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A. Fainsod et al.
experimentally supported by improvement of the patients’ night vision after dietary
vitamin A supplementation (Patek and Haig 1939; Newman and Fletcher 1941).
The inhibitory effect of ethanol on RA production was demonstrated in a number
of vertebrate experimental embryo models, including mice, fish and frogs. Using
cultured mouse embryos placed on top of monolayers of a RA reporter cell line, it was
shown that ethanol reduces RA levels at early stages of development (E7.5) (Deltour
et al. 1996). In addition, in a Xenopus embryo experimental system, expression of
known RA-regulated genes like HoxA2, HoxB3, HoxB4 and HoxB9 was reduced by
ethanol exposure (Yelin et al. 2005), supporting the hypothesis that ethanol reduced
RA levels required for normal Hox activation (Carron and Shi 2016; Parker and
Krumlauf 2017).
Additional experiments reinforced the hypothesis that ethanol partially inhibits
the production of RA. In the first set of results, Xenopus embryos were treated with
3,7-dimethyl-2,6-octadienal (citral), an RA biosynthesis inhibitor (Kikonyogo et al.
1999). The citral-mediated inhibition of RA biosynthesis recapitulated gene expression changes induced by ethanol exposure (Yelin et al. 2005). Second, reduced RA
levels obtained upon overexpression of the RA hydroxylase, Cyp26A1 (Marikar
et al. 1998; Sonneveld et al. 1998), also recapitulated the changes in gene expression induced by ethanol (Yelin et al. 2005). Third, when embryos were injected with
a RA reporter plasmid (Rossant et al. 1991) and treated with ethanol, the alcohol
exposure reduced the expression of the reporter below background levels through
inhibition of endogenous RA biosynthesis (Yelin et al. 2005). In a fourth set of experiments, Xenopus embryos were treated with 4-diethylaminobenzaldehyde (DEAB),
another RA biosynthesis inhibitor (Russo et al. 1988). Treatment of embryos with
both ethanol and DEAB produced additive effects on the induction of developmental
malformations and changes in gene expression (Kot-Leibovich and Fainsod 2009).
In conclusion, these combined molecular, pharmacological and enzymatic
approaches provided extensive support for the hypothesis that ethanol reduces RA
signaling levels.
Does Ethanol Compete with Retinol
Over the years, a number of rescue experiments have been performed to demonstrate a competitive effect of ethanol on RA biosynthesis. The rescue experiments
were usually designed on the premise that if ethanol or its metabolites compete
with the RA precursors for an enzymatic activity present in limiting amounts, then,
by increasing the amount of the RA precursor, the competition could be shifted in
favor of RA production. However, several considerations dictate the relevance of
results obtained from rescue experiments. Retinoid concentrations need to be carefully titrated, since in excess, they can induce teratogenic effects that resemble the
malformations induced by ethanol. Also, the retinoid employed determines the conclusions that can be drawn from the experiment. For instance, RA, the biologically
active product of vitamin A, should always have a rescuing effect if it is to provide
A. Fainsod et al.
experimentally supported by improvement of the patients’ night vision after dietary
vitamin A supplementation (Patek and Haig 1939; Newman and Fletcher 1941).
The inhibitory effect of ethanol on RA production was demonstrated in a number
of vertebrate experimental embryo models, including mice, fish and frogs. Using
cultured mouse embryos placed on top of monolayers of a RA reporter cell line, it was
shown that ethanol reduces RA levels at early stages of development (E7.5) (Deltour
et al. 1996). In addition, in a Xenopus embryo experimental system, expression of
known RA-regulated genes like HoxA2, HoxB3, HoxB4 and HoxB9 was reduced by
ethanol exposure (Yelin et al. 2005), supporting the hypothesis that ethanol reduced
RA levels required for normal Hox activation (Carron and Shi 2016; Parker and
Krumlauf 2017).
Additional experiments reinforced the hypothesis that ethanol partially inhibits
the production of RA. In the first set of results, Xenopus embryos were treated with
3,7-dimethyl-2,6-octadienal (citral), an RA biosynthesis inhibitor (Kikonyogo et al.
1999). The citral-mediated inhibition of RA biosynthesis recapitulated gene expression changes induced by ethanol exposure (Yelin et al. 2005). Second, reduced RA
levels obtained upon overexpression of the RA hydroxylase, Cyp26A1 (Marikar
et al. 1998; Sonneveld et al. 1998), also recapitulated the changes in gene expression induced by ethanol (Yelin et al. 2005). Third, when embryos were injected with
a RA reporter plasmid (Rossant et al. 1991) and treated with ethanol, the alcohol
exposure reduced the expression of the reporter below background levels through
inhibition of endogenous RA biosynthesis (Yelin et al. 2005). In a fourth set of experiments, Xenopus embryos were treated with 4-diethylaminobenzaldehyde (DEAB),
another RA biosynthesis inhibitor (Russo et al. 1988). Treatment of embryos with
both ethanol and DEAB produced additive effects on the induction of developmental
malformations and changes in gene expression (Kot-Leibovich and Fainsod 2009).
In conclusion, these combined molecular, pharmacological and enzymatic
approaches provided extensive support for the hypothesis that ethanol reduces RA
signaling levels.
Does Ethanol Compete with Retinol
Over the years, a number of rescue experiments have been performed to demonstrate a competitive effect of ethanol on RA biosynthesis. The rescue experiments
were usually designed on the premise that if ethanol or its metabolites compete
with the RA precursors for an enzymatic activity present in limiting amounts, then,
by increasing the amount of the RA precursor, the competition could be shifted in
favor of RA production. However, several considerations dictate the relevance of
results obtained from rescue experiments. Retinoid concentrations need to be carefully titrated, since in excess, they can induce teratogenic effects that resemble the
malformations induced by ethanol. Also, the retinoid employed determines the conclusions that can be drawn from the experiment. For instance, RA, the biologically
active product of vitamin A, should always have a rescuing effect if it is to provide