Shabtai Y et al. (2018), Acetaldehyde inhibits retinoic acid biosynthesi...

XB-ART-55483

Sci Rep January 1, 2018; 8 (1): 347.

Acetaldehyde inhibits retinoic acid biosynthesis to mediate alcohol teratogenicity.

Shabtai Y , Bendelac L , Jubran H , Hirschberg J , Fainsod A .

Abstract
Alcohol consumption during pregnancy induces Fetal Alcohol Spectrum Disorder (FASD), which has been proposed to arise from competitive inhibition of retinoic acid (RA) biosynthesis. We provide biochemical and developmental evidence identifying acetaldehyde as responsible for this inhibition. In the embryo, RA production by RALDH2 (ALDH1A2), the main retinaldehyde dehydrogenase expressed at that stage, is inhibited by ethanol exposure. Pharmacological inhibition of the embryonic alcohol dehydrogenase activity, prevents the oxidation of ethanol to acetaldehyde that in turn functions as a RALDH2 inhibitor. Acetaldehyde-mediated reduction of RA can be rescued by RALDH2 or retinaldehyde supplementation. Enzymatic kinetic analysis of human RALDH2 shows a preference for acetaldehyde as a substrate over retinaldehyde. RA production by hRALDH2 is efficiently inhibited by acetaldehyde but not by ethanol itself. We conclude that acetaldehyde is the teratogenic derivative of ethanol responsible for the reduction in RA signaling and induction of the developmental malformations characteristic of FASD. This competitive mechanism will affect tissues requiring RA signaling when exposed to ethanol throughout life.

PubMed ID: 29321611
PMC ID: PMC5762763
Article link: Sci Rep
Grant support: [+]

Species referenced: Xenopus
Genes referenced: aldh1a1 aldh1a2 aldh1a3 aldh1b1 aldh2 chrd.1 cyp26a1 dhrs3 gsc hoxa1 hoxb1 hoxb4 rdh10

Disease Ontology terms: fetal alcohol syndrome
OMIMs: ALCOHOL SENSITIVITY, ACUTE

Article Images: [+] show captions

	Figure 1. Ethanol-dependent retinoic acid inhibition requires middle-chain alcohol dehydrogenases. The enzymatic requirements for EtOH to inhibit RA biosynthesis were studied using inhibitors of the middle-chain alcohol dehydrogenases (ADH), 4-methylpyrazole (4MP), or the short-chain dehydrogenase/reductases, carbenoxolone (CBX) and chloral hydrate (CH). Late blastula stage embryos were treated with EtOH alone or in combination with 4MP (a,b), CH (c,d) or CBX (e,f). The effect on RA signaling was determined by monitoring the expression level of the known RA-regulated genes, HoxA1 and HoxB1 during early gastrula stages. n = 3, The values denote mean ± SEM. P values - p <0.05; p <0.01; p <0.001; **p <0.0001; ns, not significant.
	Figure 2. Acetaldehyde induces a reduction in retinoic acid signaling. The changes in the expression of several RA-regulated genes was determined after AcAL (5 µM), EtOH (0.5%), DEAB (100 µM), or RA (1 µM). Embryos were treated during late blastula (8.5), and RNA was extracted for analysis during early/mid-gastrula (st. 10.5). The expression level of the RA-regulated genes HoxA1 (a), HoxB1 (b), HoxB4 (c), Cyp26A1 (d), Dhrs3 (e), and Rdh10 (f) by qPCR. n = 4, The values denote mean ± SEM. P values - p <0.01; p <0.001; ***p <0.0001.
	Figure 3. Acetaldehyde phenocopies the malformations induced by ethanol. Embryos were treated with AcAL (5 µM), EtOH (0.5%) or DEAB (60 µM) and allowed to develop. At stage 34, embryos were assessed for general developmental malformations comparing controls (a) to EtOH (b), AcAL (c) and DEAB (d) treated embryos. For better qualitative length comparison, a line was drawn from the forehead to the tail-tip of the control embryo (a). This line was copied unto the treated embryos (b,c,d). For head size comparison, a bracket was drawn from the forehead to the beginning of the dorsal fin (a). A copy of the same bracket was placed at the onset of the dorsal fin in the treated embryos (b,c,d). For a more quantitative comparison of the malformations, at stage 45 the malformations in head formation were characterized. (e–i) The head region of control (e and g), AcAL (f), EtOH (h) and DEAB (i) treated embryos. The head anatomical distances measured according to Nakatsuji33 are shown. W3, inner distance between eyes; W5, head width; L1, length of head. Head malformations are shown as changes in size from control values for all the parameters, n = 70 (j–o). Two AcAL concentrations (1 µM and 5 µM) are shown (j, l and n). The size changes for EtOH and DEAB treated embryos are shown, n = 94 (k, m and o). P values - p <0.05; p <0.01; p <0.001; **p <0.0001.
	Figure 4. Acetaldehyde competes for the human RALDH2. The effect of ethanol and its oxidation product, acetaldehyde, on hRALDH2 activity was studied in vivo. Embryos injected with plasmid encoding hRALDH2 or control embryos were treated with AcAL (5 µM), EtOH (0.5%), RAL (1 µM) or DEAB (20 µM), individually or in different combinations. Treatments were initiated during late blastula, and RNA samples were prepared during early/mid-gastrula. The effect of the combined treatments was determined by analyzing the response of the RA-regulated genes, HoxA1 (a,e,i, and m), HoxB4 (b,f,j and n), Cyp26A1 (c,g,k and o) and Dhrs3 (d,h,l and p) by qPCR. (a–d) Overexpression of hRALDH2 together with EtOH treatment. (e–h) AcAL treatment together with hRALDH2 overexpression. (i–l) Combined treatment with AcAL and RAL. (m–o) Combined treatment with DEAB and AcAL. n = 3, The values denote mean ± SEM. P values - p <0.05; p <0.01; p <0.001; **p <0.0001; ns, not significant.
	Figure 5. Acetaldehyde inhibits the expression of organizer-specific genes. Embryos were treated with EtOH (0.5%) or AcAL (5 µM) alone or together with 4MP (1 mM) to inhibit the ADH activity. (a–f) In situ hybridization analysis of the effects of EtOH and AcAL treatments on gsc expression. (a) control, (b) EtOH, (c) AcAL, (d) 4MP, (e) EtOH + 4MP and (f) AcAL + 4MP treated embryos. (g) qPCR analysis of the effect of the same treatments on chordin expression. n = 3, The values denote mean ± SEM. P values - p <0.05; *p <0.01; ns, not significant.
	Figure 6. Acetaldehyde inhibits RA production by human RALDH2. The effect of ethanol and its oxidation product, acetaldehyde, was studied in vitro using a recombinant hRALDH2. (a,b) Increasing concentrations of EtOH covering the physiological range and above were added to RAL oxidation reactions in vitro. The efficiency of RAL oxidation was compared to the control sample without EtOH. (a) Kinetic analysis of the AcAL titration to determine the linear range of the oxidation reaction. (b) The initial 270 seconds of the reaction with different AcAL concentrations are shown. (c) Michaelis-Menten plot of the AcAL oxidation reaction by hRALDH2. (d) Inhibition of RAL oxidation to RA by increasing of acetaldehyde concentrations. The production of RA was determined by HPLC. (e) hRALDH2 activity in the presence of increasing EtOH concentrations. (f) The effect of increasing acetate concentrations on the activity of hRALDH2. n = 3, P values - p <0.01; p <0.001; ***p <0.0001.
	RALDH2 is the main RA-producing enzyme in the gastrula embryo. (a) Temporal pattern of expression of Raldh1 (Aldh1A1), Raldh2 (Aldh1A2), Raldh3 (Aldh1A3) and Aldh2. RNA samples were collect from embryos from the 16-cell stage to neurula stages (st. 17). Expression levels were normalized to the 16-cell RNA amounts. (b) Relative transcript abundance during early/mid gastrula (st. 10.5). The relative expression of the different aldehyde dehydrogenases was calculated relative to the level of Raldh2. n = 3, The values denote mean ± SEM. P values - ****p <0.0001.
	Figure 8. Biochemical competition between ethanol detoxification and retinoic acid biosynthesis. Schematic model of the enzymatic activities in humans involved in ethanol detoxification (left panels) and retinoic acid biosynthesis (right panels). The enzymes active in the mother are shown in the upper panel and the lower panel shows the enzymatic activities in the developing fetus.
	Figure 7. RALDH2 is the main RA-producing enzyme in the gastrula embryo. (a) Temporal pattern of expression of Raldh1 (Aldh1A1), Raldh2 (Aldh1A2), Raldh3 (Aldh1A3) and Aldh2. RNA samples were collect from embryos from the 16-cell stage to neurula stages (st. 17). Expression levels were normalized to the 16-cell RNA amounts. (b) Relative transcript abundance during early/mid gastrula (st. 10.5). The relative expression of the different aldehyde dehydrogenases was calculated relative to the level of Raldh2. n = 3, The values denote mean ± SEM. P values - ****p <0.0001.

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	Figure 1. Ethanol-dependent retinoic acid inhibition requires middle-chain alcohol dehydrogenases. The enzymatic requirements for EtOH to inhibit RA biosynthesis were studied using inhibitors of the middle-chain alcohol dehydrogenases (ADH), 4-methylpyrazole (4MP), or the short-chain dehydrogenase/reductases, carbenoxolone (CBX) and chloral hydrate (CH). Late blastula stage embryos were treated with EtOH alone or in combination with 4MP (a,b), CH (c,d) or CBX (e,f). The effect on RA signaling was determined by monitoring the expression level of the known RA-regulated genes, HoxA1 and HoxB1 during early gastrula stages. n = 3, The values denote mean ± SEM. P values - p <0.05; p <0.01; p <0.001; **p <0.0001; ns, not significant.
	Figure 2. Acetaldehyde induces a reduction in retinoic acid signaling. The changes in the expression of several RA-regulated genes was determined after AcAL (5 µM), EtOH (0.5%), DEAB (100 µM), or RA (1 µM). Embryos were treated during late blastula (8.5), and RNA was extracted for analysis during early/mid-gastrula (st. 10.5). The expression level of the RA-regulated genes HoxA1 (a), HoxB1 (b), HoxB4 (c), Cyp26A1 (d), Dhrs3 (e), and Rdh10 (f) by qPCR. n = 4, The values denote mean ± SEM. P values - p <0.01; p <0.001; ***p <0.0001.
	Figure 3. Acetaldehyde phenocopies the malformations induced by ethanol. Embryos were treated with AcAL (5 µM), EtOH (0.5%) or DEAB (60 µM) and allowed to develop. At stage 34, embryos were assessed for general developmental malformations comparing controls (a) to EtOH (b), AcAL (c) and DEAB (d) treated embryos. For better qualitative length comparison, a line was drawn from the forehead to the tail-tip of the control embryo (a). This line was copied unto the treated embryos (b,c,d). For head size comparison, a bracket was drawn from the forehead to the beginning of the dorsal fin (a). A copy of the same bracket was placed at the onset of the dorsal fin in the treated embryos (b,c,d). For a more quantitative comparison of the malformations, at stage 45 the malformations in head formation were characterized. (e–i) The head region of control (e and g), AcAL (f), EtOH (h) and DEAB (i) treated embryos. The head anatomical distances measured according to Nakatsuji33 are shown. W3, inner distance between eyes; W5, head width; L1, length of head. Head malformations are shown as changes in size from control values for all the parameters, n = 70 (j–o). Two AcAL concentrations (1 µM and 5 µM) are shown (j, l and n). The size changes for EtOH and DEAB treated embryos are shown, n = 94 (k, m and o). P values - p <0.05; p <0.01; p <0.001; **p <0.0001.
	Figure 4. Acetaldehyde competes for the human RALDH2. The effect of ethanol and its oxidation product, acetaldehyde, on hRALDH2 activity was studied in vivo. Embryos injected with plasmid encoding hRALDH2 or control embryos were treated with AcAL (5 µM), EtOH (0.5%), RAL (1 µM) or DEAB (20 µM), individually or in different combinations. Treatments were initiated during late blastula, and RNA samples were prepared during early/mid-gastrula. The effect of the combined treatments was determined by analyzing the response of the RA-regulated genes, HoxA1 (a,e,i, and m), HoxB4 (b,f,j and n), Cyp26A1 (c,g,k and o) and Dhrs3 (d,h,l and p) by qPCR. (a–d) Overexpression of hRALDH2 together with EtOH treatment. (e–h) AcAL treatment together with hRALDH2 overexpression. (i–l) Combined treatment with AcAL and RAL. (m–o) Combined treatment with DEAB and AcAL. n = 3, The values denote mean ± SEM. P values - p <0.05; p <0.01; p <0.001; **p <0.0001; ns, not significant.
	Figure 5. Acetaldehyde inhibits the expression of organizer-specific genes. Embryos were treated with EtOH (0.5%) or AcAL (5 µM) alone or together with 4MP (1 mM) to inhibit the ADH activity. (a–f) In situ hybridization analysis of the effects of EtOH and AcAL treatments on gsc expression. (a) control, (b) EtOH, (c) AcAL, (d) 4MP, (e) EtOH + 4MP and (f) AcAL + 4MP treated embryos. (g) qPCR analysis of the effect of the same treatments on chordin expression. n = 3, The values denote mean ± SEM. P values - p <0.05; *p <0.01; ns, not significant.
	Figure 6. Acetaldehyde inhibits RA production by human RALDH2. The effect of ethanol and its oxidation product, acetaldehyde, was studied in vitro using a recombinant hRALDH2. (a,b) Increasing concentrations of EtOH covering the physiological range and above were added to RAL oxidation reactions in vitro. The efficiency of RAL oxidation was compared to the control sample without EtOH. (a) Kinetic analysis of the AcAL titration to determine the linear range of the oxidation reaction. (b) The initial 270 seconds of the reaction with different AcAL concentrations are shown. (c) Michaelis-Menten plot of the AcAL oxidation reaction by hRALDH2. (d) Inhibition of RAL oxidation to RA by increasing of acetaldehyde concentrations. The production of RA was determined by HPLC. (e) hRALDH2 activity in the presence of increasing EtOH concentrations. (f) The effect of increasing acetate concentrations on the activity of hRALDH2. n = 3, P values - p <0.01; p <0.001; ***p <0.0001.
	RALDH2 is the main RA-producing enzyme in the gastrula embryo. (a) Temporal pattern of expression of Raldh1 (Aldh1A1), Raldh2 (Aldh1A2), Raldh3 (Aldh1A3) and Aldh2. RNA samples were collect from embryos from the 16-cell stage to neurula stages (st. 17). Expression levels were normalized to the 16-cell RNA amounts. (b) Relative transcript abundance during early/mid gastrula (st. 10.5). The relative expression of the different aldehyde dehydrogenases was calculated relative to the level of Raldh2. n = 3, The values denote mean ± SEM. P values - ****p <0.0001.
	Figure 8. Biochemical competition between ethanol detoxification and retinoic acid biosynthesis. Schematic model of the enzymatic activities in humans involved in ethanol detoxification (left panels) and retinoic acid biosynthesis (right panels). The enzymes active in the mother are shown in the upper panel and the lower panel shows the enzymatic activities in the developing fetus.
	Figure 7. RALDH2 is the main RA-producing enzyme in the gastrula embryo. (a) Temporal pattern of expression of Raldh1 (Aldh1A1), Raldh2 (Aldh1A2), Raldh3 (Aldh1A3) and Aldh2. RNA samples were collect from embryos from the 16-cell stage to neurula stages (st. 17). Expression levels were normalized to the 16-cell RNA amounts. (b) Relative transcript abundance during early/mid gastrula (st. 10.5). The relative expression of the different aldehyde dehydrogenases was calculated relative to the level of Raldh2. n = 3, The values denote mean ± SEM. P values - ****p <0.0001.