Consumption of Low-Dosage New Drinking Water DBPs (NAs) Mixture Disrupts Young Rats Metabolism via Gut Microbiome Alteration
Author:Zhu Jian Qiang
Supervisor:zhao mei rong zhang quan
N-nitrosamines(NAs)are an emerging group of disinfection by-products that occurs as a mixture in drinking water at home and abroad.NAs are highly carcinogenic as evidenced by the high concentration exposure in previous studies.However,detected NAs concentrations in drinking water are significantly lower than their No Observed Effect Concentrations(NOEC)that were generated from the individual exposure assessment.Therefore,the potential health risk of environmentally relevant NAs mixture exposure in drinking water needs to be carried out.In this research,six NAs were selected because of their high occurrences and in drinking water in China.In this study,the infant SD(Sprague-Dawley)rats(Postnatal day 28,PND 28)were exposed to the selected NAs mixture at environmentally relevant concentrations in drinking water(Group L)and 10 times higher in drinking water(Group M)respectively by gavage for one week.In this study,the effects of NAs mixture exposure on body weight,metabolic phenotype and intestinal flora of infant rats were analyzed comprehensively,aiming to generate new health risk data of low-dose NAs mixture exposure and provide technical and theoretical support for drinking water safety management.Main research results are stated as follows:(1)The obesogenic gut microbiota profiles responded to NAs mixture were observed in a gender-specific manner.When comparing infant female rats exposed to mixture of NAs(Group M and Group L)with the control group(Group CON),no significant differences in terms of body weight gain(WG)and serum triglyceride(TG)concentrations were noticed after one week;On the other hand,although no significant differences in terms of WG and serum TG concentrations between infant male rats of group L and group CON were observed,the WG and serum TG concentrations of infant male rats exposed to the NA mixture in group M were significantly higher.Accordingly,no significant disturbance of intestinal flora of infant female rats after NAs mixture exposure was observed.The male rats in group M,however,significant alterations were observed in the gut microbiota after exposure.Specifically,the principal coordinate analysis of weighted UniFrac distances(Weighted unifrac-PCoA)showed that,no significant separation of infant female rats’ gut microbiota among group M,group L and group CON could be observed,while gut microbiota of infant male rats in three groups could be obviously separated.Moreover,the results of linear discriminat analysis(LDA)and linear discriminant analysis effect Size(LEfSe)showed almost no differences of intestinal flora between infant female rats of the two exposure groups,namely group M and group L,and the group CON.For infant male rats,the intestinal flora of exposure groups(Group M and Group L),especially the group M,was significantly different from that of the group CON.Besides,according to the composition of intestinal flora at phylum level,no significant differences of intestinal flora between infant female rats of exposure groups(Group M and Group L)and group CON were observed.Nevertheless,though the intestinal flora of infant male rats of group L and group CON had no significant differences,the relative abundances of Firmicutes and Bacteroidetes in the intestinal tract of infant male rats significantly differed between group M and group CON.The ratio of Firmicutes and Bacteroidetes(F/B)of group M was 2.13 times the F/B ratio of group CON.Furthermore,relative abundances of two obesity-related genera,Alistipes and Ruminococcus,of infant male rats of group M was higher than that of group CON.Spearman correlation analysis indicated that 32 of the 56 genera of infant male rats in group M were significantly correlated to biological traits of the hosts(p < 0.05).(2)The gender-dimorphic metabolomic profiles in rats were also observed after NAs mixture exposure :Analysis of serum and urine metabolites could comprehensively reflect the metabolic response of organisms to exogenous stimulation.6 NAs at detectable doses in drinking water were chosen as target compounds.With NAs administered to infant rats,characteristics of laboratory animals’ serum and urine metabolism phenotype were analyzed.First of all,analysis of blood biochemical indices indicated that exposure of infant female rats of group L to mixture of NAs resulted in no significant differences in serum Aspartate aminotransferase(AST)and other blood indices in serum compared to group CON but for infant male rats of group L,AST in serum was comparatively significantly low,an obvious gender-specific differences.Secondly,principal component analysis(PCA)and partial least squaresdiscriminant analysis(PLS-DA)of serum and urine metabolites of infant female and male rats indicated that group L could be well distinguished from group CON.Finally,metabolic pathway enrichment analysis of differential metabolites in serum indicated that in serums of infant female rats exposed to mixture of NAs in group L,only 2 metabolic pathways(Taurine and hypotaurine metabolism,Glycerophospholipid metabolism)were found to be significantly disturbed while in serums of infant male rats exposed to mixture of NAs in group L,8 metabolic pathways(D-Glutamine and D-glutamate metabolism,Alanine,aspartate and glutamate metabolism,Nitrogen metabolism,Aminoacyl-tRNA biosynthesis,Arginine and proline metabolism,Glycine,serine and threonine metabolism,Valine,leucine and isoleucine biosynthesis,Glycerophospholipid metabolism)were found to be significantly disturbed.The metabolic pathway enrichment analysis of differential metabolites in urine indicated that in urine of infant female rats exposed to mixture of NAs in the group L,only 1 metabolic pathway(Glycine,serine and threonine metabolism)was found to be significantly disturbed while in urine of infant male rats exposed to mixture of NAs in the group L,3 metabolic pathways(Phenylalanine,tyrosine and tryptophan biosynthesis,Phenylalanine metabolism,Valine,leucine and isoleucine biosynthesis)were found to be significantly disturbed.These results indicated that effects of exposure to mixture of low-dose NAs on metabolism phenotype of infant male rats were more significant than that of female rats.(3)The strong correlations between gut microbiota and fecal metabolomics were observed:Different from serum and urine,changes of the levels of metabolites in faeces could reflect change of the co-metabolism between intestinal flora and hosts under exogenous stimulation.Six NAs at detectable doses in drinking water were used as target compounds.NAs were administered to infant rats by gavage,and characteristics of metabolites in faeces of laboratory animal were analyzed.PCA and PLS-DA indicated that metabolites in faeces of both infant female and male rats exposed to mixture of NAs in group L could be well distinguished from those of group CON.Levels of 7 metabolites in female rats’ faeces in group L were significantly higher than those of group CON,while 6 metabolites in faeces of male rats exposed to NAs in group L were significantly higher than those of group CON.The co-metabolic system between intestinal flora and hosts is extremely complicated.They interact with each other.Thus,the correlations between differential metabolites and differential intestinal flora in faeces of infant female and male rats exposed to NAs in group L was further analyzed.Results indicated significant negative correlations between 3 differential genera and 7 differential metabolites in female rats’ faeces.However,in male rats,though significant negative correlations between 4 differential genera and differential metabolites were also observed,2 differential genera were noticed to significantly correlate with differential metabolites in a positive manner.To sum up,we firstly use in vivo experiment to demonstrate the potential health risks of the new disinfection by-products(NAs)mixtures at environmentally relevant concentrations.The gender-dimorphic profiles were observed in gut microbiota,rats serum,and also the fecal metabolites.Based on our results,infant male rats are more sensitive and vulnerable to NAs,suggesting the necessity to apply the intestinal flora and metabonomic as important targets to the traditional mammalian risk assessment and further analyze the relevant mechanism.