Ethanol itself is a holoprosencephaly-inducing teratogen

Abstract

Ethanol is a teratogen, inducing a variety of structural defects in developing humans and animals that are exposed in utero. Mechanisms of ethanol teratogenicity in specific defects are not well understood. Oxidative metabolism of ethanol by alcohol dehydrogenase or cytochrome P450 2E1 has been implicated in some of ethanol's teratogenic effects, either via production of acetaldehyde or competitive inhibition of retinoic acid synthesis. Generalized oxidative stress in response to ethanol may also play a role in its teratogenicity. Among the developmental defects that ethanol has been implicated in is holoprosencephaly, a failure to define the midline of the forebrain and midface that is associated with a deficiency in Sonic hedgehog pathway function. Etiologically, holoprosencephaly is thought to arise from a complex combination of genetic and environmental factors. We have developed a gene-environment interaction model of holoprosencephaly in mice, in which mutation of the Sonic hedgehog coreceptor, Cdon, synergizes with transient in utero exposure to ethanol. This system was used to address whether oxidative metabolism is required for ethanol's teratogenic activity in holoprosencephaly. We report here that t-butyl alcohol, which is neither a substrate nor an inhibitor of alcohol dehydrogenases or Cyp2E1, is a potent inducer of holoprosencephaly in Cdon mutant mice. Additionally, antioxidant treatment did not prevent ethanol- or t-butyl alcohol-induced HPE in these mice. These findings are consistent with the conclusion that ethanol itself, rather than a consequence of its metabolism, is a holoprosencephaly-inducing teratogen.

Fig 1. Structures of ethanol and t-butyl alcohol.

Fig 2. t-BuOH induces HPE in Cdon mutant mice.

(A-D) Frontal views of E14 embryos. t-BuOH-treated Cdon^-/- embryos (D) developed strong facial features of HPE, including single nostril (arrow). (E-P) H&E stained coronal sections of E14 embryos. Midfacial and forebrain midline structures were missing or reduced in t-BuOH-treated Cdon^-/- embryos, including cartilage primordium of the nasal septum (H, arrow); nasal septum (L, black arrow); vomeronasal organ (L, red arrow); defective palatal shelves (L, arrowheads) flanking midline cleft; and ventral diencephalon midline structure (P, arrow).

Fig 3. t-BuOH induces eye defects in Cdon mutant mice.

t-BuOH-treated Cdon^-/- mice displayed micropthalamia and/or ventral coloboma (arrow), whereas t-BuOH-treated Cdon^+/- mice and saline-treated mice of either genotype did not.

Fig 4. Reduced expression of Shh and Nkx2.1 in forebrains of t-BuOH-treated Cdon^-/- mice.

Whole mount in situ hybridization analysis of Shh (A-D) and Nkx2.1 (E-H) expression in E10.25 embryos of the indicated genotype and treatment. Expression of Shh and Nkx2.1 were specifically reduced in the rostroventral forebrain of t-BuOH-treated Cdon^-/- embryos (D and H, arrows). Three of four t-BuOH-treated Cdon^-/- embryos had diminished Shh expression and four of five embryos had reduced Nxk2.1 expression.

Fig 5. Failure of antioxidant treatment to rescue EtOH- or t-BuOH-induced HPE in Cdon^-/- mice.

(A-E) Frontal views of E14 embryos. EtOH- and t-BuOH-treated Cdon^-/- embryos (B and D, respectively) developed strong facial features of HPE, including single nostril and smooth, pointed philtrum (arrows). These phenotypes were not rescued by treatment with N-acetylcysteine plus α-tocopherol (NAC/TCP) (C, E).

Fig 6. ROS/RNS levels in livers of mice from various treatment groups.

ROS/RNS levels were measured by production of the fluorescent compound, DCF. EtOH and t-BuOH both increased ROS/RNS levels, relative to the saline control. N-acetylcysteine plus α-tocopherol (NAC+TCP) treatment normalized ROS/RNS levels in livers after EtOH exposure, but not after t-BuOH exposure. *p<0.05 with two-tailed Fisher’s exact test; n.s., not significant; values are means ± SD, n = 3–4 mice per point.

Fig 7. Reduced glutathione levels in livers of mice from various treatment groups.

(A, B) Reduced glutathione (GSH) levels were analyzed 12 hours after EtOH (A) or t-BuOH (B) exposure. EtOH and t-BuOH both decreased GSH levels, relative to the saline control. N-acetylcysteine plus α-tocopherol (NAC+TCP) treatment partially rescued GSH levels in livers after EtOH exposure (A), but not after t-BuOH exposure (B). *p<0.05 with two-tailed Fisher’s exact test; n.s., not significant; values are means ± SD, n = 3–4 mice per point.