Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms

Sheng C. Chen, Xiaojie Wang, Guoping Xu, Lin Zhou, Jonathan L Vennerstrom, Frank Gonzalez, Harry V. Gelboin, Sidney S. Mirvish

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Abstract

Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]-MNAN and [3H-2,3-pentyl]. MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high- performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed K(m) values of 64, 610, and 170-330 μM, respectively (V(max): 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 μM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (K(i), 120 μM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7- hydroxylation by RLM and CYP2A6 (K(i), 3000 and 320 μM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. K(m) for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 μM, respectively (V(max:) 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

Original languageEnglish (US)
Pages (from-to)91-98
Number of pages8
JournalCancer Research
Volume59
Issue number1
StatePublished - Jan 1 1999

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N-amyl-N-methylnitrosamine
Liver Microsomes
Cytochrome P-450 Enzyme System
Protein Isoforms
Microsomes
Cytochrome P-450 CYP2E1

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms. / Chen, Sheng C.; Wang, Xiaojie; Xu, Guoping; Zhou, Lin; Vennerstrom, Jonathan L; Gonzalez, Frank; Gelboin, Harry V.; Mirvish, Sidney S.

In: Cancer Research, Vol. 59, No. 1, 01.01.1999, p. 91-98.

Research output: Contribution to journalArticle

Chen, Sheng C. ; Wang, Xiaojie ; Xu, Guoping ; Zhou, Lin ; Vennerstrom, Jonathan L ; Gonzalez, Frank ; Gelboin, Harry V. ; Mirvish, Sidney S. / Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms. In: Cancer Research. 1999 ; Vol. 59, No. 1. pp. 91-98.
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abstract = "Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]-MNAN and [3H-2,3-pentyl]. MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high- performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60{\%}. MNAN depentylation by REM and uninduced and induced RLM showed K(m) values of 64, 610, and 170-330 μM, respectively (V(max): 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 μM MNAN by REM was inhibited 98{\%} by CO and 65{\%} by coumarin preincubated for 15 min with REM (K(i), 120 μM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7- hydroxylation by RLM and CYP2A6 (K(i), 3000 and 320 μM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41{\%} inhibited by an antibody to CYP2C11. K(m) for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 μM, respectively (V(max:) 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.",
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T1 - Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms

AU - Chen, Sheng C.

AU - Wang, Xiaojie

AU - Xu, Guoping

AU - Zhou, Lin

AU - Vennerstrom, Jonathan L

AU - Gonzalez, Frank

AU - Gelboin, Harry V.

AU - Mirvish, Sidney S.

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N2 - Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]-MNAN and [3H-2,3-pentyl]. MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high- performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed K(m) values of 64, 610, and 170-330 μM, respectively (V(max): 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 μM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (K(i), 120 μM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7- hydroxylation by RLM and CYP2A6 (K(i), 3000 and 320 μM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. K(m) for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 μM, respectively (V(max:) 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

AB - Methyl-n-amylnitrosamine (MNAN) induces esophageal cancer in rats, probably involving activation by cytochromes P450. We studied the metabolic depentylation of MNAN. [3H-4,5-pentyl]-MNAN and [3H-2,3-pentyl]. MNAN were synthesized, purified, and incubated with rat esophageal microsomes (REM) or rat liver microsomes (RLM) to give [3H]pentaldehyde (depentylation), an indicator of MNAN activation. [3H]Pentaldehyde was determined by high- performance liquid chromatography of its 2,4-dinitrophenylhydrazone. Adding 5 mM semicarbazide to incubations increased the observed depentylation (except that due to CYP2E1) by >60%. MNAN depentylation by REM and uninduced and induced RLM showed K(m) values of 64, 610, and 170-330 μM, respectively (V(max): 20, 220, and 160-1270 pmol/mg protein/min, respectively). The depentylation of 100 μM MNAN by REM was inhibited 98% by CO and 65% by coumarin preincubated for 15 min with REM (K(i), 120 μM) but was unaffected by antibodies inhibitory to various P450s. MNAN inhibited coumarin 7- hydroxylation by RLM and CYP2A6 (K(i), 3000 and 320 μM, respectively). REM showed slight coumarin 7-hydroxylase activity. MNAN depentylation by RLM was 41% inhibited by an antibody to CYP2C11. K(m) for rat CYP2E1, human CYP2E1, and human CYP2A6 was 210, 115, and 17 μM, respectively (V(max:) 900, 570, and 120 pmol/nmol P450/min, respectively). We conclude that MNAN activation by REM is probably due to a P450 related to CYP2A3, a rodent nasal P450.

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