Effects of Exposure on Mouse Bodyweight
The results in Fig. S1 showed that the weight gradually increased over the course of exposure. Approximately 3 weeks later, the bodyweight difference increased extensively. Compared with the control check group (CK), acetamiprid (D), tebuconazole (W), and combination groups (DW) showed a 19.35–22.06% increase in bodyweight on average. This finding indicated that obvious obesity trends or even obesity occurred in the exposure-treated mice. Meanwhile, the bodyweight of the blank group showed no significant difference with that of the CK group, indicating that the corn oil used as a reagent in the administration hardly affected the basal metabolism of the mice and the bodyweight difference was mainly caused by the pesticides. Bodyweight is only the phenotype of obesity, and metabolic abnormalities occurred in the physiological reactions . Physiological observation and metabolic investigation could be performed in further studies.
Effects of Exposure on Glucose Tolerance and Insulin Resistance
Long-term obesity usually occurs along with insulin resistance (IR), and IR is a vital core in metabolic syndrome because it is closely related to obesity and directly causes T2D, NAFLD, and cardiovascular diseases [16–18]. Fasting blood glucose (FBG), glucose tolerance (GT), and IR are the commonly used indices to investigate the sensitivity of insulin and the status of body glucose metabolism. They are also believed to be the risk indicators of early-stage T2D. The results showed that the FBG in the treated groups slightly increased but less than 6 mmol/L (Fig. 1a). The mouse plasma glucose increased rapidly after glucose intake in all the groups. Then, the glucose concentrations decreased relatively slowly in the following 90 min. The treated groups maintained a higher plasma glucose than the CK group at the end of 120 min. This finding inferred that the plasma glucose in the treated mice spent more time returning to normal, indicating that pesticide exposure lowered the capacity of the body to regulate glucose .
IR shows that the target cells lowered the sensitivity to insulin. In the homeostasis model assessment of IR, higher index corresponding to more serious IR. The test results (Fig. 1b) showed that the IR indices in the treated groups were nearly 1–3 times more than those in the CK group. Significant differences in IR index levels were observed in mice pressured using two pesticides. IR is the basis of T2D, and it is the link of many other metabolism diseases. It was considered a crucial warning of metabolic diseases. The results of the present study revealed that dietary pesticide exposure induced IR to the mice and further posed metabolic health risk to them. The effect of combined pesticides on IR was apparently higher than that of their single components, indicating that the combination exposure enhanced the health risk compared with single component. This finding may be attributed to the recognized synergism in combination exposure, which strengthened the exposure effects compared with single components.
Serum Biochemistry Analysis
IR and the disorder of carbohydrate metabolism occurred, indicating that many aspects of metabolism were affected. Serum biochemistry analysis is a commonly used method in investigating physiological changes, and it is an effective evidence in clinical diagnosis [20, 21]. Pesticide exposure obviously led to a significant increase in serum cholesterol (Fig. S2) and a decrease in high-density lipoprotein cholesterol (HDL-C) in all the treated groups. Similar increasing trends were also obtained in serum triglyceride (TG). The combined exposure group displayed significantly higher serum TG than any single exposure group. Therefore, the metabolic abnormalities caused by pesticide exposure could induce risk of cardiovascular disease and fatty liver to the body.
Liver is the largest digestive gland in the body and the main metabolic organ [22, 23], and serum biochemistry analysis is an effective method to evaluate liver function in clinical diagnosis . In this work, liver function was investigated and evaluated using related indices (Fig. S3), such as aminotransferase (ALT, AST, and ALP) and serum protein (TP and ALB). Aminotransferases, especially ALT and AST, were enhanced at different degrees by pesticide exposure, indicating that minor injuries occurred in the liver. The concentration changes in serum protein were slighter in the exposure than those in aminotransferase. The evaluation results revealed that liver was slightly impaired and mild dysfunction may have occurred in the exposure . Moreover, these abnormalities were found in different exposure groups, and no obvious joint effects were obtained in the combination exposure.
Evaluation of Mouse Inflammation
According to previous reports [26, 27], chronic and systemic inflammatory responses trigger the development of IR. Thus, the inflammation status of the mice was observed in this section. The levels of several important inflammatory mediators in serum are exhibited in Fig. 2. TNF-α is one of the most crucial factors in the IR formation; it works by inhibiting insulin signal transduction. A previous study reported that the antibody against TNF-α evidently improved insulin sensitivity and alleviated IR [28, 29]. Single and combination exposures led to a massive increase in TNF-α by almost 10-fold. For example, exposure to tebuconazole increased TNF-α from 35.7 EU/L (CK) to 338.10 EU/L. No significant joint action effect was obtained in the combination exposure group compared with the single-component group.
Interleukin (IL) is a large class of cell factors that exerts considerable effects to the body’s inflammatory reaction and immunity. Numerous studies have shown that IL-1, IL-6, IL-8, and IL-10 exerted substantial effects on IR. Increased levels of IL-1β and IL-6 were detected in patients with T2D and obesity, and they promoted the inflammatory process. The combination exposure to pesticides showed an obvious synergy compared with its single counterpart. MCP-1 is another important promoter that acts as a kind of inflammatory chemokine and contributes to IR. Although MCP-1 did not respond intensively, a relatively significant increase of its concentration was observed. Different from these inflammatory mediators, IL-10 is a well-recognized multifunctional factor, and it was also negatively-related with IR in this work. No evident concentration differences were observed between CK and exposure groups. Overall, the observation of these cell factors indicated that dietary exposure to pesticides induced inflammation, and it is an important basis of IR.
Detection of Lipopolysaccharide and Intestinal Permeability
Lipopolysaccharide (LPS) is usually produced by the intestinal flora, and alteration to intestinal permeability enhances its transfer from the gut to the blood [30, 31]. Thus, investigating serum LPS and intestinal permeability is vital in examining LPS-induced chronic inflammation. The results in Fig. S4b displayed that increased FITC-dextran amount was observed in all the treated groups, indicating that the intestinal permeability significantly increased, especially in the tebuconazole and combined groups. Tebuconazole exhibited a more obvious effect than acetamiprid, and no significant difference was found between tebuconazole and combined groups. Serum LPS increased in exposure groups, and a synergistic effect was obtained in the combined exposure group (Fig. S4a).
Increase of intestinal permeability and serum LPS level can promote each other. The coordination effects of combination exposure were exhibited in the results of investigation on LPS and intestinal permeability; significant differences occurred between combined and single actions. Thus, pesticide exposure increased the intestinal permeability, and the resulting enhanced transfer of harmful gut flora and metabolites to the blood led to increased physiological dysfunction, such as low-grade inflammation.
Host tissue histology showed that lipid droplets were gradually formed and NAFLD was presenting in the treated groups (Figs. 3 and 4). It was difficult to judge the severity of fatty liver in among different groups because they were in the development of NAFLD. HE staining of colon slices manifested that aggravated inflammatory cell infiltrates occurred in the treated groups, especially in tebuconazole and combined exposure group. Cell structure of intestinal villi was loosened and intestinal barrier function was impaired in acetamiprid group, and it would enhance intestinal permeability. The results proved that dietary pesticides exposure lead to NAFLD, leaky-gut, and even enterocolitis. Metabolism health risks of chronic dietary pesticides exposure were confirmed.
Effects of Pesticide Exposure on the Diversity of Gut Flora Community Structure
As mentioned above, to investigate the changes in the intestinal flora caused by pesticide exposure were necessary in this work [30, 31]. In the present study, 16S rRNA gene sequencing for microbiome analysis was used to detect the microbial communities and evaluate the effects of pesticide exposure on mouse gut flora. Together with the host, the microflora and intestinal microflora maintain microecological balance ,. Structure and distribution are the most vital characters of a microbial community. The diversity of gut flora was investigated at alpha and beta levels. The results of alpha diversity (Shannon index) showed that no significant difference was found between the CK and treated groups, indicating that the species diversity of gut flora within a community was not destroyed by pesticide exposure (Fig. S5a). Nonmetric multidimensional scaling analysis is an appropriate method for calculating the beta diversity of the gut flora community. Fig. S5b illustrated that relatively different beta diversities occurred in different groups of treated mice, thus revealing that the gut flora of mice had a unique response to the exposure of different pesticides and that their communities were differently altered. The stress value of the model was 0.093, revealing that the model could stimulate the actual samples accurately.
Species Changes in Gut Flora
Similar to the reports [34, 35], approximately more than 95% of the gut flora of mice was mainly distributed in the phylum of Bacteroidetes and Firmicutes (Fig. S6a). It was reported that a low ratio of Bacteroidetes/Firmicutes (B/F) was usually obtained in individuals who were obese, and transplanting fecal microbes with high or low B/F was effective in making the host lose or gain bodyweight [34, 35]. In the present research, the B/F in the CK group was approximately 3-4-fold higher that in the treated groups, contributing to host obesity.
The results in Fig. 5 and Fig. S5 (c, d) showed that most significantly different gut flora species (around 18 categories) were obtained in the tebuconazole group according to LDA effect size (LEfSe) analysis when the LDA threshold was 3. The abundances of Firmicutes and Bacteroidetes were mostly obtained in the tebuconazole and control groups, respectively. In addition, the family of Peptostreptococcaceae and Lactobacillaceae showed abundant microbes in the gut, and these microbes were accumulated in the tebuconazole group. The affected groups were the combined and acetamiprid groups. A significantly different flora was only found between the tebuconazole and CK groups when the LDA threshold was 4, indicating that the gut flora was mostly affected by tebuconazole in all exposure groups. Compared with single components, the combination exposure did not perform evident synergies nor promotion effects. Further investigation was focused on the quantity alteration in the flora and its distribution in the level of family or genus.
The gut flora species of different groups at the genus level was investigated (Fig. S7). Compared with the CK group, acetamiprid enhanced the abundance of Ruminiclostridium, Roseburia, Lachnoclostridium, Marvinbryantia, Intestinimonas, Rhodococcus, and Caulobacter, while most of them were the species with low abundance. In the dominant species, Alistipes, Blautia, unidentified_Ruminococcaceae, and Oscillibacter were relatively increased and in the treated group, including harmful and beneficial bacteria. Thus, acetamiprid exposure caused varied changes to the gut flora. Compared with the CK group, Lactobacillus, Klebsiella, Streptococcus, Romboutsia, Mitsuokella, Enterococcus, and Sphingomonas were upgraded by tebuconazole, while Alloprevotella, Bacteroides, and Muribaculum were decreased. The intestinal microecology may be deteriorated due to the accumulation of major pathogenic bacteria. For example, the accumulation of Enterococcus leads to gut inflammation and lowers the butyrate-maker flora and butyrate in the gut . Klebsiella and Streptococcus are also the typical infectious bacteria , while Alloprevotella, Bacteroides, and Muribaculum are anti-inflammatory bacteria that produce short-chain fatty acids (SCFAs). A decrease in these bacteria lowers the anti-inflammatory and immune capacity of the host [38–40]. Thus, dietary tebuconazole exposure exerted negative effects on the gut flora, thus disrupting the healthy gut flora of the host and posing metabolic risk to it. However, the combination exposure of pesticides exerted multiple effects on the gut flora. Harmful bacteria, such as Helicobacter and Lachnospira, and beneficial bacteria, such as Parabacteroides and Akkermansia, were all enhanced. Meanwhile, absolutely dominant bacteria, including Alloprevotella and Bacteroides, were all decreased by the exposure. Therefore, although pesticide exposure influences to the gut flora were multifaceted, disorder still obtained for the disruption to the absolutely dominant species of bacteria. Overall, tebuconazole disrupted the gut flora more than the other treatments, and no significant synergy effects were obtained from the combination exposure group. This finding was consistent with the result of LEfSe analysis. LPS produced by Enterobacteriaceae and Desulfovibrionaceae was reported to be approximately 1000-fold higher than that from other bacteria , and it was remarkable in causing chronic inflammation. The abundance of Desulfovibrionaceae in the acetamiprid, tebuconazole, and combined groups were approximately 3.5, 1.5, and 10 times that in the CK group, and pesticide treatments significantly enhanced this abundance in the tebuconazole and acetamiprid groups. The abundance of Enterobacteriaceae in these groups was 5.3, 64, and 3.2 times higher than that in the CK group (Fig. S6b). They were highly accumulated by pesticide exposure, especially in the tebuconazole and combined groups, which could extremely promote endotoxin accumulation in the gut.
Metabolic Profiling of Gut Flora
Based on the gut structure [42, 43], the metabolites of the gut flora are the main media of interaction between the flora and the host through blood absorption . Thus, identifying the altered metabolites of the gut flora is an essential means to investigate the effects of pesticide exposure on host physiological activity. Metabolic analysis is an effective method to obtain the changes in gut flora metabolism between the CK and exposure groups.
On the basis of the metabolites, the samples of different exposure groups were distinguished significantly only by the unsupervised learning algorithm, principal component analysis (PCA, Fig. S8). The results indicated that circulating metabolism changes and differences occurred when hosts were stressed by different pesticides. Furthermore, differential metabolites were yielded via OPLS-DA model under the following statistical conditions: VIP > 1, FC > 2, FDR < 0.05, and p < 0.05. In the CK, acetamiprid, tebuconazole, and combination groups, 37, 43, and 32 different metabolites (Additional File 15: Table S1.) were investigated and used for further investigation. Different metabolites were also obtained in the single and combined exposure groups to evaluate the gut flora response to pesticide exposure.
In this work, pesticide exposure involved many significant metabolite variations in the gut. Trimethylamine N-oxide (TMAO) comes from the metabolism of intestinal microflora, and it is highly related to cardiovascular diseases. It was the main risk factor of atherosclerosis . In the present study, the acetamiprid and combined groups showed a considerable increase in TMAO in the gut by approximately thousands of times more than the CK group. However, the metabolites between the CK and tebuconazole groups did not significantly differ. Thus, acetamiprid was evidently an important dietary contaminant, and its exposure led to the risk of cardiovascular diseases via adjustment of the gut flora metabolism. Spermidine, the most effective polyamine in preventing lipid peroxidation, was lowered in the acetamiprid group. On the contrary, putrescine, another type of polyamine, increased to approximately 100 times when the mice were exposed to acetamiprid, thus considerably enhancing the risk of leaky gut and colitis. These significant variations in polyamines did not occur in the tebuconazole and combined groups. Imidazole propionate and imidazoleacetic acid were the metabolites of the gut flora. A significant decrease in imidazoleacetic acid was yielded in all pesticide-exposed groups. They were homolog in structure, with only a difference in CH2. Imidazole propionate and imidazoleacetic acid were all gut flora metabolites of histidine from totally different metabolic pathways. Imidazole propionate occurred with the action of histidine ammonia lyase, while imidazoleacetic acid was the final metabolite of histamine, which was obtained from histidine treated with histidine decarboxylase. Imidazole propionate was reported to cause T2D by disrupting GT and insulin signaling . No physiological function of imidazoleacetic acid was ever reported, but speculations could be made on the basis of their extremely similar molecular structure. Extensive work is necessary to identify how gut flora works in the pathway of imidazoleacetic acid production and what determines the metabolic method of histidine.
The metabolism of tryptophan involved a number of bulk compounds, especially indole derivatives. Many of them, including 3-Indoxyl sulphate, 3-Indolelactic acid, 5-HIAA, and indole-3-acrylic acid, were significantly different metabolites distributed in the content sequence of control > acetamiprid > combination > tebuconazole. These metabolites played substantially important signaling roles in regulating the host physiological activities, such as enhancing mucosal homeostasis by alleviating intestinal permeability (possibly mediated by pregnane X receptor), suppressing appetite, secreting insulin, and slowing gastric emptying by inducing the release of glucagon-like peptide 1 in enteroendocrine L-cells . A decrease in these metabolites by pesticide exposure could elevate the health risk to the host. The results suggested that tebuconazole lowered indole derivatives more than acetamiprid, and no combinatory effects were obtained. Many other gut intestinal metabolites that were proven to act on host metabolic activity were affected by pesticide exposure. For instance, 12, 13-DiHOME is a kind of gut lipid that activates the brown adipose tissue of the host, regulate fat metabolism, and lowers the host risk of heart disease and diabetes as a metabolic signal . In the present work, only single-component exposure significantly decreased 12, 13-DiHOME in the host gut. Trichostatin A is a metabolite of Streptomyces, and it was reported to perform the activity of histone deacetylase inhibitor, which exhibited the effects of anticancer. The pesticide exposure of tebuconazole and acetamiprid disturbed the gut flora and decreased trichostatin A in the gut, which is not beneficial to the anticancer capacity of the host. However, this phenomenon did not occur in the combination exposure group.
Association of Gut flora with Its Metabolism
Under the pressure of pesticides, the gut flora communities were affected and the metabolites were altered correspondingly. The relevance between altered gut flora and its metabolites were analyzed, and the results are shown in Fig. 6. A high relationship (r > 0.7 or r < − 0.7 and p < 0.05) between the flora at the genus level and its metabolites was subjected to network analysis, and their relevance is exhibited in Fig. 6.
The genera of Lactobacillus, Alloprevotella, Alistipes, Roseburia, Enterorhabdus, Romboutsia, Faecalibacterium, Clostridioides, Sphingomonas, Butyricimonas, Desulfovibrio, Intestinimonas, Marvinbryantia, Oscillibacter, Candidatus_Arthromitus were involved in high relevance, and Lactobacillus was associated with most of the metabolites. For example, it was positively related with N-(2-Acetylphenyl)formamide, 1,5-Dimethyl-4,5-dihydro-1H-pyrazole, Tyramine, 2-Butoxy-N-[2-(diethylamino)ethyl]nicotinamide, α-tocopheronic acid, 4-Aminopyridine, 1-[(9Z)-octadecenyl]-2-hexadecanoyl-sn-glycero-3-phosphocholine, N-(icosanoyl)ethanolamine, and negatively related with 1-Methyl-3,5,6-indolinetriol and 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine. Faecalibacterium and Sphingomonas were all positively correlated with 3-(Methylthio)propylamine. An unknown genera of Lachnospiraceae was positively related with N-Acetylserotonin and Methyl indole-3-acetate, while negatively related with Indole-3-acrylic acid. Methylimidazoleacetic acid would be decreased by Intestinimonas and Marvinbryantia, while N-Acetylputrescine was totally different with it. Apart from Methylimidazoleacetic acid, N-isopentylacetamide and 1-(4-Aminobutyl)urea were also negatively related with Oscillibacter. Although Butyricimonas and Desulfovibrio exerted different effects on host, they were all positively related with (S)-2-amino-6-oxopimelic acid. Desulfovibrio still related with it more closely than Butyricimonas. Gut flora and metabolites are all groups with large amounts and complexity, and their correlations are also extremely intricate. The pesticides altered the intestinal microflora, which changed the metabolism of the flora. This phenomenon mediated the effects of pesticides on host physiological activity. To explore their relationships is an effective way to investigate the effects of gut flora on host metabolic syndrome.
Effects of Exposure on Host Circulating Metabolism
Blood is the pool of circulating metabolism, and it characterizes the physiological activity of the host. Non-target metabolic profiling of serum was performed in this study to investigate the alterations in the host circulating metabolism. Different groups were distinguished in accordance with the identified metabolites, as shown in Fig. S9.
Wide varieties of significantly different metabolites were identified, mainly including amino acids and their derivatives, free fatty acids and their methyl esters, phospholipids, nucleotides, carbohydrates, hormones, and other physiological metabolic compounds (Fig. S10 and additional file 16: Table S2). Dysregulated metabolism of these compounds poses a great threat to the host health and is a remarkable risk factor of chronic metabolic diseases, such as obesity, T2D, and NAFLD. Shown in Table S3 (Additional File 17), different alterations in amino acids were observed; for example, the contents of branched-chain (valine and leucinein) and aromatic amino acids (phenylalanine) in the serum showed a variable degree of elevation in all the treated groups. Threonine was increased by pesticide exposure, and no obvious change trend was found in the amino acid derivatives. As reported, the disorders of fat and carbohydrate metabolism also exhibited a dysfunction of body physiological activities, especially in chronic metabolism diseases, such as obesity, T2D, and NAFLD. Fatty acids and methyl esters were accumulated by around 2–11-folds in the exposure groups. Interestingly, all of them were unsaturated fatty acids, such as docosadienoic acid, ocosatetraenoic acid, docosapentaenoic acid, and docosatrienoic acid. (5R)-5-[(1S)-1,2-Dihydroxyethyl]-alpha-D-lyxopyranose,5-O-alpha-L-Arabinofuranosyl-alpha-L-arabinofuranose and methyl 6-deoxy-2,3-O-isopropylidene-alpha-L-mannopyranoside were significantly increased by pesticide exposure in the treated groups by up to approximately 3.3 times. Increased contents of branched-chain amino acids, aromatic amino acids, fatty acids, and carbohydrate in serum are usually found in individuals with the abovementioned metabolic diseases. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and lyso PC (LPC) are the important phospholipid compounds in serum, and they were also affected by pesticide exposure. PC decreased in the pesticide-exposed groups, whereas LPC increased in the treated groups. Different results about these phospholipid compounds in a study on metabolic disorders were reported. The results obtained in the present work were not totally consistent with those from previous reports. Phospholipid-derived compounds undoubted played considerably important roles in physiological activities. For example, PC decreases blood fat and peroxide and demonstrates positive effects on the liver and heart, while LPC is an important pre-inflammatory factor of arteriosclerosis .
Spermine was reported to have a negative relationship with T2D , and it showed a 0.38–0.73-fold decrease in present study. This finding may also be an evidence for the prediction of T2D. Nucleic acids cytosine adenosine, uracil, xanthine, 7-methylxanthine, and 1-methylhypoxanthine were identified, while no obvious change trends were found. As the metabolites of purine, uric acid compounds were also reported to be closely related with obesity and T2D . They were remarkably elevated by metabolism disorders. 9-Methyluric acid and uric acid were identified in the serum, and they were decreased by exposure to acetamiprid but enhanced by exposure to tebuconazole. The effects of pesticide mixture of tebuconazole and acetamiprid were in the middle.
Similar to other fatty acids, prostaglandin A1 and E1 also increased in the treated groups, while their metabolite, 15-dehydro-prostaglandin E1, decreased. The physiological responses caused by this alteration were identified. 7-Alpha-hydroxy-17alpha-methyltestosterone was the detected androgen in the serum, and it decreased in the acetamiprid and combined groups but not in the tebuconazole group, indicating that dietary acetamiprid exposure also affected the reproductive endocrine system.
Association between Metabolites of Gut Flora and Host Circulating
Co-inertia analysis (CIA) was performed to find a covariation between serum metabolites and gut microbiota metabolites and further investigate whether the altered abundance of host metabolites correlated with the altered gut flora (Fig. S11) . A correspondence analysis model was applied to analyze the relevance between the designed significantly different metabolites of the gut flora (yield in the relevance analysis between 16 s r DNA and gut flora metabolism) and the host circulating metabolites (Additional File 18: Table S4).
Figure S11 demonstrate that the representativeness of the CIA model was obviously reflected by the first two axes, which exhibited most of the shared features of the metabolites of gut flora and serum. High consistency was obtained between the two datasets of the metabolites of the flora and serum. The relevance of the two metabolism datasets was also significant. Correlation analysis between the metabolites of gut flora and host serum exhibited quantitative relationships of the significant compounds (Fig. 7). The trimethylamine N-oxide, N-acetylsphingosine, and betaine in the gut enhanced the leucine in the serum, while 3-(2-hydroxyethyl)-1H-indol-5-yl alpha-D-glucopyranoside, N-acetyltyramine, (13alpha)-13-hydroxyspartein-2-one, N-acetylhistamine, tyramine, and 5,6-Indolinediol lowered it. Unsaturated fatty acids, except for arachidonic acid, were mostly inhibited by 2-butoxy-N-[2-(diethylamino)ethyl]nicotinamide and alpha-tocopheronic acid. Prostaglandin comes from the metabolism of arachidonic acid, and its abundance in the serum was significantly enhanced. The results of the observation proved that in the treated groups, more arachidonic acid was metabolized into prostaglandin, and its content decreased. N-methylimidazoleacetic acid, N-acetyltyramine, pyridoxamine, 3-(2-hydroxyethyl)-1H-indol-5-yl alpha-D-glucopyranoside, and nicotinamide decreased in the gut of the treated mice, whereas the contents of 4alpha-formyl-5alpha-cholest-8-en-3beta-ol, N-(2-hydroxyethyl)heptadecanamide, and N-acetylsphingosine increased. The above two groups of compounds affected the PCs in the host serum positively and negatively. Ceramide, spermine, and other amino acids related to the dysfunction of host metabolites all showed a strong correlation with the significantly different compounds in the gut. Correspondences between host and gut flora were identified via the metabolites, and it is getting more clear and close along with more action pathways or new compounds were identified.
Host Liver Metabolism and Gut-liver Dialogue under Pesticide Exposure
In this study, liver metabolism was profiled to investigate the effects of the altered metabolites of the gut flora caused by pesticide exposure on the host. These annotated significantly different compounds were subjected to the KEGG metabolic database to map and analyze the involved pathways, and the results are exhibited in Fig. 8 and Fig. S12. PCA showed that the different exposure groups were apparently distinguished in accordance with the metabolites (Fig. S13). The results indicated that pesticides exerted different effects on the body metabolism. These different compounds were annotated into many metabolic pathways, indicating that these metabolism pathways, mainly involving amino acid and derivative metabolism, glycerophospholipid and fatty acid metabolism, and vitamin and nucleotide metabolism, were intervened when the hosts were exposed to pesticides. Purine metabolism, glycerophospholipid metabolism, and vitamin B6 metabolism were shared by three different treated groups. Moreover, unique intervened pathways were obtained in the joint exposure group. For example, beta-alanine metabolism and glycine, serine, and threonine metabolism were intensively affected by the combined stress of tebuconazole and acetamiprid.
The intervened pathways were integrated together in accordance with the shared metabolites. In the metabolism of cholic acid, a decrease in choline was observed in the three treated groups, and choline deficiency impaired PC synthesis, very-low-density lipoprotein synthesis, and hepatic lipid export . Thus, this disorder posed NAFLD risk to the host. The results were consistent with the detection of serum components. Meanwhile, increased betaine was detected, and it can be speculate that the activity of choline oxidase was enhanced because betaine was obtained from the oxidation of this enzyme. While the reasons for the enhancements of enzyme were unclear. A high content of betaine led to the condition for TMAO formulation, and the results were proven in previous observations. TMAO is a high-risk factor leading to atherosclerosis and cardiovascular diseases.
Histidine could be converted into urocanic acid or histamine via two different pathways, and an abnormality in histidine and urocanic acid was found when the host was stressed by tebuconazole. The results indicated that histidine metabolism was intensively intervened. When the body was stressed by tebuconazole, downregulated urocanic acid, upregulated histidine, and accumulation of histamine occurred. Histamine was further reacted into N-methylhistamine and imidazoleacetic acid through different enzymes. In this work, more N-methylhistamine and less imidazoleacetic acid were the results of the alteration. More histamine was obviously oxidated into N-methylhistamine. Histamine is a type of key conductive chemical and one of the most widely studied inflammatory mediators that lead to inflammation and allergies to tissues. N-methylhistamine is the major metabolite of histamine produced by mast cells. Anaphylaxis and mastocytosis are typically associated with increased N-methylhistamine levels . Upgraded N-methylhistamine was detected in the blood in the above step. Thus, the histamine and N-methylhistamine in the liver could be the significant reason behind mastocytosis and hepatitis.
The downstream nucleotide metabolites of L-Asp, L-argininosuccinate, adenylosuccinate, and CDP were upregulated in all the treated groups. On the contrary, the upstream nucleotide metabolites of beta-alanine were all downregulated. Many other metabolites were also annotated into different pathways. However, an insufficient effective abnormality was found when formulating clear pathways. This finding may be resolved by detecting and identifying more metabolites in the analysis.
Effects of Interventions on Physiology of Pesticide-exposed Mice
Fructooligosaccharide (FOS) , fecal microbiota transplantation (FMT) , and FOS + FMT were used as the intervention measures in this study. IR, body inflammation status, and physiological metabolites were all detected to evaluate the variation caused by the treatments, and the results are shown in Fig. S14 and Fig. 9.
Significant differences of IR were obviously obtained between the exposure group and the dietary treatment group. The IR index was remarkably lowered by FOS and FMT, and both of them were effective means to improve IR (Fig. S14a). FMT was more effective than FOS in improving mouse IR caused by pesticide exposure, and the insulin sensitivity was basically reverted to a normal level. FOS + FMT presented similar effects with that in single treatment of FMT, indicating that FMT played a primary role in improving IR. The rapidly responded inflammatory cytokines in the serum, namely, IL-1β, IL-6, and TNF-α, were observed after the treatments, as shown in Fig. S14b. The contents of cytokines significantly decreased in the serum, indicating that host inflammatory status was remarkably released. The effects of these treatments were in the order FOS + FMT > FMT > FOS, suggesting that the combined treatments of FOS + FMT enhanced the effects of single-factor treatments, almost returning to the normal level in IL-6 and TNF-α.
The contents of some metabolites in the gut and serum were observed (Fig. 9). SCFAs, usually C2–C4, were produced by the intestinal microflora decomposing the dietary fiber. Butyrate is the most important SCFAs in the gut, and it supplies 30% energy for the host and most of the energies for the gut flora. Moreover, butyrate was reported to be inhibiting pathogens and enhancing probiotics in the gut. Pesticide exposure obviously decreased the butyrate content in the gut, while FOS, FMT, and FOS + FMT enhanced it, of which FOS + FMT was the most effective treatment. Butyrate maintains host fullness by stimulating the vagus nerve and promotes fat oxidation to restrict host diet. A high content of butyrate prevents diet-induced obesity and increases insulin sensitivity. Moreover, it was helpful in host anti-cancer activities and in improving immunity. Thus, butyrate is one of the most vital gut flora metabolites in regulating host metabolism. Propionic acid is effective in decreasing host cholesterol, relieving hypertension and inflammation, and reducing liver fat. However, in the present work, the exposed group showed increased propionic acid concentration in the feces, and the mechanism was not found. Overall, pesticide exposure altered the intestinal flora and its SCFAs, thus increasing the risk of metabolic syndrome in the host. TMAO is a high-risk pro-atherogenic metabolite produced by gut microbiota, and it was observed to be extremely accumulated in the above research. In terms of the association of gut bacteria with host circulating metabolites, TMAO had a high positive relationship with leucine in host serum, and the exposure enhanced TMAO and leucine. Both decreased in the serum after interventions. Moreover, TMAO was positively related with fat and fat acids in the serum. Significant unsaturated fat acids, such as C20:2 and C22:2, also remarkably decreased in the treated groups. High contents of branched chain amino acids (BCAAs), Aromatic amino acids (AAAs), leucine, phenylalanine and valine were reported to be T2D risk factors. They were also increased by pesticide exposure. In this step, they were remarkably reduced by the treatments, especially FOS + FMT. Tyramine was proven to be positively related with these metabolites, and similar alteration occurred. Ceramide was reported to be related with cerebral vascular diseases, IR, and HbA1c abnormality; it may be a new biomarker of adverse cardiovascular events . Lyso PE induces inflammation and increases oxidative stress . They were both the unfavorable factors in the serum increased by pesticide stress, while the conditions were improved in the intervention.