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 (PHITC), and sulforaphane (SFN), expressed as the first-order rate constant k(1) and the half-life of decomposition Dt(1/2), were measured in aqueous solutions at pH 7.4 and 37 degrees. The Dt(1/2)s for the Cys conjugates were severalfold shorter than the Dt(1/2)s for respective GSH conjugates, while the Dt(1/2)s 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 microM 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.

Consumption of Raw Cruciferous Vegetables is Inversely Associated with Bladder Cancer Risk

“…[10][11][12]. NAC-ITCs are unstable and dissociate to release isothiocyanates (4,13), and storage of urine containing NAC-ITCs in the bladder likely further enhances the exposure of bladder cells to these compounds. As a result, the bladder epithelium that gives rise to the majority of bladder cancers (90-95% in the United States; ref.…”

Radiations and biodegradation techniques for detoxifying Carica papaya seed oil for effective dietary and industrial use

“…Isothiocyanate-GSH conjugates may have been formed, which are typically metabolized to yield cysteinylglycine-, cysteine-and N-acetylcysteine conjugates that are eliminated easily and concentrated in the urine. Research has shown that N-acetylcysteine conjugates and the intermediate metabolites of ITCs, which are collectively known as dithiocarbamates (DTCs) are unstable and easily dissociate to their parent ITCs (Conaway et al 2001). This explains the sudden increase in the concentration of 1, 3-benzodithiole-2-thione formed on day 3 (Fig.…”

Electrophiles in Foods: The Current Status of Isothiocyanates and Their Chemical Biology

“…PHOX of NADPH oxidase (Nox2), 92) adenine nucleotide translocase, 93) CYP isozyme, 106) histone deacetylase, 107) a transient receptor potential (TRP) family of ion channels (TRPA1), 108) and so on. Among ITCs, phenyl ITC (PITC) is a unique inactive ITC but has electrophilic reactivity to modify protein function in vitro.…”

“…The decomposition of thiol conjugates of ITCs is also pH dependent. 106) A mechanism for the influence of hydroxyl ions on the decomposition of thiol conjugates of ITCs has been proposed: A hydroxyl ion deprotonates the conjugate nitrogen, leading to elimination of the thiol leaving group and regeneration of the ITC. The physiological implication of the influence of pH on thiol-ITC decomposition is that orally administered thiol-ITCs remain intact when absorbed through the stomach mucosa, but probably dissociate to ITCs to a much greater extent in the intestinal tract.…”

“…Although the biological activities of DTCs and the ITC-NAC conjugates are not fully understood, they are not ultimately inactive, possibly due to dissociative conversion into free ITCs. 106,111) Technically, when exposed to analytical conditions such as the neutral pH required during sample preparation, the DTCs formed between ITCs and protein cysteines might also not be stable. This instability makes it difficult to understand the biological relevance of the covalent binding of ITCs to specific cysteine residues.…”