GBHs are the most applied herbicides worldwide; humans are commonly exposed to these environmental chemicals at a wide range of doses depending upon the job setting (farming vs. food consumption) and route of exposure (ingestion vs. inhalation). Environmental contamination from GBHs is now ubiquitous and residues of glyphosate has been found in air [46], groundwater [47], drinking-water [48], crops [49], food [50] and animal feed [51]. The possible effects of GBHs on human health are the subject of an intense public debate, for both its potential carcinogenic and non-carcinogenic effects, including endocrine disruption [52, 53], neurotoxicity [54], developmental and reproductive toxicity [55], autoimmunity [56], gastrointestinal disorders [57], obesity, diabetes [58,59,60], and other metabolic and cardiovascular disorders [61] and central nervous system dysfunctions such as learning and memory impairment, anxiety, stress, depression [62] and autism [63]. These chronic pathologies (non-communicable diseases – NCDs) may occur even at doses that are much lower than the ones considered during risk assessment, in particular during sensitive periods of life (such as fetal development) [7, 22].

Recent advances in human microbiome research suggested that the gut microbiome is a key player in human metabolism [64,65,66]. It is thus reasonable to hypothesize that exposure to environmental chemicals may modify the gut microbiome and its metabolites and ultimately influence human health. Microbiota-generated metabolites and their cellular and molecular components are increasingly being recognized as an essential part of human physiology, with profound effects on the homeostasis of the host organism. Unfortunately, determining the concentrations of these biologically active substances in target cells presents serious difficulties related to the extraction and processing of samples, especially faecal material, and the limitations of currently available measurement techniques [15]. Meta-omics and evolving computational frameworks will hopefully lead to the systematic prediction and discovery of more microbial metabolites and components involved in neuroendocrine, immune, metabolic, and epigenetic pathways.

Rats are proposed to be more representative of the human gut microbiota than mice because the gut bacterial communities of humanized rats more closely reflect the gut microbiota of human donors [67, 68]. We have previously used our animal model, SD rats, to study the effect of postnatal low-dose exposure to environmental chemicals on windows of susceptibility and on the gut microbiome. The study [69] showed the low-level phthalate, paraben and triclosan exposure altered the gut microbiome of adolescent rats. These results are consistent with other studies, indicating our animal model as a suitable model for studying microbiome [70, 71].

Since glyphosate has shown enzyme inhibition activity in plants and microorganisms, we therefore postulate that low-dose exposure to glyphosate or GBHs may also modulate the composition of the gut microbiome. In this study, when compared to the adult rat dams, the gut microbiome of pups at PND 7 and 14 showed lower taxonomical richness but higher variance within sample and higher sample-to-sample dissimilarity [69]. One pitfall of our study was that direct measurements of exposure to GBHs in milk was not performed [72]. In our pilot study we simply reproduced the human exposure, which includes lactation as only source of nourishment for pups from birth until around PND 21. The shortcomings concerning the analysis of glyphosate in breast milk are mainly related to the difficulty and stressing technical procedure for collecting milk from dams and to the complex nature of the breast milk matrix. Indeed, milk is an aqueous mixture of carbohydrates, proteins and fat. Analytical methods developed for watery matrices cannot be directly transferred to breast milk. In April 2014, a non-peer-reviewed report was published, in which glyphosate in breast milk of American mothers was detected in 3 out of 10 samples ranging from 76 to 166 ng/mL. In this study, the concentration of glyphosate in milk samples was determined by enzyme-linked immunosorbent assay (ELISA) [73]. The limit of quantification (LOQ) of the assay was given as 75 μg/L in milk. Other studies, based on liquid chromatography–tandem mass spectrometry (LC-MS/MS) and a gas chromatography–tandem mass spectrometry (GC-MS/MS) methods, have found no evidence of transfer of glyphosate into milk. Both methods have been fully validated and reported as suitable for the determination of glyphosate with an LOQ of 1 ng/mL [72, 74]. Nevertheless, future independent research is needed, considering different educational and ethnic backgrounds, location of residence (e.g., urban compared with rural), occupational and dietary glyphosate exposure and adequate sample size of the cohort.

Our results revealed that both glyphosate and glyphosate formulated Roundup, at doses admitted in humans, including children and pregnant women, significantly altered the microbiota diversity and resulted in prominent changes at multiple taxon in exposed pups. However, those effects on microbiota were not significant in the adult dams. Previous evidence has shown that the gut microbiota at postnatal age is less stable than at adult age and it changes over the first several years of life [75]. The maturation of the gut microbiota has been proven to be affected by multiple factors, for instance, diet, medications, host genetics, etc. [76]. Disruption of the microbiota during its maturation by low doses of various environmental chemicals has been showed to alter host phenotypes, such as weight, metabolism and other disease risk [77]. Our data suggests that the prepubertal age microbiota is more sensitive to GBH exposure compared to the adult microbiota, therefore the postnatal age is likely a “window of susceptibility” for GBHs to modulate the gut microbiome.

Furthermore, our results showed that the overall microbiome diversity and composition were significantly different between Roundup and glyphosate, suggesting possible synergistic effects of the mixed formulation on gut microbiota. As most of GBHs contains multiple surfactants and adjuvants might act differently than glyphosate alone, it is not only important to understand the individual effects of glyphosate, but also the synergistic impact of mixed formulations. In fact adjuvants might act alone or in a synergistic manner and increase the toxic effects of glyphosate [78,79,80,81].

In addition, both clinical and experimental studies showed impact of gut microbiota on the gut-brain axis (which mainly includes the immune, neuroendocrine, and neural pathways) [82,83,84] in an age-dependent manner [85]. Gut bacteria communicating with the host through the microbiota-gut-brain axis could influence brain and behavior [86]. In particular, the changes at postnatal microbiota may affect the neurvous system, reflecting by changes in levels of pituitary hormones including ACTH [83, 87], cortisol, BNDF [88] and etc. Sprague-Dawley rats represent an excellent animal model to explore these early-life effects as their microbiome is more similar to that of humans than the microbiota profile of mice [67].

This study has some limitations. First, the actual levels of GBHs that reached the fetus during gestation or through milk consumption postnatally by the offspring cannot be accurately estimated. Second, we only collected maternal feces so that we cannot fully evaluate the role of maternal microbiota in the fetal development without the maternal sample/data collection from oral, vaginal and other body sites. Indeed, in recent years it is becoming apparent that, besides breast milk, other sources could allow maternal-offspring microbial transfer. Rodents “inherit” their microbiomes in a similar fashion to all placental mammals, including humans: through vaginal delivery and close maternal association throughout the neonatal period (vertical transmission). Maternal vaginal, skin, mammary fecal and oral microbiomes, microbial spreading in bedding are efficiently transmitted to offspring and represent other possible mechanisms of maternal influences on pups intestinal colonization [89]. Finally, the microbiome survey used a cost-effective 16S amplicon targeted sequencing approach. This technique allows us to identify differential taxa compositions by exposure only to genus level. Additional meta-genomics and meta-transcriptomic analysis may need to visualize the functional and metabolic alternations and identify bacterial features at species/strain level. In addition, given the differences in taxonomic composition of the microbiomes of rats and humans, the extent to which the results of this analysis can be relevant to humans is not clear. Future work should investigate how the route and concentration of exposure impact the rat microbiome, and quantify how these perturbations may impact subsequent health outcomes. Nevertheless, these data strongly indicate that GBHs exposure can exerts biological effects early in development which may have long-lasting health effects later in life.