One important connection between the gut and the brain is the microbiota. This means that any dysregulation of the microbiota (named dysbiosis) can affect the two parts of this axis. Oral-exposure substances that can disrupt microbiota can be in diet, drugs, antibiotics, and, most importantly in this review, pesticide residues.

Pesticide residues are food and water contaminants. Some of them alter the composition of the gut microbiota (GM)GM and disrupt and cross the IB ^[1][2][3][25,109,110]. They are defined by the Food and Agriculture Organization (FAO) as any substance or mixture of substances intended for preventing, destroying, or controlling any pest, including vectors of human or animal disease, unwanted species of plants or animals, causing harm during or otherwise interfering with human activities (production, processing, storage, transport, marketing of food, agricultural commodities, etc.) ^[4][111]. These substances are classified based on various criteria, such as the targeted pest organism type (fungicide, herbicide, insecticide, etc.), chemical composition (e.g., synthetic organic insecticides: organochlorines, organophosphates, carbamates, and pyrethroids), and mode of entry in the body ^[5][6][3,112]. The mode of action of pesticides targets the physiological systems of the pests they kill, but this can result in poisoning nontarget species, such as humans (phylogenetic similarities in digestive, respiratory, and nervous systems). Since the beginning of the 20th century, the use of pesticides and fertilizers is intensive to ensure large agriculture production to adapt with demographic growth. The most frequently used chemical families in agriculture are the synthetic organic insecticides mentioned above and triazines ^[7][5]. This means that reswearchers are not only exposed to one or two but to a mixture of environmentally persistent pesticides daily. Thus, the assessment of the impact of these substances nowadays should consider the chronic cumulative effect as well as the cocktail effect ^[8][9][10][113,114,115]. The concept of metabolization (biotransformation by microbiota bacteria and liver detoxification enzymes) is also a point of interest because the pesticide metabolite can be more harmful than the parent compound, indicating a metabolic activation. Generally, oxon-type intermediate metabolites are more hazardous than their parent pesticide. Organophosphates oxon-metabolites are an example of more toxic metabolites than the corresponding pesticide: Chlorpyrifos-oxon (CPF-oxon) is more potent than Chlorpyrifos (CPF) itself, same as for paraoxon and parathion ^[11][12][13][2,116,117]. The neurotoxic effects and impact on the gut-microbiota-blood–brain barrier (BBB) axis of the most detected pesticides in food (according to the EFSA European Food Safety Authority) ^[14][118] are detailed in Table 1. This resviearchw details the effects of the most used organophosphate insecticide, CPF, on the gut-microbiota-BBB axis.

Organophosphorus pesticides are extensively used worldwide because of the wide range of pests they kill, the broad spectrum of applications (food trees, wheat, corn, almonds, tea, etc.), and their persistence in soil ^[11][38][2,136]. Chlorpyrifos (CPF) is the most commonly used thionophosphorus organophosphate insecticide, available on the market at a low price since 1965, especially in France, one of the first pesticide consumers in Europe. In 1998, only one study was sufficient for the European Union (EU) to give use approval of CPF, but it took more than hundreds of studies showing neurotoxic, metabolic, and endocrine effects to make the decision in 2019 limiting its use. Nonetheless, CPF production has not been banned. Therefore, pollution in the environment is still present. After the ban of CPF use in the EU by the EFSA, except for the culture of spinach in France, the EPA (United States Environmental Protection Agency) made a recent regulatory decision banning CPF for food uses in the United States in February 2022 (EPA final rule) ^[39][137]. However, on one hand, it is still allowed for mosquito control, on tobacco, on plantations not for feed purposes, and on food destined to export when it complies with foreign purchaser specifications (updates on CPF uses in 2022, Iowa State University) ^[40][138]. Therefore, these limitations do not prevent the accumulation of CPF in the soil and then in water and thus its uptake by aquatic species and its entry into the food chain of human beings ^[41][139]. And still, many other regions of the world including China and India continue allowing CPF on crops. On the other hand, it has been demonstrated by many studies on the biotransformation of CPF in soil that its half-life has a very wide range, from 360 days to 17 years, because its fate depends on the initial concentration used on plants and the biodegradation rate. This means that even after limitations, measures should be taken concerning the control of CPF residues in soil, local and imported food, and most importantly in human blood or urine. In addition, strict measures should be adopted in countries where residents have easy access to this dangerous pesticide: in Iran, CPF residues are found in the milk of breast-feeding mothers, their urine, and even their children’s urine ^[42][140]. A total of 92% of these mothers confirmed pesticides’ house use, an activity banned since 2001 in the US ^[43][106]. Rathod and Garg reported a scenario in India where the commonest method of suicide (40.5%) is organophosphorus compound (OPC) intake ^[11][44][2,141]. Therefore, limitations are essential in these countries to protect residents and their future generations.

Chlorpyrifos is absorbed by all routes of exposure. Urinalyses of exposed human volunteers indicate that approximately 70% is absorbed by the oral route ^[11][45][2,142]. After CPF exposure and then distribution throughout the body, cytochrome P450 (CYP) in the liver metabolizes CPF, replacing the sulfur group with oxygen, to CPF-oxon, a metabolite that is more toxic than CPF itself. The detoxification of CPF-oxon consists of oxidase enzymes hydrolyzing it to diethylphosphate (DEP), diethyl thiophosphate (DETP), and 3,5,6-trichloro-2-pyridinol (TCP) ^[11][2]. TCP is a specific metabolite of CPF, whereas DEP and DETP can be detected after exposure to other organophosphates ^[46][47][143,144]. It is the metabolic bioactivation to CPF-oxon that leads to the irreversible inhibition of acetylcholinesterase (AChE) preventing the breakdown of acetylcholine (ACh), a neurotransmitter that ensures nerve cells communication. The accumulation of ACh in the synaptic cleft overstimulates the neuronal cells, leading to a collapse in the nervous system of insects (National Pesticide Information Center) ^[48][145]. Similarly, in higher vertebrae, in particular humans, CPF has a cholinergic effect and has been demonstrated to have a plethora of non-cholinergic effects.

The research on xenobiotics degradation in the human body mostly focuses on detoxification by CYP and other liver enzymes, but based on the literature, a minority of the studies focus on their degradation by intestinal microorganisms and specifically bacteria. A 2013 study by Harishankar and colleagues found that 70% of CPF was degraded to TCP by Lactobacillus fermentum, 61% to CPF-oxon by Lactobacillus lactis, and 16% of CPF to CPF-oxon and DEP by Escherichia coli ^[49][146]. Recently, insect experimental studies were conducted to investigate the fate of pesticides by an intestinal microorganism ^[50][147]. A 2021 study assessed the biodegradation of organophosphorus including CPF by isolating insect gut microbial species. Four potential bacterial endosymbionts such as Bacillus subtilis, Bacillus licheniformis, Pseudomonas putida, and Pseudomonas cereus used CPF as a unique source of carbon and energy for their growth and enzymatic function. They found that Pseudomonas cereus and Pseudomonas putida have more potential to degrade the CPF ^[51][148].

Similarly, in soils, the researchers suggested that CPF is totally degraded by microorganisms and especially by Pseudomonas putida MB285 to form the primary products TCP and DETP, which are further decomposed into non-toxic metabolites, such as CO₂, H₂O, and NH₃ ^[52][149]. Pseudomonas is a diverse genus with multiple degradation pathways. It was reported that Pseudomonas putida MAS-1 had the highest degradation efficiency for CPF in Pseudomonas genus, with a 90% degradation rate within 24 h ^[53][150]. Furthermore, another study published in 2008 reported that different bacteria contribute to the biodegradation of CPF in five aerobic consortia, based on antibiotic resistance survival and REP-PCR (Repetitive Extragenic Palindromic Polymerase Chain Reaction). The results illustrated that 75–87% of the CPF was degraded to TCP after 20 days of incubation by Pseudomonas aeruginosa, Pseudomonas fluorescence, Bacillus subtilis, Brucella melitensis, Klebsiella sp., Bacillus cereus, and Serratia marcescens. However, the results also showed that the TCP disappeared after 30 days of incubation ^[54][151]. A mini review published by Supreeth in 2017 evaluated the biotransformation of CPF and endosulfan by bacteria and fungi and discussed the aftereffects of their transformed byproducts (metabolites) ^[38][136]. The degradation of CPF to TCP was executed by different bacteria species, Enterobacter sp., Stenotrophomonas sp., Sphingomonas sp. ^[55][152], Pseudomonas aeruginosa, Bacillus cereus, Klebsiella sp., Serratia marscecens ^[56][153], Bacillus subtilis inaquosorum strain KCTC13429, B. cereus ATCC 14579, and B. safensis F0-36b ^[57][154], and to CPF-oxon 3,6-Dihydroxypyridine-2,5-dione and DETP by Pseudomonas putida (NII 1117) Klebsiella sp. (NII 1118) P. stutzeri (NII 1119), P.aeruginosa (NII 1120) ^[58][155], and Ralstonia sp. Strain T6 ^[59][156].

As the gut and microbiota have an impact on the absorption and metabolization of pesticides, these pesticides can have serious effects on all parts of the gut–brain axis. Prenatal pesticide exposure studies detected parent molecules of organophosphates and/or their metabolites, CPF in particular, in meconium ^{[60][61][62][63]}[157,158,159,160]. In fact, meconium has become a biomarker of in utero pollutants exposure. At this stage of rapid growth and development, especially of the brain, humans are more vulnerable and sensitive to the toxic effects of pesticides ^[64][65][161,162]. The message behind these analyses is that even before birth, which means before gut microbial colonization, reswearchers are exposed to CPF, which seems to cross the placental barrier. In addition, considering the oral route, before reaching the brain to inhibit acetylcholinesterase, the first organ to encounter CPF or any food contaminant is the digestive tract. Thus, it is necessary to investigate the organophosphate effects on the intestinal tract. Working on the rat offspring NOAEL of 1 mg/kg/day, Joly et al. studied the effect of 1 mg/kg/day or 5 mg/kg/day of CPF perinatal exposure on the GM of the rats’ progeny at two developmental time points: weaning (D21) and adulthood (D60) ^[16][34]. Their results show that CPF exposure induces microbial dysbiosis: a decrease in Lactobacillus spp. counts in the ileum, cecum, and colon as well as a decrease in Bifidobacterium spp. at D21 in the ileum and D60 in the colon, and an increase in Clostridium spp. and Staphylococcus spp. counts in the cecum and colon at D21. This means that CPF exposure reduces potentially beneficial bacteria and increases potentially pathogenic ones. The epithelial thickness of the ileum and colon was decreased by CPF treatment. Because the first study was based on classical microbiology tests, they researched further to identify more specifically intestinal bacteria by MALDI-TOF-MS and investigate bacterial translocation from intestinal segments to sterile organs ^[22][30]. By molecular typing, they were able to confirm the translocation of Staphylococcus aureus to adipose tissues, kidney, and Peyer’s patch from 12.5% of CPF-exposed rats’ intestinal segments and a 5% translocation of Enterococcus faecalis to the liver. This is logically explained by the increase in gut permeability induced by the decrease in ZO-1 and claudin 4 transcriptional expression in the ileum and colon, especially on D21 ^[66][62]. CPF exposure of an in vitro artificial human intestine (SHIME^®) combined with a Caco-2/TC-7 model was associated with a decrease in the tight junction gene expression, occludin and ZO-1, and an increase in the proinflammatory chemokine interleukin-8 (IL-8) ^[20][121]. Zhao et al. also confirmed abnormal intestinal permeability in CPF-exposed mice and reported microbiota dysbiosis (a decrease in Lactobacillaceae and Firmicutes and an increase in Bacteroidaceae and Bacteroides) and alterations in the metabolism of SCFAs that led to intestinal inflammation ^[2][109]. This is an expected result because CPF treatment alters the microbial community that produces these metabolites. In addition, an analysis of serum showed an increase in LPS by CPF treatment. J. W. Li and colleagues showed that chronic exposure to 0.3 mg CPF/ kg body weight/day induced a significant increase in TNF-α and IL-6 in the serum of exposed rats ^[67][163]. These observations might explain the alterations in the functional integrity and structure of the BBB by CPF, highlighted by Parran et al. who worked on an in vitro BBB model (bovine endothelial cells and neonatal rat astrocytes) ^[24][87]. Another study on an in vitro BBB model (rat brain endothelial cells and neonatal rat astrocytes) showed that the short-term CPF treatment at low concentrations alters the expression levels of the claudin 5, ZO-1, and TRPC4 (transient receptor potential canonical channels) genes, disrupting the BBB integrity (TRPC regulates the calcium influx that modulates the paracellular permeability of the endothelial cells of the BBB) ^[68][88]. The claudin 5 gene is the main tight junction gene involved in the BBB tightness and ZO-1 ensures the support to the TJs’ architecture ^[69][70][164,165]. Thus, CPF targets the most important actors of the BBB integrity.

Because the exposure to pesticides is often accompanied with saturated fats and refined sugars (an unbalanced diet based on fast food and processed food), other studies assessed the impact of the association of CPF to a High Fat Diet (HFD) on intestinal microbiota and the IB. Guibourdenche and colleagues confirmed that the chronic perinatal exposure to the NOAEL dose of CPF alone induced a decrease in transcriptional TJs expression and also demonstrated an increase in proinflammatory cytokines and is aggravated by the association to an HFD ^[15][23][31,122].

These experimentations all together point to the impact of CPF on the entire gut-BBB axis even though its main mechanism is the inhibition of acetylcholinesterase in neurons. Further investigations are necessary to identify molecular pathways that explain the barriers’ disruption by organophosphates.

In parallel to studies analyzing the effects of organophosphates on the gut–brain axis, there are several studies investigating the molecular pathways associated to them. It has been demonstrated that organophosphates induce apoptosis by affecting signaling molecules, including c-Jun NH2-terminal protein kinase (JNK), p38 MAP kinase, and extracellular signal regulated protein kinase (ERK1/2) ^[71][72][166,167]. ERK1/2 is activated by neurotrophic factors and growth factors, whereas environmental stresses such as reactive oxygen species (ROS) activate JNK and p38 MAP kinases. The activation of JNK and p38 MAP kinases induces apoptosis, while the activation of ERK1/2 is protective against it ^[73][168]. In fact, CPF increases JNK, ERK1/2, and p38 MAPK phosphorylation ^[74][169]. It is known that mitogen-activated protein kinases (MAPKs) regulate matrix metalloproteinases (MMPs). MMP9 is regulated by ERK1/2 ^[75][170]. MMP9 activation yet increased the IB permeability in a Caco-2 in vitro model ^[76][171]. MMP9 upregulation leads to BBB leakage as well, through the rearrangement and/or degradation of the tight junctions ^[77][78][172,173]. Moreover, MAPKs have a role in the inflammatory response. Interestingly, CPF can upregulate cyclooxygenase 2 (COX-2) through MAPK activation ^[74][169]. In addition, CPF upregulates inflammation-related genes and the protein level of NF-κB and TNF-α ^[79][174] which in turn can deteriorate the BBB permeability ^[80][84]. A possible explanation for the CPF implication in these pathways is as follows: CPF increases NADPH oxidases (NOXs) and superoxide levels which increase ROS signaling and oxidative stress in cells ^[81][175]. The increase in ROS signaling induces, on one hand, the upregulation of TNF-α and NF-κB and, on the other hand, the increase in the ASK1 expression responsible for the activation of JNK upregulation which is one of the MAPKs that regulates MMPs.

Recent studies on pesticide effects are focusing on a new concept: the recovery. In fact, a study of the effect of different doses of imazalil on mice GM evaluated the time of recovery after 2 and 15 weeks of exposure. They found that 30 or 45 days after impregnation with this fungicide, the bacterial composition at the phylum level recovered to the control level ^[36][134]. This highlights the fact that a change in the microbiota composition is flexible and tends to regain balance. In addition, new clinical trials indicated that the gut-microbiota–brain axis pathways and mechanisms are prone to dietary modulation and are of vital interest in clinical nutrition. As a matter of fact, dietary interventions and supplementation with probiotics and prebiotics can reshape the bacterial composition and are now administered as “psychobiotics” to treat neurological disorders because of their beneficial effects on the brain ^[82][176]. This means that by reshaping the GM, the whole gut-microbiota-BBB axis is positively modulated. The concept of probiotic use in the modulation of gut microflora was initiated in the 19th century by Elie Metchnikoff who theorized that “health and longevity could be achieved by manipulating intestinal microflora, i.e., replacing harmful microbes with beneficial microbes” while prebiotics were introduced by Gibson and Roberfroid in 1995 ^[83][177].

Their supplementation is now considered a promising approach that alleviates the negative effects of food contaminants ^[84][178]. In effect, bacterial strains that are considered probiotic strains (mostly Lactobacillus strains) can bind to xenobiotics and reduce their toxicity (through biotransformation) and the amount absorbed by the host ^[85][179]. Lactobacilli spp. are known to have the highest anti-inflammatory effects ^[86][52]. To exert their beneficial roles, probiotic strains need substrates. Prebiotics partially provide probiotic strains with substrates that are “nondigestible food ingredients which selectively stimulate the growth and activity of beneficial bacterial species already implanted in the colon, and thus improve the health of the host” ^[87][180]. Prebiotics are fructo-oligosaccharides (FOS) such as inulin, galacto-oligosaccharides (GOS), trans-galacto-oligosaccharides (TOS), and resistant starch which can be found in many fruits, vegetables, grains, and milk ^[88][89][181,182].

Arabinoxylo-oligosaccharides and inulin induced an increase in some SCFAs (acetate, propionate, and butyrate) and a shift in the microbial composition from Firmicutes to Bacteroidetes ^[90][183]. Similarly, Sialyllactose (isolated from milk) and GOS induced the differentiation of the epithelial cells in the Caco-2 model, the modulation of the microbial composition (an increase in Bacteroides and Bifidobacteria), and consequently the production of SCFAs ^[91][184]. In fact, the fermentation of prebiotics by gut bacteria produces SCFAs which can reach the bloodstream by diffusing through gut enterocytes and have beneficial effects on the host ^[88][181]. In effect, an in vitro study linking the Caco-2 model and SHIME demonstrated that arabinogalactan and FOS decreased proinflammatory cytokines (IL-6 and IL-8), increased the anti-inflammatory cytokine IL-10, and improved the gut barrier permeability (the TEER measurements of the Caco-2 model) ^[92][185]. These findings emphasize the direct effect of prebiotics on IB function through the modulation of the microbiota composition. However, these oligosaccharides and the products of their fermentation (SCFAs) can improve IB function through TJs modulation ^[93][186]. Many studies underline the role of inulin ^[94][187] and FOS ^[95][188] in enhancing IB through TJs assembly.

Because they have a beneficial effect on the IB and microbial composition, researchers investigated if pretreatment or supplementation with prebiotics could attenuate damage in an inflammatory environment or intestinal harm caused by food contaminants exposure. As an example, GOS pretreatment can alleviate damage of the IB in an inflammatory environment (in LPS-challenged mice) ^[96][189]. Additionally, inulin supplementation to rats exposed to CPF, an HFD, or the association of both CPF and a HFD and in in vitro models (SHIME and Caco-2) reversed their effects on microbial composition by increasing potentially beneficial flora (Lactobacillus and Bifidobacterium), decreasing potentially pathogenic ones (Enterococcus and Enterobacteriacea) and improving the IB integrity ^{[19][20][21][23]}[26,120,121,122]. Thus, prebiotics use through nutrition can modulate the GM with beneficial outcomes. New methods are now emerging such as fecal transplantation, a strategy to deal with dysbiosis, that has been shown to have a positive effect on the treatment of Parkinson’s disease ^[97][190].