Molecular and organismal changes in offspring of male mice treated with chemical stressors

Abstract

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

Fig. 1.

Diagram of the 45S rDNA gene in the mouse strain used in this study, showing SNP sites and the genotypes/haplotypes created by these SNPs. NTS = non-transcribed upstream spacer-promoter region. ETS = external transcribed sequences. ITS = internal transcribed sequences. Methylation at sites 19–23, representative of original 27 CpG dinucleotides in the spacer promoter (Shiao et al., 2011), was measured in the present study. SNPs at −2214 in the spacer-promoter and at −218, −178, and −104 in the main promoter are shown.

Fig. 2.

Effects of paternal treatments on methylation of rDNA in offspring tissues. A. Methylation at CpG sites in male offspring lung. Compared with UT-lineage, a, P = 0.012, b, P = 0.039, c, P = 0.037, d, P = 0.023, e, P = 0.042, all sites together, p = 0.028, after correction for variables (see Methods). B. Methylation level was higher in lung than in liver, and this difference was greater in Cr-lineage offspring, b, P = 0.0047, c, P = 0.0069 compared with UT-lineage, based on within-animal comparisons (see Methods). C. Methylation level was higher in sperm than in lung, and this difference was greater in Cr-lineage offspring, d, P < 0.0001, and in AS-lineage offspring, e, P = 0.0002, compared with UT-lineage. D. Methylation was higher in sperm than in liver, a difference that was not significantly affected by paternal treatments.

Fig. 3.

Effects of paternal treatments on relative percentages of rDNA genotypes. A. Overall averages of percent CGC genotype were lower in lungs of offspring of treated fathers. a, P = 0.0053; b, P =0.0074; c, P = 0.013; d, P = 0.056 compared with the UT-lineage. B. Within-animal comparison of percent genotypes in lung compared with liver confirm the effect of paternal treatments on percent CGC in lung, with reciprocal changes in percent CCC. e, P = 0.0004; f, P = 0.0035; g, P < 0.0001; h, P = 0.028, i, = 0.0079 compared with UT-lineage. C. Within-animal comparison of percent genotypes in lung compared with sperm revealed a tissue differential for effects of paternal treatments on all genotypes. j, P = 0.0058; k, P < 0.0001; l, P = 0.025; m, P = 0.0001; n, P = 0.0045; o, P = 0.0073 compared with UT-lineage. D. Within-animal comparison of percent genotypes in liver compared with sperm showed a tissue differential for effects of paternal treatments on CGC and ACC genotypes. p, P = 0.0080; q, P = 0.015; r, P = 0.0098; s, P = 0.031 compared with UT-lineage.

Fig. 4.

Relationships between rDNA methylation and genotypes and effects of paternal treatments as indicated by analysis of population regression slopes from random coefficient regression models in offspring tissues. A. Significant negative associations between % T genotype and % methylation are illustrated for meCpG 21 and female lung. Line slope was less negative for AS-lineage compared with Cr-lineage offspring (P value on graph). B. There was a strong negative correlation between % methylation and % CGC genotype (P<0.001) that was not affected by paternal treatments. Male and female offspring lung combined are illustrated. C. In UT-lineage lungs % methylation was not significantly associated with % ACC genotype (P = 0.86), but this relationship was significantly negative for both Cr-lineage (P = 0.0048) and AS-lineage (P < 0.0001) offspring lungs, and these slopes were different from the UT-lineage slope (P values on graph). D. In UT-lineage lungs % methylation was not significantly associated with % CCA genotype (P = 0.53), but this relationship was significantly positive for both Cr-lineage (P = 0.028) and AS-lineage (P=0.0002) lungs, and these slopes were different from the UT-lineage slope (P values on graph). E. In UT-lineage female embryos % methylation was not significantly associated with % CGC genotype (P = 0.26), but this relationship was significantly negative for both Cr-lineage (P = 0.0031) and AS-lineage (P = 0.0078) female embryos, and these slopes were different from the UT-lineage slope (P values on graph).

Fig. 5.

Effects of paternal treatments on organismal parameters. A. Reduction in serum glucose in offspring of treated fathers compared with UT-lineage. a, P = 0.0083; b, P = 0.0013; c, P = 0.018. B. Increase in serum leptin in AS-lineage females. d, P = 0.016 compared with Cr-lineage. C. Increased body weights in offspring of treated fathers compared with UT-lineage. e, P = 0.052; f, P = 0.0081; g, P = 0.097; h, P = 0.085. D. Increased liver weights in female Cr-lineage offspring compared with UT-lineage, i, P = 0.026. E. Increased liver/body weight ratios in female Cr-lineage offspring compared with UT-lineage, j, P = 0.039.

Fig. 6.

Effects of paternal treatments on correlations between serum Igf-1 and rDNA genotype and methylation profiles. A. The % T SNP in sperm was positively correlated with serum Igf-1 in the UT-lineage (P = 0.033) and negatively correlated in the Cr-lineage (P = 0.033); these slopes were significantly different (see P value on graph). B. The % CGC SNP in lungs of female offspring was significantly, positively correlated with serum Igf-1 in the AS-lineage (P = 0.012) but negatively (P = 0.15) correlated in the Cr-lineage; these slopes were significantly different (see P value on graph). C. The % meCpG 19 in female offspring lung of AS-lineage was significantly and negatively associated with serum Igf-1 (P = 0.0017), whereas the line slopes for the UT- and Cr-lineage tissues did not differ from zero. Slopes for the AS-lineage and Cr-lineage results were significantly different (see P value on graph). There were no other significant associations between organismal parameters and rDNA parameters (not shown).

Fig. 7.

Effects of direct i.p. treatments of male Swiss mice on serum parameters. Cr(III) caused an immediate, large increase in serum glucose by 1 hr (a, P <0.001), followed by significant decreases at 6 and 24 hrs (b, c, P<0.001). Both insulin and leptin were decreased 1 hr after AS (d, i, P <0.01), with a significant decrease in leptin at this time after Cr(III) (P<0.0001). At 6 hrs both insulin and leptin increased relative to controls after Cr(III) (e, P<0.001, j, P < 0.01), and a similar response was significant for insulin after AS (f, P < 0.05). Cr(III) treatment resulted in reduction in serum insulin at 24 hrs (g, P < 0.0001). Results are presented relative to the average of controls at each time point, because of the well-known diurnal and environment-dependent variability in baselines for these parameters. Raw values are given in Supplementary Table 4.