8 Fetal Alcohol Spectrum Disorder: Embryogenesis Under …
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of this enzyme for normal vertebrate embryonic development (Niederreither et al.
1999; Begemann et al. 2001; Grandel et al. 2002; Niederreither and Dollé 2008;
Rhinn and Dollé 2012).
To directly address the involvement of RALDH2 as a target for the ethanol
inhibitory effect, a series of combined treatments were performed in Xenopus
embryos (Kot-Leibovich and Fainsod 2009; Shabtai et al. 2018) Partially blocking the endogenous RALDH activity with DEAB in parallel to ethanol exposure
led to the induction of developmental malformations and abnormal gene expression
profiles that were more severe than those observed after each individual treatment
(Kot-Leibovich and Fainsod 2009). However, although these results supported a link
between the ethanol effect and RALDH activity, they did not rule out the possibility
that both could still be functioning in parallel.
A more direct approach, showed that supplementation of RALDH activity reduces
the teratogenicity of ethanol or its oxidation product, acetaldehyde. Embryos treated
with ethanol and manipulated to overexpress RALDH2 exhibited milder developmental malformations when compared to the single treatments. Moreover, gene
expression levels and the activity of the RA reporter plasmid returned to almost normal levels (Kot-Leibovich and Fainsod 2009). Similar results were obtained when
instead of ethanol, acetaldehyde was used to induce teratogenic effects (Shabtai et al.
2018). Direct involvement of RALDH2 in the ethanol-promoted competition with
RA biosynthesis was demonstrated by knock-down of the aldh1A2 gene with antisense morpholino oligonucleotides (Kot-Leibovich and Fainsod 2009). As expected,
loss of RALDH2 activity worsened the effects of alcohol exposure.
Evidence for the sequestering of the RALDH2 activity by acetaldehyde was
obtained by exposing embryos to this ethanol oxidation intermediate (Shabtai et al.
2016, 2018). Treatment of embryos with acetaldehyde concentrations common in
serum of intoxicated individuals (Tsukamoto et al. 1989; Halvorson et al. 1993;
Brecher et al. 1997) recapitulated the ethanol-induced abnormal gene expression
patterns and developmental malformations that affect embryo size, microcephaly
and microphthalmia (Shabtai et al. 2018). Importantly, combined treatments of
acetaldehyde with retinaldehyde or RALDH2 rescued the expression levels of known
RA-regulated genes. These observations supported the role of acetaldehyde as the
teratogenic derivative of ethanol.
Additional conclusive proof that acetaldehyde is the teratogenic component in
the induction of FASD as a competitor and inhibitor of RA production was obtained
by enzymatic kinetic analysis. Acetaldehyde was studied as a substrate of human
RALDH2 (Shabtai et al. 2018). This study identified acetaldehyde as an efficient
substrate of hRALDH2 with a Km of 8.47 μM. In a previous study, the Km for alltrans retinaldehyde was determined to be about 16 μM (Shabtai et al. 2016). The Km
values for both substrates with similar Vmax values suggested that the enzymatic efficiency of hRALDH2 for oxidation of acetaldehyde is higher than for retinaldehyde.
To formally demonstrate the competition between acetaldehyde and retinaldehyde,
HPLC analysis of hRALDH2 oxidation reactions containing both substrates were
performed (Shabtai et al. 2018). HPLC analysis allowed the direct measurement of
the utilization of the all-trans retinaldehyde substrate and the production of RA in
209
of this enzyme for normal vertebrate embryonic development (Niederreither et al.
1999; Begemann et al. 2001; Grandel et al. 2002; Niederreither and Dollé 2008;
Rhinn and Dollé 2012).
To directly address the involvement of RALDH2 as a target for the ethanol
inhibitory effect, a series of combined treatments were performed in Xenopus
embryos (Kot-Leibovich and Fainsod 2009; Shabtai et al. 2018) Partially blocking the endogenous RALDH activity with DEAB in parallel to ethanol exposure
led to the induction of developmental malformations and abnormal gene expression
profiles that were more severe than those observed after each individual treatment
(Kot-Leibovich and Fainsod 2009). However, although these results supported a link
between the ethanol effect and RALDH activity, they did not rule out the possibility
that both could still be functioning in parallel.
A more direct approach, showed that supplementation of RALDH activity reduces
the teratogenicity of ethanol or its oxidation product, acetaldehyde. Embryos treated
with ethanol and manipulated to overexpress RALDH2 exhibited milder developmental malformations when compared to the single treatments. Moreover, gene
expression levels and the activity of the RA reporter plasmid returned to almost normal levels (Kot-Leibovich and Fainsod 2009). Similar results were obtained when
instead of ethanol, acetaldehyde was used to induce teratogenic effects (Shabtai et al.
2018). Direct involvement of RALDH2 in the ethanol-promoted competition with
RA biosynthesis was demonstrated by knock-down of the aldh1A2 gene with antisense morpholino oligonucleotides (Kot-Leibovich and Fainsod 2009). As expected,
loss of RALDH2 activity worsened the effects of alcohol exposure.
Evidence for the sequestering of the RALDH2 activity by acetaldehyde was
obtained by exposing embryos to this ethanol oxidation intermediate (Shabtai et al.
2016, 2018). Treatment of embryos with acetaldehyde concentrations common in
serum of intoxicated individuals (Tsukamoto et al. 1989; Halvorson et al. 1993;
Brecher et al. 1997) recapitulated the ethanol-induced abnormal gene expression
patterns and developmental malformations that affect embryo size, microcephaly
and microphthalmia (Shabtai et al. 2018). Importantly, combined treatments of
acetaldehyde with retinaldehyde or RALDH2 rescued the expression levels of known
RA-regulated genes. These observations supported the role of acetaldehyde as the
teratogenic derivative of ethanol.
Additional conclusive proof that acetaldehyde is the teratogenic component in
the induction of FASD as a competitor and inhibitor of RA production was obtained
by enzymatic kinetic analysis. Acetaldehyde was studied as a substrate of human
RALDH2 (Shabtai et al. 2018). This study identified acetaldehyde as an efficient
substrate of hRALDH2 with a Km of 8.47 μM. In a previous study, the Km for alltrans retinaldehyde was determined to be about 16 μM (Shabtai et al. 2016). The Km
values for both substrates with similar Vmax values suggested that the enzymatic efficiency of hRALDH2 for oxidation of acetaldehyde is higher than for retinaldehyde.
To formally demonstrate the competition between acetaldehyde and retinaldehyde,
HPLC analysis of hRALDH2 oxidation reactions containing both substrates were
performed (Shabtai et al. 2018). HPLC analysis allowed the direct measurement of
the utilization of the all-trans retinaldehyde substrate and the production of RA in