Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome P450 enzymes

C. C. Conaway, Jacek Krzeminski, Shantu Amin, F. L. Chung

Research output: Contribution to journalArticle

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Abstract

Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHI-TC), and sulforaphane (SFN), expressed as the first-order rate constant k1 and the half-life of decomposition Dt1/2, were measured in aqueous solutions at pH 7.4 and 37°. The Dt1/2s for the Cys conjugates were severalfold shorter than the Dt1/2s for respective GSH conjugates, while the Dt1/2s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 μM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.

Original languageEnglish (US)
Pages (from-to)1170-1176
Number of pages7
JournalChemical Research in Toxicology
Volume14
Issue number9
DOIs
StatePublished - Oct 10 2001

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Isothiocyanates
Sulfhydryl Compounds
Cytochrome P-450 Enzyme System
Dealkylation
Decomposition
Acetylcysteine
Tumors
Enzyme inhibition
isothiocyanic acid
Cytochrome P-450 Enzyme Inhibitors
Mammals
Bioassay
Metabolites
Biological Assay
Carcinogens
Half-Life
Rate constants
Neoplasms
Chemical activation
Tissue

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

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title = "Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome P450 enzymes",
abstract = "Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHI-TC), and sulforaphane (SFN), expressed as the first-order rate constant k1 and the half-life of decomposition Dt1/2, were measured in aqueous solutions at pH 7.4 and 37°. The Dt1/2s for the Cys conjugates were severalfold shorter than the Dt1/2s for respective GSH conjugates, while the Dt1/2s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 μM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.",
author = "Conaway, {C. C.} and Jacek Krzeminski and Shantu Amin and Chung, {F. L.}",
year = "2001",
month = "10",
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doi = "10.1021/tx010029w",
language = "English (US)",
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pages = "1170--1176",
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Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome P450 enzymes. / Conaway, C. C.; Krzeminski, Jacek; Amin, Shantu; Chung, F. L.

In: Chemical Research in Toxicology, Vol. 14, No. 9, 10.10.2001, p. 1170-1176.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome P450 enzymes

AU - Conaway, C. C.

AU - Krzeminski, Jacek

AU - Amin, Shantu

AU - Chung, F. L.

PY - 2001/10/10

Y1 - 2001/10/10

N2 - Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHI-TC), and sulforaphane (SFN), expressed as the first-order rate constant k1 and the half-life of decomposition Dt1/2, were measured in aqueous solutions at pH 7.4 and 37°. The Dt1/2s for the Cys conjugates were severalfold shorter than the Dt1/2s for respective GSH conjugates, while the Dt1/2s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 μM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.

AB - Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHI-TC), and sulforaphane (SFN), expressed as the first-order rate constant k1 and the half-life of decomposition Dt1/2, were measured in aqueous solutions at pH 7.4 and 37°. The Dt1/2s for the Cys conjugates were severalfold shorter than the Dt1/2s for respective GSH conjugates, while the Dt1/2s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 μM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.

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