers of low potency is required. Previously this separation required two successive high-performance liquid chromatography procedures. We now report that the separation of the zinc complexes of the NA and NB regioisomers from the NC and ND regioisomers can be achieved by a rapid and inexpensive thin-layer chromatography procedure.

Evidence for the stereoselective inhibition of chick embryo hepatic ferrochelatase by N-alkylated porphyrins. II.

N-Ethylprotoporphyrin (N-ethyl-PP) was isolated from the livers of phenobarbital-pretreated rats after the administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine, separated into its four regioisomers by high performance liquid chromatography, and quantitated. The percentage ratio, in the chromatogram, of the peak areas of the ring A-substituted (NA) and the ring B-substituted (NB) regioisomers was 80:20, compared with 50:50 for synthetic N-ethyl-PP. The NA regioisomer of N-ethyl-PP isolated from rat liver was found to be approximately 5 times more potent an inhibitor of ferrochelatase than was the NB regioisomer. Because the synthetic NA regioisomer (an equal mixture of the NA and the epi-NA enantiomers) is equipotent with the synthetic NB regioisomer (an equal mixture of the NB and the epi-NB enantiomers), epi-NB must be more potent than epi-NA. The higher potency previously observed with the NA plus NB regioisomers of N-ethyl-PP isolated from rat liver, compared with the NA plus NB regioisomers of synthetic N-ethyl-PP, is explained by the fact that the biological preparation contains 80% of the potent NA, compared with 25% of the potent NA and 25% of the potent epi-NB in the synthetic preparation. The critical features for optimal ferrochelatase-inhibitory activity are the ring A N-ethyl group facing downward in the NA isomer and the ring B N-ethyl group in the epi-NB isomer being rotated through 180 degrees to occupy the same position. According to one proposed mechanism, N-alkylprotoporphyrins inhibit ferrochelatase by serving as transition state analogues for the iron insertion step. X-ray crystallography shows that the N-alkyl group-bearing pyrrole ring and the pyrrole ring opposite to the N-alkyl group are tilted out of planarity in opposite directions. We suggest that this tilting reflects the normal conformational changes required for the insertion of iron into the protoporphyrin IX ring by ferrochelatase and that the greater inhibitory activity of NA and epi-NB isomers, compared with epi-NA and NB isomers, is due to the fact that the normal mechanism for ferrochelatase-catalyzed iron insertion has preference for an A-C ring tilt over a B-D ring tilt.

Chick embryo liver microsomal steroid hydroxylations: induction by dexamethasone, phenobarbital, and glutethimide and inactivation following the in ovo administration of porphyrinogenic compounds.

Metabolites of [4-(14)C]androstenedione (AD) and [4-(14)C]progesterone (PG) were separated and quantitated following incubation with hepatic microsomes from chick embryos. PG 2 alpha-hydroxylase and AD 7 alpha-hydroxylase, which are diagnostic markers in rat liver for cytochrome P450 (P450) 2C11 and 2A1/2, respectively, were not identified in chick embryo liver. PG 6 beta-hydroxylase and AD 6 beta-hydroxylase, diagnostic markers for P450 3A1/2 activity in rat liver, were identified in chick embryo liver, and we were able to show that radiolabeled 6 beta-hydroxyprogesterone and 6 beta-hydroxyandrostenedione, the metabolites of PG and AD 6 beta-hydroxylase, respectively, were homogeneous and identical with authentic standards. Dexamethasone, phenobarbital, and glutethimide were found to be significant inducers of PG and AD 6 beta-hydroxylases in chick embryo liver. The in ovo administration of the porphyrinogenic compounds 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) and 3-[2-(2,4,6-trimethylphenyl)-thioethyl]-4-methylsydnone (TTMS) caused inactivation of chick embryo hepatic PG and AD 6 beta-hydroxylases. Therefore, we suggest that PG and AD 6 beta-hydroxylases may serve as diagnostic markers for a P450 3A-like isozyme in the chick embryo, and that this isozyme is one of the targets for inactivation by 4-ethyl DDC and TTMS.

Mechanism-based inactivation of hepatic cytochrome P450 2C6 and P450 3A1 following in vivo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine to rats: differences from previously observed in vitro results.

Using progesterone 21-hydroxylase as a selective substrate for P450 2C6 in phenobarbital-treated male rats, and androstenedione and progesterone 6 beta-hydroxylases as well as erythromycin N-demethylase as selective markers for P450 3A1 in dexamethasone-treated female rats, we have shown that these P450 isozymes undergo mechanism-based inactivation after in vivo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC). These results differ from our previous studies where no inactivation was observed after in vitro administration of 4-ethyl DDC to rat hepatic microsomes. We show that the differences between the in vivo and in vitro effects of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) analogues are due to the presence of residual 4-ethyl DDC in the in vitro experiments causing time-independent competitive inhibition and obscuring observation of mechanism-based inactivation.

Isolation of N-vinylprotoporphyrin IX after hepatic cytochrome P450 inactivation by 3-[(arylthio)ethyl]sydnone in chick embryos pretreated with phenobarbital, glutethimide, dexamethasone, and beta-naphthoflavone: differential inhibition of ferrochelatase by N-vinylprotoporphyrin regioisomers.

Several xenobiotics caused hepatic porphyrin accumulation through mechanism-based inactivation of cytochrome P450(P450) and heme alkylation. Loss of iron from the alkylated heme results in formation of an N-alkylporphyrin, which is a potent inhibitor of ferrochelatase. N-Vinylprotoporphyrin IX (N-vinylPP) was identified in chick embryo liver after in ovo administration of 3-[(arylthio)ethyl]sydnone (TTMS). Pretreatment of chick embryos with beta-naphthoflavone, which causes a 90-fold increase in P450 1A levels, did not increase the formation of N-vinylPP after TTMS administration, showing that the heme moiety of P450 1A does not contribute to the formation of N-vinylPP. Increased amounts of N-vinylPP were isolated from dexamethasone-, phenobarbital-, and glutethimide-pretreated chick embryos, and it is possible that P450 2H and/or a P450 3A-like isozyme contributes to the formation of N-vinylPP. The ring B-substituted (NB) regioisomer of N-vinylPP constituted the lowest percentage of the total regioisomers (9-13%) in untreated and drug-induced chick embryos, thus supporting the concept that ring B of heme is occluded by a protein residue in the P450 active site. Previously, the finding that the NB regioisomer of N-ethylprotoporphyrin IX had one fifth the potency of the ring A-substituted (NA) regioisomer as a ferrochelatase inhibitor led to a proposal that an A-C ring tilt was important in N-alkylprotoporphyrins for ferrochelatase inhibition. The finding in the present study that the NA and NB regioisomers of N-vinylPP are equipotent does not support the above proposal. The ring C-substituted (NC) and ring D-substituted (ND) regioisomers of N-vinylPP had low potency.

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

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.

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.