From the Gene Regulation Program, Burnham Institute, La Jolla, California 92037
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ABSTRACT |
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Targeting of mouse alcohol dehydrogenase genes
Adh1, Adh3, and Adh4 resulted in
null mutant mice that all developed and reproduced apparently normally
but differed markedly in clearance of ethanol and formaldehyde plus
metabolism of retinol to the signaling molecule retinoic acid.
Following administration of an intoxicating dose of ethanol,
Adh1 The alcohol dehydrogenase
(ADH)1 family consists of
numerous enzymes able to catalyze the reversible oxidation of a wide
variety of xenobiotic and endogenous alcohols to the corresponding
aldehydes (1). Several distinct classes of vertebrate ADH have been
described, all of which are cytosolic and zinc-dependent
but differ in substrate specificities and gene expression patterns (2,
3). Three forms that are highly conserved in mammals and other
vertebrates are class I ADH (ADH1), class III ADH (ADH3), and class IV
ADH (ADH4); see Ref. 1 for ADH nomenclature. Biochemical studies indicate that these three ADHs are able to utilize a wide variety of
alcohol and aldehyde substrates in vitro ranging from
ethanol to formaldehyde to retinol. However, the precise functions of these enzymes are not yet well established. In humans, ADH4
demonstrates higher retinol dehydrogenase activity than ADH1 with ADH1
having higher ethanol dehydrogenase activity than ADH4 and ADH3 having insignificant retinol or ethanol dehydrogenase activity (4-6). Instead, ADH3 has glutathione-dependent formaldehyde
dehydrogenase activity, i.e. upon reaction of formaldehyde
with glutathione to produce S-hydroxymethylglutathione, ADH3
oxidizes the hydroxymethyl group to a formyl group to produce
S-formylglutathione, which is then the substrate for a
hydrolase that regenerates glutathione and produces formate (7).
The potential role of ADH1 and ADH4 in retinol metabolism is
particularly interesting because this pathway produces retinoic acid,
which is a physiological ligand controlling numerous retinoic acid
receptor signaling pathways (8). Also, the potential dual roles of ADH1
and ADH4 as ethanol and retinol dehydrogenases have led us to propose
that alcohol abuse may lead to ethanol inhibition of ADH-catalyzed
retinol oxidation, hence reduced retinoic acid synthesis, and that this
may contribute to ethanol damage such as that seen in fetal alcohol
syndrome (9, 10). Physiological data are now needed to complement the
available biochemical data to effectively address the in
vivo functions of these enzymes, particularly the role in retinoic
acid synthesis, which is presently a controversial issue (3).
Genes encoding ADH1, ADH3, and ADH4 have been identified in the mouse,
i.e. Adh1, Adh3, and Adh4,
respectively (11, 12). An involvement of mouse Adh1 in
ethanol metabolism has been proposed (13), but no role in retinol
metabolism has been shown. We are not aware of any data linking mouse
Adh3 to formaldehyde metabolism. A role for Adh4
in retinoic acid synthesis was first proposed when a retinol
dehydrogenase isolated from mouse epidermis was found to be identical
to ADH4 (14). Additional evidence linking both Adh1 and
Adh4 to retinoic acid synthesis consists of gene expression
data where these enzymes show localization in numerous adult
retinoid-responsive epithelia including the epidermis (15), male
reproductive tract (16), and gastrointestinal tract (17) as well as in
retinoid target tissues of embryos at stages E8.5-E9.5 (18, 19) and
later embryonic stages (20). Also, retinoic acid and Adh1
plus Adh4 expression have been colocalized in the adult and
embryonic adrenal gland, which may function as an endocrine source of
retinoic acid (21, 22). We have now generated mice carrying targeted
null mutations in Adh1, Adh3, and
Adh4. Our results provide genetic evidence demonstrating
in vivo functions for these genes in the metabolism of
ethanol, formaldehyde, and retinol, with significant overlap of
Adh1 and Adh4 in ethanol and retinol metabolism.
Construction of Adh-targeting Vectors--
Gene replacement
targeting vectors were produced for all three mouse Adh
genes. Genomic clones for mouse Adh1 previously have been
described (23, 24). We screened a mouse 129/SvJ genomic library
(Stratagene, La Jolla, California) using a mouse Adh1 cDNA (25) and isolated a
A 129/SvJ genomic clone ( Creation of Adh Null Mutant Mice--
Adh1,
Adh3, and Adh4 gene-targeting vectors were all
linearized with NotI and introduced by electroporation into
mouse embryonic stem cells (R1 cells from 129/Sv strain) using standard
methodology (29). To enrich the cells incorporating the constructs by
homologous recombination, positive selection was with G418, and
negative selection was with ganciclovir. Identification of cells
carrying Adh deletions was accomplished by Southern blot
analysis (30) of genomic DNA isolated from surviving cell clones.
External DNA probes for Southern blot analysis consisted of a 0.2-kb
EcoRV-HindIII fragment from the 3'-flanking
region of Adh1 (24), a 0.7-kb HindIII fragment
from intron 6 of Adh3 (27), and a 1.5-kb XbaI fragment containing exon 9 of Adh4 (31).
Southern blot positive clones were subjected to karyotype analysis to
identify those having a normal karyotype for blastocyst injection.
Mutant embryonic stem cells were microinjected into C57Bl/6
blastocysts, which were then implanted into pseudopregnant females
resulting in chimeric mice (29). For each Adh mutant several
male chimeric mice were mated to wild-type female Black Swiss mice, and
germ-line transmission was identified by agouti coat color. Agouti
offspring were subjected to Southern blot analysis of tail DNA (30) to
identify individuals heterozygous for each Adh mutation.
Heterozygous matings were performed to produce homozygous mutant mice
and wild-type mice (as identified by Southern blot analysis of tail
DNA), which were then expanded to form permanent mouse lines maintained
under standard laboratory conditions on a Purina basal diet 5755.
Northern blot analysis was performed on 10 µg of total RNA from mouse
tissues using Adh cDNA probes as described previously (12). Polyclonal antibodies against mouse ADH1, ADH3, and ADH4 were
used to probe Western blots of mouse tissues containing 20 µg of
total protein as reported (17).
Ethanol Treatment--
For all treatments ethanol was
administered intraperitoneally as one acute dose at 3.5 g/kg (18 µl
of 25% ethanol in physiological saline/g of body weight). Control
injections consisted of the same volume of physiological saline. All
mice examined were female and were matched for approximate age and
weight. Blood ethanol clearance was performed as described previously
(32, 33) using a Sigma Alcohol Reagent Kit (Sigma) to measure the
concentration of ethanol in blood (20 µl) collected from the hind leg
vein at 5, 30, 60, 120, 240, and 360 min after administration of ethanol.
Ethanol-induced loss of righting response (LORR) was performed as
described (32) by placing animals upside down in a V-shaped trough
immediately upon induction of ethanol-induced sleep and measuring the
time until the animal could regain its righting response by being able
to right itself three times within a 30 s period.
For examination of ethanol-induced embryonic resorption, the day of
pregnancy and stage of embryonic development were determined by vaginal
plug appearance with noon on the day of plug detection being considered
E0.5 (34).
Formaldehyde Treatment--
All mice treated with formaldehyde
for determination of LD50 values were male and were matched
for approximate age and weight. Mice were given an intraperitoneal
injection of 10% formalin in physiological saline (Sigma) in doses
ranging from 0.09-0.22 g/kg (2.5-6.0 µl of 10% formalin/g of body
weight). A lethal dose of formaldehyde was defined as an amount able to
result in death within 90 min.
Retinol Treatment and Measurement of Retinoic Acid--
All
trans-retinol (Sigma) was dissolved in a vehicle consisting
of acetone/Tween 20/water (0.25:5:4.75) and administered at a dose of
100 µg/kg to adult female mice (age and weight matched) by oral
intubation as described previously (35). Retinol solution or vehicle
solution (same as above but without retinol added) was administered at
5 µl/g body weight. Two h after administration, tissues were
dissected, and retinoic acid was monitored either qualitatively or
quantitatively using an F9-RARE-lacZ reporter cell bioassay,
which detects the sum of all active carboxylated retinoids including
all trans-retinoic acid (21, 36). Qualitative analysis was
performed by incubating the tissues as explants on a monolayer of
F9-RARE-lacZ reporter cells and examining retinoic acid
diffusion by monitoring induction of lacZ expression in the adjacent reporter cells in situ (21). Retinoic acid levels
in kidney homogenates were quantitated essentially as described
previously (20) by performing a spectrophotometric variation of the
reporter cell bioassay with all trans-retinoic acid (Sigma)
as the standard; kidney tissue was homogenized for 30 s with a
Tissue-Tearor homogenizer (Biospec Products, Inc.)and immediately
centrifuged, and then the supernatant was applied to the reporter cells.
Statistics--
Statistical significance was determined for raw
data using Fisher's exact test (two-sided), two-way analysis of
variance (ANOVA), or the unpaired Student's t test
(two-tailed) (GraphPad Prism version 2.0b, GraphPad Software, Inc., San
Diego, California).
Production of Adh Null Mutant Mice--
Shown are the targeting
vectors used to inactive Adh1 (Fig.
1A) and Adh3 (Fig.
2A) as well as maps of the
wild-type and mutant loci; a null mutant for Adh4 has been
previously described (28). For each mutation a gene replacement
strategy was employed that deletes a portion of the gene,
i.e. deletion of exons 7-9 in Adh1 and deletion
of exons 1-4 in Adh3. Following introduction of each gene-targeting vector into embryonic stem cells and Southern blot analysis of approximately 100 independently selected clones for each
targeting event, we retrieved two clones carrying the expected Adh1 heterozygous mutation identified by a 4.1-kb
BamHI fragment (Fig. 1B) and fifteen clones
carrying the expected Adh3 heterozygous mutation identified
by a 6.9-kb BamHI fragment (Fig. 2B).
Mutant embryonic stem cell clones with a normal karyotype were used for
blastocyst injection to introduce the mutations into mice. For each
Adh mutation, we generated at least four chimeric males, and
at least two males demonstrated germ line transmission of the mutation
to their offspring as identified by Southern blot analysis of tail DNA
at weaning. Crossing of heterozygous Adh +/
To determine whether the Adh1 and Adh3 mutations
generated null phenotypes, Northern and Western blot analyses were
performed. In contrast to wild-type mice Adh1
Thus, generation of null mutations in Adh1, Adh3,
or Adh4 was not obviously hazardous to prenatal or postnatal
survival of mice raised under standard laboratory conditions.
Homozygous lines for each Adh mutant were derived, and all
further studies were focused upon a comparison of these three Effect of Adh1, Adh3, and Adh4 Null Mutations on Ethanol Clearance
and Toxicity--
As ethanol is often thought to be the main substrate
for ADHs, we tested the ability of each null mutant to metabolize
ethanol following a large acute dose consisting of 3.5 g of
ethanol/kg of body weight. A comparison of blood alcohol clearance in
the three Adh mutants and wild-type mice demonstrated that
Adh1
The duration of ethanol-induced sleep following an ethanol dose of 3.5 g/kg was measured by examining the LORR. LORR was significantly longer
only in Adh1
We also examined the effect of a 3.5 g/kg ethanol dose given at day
E8.5 of gestation on ethanol-induced embryonic resorption at day E12.5.
Whereas the rate of resorption in control-treated mice of each genotype
was only 5-10%, the rate of resorption in ethanol-treated
Adh1
Previous studies on the genetics of alcohol abuse in humans have shown
that polymorphic variants of the human ADH1B gene play a
role in ethanol susceptibility (reviewed in Refs. 37 and 38). In
particular, the ADH1B*2 allele found commonly in Oriental
populations encodes an enzyme with higher activity for ethanol
oxidation than the enzyme encoded by the ADH1B*1 allele
found commonly in Caucasian populations. A lower incidence of
alcoholism is associated with the ADH1B*2 allele, presumably
because increased production of acetaldehyde following ethanol
consumption leads to alcohol aversion. Our genetic studies in the mouse
indicate that a reduction in ethanol clearance because of knockout
mutations of ADH1 activity (and to a lesser extent ADH4 activity) will
also be important for understanding the mechanism of alcohol abuse.
Thus, the persistence of ethanol itself, in addition to its more toxic
metabolite acetaldehyde, plays a role in alcohol damage as demonstrated
by our studies of ethanol-induced resorption during pregnancy.
Effect of Adh Null Mutations on Formaldehyde Toxicity--
As we
observed no role for Adh3 in ethanol clearance or toxicity,
we examined formaldehyde toxicity because human ADH3 is known to
function as a glutathione-dependent formaldehyde
dehydrogenase. For Adh3 Metabolism of Retinol to Retinoic Acid in Adh Null
Mutants--
Treatment of adult mice with a large dose of retinol has
previously been found to result in production of excess retinoic acid
(35). We have previously shown that the mouse kidney normally has
undetectable levels of retinoic acid using a bioassay (20) and that
retinol treatment dramatically increases the level of retinoic acid in
wild-type mice but leads to a much smaller increase in Adh4
The above findings provide in vivo evidence that ADH1
and ADH4 can metabolize retinol to retinoic acid when retinol is
administered under superphysiological conditions, i.e. a
dose that is teratogenic to developing embryos. Thus, these studies do
not directly address the ability of ADHs to utilize retinol under
conditions of sufficiency to produce the small amount of retinoic acid
needed for normal growth and development. However, we have also
previously shown that Adh4
If Adh1 and Adh4 have overlapping roles in the
conversion of physiological levels of retinol to retinoic acid, then
the apparently normal survival of each mutant when maintained on a
standard mouse diet could be explained by one gene compensating for the
loss of the other. However, in addition to ADHs, which function as cytosolic retinol dehydrogenases, there exist microsomal retinol dehydrogenases that are members of the short-chain
dehydrogenase/reductase enzyme family (42-45). It is possible that one
or more members of the short-chain dehydrogenase/reductase family may
also function physiologically in the conversion of retinol to retinoic
acid, hence allowing Adh1 and Adh4 mutants to
grow and reproduce apparently normally. The relative importance of ADHs
and short-chain dehydrogenase/reductases may be different under
different states of retinol deficiency, sufficiency, or excess.
Production of Adh1/Adh4 homozygous double mutants
will serve to further decipher the role of ADHs in physiological
retinol utilization, but this will be difficult because these genes
have been mapped to the same general region of mouse chromosome 3 (46). We attempted to generate such double mutants by mating Adh1
and Adh4 null mutant mice and looking for crossover events
in mice heterozygous for both Adh1 and Adh4
mutations. Examination of approximately 300 individual offspring by
Southern blot analysis resulted in no detection of crossover events
that would place both mutations on the same chromosome and thus allow
generation of double homozygous mutant mice (data not shown). This
indicates that the genes are indeed very closely linked, requiring the
use of alternative approaches to generate
Adh1/Adh4 double mutants.
Conclusions--
In summary, genetic studies provide evidence that
Adh1, Adh3, and Adh4 null mutant mice
have clear defects in ethanol clearance, formaldehyde toxicity, and
metabolism of retinol to retinoic acid, respectively. Overlap in
function is seen because Adh4 contributes secondarily to
ethanol metabolism and Adh1 contributes secondarily to
retinol metabolism. Adh3 plays no role in the metabolism of either ethanol or retinol but does play a role in formaldehyde metabolism. The functional overlap noticed between Adh1 and
Adh4 is likely to be important both for the utilization of
vitamin A and for the mechanism of alcohol abuse. Further genetic
studies such as the generation of Adh1/Adh4
double knockouts should provide important information on these topics.
/
mice, and to a lesser extent Adh4
/
mice, but not Adh3
/
mice, displayed significant
reductions in blood ethanol clearance. Ethanol-induced sleep was
significantly longer only in Adh1
/
mice. The incidence
of embryonic resorption following ethanol administration was increased
3-fold in Adh1
/
mice and 1.5-fold in Adh4
/
mice but was unchanged in Adh3
/
mice. Formaldehyde toxicity studies revealed that only Adh3
/
mice had a significantly reduced LD50 value. Retinoic acid
production following retinol administration was reduced 4.8-fold in
Adh1
/
mice and 8.5-fold in Adh4
/
mice. Thus, Adh1 and Adh4 demonstrate overlapping functions in ethanol and retinol metabolism in
vivo, whereas Adh3 plays no role with these
substrates but instead functions in formaldehyde metabolism. Redundant
roles for Adh1 and Adh4 in retinoic acid
production may explain the apparent normal development of mutant mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
clone containing the region spanning from exon 4 to the 3'-flanking region. A
ScaI-EcoRV DNA fragment of 2.2 kb containing the
3'-flanking region of Adh1 was blunt end cloned into the
XbaI site of plasmid pTK-neo, which lies between the PGK-neo
and PGK-tk gene cassettes (26). A 4.2-kb HindIII fragment
containing exon 6 was subcloned into pBluescript II KS, excised with
XhoI and NotI, and inserted between
XhoI-NotI located downstream of PGK-neo to
produce the Adh1 gene-targeting vector.
2-2) for mouse Adh3 containing
the 5'-flanking region and exons 1-6 has previously been described (27). A 4.2-kb SalI-XbaI fragment containing the
5'-flanking region was liberated from
2-2 and inserted into the
SalI-XbaI sites of pTK-neo located between
PGK-neo and PGK-tk. A 3.7-kb XhoI-NotI fragment
containing exons 5 and 6 was liberated from
2-2 and inserted between
XhoI-NotI located downstream of PGK-neo to
produce the Adh3 gene-targeting vector. The
Adh4-targeting vector has been previously described
(28).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Adh1 gene targeting. A, the
wild-type Adh1 gene contains nine exons. Gene targeting with
the replacement vector shown creates a mutant Adh1 locus in
which exons 7-9 have been deleted. B, Southern blot of
Adh1-targeted embryonic stem cells using BamHI
digestion shows a heterozygous (+/ ) Adh1 mutant cell line
carrying the mutant 4.1-kb BamHI fragment. C,
Northern blot analysis of 10 µg of RNA derived from the liver
(Liv), kidney (Kid), and small intestine
(S.I.) of homozygous Adh1
/
mutant mice
(
) and wild-type mice (+) indicates a total
lack of Adh1 mRNA in the mutant; sample integrity is
shown by intact rRNA species in both mutant and wild-type samples.
D, Western blotting of 20 µg of total protein from
epididymis (Epi), kidney (Kid), and liver
(Liv) homogenates demonstrates a lack of ADH1 protein in
homozygous Adh1
/
mutant mice (
) compared
with wild-type mice (+).
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Fig. 2.
Adh3 gene targeting. A, gene
replacement targeting of Adh3 creates a mutant locus in
which exons 1-4 have been deleted. B, Southern blot of
Adh3-targeted mice using BamHI digestion shows
both heterozygous (+/ ) and homozygous (
/
) Adh3 mutant
individuals carrying the mutant 6.9-kb BamHI fragment.
C, Northern blot analysis of 10 µg of RNA derived from
liver (Liv), kidney (Kid), and epididymis
(Epi) of homozygous Adh3
/
mutant mice
(
) and wild-type mice (+) indicates a total
lack of Adh3 mRNA in the mutant; sample integrity is
shown by intact rRNA species. D, Western blotting of 20 µg
of total protein from homogenates of epididymis (Epi), liver
(Liv), testis (Tes), stomach (Sto),
and kidney (Kid) demonstrates a total lack of ADH3 protein
in homozygous Adh3
/
mutant mice (
) compared
with wild-type mice (+).
mutant mice
resulted in each case in the production of homozygous Adh
/
mutant mice in the following ratios: Adh1, +/+ = 33%, +/
= 57%,
/
= 10% (from 21 total progeny); Adh3, +/+ = 22%, +/
= 45%,
/
= 33% (from 27 total progeny);
Adh4, +/+ = 29%, +/
= 50%,
/
= 21% (from 90 total
progeny). All homozygous and heterozygous mutant mice appeared to
develop normally compared with wild-type +/+ mice. Adh1
/
, Adh3
/
, and Adh4
/
mice of both
sexes were fertile, and when mated to produce litters of second
generation
/
mice, all gave rise to normal-sized litters (7.7-9.7
weaned mice/litter) that appeared to develop normally.
/
mice had
no detectable Adh1 mRNA in liver, kidney, or small
intestine (Fig. 1C). Adh3
/
mice had no
detectable Adh3 mRNA in liver, kidney, or epididymis, whereas wild-type mice had easily detectable Adh3 mRNA
in all these tissues (Fig. 2C). Western blot analyses of
tissues that normally contain ADH1 or ADH3 in wild-type mice
demonstrated that Adh1
/
mutants lack detectable ADH1
protein in epididymis, kidney, and liver (Fig. 1D) and that
Adh3
/
mutants lack ADH3 protein in epididymis, liver,
testis, stomach, and kidney (Fig. 2D). A null phenotype for
Adh4
/
mice (i.e. no detectable
Adh4 mRNA or protein in either stomach or skin) has been
previously described (28). Western blot studies also demonstrated that
each Adh mutation had no effect on the expression of the
other two Adh genes examined here (28) (data not shown).
/
lines compared with the +/+ wild-type mice generated during the
heterozygous matings.
/
mice have a severe defect in clearing ethanol,
whereas Adh4
/
mice demonstrated a noticeable but less
severe defect. 360 min after ethanol administration blood ethanol
concentrations in Adh1
/
mice were approximately 0.30%
(w/v) with Adh4
/
mice having levels of about 0.15%,
whereas wild-type and Adh3
/
mice both had levels near
the lower limit of detection at 0.03% (Fig.
3). Thus, it can be concluded that
Adh3 plays no role in blood ethanol clearance, whereas both
Adh1 and Adh4 do with Adh1 playing a
much more significant role.
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Fig. 3.
Blood ethanol clearance in Adh
null mutants compared with wild-type mice following an ethanol
dose of 3.5 g/kg. Five mice of each genotype were tested.
Asterisks indicate significant differences between wild-type
mice and either Adh1 /
or Adh4
/
mice. *,
p < 0.0001; **, p = 0.001 (two-way
ANOVA).
/
mice (85 ± 10 min) as compared with
wild-type (45 ± 10 min), Adh3
/
(45 ± 3 min), or Adh4
/
(50 ± 5 min) mice, which had
similar LORR values (Fig. 4). The
significantly increased length of ethanol-induced sleep in
Adh1
/
mice correlates with their greatly decreased
blood ethanol clearance relative to the other Adh genotypes.
The reduced blood ethanol clearance noticed in Adh4
/
mice may be responsible for the slight increase in the duration of
LORR, but this effect was not statistically significant. The 1.9-fold
increase in LORR that we notice in Adh1
/
mice relative
to wild-type mice is similar to the 1.8-fold increase in LORR observed
in the Adh1 deficient deer mouse (138 ± 9 min)
relative to the wild-type deer mouse (76 ± 4 min); these deficient deer mice also have reduced blood ethanol clearance (32).
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Fig. 4.
Effect of Adh1,
Adh3, and Adh4 null mutations on LORR
following ethanol intoxication. Sensitivity was evaluated by
measuring the duration of LORR following administration of ethanol at a
dose of 3.5 g/kg. The asterisk indicates a significant
difference between Adh1 /
and wild-type mice. *,
p = 0.0085 (unpaired Student's t test). The
number of animals tested (n) is indicated below each
column.
/
mice rose to 30% (p = 0.003), with
ethanol-treated Adh4
/
mice exhibiting 18% resorption
(p = 0.21) and with ethanol-treated Adh3
/
mice plus wild-type mice having similar resorption rates to control
animals (8-10%) (Fig. 5). These results
show that the Adh1
/
genotype correlates more strongly
than the Adh4
/
genotype with ethanol damage and that
the Adh3
/
genotype does not lead to increased ethanol
damage.
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Fig. 5.
Ethanol-induced embryonic resorption as a
function of Adh genotype. On E8.75 of gestation,
pregnant mice were treated with 3.5 g/kg ethanol (6-8 dams and 40-76
implantations for each genotype) or saline control (2-3 dams and
10-33 implantations for each genotype). Dams were dissected on E12.5,
and each implantation was categorized as either a resorption or an
intact embryo. The asterisk indicates a significant
difference between the resorption rate of Adh1 /
and
wild-type mice. *, p = 0.003 (Fisher's exact test). A
small difference was noted between Adh4
/
and wild-type
mice, but this was not statistically significant (p = 0.21 by Fisher's exact test).
/
mice the LD50 for
formaldehyde was 0.135 g/kg, significantly less than the
LD50 for wild-type (0.200 g/kg) (Fig.
6). The LD50 values for both
Adh1
/
mice (0.175 g/kg) and Adh4
/
mice
(0.190 g/kg) were not significantly different from that of wild-type. Thus, our findings support a role for mammalian ADH3 in the clearance of formaldehyde, a role which has apparently been conserved in most if
not all organisms including microorganisms (39), plants (40), and
invertebrate animals (41).
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Fig. 6.
Formaldehyde toxicity in Adh
null mutants compared with wild-type mice. Mice were
challenged with five doses of formaldehyde ranging from 0.09 to 0.22 g/kg (31-73 individuals from each genotype) and monitored for death
within 90 min. Plots shown are linear regression analyses of the
results.
/
mice (28). Here, mice of each Adh genotype were treated with retinol (100 µg/kg) or vehicle control, and 2 h
later kidney retinoic acid levels were monitored using the bioassay. Whereas vehicle-treated mice of each genotype had undetectable levels
of retinoic acid in kidney tissue explants examined using a qualitative
version of the bioassay, retinol-treated wild-type and Adh3
/
mice exhibited comparably high levels of kidney retinoic acid;
retinol-treated Adh1
/
and Adh4
/
mice
had significantly lower levels of kidney retinoic acid than wild-type
mice (data not shown). Retinoic acid quantitation was performed on
kidney homogenates from treated Adh1
/
, Adh4
/
, and wild-type mice using a spectrophotometric variation of the
bioassay (20). Kidney retinoic acid levels in all vehicle-treated mice
were below the limit of detection (<1 pmol/g). Retinol treatment
resulted in kidney retinoic acid levels of 273 ± 186 pmol/g for
wild-type mice but only 57 ± 15 pmol/g for Adh1
/
mice and 34 ± 16 pmol/g for Adh4
/
mice (Fig.
7). Thus, Adh1
/
and
Adh4
/
mutations lead to reductions of 4.8- and
8.5-fold, respectively, in the ability to metabolize retinol to
retinoic acid. Our finding of a larger role for Adh4 than
Adh1 in the metabolism of retinol is consistent with the
higher in vitro catalytic activity of ADH4 as a retinol
dehydrogenase relative to ADH1 (4-6).
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Fig. 7.
Effect of Adh1 and
Adh4 null mutations on metabolism of retinol to
retinoic acid. Two h after administration of retinol (100 µg/kg)
or vehicle control, animals were sacrificed and retinoic acid levels
were measured in kidney homogenates using the retinoic acid bioassay.
The number of mice (n) treated for each Adh
genotype is shown in parentheses. Values are represented as
the mean ± S.E.; for all vehicle-treated mice the level was below
the limit of detection for the bioassay (<1 pmol/g).
Asterisks indicate significant differences in retinoic acid
levels between wild-type mice and either Adh1 /
mice or
Adh4
/
mice when treated with retinol (*,
p = 0.028; **, p = 0.009; unpaired
Student's t test).
/
mice have an increased risk
of embryonic lethality during vitamin A starvation, suggesting that
ADH4 may facilitate retinol utilization when levels are low (28). Thus,
ADH4 is likely to participate in retinol utilization for normal growth and development.
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ACKNOWLEDGEMENTS |
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We thank M. Wagner for the F9-RARE-lacZ reporter cell line, D. Abbe and W. Charbono for help with mouse dosing and retrieval of blood samples, R. J. Haselbeck for antibodies against mouse ADH1, ADH3, and ADH4, and the Burnham Institute Mouse Genetics Facility.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AA09731 (to G. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Institut Cochin de Genetique Moleculaire,
Institut National de la Sante et de la Recherche Medicale U257, 24 rue
du Faubourg Saint Jacques, 75014 Paris, France.
§ To whom correspondence should be addressed: Gene Regulation Program, Burnham Inst., 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3138; Fax: 619-646-3195; E-mail: duester{at}burnham-inst.org.
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ABBREVIATIONS |
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The abbreviations used are:
ADH, alcohol
dehydrogenase;
kb, kilobase(s);
E, embryonic day;
LORR, loss of
righting response;
Adh1, mouse class I ADH gene;
Adh3, mouse class III ADH gene;
Adh4, mouse class
IV ADH gene;
ADH1B, human class I ADH gene;
tk, thymidine kinase.
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REFERENCES |
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