Fig 1.
Structures of ethanol and t-butyl alcohol.
Table 1.
Effects of t-BuOH treatment on embryos at E10.0.
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.
Table 2.
Frequency of HPE defects in t-BuOH-treated mice at E14.
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).
Table 3.
Frequency of HPE defects in EtOH- plus antioxidant-treated mice at E14.
Table 4.
Frequency of HPE defects in t-BuOH- plus antioxidant-treated mice at E14.
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.