The mechanisms of 2,4-D toxicity in animals and human are not quite definite and oxidative stress may be one of them27,28,29,30. In the present study, metagenomic sequencing and metabolomic profiling were applied to examine the impact of subchronic low-dose 2,4-D exposure on the host and gut microbiome. Clearly, the host metabolic profiles were differentiated by 2,4-D exposure indicating low-dose toxicity. Meanwhile, the results also demonstrate that 2,4-D perturbed not only the composition and diversity, but also functional pathways and metabolic profiles of the gut microbiota. Accumulating evidence suggests that the alterations of microbiome-related pathways and metabolites would lead to a disturbed gut-host homeostasis hence increasing disease risk18. For example, perturbations in gut microbial metabolic activities involved in protein fermentation and amino acid metabolism can lead to the elevation of toxic metabolites such as polyamines, hydrogen sulphide and ammonia, which play an role in the progression of colorectal cancer by inducing inflammation, ROS production, or genotoxicity31. Likewise, microbiome-derived uremic toxins such as p-cresol sulfate and indoxyl sulfate resulting from gut dysbiosis are associated with chronic kidney disease32,33. Given the strong association between gut microbiome perturbations and a variety of human diseases coupled with the capacity of 2,4-D to induce gut microbiome alterations, the findings of the present study might provide insights regarding the mechanistic basis by which 2,4-D adversely affects human health.

One of the intriguing findings from our study was that 2,4-D induced remarkable abundance changes in gut microbial pathways and gene families. We found 35 microbial pathways (Level 3 Subsystems) and 82 microbial genes (Functional Level Subsystems) at significantly different abundances in the treatment group compared to control group. A reduction of genes involved in plant hormones was observed in mouse gut microbiome after 2,4-D exposure (Fig. 2A). Even though plant hormone-related pathways do not exist in animals and human, they are commonly present in gut bacteria. Thus, the alterations to bacterial metabolic activities and products resulting from the impact of 2,4-D on plant hormone metabolism may indirectly affect host health, which contradicts the notion that certain environmental chemicals are safe to human just because their targeting pathways are not found in the human body. We also discovered a consistent enrichment of pathways and genes involved in urea degradation in treatment group compared to control group (Fig. 3). It is estimated that almost up to one third of urea produced endogenously by human body is degraded by microbial urease34. More importantly, microbial urease has been recognized as a virulence factor for gastrointestinal pathogenesis35. In addition, a significant shift in gene abundances of microbial amino acid metabolism as well as carbohydrate metabolism occurred in the gut microbiome of 2,4-D treated mice. To be more specific, for amino acids, the metabolism of alanine, serine, and glycine was enhanced whereas lysine, threonine, methionine, cysteine and branched-chain amino acids was diminished; for carbohydrates, the metabolism of polysaccharides was enhanced whereas monosaccharides was diminished. Together this may indicate the change of utilization preference in amino acids and carbohydrates in the 2,4-D-altered gut microbial community, and evidence shows that microbial amino acids and carbohydrate metabolism can influence host amino acid and energy homeostasis, respectively36,37. Therefore, the fundamental metabolism and the functional role of the gut microbiome were different in 2,4-D-treated mice compared to controls due to changes in diverse microbial metabolic pathways and functions.

A less resilient gut microbial community indicated by a lower alpha diversity along with a variety of shifted gut microbial species was found in mice treated with 2,4-D (Fig. 1). The relative abundances of Dehalococcoides ethenogenes increased in the gut microbiome of treatment group compared to control group. Dehalococcoides ethenogenes has been shown to play a major role in the degradation of chlorinated hydrocarbons in contaminated environments38,39. It is suggested that Dehalococcoides ethenogenes strain 195 has the dehalogenation potential to a diversity of chlorinated hydrocarbons based on up to 17 putative dehalogenase gene homologues found in its genome40. Thus, the enrichment of Dehalococcoides ethenogenes in 2,4-D-treated mouse gut microbiome likely reflects the selective pressure exerted by 2,4-D in the gut environment, and the competitive advantage held by Dehalococcoides ethenogenes resulting from its dehalogenation ability. In addition, an increased bacterial population in Phylum Spirochaetes has been found to be associated with 2,4-D exposure. Many members from Phylum Spirochaetes are involved in prevalent pathogenic diseases, such as Brachyspira pilosicoli and Brachyspira aalborgi in human intestinal spirochaetosis41, and Leptospira genus in Leptospirosis42. Various Spirochaetes species are also associated with the development of dementia and could be involved in the pathogenesis of Alzheimer’s disease43.

The communication between the gut microbiota and host through production of gut metabolites is one essential aspect of the gut-host cross-talk. On one hand, the gut microbial profiles are under regulations of host-produced metabolites, for instance, antimicrobial peptides44. On the other hand, gut microbiome-derived metabolites can contribute to the rise of either host fitness or disease risk45. For example, the gut microbiome is an important source for vitamins such as vitamin K, vitamin B12, biotin, folate and so forth22,46. Meanwhile, trimethylamine N-oxide, derived from the gut bacterial metabolite trimethylamine, is highly correlated with cardiovascular disease and kidney disease47. In the present study, we discovered distinct gut metabolic profiles between treatment and control groups, demonstrating the capacity of 2.4-D to alter the metabolic functions and metabolite fingerprints in the gut microbiome. Prostaglandins were found to be enriched in fecal samples after 2,4-D exposure in mice. Prostaglandins are a class of lipid autacoids involved in inflammatory response48, which can be found in intestinal mucosa49. It is speculated that the increase of prostaglandins might be one of the coping strategies with gut inflammation. However, the mechanisms by which the abundances of prostaglandins being elevated and the physiological effects on host are unknown and merit further studies. We also found significant perturbations in nitrogen metabolites in the gut microbiome. In concert with the alterations in gene abundances of amino acid metabolism, differentiated metabolites including several amino acids and their metabolic intermediates supported that 2,4-D treatment induced functional alterations in gut microbial amino acid metabolism. Additionally, the differences in purines, pyrimidines and their derivatives also indicated a perturbed nitrogen metabolism. Taken together, the alterations in the metabolite profiles of mouse fecal samples testify to 2,4-D-induced functional changes in the mouse gut microbiome.

Furthermore, the metabolite profiles in host plasma were differentiated by 13-week low-dose 2,4-D treatment, suggesting the capability of low-dose 2,4-D exposure to directly or indirectly impact host metabolism. In particular, the plasma levels of a series of acylcarnitines were observed to be significantly lower in 2,4-D-treated mice. The NOAEL for subchronic (13-week) exposure of 2,4-D in mice is 15 mg/kg body weight per day according to previous studies5, which is equivalent to 90 ppm 2,4-D in drinking water using the daily water intake as 8 ml per 30 g body weight50. The dose used in the present study was approximately 60 times lower. However, strong toxic effects of 2,4-D manifested by marked perturbations in the host plasma acylcarnitine levels were observed at this very low dose, indicating that the 2,4-D toxicity can be exerted at doses that are far lower than NOAEL. In this context, acylcarnitines have never been associated with 2,4-D exposure and/or 2,4-D toxicity before. Acylcarnitines are involved in the beta-oxidation of fatty acids51, and the alterations in the plasma levels of acylcarnitines suggested perturbations in the fatty acid beta-oxidation pathway. It is reported that the daily doses of 2,4-D exposure in professional turf applicators were predicted to be up to 20 mg per day, which is comparable to the dose in the present study20. Thus, monitoring the changes of plasma acylcarnitine levels could help to identify occupational toxicity of 2,4-D. In addition, growing evidence suggests an important role that acylcarnitines play in neurological diseases52. For instance, a decrease of serum acylcarnitines is reported to be associated with neurological disorders including Parkinson’s disease and Alzheimer’s disease53,54. Thus, lower levels of plasma acylcarnitines observed in 2,4-D-treated mice may be associated with the potential neurotoxicity of 2,4-D. Although the link between 2,4-D-induced gut microbiome perturbations and low plasma acylcarnitines cannot be built based on the results of the present study, clear correlations could be identified between the perturbed gut microbial species, Xylanimonas cellulosilytica and plasma acylcarnitine levels (Fig. S1). The association between low-dose 2,4-D exposure-induced gut microbiome perturbations and lower plasma acylcarnitine levels provided novel insights upon the mechanisms for 2,4-D toxicity, and the plasma acylcarnitines can serve as a novel biomarker for 2,4-D toxicity at low doses.

Despite the relatively limited sample size in this study, the results clearly demonstrated the capacity of 2,4-D to induce both phylogenetic and functional changes in mouse gut microbiome with differentiated metabolic profiles. Significant distinction was also shown in host plasma metabolic profiles indicating perturbations in host metabolism, even though direct causal effects of 2,4-D-disturbed gut microbiome on host metabolic outcomes cannot be inferred from the present study. Ours is the first study to investigate the impact on the gut microbiome by 2,4-D exposure using multi-omics approaches, the interplay between the alterations in microbial pathways and metabolites and their resultant health effects on host warrants further investigation.

To conclude, this is the first study that demonstrated the alterations in the gut microbiome driven by subchronic low-dose 2,4-D exposure. The metagenomics indicated significant perturbations by 2,4-D to the gut microbial composition and functionality, in concert with distinct metabolic profiles in mouse fecal samples revealed by metabolomics. Meanwhile, the metabolomic profiling of host plasma samples revealed altered host metabolic profiles suggesting 2,4-D toxicity at occupationally relevant doses. These findings support the notion that the homeostasis of gut microbiome can be readily impacted by environmental chemicals, suggesting reconsideration for the health effects and toxicity of wildly-used chemicals including 2,4-D.