Pleiotropic effects of a methyl donor diet in a novel animal model

Abstract

Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation). Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (A(vy)). In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = A(Nb)) that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts) and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

Figure 1. Effects of methyl-donor diet on coat-color/pattern.

(A) Whole pelts and (B) corresponding hair tufts from representative six-month old female A^Nb methyl diet (#1) and control diet (#2) animals. Note the visible differences in yellow band length in hair tufts and size. (C) Distribution of yellow band lengths (in mm) in tufts of hair. T test was used to determine significance between methyl diet animals and control animals: t(107) = 15.9, p<0.005, d = 2.2. The calculated Cohen’s D value of 2.2 indicates a large treatment effect.

Figure 2. Weight distributions of methyl-diet vs control diet animals.

We weighed 68 experimental animals (40 ♀ & 28 ♂) and 40 controls (12 ♀ & 18 ♂) at six months of age. The difference between female experimental & female control (ctrl) was significant (p<0.05; t-test), male averages were not significant. However, there were two methyl-diet males that were much larger than the control population.

Figure 3. Representative abnormalities observed in methyl diet animals.

(A) Hemorrhagic ovarian cyst in a methyl diet female. (B) Normal diet animal’s ribcage, heart, and lungs (left) compared to one methyl diet animal’s ribcage, heart and lungs; note abnormalities in size and shape of lungs and heart. (C) Cataracts were visible in the left eye of some animals. (D) Left and right testes from a control diet male (top) and a methyl diet male (bottom). Chi squared tests suggest significant size differences between right and left testes in these three methyl diet males.

Figure 4. Effects of methyl-donor diet on behavior.

(A) Repetitive behaviors in each group tested. Repetitive behaviors included jumping, back-flipping, and running in circles. Female methyl diet animals performed significantly higher numbers of repetitive behaviors than control diet females (p<0.01, Kruskal-Wallis test). (B) Social behaviors and aggressive behaviors for each group tested. Social behaviors included sniffing, following, and allogrooming. Female methyl diet animals were, on average, less social, but this was statistically insignificant (p = 0.064, Kruskal-Wallis). Aggressive behaviors included biting, boxing, mounting, and chasing. Male methyl diet animals were, on average, more aggressive than control diet males, but this was statistically insignificant (p = 0.069). In both cases, bars represent standard error.