Báo cáo Y học: Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3 docx

Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3 Andrei Molotkov, Xiaohong Fan and Gregg Duester Gene Regulation Program, Burnham Institute, La Jolla, CA, USA Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physio- logical roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3,andAdh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mgÆkg )1 ), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD 50 value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one gen- eration on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild- type (92–95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity. Keywords: alcohol dehydrogenase; retinol; retinoic acid; vitamin A; toxicity. Mammalian alcohol dehydrogenase (ADH, EC 1.1.1.1) is encoded by a family of genes closely linked on human chromosome 4 and mouse chromosome 3, with all genes in the same transcriptional orientation [1]. Five distinct classes of mammalian ADH have been identified that differ significantly in primary structure, catalytic activity with various alcohols, and gene expression patterns [2]. The physiological roles of these several classes of ADH are not fully established, but studies along this line have led to the hypothesis that retinoid metabolism is one of these roles [3]. The ability of horse liver ADH to oxidize retinol to retinal in vitro was recognized early [4] and further studies demonstrated retinol activity using purified class I (ADH1), class II (ADH2), and class IV (ADH4) enzymes [5–9]. Class III (ADH3) was not originally associated with activity for retinol oxidation, but recent studies using a more sensitive assay have demonstrated its ability to catalyze retinol oxidation [10]. ADHs are cytosolic enzymes with many forms having relatively high catalytic activity for retinol oxidation compared with microsomal enzymes. Several microsomal short-chain dehydrogenase/reductase (SDR) enzymes have been reported to oxidize retinol to retinal, but with activities that are 100-fold less than that of ADH1 [3,11]. The physiological functions of ADHs in retinoid meta- bolism are now being examined genetically in null mutant mice. A role for ADH4 in protection against vitamin A deficiency has been demonstrated in Adh4 –/– mice that suffer an increased rate of stillbirths relative to wild-type mice when maintained on a vitamin A deficient diet during gestation [12]. Adh1 –/– mice have been shown to have reduced metabolism of both ethanol and retinol, indicating that ADH1 is likely to play a role in retinoid metabolism in vivo [13]. In addition to functioning in the production of retinoic acid (RA) for development, which is particularly critical during vitamin A deficiency as pointed out by studies on Adh4 –/– mice [12], it is possible that ADHs also function in the oxidative elimination of excess retinol to prevent vitamin A toxicity. The toxicity of excess vitamin A has been well established [14–16] resulting in recommendations that consumption of liver or vitamin A supplements be limited to avoid excess exposure to retinol [17]. The pathways for clearance of excess retinol have been proposed to involve both oxidative and nonoxidative mechanisms as reviewed [18]. The main oxidative pathway for retinol turnover has been suggested to involve the oxidation of retinol to retinal, then oxidation of retinal to RA followed by glucuronide conjugation of the acid and/or 4-hydroxylation of RA. The key enzyme initiating this catabolic pathway, i.e. the one responsible for oxidizing excess retinol to retinal, has not been identified. In order to further investigate this metabolic pathway using genetic means, we have compared several strains of ADH Correspondence to G. Duester, Gene Regulation Program, Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Fax: + 1 858 646 3195, Tel.: + 1 858 646–3138, E-mail: duester@burnham.org. Abbreviations: ADH, alcohol dehydrogenase; Adh1, mouse class I ADH gene; Adh3, mouse class III ADH gene; Adh4, mouse class IV ADH gene; AST, aspartate aminotransferase; RA, retinoic acid; SDR, short-chain dehydrogenase/reductase. Enzyme: alcohol dehydrogenase (EC 1.1.1.1). (Received 9 January 2002, revised 15 April 2002, accepted 16 April 2002) Eur. J. Biochem. 269, 2607–2612 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02935.x null mutant mice (Adh1 –/– , Adh3 –/– ,andAdh4 –/– ) with wild- type mice for the effects of acute and chronic retinol treatment. The results demonstrate that ADH1 is the key enzyme essential for efficient elimination of excess retinol, thus indicating that it functions as the initiator of the oxidative pathway. These findings also have implications for the mechanism of vitamin A toxicity. MATERIALS AND METHODS Maintenance of mouse strains Mice carrying targeted disruptions of Adh1, Adh3 [13] and Adh4 [12] have been previously described. These null mutant mice as well as wild-type litter-mates were propagated on Purina 5015 Mouse Chow unless specified otherwise. This is a standard mouse diet containing 30 IUÆg )1 vitamin A. Dietary retinol supplementation Mice were propagated on Purina 5755 Basal Diet supple- mented with additional retinyl acetate to bring the total vitamin A concentration to 300 IUÆg )1 , all in the form of retinyl acetate, which is quickly hydrolyzed to retinol in the digestive tract. Adult female mice were placed on the retinol- supplemented diet for 2 weeks, then mated with a male while still on this diet to generate offspring. Offspring were maintained on the retinol-supplemented diet after weaning. Lethal dosing of retinol For lethal dose evaluation, mice were given oral doses of retinol, as described previously [14]. Male 14-week-old mice were used for all strains examined. All-trans-retinol (Sigma Chemical Co., St Louis, MO, USA) was dissolved in corn oil and administered by oral intubation at 0.2 mL per 10 g of body weight. Doses ranged from 0.5 to 3.5 gÆkg )1 . Lethality was monitored daily over 14 days after retinol administration. Doses resulting in the death of 16% (LD 16 ), 50% (LD 50 ), or 84% (LD 84 ) of the mice by day 14, plus the 95% confidence limits for the LD 50 dose, were calculated using the methods of Litchfield & Wilcoxon [19]. Acute retinol administration for tissue retinoic acid determination Retinol was administered essentially as described previously [20]. All-trans-retinol (Sigma) was dissolved in acetone/ Tween 20/water (0.25 : 5 : 4.75, v/v) and a dose of 50 mgÆkg )1 was injected orally to age- and weight-matched female mice. After 2 h, liver was collected and stored at )20 °C until HPLC analysis. HPLC quantitation of retinoic acid and retinol For tissue retinoic acid determination, liver (250 mg) was homogenized on ice in 2 mL of methanol/acetone (50 : 50, v/v). For serum retinol determination, blood was collected andstoredat)20 °C until analysis. Serum (200 lL) was extracted with 2 mL of methanol/acetone (50 : 50, v/v). After centrifugation at 10 000 g for 10 min at 4 °C, the organic phases from liver or serum extracts were evaporated under vacuum. Residues were dissolved in 200 lLof methanol/dimethylsulfoxide (50 : 50, v/v) and injected into the HPLC system to quantitate retinoids using all-trans- retinol and all-trans-retinoic acid (Sigma) as standards. Reverse-phase HPLC analysis was performed using a MICROSORB-MVTM 100 C18 column (4.5 · 250 mm; Varian) at a flow rate of 1 mLÆmin )1 . UV detection was carried out at 340 nm. Mobile phase consisted of 0.5 M ammonium acetate/methanol/acetonitrile (25 : 65 : 10, v/v/ v; solvent A) and acetonitrile (solvent B). The gradient composition was (only solvent B is mentioned): 0% at the time of injection; 30% at 1 min; 35% at 14 min; and 100% at 16 min. Measurement of aspartate aminotransferase levels in serum Aspartate aminotransferase/glutamic oxalacetic transami- nase (AST/GOT) activity was measured in mouse serum using the Sigma Diagnostics Transaminase kit following manufacturer’s procedure. In brief, 200 lLofserumwas mixed with substrate and incubated for 1 h at 37 °C. After 1 h, colour reagent was added and samples were left at room temperature for 20 min. The reaction was stopped by adding 0.4 N NaOH and the A 505 was then measured. Data are reported as Sigma–Frankel (SF) units per mL of serum. Statistics Statistical significance was determined for raw data using the unpaired Student’s t-test ( STATISTICA version 5.0). RESULTS Metabolism of acute dose of retinol The main pathway for retinol turnover begins with the oxidation of retinol to retinal followed very quickly by further oxidation of retinal to RA, which then accumulates before it is further metabolized [18]. In order to examine the in vivo contribution of ADH1, ADH3, and ADH4 to retinol metabolism, wild-type mice as well as null mutant mice deficient in these ADHs were treated orally with a 50-mgÆkg )1 dose of all-trans-retinol and 2 h later, all-trans- RA was quantitated in liver. Under these conditions, wild- type mice produced a large amount of RA (2.0 lgÆg )1 ) whereas Adh1 –/– mice produced 10-fold less RA (0.21 lgÆg )1 ) (Fig. 1). Adh3 –/– mice exhibited a 3.8-fold reduction in RA production (0.53 lgÆg )1 ) compared to wild-type, and Adh4 –/– mice exhibited a small decrease in RA (1.49 lgÆg )1 ) that was not statistically significant (Fig. 1). These results indicate that ADH1 plays a dominant role in clearance of an acute dose of retinol, and that ADH3 also contributes to a lesser extent, but that ADH4 plays little or no role in liver retinol metabolism. Retinol lethal dose In order to determine if ADH reduces the toxicity of a supraphysiological dose of retinol, we determined the LD 50 values for each mutant strain. Our wild-type mice exhibited a retinol LD 50 value of 2.72 gÆkg )1 , very close to the value of 2.52 gÆkg )1 previously reported for mice [14]. The retinol LD 50 value for Adh1 –/– mice was reduced threefold to 2608 A. Molotkov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 0.9 gÆkg )1 ,whereastheLD 50 value for Adh3 –/– mice was reduced 1.8-fold to 1.55 gÆkg )1 (Table 1). For Adh4 –/– mice the LD 50 was reduced only 1.5-fold to 1.74 gÆkg )1 , thus on the border of being statistically signficant when considering the confidence limits (Table 1). Thus, ADH1 plays a dominant role in providing protection against large acute doses of vitamin A that are life-threatening, with ADH3 playing a significant secondary role, and ADH4 playing a very minor role. Chronic retinol treatment during development The effect of a chronic modest increase in vitamin A was examined by propagating mice for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal mouse chow (300 IUÆg )1 in the supplemen- ted diet vs. 30 IUÆg )1 in normal chow). The amount of vitamin A present in the supplemented diet is not beyond the range that could be ingested naturally if one considers that it could also be obtained from a diet high in liver, known to contain 660–1300 IUÆg )1 vitamin A [17]. This level of retinol supplementation did not have a negative effect on development of Adh3 –/– and Adh4 –/– mice, which behaved similarly to wild-type mice with respect to survival to adulthood (92–95% survival for all three strains), but Adh1 –/– mice exhibited a large reduction in survival to adulthood (36% survival) (Fig. 2A,B). Adh1 –/– mice that did not survive were effected very early after birth as they were found to have decreased maternal suckling resulting in death by postnatal day 3. No gross malforma- tions were observed in any of the mice that died (including limbs and craniofacial region) indicating that overt retinoid teratogenicity had not occurred with this moderate retinol treatment. Instead, death was likely due to more subtle effects of general vitamin A toxicity, perhaps leading to reduced intake of maternal milk resulting in dehydration and lack of nourishment. Vitamin A toxicity is known to reduce food intake leading to weight loss and eventually death [16]. Liver toxicity following chronic retinol treatment Adh1 –/– mice that survived development on the retinol- supplemented diet had growth rates that were similar to those of wild-type, Adh3 –/– ,andAdh4 –/– mice through 7 weeks of age (Fig. 2C). Thus, we examined surviving mice further to see if any differences could be found. Compared to mice generated on normal mouse chow, all strains of mice generated on the retinol-supplemented diet exhibited higher serum retinol levels (Fig. 3). Compared to wild-type mice generated on the retinol-supplemented diet, Adh1 –/– mice, and to a lesser extent Adh3 –/– mice, exhibited significantly higher serum retinol levels; Adh4 –/– mice were not significantly different to wild-type (Fig. 3). These findings indicate that ADH1 provides the greatest protec- tion against retinol accumulation in the serum when the diet contains excess vitamin A. We also examined liver toxicity in these mice by examination of aspartate aminotransferase (AST) levels in serum. Serum AST levels were elevated in all mice generated on the retinol-supplemented diet relative to normal chow, but the elevation was particularly high in Adh1 –/– mice (Fig. 4). Relative to serum AST levels in wild-type mice generated on the retinol-supplemented diet, Adh1 –/– mice displayed a 92% increase, whereas Adh3 –/– mice exhibited a 37% increase and Adh4 –/– mice had no significant difference (Fig. 4). These results essentially mirror the serum retinol results discussed above. Thus, an increase in serum retinol due to an absence of ADH1 (and to a lesser extent ADH3) leads to an increase in liver toxicity. DISCUSSION The results reported here establish that ADH1 functions as a major protective factor against vitamin A toxicity. The ability of several classes of ADH to perform retinol WT Adh1 -/- Adh3 -/- Adh4 -/- 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 RA (µg/g) * ** Fig. 1. Metabolism of retinol to RA in liver of Adh deficient mice. Liver all-trans-RA levels were quantitated by HPLC 2 h after a 50 mgÆkg )1 oral dose of all-trans-retinol for wild-type mice (WT) and each null mutant strain indicated; all were age-matched adult female mice (n ¼ 4). Values are mean ± SEM. *P <0.01; **P < 0.03 (null mutant vs. wild-type). Table 1. Effect of Adh genotype on retinol lethal dose. *P <0.05(Adh1 –/– , Adh3 –/– ,andAdh4 –/– vs. wild-type). The confidence limits for LD 50 are in parentheses. Genotype n LD 16 (gÆkg )1 )LD 50 (gÆkg )1 )LD 84 (gÆkg )1 ) Wild-type 15 2.31 2.72 (2.32/3.18) 3.25 Adh1 –/– 12 0.73 0.90 (0.65/1.25)* 1.12 Adh3 –/– 24 1.22 1.55 (1.18/2.03)* 1.97 Adh4 –/– 12 1.37 1.74 (1.32/2.31)* 2.24 Ó FEBS 2002 Vitamin A toxicity and alcohol dehydrogenase (Eur. J. Biochem. 269) 2609 oxidation has been apparent for many years [4–9]. However, it has previously been unclear what in vivo functions the several classes of ADH might perform. Also, identification of microsomal enzymes of the SDR family that can oxidize retinol to retinal has produced conflicting results as to whether ADHs or SDRs contribute significantly to retinol metabolism [3,11]. Previous genetic studies have identified a physiological role for ADH4 in maintaining sufficient retinol oxidation during vitamin A deficiency to generate RA for development [10,12]. It is now apparent from the genetic studies presented here that ADH1 is responsible for most of the in vivo oxidation of retinol to RA in liver during vitamin A excess. We demonstrated a 10-fold reduction in metabolism of retinol to RA in liver of Adh1 –/– mice. Thus, ADH1 is important as the main initiator of the oxidative pathway for retinol turnover. That this is significant in vivo is shown by results indicating that Adh1 –/– mice are much more sensitive to vitamin A toxicity as demonstrated by a greatly reduced LD 50 value for retinol as well as greatly reduced survival during development on a retinol-supple- mented diet. The LD 50 studies reported here indicate that ADH3 and to a lesser extent ADH4 also provide some protection against death induced by massive nonphysiological doses of retinol, but that both provide much less protection than that afforded by ADH1. Our results with Adh3 –/– and Adh4 –/– WT Adh1 Adh3 Adh 4 WT Adh1 Adh 3 Adh 4 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 normal mouse chow serum retinol (µg/ml) * ** retinol-supplemented Fig. 3. Serum retinol levels in mice generated on a retinol-supplemented diet. For wild-type and each Adh null mutant strain indicated, HPLC was used to quantitate serum retinol levels in mice generated on nor- mal mouse chow (30 IUÆg )1 vitamin A) or a retinol-supplemented diet (300 IUÆg )1 vitamin A). All mice were first generation 10-week-old females (n ¼ 5). Values are mean ± SEM. *P < 0.02; **P <0.01 (null mutant vs. wild-type, retinol supplemented). WT Adh1 Adh3 Adh4 WT Adh1 Adh3 Adh4 0 20 40 60 80 100 120 normal mouse chow * ** AST (SF units/ml) retinol-supplemented Fig. 4. AST levels in wild-type and Adh null mutant mice. Serum AST levels are shown for mice generated on either normal chow or a retinol- supplemented diet. All mice were first generation 10-week-old males (n ¼ 4). Values are mean ± SEM. *P < 0.03; **P < 0.05 (null mutant vs. wild-type, retinol-supplemented). A B 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 Adh1 WT Adh3 Adh4 0 5 10 15 20 25 30 35 40 45 0 20 40 60 80 100 Adh1 WT Adh3 Adh4 0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 Adh1 WT Adh 4 Adh 3 C Age (days) A g e (da y s) Number of mice alive % survival Weight (g) A g e (da y s) Fig. 2. Postnatal lethality in Adh1 –/– mice generated on a retinol-supplemented diet. (A) Shown is the number of first generation off- spring born for wild-type and each Adh null mutant strain maintained on a retinol-sup- plemented diet, plus the numbers of offspring surviving until postnatal day 40 (P40); exten- sive postnatal death occurred in Adh1 –/– mice between birth and P3. (B) The percentage survival for each mouse strain is shown; only 36% survival was observed for Adh1 –/– mice by P40. (C) Shown is the weight gain for the above wild-type and Adh null mutant mice. Data for some of the mice reported here is also described elsewhere [21]. 2610 A. Molotkov et al. (Eur. J. Biochem. 269) Ó FEBS 2002 mice also indicate that ADH3 plays a significant role in metabolism of a dose of retinol to RA in the liver, albeit less than that of ADH1, but that ADH4 does not effect retinol turnover significantly in the liver perhaps due to its lack of expression in liver as detailed further below. These findings are therefore in agreement with the retinol LD 50 values observed for these mice. The data presented here on retinol toxicity provide additional evidence that ADH3 functions in retinol meta- bolism. Several studies had originally failed to recognize a role for ADH3 in retinol metabolism [5,6,8], but recent studies using a more sensitive assay have shown that ADH3 can oxidize retinol in vitro, but with much lower activity than ADH1 or ADH4 [10]. Also, Adh3 –/– mice have a growth deficiency that can be rescued by dietary retinol supplemen- tation, and they have greatly reduced survival during vitamin A deficiency compared to wild-type mice [10]. Thus, there is now much evidence of a role for ADH3 in metabolism of retinol to RA under physiological conditions as well as during vitamin A toxicity and deficiency. In contrast to the LD 50 studies, which examine vitamin A toxicity under nonphysiological conditions, our results using the retinol-supplemented diet are applicable to physiological conditions as the amount of retinol used could be obtained by natural diets rich in vitamin A (i.e. liver). Under these dietary conditions we observed that ADH1 provided the most protection (against both postnatal lethality and liver toxicity assessed by serum AST), ADH3 provided less protection (against liver toxicity only), and ADH4 provided no protection. Thus, our results suggest that ADH1 has evolved to be more efficient than ADH3 for retinol turnover, and that ADH4 has not evolved for this function. In addition, a recent description of mice deficient in both Adh1 and Adh4 has demonstrated that the loss of both activities does not result in increased vitamin A toxicity over that seen for mice deficient in only Adh1 [21]. The role observed for ADH1 in prevention of vitamin A toxicity also suggests that the microsomal SDRs reported to metabolize retinol probably do not play major roles in retinol turnover or protection against vitamin A toxicity, as their activities and expression are relatively low compared to ADH1. The expression patterns of the ADH gene family provide further understanding into the roles of these enzymes in retinol turnover observed in the null mutant mice. ADH1 mRNA and protein is expressed at very high levels in mouse liver, intestine, and kidney [22,23] and it accounts for 0.9% of mouse liver protein [24]. ADH3 is expressed ubiquitously [22,23] and accounts for 0.2% of mouse liver protein [24]. ADH4 is not expressed in liver, but is found at highest levels in the stomach, esophagus, and skin [22,23] and accounts for 0.07% of mouse stomach protein [24]. Thus, high expression of ADH1 in liver makes it well-equipped to handle turnover of large amounts of retinol as we observed. The ubiquitous expression of ADH3, with a high concen- tration in the liver, allows it to also contribute significantly to retinol turnover as we observed, but the lack of ADH4 expression in liver and relatively low expression in other organs precludes it from being a major player in systemic retinol turnover consistent with the results provided here. Mammalian ADH genes were derived from duplications of an ancestral ADH3 gene conserved in lower vertebrates (cartilaginous fishes) and invertebrates including Amphi- oxus [25,26]. As ADH1 did not appear until bony fishes [27] and ADH4 until amphibians [28], early vertebrates could have relied upon ADH3 to oxidize retinol to retinal. As it has now been demonstrated that retinol metabolism by mouse ADH3 functions to catalyze turnover of excess retinol, ADH3 can be thought of as a prototype retinol dehydrogenase that was expanded upon later in evolution. In particular, evolution of ADH1 may have allowed more efficient turnover of excess retinol providing a selective advantage against vitamin A toxicity observed in mice. In the case of ADH4, evolution of this enzyme may have provided added protection against vitamin A deficiency encountered by land animals, as it did not appear until amphibians and does give protection against vitamin A deficiency in mice [10,12]. It has been proposed that both the toxic and teratogenic properties of retinol may be due to its conversion to RA, which is known to control a signaling pathway involving RA receptors [16]. Studies in mice have shown that retinol teratogenicity is associated with increased production of RA, and that the teratogenicity of retinol can be reduced by treatment with the ADH inhibitor 4-methylpyrazole, which also reduces RA production [20]. Interestingly, among the several classes of ADH, ADH1 is by far the most sensitive to 4-methylpyrazole [24,29]. Combining these inhibitor find- ings with those here using Adh1 –/– mice, it is now clear that when retinol is given at very high doses, it is metabolized primarily by ADH1 to produce high levels of RA, which are teratogenic for embryonic development. However, our data show that when metabolism of retinol to RA is greatly reduced in Adh1 –/– mice, there is an increase in retinol toxicity (rather than teratogenicity) as demonstrated by a decrease in the lethal dose for retinol in adult mice as well as reduced survival of newborn mice generated on a retinol- supplemented diet. In our developmental studies, we provided a very modest increase in dietary retinol, much less than that needed to produce retinoid teratogenicity, but enough to produce toxicity when ADH1 is missing, as shown by decreased survival of newborn mice and increased serum AST in those that did survive to adulthood. Thus, retinol toxicity, as opposed to teratogenicity, occurs when there is a defect in the ability to turnover retinol oxidatively. Our findings demonstrate that in order to avoid retinol toxicity, it is more beneficial to metabolize retinol oxida- tively to RA than to allow it to be disposed of in any other way. When oxidation of retinol to RA is severely impaired as it is in Adh1 –/– mice, retinol may instead become a substrate for P450s known to metabolize retinol to 4-hydroxyretinol [30]. This may lead to toxicity as P450- mediated metabolism requires molecular oxygen and pro- duces oxygen free radicals that can cause liver damage [31,32]. In contrast, ADH-mediated metabolism occurs via dehydrogenation with the cofactor NAD, thus does not produce oxygen free radicals. Also, retinol toxicity has been shown to be associated with formation of large amounts of glucuronidated retinol leading to reduced levels of uridine diphosphoglucuronic acid suggesting that retinol toxicity may be due in part to reduction of the substrate needed to perform other essential glucuronidations [33]. Our findings point out a fundamental difference in retinol toxicity and retinol teratogenicity, as the former is mediated by retinol (or metabolites other than RA) whereas the latter is mediated by excessive production of RA that leads to aberrant retinoid signaling during embryogenesis. Ó FEBS 2002 Vitamin A toxicity and alcohol dehydrogenase (Eur. J. Biochem. 269) 2611 ACKNOWLEDGEMENTS We thank H. Freeze for providing access to an HPLC system and F. Mic for useful discussions. This work was supported by National Institutes of Health Grant AA09731 (G.D.). REFERENCES 1. Szalai, G., Duester, G., Friedman, R., Jia, H., Lin, S., Roe, B.A. & Felder, M.R. 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Measurement of aspartate aminotransferase levels in serum Aspartate aminotransferase/glutamic oxalacetic transami- nase (AST/GOT) activity was measured. providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have