Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of n-alkyl and n-alkenyl heme adduct formation

Grab, Lawrence A.; Swanson, Barbara A.; Montellano, Paul R. Ortiz de

doi:10.1021/bi00413a034

Cited by 31 publications

(8 citation statements)

References 29 publications

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“…It is conceivable that this spectrum reflects the presence of SB-N-heme (Fe 3ϩ ) adduct in this in vitro system, because it is similar to that of the purified iron-complexed N-ethylporphyrins, that exhibit absorbance maxima at 442 nm (25). Furthermore, consistent with this possibility, the absolute spectra of phenylacetylene-or 3-alkylsydnone-inactivated P450 2B1 preparations that yield N-modified heme adducts also exhibit a time-dependent increase in a 445 nm shoulder (26,27). It is interesting that no corresponding spectra are detected after inactivation of P450 2B1 by allylisopropylacetamide, or of other P450s by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridene, in spite of the fact that much larger quantities of N-alkylated porphyrins are formed and isolated from such inactivation systems.…”

Section: Sb Epoxidation By P450 2b1 and Its Mutants-supporting

confidence: 53%

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

Szklarz

et al. 1996

Journal of Biological Chemistry

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Secobarbital (SB) is a relatively selective mechanismbased inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SBprotein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme Nalkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

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Section: Sb Epoxidation By P450 2b1 and Its Mutants-supporting

confidence: 53%

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

Szklarz

et al. 1996

Journal of Biological Chemistry

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“…are potent porphyrinogenic xenobiotics capable of eliciting mechanism-based inactivation of cytochrome P450 (CYP), with concomitant N-alkylprotoporphyrin IX (N-alkylPP) formation and subsequent ferrochelatase (FC) inhibition in rats (Grab et al 1988;Tephly et al 1979). Previously, we have shown that the rat CYP isozymes responsible for N-alkylPP formation, and thus the critical mediators of hepatic porphyrin accumulation, are different for TTMS and 4-ethylDDC (Wong et al 1998).…”

Section: -[(Arythio)ethyl]sydnone (Ttms) and 35-diethoxycarmentioning

confidence: 99%

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

Gamble¹,

Wong²,

Dailey³

et al. 2000

Can. J. Physiol. Pharmacol.

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Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV-visible spectrophotometry to isolate and identify N-alkylPPs after incubating porphyrinogenic compounds with rat liver microsomes. However, the overall yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA-expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present study we demonstrate that purified recombinant human ferrochelatase (FC) provides an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10(-6) nmol range. Therefore, we propose that this bioassay system might allow the use of human lymphoblastoid microsomal preparations containing single cDNA-expressed human CYP isozymes to detect N-alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potentially porphyrinogenic drugs prior to administration to humans.

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“…The residue was resuspended in tetrahydrofuran, the radioactivity was measured, and an aliquot was analyzed by reverse-phase HPLC. The heme fraction that contained radioactivity was treated with H2SO4/ CH3OH to derivatize the carboxylic acid groups on the heme moiety to methyl esters, followed by treatment with zinc acetate to form methylated zinc metalloporphyrins (23)(24)(25)(26); it was then analyzed by HPLC. As an alternative approach, the whole hemoglobin was treated in a similar manner; this simultaneously precipitates the protein and derivatizes the heme moiety to the corresponding methyl ester.…”

Section: Chemicalsmentioning

confidence: 99%

“…Concentrated acid and base treatments of the heme fraction did not release any detectable derivative of 1-NP, suggesting that the radioactivity was strongly bound to the heme moiety. Other compounds (for example phenylhydrazine, 3-alkylsydnones, diethylnitrosamine, aminobenzotriazole, and norethindrone) have also been shown to form adducts with the heme moiety of hemoglobin as well as with heme-containing enzymes (23)(24)(25)(26)(43)(44)(45) …”

Section: Analysis Of Hemoglobin Adducts Fliowig the Or Adnistration Omentioning

confidence: 99%

Development of Methods to Monitor Exposure to 1-Nitropyrene

El‐Bayoumy¹,

Johnson²,

Roy³

et al. 1994

Environmental Health Perspectives

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On the basis of 32P-postlabeling analysis, treatment of rats with 1-nitropyrene (1-NP) resulted in the formation of multiple DNA adducts in the liver, mammary glands, and peripheral lymphocytes. The one adduct resulting from nitroreduction, N-(deoxyguanosin-8-yl)-1-aminopyrene, constitutes only a minor component among the adducts. In the present study, incubation of calf thymus DNA with mutagenic ring-oxidized metabolites of 1-NP in vitro in the presence and absence of xanthine oxidase also resulted in the formation of multiple adducts. On the basis of their chromatographic behavior, it appears that DNA adducts derived from such metabolites may have been formed in vivo; however, this needs to be confirmed.[3H]1-NP was given to male and female F344 rats and Sprague-Dawley rats by gavage at five dose levels in the range of 0.1 to 1000 pg/kg bw. This led to stable hemoglobin adducts accounting for 0.08 ± 0.05% of the dose (n = 3 rats). The radioactivity associated with hemoglobin following administration of [3H]1-NP was cleared with a half-life of about 14 days, which is faster than that of unmodified erythrocytes in the rat (t1/2= 30 days).Treatment of the hemoglobin with 1% HCI in acetone, to precipitate the globin, released the radioactivity; it was all bound to the heme moiety. The structures of the heme adducts have not been elucidated; yet, because of their stability, they may be useful as dosimeters for human exposure to 1-NP. The results of this study demonstrate the potential of using hemoglobin adducts of 1-NP as dosimeters of uptake and metabolic activation of nitropolynuclear aromatic hydrocarbons (NO2-PAH). These indicators are a prerequisite for cancer risk assessment of N02-PAH. -Environ Health Perspect 102(Suppl 6): 31-37 (1994)

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Cytochrome P-450 inactivation by 3-alkylsydnones. Mechanistic implications of n-alkyl and n-alkenyl heme adduct formation

Cited by 31 publications

References 29 publications

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

Development of Methods to Monitor Exposure to 1-Nitropyrene

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