Preconception Alcohol Increases Offspring Vulnerability to Stress

Abstract

The effect of preconception drinking by the mother on the life-long health outcomes of her children is not known, and therefore, in this study using an animal model, we determined the impact of preconception alcohol drinking of the mother on offspring stress response during adulthood. In our preconception alcohol exposure model, adult female rats were fed with 6.7% alcohol in their diet for 4 weeks, went without alcohol for 3 weeks and were bred to generate male and female offspring. Preconception alcohol-exposed offsprings' birth weight, body growth, stress response, anxiety-like behaviors, and changes in stress regulatory gene and protein hormone levels were evaluated. In addition, roles of epigenetic mechanisms in preconception alcohol effects were determined. Alcohol feeding three weeks prior to conception significantly affected pregnancy outcomes of female rats, with respect to delivery period and birth weight of offspring, without affecting maternal care behaviors. Preconception alcohol negatively affected offspring adult health, producing an increased stress hormone response to an immune challenge. In addition, preconception alcohol was associated with changes in expression and methylation profiles of stress regulatory genes in various brain areas. These changes in stress regulatory genes were normalized following treatment with a DNA methylation blocker during the postnatal period. These data highlight the novel possibility that preconception alcohol affects the inheritance of stress-related diseases possibly by epigenetic mechanisms.

Figure 1

Effects of preconception alcohol feeding on pregnancy outcomes. Physiological parameters and pregnancy outcomes of preconception alcohol feed rats are shown. Time-dependent changes in blood concentrations of ethanol in female rats who had free access to alcohol in liquid diet for a 4-week period and were used in preconception alcohol feeding study (a). Changes in body weight before and two weeks after ethanol withdrawal (b). Estrous cycle patterns two weeks after ethanol withdrawal (c). Pregnancy outcome of the alcohol-fed rats (AF) were compared with those of pair-fed (PF) and ad lib-fed (AD) control rats by pregnancy days (d), total pups/litter count (e), % male or female pups/litter count (f and g), and birth body weight of male or female offspring pups (h and i). Data are mean±SEM (n=7) and were compared by one-way analysis of variance (ANOVA) and Newman-Keuls post-test. *p<0.05, **p<0.01, ***p<0.001, AF vs PF and AD. The body growth curves of male offspring (AF-M, PF-M, and AD-M) or female offspring (AF-F, PF-F, and AD-F) of dams fed with different diets (AF, PF, and AD) were shown in panels j and k, respectively. Data were analyzed by two-way ANOVA (treatment × time) followed by the Bonferroni post-test. A significant time × treatment effect on body growth was noted for both male (p<0.001) and female offspring (p<0.001).

Figure 2

Consequences of preconception alcohol feeding on stress axis responses in adult male and female offspring. The stress axis function tests include measurement of plasma levels of corticosterone (a, b) and ACTH (c, d) measured before (0 time) and 2 h after LPS injection and the basal hypothalamic protein level of CRF (e, f), β-endorphin (g, h), and AVP (i, j) in adult male offspring (AF-M, PF-M, AD-M) or female offspring (AF-F, PF-F, AD-F) of dams fed with different diets (AF, PF and AD). Data are mean±SEM (n=6). Data were analyzed using one-way ANOVA followed by the Newman–Keuls post-test. *p<0.05, **p<0.01,***p<0.001, AF (both M and F) vs AD and PF (both M and F) groups. (a) P<0.001, within treatment group-corticosterone levels at 0 time vs 2 h post LPS.

Figure 3

Consequences of preconception alcohol feeding on anxiety-like behaviors in adult male and female offspring. Adult AF-M rats spent less time in the center of the OF compared with AD-M offspring (a; *p<0.05) indicating increased anxiety-like behavior in AF-M offspring. AF-F rats did not differ from PF-F and AD-F in time spent in the center zone (b; p>0.05). AF-M also showed significantly more anxiety-like behavior in the EPM, since they spent significantly less time in the open arms compared with AD-M (c) and made less entries into the open arms compared with AD-M (d). In males, there were no significant differences between groups in total arm entries (e). There were no significant differences between AF-F, PF-F, and AD-F females in anxiety-like behavior in the EPM (f, g, h). Data are mean±SEM (n=7–14; AD-M=9, PFM=7; AFM=14) and were analyzed using one-way ANOVA and Newman–Keuls post-test. *p<0.05, **p<0.01, compared with the AD group.

Figure 4

Effects of preconception alcohol drinking on expression and methylation of stress regulatory genes in various brain areas. Adult AF-M rats showed tissue specific changes in mRNA expression, as determined by q-RT-PCR, of Crf (a, b), Crfr1 (c–f) and Pomc (g, h) in the hypothalamus, hippocampus, and/or amygdala. AF-M rats had increased Crf mRNA compared with PF-M and AD-M rats in the hypothalamus (a, b). In addition, AF-M rats had increased Crfr1 mRNA compared with PF-M and AD-M rats in the hippocampus (c, d) and amygdala (e, f). Pomc mRNA levels in the hypothalamus were lower in AF-M compared with AD-M and PF-M offspring (g, h). AF-M rats also showed differential methylation patterns of Crf (i), Crfr1 in hippocampus (j) and amygdala (k) and Pomc (l, m) genes in hypothalamus. AF-M rats had suppressed CRF methylation in the hypothalamus (i) of the CpG dinucleotides at −232 in the proximal promoter as determined by pyrosequencing methods. AF-M rats also showed a reduction in Crfr1 methylation in the hippocampus (j) and amygdala (k) as determined by MSPR assay. In contrast, the methylation of the POMC gene was increased in the hypothalamus of AF-M as compared with AD-M and PF-M (l, m) as determined by MSPR assay and or pyrosequencing assay. Data are mean±SEM (n=7, except n=12 in AF group shown in i) and were compared by a one-way ANOVA followed by the Newman–Keuls post-test. *p<0.05, **p<0.01, ***p<0.001, group with this symbol vs AF-M.

Figure 5

Suppression of DNA methylation prevents preconception alcohol effect on stress response, anxiety behaviors and stress regulatory gene expression. Treatment of AZA prevents the stress response (a, b, c, d), anxiety behaviors measured in OF (e) and in EPM (f, g, h), and the regulatory genes expressions (i, j, m, n, q, r), and methylation (k, l, o, p, s) in AF-M or AF-F rats. Data are mean±SEM (n=8, except n=18 in all groups shown in f-h) and were analyzed by two-way ANOVA (alcohol × drug treatment) followed by Bonferroni post-test comparisons. *p<0.05, **p<0.01, ***p<0.001, compared with saline-treated but differentially fed offspring for the gene measurements. *p<0.05, compared with saline-treated AFM for the gene measurements.