PILOT: Center for Appalachian Research in Environmental Sciences: Epigenetic Rearrangement in Sperm Caused by Chronic Cadmium Exposure

The highly persistent heavy metal, cadmium, a known carcinogen, occurs naturally in the environment. In addition to background levels in soil, application of phosphate fertilizers and sewage sludge to cropland is a critical link in the cadmium exposure pathway because it is taken up by food1. As a result, cadmium is present in virtually all foods, with more than 80% of food-cadmium coming from cereals, vegetables, and potato2; mollusks and crustaceans are also an important source of cadmium. Average cadmium intake in food generally varies from 8-25 ..g/day1; 3. The US Food and Drug Administration’s (FDA) Total Diet Study (TDS) update reported a 26% increase in dietary cadmium exposure from 1990-2003, from 8.81 to 11.06 ..g/person/day4. Though a common misconception, tobacco smoke exposure is not the only source of cadmium where each cigarette contains only 1-2 ..g of cadmium 1; 3. Further, cadmium exposure is a major concern in Appalachia because of its presence in coal and subsequent release during mining operations5. Cadmium has been shown to cause decreased sperm production and increased oxidative stress6-8. However, as cadmium is not a mutagen, we hypothesize that it exerts its pathogenic potential through the reprogramming of the epigenome. Epigenetic reprogramming allows cells to adapt to their ever-changing environment resulting in the accessibility of chromatin DNA, to the many regulatory factors that impact gene regulation (including toxic exposures). Cadmium exposure modifies epigenetic alterations in various cell types and is likely to occur in sperm as well. However, whether these hypothesized changes occur, and whether they will cause transgenerational influences on offspring disease risk are unknown. It has long been thought that DNA methylation in the sperm is erased after fertilization. However, recent studies now refute these long held beliefs and it is now clear that some of these methylation patterns are indeed retained and transgenerational in nature9. Our goal for this project is to determine the impact of cadmium on DNA methylation patterns using a mouse model of chronic cadmium exposure at environmentally relevant doses. Our hypothesis is that chronic cadmium exposure induces DNA methylation alterations that cause an epigenetic rearrangement of the chromatin that ultimately impact mRNA and protein expression. We then predict future long-term developmental detriments such as obesity, diabetes, and cardiovascular disease will occur in offspring from those paternally exposed sires. While investigating the long-term outcomes in the next generation are outside of the scope of this P30 pilot proposal, we feel epigenetics and developmental programming are highly impactful and will help improve our chances of funding success with a future R01 application. To test our hypothesis, we will conduct exposure studies and measure gene expression and DNA methylation in the exposed sperm. Aim 1. We hypothesize that epigenetic modifications caused by cadmium exposure will, in part, affect not only phenotypically but epigenetically alter sperm cells. In laboratory models, cadmium has been shown to cause gene specific hypo- and hypermethylation events in multiple cell lines. Furthermore, recent studies have identified differences in global methylation and gene specific promoter CpG island methylation in cord blood, placenta, newborns and children exposed in utero to cadmium. However, the impact of cadmium on DNA methylation in the sperm to drive epigenetic inheritance is unknown. Numerous studies in rodents have shown that chronic cadmium exposure in the drinking water at doses relevant to human exposures can cause decreased sperm numbers and increase oxidative stress. Here, we will use a similar and environmentally relevant exposure paradigm where cadmium chloride (3 mg/kg body weight dose) will be added to the drinking water in the exposed group of C57BL/6 male mice for 8 weeks in order to cover the entire period of spermatogenesis and maturation. We will monitor food and water intake as well as body weight prior to collection of sperm. Live sperm will be isolated, followed by nuclei separation, and RNA and DNA purification. We will perform RNA sequencing and use high-resolution methyl-seq to map DNA methylation changes in mouse spermatozoa from mice exposed to cadmium and non-exposed vehicle controls. We will analyze only viable sperm, which are most likely to fertilize the egg and carry-on the inherited epigenetic change