The Effects of Di-(2-ethylhexyl)-phthalate Exposure on Fertilization and Embryonic Development In Vitro and Testicular Genomic Mutation In Vivo

Abstract

The present study was undertaken to determine the reproductive hazards of Di-(2-ethylhexyl)-phthalate (DEHP) on mouse spermatozoa and embryos in vitro and genomic changes in vivo. Direct low-level DEHP exposure (1 μg/ml) on spermatozoa and embryos was investigated by in vitro fertilization (IVF) process, culture of preimplanted embryos in DEHP-supplemented medium and embryo transfer to achieve full term development. Big Blue® transgenic mouse model was employed to evaluate the mutagenesis of testicular genome with in vivo exposure concentration of DEHP (500 mg/kg/day). Generally, DEHP-treated spermatozoa (1 μg/ml, 30 min) presented reduced fertilization ability (P<0.05) and the resultant embryos had decreased developmental potential compared to DMSO controls (P<0.05). Meanwhile, the transferred 2-cell stage embryos derived from treated spermatozoa also exhibited decreased birth rate than that of control (P<0.05). When fertilized oocytes or 2-cell stage embryos were recovered by in vivo fertilization (without treatment) and then exposed to DEHP, the subsequent development proceed to blastocysts was different, fertilized oocytes were significantly affected (P<0.05) whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). Testes of the Big Blue® transgenic mice treated with DEHP for 4 weeks indicated an approximately 3-fold increase in genomic DNA mutation frequency compared with controls (P<0.05). These findings unveiled the hazardous effects of direct low-level exposure of DEHP on spermatozoa's fertilization ability as well as embryonic development, and proved that in vivo DEHP exposure posed mutagenic risks in the reproductive organ – at least in testes, are of great concern to human male reproductive health.

Effects of long-term endocrine disrupting compound exposure on Macaca mulatta embryonic stem cells

“…Treatment groups included a vehicle control (methanol) and each of five EDCs at environmentally relevant doses [10 nM BPA (36 nM fetal serum, [35]), 5 μM DEHP (7.7μM pubertal serum, [12]), 30 μM ATR (up to 7 μM drinking water reported, [36]), 100 nM PFOA (average 94 nM ≥12 year old serum, [37]), and 25 nM TBT (0.17 – 534 adult nM serum, [38]). These concentrations are within the range those reported for human serum, for drinking water, or studies reported for mammalian oocytes and preimplantation stage embryos or pluripotent cells in vitro [22, 23, 27, 39–41]. The concentration of ATR used was ~4-fold higher than the maximum concentration reported in one study in drinking water [36], and one study of occupational human serum level (up to 245 nM [42]).…”

“…Consequently, although rodent stem cell and rodent developmental studies provide important information about the potential developmental consequences of EDC exposure, the possibility of species differences needs to be addressed by determining EDC effects in model organisms more closely resembling humans. Some other studies reported that some EDCs affect germ cells and early embryos, but others reported little or no effect [22–32]. Recent studies also illustrate the effects of prolonged low-level exposure on embryonic stem cell (ESC) differentiation [33].…”

Di-(2-ethylhexyl) phthalate inhibits DNA replication leading to hyperPARylation, SIRT1 attenuation and mitochondrial dysfunction in the testis

“…The finding that DEHP inhibited DNA replication and induced cellular oxidative stress is reminiscent of previous work suggesting that DEHP treatment may induce DNA damage303132. Synthesis and conjugation of poly(ADP-ribose) polymers (PARs) to proteins are common response to DNA damage.…”

Testicular Dysgenesis Syndrome and Long-Lasting Epigenetic Silencing of Mouse Sperm Genes Involved in the Reproductive System after Prenatal Exposure to DEHP

“…In addition, an increased expression of LXRα and its downstream genes involved in lipid and cholesterol synthesis was observed in both male and female gonads [9]. Treatment of Big Blue ® transgenic mice with DEHP for 4 weeks induced a 3-fold increase in genomic DNA mutation frequency [10]. Furthermore, a 96 h exposure to 10 μM MEHP of cultured chicken postnatal testis cells altered seminiferous tubule differentiation [11].…”