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

Following an acute dose of retinol 50 mgÆkg1, 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 r

Excessive vitamin A toxicity in mice genetically deficient in either

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, and Adh4 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 LD50value, was

greatly increased in Adh1 mutants and moderately increased

in Adh3 mutants, but only a minor effect was observed in

Adh4mutants 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)

null mutant mice (Adh1–/–, Adh3–/–, and Adh4–/–) 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

M A T E R I A L S A N D M E T H O D S

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)1vitamin 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% (LD16),

50% (LD50), or 84% (LD84) of the mice by day 14, plus the

95% confidence limits for the LD50dose, 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)1was 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

and stored at )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 4C, the

organic phases from liver or serum extracts were evaporated

under vacuum Residues were dissolved in 200 lL of

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 0.5M 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 lL of serum was mixed with substrate and incubated for 1 h at 37C 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 A505was 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 (STATISTICAversion 5.0)

R E S U L T S 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 vivocontribution 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)1dose of 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 LD50 values for each mutant strain Our wild-type mice exhibited

a retinol LD50value of 2.72 gÆkg)1, very close to the value of 2.52 gÆkg)1previously reported for mice [14] The retinol

LD value for Adh1–/– mice was reduced threefold to

0.9 gÆkg)1, whereas the LD50 value for Adh3–/– mice was

reduced 1.8-fold to 1.55 gÆkg)1(Table 1) For Adh4–/–mice

the LD50was 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)1in 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)1vitamin 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–/–, and Adh4–/– 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 accumulaprotec-tion 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

D I S C U S S I O N 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

*

**

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–/–, and Adh4–/–vs wild-type) The confidence limits for LD 50 are in parentheses.

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 LD50 value for retinol as well as greatly reduced survival during development on a retinol-supple-mented diet

The LD50studies 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–/–

4

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

*

**

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)1vitamin A) or a retinol-supplemented diet

(300 IUÆg)1vitamin 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).

0 20 40 60 80 100 120

normal mouse chow

*

**

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

Adh1

WT

Adh3 Adh4

0

20

40

60

80

100

Adh1

WT

Adh3

0 5 10 15 20 25 30

Adh1

WT

Adh4 Adh3

C

Age (days)

Age (days)

Age (days)

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].

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 LD50 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 LD50studies, 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

A C K N O W L E D G E M E N T S

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.).

R E F E R E N C E S

1 Szalai, G., Duester, G., Friedman, R., Jia, H., Lin, S., Roe, B.A &

Felder, M.R (2002) Organization of sixfunctional mouse alcohol

dehydrogenase genes on two overlapping bacterial artificial

chromosomes Eur J Biochem 269, 224–232.

2 Duester, G., Farre´s, J., Felder, M.R., Holmes, R.S., Ho¨o¨g, J.-O.,

Pare´s, X., Plapp, B.V., Yin, S.-J & Jo¨rnvall, H (1999)

Recommended nomenclature for the vertebrate alcohol

dehy-drogenase gene family Biochem Pharmacol 58, 389–395.

3 Duester, G (2000) Families of retinoid dehydrogenases regulating

vitamin A function: production of visual pigment and retinoic

acid Eur J Biochem 267, 4315–4324.

4 Bliss, A.F (1951) The equilibrium between vitamin A alcohol and

aldehyde in the presence of alcohol dehydrogenase Arch

Bio-chem 31, 197–204.

5 Boleda, M.D., Saubi, N., Farre´s, J & Pare´s, X (1993)

Physiolo-gical substrates for rat alcohol dehydrogenase classes: aldehydes of

lipid peroxidation, omega-hydroxyfatty acids, and retinoids Arch.

Biochem Biophys 307, 85–90.

6 Yang, Z.-N., Davis, G.J., Hurley, T.D., Stone, C.L., Li, T.-K &

Bosron, W.F (1994) Catalytic efficiency of human alcohol

dehy-drogenases for retinol oxidation and retinal reduction Alcohol.

Clin Exp Res 18, 587–591.

7 Kedishvili, N.Y., Bosron, W.F., Stone, C.L., Hurley, T.D., Peggs,

C.F., Thomasson, H.R., Popov, K.M., Carr, L.G., Edenberg, H.J.

& Li, T.-K (1995) Expression and kinetic characterization of

recombinant human stomach alcohol dehydrogenase Active-site

amino acid sequence explains substrate specificity compared with

liver isozymes J Biol Chem 270, 3625–3630.

8 Han, C.L., Liao, C.S., Wu, C.W., Hwong, C.L., Lee, A.R & Yin,

S.J (1998) Contribution to first-pass metabolism of ethanol and

inhibition by ethanol for retinol oxidation in human alcohol

dehydrogenase family – implications for etiology of fetal alcohol

syndrome and alcohol-related diseases Eur J Biochem 254,

25–31.

9 Allali-Hassani, A., Peralba, J.M., Martras, S., Farre´s, J & Pare´s,

X (1998) Retinoids, omega-hydroxyfatty acids and cytotoxic

aldehydes as physiological substrates, and H 2 -receptor antagonists

as pharmacological inhibitors, of human class IV alcohol

dehy-drogenase FEBS Lett 426, 362–366.

10 Molotkov, A., Fan, X., Deltour, L., Foglio, M.H., Martras, S.,

Farre´s, J., Pare´s, X & Duester, G (2002) Stimulation of retinoic

acid production and growth by ubiquitously-expressed alcohol

dehydrogenase Adh3 Proc Natl Acad Sci USA 99, 5337–5342.

11 Napoli, J.L (1999) Interactions of retinoid binding proteins and

enzymes in retinoid metabolism Biochim Biophys Acta Mol Cell

Biol Lipids 1440, 139–162.

12 Deltour, L., Foglio, M.H & Duester, G (1999) Impaired retinol

utilization in Adh4 alcohol dehydrogenase mutant mice Dev.

Genet 25, 1–10.

13 Deltour, L., Foglio, M.H & Duester, G (1999) Metabolic

defi-ciencies in alcohol dehydrogenase Adh1, Adh3, and Adh4 null

mutant mice: overlapping roles of Adh1 and Adh4 ethanol

clear-ance and metabolism of retinol to retinoic acid J Biol Chem 274,

16796–16801.

14 Kamm, J.J (1982) Toxicology, carcinogenicity, and teratogenicity

of some orally administered retinoids J Am Acad Dermatol 6,

652–659.

15 Biesalski, H.K (1989) Comparative assessment of the toxicology

of vitamin A and retinoids in man Toxicology 57, 117–161.

16 Armstrong, R.B., Ashenfelter, K.O., Eckhoff, C., Levin, A.A & Shapiro, S.S (1994) General and reproductive toxicology of retinoids In The Retinoids: Biology, Chemistry, and Medicine, 2nd edn (Sporn, M.B., Roberts, A.B & Goodman, D.S., eds), pp 545–572 Raven Press, Ltd., New York.

17 Nelson, M (1990) Vitamin A, liver consumption, and risk of birth defects Liver is a cheap source of many nutrients Br Med J 301, 1176.

18 Frolik, C.A (1984) Metabolism of retinoids In The Retinoids, Vol 2 (Sporn, M.B., Roberts, A.B & Goodman, D.S., eds), pp 177–208 Academic Press, Orlando.

19 Litchfield, J.T Jr, & Wilcoxon, F (1949) A simplified method of evaluating dose–effect experiments J Pharmacol Exp Ther 96, 99–117.

20 Collins, M.D., Eckhoff, C., Chahoud, I., Bochert, G & Nau, H (1992) 4-methylpyrazole partially ameliorated the teratogenicity of retinol and reduced the metabolic formation of all-trans-retinoic acid in the mouse Arch Toxicol 66, 652–659.

21 Molotkov, A., Deltour, L., Foglio, M.H., Cuenca, A.E & Duester, G (2002) Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice J Biol Chem 277, 13804–13811.

22 Zgombic-Knight, M., Ang, H.L., Foglio, M.H & Duester, G (1995) Cloning of the mouse class IV alcohol dehydrogenase (retinol dehydrogenase) cDNA and tissue-specific expression patterns of the murine ADH gene family J Biol Chem 270, 10868–10877.

23 Haselbeck, R.J & Duester, G (1997) Regional restriction of alcohol/retinol dehydrogenases along the mouse gastrointestinal epithelium Alcohol Clin Exp Res 21, 1484–1490.

24 Algar, E.M., Seeley, T.-L & Holmes, R.S (1983) Purification and molecular properties of mouse alcohol dehydrogenase isozymes Eur J Biochem 137, 139–147.

25 Danielsson, O & Jo¨rnvall, H (1992) Enzymogenesis: classical liver alcohol dehydrogenase origin from the glutathione-depend-ent formaldehyde dehydrogenase line Proc Natl Acad Sci USA

89, 9247–9251.

26 Can˜estro, C., Hjelmqvist, L., Albalat, R., Garcia-Ferna`ndez, J., Gonza`lez-Duarte, R & Jo¨rnvall, H (2000) Amphioxus alcohol dehydrogenase is a class 3 form of single type and of structural conservation but with unique developmental expression Eur.

J Biochem 267, 6511–6518.

27 Danielsson, O., Eklund, H & Jo¨rnvall, H (1992) The major pis-cine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III Biochemistry 31, 3751–3759.

28 Hoffmann, I., Ang, H.L & Duester, G (1998) Alcohol dehy-drogenases in Xenopus development: conserved expression of ADH1 and ADH4 in epithelial retinoid target tissues Dev Dyn.

213, 261–270.

29 Boleda, M.D., Julia`, P., Moreno, A & Pare´s, X (1989) Role of extrahepatic alcohol dehydrogenase in rat ethanol metabolism Arch Biochem Biophys 274, 74–81.

30 Roberts, E.S., Vaz, A.D.N & Coon, M.J (1992) Role of isozymes

of rabbit microsomal cytochrome P-450 in the metabolism of retinoic acid, retinol, and retinal Mol Pharmacol 41, 427–433.

31 Lieber, C.S (1994) Mechanisms of ethanol-drug–nutrition inter-actions J Toxicol Clin Toxicol 32, 631–681.

32 Kono, H., Bradford, B.U., Yin, M., Sulik, K.K., Koop, D.R., Peters, J.M., Gonzalez, F.J., McDonald, T., Dikalova, A., Kadiiska, M.B., Mason, R.P & Thurman, R.G (1999) CYP2E1

is not involved in early alcohol-induced liver injury Am J Physiol Gastrointest Liver Physiol 277, G1259–G1267.

33 Bray, B.J & Rosengren, R.J (2001) Retinol potentiates acet-aminophen-induced hepatotoxicity in the mouse: Mechanistic studies Toxicol Appl Pharmacol 173, 129–136.