Study On The Oxidative Stress Effect And Combined Toxicity Of PAH4 Using Soybean Oil As A Carrier On Liver Cells

In recent years,the harm of polycyclic aromatic hydrocarbons(PAHs)to the human body has attracted more and more attention.Due to the lipophilic properties of PAHs,the amount of PAHs consumed by the human body with oil accounts for one-third of the total amount of PAHs intake.Therefore,co-exposure of PAHs with oil is more in line with the exposure mode of PAHs to the human body.Due to the high detection rates of benzo[a]anthracene(BaA),chrysene(Chr),benzo[b]fluoranthene(BbF),and benzo[a]pyrene(BaP)in food contaminated with PAHs,and they are being prioritized by the international cancer agency,this study mainly focuses on the cytotoxicity of these four types of PAHs(PAH4).PAHs are mostly exposed to the human body in the form of mixtures,but the interaction between PAH4 is not clear.Therefore,this work studied the cytotoxicity and oxidative stress effects of PAH4 using oil as a carrier and studied the combined toxicity and interaction of mixtures of oil-based PAH4 using a combined toxicity prediction model.The difference in toxicity between oil-based PAH4 and pure PAH4 on HepG2 and HL-7702 cells was investigated.The results showed that the toxic effect of oil-based PAH4 on liver cells was concentration-dependent and time-dependent,with the toxicity order being BaP>BbF>Chr>BaA.The results of intracellular lipid accumulation indicated that PAH4 had a synergistic effect with oil at 1 μmol/L,resulting in greater cell toxicity of oil-based PAH4 compared to pure PAH4.The mitochondrial membrane potential and apoptosis rate of HepG2 and HL-7702 cells were measured.The results showed that oil-based PAH4 induced the decrease of mitochondrial membrane potential and the increase of apoptosis rate in HepG2 and HL-7702 cells in a concentration-dependent manner.The sequence of changes in mitochondrial membrane potential and apoptosis rate induced by PAH4 was BaP>BbF>Chr>BaA.The above results indicated that oil and PAH4 had a synergistic effect on cell toxicity,causing the collapse of mitochondrial membrane potential and promoting cell apoptosis.In addition,oil-based PAH4 had a higher impact on HL-7702 cells than HepG2 cells,indicating that HL-7702 cells are more sensitive to oil-based PAH4.The process of apoptosis is usually accompanied by the occurrence of intracellular oxidative stress.To investigate the oxidative stress effect and its mechanism of oil-based PAH4 on HepG2 and HL-7702 cells,the oxidative stress effect was described by measuring the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH Px),reactive oxygen species(ROS),and malondialdehyde(MDA)levels.The expression levels of related genes and proteins,AHR,AKR,EH,KEAP1,Nrf2,and HO-1,on the oxidative stress pathway were detected by qRT-PCR and Western blot to elucidate the mechanism of oxidative stress induced by oil-based PAH4.The results showed that oil-based PAH4 activated the AHR pathway,resulting in increased expression levels of genes and proteins of AHR,AKR,and EH,increased ROS level and MDA content,and increased activity of CYP1A,the key metabolic enzyme of PAHs.In addition,the increase in ROS levels inhibited the enzyme antioxidant system,resulting in a decrease in gene and protein expression levels of KEAP1,Nrf2,and HO-1,leading to a significant decrease in the activity of antioxidant enzymes,SOD,CAT,and GSH-Px.The expression levels of genes and proteins in HepG2 cells,as well as the activity of CYP1A,were lower than those in HL-7702 cells,indicating that HL-7702 cells were more sensitive to oil-based PAH4 than HepG2 cells.The MTT method was used to determine the effects of binary,ternary,and quaternary mixtures of oil-based PAH4 on the survival rate of HL-7702 cells.The concentration addition(CA)model and the combination index(CI)model were used to predict the combined toxicity of oil-based PAH4 mixtures on cells.The results showed that the activity of HL-7702 cells was significantly reduced after 48 hours of treatment with a mixture of oil-based PAHs,and there was a concentration-dependent relationship between the mixture of oil-based PAHs and the cell inhibition rate.In addition,the combined toxicity of BaA+BaP and BaA+Chr+BbF+BaP within the entire concentration range was antagonistic.The combined toxicity of BaA+Chr+BbF,BaA+Chr,BaA+BbF,and Chr+BbF within the entire concentration range was synergistic.The combined toxicity of Chr+BaP,BbF+BaP,BaA+Chr+BaP,BaA+BbF+BaP,and Chr+BbF+BaP had shifted from antagonism to synergy at a concentration of 15.83 μmol/L,20.56 μmol/L,8.45 μmol/L,14.07 μmol/L,and 13.60μmol/L,respectively.By comparing the dose reduction index(DRI)values of some binary and ternary combinations with synergistic effects,it was found that the DRI value of BbF was the largest,indicating that when the combined toxicity of oil-based PAH4 mixture was synergistic,the PAH that played a decisive role was BbF.This work is the first to investigate the toxicity and oxidative stress effects of oil-based PAH4 on human liver cells and elucidate the combined toxicity of oil-based PAH4 mixtures on human liver cells,providing a theoretical basis for in-depth research on the toxic effects of PAHs on humans.