Chronic paternal alcohol exposures induce dose-dependent changes in offspring craniofacial shape and symmetry

Abstract

Although dose-response analyses are a fundamental tool in developmental toxicology, few studies have examined the impacts of toxicant dose on the non-genetic paternal inheritance of offspring disease and dysgenesis. In this study, we used geometric morphometric analyses to examine the impacts of different levels of preconception paternal alcohol exposure on offspring craniofacial shape and symmetry in a mouse model. Procrustes ANOVA followed by canonical variant analysis of geometric facial relationships revealed that Low-, Medium-, and High-dose treatments each induced distinct changes in craniofacial shape and symmetry. Our analyses identified a dose threshold between 1.543 and 2.321 g/kg/day. Below this threshold, preconception paternal alcohol exposure induced changes in facial shape, including a right shift in facial features. In contrast, above this threshold, paternal exposures caused shifts in both shape and center, disrupting facial symmetry. Consistent with previous clinical studies, changes in craniofacial shape predominantly mapped to regions in the lower portion of the face, including the mandible (lower jaw) and maxilla (upper jaw). Notably, high-dose exposures also impacted the positioning of the right eye. Our studies reveal that paternal alcohol use may be an unrecognized factor contributing to the incidence and severity of alcohol-related craniofacial defects, complicating diagnostics of fetal alcohol spectrum disorders.

Keywords: craniofacial dysgenesis; dose-response; epigenetics; geometric morphometric; paternal epigenetic inheritance; toxicology.

FIGURE 1

Average daily ethanol dose for males in each treatment group. We exposed males to different ethanol treatments for 6 weeks (Thomas et al., 2022), then determined the average daily dose of the males that sired the offspring we examined in this study as grams per kilogram per day (g/kg/day), consistent with clinical studies (Leeman et al., 2010). We used a one-way ANOVA to contrast differences between treatments; error bars represent the standard area of the mean, n = Low 8, Medium 4, and High 4.

FIGURE 2

Preconception paternal alcohol exposures induce dose-dependent changes in offspring craniofacial shape and symmetry. (A) We used geometric morphometrics, followed by canonical variate analysis to compare alcohol-induced shifts in the facial shape of the gestational day 16.5 male offspring sired by Low- (3%), Medium- (6%), and High-dose (10%) males. Canonical variant one accounted for 67.3%, and variant two accounted for 20.9% of the observed variance. (B) Lollipop diagrams reporting relative shifts in facial landmarks (compared to Controls) induced by preconception paternal alcohol exposures between offspring derived from Low- (top), Medium- (middle), and High-dose (bottom) sires. (C) Wire diagrams comparing the shift in facial landmarks between the offspring of Low- (top), Medium- (middle), and High-dose (bottom) males. The red lines demarcate the alcohol-induced change, while the grey lines demarcate the average of the Control population. Note that representative pictures of the fetal face do not perfectly align with the population average, particularly in the High-dose treatments, where we observed wide variation in facial shape. (D) A selection of craniofacial images shows the observed dysmorphology range across treatment groups. We used digital images from 24 Control, 43 Low-, 17 Medium-, and 14 High-dose male fetuses, sampled from at least four litters.