Application Of Bioinformatics In The Toxicology Of Fluoride And Antibiotic Resistance Of Water Environments Based On High-throughput Sequencing Technologies

[Objective] Previous studies have indicated that fluoride can affect spermatogenesis in humans and rodents. However, the molecular mechanisms underlying fluoride-induced spermatogenesis dysfunction remain largely unknown. The primary goal of this study was to identify differentially expressed genes and enriched pathways by using RNA deep sequencing and Ingenuity Pathway Analysis, and then evaluate the spermatogenesis and testicular immunotoxicity in male mice following fluoride exposure.[Method] We sequenced RNA transcripts in the testicles of 60 healthy Kunming male mice (8 weeks old), which were randomly divided into three groups:one control group with distilled water and two experimental groups with sodium fluoride (NaF) at 50 and 100 mg/L in the drinking water for 56 days. Following deep-sequencing analysis of 50-bp paired-end reads of RNA-Sequencing, we used Bowtie/Tophat/Cufflinks suites to align these reads into transcripts based on the mouse reference genome and to measure the relative abundance of each transcript. [Result] After sequencing and mapping, an average of approximately 29 million raw reads were gained. About 82.91% raw reads were uniquely aligned to mouse genome sequence among samples. Enriched biochemical pathways were altered in the testicles in the 50 mg/L and 100 mg/L NaF-treated groups, acting on immune responses, neuron signal transduction, oxidative stress and cellular metabolism. There were four pathways highly expressed to be relevant for IL-17. Several pathways related to toxicology, such as TGF-? signaling pathway, highly expressed in 100 mg/L NaF-treated group. In the perspective of gene functions,56.38%, 42.78%,13.62% and 12.80% of the genes participated in the process of cell metabolism, nervous system function, skeletal and muscular disorders and reproductive system development, respectively. Among them, all of IL-17A, IL-17RA, IL-17RC, MAP2K3, MAP2K6, PIK3R1, MAPKAPK2, MAP2K1 and MAP2K2 participated in the IL-17 intracellular metabolic processes, and were selected to monitor their expression by qRT-PCR. Compared with the control group, in the 100 mg/L NaF group the mRNA expression level of IL-17RA, IL-17RC, MAP2K1, MAP2K2, MAP2K3 and MAPKAPK2 increased remarkably, and coincided with the result of RNA-Sequencing.[Conclusion] Taken together, fluoride exposure disrupted spermatogenesis and testicles in male mice by influencing a large number of signaling pathways and genes, which were working on immune responses, reproductive development, neuron signal transduction, oxidative stress and cellular metabolism. The high expression of the IL-17 signal pathway, cytokines of the TGF-? family and other proinflammatory cytokines were the response to the invasion of the testicular immune system by extracellular fluoride, which may act on the maintenance of immune privilege and spermatogenesis.[Objective] The blood-testis barrier (BTB) is formed by the basement membrane, the tight junctions, and the biochemical activity of the Sertoli cells, which can protect spermatids from the attack of the self-immune during spermatogenesis. High levels of fluoride in the water results in the lack of maturation and differentiation of spermatocytes and loss of spermatogenesis, but the changes of BTB under fluoride exposure is rarely reported. To overview of the effect of fluoride on gene expression in the BTB of testicle, High-throughput cDNA sequencing technology were applied in our study.[Method] Ten healthy, adult male Kunming mice (aged 8 weeks,25-26 g b.w.) were divided into two equal groups of 5:a control group with distilled water and a fluoride group with 100 mg NaF/L in their drinking water. After 56 days, testicles were isolated and testicular total RNA of each mouse was extracted for RNA-Sequencing. TopHat and Cufflinks were used in the analysis of raw data from RNA-Sequencing. Ingenuity Pathway Analysis (IPA) was available to the biological complexity of the differentially expressed genes and find the main biological processes associated to the experimental system. The related molecules MAGI1, ARPC5, SSX2IP, CLDN14, PVRL2 and DNM3 were selected, and the mRNA expression in testicle was tested using qRT-PCR.[Result] Studies have shown that among the 103 signaling pathways, four different pathways were identified among the NaF-treated testicle of fluorosis, connected with tight junctions of the BTB, particularly Epithelial Adherens Junction Signaling expressed significantly. The SSX2IP and PVRL2 gene expression was up-regulated obviously, but the expression levels of ARPC5 and CLDN14 decreased significantly and the others had no meaningful variation.[Conclusion] It was concluded that high levels of fluoride could cause a distinct influence on the signaling pathways and molecules to coordinate the opening and closing of the tight junctions among the blood-testis barrier, because of the structure and function destruction of cell membrane, membrane protein and other related phosphokinase. Effects of fluoride on testicles injury and reproductive impairment need to be further explored via to the exploration of the physiological and immunological response to BTB.[Objective] The utilization of antibiotics in the feeding, anthropogenic activities, and sewage discharged and the spread and transfer of antibiotic resistance have resulted in fears of the effect on environment and biology. Water environment is an important site for the storage and evolution of the antibiotic resistant genes (ARGs). Oceans, lakes and rivers are complex and changeful environments, which can bring about the diversity of the microbe in species composition, ecological function and genetics.[Method] In order to investigate the prevalence of ARGs and detect the characterization of integrons in the antibiotic resistance, the surface sediments from 13 typical aquaculture areas in China's coastal aquaculture sites were collected during dry season. Total DNA from sediment samples was extracted and sequenced by Illumina HiseqTM2000 platform. Metagenome technology was used to determine the occurrence and quantities of ARGs in each different point.[Result](1) Statistical analysis showed that up to 322 kinds of drug-resistant genes have been checked out, resistance to aminoglycoside(60), beta-lactam(60), macrolide-lincosamide-streptogramin B (MLSB)(45), multidrug resistant genes(44), tetracycline(23), tuberculostatics(18), polypeptide(16), chloramphenicol(13), quinolone(13), sulfonamide(12), aminocoumarin(5) and other rare but distinctive resistant genes(13), respectively. The most abundant resistance type was multidrug resistance genes, as many as 6656 reads.(2) The Pseudomonas aeruginosa, Escherichia coli and Mycobacterium tuberculosis were predominant bacteria among the 175 recorded species,56 genus of drug resistant bacteria.(3) Bacterial resistance was mainly caused by the mechanism of drug enzymatic destruction and drug efflux pumps.(4) By comparison, Dalian and Laiyang showed low levels of antibiotic resistance pollution; conversely, Hangzhou Bay was characterized by the highest abundance of both resistance gene types and total gene reads. The abundance of sulfonamides-resistant genes was prevalent in Shanhai. Genes resistant to the aminoglycosides were popular in Xiangshan Harbor. The multiple drug resistant genes were dominant around the South China Sea coast.[Conclusion] The application of metagenomic technology provided a comprehensive overview of the antibiotic resistant gene reservoir in coastal aquaculture sites. The distribution of antibiotic resistant genes in the sediments was obviously different and varied along the coast of Bohai, Yellow, East and South China Seas. Environmental microbial drug resistance is worthy of attention, not just the residues of antibiotics in aquatic food. Relevant regulations and evaluating systems should be well established to keep the balance of environmental security and curb the emission of veterinary drugs.