Previous studies suggest that the microbiome and metabolome of the gut content could be affected by arsenic.11, 12 The present study not only demonstrates more comprehensive evidences for the arsenic impact on gut microbiome and metabolome, but also shows the partial reversibility of the effects after the 30-days recovery duration.
Arsenic induced gut microbiome and its metabolic profile alterations in rats
The microbiome data clearly shows that arsenic exposure induced the profound changes in rat gut microbiome, namely the changes of composition and diversity of the microbiota. Firstly, the microbial diversity significantly increased and this seems to be a counterintuitive situation, because the higher diversity is usually thought as the healthier in microbiota. However, the risk of malnutrition has been associated with the increased microbiota diversity.20 Therefore, it is not sure these changes are supportive for health or not. The disturbances of gut microbiota by arsenic may induce some diseases. Under arsenic stress, taxonomic assignments of Firmicutes decreased, while Proteobacteria and Bacteroidetes significantly increased. The changes of these phyla and increased bacterial diversity have been observed in the feces from the children with autism.21 The gut microbiota was more diverse in the autism spectrum disorder (ASD) children than in the control group,22,23 which also may explain the counterintuitive result of our study. In addition to the gut microbiome changes, the arsenic levels in the ASD children also increases significantly, 22 indicating the gut microbiome disruption may be one of the potential mechanisms of arsenic-induced autism.
The liver is a major target organ for arsenic toxicity.24 Arsenic concentration ≥ 0.3 mg/L in drinking water significantly increase risk of hepatitis or cirrhosis in people without chronic viral hepatitis.25 Liver disorders have been associated with microbiome disruption.26 Compared to healthy people, lower Firmicutes and higher Bacteroidetes, Proteobacteria proportions were detected in the faeces of non-alcoholic steatohepatitis patients.27 Moreover, increased Proteobacteria abundance is even a remarkable maker of liver cirrhosis.28,29 The same change trends of these bacteria species were also observed in the rats exposed to arsenic. In our study, certain arsenic treatment increased the levels of liver injury markers (i.e. serum total protein TP, albumin ALB, total bile acid TBA and lactic dehydrogenase LDH) (Additional file 1: Figure S5), indicating the potential association between arsenic exposure, microbiome disruption and hepatic injury.
The altered gut microbiota is also a potential indicator of cardiovascular disease (CVD).30 Bacterial metabolic products can penetrate intestinal barrier into systemic circulation, and induce low-grade chronic inflammation and elevate CVD risk.31 Arsenic exposure is one of the common causes of CVD.32,33 The elevated biochemical parameters of serum LDH, alpha-hydroxybutyric dehydrogenase (α-HBDH) and creatine kinase MB isoenzyme (CK-MB) in this study suggested the possibility of myocarditis or myocardial infarction (MI) after arsenic exposure (Additional file 1: Figure S5). Previous evidences show that the disordered gut microbial communities determine the susceptibility to MI in rats and human.34,35 In this study, treatment groups presented increased Proteobacteria, which is consistent with the findings from the patients with MI.36 Gut microbes, through trimethylamine N-oxide generation, directly contribute to enhance the risk of platelet hyperreactivity and thrombosis, such as MI.37 There are similar results for an elevation of Proteobacteria in myocarditis and atherosclerosis.38,39 Moreover, the high level of Proteobacteria was associated with atherosclerotic plaques,40 thus it was speculated that the arsenic-induced elevated Proteobacteria may have pro-inflammatory effects and further contribute to plaque progression. These evidences increase the possibility of gut microbiome to predict arsenic-induced CVD risk.
In order to uncover the metabolic information behind arsenic-induced microbiome alteration, we performed untargeted analysis of gut microbial metabolome. Lachnospiraceae is an important gut microbial species and suspected to be a specific indicator of arsenic-induced gut bacteria imbalance. Lachnospiraceae significantly associated with the disruption of lipid metabolism. The specie has positive correlation with phosphatidylcholine metabolites, such as lysoPC (20:4) and lysoPE (15:0), which also could promote CVD.5,41 Additionally, as important components of cell membranes, phospholipids play critical roles in maintaining cell membrane fluidity and enhancing solubility of carrier systems due to their satisfactory biocompatibility.42 Arsenic bioavailability is elevated by phospholipids; with the supportive agent phospholipids, arsenic can go through cell layers with high transport rate. Arsenic-induced positive correlation between Lachnospiraceae and phospholipids may be another way for arsenic impact on health.
Arsenic-induced gut microbiome and its metabolic profile alterations are reversible to some extent after a recovery period
Human hosted microbiomes can metabolize arsenic when cultured in vitro.43,44 Microbes in human gut protect their host against the arsenic toxicity.45 Thus, it is questioned, whether the gut microbiota will relieve the symptoms of arsenic poisoning or not? Our results show that the taxonomic richness did not, but the diversity significantly recovered after the 30-days recovery following the arsenic treatment. We infer that is caused by a decrease in the taxonomic evenness of the community. Arsenic may not impact all species equally, but weaken the few dominant species (i.e., the hub nodes in the free-scale network theory) who have controlled the many disadvantage species in the system.46 When the species balance is disrupted, the components and correlations in a system will lose the original orders and add the evenness. This effect lessens after a recovery period, resulting in a decrease in the taxonomic evenness of the community. Microbiomes from the As-treated groups and As-recovery groups are separated in the PCoA, which may indicate a restoration effect, but this restoration effect is not dose-depend.
At phylum level, the taxonomic assignments of Firmicutes, Proteobacteria and Bacteroidetes were closely resembled to its baseline state by 30 days after the end of arsenic treatment. Firmicutes can ferment indigestible food into substances such as short chain fatty acids (SCFA) in the intestine, and these metabolites can be absorbed through specific absorption processes and provide maximum energy to the body.47 Their restoration can effectively reduce the damage of arsenic to the body. These restorations can also be proved by the recovery of serum biochemical parameters (Additional file 1: Figure S5). However, Bacteroidetes failed to recover within 30 days in the 6.25 mg/L group, we could not determine whether it was caused by the arsenic-exposed concentration or insufficient recovery time.
Lactobacillus was identified as characteristic mircobiota after recovery. It is an antimicrobial and antioxidative probiotic strain with protective effect by promoting a stable gut microbial community. Lactobacillus strains in vitro partially revert the oxidative stress, the response of pro-inflammatory cytokines, the alterations in tight junction proteins distribution, and the cell permeability increases caused by inorganic arsenic.48 These evidences illustrate the important role of Lactobacillus to protect their host against the arsenic toxicity.
The five mainly perturbed metabolic pathways, especially glycerophospholipid metabolism were observed after arsenic treatment, which are also the predicted functional metabolic pathways affected by gut microbiota (Additional file 2: Table S8). After the 30-days recovery, the numbers of significant differential metabolites were less identified (Fig. 6), which indicate the perturbations of metabolic profiles have a great recovery. However, there are still four enrichment metabolic pathways were found. For example, the purine metabolism was significantly changed, number of disorders of purine metabolism may lead to immunodeficiency.49 A persistent impact of arsenic on glycerophospholipid metabolism was observed either a recovery period. Previously, the metabolism of gut microbial glycerophospholipids can promote CVD.5 These implied the lasting risk of arsenic.
Interestingly, glycerophospholipid metabolism was the significantly disturbed metabolic pathway, but it partially recovered after the 30-day recovery period. Lactobacillus was the unique representative gut species, and it has a negative correlations with PE(34:1), which increased significantly after the 30-day recovery. Therefore, we infer that the therapeutic effect of Lactobacillus on arsenic exposure may be achieved by reducing excessive PE(34:1) and thereby restoring the abnormal disturbance of glycerophospholipid metabolism. Lactobacillus strains may work as protective agents against the arsenic-induced health damage.