Original ContributionInhibition of Embryonic Retinoic Acid Synthesis by Aldehydes of Lipid Peroxidation and Prevention of Inhibition by Reduced Glutathione and Glutathione S-Transferases
Introduction
Recently it was demonstrated1, 2that additions of various polyunstaturated fatty acids (PUFAs) to the culture medium could alter the normal development of whole mouse embryos cultured in vitro. More importantly, the deleterious effects of PUFAs on embryonic development could be prevented by adding antioxidants to the culture medium. These observations strongly suggested that lipid peroxidation can play an important role in the causation of abnormal development of embryos, at least in vitro. However, the biochemical or molecular mechanisms whereby lipid peroxidation induces such abnormal development have not been elucidated.
All-trans-retinoic acid (t-RA), an active metabolite of all-trans-retinol (t-ROH) and all-trans-retinal (t-RAL), triggers a cascade of retinoid-mediated signal transaction pathways by activating nuclear retinoic acid receptors (RARs) that subsequently regulate the transcription of many developmental genes.3, 4, 5, 6Consequently, control of the biosynthesis of t-RA in embryonic tissues from its alcohol/aldehyde precursors would be expected to play a crucial role in embryonic development. This aspect, however, has received relatively little attention.
In mammalian tissues, oxidative biotransformation of t-ROH/t-RAL catalyzed by dehydrogenases represents a major pathway for generation of t-RA.7, 8, 9Class I and IV alcohol dehydrogenases (ADH I and IV) appear to be the primary enzymes for the catalyses of oxidations of t-ROH and t-RAL9, 10although several additional enzymes also participate.11, 12, 13, 14Interestingly, kinetic studies have shown that rodent ADH I and ADH IV also exhibited low Km and high catalytic efficiency (kcat/Km) for a variety of aldehydes generated from lipid peroxidation, suggesting that biosynthesis of t-RA and oxidation of these aldehydes were catalyzed by common enzymes.[9]Therefore, we hypothesized that abnormal embryonic development caused by lipid peroxidation, at least in part, could be due to the inhibition by aldehydes generated from lipid peroxidation of biosynthesis of t-RA. First, t-RA is a key factor in retinoid receptor-mediated signal transduction pathways, and the homeostasis of t-RA in embryonic tissues is crucial for normal development. Inhibition of t-RA biosynthesis can result in a deficiency of t-RA, and is thus likely to cause a blockage of the retinoid-mediated signal transduction. Second, as demonstrated in our recent studies,15, 16, 17, 18the conceptal retinol/retinal dehydrogenases exhibited very similar functional properties with those of stomach ADH IV. This would suggest that the conceptal retinol/retinal dehydrogenases may also have high affinities for aldehydic products of lipid peroxidation. If this assumption is correct, the aldehydes should act as competitive inhibitors for biosynthesis of t-RA. Finally, studies have shown that malondialdehyde, an aldehyde generated from lipid peroxidation, can be measured quantitatively in cultured embryos in vitro under conditions predisposing to oxidative stress.[19]This certainly supports the possibility of inhibition of t-RA synthesis by the aldehydes produced by lipid peroxidation in embryonic tissues.
In this study, we tested our hypothesis that embryonic biosynthesis of t-RA can be inhibited by aldehydic products of lipid peroxidation such as hexanal, nonyl aldehyde (NA), trans-2-nonenal (tNE), and 4-hydroxy-2-nonenal (4HNE). To test the hypothesis, we demonstrated first that the generation of t-RA in conceptal tissues was inhibited by the aldehydes; and, second, that the inhibition of generation of t-RA can be prevented by addition of reduced glutathione (GSH) and/or glutathione S-transferases (GST), which effectively conjugate aldehydes thereby converting them to inactive compounds. t-RAL was used as substrate for the generation of t-RA, and cytosolic fractions prepared from rat conceptal tissues at gestational day 12.5 (a crucial period for organogenesis) were used as enzyme sources. Reactions catalyzed by adult rat hepatic cytosol (ARHC) were carried out to investigate the potential protection against inhibition of aldehydes via GSH-dependent conjugation reactions.
Section snippets
Materials
Retinoids, NAD, GSH, GST (EC 2.5.1.18, from rat liver), and 4-hydroxy-2-nonenal (4HNE) were purchased from Sigma Chemical Co. (St. Louis, MO). Hexanal, trans-2-nonenal (tNE), nonyl aldehyde (NA), and triethyltin bromide (TEB) were purchased from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and reagents utilized were of the highest purity commercially available.
Preparation of Cytosol
Time-mated pregnant rats (Sprague-Dawley, Wistar-derived) were obtained from Tyler Laboratories (Bellevue, WA) on days 12.5
Results
Fig. 1 shows the inhibition by aldehydes generated from lipid peroxidation of RCC-catalyzed oxidative conversion of t-RAL to t-RA. Both one-way and two-way ANOVA indicated that the inhibitory effects of all aldehydes tested were dose dependent, and that there was a strong interaction between the effects of dose and the effects of individual aldehydes (p < .001). Group-to-group comparisons with a t-test also indicated that tNE significantly inhibited the reactions at all initial molar ratios
Discussion
Mammalian ADH and ALDH are important in catalysis of oxidative conversion of both t-RAL and aldehydes derived from lipid peroxidation to their corresponding carboxylic acids.7, 8, 9, 10, 11, 12The former reaction is crucial for retinoid mediated signal transduction pathways because generated t-RA regulates expression of many developmental genes.5, 6Consequently, both excess and deficiency of t-RA can cause severe embryonic malformations.[21]The latter reaction eliminates both endogenous and
Acknowledgements
This work was supported by NIEHS Grants (ES-04041 and ES-05861) and H.C. was supported by NIEHS Training Grant (ES-07032).
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