2-hexenal--z-isomer and cinnamaldehyde

Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to <20% of control (viability >85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals.

Topics: Acrolein; Aldehydes; Alkadienes; Animals; Caco-2 Cells; Cell Survival; Cells, Cultured; Cricetinae; Cyclohexanones; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Glutathione; Humans; Mammals; Oxidative Stress; Structure-Activity Relationship; Time Factors

In the present study the irreversible inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by alpha, beta-unsaturated aldehydes and ketones was studied. When GSTP1-1 was incubated with a 50-fold molar excess of the aldehydes acrolein (ACR) and 4-hydroxy-2-nonenal (HNE) and the ketones curcumin (CUR) and ethacrynic acid (EA) at 22 degrees C, all of them inactivated GSTP1-1. The remaining activity after 4 h of incubation in all cases was lower than 10%. The aldehydes crotonaldehyde (CRA), cinnamaldehyde (CA) and trans-2-hexenal were found to inhibit GSTP1-1 only at a 5000-fold molar excess and even then, for example, for CA a higher remaining activity of 17% was observed. The same inhibition experiments were conducted with 3 mutants of GSTP1-1: the C47S and C101S mutants and the double mutant C47S/C101S. Remaining activity for C47S varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. For C101S it varied between 0 and 9% and for the double mutant C47S/C101S, activity after 4 h of incubation was variable. Again it varied between +/- 40% for CRA, CA, CUR and HEX and +/- 80% for ACR, EA and HNE. EA is known to react almost exclusively with cysteine 47. When [14C]EA was incubated with the GSTP1-1, modified by the alpha, beta-unsaturated carbonyl compounds, no [14C]EA was incorporated in the enzyme, indicating that in all cases this cysteine residue was one of the major targets. Since Michael addition with these reagents is known to be reversible, the results of incubation of the inactivated enzymes with an excess of glutathione (GSH) were determined. For all compounds, a restoration of the catalytic activity was observed. The results indicate that alpha, beta-unsaturated carbonyl derivatives inhibit GSTP1-1 irreversibly mainly by binding to cysteine residues of GSTP1-1, especially Cys-47, This means that some of these compounds (e.g. CUR) might modify GST activity in vivo when GSH concentrations are low by covalent binding to the enzyme.

Topics: Acrolein; Aldehydes; Curcumin; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Ethacrynic Acid; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Isoenzymes; Ketones; Mutagenesis, Site-Directed

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.

Topics: Acrolein; Acyclic Monoterpenes; Aldehydes; Chromatography, High Pressure Liquid; Curcumin; Enzyme Inhibitors; Ethacrynic Acid; Glutathione; Glutathione Transferase; Humans; Melanoma; Monoterpenes; Skin Neoplasms; Terpenes; Tumor Cells, Cultured