Potassium bromate, a potent DNA oxidizing agent, exacerbates germline repeat expansion in a fragile X premutation mouse model

Abstract

Tandem repeat expansion is responsible for the Repeat Expansion Diseases, a group of human genetic disorders that includes Fragile X syndrome (FXS). FXS results from expansion of a premutation (PM) allele having 55-200 CGG.CCG-repeats in the 5' UTR of the FMR1 gene. The mechanism of expansion is unknown. We have treated FX PM mice with potassium bromate (KBrO(3)), a potent DNA oxidizing agent. We then monitored the germline and somatic expansion frequency in the progeny of these animals. We show here that KBrO(3) increased both the level of 8-oxoG in the oocytes of treated animals and the germline expansion frequency. Our data thus suggest that oxidative damage may be a factor that could affect expansion risk in humans.

Fig. 1

8-oxoG in the ovaries of untreated and bromate-treated mice. Panel A. 8-oxoG in the oocytes of untreated and bromate-treated mice. Sections of the ovaries of untreated mice (i) and bromate-treated animals of the same age (ii and iii) were stained with antibodies to 8-oxoG as described in the Materials and Methods. The DNA was stained with Hoechst 33258. The 8-oxoG staining regions are shown in red with the DNA shown in blue. Bars = 100 microns. The white arrows indicate 8-oxoG-positive signals in the oocyte nuclei. Note that occasional cytoplasmic staining is also seen in bromate-treated ovaries, most likely due to oxidation of mitochondrial DNA and RNA. Note that the oocyte shown in (iii) demonstrates the typical perinucleolar clumping of DNA sometimes seen in oocytes of this stage. The non-uniform distribution of 8-oxoG seen in the nuclei of some cells may reflect the known clustering of 8-oxoG lesions in the human genome (Ohno, et al., 2006). Panel B. Relative 8-oxoG levels in the ovaries of 2 bromate-treated mice and their age matched controls raised on unadulterated water. The 8-oxoG levels in treated animals are expressed relative to the amount of 8-oxoG in the appropriate age-matched control animals. Each bar represents the average of 3 independent determinations.

Fig. 2

The effect of KBrO3 on the frequency of expansions and deletions seen on maternal and paternal transmission of the PM allele in animals. The single asterisks on the first pair of dark gray bars indicates that the difference between the expansion frequency seen in the offspring of bromate-treated and untreated mothers is significant at a P value of <0.0001. The double asterisks in the 2^nd pair of dark gray bars indicates that the difference between the expansion frequency seen in the offspring of bromate-treated and untreated fathers is significant at a P value of 0.0114.

Fig. 3

Distribution of repeat length changes on maternal and paternal transmission of the PM allele with and without KBrO₃ treatment. The product of the total number of alleles of each repeat size and the number of repeats added or lost to that particular allele class was divided by the total number of expanded alleles examined. A) Transmission of the PM by KBrO₃-treated males leads to an increase in the number of larger expansions. The profile of repeat length changes on paternal and maternal transmission in untreated animals is indistinguishable (Entezam, et al., 2007; Entezam and Usdin, 2008). B) Paternal exposure to KBrO₃ results in larger repeat length changes than maternal exposure.