The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes

Michael A. Malfatti, Michael H. Buonarati, Ken W Turteltaub, Nancy H. Shen, James S. Felton

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b] pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor α-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 μM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4′-(2-amino-1-methylimidazo[4,5-6]pyrid-6-yl)phenyl sulfate(4′-PhIP-sulfate),2-amino-1-methyl-4′-hydroxy-6- phenylimidazo[4,5-b]pyridine(4′-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4′-hydroxy-PhIP to 4′-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP. Pretreatment of cells with ANF reduced the formation of all detectable metabolites by at least 50%, yet increased DNA adduct levels by nearly 4-fold. The lack of significant effect by PCP or DCNP on binding of PhIP to DNA or protein may suggest involvement of other metabolic pathways besides sulfation and/or acetylation in the activation of PhIP to reactive intermediates. The increase in DNA binding by ANF pretreatment suggests that other cytochrome P450 enzymes not inhibited by ANF may be involved in the bioactivation of PhIP in rat hepatocytes.

Original languageEnglish (US)
Pages (from-to)139-147
Number of pages9
JournalChemical Research in Toxicology
Volume7
Issue number2
StatePublished - 1994
Externally publishedYes

Fingerprint

Acetylation
Salmonella
Mutagens
Salmonella typhimurium
Metabolism
Pentachlorophenol
Rats
Hepatocytes
Food
Atrial Natriuretic Factor
Chlorodiphenyl (54% Chlorine)
Metabolites
DNA Adducts
Cytochrome P-450 Enzyme System
Sulfates
Chemical activation
2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine
Sulfotransferases
Cytochrome P-450 CYP1A1
Proteins

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Chemistry(all)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes. / Malfatti, Michael A.; Buonarati, Michael H.; Turteltaub, Ken W; Shen, Nancy H.; Felton, James S.

In: Chemical Research in Toxicology, Vol. 7, No. 2, 1994, p. 139-147.

Research output: Contribution to journalArticle

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abstract = "Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b] pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor α-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 μM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4′-(2-amino-1-methylimidazo[4,5-6]pyrid-6-yl)phenyl sulfate(4′-PhIP-sulfate),2-amino-1-methyl-4′-hydroxy-6- phenylimidazo[4,5-b]pyridine(4′-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4′-hydroxy-PhIP to 4′-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP. Pretreatment of cells with ANF reduced the formation of all detectable metabolites by at least 50{\%}, yet increased DNA adduct levels by nearly 4-fold. The lack of significant effect by PCP or DCNP on binding of PhIP to DNA or protein may suggest involvement of other metabolic pathways besides sulfation and/or acetylation in the activation of PhIP to reactive intermediates. The increase in DNA binding by ANF pretreatment suggests that other cytochrome P450 enzymes not inhibited by ANF may be involved in the bioactivation of PhIP in rat hepatocytes.",
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T1 - The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes

AU - Malfatti, Michael A.

AU - Buonarati, Michael H.

AU - Turteltaub, Ken W

AU - Shen, Nancy H.

AU - Felton, James S.

PY - 1994

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N2 - Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b] pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor α-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 μM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4′-(2-amino-1-methylimidazo[4,5-6]pyrid-6-yl)phenyl sulfate(4′-PhIP-sulfate),2-amino-1-methyl-4′-hydroxy-6- phenylimidazo[4,5-b]pyridine(4′-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4′-hydroxy-PhIP to 4′-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP. Pretreatment of cells with ANF reduced the formation of all detectable metabolites by at least 50%, yet increased DNA adduct levels by nearly 4-fold. The lack of significant effect by PCP or DCNP on binding of PhIP to DNA or protein may suggest involvement of other metabolic pathways besides sulfation and/or acetylation in the activation of PhIP to reactive intermediates. The increase in DNA binding by ANF pretreatment suggests that other cytochrome P450 enzymes not inhibited by ANF may be involved in the bioactivation of PhIP in rat hepatocytes.

AB - Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b] pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor α-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 μM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4′-(2-amino-1-methylimidazo[4,5-6]pyrid-6-yl)phenyl sulfate(4′-PhIP-sulfate),2-amino-1-methyl-4′-hydroxy-6- phenylimidazo[4,5-b]pyridine(4′-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4′-hydroxy-PhIP to 4′-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP. Pretreatment of cells with ANF reduced the formation of all detectable metabolites by at least 50%, yet increased DNA adduct levels by nearly 4-fold. The lack of significant effect by PCP or DCNP on binding of PhIP to DNA or protein may suggest involvement of other metabolic pathways besides sulfation and/or acetylation in the activation of PhIP to reactive intermediates. The increase in DNA binding by ANF pretreatment suggests that other cytochrome P450 enzymes not inhibited by ANF may be involved in the bioactivation of PhIP in rat hepatocytes.

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