Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N- acetyl-L-cysteine conjugates

Ding Jiao, C. Clifford Conaway, Mong-Heng Wang, Chung S. Yang, Werner Koehl, Fung Lung Chung

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 μM were used to probe the activity of cytochrome P4502E1 isozyme through the α-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 μM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 μM) and with glutathione (IC50 = 4.0 μM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 μM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

Original languageEnglish (US)
Pages (from-to)932-938
Number of pages7
JournalChemical Research in Toxicology
Volume9
Issue number6
DOIs
StatePublished - Sep 23 1996
Externally publishedYes

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Isothiocyanates
Cytochrome P-450 CYP2E1
Acetylcysteine
Liver Microsomes
Liver
Glutathione
Cysteine
Rats
Inhibitory Concentration 50
Cytochromes
Sulfhydryl Compounds
Isoenzymes
Hydroxylation
Tobacco
Half-Life
Assays
Chemical activation
High Pressure Liquid Chromatography
Substrates
phenethyl isothiocyanate

ASJC Scopus subject areas

  • Toxicology

Cite this

Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N- acetyl-L-cysteine conjugates. / Jiao, Ding; Clifford Conaway, C.; Wang, Mong-Heng; Yang, Chung S.; Koehl, Werner; Chung, Fung Lung.

In: Chemical Research in Toxicology, Vol. 9, No. 6, 23.09.1996, p. 932-938.

Research output: Contribution to journalArticle

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title = "Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N- acetyl-L-cysteine conjugates",
abstract = "Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 μM were used to probe the activity of cytochrome P4502E1 isozyme through the α-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 μM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 μM) and with glutathione (IC50 = 4.0 μM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 μM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.",
author = "Ding Jiao and {Clifford Conaway}, C. and Mong-Heng Wang and Yang, {Chung S.} and Werner Koehl and Chung, {Fung Lung}",
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T1 - Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N- acetyl-L-cysteine conjugates

AU - Jiao, Ding

AU - Clifford Conaway, C.

AU - Wang, Mong-Heng

AU - Yang, Chung S.

AU - Koehl, Werner

AU - Chung, Fung Lung

PY - 1996/9/23

Y1 - 1996/9/23

N2 - Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 μM were used to probe the activity of cytochrome P4502E1 isozyme through the α-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 μM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 μM) and with glutathione (IC50 = 4.0 μM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 μM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

AB - Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 μM were used to probe the activity of cytochrome P4502E1 isozyme through the α-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 μM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 μM) and with glutathione (IC50 = 4.0 μM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 μM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

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