Original Contribution
Inhibition of Embryonic Retinoic Acid Synthesis by Aldehydes of Lipid Peroxidation and Prevention of Inhibition by Reduced Glutathione and Glutathione S-Transferases

https://doi.org/10.1016/S0891-5849(97)00272-4Get rights and content

Abstract

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 μM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 μM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.

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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