Preliminary Study On Toxicity And Internal Exposure Markers Of Low Dose Combined Exposure Of Four Polycyclic Aromatic Hydrocarbons In Rats

Polycyclic aromatic hydrocarbons（PAHs）are semi-volatile organic compounds formed by the consistence of two or more benzene rings.PAHs in food are mainly produced in the process of production,such as being polluted,fried,smoked and baked.The dietary exposure of people is a combination of PAHs,and the total content of four PAHs（benzo[a]pyrene,benzo[a]anthracene,chrysene and benzo[b]fluoranthene,hereinafter referred to as PAH4）has been proposed as the limit standard of PAHs in food,but the limit standard of PAHs in food in China is limited to benzo [a] pyrene.At present,there are few studies on the toxicity of low-dose combined exposure of various PAHs.Therefore,it is important to explore the toxic effect of low-dose combined exposure and the internal exposure markers for the formulation and monitoring of PAHs limit in food.This study is divided into two parts: first,the preliminary study on the toxicity of four PAHs combined low dose exposure;the second is to screen the internal surface exposure markers of four kinds of PAHs.Section 1 : Toxic damage to rats induced by low dose combined exposure of PAH4.Objective: To explore the toxic effect of combined exposure to low dose of PAH4 on SD rats.Methods:（1）Two batches of 8-week-old male Sprague Dawley rats were purchased and randomly divided into 5 groups with 10 rats in each group,and the rats were treated with 0,10 μg/kg·bw,50 μg/kg·bw,250 μg/kg·bw and 1000μg/kg·bw respectively.Based on the content of PAH4 detected in food,the proportion of PAH4 in intragastric solution was determined as benzo[a]pyrene: benzo[a]anthracene: chrysene: benzo[b]fluoranthene =0.99:2.92:2.68:1.68.Two groups of rats were killed at 7 days and 30 days after exposure respectively,and biological samples such as serum,urine and organs were taken.（2）The pathological changes of rat liver were observed.（3）Serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）were detected.（4）Catalase（CAT）,malondialdehyde（MDA）,glutathione peroxidase（GSH PX）and superoxide dismutase（SOD）were detected.（5）Total cholesterol（TC）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）in serum and TC,TG in liver were detected.（6）The serum samples of rats were collected after 30 days of exposure,and a full spectrum metabonomics method was established based on Ultra Performance Liquid Chromatography Tandem mass spectrometry（UPLC-MS/MS）to detect the different metabolites between each exposure group and the control group.Results:（1）Compared with the control group,the liver structure of rats in the exposed group was significantly abnormal,the hepatic sinuses were enlarged,and the hepatocytes showed balloon like changes;the liver organ ratio in the 1000 μg/kg·bw group was significantly increased,but the overall body weight had no significant change.（2）After 7 days and 30 days of exposure,the serum ast of rats in 1000μg/kg·bw group increased significantly（P < 0.05）,but the ALT did not change significantly.（3）GSH-PX in serum of rats in the exposure group decreased significantly at 7 days,but increased significantly at 30 days;SOD in the exposure group increased significantly at 7 days,but there was no difference at 30 days;there was no significant change in CAT,MDA,and SOD at 30 days.（4）After 7 days of exposure,the serum HDL-C in 50 μg/kg·bw and above groups decreased significantly（P < 0.05）and the liver TG increased significantly in 50μg/kg·bw and above groups;after 30 days of exposure,HDL-C in serum of rats in1000 μg/kg·bw group decreased significantly and TC in liver increased significantly,while TG in serum of rats in 250 μg/kg·bw group increased significantly（P < 0.05）,and the TG in liver of 50 μg/kg·bw and above groups increased significantly.（5）After 30 days of exposure,a total of 1349 metabolites were detected in each exposure group.Compared with the control group,10,14,19 and 10 different metabolites were selected in the four dose groups.The enriched pathways included glyceride metabolism pathway,glycerophospholipidipid metabolism pathway,phosphoinositide metabolism pathway,phosphatidylinositol signaling system,steroid hormone biosynthesis and GPI anchored protein biosynthesis The common differential metabolite is DG（16:1₁8:2）.Conclusion: Low dose PAH4 combined exposure can cause some observable liver damage,oxidative stress and dyslipidemia,and can lead to significant metabolic changes,which mainly affect the related pathways of lipid metabolism,and the changes are complex and changeable.Section 2 :Study on exposure markers in low dose combined exposure of PAH4Objective: To explore the metabolites of internal exposure markers after low dose combined exposure to PAH4 in rats.Methods:（1）The animals were reared and grouped as above.24-hour urine was collected at 7 and 30 days after exposure.（2）Based on UPLC-MS/MS,a metabonomics method was established to detect1-hydroxypyrene（1-OHP）and 3-hydroxychrysene（3-OH-chrysene）in urine.The contents of chrysene,3-hydroxybenzo[a]anthracene（3-OH-Ba A）and3-hydroxybenzo[a]pyrene（3-OH-Ba P）in urine were determined.Results:Compared with the control group,there was no significant difference in the content of 1-OHP in urine after 7 days and 30 days of exposure.The content of3-oh-chrysene,3-oh-baa and 3-oh-bap in urine of the exposure group had a significant dose-dependent effect,in which the content of 3-oh-chrysene was relatively high and had a good time-dependent effect.Conclusion: 3-OH-chrysene in urine metabolites is a relatively good internal exposure marker for PAH4 low dose combined exposure.