اثر اسید فولیک غذایی بر تعداد اسپرم، آسیب DNA و جهش

ABSTRACT Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

Germline mutation may lead to various detrimental outcomes including embryonic lethality, inherited genetic disease or transgenerational genetic instability [1]. Since the germline has a very low spontaneous mutation frequency, germ cell mutation assays generallyrequirelargenumbersofanimalsandhighchemicaldoses to establish an agent as mutagenic [2]. As a result, much less is known about the environmental variables that influence germ cell mutation rates compared to somatic cells. To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations [3]. Existing evidence supports the germline mutagenicity of various agents including radiation, mutagenic anti-cancer drugs, and specific environmental mixtures (tobacco smoke, urban air pollution) [4–11]. However, relatively little is known about dietary variables that affect germ cell mutation rates.

Folate, an essential B vitamin, is required for the de novo synthesis of purines, thymidylate and methionine [12]. As such, folate is required for the maintenance of DNA synthesis and methylation, and consequently chromatin structure and gene expression (Fig. 1). In somatic cells, folate deficiency can lead to increased uracil incorporation into DNA, DNA double strand breaks, genome instability and DNA hypomethylation [13–17]. However, few studies have examined the effects of folic acid deficiency on germ cells.

Folate deficiency is associated with indicators of reduced male fertility. For example, low folate in seminal plasma is correlated with decreased sperm counts and increased sperm DNA damage in humans [18,19]. An inverse relationship between total daily folate intake and the frequency of aneuploid sperm in humans has also been observed [20]. In mice, treatment with methotrexate, an antifolate chemotherapeutic, leads to decreased sperm counts and increased sperm DNA damage [21,22]. These studies demonstrate that folate impacts sperm health and DNA integrity, suggesting that folate deficiency may be mutagenic to germ cell DNA.

In 1998, Canada and the US mandated fortification of white flour with folic acid to reduce the incidence of neural tube defects [23]. Folic acid fortification has successfully eliminated folate deficiency in Canada; however, the folate status of a significant proportion of the general population is indicative of folic acid intakes at or above the tolerable upper intake level [24,25]. High folic acid intake can overwhelm enterocyte metabolism and result in the presence of unmetabolized folic acid in circulation [26,27]. Circulating folic acid must be metabolized by the tissues and the presence of unmetabolized folic acid could result in the accumulation of cellular dihydrofolate, an inhibitor of methylenetetrahydrofolate reductase [28] and thymidylate synthase [29]. Inhibition of these enzymes has been proposed to decrease methionine and thymidylate synthesis [30]. Consequently, high folic acid intake may, paradoxically, result in a functional folate deficiency, resulting in increased mutation rates and genome instability.

In the present study we explore the effects of folic acid deficiency and supplementation on male germ cell integrity. We evaluated three measures of sperm health including sperm count, DNA damage using the sperm chromatin structure assay (SCSA), and DNA sequence mutations at tandemly repeated sequences. The effects of dietary folic acid in utero and during lactation (early development) or from weaning for 15 weeks were investigated. Three dietary groups representing folic acid deficiency (0 mg/kg), the recommended daily intake of folic acid (control, 2 mg/kg), and folic acid supplementation (6 mg/kg) at three times the daily recommended intake and approximating the consumption of a 1 mg/day folic acid supplement and folic acid from fortified foods were used.