Glifosato e micotossine nella catena alimentare

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Glyphosate, mycotoxins and antibiotics in the food chain, effects on the gastrointestinal apparatus and immune system.  May it be a predisposing condition ASD?

Maurizio Proietti M.D.
President of scientific committe ASSIS, Pisa Italy. Independent researcher.
E-Mail: drproietti@gmail.com

Abstract

Today we have pollution levels out of control; the most important problem is the pollution of the human and animal food chains. The most recognized toxic substances is glyphosate, including involuntary biological contaminants mycotoxins. Each species has its own peculiarities in inducing effects on health. Mycotoxins is an important stressor factor, in particular deoxynivalenol (DON) that disrupted the expression of the Zonulin-1(ZO-1), occludin and claudin of porcine gut epithelial TJs. Mycotoxins are immunotoxic and teratogenic. Antibiotics imbalance microbiota. Pollutants, including involuntary, and antibiotics have a negative effect on cell junctions, the microbiota and the microbiome, which ultimately lead to dysbiosis and leaky gut. Glyphosate is another emerging problem; it appears that the damage is caused by the surfactant, TN-20. Surfactant and glyphosate led to mitochondrial damage, apoptosis, and necrosis, disrupts the integrity of the cellular barrier and blocks out microbial growth at lower concentrations. Furthermore, glyphosate induces an increased activity of PAL that leading to a hyperammonemic environment in the gut, incresed ammonia level is believed to be intestinal bacteria in small and large intestine. There is strong correlation between gut dysbiosis and autism spectrum disorder (ASD).  There is strong relationship between intestinal gene expression and bacterial structure.  This is the future challenge for research.

Keywords: autism spectrum disorder (ASD); deoxynivalenol (DON); dysbiosis; tight Junction (TJ) glyphosate; microbiota; mycotoxin; zearalenone (ZEN).

 

 

Introduction

 

Today we have pollution levels out of control, with negative effects on human health; the most important problem is the pollution of the human and animal food chains. The indiscriminate use of pesticides, herbicides and many other chemicals in agriculture helped create this situation. 1 It is the first time in history that man is exposed in a manner so massive in this type of foreign substances, and especially non-biodegradable. Toxic substances most responsible are the chemicals used in agriculture, in particular glyphosate, involuntary biological contaminants mycotoxins and finally noteworthy antibiotics. Antibiotics are xenobiotics and are notorious for their destructive action on the normal intestinal flora. The presence of mycotoxins is a real and serious risk to health, particularly for children under 3 years. As for durum wheat, the mycotoxin that is one of the risk parameters recognized by the scientific literature is the deoxynivalenol (DON). 2  Also zearalenone (ZEN) also known as F-2 toxin, is one of the must studied  myvotoxins, is a non-steroidal estrogenic mycotoxin synthesized by a variety of Fusarium fungi that are commonly found in feed and foodstuffs. 3 Contamination of food matrices is already on the field, trichothecenes, fumonisin, zearalenone (ZEN). The factors that account for a predominant development of mycotoxins are: temperature and humidity. At risk are all products of the crops that are the basis of human food and animal cereals, to mention the most important crops, but are not exempt coffee, cocoa, spices, fruit. Most harmful microorganisms for human beings and animals belong mostly to the genera Aspergillus, Penicillium and Fusarium, most spread between 15 and 30 degrees Celsius with optimum around 22degrees. Exceptions are microorganisms that can withstand temperatures that go below zero, it is above 100 degrees; this depends mostly on the substrates and on the availability of water. 4 5

Mycotoxins and health damage

 

  • Aspergillus flavus and parasiticus produce aflatoxins that infest mainly peanuts, legumes, corn and other grains, oil seeds, nuts, almonds, dried figs, milk and derivatives. There is the risk of developing hepatocellular carcinoma. 6
  • Aspergillus ochraceus, Penicillium viridicatum, other Aspergillus spp. and Penicillium spp produce ochratoxins that infest peanut, rice, coffee, corn, barley, other grains therefore bread and pasta; it is possible have kidney diseases, enteritis, hepatitis, teratogenic and mutagenic diseases. 7
  • Fusarium graminearum, roseum, tricinctum, moniliforme and other Fusarium spp, which infest corn and other cereals ZEN produce derivatives that are capable of causing abortions, infertility, dysregulation ormonale. A consumer product that may contain ZEN is beer. 8
  • Fusarium moniliforme produces Fumonisin that infest corn and sorghum and nephrotoxic, cytotoxic, carcinogenic and teratogenic. The main toxicological effect of Fumosin B(1) is the accumulation of sphingoid bases associated with the depletion of complex sphingolipids. These problems on the sphingolipid biosynthesis explain the toxicological effects such as an alteration of enterocytes vitality and proliferation. There is also an imbalance on cytokine production. 9 To note that very frequently in ASD children is found an alteration of membrane fatty acids, in particular, there is a decrease of Dihomo-γ-linolenic acid (DGLA) an ω-6 fatty acid. These data are recorded in my case reports, and it will be the subject of a future article.
  • Poae Fusarium, Fusarium tricinctum, Fusarium nivale, Trichoderma lignorum, other Fusarium spp. Infest corn, barley, rice, other cereals, and their derivatives, for example oat flakes. These cereals produce Trichothecenes that cause pathologies of the skin and mucous membranes, leukopenia, bleeding, vomiting, diarrhea as well as being cytotoxic. 10
  • Penicillium citrium, purpurogenum, rubrum and other Penicillium spp. that infest corn, other grains, rice, nuts and tomatoes can cause bleeding, liver and kidney damage of varying severity; are neurotoxic as well as being teratogenic. 11 Expansus Penicillium, Penicillium clavatus, Byssoclamys Egret, Penicillium verrucosum and Penicillium patulum infest fruit and vegetables, peppers, tomatoes, cucumbers, carrots, apples, pears and juices, produce patulin which is able to give toxicity in different organs, is toxic to skin, neurotoxic and cytotoxic.
  • Nidulans, Aspergillus versicolor, Chaetomium thielavoideum and other ascomycetes produce Sterigmatocystin (IARC class 2B) that we find in grains in nuts and cheese, cause damage to the body in dependence of the route of administration. There are insufficient data to assess the carcinogenicity in humans. Oral administration produces lung tumors in mice and liver tumors in rats. The cutaneous application in rats produces liver tumors; subcutaneous injection local sarcomas. 5

Mycotoxins and immune system

The immune system puts in being the first defenses through physical barriers; the most important are the skin and mucous membranes. Such barriers are exposed to xenobiotics, inadvertent contaminants such as mycotoxins, most exposure occurs through contamination of the food chain. The intestinal permeability is the starting point of several human diseases that involves the immune system that undergoes “dysregulation”. 12  Glyphosate has a negative effect on the microbiota, mycotoxins and glyphosate on cell junctions; this will lead to dysbiosis and intestinal permeability. As already mentioned, mycotoxins are toxic secondary metabolites produced by fungal species (aflatoxins, zearalenone, ochratoxins, trichothecenes, patulin, fumonisins, and ergot alkaloids). 13 They are the most frequently occurring natural food contaminants, mycotoxins have mutagenic, genotoxic and potential carcinogenic effects. Mycotoxins especially trichothecenes and patulin, strike the intestinal barrier because affecting the tight junctions with loss of integrity of the barrier.  14  Tight junctions (TJs) are structural components that influence epithelial polarization and barrier functions, claudins  are the most important integral membrane proteins of TJs. 15 16 In inflammatory bowel disease (IBD) is detected  an elevation of Claudin-2 and a defective epithelial barrier function; furthermore  Claudin-2  are important for cellular functions and for its effects on TJ function. Moreover claudin-1 and claudin-2 expression are involved in IBD-associated transformation neoplasia, since the early stages. 17 18 Fungal spores are ubiquitous in the environment, its exposure can cause allergies and toxic mold syndrome in humans. Particularly deoxynivalenol (DON) play an important role in etiology of many chronic intestinal inflammatory diseases, food allergies (particularly in children), and toxic mold syndrome.  14 19  It was found that in addition to the integrity of cell junctions, mycotoxins have a negative impact on the gut-associated immune system as a consequence in poor intestinal health and integrity. 13 The tight junctions are important because control the equilibrium between tolerance and immunity to nonself-antigens, if macromolecules is dysregulated for individual genetically susceptibility autoimmune disorders can occur. Equilibrium on intestinal barrier function resulting from a correct interplay between genes and environmental triggers can prevent the autoimmune process. For some years, there is considerable interest in understanding the role of a leaky gut in the pathogenesis of gastrointestinal diseases in virtue of the fact that can induce autoimmunity. 20  Gastrointestinal tract is controlled by the immune system and neuro-endocrine system, in animals it was found that a reduction of stress factors improve gut homeostatic balance of livestock. 21  On animals, contaminated feed determines imbalance of intestinal pro-inflammatory cytokines and   barrier function, but the immunological mechanism that underlie to this nutritional stress is yet unclear. Mycotoxin is an important stressor factor, in particular deoxynivalenol (DON) disrupted the expression of the Zonulin-1(ZO-1), occludin and claudin of porcine gut epithelial TJs, within two days. 22 The ZO-1 expression is up regulated in enterocytes of pigs treated with Bacillus subtilis, and protect them by a damage DON-induced, therefore B. subtilis is an important supplementation to prevent DON-induced barrier dysfunction. 23 DON determines an increase of the pro-inflammatory cytokines production, like TNF-α, IL-6, and IL-1β in jejunum and ileum of these animals. 24 In animals feeding with aflatoxin and DON together within about a month, there is an increase of monocytes, which coincides with high concentration of TNF-α, but if the animals are fed a diet containing only one of the toxic does not have these changes on cytokine. With the feed containing DON and ZEN has been detected an high level of 8-hydroxy-deoxyguanosine, significant of oxidative stress on DNA and consequent structural damage. 22  Depending on the mycotoxin ingested, in the animals are detected IL-12/IL-23p40 and IL-1β, vasoactive intestinal peptides (42 days feeding of ZEN – 0.1 mg/kg). Furthermore, there are reduction of CD21+ B cells, and high levels of interferon-γ (IFN-γ) in ileocecal lymph nodes. 25 Animal consumption of feed contaminated with ZEN causes serious defects in growth, and reproductive performance, pigs are sensitive to this mycotoxin.  26 In the liver ZEN is metabolized to α- and β-zearalenol by 3α- and 3β-hydroxysteroid dehydrogenases, the α- zearalenol is the dominant ZEN derivative also in humans. 27  28  ZEN is implicated in reproductive disorders of farm animals, but also in hyperoestrogenic syndromes of humans, its adverse effects is observed during pregnancy. This mycotoxin can change the intrauterine environment in early gestation because there is an imbalance of the secretory mechanism of the endometrium, and delayed fetal development. The fetuses are most susceptible to toxins because for its fragile developmental. 29   Also Ochratoxin A (OTA) is one of the most widespread mycotoxins, is considered a possible carcinogen and present a potent nephrotoxicit, this mycotoxin, as many other, is stable to higher-temperature treatment. 30 Aflatoxins (AF) cross the placental barrier, and concentrations in the umbilical cord are greater than the maternal plasma, for this are teratogenic. The ingestion of aflatoxins (AF) would have an immunosuppressive effect, alter the metabolism of interferons. 31 Ochratoxin A contaminates food, it is capable to triggering modulation of survivin, interleukin 2 (IL-2) and tumor necrosis factor α (TNF-α). it is also able to activate caspase-8 with the consequent death receptor pathway activation. Probably there is an autocrine pro-apoptotic effect of TNF-α on H9 cells. 32 Currently it is given much attention to the harmful effects of mycotoxins on intestinal health, it is known that the levels of mycotoxins is critical for food and feed safety.  Research is careful to study the adverse effects of mycotoxins on intestinal integrity and the gut-associated immune system. Aflatoxins, ochratoxins, patulin, fumonisins, zearalenone, trichothecenes, and ergot alkaloids attack intestinal epithelium, with negative effects on the intestinal barrier, the gut microbiome, intestinal local immune system, and TJs proteins. Finally, it can lead to intestinal permeability.  13 One of the most important negative effect of mycotoxin is the imbalance of microbiota, which can be aggravated by the indiscriminate use of antibiotics.

 

 

Antibiotics

Bacteria exposed to antibiotics produce increased levels of reactive oxygen species (ROS) that, in turn, cause mutations in the affected bacterium. ROS also induces the “SOS response,” mediated by error-prone DNA polymerases, which creates additional mutations. Today are available a broad-spectrum antibiotics, it is not know all effects on bacterial evolution. Chambers say that they represented “the strongest—not the only, but by far the strongest—selective pressure ever encountered by the human microbiome”. Casadevall observed that:” broad-spectrum anti-microbials have created a culture of empiricism in medicine, in which treatment choices are determined by guesswork rather than by diagnosis”. 33 Microbiota assumes importance in the unborn child, because is an important determinants of health and disease, but the factors that determine initial human oral microbiota development are unknown. Most of the microbiota of the newborn,  (65.35%), comes from maternal oral, while 3.09% is sourced of placental, and 31.56% is unknown. No one microorganism comes from the maternal gut. The newborn microbiota profile depend also from maternal exposure to intrapartum antibiotics. In fact after maternal antibiotics exposure there is a prevalence of Proteobacteria, while Streptococcaceae, Gemellaceae and Lactobacillales are prevalent in unexposed neonates. About a quarter of the exposed newborns expresses the Vim-1 antibiotic resistance gene, which is the predominant gene among the positive antibiotic resistant species. In the light of the above, maternal antibiotic treatment is an important factor for the regulation of the initial neonatal oral microbiome. 34  The antibiotic are needed in therapy of infections but perturbs commensal microbial communities,  in the last few years there are a lot of studies on the effects of antibiotic on gut microbiota and immunity. Eg long-term ceftriaxone administration induces gut microbiota dysbiosis and histological lesions on gut, immune regulation and immune defense ability is reduced: serum IgG and mucus IgA decreased significantly. In the weeks following administration of this antibiotic we witness to an increase of  IFN-γ/IL-4 and CD4/CD8 ratios and a reduction of CD4+CD25+ cells. Many studies are still needed to understand how antibiotic influencing gut microbiota and immunity. 35 Gut microbiota is an important regulator of Th1/Th2 balance,e g. Bacteriodes fragilis restored imbalanced Th1/Th2 ratio in germ free mice. Also homeostatic T cell proliferation is regulated by microbiota or their penetrant molecules. Furthermore intestinal-derived antigens shape colonic T regulatory cell. 36 37  A dysbiosis by antibiotic can lead to abnormal growth of pathogenic species such as Clostridium difficile, this infections can cause severe  diarrhea when competing members of the intestinal microbiota are killed from antibiotics. 33

Biopesticides and glyphosate

Around 135 products are commercialized worldwide as biocontrol agents crop protection tools, known as biopesticides; but what worries us most is the increased production of crop and consequently the increased use of pesticides and herbicides. Since from the second half of last century, in the industrialized countries there was an intensive and growing use of synthetic chemical pesticides. Excessive use of pesticides can lead to the development of heritable resistance; in fact, over 500 species of arthropod pests have resistance to one or more insecticides. As regards herbicide resistance, there are approximately 200 varieties of weeds. 38 To give the resistance to chemical herbicides were produced genetically modified plants, in which it is present bacterial RNA and DNA, but their biological effects have not yet fully understood. Today more attention is now paid to glyphosate that is the active ingredient of a known commercial herbicide, it is a broad-spectrum herbicide, considered to be nearly nontoxic to humans.39 The United States Environmental Protection Agency (US EPA) originally classified glyphosate as possibly carcinogenic to humans (Group C) in 1985. Glyphosate caused DNA and chromosomal damage in human cells. 40  In glyphosate-treated plants there is an excess of ammonia, which increases the activity of phenylalanine ammonia lyase (PAL), an enzyme that catalyzes the reaction that converts phenylalanine to trans-cinnamate, releasing ammonia. PAL produced  phenolic compounds and glyphosate achieves its growth-retardation effects on plants through induction of PAL activity. The inhibition of growth could be due either to toxicity of the derived phenolic compounds or to toxicity of the ammonia. Glyphosate is able to inhibits the enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSP synthase), involved in the synthesis of aromatic amino acids in the shikimate pathway in plants. The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. 41   The increased phenylalanine ammonia lyase (PAL) activity, induced by glyphosate, creates an unfavorable environment in the gut that may lead to various diseases.  Autism is associated with dysbiosis, increased levels of ammonia and short chain fatty acids and this could be encouraged by dysbiosis induced by glyphosate, which promotes overgrowth of Clostridia spp. Clostridium difficile,  induces the production of p-cresol through the breakdown of tyrosine, then,  there is the depletion of sulfate. p-hydroxyphenylacetic acid (PHPA) is the intermediary (the body decarboxylates PHPA to p-cresol). Be emphasized that low levels of sulphates are associated with autism. 39  It was observed that in autistic children there is an overgrowth of unusual gut bacterial strains, capable of   increasing the production of p-cresol by the fermentation of tyrosine. The urinary p-cresol, and   p-cresylsulfate  can be considered a biomarker of autism in small children, especially females. 42  There are other urinary bacterial metabolites of phenylalanine were detected in the urine, they are benzoic and phenylacetic acids, and of tyrosine (p-hydroxybenzoic acid and p-hydroxyphenylacetic acid) that are elevated in several diseases. They reflect impaired intestinal resorption, as coeliac disease, cystic fibrosis, and unclassified diarrhoea.  High levels of phenylalanine urinary metabolites, about 300 times the values of the control groups, are also been found in autistic and schizophrenic patients. This situation reflects the underlying dysbiosis, due to multiple species of anaerobic bacteria in the Clostridium spp. 39  C. difficile produces p-cresol through the breakdown of tyrosine and has suggested that p-hydroxyphenylacetic acid (PHPA) is the intermediary, the organism decarboxylates PHPA to p-cresol. 43 The beneficial bacteria reduction in the gastrointestinal tract is possible after ingestion of glyphosate; this herbicide has demonstrated toxicity to Enterococcus spp. A gut dysbiosis favoring overgrowth of the Clostridium species. The following pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum show resistance to glyphosate. 44 Now there is evidence that gut microbiome influences cognitive function via the gut-brain axis, and the diet in westernized countries is an important factor because it alters the commensal bacteria in the gastrointestinal tract. 45  There is a lot of documentation that proves the correlation between high levels of fat, added sugars and cognitive dysfunction in westernized countries diets. 46 47 The hippocampus is particularly vulnerable to the deleterious effects of this type of diets. Hippocampus is the region dedicated to control of learning and memory processes. 48 How this can happen are not completely understood, but there are evidences that the gut-brain axis plays an important role, and the gut microbiome, which is the collective genome of microbes residing in the gastrointestinal tract,  has a primary impact on brain function. 49 50 Twin studies indicate a strong genetic component, while the rise in the incidence of autism in the last period suggests involvement of environmental factors, and a heritable epigenetic effect as the primary factor. Are very important the epigenetic effects taking place during gestation that can influencing the phenotypical expression.  Sulfates deficiency in both the mother and the child, which derives by excess exposure to environmental toxins, cause   hypo methylation in the fetal brain with devastating consequences. 51 As already mentioned, the gut microbiota alterations have potent effects on gut permeability and blood-brain barrier (BBB) integrity. Gut permeability, which in turn allows for the influx of substances, which that under normal conditions does not pass the intestinal barrier, contributes to the development of cognitive dysfunction. 52 53 For example  in Italian children  there is  association between obesity, gut permeability, systemic inflammation, consequent to the intestinal permeability and dysbiosis (contemporaneously associated) and type 1 diabetes (T1D). To confirm microbial markers in these children more studies are needed, also to clarify the involvement of dysbiosis in the mechanisms leading to increased permeability and the autoimmune mechanisms that leads to T1D.  54 55 It was observed that gut microbiota change, increases endogenous glucagon-like peptide-2 (GLP-2) production, and consequently improves gut barrier functions, contributing to the improvement of gut barrier functions during obesity and diabetes. 52 In animal study models has been observed that westernized countries diet, induces a decreases levels of the TJs protein ZO-1 and also a decrease of transepithelial resistance in the colon, these are markers of increased gut permeability. 56 The aforementioned results describe a higher vulnerability to the gram-negative bacteria-derived lipopolysaccharides (LPS) that promote endotoxemia and systemic inflammation. 57 58 Even BBB damage is associated with the above-mentioned diet and which may be causally related with cognitive dysfunction. 59   Neuroinflammation is supported by bacterial endotoxins, but also by diet, I think more to pollutants in food, that leads to a reduction of saprophytic microorganisms with anti-inflammatory action, and leads to impaired cognitive function. 50 Neuroinflammation  affects the hippocampus, and cortical areas due in part from dysbiosis, in fact it is known that some gut bacteria stimulate the innate immune system to elevate inflammatory cytokines in the brain.  These mechanisms that lead to inflammation are the basis of many diseases such as: gastrointestinal disorders, diabetes, heart disease, autism, cancer and Alzheimer’s disease, the glyphosate may be the cause. 39 60

 Discussion

We have seen that the pollutants, including involuntary, and antibiotics have a negative effect on cell junctions, the microbiota and the microbiome, which ultimately lead to dysbiosis and leaky gut. All this can be the starting point for various diseases, surely imbalance the immune system, and leading to immune dysregulation.  We have also seen that gut microbiota has an important effect on immune system, furthermore is important for the development and differentiation of the immune system. Both the immune response and immune homeostasis are affected by gut microbiota, this could be the starting point of an immune dysregulation, as I have writed in my article published in 2013. 35 61 Article that followed to my intuition in my 1982 thesis, published in the forensic “quaderni camerti”, which led me to address the issue of the relationship between mycotoxins and pathology known as farmer’s lung. In the previously mentioned article, I assumed that the possible immune dysregulation resulting in the ingestion of mycotoxins and the use of antibiotics might be one of the causes that lead to ASD. Today I add that pesticides and herbicides that pollute the Food Chain add additional risk factor, as it is clear from the increasing number of studies. 12 Currently the misuse of antibiotics, especially abuse treatment, disrupting the beneficial gut bacteria, and glyphosate can enhance the destructive effects on the microbiota, both lead to an increased intestinal permeability. The same type of damage is caused by mycotoxins. In ASD gut we observe high levels of short chain fatty acids and ammonia, they are by-products of anaerobic fermentation, which suggests that is possible an overgrowth of anaerobic bacteria such as Clostridia, that is most common in ASD gut. 39   There are also Bacteriodetes, and Desulfovibrio, this microorganism has a metabolism based on the reduction of sulphate. If there is a proliferation of Archaea it is possible an inhibition on Desulfomicrobium and Desulfovibrio.  Methanogens Archaea colonize the human intestine after the second year of life, they have a peak between the third and and fifth year in the following years the percentage depends on the nationality or race of the subjects. In so-called industrialized countries the presence tends to decrease in the countries with the poorest population can be up to 80%, after the tenth year we witness a stabilization regardless of the diet, and the administered antibiotics. Several Archaea are considered toxigenic, the toxic substances produced generally controls the active competition within these microorganisms, are ineffective against Bacteria and Eukarya. Some of the substances that constitute the cell wall dell’Archeon Sulfolobus are toxic, have toxicity similar to that of LPS. The Methanococcus jannaschii and the Archaeglobus fulgidus have genes coding for molecules toxic for the human organism. In summary the archaea are not pathogenic for man, the genomic analysis revealed the horizontal transmission of genes among Archeae and the Bacteria, this information flow has contributed to the evolution of metabolic pathways.  62 There is strong correlation between gut dysbiosis and autism spectrum disorder (ASD), in particular it has been detected a strong relationship between intestinal gene expression and bacterial structure. This has stimulated research to discover the brain-gut connection. 63 Serotonin (5-HT) is a well know neurotransmitter and vasoactive substance, it is a central physiologic mediator of many gastrointestinal (GI) functions and a mediator of the brain-gut connection. 64  Gliphosate is another emerging problem, it appears that the damage is caused by the surfactant, TN-20 used in combination with glyphosate, surfactant and glyphosate led to mitochondrial damage, apoptosis, and necrosis. This type of surfactant disrupts the integrity of the cellular barrier and blocks out microbial growth, also at lower concentrations. 39  Glyphosate induces an increased activity of PAL that leading to a  hyperammonemic environment in the gut, incresed ammonia level is believed to be intestinal bacteria in small and large intestine. 65 A correlation has been highlighted between autism and hepatic encephalitis, to be attributed to ammonia  excess, derived also from urea pathway, in this case it is important as well a possible single nucleotide polymorphism in the methionine cycle. 66 Also by-products of fermentation by anaerobes, such as phenols, ammonia, and hydrogen sulfide, can be toxic to the large bowel. 67 Also the sulfate deficiency associated with autism, is justified by the depletion of sulfate deriving from tyrosine by Clostridium difficile, which develops in consequence of dysbiosis. 68 69 In ASD children are frequently observed high concentrations of urinary metabolites of phenylalanine; the values are hundreds of times larger  of the standard, this is due to the proliferation of Clostridium spp. 70 The infiltration of intestinal epithelial lymphocytes is characteristic in ASD, and gives an inflammatory mucosal immunopathology, it is a direct consequence of increased intestinal permeability. 71 Also mycotoxins contribute to aggravating the damage because affecting the TJs and have detrimental action on the microbiota.

Conclusion

In the light of the above, a working hypothesis could be: to deepen the precise correlations between dysbiosis induced by the use of antibiotics, pollution of the food chain as part of the so-called phyto drugs and mycotoxins. Understanding the mechanisms that lead to dysregulation of the immune system and the eventual rupture of tolerance, which could lead to autoimmunity. To study oxidative stress, lipid peroxidation, metabolites and their intermediates, such as those of the metabolism of the vitamins involved in the methionine cycle, vitamin B and folic acid, just to mention the most important. Another fundamental step study of single nucleotide polymorphisms (SNPs) involved in the cycle of methionine, folate, urea and tetrabetabiopterin (BH4), bearing in mind the epistatic approach, which is crucial in a multifactorial disease like ASD. This is the future challenge for research.

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