Endocrine Disruption Potentials and Related Mechanisms of Several Organophosphate Flame Retardants

Abstract

Organophosphate flame retardants (OPFRs) have been widely used as alternatives to polybrominated diphenyl ethers (PBDE) to prevent fire. Several OPFRs have been frequently detected in environment and biota, however knowledge on their potential toxicological effects are limited. Recently, these compounds are reported to be associated with altered hormones like sex steroid, thyroid hormone and prolactin, and decreased semen quality in adult men. In the present study, various cell lines and a zebrafish (Danio rerio) model were employed to investigate the endocrine disruption potency and underlying mechanisms of several OPFRs. The results of this study will provide essential toxicity information for ecological risk management about this re-emerging group of environmental contaminants. For this purpose, four separate sets of experiments were conducted. Firstly, two human cell lines (H295R and MVLN) and zebrafish were employed to screen the endocrine disrupting potentials of six OPFRs, i.e., tris-(2-chlorethyl) phosphate (TCEP), tris-2-chloroisopropyl phosphate (TCPP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris-(2-butoxyethyl) phosphate (TBEP), triphenyl phosphate (TPP), and tricresylphosphate (TCP). By all six OPFRs, both 17β-estradiol (E2) and testosterone (T) concentrations increased in H295R cells. In addition, transcription of four major steroidogenic genes (CYP11A1, CYP11B2, CYP19A and 3βHSD2) was up-regulated and that of two sulfotranferase genes (SULT1E1 and SULT2A1) was down-regulated. In MVLN cells, no OPFRs acted as estrogen receptor (ER) agonists, while TDCPP, TPP, and TCP acted as ER antagonists inhibiting binding of E2. Following 14 d exposure to adult zebrafish, TDCPP, TPP, or TCP significantly increased plasma T and E2 concentrations. Among males, both T and 11-KT decreased and E2 increased. In general, transcription of CYP17 and CYP19a genes in gonad was significantly up-regulated in both sexes, while VTG1 gene was down-and up-regulated in female and male fish, respectively. In second experiment, a 21 d reproduction test was conducted using adult zebrafish to understand the effects of two major OPFRs on reproduction performances. The study OPFRs included TDCPP and TPP, which are most widely used and also exhibited clear evidence of hormone alterations previously in vitro cell assay. In this experiment, paired adult zebrafish were exposed to various concentrations of TDCPP and TPP (0, 0.04, 0.2, and 1.0 mg/L) for 21 d, and the effects on gene transcription, proteins, and several reproduction related parameters were evaluated. Fecundities were significantly decreased by both OPFRs, and these reproduction changes were accompanied with significant increases of plasma E2, E2/T and E2/11-KT, and decreases of T and 11-KT in male and female fish. Significant increase of plasma vitellogenin (VTG) level was also observed. Altered transcription of genes along HPG axis was sex-dependent, and was supportive of the protein levels observations. The results of the second experiment demonstrated that both TDCPP and TPP could perturb the function of HPG axis, and then influence the sex hormone balance as well as reproduction performance. In third experiment, the effects on fish thyroid hormones (THs) were investigated after 14 d exposure. Plasma concentrations of THs, and transcription of related genes involved in thyroid synthesis, transport, binding, or regulation were examined in brain, thyroid, and liver of zebrafish. The concentrations of peripheral triiodothyronine (T3) and thyroxine (T4) decreased significantly in male fish following the exposure to both OPFRs, but the trend was opposite in female fish. The transcription of genes along hypothalamus-pituitary-thyroid (HPT) axis by the exposure was sex-dependent: In male fish, transcriptions of CRH and TSH in brain were significantly greater as a compensation of hypothyroidism, but TG, or Dio2 were decreased in thyroid or liver. In contrast, transcriptions of CRH and TSH genes were significantly down-regulated in females, but increases in transcription of other genes did not support the compensatory efforts of the brain. In the fourth experiment, a longer term exposure from the fertilization of the eggs was conducted to understand the effects from very early stage of the life, and possibly to assess cross-talks among the HPG, HPT and hypothalamic-pituitary-adrenal (HPA) axes. During this test, zebrafish embryos were exposed to 0.005, 0.05 and 0.5 mg/L TDCPP or TPP for 120 d, and the growth parameters, hormone levels, as well as the transcriptional profiles along the HPG, HPA, and HPT axes were examined. Following the exposure, conditional factor (CF), gonad somatic index (GSI), and liver somatic index (LSI) were significantly decreased in exposed fish. In female fish, exposure to TDCPP or TPP led to increase of cortisol, follicle stimulating hormone (FSH), luteinizing hormone (LH), T4, T3, and E2, and lesser concentration of 11-KT. In males, exposure to these two chemicals resulted in decrease of cortisol, FSH, LH, T4, T3, T, and 11-KT. Sex dependent changes were also evident: Transcription of genes along HPG, HPA and HPT axis were mostly up-regulated in female, while down-regulated in males. The observations of a series of experiments showed that (1) all six selected OPFRs could disturb the synthesis, metabolism or activation of sex hormone, and that (2) TDCPP and TPP influenced balances of sex hormones and THs, and could cause reproductive impairments in zebrafish. In addition, (3) Life cycle exposure to TDCPP or TPP caused developmental retardation, and disturbed hormone balance at concentrations that could occur in the environment. Ecological implications of these observations deserve further investigation.