Variation And Regulation Of Cadmium Relative Bioavailability In Leafy Vegetables

Cadmium（Cd）is the primary environmental pollutant of agricultural soil contamination in China,and it has become a major environmental pollution problem.Soil Cd can be taken up by crops and enter the food chain,causing profound and long-lasting health effects including chronic diseases and acute diseases.However,most studies were conducted focusing on Cd in rice,and Cd in vegetables has received less attention,while vegetable consumption is identified as a non-negligible contributor to Cd exposure.Moreover,among vegetables,leafy vegetables are of the greatest concern since they tend to accumulate higher Cd concentrations than rootstalk and legume vegetables.More importantly,previous studies mainly focused on the process of Cd being transferred from soil to crops in the‘soil-plant’system,while limited studies have evaluated to what extent Cd in food can be absorbed by humans when ingested and what factors drive this process in the‘plant-human’system,which has effects on the accurate evaluation of human health and the development of regulation measures.Besides,studies have shown that mineral element（calcium,iron,zinc,etc.）and organic acid（phytic acid,etc.）may affect Cd absorption in the gastrointestinal tract,but the mechanisms influencing Cd relative bioavailability（Cd RBA,relative to Cd Cl₂）variation in leafy vegetables remains unclear.Thus,Cd-contaminated soil was collected from a crop vegetable growing farmland near a mining area,and 14 vegetables of 3species varying in genotypes were grown in a greenhouse in the Cd-contaminated soil(7.30mg Cd g^–1)and harvested for Cd RBA measurement using a mouse assay.Leafy vegetables were amended into basal mouse feed at 1:1.After 10-days exposure,kidneys were collected to evaluate Cd RBA in various leafy vegetables.Weekly Cd intake via vegetable consumption was calculated based on Cd RBA.Furthermore,a Ca biofortification trial was performed by coupling in vivo mouse bioassay to reveal that Ca is identified as a driver of controlling Cd RBA.According to the In Vitro Gastrointestinal（IVG）method,an in vitro test was developed to determine Ca bioaccessibility,exploring the mechanism of phytic acid in mitigating Cd RBA.This indicates a possibility of applying a novel strategy of Ca manipulation to reduce Cd exposure and potential health risk.The main findings are as follows:（1）Significant variation in vegetable Cd accumulation was observed with Cd concentrations in the shoot of 4 water spinach,4 amaranth and 6 pakchoi varieties ranging from 2.56±0.12 to 3.92±0.21 mg kg^–1 dw,from 2.20±0.09 to 4.26±0.02 mg kg^–1dw,and 2.88±0.14 to 6.21±0.06 mg kg^–1dw,respectively,which indicates that long-term dietary consumption of vegetables grown in the soil may cause health risks of Cd exposure.In addition,Ca contents are high in leafy vegetables.Similar to variation in shoot Cd accumulation,shoot Ca concentrations were also higher in pakchoi(27,000–31,000 and 94–144 mg kg^–1)than in amaranth(13,000–26,000 and 75–112 mg kg^–1)and water spinach(13,000–18,000mand 51–83 mg kg^–1).This was likely due to Cd uptake and transport in plants via cation channels which are responsible for Ca uptake,distribution,and accumulation,leading to correlated mineral Ca and Cd accumulation in the edible portions in the‘soil-plant’system.（2）To evaluate the variation in Cd oral bioavailability in leafy vegetables,an in vivo mouse bioassay was conducted to determine Cd RBA.Cd RBA varied considerably among vegetables from 22.9±2.12%to 77.2±4.46%,and it in water spinach,amaranth,and pakchoi were 61.0±11.2%to 77.2±4.46%,36.9±6.98%to75.7±6.58%,22.9±2.12%to 51.1±10.7%,respectively.Unlike results in rice by Zhao et al.,who reported a strong positive correlation（R²=0.37）between Cd-RBA and Cd concentration of rice,a negative correlation（R²=0.43）was observed between Cd RBA and Cd concentration.In addition,a strong negative correlation（R²=0.62）between Cd RBA and Ca concentration was observed,suggesting 62%of the variance in Cd RBA was explained by Ca concentration.The results suggested Ca concentration was likely an important driver of Cd RBA,confirming the role of Ca in reducing Cd bioavailability.Sharing the same absorption and transport cation channels in intestinal epithelium might explain the observed positive correlation between Cd RBA and Ca due to the similar ionic radius.Ca²⁺would compete with Cd²⁺for binding to transporter molecules on the apical side of the small intestinal epithelium,reducing Cd absorption via cation channels,thereby leading to decreased Cd RBA.Based on total Cd concentrations,consumption of all 14 vegetables would result in Cd intakes in adults exceeding the threshold of 5.8mg Cd kg^–1 bw week^–1 proposed by JECFA,in the order of water spinach(8.52–13.1,mean 10.4mg kg^–1 bw week^–1)<amaranth(7.35–15.6,mean 12.2mg kg^–1 bw week^–1)<pakchoi(9.59–20.7,mean 14.0mg kg^–1 bw week^–1),suggesting pakchoi would pose a higher Cd health risk to consumers when assessed based on total Cd concentration.However,when Cd RBA was considered,Cd intake varied in the reverse order of water spinach>amaranth>pakchoi,being 5.29–10.1,4.85–9.18,and4.19–7.26mg kg^–1 bw week^–1,mean 7.64,6.91,and 5.64mg kg^–1 bw week^–1)m,respectively.Overall,although pakchoi contained a higher Cd concentration in the edible portion than water spinach and amaranth,it exhibited lower Cd oral bioavailability,thus leading to lower Cd exposure.This proves that to screen vegetable species or varieties of low Cd risk,the commonly used Cd concentration-based assessment may produce overestimated evaluations.To improve Cd exposure and risk assessment process,determination of Cd RBA in vegetables is required.（3）To ascertain the identified role of Ca in decreasing vegetable Cd RBA,4varieties of leafy vegetables were selected for Ca bioforification trial and an in vivo mouse bioassay was conducted.Foliar application of Ca fertilizer significantly increased Ca concentration,while low increase was observed for Cd concentration,which suggested that with different absorption pathways,i.e.,absorption of foliar-applied Ca by leaves,and absorption of Cd by roots,there may be weak competition between Ca and Cd for the absorption and translocation channels.For a water spinach species,Ca concentration increased by 30%(11.5 mg g^-1 to 14.8 mg g^-1),while a particular decrease in Cd RBA from 90.5±12.0%to 63.5±5.53%was observed for the water spinach.The results suggested that when elevating Ca in vegetables at a high degree,Cd RBA in vegetables can be reduced by antagonism between Ca²⁺and Cd²⁺in intestine.For other 3 leafy vegetables,low levels of Ca increase（4.89–10.7%）did not significantly decrease Cd RBA.The study further proved that when elevating Ca in vegetables at a high degree,Cd bioavailability can be reduced.Thus,dietary supplementation with Ca can reduce Cd oral bioavailability in vegetables.（4）To explain the reason that Cd RBA in vegetables with higher Ca contents were far lower than that in rice,an in vitro gastrointestinal（IVG）Ca bioaccessibility test was performed.By comparing with the contents of phytic acid,another factor affecting Cd oral bioavailability was ascertained.In this study,water spinach(4.10–5.96,mean 5.23mg g^–1)and amaranth(3.14–32.8,mean 19.6 mg g^–1)contained higher phytic acid than pakchoi(1.45–3.04,mean 2.51 mg g^–1).Furthermore,results of in vitro method showed that low Ca solubility in simulated intestinal fluids for water spinach（20.5–26.4%）and amaranth（0.29–0.82%）,while Ca solubility was high in pakchoi（61.8–67.3%）.The high phytic acid contents in water spinach and pakchoi may significantly decrease Ca bioactivity in the gut,lowering its role in competing with Cd for absorption transporters,thereby contributing to the observed high values of Cd RBA.It should be noted that for vegetables with low Ca concentrations,applying Ca biofortification trial can reduce Cd RBA effectively.As for vegetables with initial high Ca and phytic acid concentrations,taking measures to activate endogenous Ca by counteracting the effects of phytic acid in decreasing Ca intestinal solubility might be an alternative strategy to decrease Cd bioavailability and Cd exposure.This study is important for proposing a possibility of applying a novel strategy to decrease Cd exposure,which provides theoretical basis and supporting information to reduce potential health risks.