Toxic Effects Of 2-Bromo-4,6-Dinitroaniline On Rats Based On Intestinal Microbiome And Serum Metabolism

China is the world’s largest producer,exporter and consumer of dyes.Due to the advantages of easy synthesis,low cost,diverse colors and outstanding dyeing properties,azo dyes have become the most widely used dyes.Azo dyes and their metabolites can induce some toxic effects such as genotoxicity,carcinogenicity,allergy,and endocrine disruption.However,due to their diverse varieties and complex compositions,the toxic effects and molecular mechanisms of many azo dyes and their metabolites are not yet known.BDNA is an important metabolite of some azo dyes with mutagenicity and endocrine disruption effects on microorganisms and aquatic organisms,and has been detected in a broad variety of environmental matrices with high concentrations.However,its toxic effects on mammals and toxic molecular mechanisms are still unclear.Objective: To explore the toxicity and potential mechanisms of BDNA,intestinal flora and metabolic molecules were determined by combining general toxicological examination,16 S rDNA sequencing and serum non-targeted metabolomics.Methods:(1)Animal exposure: 80 male and female SPF-grade SD rats were randomly divided into 4 groups and housed in a constant temperature animal room,and fed with different concentrations of BDNA(0,1,10,100 mg/kg bw/d).The physiological status of the rats was observed and the changes in diet and weight were measured.After 28 days,they were sacrificed and the organs were dissected.Samples of serum,tissues,organs and intestinal faces were stored in-80 ° C refrigerators for future use.(2)General toxicological examination: the weight of each tissue and organ was measured and organ coefficients was calculated,and blood routine,blood biochemistry,and levels of cytokine and hormone were detected.(3)16S rDNA sequencing analysis: intestinal stool samples were used to extract total DNA,the sequences were amplified by PCR and a library was constructed,and sequencing was perform after quantification and library detection.Reads data was filtered and dechimerized.Species annotation and analysis of alpha diversity and beta diversity were performed.(4)Non-targeted metabolomics analysis: serum samples were used for LC/MS analysis,and endogenous MS database was used to identify metabolites.MELI,OPLS-DA,PCA,and NMDS analysis were performed.The significantly changed metabolites were identified according to P <0.05 and VIP> 1 and Metabo Analyst was used for metabolic pathway analysis.(5)Correlation analysis: Corrplot package of R software was used to analyze weight gain,food utilization rate,visceral coefficient(except for testicular and testicular body ratio),blood routine,blood biochemistry,cytokines and hormones.Correlation analysis was performed on indicators such as the top ten mycobacteria/genus and significantly changed metabolites.Results:(1)Compared with the control group,the weight and food utilization rate of rats after BDNA exposure decreased slightly,and the hair fell off;the testes and brain weights of male rats were significantly reduced,and the liver-body ratio of female rats was significantly increased;The number decreased significantly,the hemoglobin concentration and hematocrit decreased significantly;the total protein and albumin in serum decreased significantly,and triglycerides and cholesterol increased significantly.Rat liver injury indicators(arginase I,aspartate aminotransferase,glutathione S transferase and sorbitol dehydrogenase)and inflammation indicators(G-CSF,IL-1 beta,IL-2,IL-13,IL-10,IL-5,IL-17,IL-18,MCP-1,VEGF,Fractalkine,LIX,and MIP-2)generally increased significantly,and the levels of pituitary-related hormones generally declined.(2)The results of 16 S rDNA sequence analysis showed that after BDNA exposure,the relative abundance of Actinobacteria in male rats increased significantly(P <0.05),and the relative abundance of Verrucomicrobia and Tenericutes decreased significantly(P <0.05);the relative abundance of female Firmicutes increased High(P <0.05),the relative abundance of Bacteroidetes decreased.BDNA exposure leads to a significant increase in the relative abundance of gut microbiota that contributes to liver detoxification,lipid degradation,and immune inflammation,such as Bifidobacterium spp.,Dubosiella spp.,Faecalibaculum spp.,and Helicobacter spp.,While Ruminococcaceae and Bacteroides spp.decreased significantly.Alpha diversity analysis showed that BDNA exposure reduced the intestinal flora richness of rats and reduced the difference between male and female flora.Beta diversity analysis showed significant differences in intestinal flora between male and female rats exposed to BDNA.(3)Compared with the control group,BDNA exposed male rats had 147 significantly changed metabolites,and female rats had 149 different metabolites,of which 47 were shared metabolites.Male rats mainly show changes in metabolism-related pathways such as amino acids,linoleic acid,glyoxylic acid,butyric acid,and pyrimidine;female rats mainly show changes in metabolism-related pathways such as amino acid,glycerophospholipid,pyrimidine,and pantothenic acid and coenzyme A biosynthesis.The difference in metabolic levels caused by BDNA exposure is smaller than that between sexes.Conclusions: Based on the above data,we believe that BDNA mainly causes liver damage and abnormal hormone secretion in rats,which leads to lipid metabolism disorders and triggers inflammation.BDNA exposure will reduce the abundance of intestinal flora in rats,reduce the differences between male and female flora,and cause metabolic disorders.Intestinal flora structure,serum metabolic level and physiological health are closely related.