Oxidative Metabolism of 1-Nitropyrene by Rabbit Liver Microsomes and Purified Microsomal Cytochrome P-450 Isozymes

Paul C. Howard, Kimberly A. Reed, Dennis Koop

Research output: Contribution to journalArticle

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Abstract

Rabbit liver (male) microsomal metabolism of 10 μM[4,5,9,10-3H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 ± 0.45, 3.98 ± 0.19, 3.90 ± 0.16, and 3.75 ± 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored l-nitropyren-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored l-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and α-naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (1 μM), and an activator of form 3c at high concentrations. The presence of a-naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54% of the metabolites were accounted for as l-nitropyren-3-ol, while with form 3b, 73% of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.

Original languageEnglish (US)
Pages (from-to)4256-4260
Number of pages5
JournalCancer Research
Volume48
Issue number15
StatePublished - 1988
Externally publishedYes

Fingerprint

1-nitropyrene
Liver Microsomes
Cytochrome P-450 Enzyme System
Isoenzymes
Rabbits
Chlorodiphenyl (54% Chlorine)
Phenobarbital
Microsomes
Ethanol
Epoxide Hydrolases
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Oxidative Metabolism of 1-Nitropyrene by Rabbit Liver Microsomes and Purified Microsomal Cytochrome P-450 Isozymes. / Howard, Paul C.; Reed, Kimberly A.; Koop, Dennis.

In: Cancer Research, Vol. 48, No. 15, 1988, p. 4256-4260.

Research output: Contribution to journalArticle

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title = "Oxidative Metabolism of 1-Nitropyrene by Rabbit Liver Microsomes and Purified Microsomal Cytochrome P-450 Isozymes",
abstract = "Rabbit liver (male) microsomal metabolism of 10 μM[4,5,9,10-3H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 ± 0.45, 3.98 ± 0.19, 3.90 ± 0.16, and 3.75 ± 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored l-nitropyren-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored l-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and α-naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (1 μM), and an activator of form 3c at high concentrations. The presence of a-naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54{\%} of the metabolites were accounted for as l-nitropyren-3-ol, while with form 3b, 73{\%} of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.",
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T1 - Oxidative Metabolism of 1-Nitropyrene by Rabbit Liver Microsomes and Purified Microsomal Cytochrome P-450 Isozymes

AU - Howard, Paul C.

AU - Reed, Kimberly A.

AU - Koop, Dennis

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N2 - Rabbit liver (male) microsomal metabolism of 10 μM[4,5,9,10-3H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 ± 0.45, 3.98 ± 0.19, 3.90 ± 0.16, and 3.75 ± 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored l-nitropyren-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored l-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and α-naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (1 μM), and an activator of form 3c at high concentrations. The presence of a-naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54% of the metabolites were accounted for as l-nitropyren-3-ol, while with form 3b, 73% of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.

AB - Rabbit liver (male) microsomal metabolism of 10 μM[4,5,9,10-3H]-1-nitropyrene (1NP) was investigated. The total metabolism was not appreciably different with rates of 4.44 ± 0.45, 3.98 ± 0.19, 3.90 ± 0.16, and 3.75 ± 0.27 nmol/min/mg protein, respectively, for microsomes from phenobarbital, Aroclor-1254, ethanol-treated, and untreated rabbits. However, a more noticeable difference was found in the formation of specific metabolites. Phenobarbital treatment induced changes which favored l-nitropyren-3-ol formation, and Aroclor-1254 and ethanol-induced changes which favored l-nitropyren-6-ol and 1-nitropyren-8-ol formation. 1NP was incubated with untreated microsomes and α-naphthoflavone, an inhibitor of rabbit cytochrome P-450 form 6 at low concentrations (1 μM), and an activator of form 3c at high concentrations. The presence of a-naphthoflavone changed the profile of metabolites while not affecting the total metabolism. Using purified isozymes of rabbit P-450, we found the constitutive form 3b metabolized 1NP at the highest rate with a catalytic activity of 26.8 nmol/min/nmol P-450. Forms 2 and 6 exhibited rates of 2 and 2.2 nmol/min/nmol P-450. Forms 3a, 3c, and 4 had rates about 50- to 300-fold lower than form 3b. High performance liquid chromatography was used to identify the metabolites when the incubations were carried out in the presence of purified rabbit epoxide hydrolase. With form 6, 54% of the metabolites were accounted for as l-nitropyren-3-ol, while with form 3b, 73% of the metabolites were 1-nitropyren-6-ol and 1-nitropyren-8-ol. The K-region dihydrodiols were formed by forms 2 and 3b, but not by forms 3c or 6. These results demonstrate that 1NP is a preferential substrate for form 3b, and that a preponderance of the metabolism with untreated rabbit liver microsomes can be attributed to this isozyme.

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