Detrimental effects of flame retardant, PBB153, exposure on sperm and future generations

Abstract

In 1973, the Velsicol Chemical Company, which manufactured FireMaster, a brominated flame retardant, and NutriMaster, a nutritional supplement, mistakenly shipped hundreds of pounds of FireMaster to grain mills around Michigan where it was incorporated into animal feed and then into the food chain across the state. An estimated 6.5 million Michigan residents consumed polybrominated biphenyl (PBB)-laced animal products leading to one of the largest agricultural accidents in U.S. history. To date, there have been no studies investigating the effects of PBB on epigenetic regulation in sperm, which could explain some of the endocrine-related health effects observed among children of PBB-exposed parents. Fusing epidemiological approaches with a novel in vitro model of human spermatogenesis, we demonstrate that exposure to PBB153, the primary component of FireMaster, alters the epigenome in human spermatogenic cells. Using our novel stem cell-based spermatogenesis model, we show that PBB153 exposure decreases DNA methylation at regulatory elements controlling imprinted genes. Furthermore, PBB153 affects DNA methylation by reducing de novo DNA methyltransferase activity at increasing PBB153 concentrations as well as reducing maintenance DNA methyltransferase activity at the lowest tested PBB153 concentration. Additionally, PBB153 exposure alters the expression of genes critical to proper human development. Taken together, these results suggest that PBB153 exposure alters the epigenome by disrupting methyltransferase activity leading to defects in imprint establishment causing altered gene expression, which could contribute to health concerns in the children of men exposed to PBB153. While this chemical is toxic to those directly exposed, the results from this study indicate that the epigenetic repercussions may be detrimental to future generations. Above all, this model may be expanded to model a multitude of environmental exposures to elucidate the effect of various chemicals on germline epigenetics and how paternal exposure may impact the health of future generations. In recent years, a major focus of research has been the Developmental Origins of Health and Disease (DOHaD) 1. A component of DOHaD research examines the impact of pre-and post-conception risk factors on long-term health and disease progression in adulthood. In terms of pre-conception risk factors, many researchers report that toxicants and environmental exposures can potentially alter the epigenome in gametes, giving these exposures the potential to ultimately impact the health of the next generation 2-5. In 1973, farms across Michigan mistakenly received cattle feed mixed with FireMaster BP-6, a chemical flame retardant composed of a mixture of polybrominated biphenyl compounds (PBBs), rather than NutriMaster, a food additive that increased milk production in farm animals 6-9. PBB-laced animal products were consumed by millions of residents, and the decline in animal

Blastocyst development after fertilization with in vitro spermatids derived from nonhuman primate embryonic stem cells

“…This work has since been independently replicated (15), highlighting the reproducibility of our methodology. To first determine whether nhpESCs differentiate into spermatogenic cells similar to hESCs and hiPSCs, we differentiated nhpESCs in mouse SSC culture conditions as previously described (10,15,30,(32)(33)(34)(35)(36). We chose nhpESC H2B-GFP line for downstream analyses, but all lines tested (Supplemental Fig.…”

“…Genomic DNA from diploid nhpESCs and haploid cells obtained from FACS was isolated using the DNeasy Blood and Tissue kit from Qiagen following the manufacturer’s instructions. Samples were prepared for imprint control region methylation analyses as previously described ( 30 ) with the EpiTect Methyl DNA Restriction Kit (Qiagen) as per the manufacturer’s instructions. For quantitative polymerase chain reaction (qPCR)-based analyses of imprint control region methylation for H19 and SNRPN for each sample, the following EpiTect qPCR Methyl Promoter Primers were used: H19 , catalog number 335002 EPHS102101–1A, and SNRPN , 335002 EPHS104389–1A.…”

“…Nonhuman primate embryonic stem cells cultured in conditions defined earlier were immunostained as previously described (30). Briefly, samples were fixed in 4% paraformaldehyde (Sigma) for 15 minutes and then blocked with buffer containing 1Â PBS (Thermo Fisher), 0.25% Triton-X (Sigma), 5% bovine serum albumin (Thermo Fisher), and 5% normal goat serum or donkey serum (Sigma) overnight at 4 C. Primary antibody incubation occurred overnight at 4 C in blocking buffer followed by three washes in 1Â PBS with 0.25% Triton-X for 10 minutes each at RT.…”

Exposures during the prepuberty period and future offspring’s health: evidence from human cohort studies†

“…None the less, some evidence in humans does exist. For example, paternal toxicant exposures and even endurance training have been shown to result in altered ncRNA content of spermatozoa and methylation [110,[115][116][117][118][119]. Involvement of epigenetic mechanisms transferred from father to offspring was suggested by Knudsen et al based on a preliminary EWAS analyses of 195 Bergen RHINESSA and ECRHS participants, showing that father's smoking was associated with specific DNA methylation patterns in adult offspring [120].…”

Sperm biology and male reproductive health

“…Greeson et al examined environmental exposure to PBB153, a polybrominated biphenyl compound used as a flame retardant that accidentally entered the food chain in 1973, and led to alterations in liver and thyroid functions, as well as to increased odds of lymphomas and cancers of the digestive tract. The authors demonstrated that higher serum PBB153 levels were associated with decreased methylation of imprint control regions, which in turn demonstrated that this contaminant determines alterations to the germ line which are then potentially transferred to the offspring of these men 13 . Other environmental effects, such as diets, are more difficult to measure.…”