In vitro immunotoxicity and possible mechanisms of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on Ruditapes philippinarum hemocytes

Highlights

• BDE-47 declined the hemocyte cell viability, reduced the granulocyte ratio.

• BDE-47 decreased the hemocytes phagocytosis and bacteriolytic activity.

• BDE-47 destroyed the antioxidant system and induced an obvious overproduction of ROS in hemocytes.

• BDE-47 increased lysosomal membrane permeability of hemocytes.

• BDE-47 exposure altered the ERK and p38 phosphorylation expression.

Abstract

Marine bivalves can accumulate large amounts of pollutants from sea water, sediments and microalgae due to their filter-feeding habits. BDE-47 is often the most highly concentrated congener in bivalves. BDE-47 has been found to have toxic effects on bivalves, however, the immunotoxicity and the underlying mechanisms of BDE-47 on bivalves are not well understood yet. In this study, isolated hemocytes of Manila clam Ruditapes philippinarum were exposed to five concentrations of BDE-47 (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM), the effects of BDE-47 on hemocyte survival rate, cell viability, granulocyte ratio, phagocytosis, bacteriolytic activity, reactive oxygen species (ROS), lysosomal membrane permeability (LMP), superoxide dismutase (SOD), and phosphorylation state of extracellular regulated protein kinase (ERK) and p38 at 2 h, 6 h and 12 h were studied. The results indicated that BDE-47 exposure declined the hemocyte cell viability, reduced the granulocyte ratio, hampered the hemocyte phagocytosis and bacteriolytic activity, elevated the ROS levels, increased the LMP, significantly changed SOD expression and depressed the phosphorylation levels of ERK and p38. Taken together, the results demonstrated that BDE-47 had significant toxic effects on the immune function, and the immunotoxicity may partly via the overproduction of ROS and the alteration of MAPK signaling pathways.

Introduction

Polybrominated diphenyl ethers (PBDEs) are widely used as brominated flame retardant in various products, such as electrical appliances, household products, cables, furniture industries and so on [1,2]. The PBDEs are not strongly chemically bounded to the products, prone to leach out to surrounding environments during production, use and disposal. Due to the environmental persistence, lipophilicity and bioaccumulation characteristics, PBDEs have been generally detected in various matrices, including atmosphere, water, sediment, soil, biota and even in the human [[3], [4], [5]]. Production of penta- and octa-brominated diphenyl ether (BDE) formulations was first phased out in the U.S. in 2004 [6]. The most widely used PBDE formulation, deca-BDE, was banned in the European Union in 2008 [7]. Although the mass production and usage of these PBDEs have been banned, high PBDEs levels can still be detected in aquatic ecosystem, and BDE-47 is the most predominant PBDE congeners [[8], [9], [10], [11]]. Table 1 listed some investigations of high concentrations of PBDEs and BDE-47 in sediments and organisms. It's easy to see that the pollution of PBDEs in aquatic environments has become a serious and widespread problem.

BDE-47 is often the most highly concentrated congener in marine organism especially in bivalves [14,[16], [17], [18]]. Toxicity of BDE-47 are listed in Table 2, including developmental [[19], [20], [21]], reproductive [22,23], immunological [[24], [25], [26], [27]] and other adverse effects [2,28,29]. Mussels are the most commonly used as sentinel organisms for the assessment of environmental pollution in marine ecosystems. To date, there are only three researches on the immunotoxicity of BDE-47 in mussels [[25], [26], [27]], the specific toxicity effects are listed in Table 2. However, the study of BDE-47 on R. philippinarum is still scarce, and the underlying mechanisms of the immunotoxicity of BDE-47 on bivalves remain limited.

R. philippinarum is one of the most important economic bivalves in marine aquaculture of China. The immune system of the bivalves includes hemocytes and humoral components. The circulating hemocytes, acting as both the undertaker of the cellular immunity and the supplier of the humoral immunity, play a central role in the immune defense [30,31]. Previous studies showed that BDE-47 exposure could provoke ROS production, cause oxidative stress, change antioxidant enzyme activities [27,32,33] and alter mitogen-activated protein kinases (MAPKs) signaling pathways [21,34,35]. Whether BDE-47 exposure could raise ROS production, cause oxidative stress or affect MAPK signaling pathways in R. philippinarum hemocytes are still ambiguous.

In vitro tests with mussel hemocytes have been demonstrated to be sensitive tools to evaluate the toxicity and relative toxicity mechanisms of the marine pollutant [30,36,37]. Due to the devastating effects of BDE-47 on marine organisms and insufficient database on immunotoxicity, in this paper, the in vitro effects of BDE-47 on hemocytes survival rate, cell viability, granulocytes ratio, phagocytosis, bacteriolytic activity, ROS production, SOD protein expression, LMP as well as phosphorylation level of MAPKs (ERK and p38) were evaluated in R. philippinarum hemocytes. This study would provide a better understanding of the immunotoxicity and the relative mechanisms of BDE-47 on R. philippinarum hemocytes.