| Cadmium(Cd)is a ubiquitous environmental contaminant.Cd could cause adverse effects on kidney,bone,lung and reproductive system.Because of the wide distribution of Cd in the environment and food contamination with Cd,the whole population is exposed to this metal from birth up until death.Diet is the main route of Cd exposure in the general population(in unpolluted areas,non-occupational exposure,non-smoking population).Studies have predicted that Cd exposure in the general population will increase in the coming decades and Cd as an environmental risk factor for the health of the general population should be drawn more attention.Hazard identification is the first key step in risk assessment.Kidney-based risk assessment establishes the urinary Cd threshold at 5.24 ?g/g creatinine,and tolerable dietary intake of Cd at 62 ?g/day per 70-kg person.There has been growing evidence that Cd has a direct toxic effect on bone and that exposure to this metal via the diet may occur at exposure levels lower than the threshold limits of dietary Cd intake and urinary Cd threshold.Whether to impose stricter dietary intake guidelines to strengthen health protection against Cd-induced bone injury is a particular concern in Cd health-risk assessment.Low calcium(Ca)intake is also one of etiological factors contributing to osteoporosis.Because Cd exposure and Ca intake occurs in the daily diet in the general population,the effect of Cd on osteotoxicity may be overestimated due to insufficient Ca intake.Few epidemiological studies on the association of low Cd exposure and decreased bone mineral density(BMD),osteoporosis,and increased fracture risk have taken into consideration the protective effect of Ca.Although several studies have adjusted the intake of Ca,it remained between 902–1081 mg,less than the Ca intake for adults aged 50 years and older(1000–1200 mg)recommended by the American Association of Clinical Endocrinologists and the American College of Endocrinology Clinical Practice Guidelines for the Diagnosis and Treatment of Postmenopausal Osteoporosis—2016.Therefore,the association of low dose exposures to cadmium below renal damage level and bone injury in population studies remains uncertain.Ca intake is particularly important in acquiring peak bone mass during young adulthood,which lowers risk of bone fracture later in life.Furthermore,excessive Ca consumption may have potential adverse effects on health.In addition,there are a few individuals in the general population who took more than the recommended upper limit of Ca through diet and supplements.The kidney is the sensitive target organ of Cd toxicity.At the same time,it is also the major Ca excretion and reabsorption organ.However,little is known about Ca exposure to Cd in the general population.Such data are difficult to obtain from population studies,and similar results from animal studies have not yet been obtained.Dietary Cd exposure level is different in the general population due to dietary habits,dietary structure.At present,it is not clear whether Ca supplementation has a protective effect against Cdinduced bone damage under different Cd level in the general population.Furthermore,previous studies have shown that enhanced Ca intake may prevent absorption of this toxic element and reduce accumulation of this metal in the organism.Interactions between Ca and Cd are regulated by hormones,nutrients,and other factors,including Ca and Cd dose.However,whether Ca supplementation can prevent Cd absorption and accumulation in the general population is unclear.Taking the above into account,the aim of this study was to identify whether Ca supplementation during exposure to different population-relevant doses of Cd can prevent Cd-induced bone damage under the tolerable upper intake level of Ca supplementation,and explore the mechanism from the FGF23/ Klotho axis.Results of this study have important implications for Cd health-risk assessment and development of effective dietary strategies to reduce the adverse health effects of Cd exposure.The main research contents are as follows: Chapter 1: Subchronic Toxicity Study of Calcium Intake Under Cadmium Exposure of the General Population: A Study on a Rat Model Exposure to Cadmium Objective: To evaluate the sub-chronic toxicity and lowest observed adverse-effect level(LOAEL)of Ca supplementation with normal dietary Cd levels in the general population using a rat model and to know whether dietary Ca supplementation may influence the body status of iron(Fe),zinc(Zn)and copper(Cu).Methods: According to procedures and methods for toxicological assessment of food,fifty young female Sprague–Dawley rats randomly were divided into 5 groups according to body weight.Rats were fed one of the five experimental AIN-93 G diet for 90 days: 0+0(Control);1mg Cd/kg+0(Cd group),1mg Cd/kg+0.15%Ca(low-Ca supplementation group);1mg Cd/kg+0.4%Ca(medium-Ca supplementation group);1mg Cd/kg+0.6% Ca(high-Ca supplementation group).At the end of the experiment,samples of blood were collected for hematological and biochemical analysis,respectively.The heart,liver,spleen,and kidneys(paired)were excised,examined,and weighed.The kidneys were removed for histopathological examinations.And serum Ca,Fe,Zn,Cu,the apparent absorption rate of Ca,Fe,Zn,Cu,the concentration of Ca,Fe,Zn,Cu in the liver and the kidney were detected.Results: Compared with the control group,(1)all Ca supplement group showed there were no statistically significant differences in body weight,total intake,feed efficiency,absolute organ weight or relative organ weight(p>0.05).(2)Significant increases,or a tendency thereto,in blood urea nitrogen was noted in the medium-and high-Ca supplement groups(p<0.05).But there were no statistically significant differences in the low-Ca supplement group(p>0.05).(3)Kidney histopathology results showed increased diffuse tubular epithelial cell calcification in the proximal tubules,with higher grade and incidence in the medium-and high-Ca supplement groups.(4)Significant increases,or a tendency thereto,in white blood cell count,monocyte count,and lymphocyte count were noted for the high-Ca supplementation group(p<0.05).But there were no statistically significant differences in the low-and medium-Ca supplement group(p>0.05).(5)Significant decreases,or a tendency thereto,were noted in the high-Ca supplementation group in Fe content of the kidney and liver(p<0.05).But there were no statistically significant differences in lowand medium-Ca supplement group(p>0.05).Conclusion: According to the results of the kidney histopathology and serum blood urea nitrogen in the medium-Ca supplementation group,the lowest observed adverse-effect level of calcium carbonate exposure to a 1 mg Cd/kg diet in this 90-day oral toxicity study was considered to be lower than the Ca content in the diet of medium-Ca supplementation group(+0.4% Ca)for female rats.Current findings on the LOAEL of Ca exposed to Cd are lower relative to those for Ca alone(+0.6% Ca).Chapter 2: Effect of Different Dietary Ca supplementation on the Cadmium Absorption under General Population-Relevant Doses Dietary Cadmium in Young Female Rats Objective: To investigated whether Ca supplementation may reduce the Cd absorption and kidney Cd accumulation in the sub-chronic rat model of treated with Cd at the general population dietary exposure.Methods: Through the detection of the apparent absorption rate of Cd and the Cd concentration in liver and kidney in the animal model mentioned above,to determine whether Ca supplementation may reduce the Cd absorption in the sub-chronic rat model of treated with Cd at the general population dietary exposure.The expression of calcium binding protein(CaBP)gene in small intestine and kidney was detected to explain the possible mechanism of Ca supplementation on Cd absorption.Results: There was no significant difference in the apparent absorption of Ca between the Ca supplementation group and the Cd group(p>0.05),but there was a decreasing trend in the high-Ca supplementation group.There was no significant difference in apparent absorption of Cd,iron,zinc and copper(p>0.05).The concentration of Cd in the kidney cortex in the control group was lower than the detection limits.Compared with the Cd group,the concentration of Cd in the kidney cortex and liver increased in the low-Ca supplement group(p<0.05).However,there was no significant difference in renal Cd concentration in the medium-and high-Ca supplementation group compared with the Cd group(p>0.05).The expression of small intestine calcium binding protein(CaBP)gene was consistent with the trend of renal Cd concentration.The expression of small intestine CaBP in the low-Ca supplement group was significantly higher than that in the Cd group(p<0.05).But there were no statistically significant differences in low-and medium-Ca supplement group compared with the Cd group(p>0.05).Conclusion: Dietary Ca supplement(0.65%~1.1%)does not decrease the absorption and the concentration of Cd in liver and kidney.CaBP in small intestine may be associated with this process.Chapter 3: Protective Effects of Moderate Ca Supplementation against Cd-Induced Bone Damage Objective: To identify whether Ca supplementation during exposure to different population-relevant doses of Cd can prevent Cd-induced bone damage under the tolerable upper intake level of Ca supplementation.Methods: Seventy young female Sprague–Dawley rats randomly were divided into 7 groups according to body weight.Rats were fed one of the seven experimental AIN-93 G diet for 90 days: 0+0(control);1mg Cd/kg+0(low-Cd group);1mg Cd/kg+ 0.4% Ca(low-Cd plus Ca group);5mg Cd/kg+0(medium-Cd group);5mg Cd/kg+ 0.4% Ca(medium-Cd plus Ca group);50mg Cd/kg+0(high-Cd group);50mg Cd/kg+ 0.4% Ca(high-Cd plus Ca group).At the end of the experiment,information on the Cd exposures in rats(the average daily Cd intake,the Cd content in the kidney,liver,and femur)were collected.Bone toxicity effect indicators(femoral bone Ca content,bone biomechanics,serum bone formation markers,bone histology and the OPG/RANKL system of bone metabolism gene and protein expression)were examined.Results: Ca supplementation significantly decreased Cd-induced bone microstructure damage,increased bone biomechanics(p<0.05),serum bone formation marker level(p<0.05)and increase in OPG gene and protein expression(osteogenic markers)exposure to the 5 and 50 mg Cd/kg diets.However,it had no impact on these indicators under the 1 mg Cd/kg diet,with the exception of expression of osteogenic marker genes and protein in the OPG/RANKL system.Conclusion: Ca supplementation has a positive effect on bone formation and bone quality against the damaging impact of Cd,especially with exposure to the 5 mg and 50 mg Cd/kg diet.However,to 1 mg Cd/kg diet low Cd group,Ca supplementation had no effect on bone biomechanics(a macro sensitive indicator).Chapter 4: Exploration of Potential Mechanisms underlying the Effect of Ca in Protecting against Cd-induced Bone Damage via the FGF23/ klotho axis Objective: To investigate whether protective influence of Ca against Cd-induced disorders in bone metabolism may be related to the FGF23/Klotho axis.Methods: Serum 1,25-dihydroxy vitamin D3,serum Klotho protein and FGF23 protein in the above animal models were detected by enzyme-linked immunosorbent assay,and the expressions of Klotho and Fgfr1 in kidney tissues were detected by real-time quantitative PCR and immunohistochemical staining.Femur FGF23 gene and protein was detected by real-time quantitative PCR,immunohistochemical staining and Western blot.As well as Wnt/?-catenin pathway,genes and proteins related to proliferation and differentiation of osteoblasts were detected by real-time quantitative PCR and immunohistochemical staining.Results: The expression of Klotho gene,serum Klotho protein and kidney tissue Klotho protein was decreased after Ca supplementation intervention.Moreover,gene expression of Fgfr1 significantly increased with exposure to the 5 and 50 mg Cd/kg diets.Gene expression of Napi2 a significantly decreased in rats treated with Cd plus Ca compared with rats treated with Cd alone with low,moderate,and high Cd exposures.There was a significant increase in the expression of Cyp27b1 in rats exposed to Cd plus Ca compared with rats treated with Cd alone when fed the 1 and 50 mg Cd/kg diets.The gene expression of Cyp24a1 showed a tendency to increase in rats treated with high level Cd plus Ca compared with those treated with high level Cd alone,but this was not statistically significant.After Ca supplementation intervention,a significant increase in FGF23 gene and protein expression in femur was observed.Increased expression of the Wnt target genes Tcf1 and Axin2 was observed in rats treated with Cd plus Ca compared with those treated with Cd alone in the 50 mg Cd/kg diet group.Increased expression of C-myc protein,Runx2 protein and Col1a1 gene and protein was observed in rats treated with Cd plus Ca compared with those treated with Cd alone.Conclusion: The protective effect of Ca supplementation against the damaging impact of Cd on the skeleton may be related to its impact on the FGF23/Klotho axis,including down-regulation of Klotho mediation of the Wnt/?-catenin pathway in osteoblasts and the function of Klotho as a specific receptor complex with FGF23 in vitamin D synthesis in the kidney and as a coordinator of renal phosphate handling to match bone mineralization.The conclusions of this paper(1)The lowest observed adverse-effect level of calcium carbonate exposure to a 1 mg Cd/kg diet in this 90-day oral toxicity study was considered to be lower than the Ca content in the diet of mediumCa supplementation group for female rats.The LOAEL of Ca exposed to Cd are lower relative to those for Ca alone.(2)Dietary Ca supplement(0.65%~1.1%)does not decrease the absorption and the concentration of Cd in liver and kidney.(3)Ca supplementation has a positive effect on bone formation and bone quality against the damaging impact of Cd,especially with exposure to the 5 mg and 50 mg Cd/kg diet.However,to 1 mg Cd/kg diet low Cd group,Ca supplementation had no effect on bone biomechanics(a macro sensitive indicator).(4)Protective effect of Ca supplementation against Cd-induced bone damage may be mediated by the FGF23/klotho axis and the OPG/RANKL system. |
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