Studies On Metabolism And Transformation Of Typical Bisphenols In Eggs And Laying Hens

Environmental endocrine disrupting chemicals（EDCs）were defined by the U.S.Environmental Protection Agency（EPA）as“exogenous agent that interferes with synthesis,secretion,transport,metabolism,binding action,or elimination of natural blood-borne hormones that are present in the body and are responsible for homeostasis,reproduction,and developmental process.”EDCs have toxic effects such as interfering with the endocrine process of animals and human,"three causes"（carcinogenic,teratogenic,mutagenic）and inducing diabetes,which can harm human health through the food chain,even at very low concentration levels.Recently,the international research on the impact of EDCs on food safety is in full force,but there is still a blind spot in the whole chain migration and transformation of EDCs in"environment-feed-farmed animals-livestock and animal-derived foods".In this study,we develop a highly sensitive analytical method for the quantification of bisphenols,and then reveal the occurrence and fate of bisphenols in feeds and animal-derived foods.Furthermore,we reveal the transformation and metabolism of BPF in laying hens,and define the carry-over and transfer factor of bisphenols in the whole chain of"environment-feed-farmed animals-livestock and animal-derived foods".The quantity of data can therefore be helpful in the frame of risk assessment,especially for a comprehensive estimation of consumer exposure to the residues of bisphenols.Herein,we described a combination of enzymatic probe sonication（EPS）and ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）for quick extraction and simultaneous determination of eight important bisphenols（BPA,BPS,BPF,BPAF,BPB,BPP,BPAP,BPBP）in feeds and animal-derived foods.The limits of detection（LODs）were 0.02-0.05?g/kg,limits of quantification（LOQs）were 0.1-0.2?g/kg,mean recoveries were 81.2-106.0%with intra-day RSD0.6-9.7%and inter-day RSD≤12.5%.The linearity was acceptable,with R²≥0.996 in the concentration ranges of 0-50?g/L.Widespread occurrence of BPA,BPS,BPF,BPAF,and BPP were found,with detection frequencies of 65.2%,42.4%,33.7%,29.4%,28.3%,and 27.2%in the animal-derived foods,respectively.Two-tailed Pearson correlation analysis was also used to test the correlations among the concentration levels of individual bisphenols analyzed,and significant positive correlations were found between BPA and BPAF,BPS and BPF,BPS and BPAF,and BPF and BPAF,suggesting the co-occurrence and similarity in the sources of the animal-derived foods.Our studies demonstrated that BPF exposure did show a significant influence on egg production,with average laying rates of 82.8±2.3%,89.3±3.8%,82.2±4.3%and 75.6±3.3%for the control,low,medium,and high level groups,respectively.The effects of different levels of BPF exposure on laying performance followed a non-monotonic dose-response curve,since low level BPF（0.1 mg/kg）exposure did increase the laying rate,mean egg weight and daily feed intake,while high level BPF（2.5 mg/kg）exposure showed a decreasing trend.Since the non-monotonic dose-response curve is one of the typical characteristics of endocrine disruptors and is consistent with the natural hormonal effects in animals and humans.Our results revealed that BPF exposure may have endocrine disrupting effects on laying hens.BPF was detected in egg yolks on the second day after administration of BPF contaminated feed.Peak concentrations(C_max)were observed after 11 days,while the plateau levels of 4.52±1.21?g/kg,19.13±1.98?g/kg,and 69.12±5.12?g/kg for the low,medium,and high level groups,respectively,were achieved after 9 days of exposure and lasted until 4th day of the depuration period（18th of the experimental period）.BPF-contaminated diet caused the BPF concentration levels to be up to 4?g/kg（day 8）in the whole eggs.The tolerable daily intake（TDI）of BPA recommended by European Food Safety Authority（EFSA）was therefore exceeded;while for the high level group,only 4 days of exposure was needed to exceed this level.The residues of BPF in egg yolks were much higher than in whites,with concentration levels from the plateau phase of about 69.1±7.4?g/kg and 3.0±0.4?g/kg,both for the high level group.The residue levels of BPF in egg yolks were approximately 23 times higher than in egg whites.The concentration levels of BPF in liver of laying hens were much higher than that in blood,eggs and muscle,suggesting that liver was the major organism for bisphenols metabolism.The half-life time of BPF for the low,medium and high level groups were approximately5.8,4.5 and 4 days,respectively.The residues of BPF were below the LOQ level at day 14 of the depuration period for the low level group,and at day 28 for the medium and high level groups.Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry（UPLC-HRMS）Thermo QE HF was used to comparative study of the in vitro and in vivo metabolism of BPF.It was found that BPF was mainly hydroxylated,hydrolyzed and oxidized by cytochrome P450enzyme in chicken liver microsomes.Five phase I metabolites were identified,namely4-（Hydroxymethyl）phenol（m/z 123）,o-OH BPF（m/z 215）,GSH conjugated BPF（m/z 504）,and GSH conjugated o-OH BPF（m/z 520）.While the in vivo study of BPF in laying hens were demonstrated to be hydroxylation and binding reactions based on the glucuronidase and sulfate esterase enzyme.Six phase II metabolites were identified,namely hydroxylated BPF（m/z 215）,BPF-sulfate（m/z 279）,BPF-disulfate（m/z 359）,BPF-glucuronide（m/z 375）,BPF-diglucuronide（m/z 551）,and BPF-sulfate/glucuronide（m/z 455）.