Bioaccessibility And Health Risks Of 2,6-Di-Tert-Butyl-Hydroxytoluene

2,6-Di-tert-butyl-hydroxytoluene（BHT）,as one of the most commonly used antioxidants,is widely used in foods,cosmetics and industrial products.The widespread use of BHT results in its release into environment,such as indoor dust,sediment,and water.In addition,more and more toxicological evidence shows that BHT has endocrine disrupting effects,organ toxicity,carcinogenicity,and reproductive and developmental toxicity,and its metabolites are more toxic than the parent compound.Based on its widespread use and toxic effects,it is important to understand its human exposure level and assess its health risk.Understanding environmental occurrence of BHT and its metabolites can accurately assess their environmental exposure level.Bioaccessibility can accurately assess the human exposure level of pollutants and is an important parameter in health risk assessment.Tissue distribution and in vivo metabolism of BHT are essential for assessing its health risks.The impact of BHT on the bioavailability of co-ingested persistent organic pollutants is also an important category for evaluating the health risks of BHT.In this work,the target pollutant is BHT,and the following studies are carried out:（1）Analytical method establishment and environmental occurrence:The analytical method of pre-column derivatization combined with gas chromatograph-mass spectrometer（GC-MS）was established.The analytical method had high sensitivity（method detection limit:0.65-2.67 ng/g）,good reproducibility（relative standard deviation<10.6%）,and satisfactory recovery（71.1-118%）.The method was applied to analyze BHT and its four main transformation products in 11 indoor dust samples from Nanjing,China,and 26 sediment samples from Tai Lake,China.The concentrations of BHT in indoor dust and sediment were 0.22-47.4μg/g and 0.09-6.93μg/g,respectively.2,6-Di-tert-butylcyclohexa-2,5-diene-1,4-dione（BHT-Q）was the dominant transformation product of BHT in indoor dust and sediment,with concentrations of0.28-1.77μg/g and 0.02-1.36μg/g,followed by 3,5-di-tert-butyl-4-hydroxybenzaldehyde（BHT-CHO）,3,5-di-tert-butyl-4-hydroxybenzyl alcohol（BHT-OH）and 3,5-di-tert-butyl-4-hydroxybenzoic acid（BHT-COOH）.In addition,correlation analysis was conducted between BHT and its transformation products in indoor dust and sediment.It was shown that a strong positive correlation was found between BHT and its transformation products-BHT-OH and BHT-COOH in indoor dust（r=0.79,r=0.76,p<0.01）,indicating that the degradation of BHT may be the main source of BHT-OH and BHT-COOH in indoor dust.Correlation between BHT and its transformation products in sediment was poor（r=0.05-0.29,p>0.05）,indicating that the source of BHT transformation products in the sediment was not only from the degradation of BHT,but also from other sources.（2）BHT bioaccessibility measured by in vitro gastrointestinal test coupled with Caco-2 cell model:An in vitro gastrointestinal simulation method combined with Caco-2 cell model was used to investigate the effects of nutrient states（i.e.,fasted（as control）,lipid,carbohydrate,fiber,and protein）on BHT intestinal absorption,metabolism,and bioaccessibility.It was found that nutrition constituents decreased the intestinal absorption rate.The absorption rate of BHT by Caco-2 cells was fasted(4.26±0.72 h^-1)>carbohydrate(2.36±0.11 h^-1)>fiber(1.39±0.09 h^-1)≈lipid(1.34±0.05 h^-1)>protein(1.15±0.05 h^-1).In addition,metabolism of BHT only occurred in fasted and lipid treatments.In the fasted state,the two metabolites 2,6-di-tert-butyl-4-hydroxy-4-methyl-2,5-cyclohexadione（BHT-quinol）and BHT-Q were detected and mainly distributed in the cells,accounting for 12.4±1.01%and 13.5±1.07%of initial BHT dose.In the lipid state,only BHT-Q was detected,and the content of BHT-Q in the cells was 7.56±0.30%of initial BHT dose.Nutrition constituents intake also reduced the bioaccessibility of BHT.Bioaccessibility of BHT was fasted（100±11.5%）>protein（83.1±2.69%）>fiber（65.8±2.67%）>carbohydrate（56.8±1.58%）≈lipid（56.7±0.82%）.Solid phase microextraction test combined with a self-constructed computational in vitro kinetic model suggested that the nutrition constituents may bind to BHT to reduce its free concentration,and reduce the bioaccessibility.Understanding the effects of nutrition constituents on the bioaccessibility of BHT can provide a theoretical basis for reducing the health risks of BHT through dietary approaches.（3）BHT bioavailability-the distribution,metabolism,and excretion of BHT in mammals:Studying on the in vivo distribution and metabolism of BHT can clarify the biological end points,and accurately calculate its bioavailability;meanwhile,the study of the distribution and metabolism of BHT in mammals is also an important category for evaluating its health risks.Tissue distribution,excretion,and metabolism of BHT in female Balb/c mice by a single gavage were investigated.It was found that BHT was mainly accumulated in metabolism-related organs（ie.,liver and kidney）with the order of AUC_{0-120 h}（area under the concentration-time curve from 0 to 120 h）of BHT in various tissues as liver（206 h·μg/g）>kidney（162 h·μg/g）>adipose（59.0 h·μg/g）>blood（0.30 h·μg/m L）.For metabolites,BHT-quinol was the dominant and preferentially accumulated in liver with a AUC_{0-120 h} value of 150 h·μg/g vs 0.01-11.5h·μg/g in other tissues.BHT-COOH was the major metabolite in excreta,with total excretion of 1.77±0.16%of initial BHT dose,which was 3-10 times higher than those of other four metabolites BHT-quinol,BHT-Q,BHT-CHO and BHT-OH（0.17-0.59%）.The excretion of BHT and the five main metabolites was prone to via feces with value of 25.1±0.16%of the initial dose compared with urine of 1.28±0.05%.Other possible BHT metabolites in excreta were also identified through high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry（HPLC-Q-TOF-MS/MS）analysis,and the possible metabolic pathways of BHT were elucidated as the oxidation of the para-methyl,tert-butyl groups,and benzene ring.The preferred accumulation of BHT and its metabolites in liver and kidney indicates that liver and kidney can be considered as biological endpoints for bioavailability determination.At the same time,the preferred accumulation of BHT and metabolites in liver and kidney implies potential hepatotoxicity and nephrotoxicity.（4）The effect of food antioxidants represented by BHT on the bioavailability of coingested organic contaminants:As a typical food antioxidant,BHT and persistent organic pollutants can be exposed to humans through ingestion pathways,and whether BHT affects the bioavailability of co-ingested persistent organic pollutants is also an important component in assessing BHT health risks.Bioavailability and excretion of perfluorooctanoic acid（PFOA）under the influence of co-ingested with BHT and other commonly used antioxidants（e.g.,syntheticα-tocopherol（αT）and tea polyphenol（TP））were investigated by use of mouse model.The results showed that the relative bioavailability of PFOA（PFOA-RBA）in control group（i.e.,without antioxidant exposure）was 25.0±6.25%,while high dose ofαT（100 mg/kg）treatment resulted in significantly（p<0.05）increased PFOA-RBA（36.1±4.92%）.This may be associated with significantly elevated RNA expression of organic anion transporter 2（Oat2）in liver under high dose ofαT exposure,which in turn promoted the transport of PFOA from the blood to the liver,and increased PFOA-RBA.Compared with control group（4.65±0.92%）,low dose of TP（12.5 mg/kg）treatment significantly（p<0.05）decreased urinary excretion of PFOA（1.40±0.37%）.This may be associated with enhanced renal reabsorption of PFOA through organic anion transporting polypeptide1a1（Oatp1a1）in kidney.In addition,low dose of BHT（1.56 mg/kg）andαT（12.5mg/kg）treatments reduced intestinal permeability,which to some extent may be attributed to the increase of PFOA excretion.