Evaluating Effects And Mechanism Of Mineral Elements In Three Drinking Waters On Growth And Development And Physiological Function Of Body At Different Stages Based On Zebrafish Model

The incidence of chronic non-communicable diseases worldwide increases year by year,becoming an invisible"killer"threatening national health.More and more studies have found that daily intake of appropriate mineral elements especially K,Na,Ca,Mg and Zn plays an important role in the prevention of some chronic diseases,such as cardiovascular and cerebrovascular diseases,hypertension and hyperlipidemia.WHO points out that 5%to 20%of essential minerals must be obtained from drinking water.Since the beginning of the 21st century,the consumption of bottled water in China has been increasing rapidly.In 2018,the average consumption of bottled water reached 120 bottles per person（550 mL per bottle）.It is worth noting that the level of mineral elements in both bottled natural water and bottled purified water is lower than that in traditional tap water.Therefore,it is worth discussing whether long-term drinking bottled water will have adverse effects on the body.Based on this,the contents of mineral elements in three popular drinking waters（tap water TW,bottled natural water NW,bottled purified water PW）were determined.Zebrafish were used as the model animal to study the characteristics in different stages（（1）embryonic:cardiac development,（2）adolescence:growth and lipid metabolism,（3）pregnancy and offspring:reproductive ability and learning and memory ability）.The related mechanism of mineral elements on zebrafish cardiac development and reproductive ability were also discussed.Main research contents and conclusions are as follows:（1）Determination of mineral elements in three drinking watersAccording to GB/T 5750-2006,the conventional water quality indexes,zebrafish water indexes and mineral contents in three drinking waters were determined.The results showed that the water quality of the three drinking waters all met the national standard and they were all suitable for short-term breeding of zebrafish.Except Zn level,the contents of five main mineral elements（K,Na,Ca,Mg and Zn）:tap water>bottled natural water>>bottled purified water.The contents of five mineral elements in bottled purified water were 0.1503 mg/L,0.3022 mg/L,0.1192mg/L,0.0093 mg/L and 0.0341 mg/L respectively.（2）Effects of three drinking waters on growth and development of zebrafish in embryonic stage and mechanism of heart developmentThe toxicological model of zebrafish embryonic development was used.With embryonic water as control,three drinking waters were used to hatch zebrafish embryos for 3 days.The results showed that 3 dpf zebrafish in PWG showed delayed growth（larger yolk sac,shorter body length）and abnormal development（egg coagulation rate increased by 22%,hatching rate decreased by26%,heart rate decreased by 19.71%,spinal curvature,etc.）.After reducing Ca²⁺,K⁺,Na⁺levels in embryonic water,the heart rate of zebrafish in[Ca²⁺]=0 mM water decreased significantly（p<0.01）,suggesting that Ca deficiency is responsible for abnormal cardiac development in zebrafish hatched in PWG.The possible mechanism were as follows:（1）up-regulation of aldh1a2expression,followed by up-regulatiion of cspg2 expression through RA pathway;（2）down-regulation of grk5 and tmp4-tv1 expression,but not stc-1 expression and T3 level.（3）Effects of three drinking waters on growth and development,lipid metabolism and antioxidant capacity of zebrafish in adolescent stageUnder the state of feeding,three drinking waters were used to intervene the juvenile zebrafish for three months.The results showed that compared with NWG,Ca and Zn levels in 90 dpf zebrafish in PWG decreased,survival rate decreased by 26%,body length shorten 0.84 mm（p<0.01）,TG level lower 52.57%（p<0.05）,HDL-C level lower 32.07%（p<0.05）,MDA level higher 50.78%（p<0.01）.Positive correlation were found between body length and Ca,Mg levels in water（r=0.273,0.307,p<0.05）,TG level was negatively correlated with Ca level（r=-0.522,p<0.01）and Mg level（r=-0.540,p<0.001）,HDL-C level was positively correlated with Ca and Mg levels（r=0.229,0.227,p<0.05）.It is suggested that the lack of Ca and Mg in bottled purified water may lead to growth and development dysplasia,and there is a risk of lipid metabolism disorder at high TG and low HDL-C levels of zebrafish in adolescent stage.（4）Effects of three drinking waters on reproductive performance of zebrafish in maturation stage and learning and memory ability of offspringThe reproductive intervention experiment of pregnant zebrafish parents was conducted for 21days with three drinking waters,fish water as control.The results showed that the spawning amount of zebrafish in PWG decreased by 68%（p<0.05）,the number of live eggs decreased by83%（p<0.01）.Compared with control group,the Ca level in female zebrsfish was 17.51%lower（p<0.01）,the fecundity and pregnancy index decreased.VTG level,the expression of vtg-1 and vtg-3 in female zebrafish in PWG was 23%（p<0.05）,97.20%（p<0.05）,82.73%（p<0.001）lower than that in control group by q-PCR.The lack of Ca in bottled purified water down-regulated the expression of vtg-1 and vtg-3 in female zebrafish,resulting in the decline of reproductive capacity.When the offspring of zebrafish grew up to 90 dpf in different water,after a week of learning and memory association training,T maze experiment was carried out.The results showed that latency time of 90 dpf zebrafish offspring in NWG were 30.69 s longer than that in TWG（p<0.05）,and their trajectories were complex,which means they failed to form a good association between learning and memory.The latency time of the offspring of zebrafish transferred to tap water at 5dpf was 21.4 s,which was not significantly different from TWG（p>0.05）,indicating that the learning and memory deficiency of zebrafish in bottled water could be compensated by transferring to tap water at 5 dpf,suggesting that zinc was essential for the formation of learning and memory abilities of offspring zebrafish.