Apoptosis induction on human hepatoma cells Hep G2 of decabrominated diphenyl ether (PBDE-209)

Abstract

Polybrominated diphenyl ethers (PBDEs) are an important class of halogenated organic brominated flame retardants. Because of their presence in abiotic and biotic environments widely and their structural similarity to polychlorinated biphenyls (PCBs), concern has been raised on their possible adverse health effects to humans. This study was designed to determine the anti-proliferative, apoptotic properties of decabrominated diphenyl ether (PBDE-209), using a human hepatoma Hep G2 line as a model system. Hep G2 cells were cultured in the presence of PBDE-209 at various concentrations (1.0–100.0 μmol/L) for 72 h and the percentage of cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results showed that PBDE-209 inhibited the cells viability in time and concentration-dependent characteristics at concentrations (10.0–100.0 μmol/L). We found that anti-proliferative effect of PBDE-209 was associated with apoptosis on Hep G2 cells by determinations of morphological changes, cell cycle and apoptosis. Mechanism study showed that PBDE-209 could increase the generation of intracellular reactive oxygen species (ROS) concentration-dependently. Antioxidant N-acetylcyteine partially inhibited the increase of ROS. The mechanism for its hepatoma-inhibitory effects was the induction of cellular apoptosis through ROS generation. In addition, activity of lactate dehydrogenase (LDH) release increased when the cells incubated with PBDE-209 at various concentrations and times. These results suggested that PBDE-209 had the toxicity activity of anti-proliferation and induction of apoptosis in tumor cells in vitro.

Introduction

Polybrominated diphenyl ethers (PBDEs) are one class of halogenated organic brominated flame retardants (BFRs), and have been used industrially in large volumes for flame protection purposes in various commercial products such as electronic equipment and textiles. The commercial PBDE products predominantly consist of so-called penta-, octa- and decabromodiphenyl ether products. Today, decabromodiphenyl ether (DecaBDE) is the largest product on the market and makes up over 80% of the total production of PBDEs, whereas pentabromodiphenyl ether (PentaBDE) and octabromodiphenyl ether (OctaBDE) products constitute about 12% and 6%, respectively, of the total PBDEs production (De Wit, 2002). Because of their physical, chemical and bio-accumulative characteristics, such as environmental persistence and high lipophilicity, they have become widely distributed in the environment, where they are found to persist for a long time (De Wit, 2002, Watanabe and Sakai, 2003, Alaee et al., 2003, Alcock et al., 2003). PBDEs have recently been found in indoor and outdoor air (Wilford et al., 2004) and dust samples (Stapleton et al., 2005). Furthermore, time trend studies from several regions have indicated increasing concentrations in the biotic environment, such as gull eggs, wildlife and fish (Norstrom et al., 2000, Law et al., 2003, Sellstrom et al., 2003, Kirkegaard et al., 2004), and in human milk, body tissue, and serum samples (Meironyte et al., 1999, Petreas et al., 2003, Sjodin et al., 2003, Hites, 2004, Noren and Meironyte, 2000, Meneses et al., 1999, Strandman et al., 2000, Ryan and Party, 2000, She et al., 2002, Mazdai et al., 2003). Generally, in most cases of abiotic environments, such as sediment, sewage sludge and air, the dominant congeners are PBDE-209 and PBDE-47. And the most prevalent PBDE congeners in humans and biotic samples are PBDE-47 and PBDE-99. So the widespread uses of PBDEs and increased contamination in the environment have led to the rising concern about the possible adverse health effects to humans.

Although the toxicology of PBDEs is still under investigation, toxicity studies indicate that the liver, thyroid gland and possibly also developing reproductive organs are particular targets of PBDEs toxicity (Darnerud et al., 2001, Kuriyama et al., 2005). Evidence is emerging that PBDEs may be developmental neurotoxicants, as behavioural, neurochemical and hormonal deficits have been found following perinatal exposure (Eriksson et al., 2001, Viberg et al., 2002, Zhou et al., 2002, Branchi et al., 2002, Branchi et al., 2003). Carcinogenicity studies on PBDE-209, revealing some effects at very high doses, have resulted in an IARC (International Agency for Research on Cancer) classification stating limited evidence for carcinogenicity of PBDE in experimental animals (IARC, 1990). PBDEs are capable to induce cell death of cerebellar granule cells in culture (Reistad et al., 2006). Madia et al. reports PBDE-99 can induce apoptosis in astrocytoma cells assessed by the TUNEL method and by Hoechst 33258 staining, via a p53 dependent mechanism (Madia et al., 2004). Shin et al. reports polychlorinated biphenyls (PCBs) induce apoptotic cell death of human promyelocytic leukemia line (HL-60) with the involvement of PKC activity (Shin et al., 2002).

Human hepatoma cells Hep G2 (American Type Culture Collection, ATCC, Rockville, MD, USA) is a human hepatoblastoma cell line with a wide variety of signal responses to different kinds of drugs (Knowles et al., 1980). Based on increased contamination of PBDEs in the environment, its possible adverse health effects to humans and its structural similarity to PCBs, in this paper, Hep G2 cells are selected as a model system and apoptosis induction and mechanism of a purified single congener PBDE-209 of PBDEs isomers on the cells are investigated.