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Effects of 4-hydroxynonenal on isolated hepatocytes. Studies on chemiluminescence response, alkane production and glutathione status.

ABSTRACT: The effect of 4-hydroxy-2,3-trans-nonenal, a diffusible product of lipid peroxidation, on isolated hepatocytes was evaluated with two non-invasive techniques measuring low-level chemiluminescence and alkane evolution. Oxygen-induced low-level chemiluminescence and ethane and n-pentane formation by hepatocytes is enhanced over 7-fold in the presence of 4-hydroxynonenal (2 mM). Glutathione-depleted hepatocytes show a higher increase than controls in both low-level chemiluminescence and alkane formation upon supplementation with 4-hydroxynonenal. The effects on both parameters are diminished by vitamin E pretreatment of rats and are absent under anaerobiosis. At variance with chemiluminescence and alkane formation, 4-hydroxynonenal does not elicit a concomitant increase in malonaldehyde or diene-conjugate formation. Addition of 4-hydroxynonenal to a suspension of hepatocytes causes a rapid loss of cellular glutathione in the form of a glutathione conjugate with the alkenal as observed with high-pressure liquid-chromatographic analysis. The reaction between glutathione and 4-hydroxynonenal proceeds also spontaneously in vitro at 1:1 stoichiometry. The cellular effects of 4-hydroxynonenal evaluated by low-level chemiluminescence and alkane formation are independent of the formation of a glutathione conjugate and seem to rely on the remaining not-bound 4-hydroxynonenal. The sensitivity of 4-hydroxynonenal-enhanced chemiluminescence and alkane formation to free-radical quenchers suggests the participation of a free-radical propagation process.

SUBMITTER: Cadenas E

PROVIDER: S-EPMC1152270 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

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Project description:The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.

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Effects of 4-hydroxynonenal on isolated hepatocytes. Studies on chemiluminescence response, alkane production and glutathione status.

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