Biphasic Dose-response Induced By Polychlorinated Biphenyls In Human Lung Fibroblast Cell

The central pillar in toxicology is that of dose response relation to determine risk assessment and regulation, for which two models threshold model and linear non-threshold model have traditionally been used. Recently, the biphasic (hormetic) dose response model has attracted considerable interest as it describes stimulatory effects at a low dose and toxic effects at a high dose. Hormetic model rejects the standard toxicological assumption that effects at low doses can be extrapolated from data obtained from high doses, thus challenge the current risk assessment practices. During the past decades, growth, reproduction, life span and bioluminescence etc has been used as in-vivo and in-vitro biological models (rats, bacteria, microalgae, cell cultures and other organisms etc) for toxicological studies, and made the toxicity testing rapid, cost effective, reproducible, robust, sensitive and reliable, and have practical applications in toxicity testing and ecological risk assessment. The lung is a highly vascularized organ and is exposed to a wide variety of xenobiotics such as polychlorinated biphenyls (PCBs) that may enter via inhalation and blood stream; hence lungs may be more vulnerable to toxic chemicals. In this study, four PCB congeners including 2,2’,3,3’-tetrachlorobiphenyl (PCB40),3,3’,4,4’-tetrachlorobiphenyl (PCB77),2,2’,4,5,5’-pentachlorobiphenyl (PCB101) and 2,3’,4,4’,5-pentachlorobiphenyl (PCB118) were studied with different chlorination and different structures. We focused growth as end point, and used human lung fibroblast cells (HELF) testing model to investigate biphasic dose response of low(4Cl) and high(5CL) chlorinated PCB congeners and their potential molecular mechanisms. Further, structure dependent coplanar (PCB77 and PCB 118) and non-coplanar (PCB40 and PCB101) PCB congener’s biphasic dose response variations were also investigated.The thesis concluded that both coplanar and non-coplanar PCBs congeners with four chlorination and five chlorination exhibited toxic effects at higher concentrations on HELF cells proliferation, and these were related with oxidative stress, cell cycle protein and mitogen activated protein kinase (MAPK) signaling pathway. However, less toxic effects were recorded for the non-coplanar four chlorinated PCB40 and five chlorinated PCB101 as compared to the coplanar four chlorinated PCB77 and five chlorinated PCBl 18. PCB40 and PCB77 toxic effects on HELF cell proliferation at high concentrations were 4-34.97% and 6.98-45% while PCB101 and PCB118 showed 9.51-46.03% and 11.48-71.01%, respectively in different time periods. These chemicals revealed biphasic dose response effects, which stimulated HELF cell proliferation at low doses (10-4,10-3,10-2 and 10-1μg/mL), suggesting theses as threshold levels for risk assessment and cell death at high doses (5,10 and 20 μg/mL), suggested that biphasic effects were concentration dependent. The biphasic nature of oxidative stress and antioxidant response in HELF cell culture manifested evidence of PCB congener’s (PCB101 and PCB118) toxicity. The results revealed that reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) contents increased significantly at higher concentrations of PCB101 and PCB118 compared to the lower concentrations with the passage of time. Glutathione (GSH-Px) contents showed decreasing trends after PCB101 and PCB118 exposure in dose and time dependent manner. Moreover, comet assay indicated that PCB101 and PCB118 at high doses posed genotoxic effects in HELF cell compared to low doses and control. Overall, we found that HELF cell proliferation was higher at low ROS level and vice versa, which revealed activation of cell signaling-mediated hormetic mechanisms.The difference between the tetrazolium-based colorimetric assay (MTT) and cellular processes (apoptosis and cell cycle) alteration revealed that different cytotoxic mechanisms might operate in the induction of apoptosis between coplanar PCB77 and non-coplanar PCB40 congeners treated cells. Induction of apoptosis by PCB40 (12%) through alteration of membrane integrity confirmed by flow cytometry analysis, suggested mitochondrial apoptic pathway involved for non-coplanar PCB40 while nuclear apoptotic pathway for PCB77 (25%), and it was structure dependent.The modulation of cell cycle with exposure to PCB40 or PCB77 might be one mechanism involved in biphasic dose response. Low doses of PCB40 or PCB77 significantly increased the percentage of cells in the S Phase, which might be associated with cell proliferation caused by low doses PCBs exposure. Meanwhile, the massive accumulation of cell in G1 Phase might reflect an arrest of cells in G1 Phase, which might response for the cytotoxicity of high concentration PCBs treatment. Western blot analysis indicated that PCB40 and PCB77 increased the expression of cycline E, CDK2 and p21 (activated by growth factors receptors, express in later G1 phase and initial S phase), and caspases-9 (apoptic protein, activated by cytokines and death factor receptors), suggested that apoptosis was caspase-dependent in both PCB congeners. Mitogen activated protein kinase pathway (MAPK) was further studied for PCB101 and PCB118 to understand the response of responsible proteins for cell proliferation and apoptosis in HELF cells. The increase capacity of ERK1/2 signal transduction pathways activation at low doses of PCB101 and PCB118 was response for cell proliferation stimulation. The regulation of cellular process with low dose coplanar and non-colpnanr PCB congener’s exposure might ascribe to the release from contact inhibition caused by the AhR and CAR agonist characteristic, respectively.