Preliminary Study Of Hepatotoxicity And Liver Metabolic Mechanism On Decabromodiphenyl Ethane (DBDPE)

[Objective] DBDPE was developed in the early1990s and has became animportant commercial replacement to PolyBrominated Diphenyl Ethers (PBDEs).DBDPE would become one of the most widely used flame retardants. For a long time,DBDPE was thought to be released minimally into the environment during all phasesof its use and not available biologically due to its large molecular size and lowaqueous solubility. In2003, DBDPE was discovered for the first time to be presentwithin sewage sludge. Then, it was identified in sediment and indoor ai. Since thattime, it has also been found in organism. It was demonstrated that DBDPE, likestructural analogues Decabromodiphenyl ether (BDE-209), is leaking out of thetechnosphere and is present in the environment, and DBDPE can accumulate in theenvironment, food chain and organism. Related research showed that crowdenvironmental exposure levels of DBDPE continues to increase and the accumulationlevel of the bodys showed faster increasing trend. This observation has raised severalconcerns regarding its potential adverse health effects on organism. Less researcheswere carried out for toxicological evaluation of DBDPE. The research results ofHardy et.al indicated DBDPE was difficult to be degraded by rodent and presentedlittle risk to sediment organisms and animals. The results of Nakari et.al aimed ataquatic organism demonstrated that DBDPE could be biodegradable by aquaticorganism and was acutely toxic, estrogenic effects and reproductive toxicity to aquaticorganism. But there are some defects in Narari's experimental design, which was thattoluene was used as solvent. In addition, metabolic and toxic mechanism of DBDPEare not clear. DBDPE structural analogues of PBDEs, may act on endocrine-relatedreceptor, interfere with the endocrine system and metabolic balance. Based on theabove, combined with the key role of liver metabolism in the toxic effects ofbrominated flame retardants (BFRs), it is necessary to carry out hepatotoxicity studiesabout DBDPE, which would solve the problems in the present study, obtain accurateexperimental data and provide a reliable basis for further researches.[Contents and Methods] Base on experimental design of DBDPE structuralanalogues BDE-209, human hepatoma cell line HepG2cells were used as the experimental subject.0-100mg/L DBDPE were selected as HepG2cells exposeddoses. DBDPE was dissolved in DMSO. And DMSO concentration was held constantat0.5%(v/v). The exposure times separatedly were24h,48h and72h. After the end ofexposure, cell viability was measured by the3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and cell damage, L-lactatedehydrogenase (LDH) assay. Nuclear staining with Hoechst33258was performed,nuclear morphology was then observed under using an inverted fluorescencemicroscope and recorded using an imaging system. Induction of apoptosis wasdetected by the method of propidium iodide (PI) staining-flow cytometry detection.To explore the mechanism of cell damage and apoptosis, reactive oxygen species(ROS) was measured after exposure of DBDPE. To verify the relationship betweenROS and cell damage/cell apoptosis, ROS scavenger N-acetylcysteine (NAC) wasmixed in the cell culture medium before exposure of DBDPE, then cell viability andapoptosis separately were detected by MTT assay and PI staining-flow cytometrydetection. Before and after NAC joined, ROS generation, cell viability and apoptosischanges were analyzed so that the relationship between ROS and cell damage couldbe verified.Wistar rats were used as the experimental subject. A technical mixture ofDBDPE was obtained from Albemarle Corporation, which consisted of98.5%DBDPE.0-1000mg/kg.d DBDPE were selected asWistar rats exposed doses. Therats were orally administered DBDPE daily via gavage for28consecutive days.Determination of the weight of rats, liver weight, organ coefficient and biochemicalindicators related with liver function damage were carried out in order to exploreliver damage induced by DBDPE. With a view of HepG2cells ROS induced byDBDPE in cytotoxicity assay, oxidative damage indicators including glutathione(GSH), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), total superoxidedismutase (T-SOD) were measured in order to verify the relationship betweenoxidative damage and liver toxicity in the animal level. In this study, Different liverCYP450enzyme mRNA levels of rat exposed to different doses were determinedusing real time-PCR technology, and then the protein level change of CYP450enzyme which has a significant change on mRNA levels was assayed by Western blotexperiments. Rat liver microsomes were prepared by ultra-high-speed centrifugation technique. Activity of uridinediphosphate-glucuronosyltransferase (UDPGT),pentoxyresorufin O-dealkylation(PROD) related with CYP2B enzyme and luciferinbenzylether debenzylase (LBD) activity related with CYP3A enzyme were analyzed,and infer of DBDPE metabolism in the liver and mechanism. Accordingly,metabolism and mechanism of action of DBDPE in the liver were analyzed andinfered.[Results] In the cell toxicity studies, HepG2cells were cultured in the presenceof DBDPE at various concentrations (0-100.0mg/L) for24h,48h and72h. The resultsshowed that the change of cell viability, cell damage and cell morphology have nosignificant difference between DMSO group and control group, the same between0-6.25mg/L group and control group. DBDPE inhibited HepG2viability in a time anddose-dependent manner from12.5mg/L to100mg/L, at48h and72h, as evaluated bythe3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactatedehydrogenase assays and nuclear morphological changes. Induction of apoptosis wasdetected at12.5-100mg/L at48h and72h by propidium iodide staining, accompaniedby overproduction of ROS. The study also found that DBDPE induced cell apoptosisand cell damage were related with ROS.In animal toxicity studies, Wistar rats were orally administered0-1000mg/kgDBDPE daily via gavage for28consecutive days. There were no significantdifferences of body weight, liver weight, organ coefficient index between DBDPEexposed Wistar rats and control group. According to serological test results, it wasfound that higher DBDPE dose group could induce significant changes of alanineaminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBA) andglucose (Glu) levels in male rats.In addition, significant differences of glutamyltranspeptidase (GGT), total protein (TP), triglyceride (TG), urea nitrogen (UN),creatinine (Cr)were found in parts of dose group of male group. Higher DBDPE dosegroup could induce significant changes of alkaline phosphatase (ALP), AST, TBA andGlu levels in female rats. In addition, significant differences of TBA, TG and Cr werefound in parts of dose group of female group. The results indicated that thedifferences of injury and metabolic function effects of lived induced by DBDPEexisted between male Wistar rats and female Wistar rats. And male Wistar rats wereseriously affected by DBDPE. DBDPEcan affect the normal rat sugar and fat metabolism, suggesting that DBDPE may has endocrine disrupting activities. AndDBDPE could cause liver damage of Wistar rats, which may be associated withoxidative damage.The levels of mRNA, protein and enzyme activity of rat liver CYP450metabolicenzymes were detected after Wistar rats were exposed to DBDPE. No significantchange was found in CYP1A1mRNA expression induced by DBDPE, which shownthat DBDPE has no or little dioxin-like activity. No significant differences ofCYP2B1and CYP3A1/3mRNA levels between dose group and control group werealso found. DBDPE treatment led to a significant increase in CYP2B2mRNA,CYP2B1/2protein, and7-pentoxyresorufin O-depentylase (PROD) activity atrelatively high doses in male rats. There was also a significant induction in CYP3A2mRNA, CYP3A2protein, and luciferin benzylether debenzylase (LBD) activity foundwith higher dose DBDPE treatment in male rats. In female rats, the differences ofmRNA level, protein level and activity level were only found in indivisual dose group.UDPGT activity increased with increasing exposure levels in male rats. In female rats,significant difference of UDPGT activity only was found at500mg/kg.d dose group.Accordingly, it was deduced that DBDPE may activate constitutive androstanereceptor (CAR) and pregnane xenobiotic receptor (PXR) signaling pathway, and thusliver metabolic enzymes were induced for DBDPE metabolism, and endocrine systemof Wistar rat was affected, which could disturb metabolic balance.[Conclusions] Results suggest that DBDPE has hepatotoxicity, and ROSoxidative damage play an important role in cell toxicity and rat liver injury. DBDPEshould contribute little dioxin-like or non-dioxin-like properties. DBDPE inducesdrug-metabolizing enzymes in rats most likely via the CAR/PXR signaling pathway.The induction of CYPs and co-regulated enzymes of phase II biotransformation mayaffect the homeostasis of endogenous substrates. DBDPE has certain endocrinedisrupting activity.