Cytochrome P450 3A1 mediates 2,2',4,4'-tetrabromodiphenyl ether-induced reduction of spermatogenesis in adult rats

Abstract

Background: 2,2',4,4'-tetrabromodiphenyl ether (BDE47) is the dominant PBDE congener in humans, wildlife, and the environment. It has been reported to be metabolized by cytochrome P450 (CYP) enzymes. Still, the effects of BDE47 on spermatogenesis failure are attracting an increasing amount of attention. However, it is unclear whether CYP-mediated metabolism contributes to BDE47-induced reproductive toxicity.

Methodology and principal findings: The role of cytochrome P450 3A1 (CYP3A1) in the formation of oxidative metabolites of BDE47 and its induced spermatogenesis failure was investigated in SD rats. BDE47 significantly increased the expression and activity of CYP3A1 in rat liver, and 3-OH-BDE47, the major oxidative metabolite of BDE47, dose-dependently increased in rat liver, serum, and testis, which was aggravated by dexamethasone (DEX), an inducer of CYP3A1. Additionally, testicular 3-OH-BDE47 and reactive oxygen species (ROS) in seminiferous tubules increased especially when BDE47 was administered in combination with DEX, which was confirmed in GC-1 and GC-2 cells that 3-OH-BDE47 induced more ROS production and cell apoptosis via the upregulation of FAS/FASL, p-p53 and caspase 3. As a result, daily sperm production dose-dependently decreased, consistent with histological observations in giant cells and vacuolar spaces and increase in TUNEL-positive apoptotic germ cells.

Conclusion: CYP3A1-mediated metabolic activation of BDE47 and the active metabolite 3-OH-BDE47 and consequent ROS played an important role in reduction of spermatogenesis by germ cell apoptosis. Our study helps provide new insights into the mechanism of reproductive toxicity of environmental chemicals.

Figure 1. Induction of CYP3A1 by BDE47 in primary hepatocytes and liver in rats.

(a) mRNA expression of several CYP genes that encode potential metabolic enzymes in primary rat hepatocytes treated with 10 µM BDE47. cDNA (0.1 µg) for each gene was used in the quantitative real-time RT-PCR. The data are expressed as the mean ± SD of three independent experiments with triplicate samples. (b) Expression of CYP3A1 protein in rat liver with BDE47 treatment in saline or dexamethasone (DEX). Hepatic microsome protein (10 µg) for each sample was used in the immunoblotting assay. GAPDH was used as an internal reference. (c) Hepatic CYP3A-related 7-ethoxyresorufin O-deethylase (EROD) activity induced by treatment with BDE47 in saline or DEX, assessed using a fluorescence assay. The data are expressed as the mean ± SD from five rats in each subgroup. ^## P<0.01, compared with corresponding vehicle control, *P<0.05, **P<0.01, compared with corresponding BDE47 treatment in saline group.

Figure 2. Cell apoptosis of BDE47 and 3-OH-BDE47 in GC-1 and GC-2 cells.

Cell apoptosis were determined using the Hoechst 33258 assay and flow cytometry assay. (a–b) GC-1 cells or GC-2 cells were treated with BDE47 (0–50 µM) or 3-OH-BDE47 (0–50 nM) for 24h and then subjected to Hochest 33258 assay to detect apoptotic cell using fluorescence microscope. (c–d) Cell apoptosis of GC-1 cells treated with BDE47 (0, 10, 50 µM) or 3-OH-BDE47 (0, 10, 50 nM) for 24h and then subjected to flow cytometry assay. (e–f) Cell apoptosis of GC-2 cells treated with BDE47 (0, 10, 50 µM) or 3-OH-BDE47 (0, 10, 50 nM) for 24 h and then subjected to flow cytometry assay. The data are expressed as the mean ± SD of three independent experiments from triplicate samples. *P<0.05, compared with corresponding BDE47 treatment in saline group; ^# P<0.05, compared with corresponding vehicle control.

Figure 3. Effects of BDE47 on testicular morphological, daily sperm production and serum testesterone levels.

(a) The testis tissue was stained with hematoxylin and eosin (H&E), and testicular morphological changes and disrupted seminiferous tubule architecture were assessed by optical microscopy. Horizontal arrow, giant cell; vertical arrow, vacuolar spaces in the epithelium. (b) Daily sperm production (DSP) in rats exposed to BDE47. (c) Serum testosterone (T) level in rats exposed to BDE47. Bars show the mean ± SD of 10 animals per group. *P<0.05, compared with corresponding BDE47 treatment in saline group; #P <0.05; ##P<0.01; compared with corresponding vehicle control.

Figure 4. Production of reactive oxygen species (ROS) in seminiferous tubules in rats exposed to BDE47.

Testicular ROS were measured fluorimetrically with the probe CM-H2DCFD (6-chloromethyl-2′,7′-dichlorodihyrofluorescin diacetate, acetyl ester). (a) Representative photograph of ROS in seminiferous tubules. (b) Fluorescence intensity quantified by LSM software. The data are expressed as the mean ± SD of five rats in each subgroup. *P<0.05, compared with corresponding BDE47 treatment in saline group; ^# P<0.05, ^## P<0.01, compared with corresponding vehicle control.

Figure 5. Effects of BDE47 on the production of intracellular ROS and the expression of apoptosis-related proteins in GC-1 cells.

(a) Representative fluorescence images of ROS in GC-1 cells. GC-1 cells were incubated with BDE47 (10 µM) or 3-OH-BDE47 (10 nM) in presence or absence of 400 U/ml catalase (CAT) for 24h. (b) Expression of apoptosis-related proteins in GC-1 cells after BDE47 (10 µM), 3-OH-BDE47 (10 nM) in presence or absence of CAT (400 U/ml) for 24h using immunoblotting assay. GAPDH was used as an internal reference.

Figure 6. Effect of BDE47 on daily sperm production and germ cell apoptosis in seminiferous tubules.

(a) TUNEL assay of germ cell apoptosis in rat testes. Arrows, apoptotic cells. (b) Average number of apoptotic nuclei per tubule. The data are expressed as the mean±SE of three rats per subgroup. (c) Expression of apoptosis-related proteins in rat testis after BDE47 treatment using immunoblotting assay. GAPDH was used as an internal reference. ^# P<0.05, ^## P<0.01, compared with vehicle control.

Figure 7. Schematic of the process and mechanism of CYP3A1-mediated BDE47-induced reduction of spermatogenesis.