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

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

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

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

) (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

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

–/–
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

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

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

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

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

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-

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

¨
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

ciencies in alcohol dehydrogenase Adh1, Adh3,andAdh4 null
mutant mice: overlapping roles of Adh1 and Adh4.ethanolclear-
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

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-