Because germ cells are exclusively capable of passing the genetic materials to the progeny, damages to their genome – either involving DNA mutations or only affecting epigenetic machineries – impose a unique risk of initiating heritable disorders. Whereas toxicants that reduce viability of germ cells may diminish fertility, lesions in the genome of survived germ cells may be persistent beyond generations to harm health of the offspring. Because the risk of creating hereditary diseases would increase when the genome of embryonic precursors of germ cells is impaired, and because sensitivities and genotoxic mechanisms of a toxic substance may vary among different cell types, assessments of such a risk would be best performed using human germline precursor cells. However, the lack of cell culture models of human germ cell precursors suitable for in vitro assessments has been a significant obstacle to understanding the realistic risk that chemical exposure initiates heritable diseases as well as to develop strategies for protecting the genome of human germ cells. The large and ever-increasing number of synthetic compounds further raises the hurdle of comprehensive understanding of such exposure-initiated heritable health threats. The Primordial germ cells (PGC) is the most upstream precursor of all germ cells. The human PGC-like cell (hPGCLC) is a pluripotent stem cell-derived cell culture model of human PGCs, but technical difficulties with its in vitro expansion has been preventing its practical use for toxicological assessments. Our laboratory has recently overcome this hurdle by successfully establishing the Long-Term Culture hPGCLC (LTC-hPGCLC), which perpetually expands in a serum/feeder layer-free cell culture condition as a highly homogeneous, senescence-free cell population without losing their PGC-like characteristics. Taking advantage of LTC-hPGCLCs, this project will obtain critical mechanistic insights into how environmental chemicals affect genetic and epigenetic integrity of human PGCs through creating novel nucleotide base mutations (Aim 1) or disrupting the epigenetic integrity (Aim 2). A genetically engineered variant of LTC-hPGCLCs that are resistant to stress-induced cell death will be exposed to toxicants directly or in the context of organ cultures mimicking the micro-environment of human embryonic testis. Aim 1 will examine DNA damaging effects of cigarette smoke mutagens, drugs commonly used for cancer chemotherapy, and nanoparticles whereas Aim 2 will examine epigenetic effects of endocrine disrupting chemicals and the PFAS chemicals, which are used as stain repellants, polishers, paints, and coatings. Attempts will also be made to develop high-throughput compatible assays for evaluation of these genotoxic actions of chemicals in LTC-hPGCLCs. Outcomes of our research will directly inform not only the scientific research community but also the industry and the governmental programs regulating chemical safety about the risk of c...