Effects of several low molecular weight phthalates and their metabolites on sex hormone system in male zebrafish (Danio rerio) and H295R cell line

Abstract

Phthalates are commonly used to increase flexibility of plastics, and are widely used in PVC, commercial products, wrapping food, cosmetics, medical devices and personal care products. Some phthalates with low molecular weight such as diethyl phthalate (DEP), benzyl butyl phthalate (BBzP), or diisobutyl phthalate (DiBP) are suspected as an endocrine disruptor. It is generally known that they possess higher toxicity than other long chain phthalates and can be quickly metabolized in organism. However, their adverse effects on sex steroid production and underlying mechanism are not fully understood. The aim of this study is to investigate the effects of three low molecular weight phthalates (LMWPs), DEP (0, 0.08, 0.4, 2, or 10 mg/L), BBzP (0, 0.02, 0.1, 0.5, or 2.5 mg/L), and DiBP (0, 0.0008, 0.004, 0.02, or 0.1 mg/L), on sex steroid hormone system in male zebrafish. In addition, these three phthalates as well as their major metabolites, monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), or monoisobutyl phthalate (MiBP), were individually exposed to a human adrenocortical carcinoma (H295R) cell to understand their effects on sex steroidogenesis. In both zebrafish and H295R cells, changes in sex steroid hormone and gene transcriptions involved in steroidogenic pathway were evaluated. For male zebrafish, 14 days exposure to DEP, BBzP, or DiBP significantly decreased testosterone (T) concentrations at higher exposure concentrations of each compound. For DEP, 17β-estradiol (E2) concentrations were also significantly decreased at 10 mg/L. In addition, transcriptions of the genes involved in steroidogenesis pathway were also affected. DEP or DiBP exposure led to significant down-regulation of star gene and similar decreasing trend was also found in male zebrafish exposed to BBzP. All test compounds significantly up-regulated cyp19a gene transcription in a concentration dependent manner. In H295R cell bioassay, five test compounds, i.e. DEP, BBzP, DiBP, MEP, or MBzP, decreased T concentrations, except MiBP which showed opposite direction of change. E2 levels were slightly decreased by MEP, and increased by BBzP, or MBzP in H295R cells. Changes in gene transcription level were in good agreement with the zebrafish exposure data where significant down-regulation of StAR gene and up-regulation of CYP19A gene were observed. On the other hand, DEP and MEP exposure significantly down-regulated the transcriptions of 3βHSD2 gene respectively in H295R cells unlike in male zebrafish. The results of our present study show that some LMWPs could affect sex hormone system in both zebrafish and cell lines by alteration of steroidogenesis pathway.