Celiac disease, and, more generally, gluten intolerance, is a growing problem worldwide, but especially in North America and Europe, where an estimated 5% of the population now suffers from it. Symptoms include nausea, diarrhea, skin rashes, macrocytic anemia and depression. It is a multifactorial disease associated with numerous nutritional deficiencies as well as reproductive issues and increased risk to thyroid disease, kidney failure and cancer. Here, we propose that glyphosate, the active ingredient in the herbicide, Roundup®, is the most important causal factor in this epidemic. Fish exposed to glyphosate develop digestive problems that are reminiscent of celiac disease. Celiac disease is associated with imbalances in gut bacteria that can be fully explained by the known effects of glyphosate on gut bacteria. Characteristics of celiac disease point to impairment in many cytochrome P450 enzymes, which are involved with detoxifying environmental toxins, activating vitamin D3, catabolizing vitamin A, and maintaining bile acid production and sulfate supplies to the gut. Glyphosate is known to inhibit cytochrome P450 enzymes. Deficiencies in iron, cobalt, molybdenum, copper and other rare metals associated with celiac disease can be attributed to glyphosate's strong ability to chelate these elements. Deficiencies in tryptophan, tyrosine, methionine and selenomethionine associated with celiac disease match glyphosate's known depletion of these amino acids. Celiac disease patients have an increased risk to non-Hodgkin's lymphoma, which has also been implicated in glyphosate exposure. Reproductive issues associated with celiac disease, such as infertility, miscarriages, and birth defects, can also be explained by glyphosate. Glyphosate residues in wheat and other crops are likely increasing recently due to the growing practice of crop desiccation just prior to the harvest. We argue that the practice of “ripening” sugar cane with glyphosate may explain the recent surge in kidney failure among agricultural workers in Central America. We conclude with a plea to governments to reconsider policies regarding the safety of glyphosate residues in foods.
Gluten intolerance is a growing epidemic in the U.S. and, increasingly, worldwide. Celiac sprue is a more specific disorder, characterized by gluten intolerance along with autoantibodies to the protein, transglutaminase, which builds crosslinks in undigested fragments of gliadin, a major constituent of gluten (Green & Cellier, 2007). The autoantibodies are produced as an immune response to undegraded fragments of proteins in gluten. A remarkable set of symptoms develop over time in association with celiac disease, including weight loss, diarrhea, chronic fatigue, neurological disorders, anemia, nausea, skin rashes, depression, and nutrient deficiencies. Usually, but not always, a strict gluten-free diet can alleviate many of the symptoms. A key associated pathology is an inflammatory response in the upper small intestine, leading to villous atrophy, a flattening of the microvilli which impairs their ability to function in their important role in absorbing nutrients.
Some have suggested that the recent surge in celiac disease is simply due to better diagnostic tools. However, a recent study tested frozen sera obtained between 1948 and 1954 for antibodies to gluten, and compared the results with sera obtained from a matched sample from people living today (Rubio-Topia et al., 2009). They identified a four-fold increase in the incidence of celiac disease in the newer cohort compared to the older one. They also determined that undiagnosed celiac disease is associated with a 4-fold increased risk of death, mostly due to increased cancer risk. They concluded that the prevalence of undiagnosed celiac disease has increased dramatically in the United States during the past 50 years.
Transglutaminases play many important roles in the body, as they form covalent crosslinks in complex proteins in connection with blood coagulation, skin-barrier formation, extracellular matrix assembly, and fertilization, endowing the substrate with protection from degradation by proteases (Lorand & Graham, 2003). They also form crosslinks in undigested fragments of gliadin derived from wheat, and sensitivity to certain of these fragments leads to the development of autoantibodies to tissue transglutaminase (Esposito et al., 2002) that inhibit its activity.
Glyphosate is the active ingredient in the herbicide Roundup. It is a broad-spectrum herbicide, considered to be nearly nontoxic to humans (Williams et al., 2000). However, a recent paper (Samsel & Seneff, 2013), argued that glyphosate may be a key contributor to the obesity epidemic and the autism epidemic in the United States, as well as to several other diseases and conditions, such as Alzheimer's disease, Parkinson's disease, infertility, depression, and cancer. Glyphosate suppresses 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSP synthase), the rate-limiting step in the synthesis of the aromatic amino acids, tryptophan, tyrosine, and phenylalanine, in the shikimate pathway of bacteria, archaea and plants (de María et al., 1996). In plants, aromatic amino acids collectively represent up to 35% of the plant dry mass (Franz, 1997). This mode of action is unique to glyphosate among all emergent herbicides. Humans do not possess this pathway, and therefore we depend upon our ingested food and our gut microbes to provide these essential nutrients. Glyphosate, patented as an antimicrobial (Monsanto Technology LLC, 2010), has been shown to disrupt gut bacteria in animals, preferentially killing beneficial forms and causing an overgrowth of pathogens. Two other properties of glyphosate also negatively impact human health – chelation of minerals such as iron and cobalt, and interference with cytochrome P450 (CYP) enzymes, which play many important roles in the body. We will have much more to say about these aspects in later sections of this paper.
A recent study on glyphosate exposure in carnivorous fish revealed remarkable adverse effects throughout the digestive system (Senapati et al., 2009). The activity of protease, lipase, and amylase were all decreased in the esophagus, stomach, and intestine of these fish following exposure to glyphosate. The authors also observed “disruption of mucosal folds and disarray of microvilli structure” in the intestinal wall, along with an exaggerated secretion of mucin throughout the alimentary tract. These features are highly reminiscent of celiac disease. Gluten peptides in wheat are hydrophobic and therefore resistant to degradation by gastric, pancreatic and intestinal proteases (Hershko & Patz, 2008). Thus, the evidence from this effect on fish suggests that glyphosate may interfere with the breakdown of complex proteins in the human stomach, leaving larger fragments of wheat in the human gut that will then trigger an autoimmune response, leading to the defects in the lining of the small intestine that are characteristic of these fish exposed to glyphosate and of celiac patients. As illustrated in Figure 1, the usage of glyphosate on wheat in the U.S. has risen sharply in the last decade, in step with the sharp rise in the incidence of Celiac disease. We explain the reasons for increased application of glyphosate to wheat in Section 13.
In the remainder of this paper, we will first show that gut dysbiosis, brought on by exposure to glyphosate, plays a crucial role in the development of celiac disease. Many CYP enzymes are impaired in association with celiac disease, and we show that glyphosate's known suppression of CYP enzyme activity in plants and animals plausibly explains this effect in humans. In Section 4, we describe the role of excess retinoic acid in celiac disease, and show how this ties also to reproductive problems. We link this to the known effects of glyphosate on retinoic acid, mediated by its suppression of CYP enzymes. Section 5 addresses cobalamin deficiency, a known pathology associated with celiac disease that leads to macrocytic anemia. We argue that this follows as a direct consequence of glyphosate's ability to chelate cobalt. Section 6 discusses in more depth the role of anemia in celiac disease, a consequence of both cobalamin and iron deficiency. Section 7 discusses molybdenum deficiency and its link to microcephaly, which is associated with celiac disease. Section 8 discusses the link between selenium deficiency and autoimmune thyroid disease. Section 9 discusses kidney disease in connection with celiac disease and glyphosate. Section 10 discusses various nutritional deficiencies associated with celiac disease, and shows how these can directly be explained by glyphosate. Section 11 discusses the link between celiac disease and certain rare cancers that have also been linked to glyphosate. Section 12 goes into an in-depth discussion of how glyphosate might promote autoantibodies to transglutaminase. Following a section which presents compelling evidence that glyphosate residues in wheat, sugar and other crops are likely increasing in recent decades, and a section discussing the increased risk to kidney failure in agricultural workers exposed to excess glyphosate occupationally, we close with a discussion section that summarizes our findings, and a conclusion which implores governments to pay more attention to the damaging consequences of the escalation in chemical warfare on weeds that characterizes current agricultural practices.
2 Gut bacteria
In this section, we first discuss the role of pathogens in inducing the breakdown of tight junctions in enterocytes lining the small intestinal wall. We then show that glyphosate is associated with an overgrowth of pathogens along with an inflammatory bowel disease in animal models. A parallel exists with celiac disease where the bacteria that are positively and negatively affected by glyphosate are overgrown or underrepresented respectively in association with celiac disease in humans. We also discuss how the beneficial bacteria that are negatively impacted by glyphosate can protect from celiac disease through their enzymatic activities on gluten, and point to several articles recommending treatment plans based on probiotics.
Pathogens, through their activation of a potent signaling molecule called zonulin, induce a breakdown of the tight junctions in cells lining the gut, leading to “leaky gut” syndrome (Fasano, 2011). Concentrations of zonulin were sharply elevated (p/em>0.000001) in subjects with celiac disease during the acute phase (Fasano et al., 2000). As many as 30% of celiac patients continue to experience GI symptoms after adopting a gluten-free diet, despite optimal adherence, a condition that was attributed to bacterial overgrowth in the small intestine (Tursi et al.,2003). Figure 2 shows that there is a correlation between glyphosate application to wheat and the incidence of intestinal infections.
Evidence of disruption of gut bacteria by glyphosate is available for poultry (Shehata et al., 2013), cattle (Krügeret al., 2013), and swine (Carman et al., 2013). Glyphosate disrupts the balance of gut bacteria in poultry (Shehata et al., 2013), increasing the ratio of pathogenic bacteria to other commensal microbes. Salmonella and Clostridium are highly resistant to glyphosate, whereas Enterococcus, Bifidobacteria, and Lactobacillus are especially susceptible. Glyphosate was proposed as a possible factor in the increased risk to Clostridium botulinum infection in cattle in Germany over the past ten to fifteen years (Krüger et al., 2013b). Pigs fed GMO corn and soy developed widespread intestinal inflammation that may have been due in part to glyphosate exposure (Carmanet al., 2013).
Celiac disease is associated with reduced levels of Enterococcus, Bifidobacteria and Lactobacillus in the gut and an overgrowth of pathogenic gram negative bacteria (Sanz et al., 2011; Di Cagno et al., 2011; Collado et al.,2007). In (Di Cagno et al., 2011), Lactobacillus, Enterococcus and Bifidobacteria were found to be significantly lower in fecal samples of children with celiac disease compared to controls, while levels of the pathogens, Bacteroides, Staphylococcus, Salmonella, a Shighella were elevated. In (Collado et al., 2007), another study comparing the fecal material of celiac infants to healthy controls, Bacteroides, Clostridium and Staphylococcus were all found to be significantly higher (p/em>0.05). Sulfate-reducing bacterial counts were also elevated (p/em>0.05) (Nadal et al., 2007; Collado et al., 2007), an interesting observation which we will return to later in this paper. A significant reduction in Bifidobacteria was also found in (Nadal et al., 2007). An increased excretion of the bacterial metabolites p-Cresol and phenol has also been recognized in association with celiac disease (Tamm,1984). p-Cresol is produced via anaerobic metabolism of tyrosine by pathogenic bacteria such as Clostridium difficile (D'Ari and Barker, 1985). It is a highly toxic carcinogen, which also causes adverse effects on the central nervous system, the cardiovascular system, lungs, kidney and liver (Kelly et al., 1994).
Probiotic treatments are recommended to aid in digestive healing in celiac disease. The proteolytic activity of Lactobacilli aids the breakdown of wheat into less allergenic forms. Ongoing research aims to produce gluten-containing sourdough breads fermented by Lactobacilli that can then serve as probiotics to help ameliorate the symptoms of celiac disease and allow celiac patients to consume wheat (Gobbetti et al., 2007). Probiotic Lactobacilli produce the enzyme phytase which breaks down phytates that would otherwise deplete important minerals and other cations through chelation (Famularo et al., 2005). Their activities would therefore improve absorption of these micronutrients, a known problem in celiac patients (Cavallaro et al., 2004). Glyphosate itself also chelates rare minerals, a subject we will address in the section on nutritional deficiencies.
Probiotic treatment with Bifidobacteria has been shown to alleviate symptoms associated with celiac disease (Smecuol et al., 2013; Whorwell et al., 2006). Bifidobacteria suppress the pro-inflammatory milieu triggered by the microbiota of celiac patients (Medina et al., 2008). Live cultures of Bifidobacterium lactis would promote healing of the gut if offered as treatment in conjunction with the gluten-free diet, or might even allow the celiac patient to consume modest amounts of gluten without damaging effects (Lindfors et al., 2008). In this in vitro study, it was demonstrated that B. lactis reduced epithelial permeability and improved the integrity of the tight junctions in human colon cells.
In summary, celiac disease is associated with a reduced presence in the gut of commensal bacteria such as Lactobacilli and Bifidobacteria, which are known to be preferentially killed by glyphosate, and with an overabundance of C. difficile, which is known to be promoted by glyphosate exposure. Bifidobacteria and Lactobacilli are both capable of modifying gluten in such a way as to make it less allergenic, a feature that is being exploited in recent efforts to develop gluten-containing foods that may be safe for consumption by celiac patients. Probiotics containing live forms of these bacteria are also being actively marketed today.
3 CYP Enzyme impairment and sulfate depletion
As mentioned previously, glyphosate has been shown to suppress CYP enzymes in plants (Lamb et al., 1998) and animals (Hietanen et al., 1983). A study on rats demonstrated that glyphosate decreased the levels of CYP enzymes and monooxygenase activities in the liver and the intestinal activity of aryl hydrocarbon hydroxylase (Hietanen et al., 1983).
CYP enzymes are essential for detoxification of many compounds in the liver (Lindros, 1997). Intraperitoneal exposure of rats to Roundup in acute doses over a short time interval induced irreversible damage to hepatocytes and elevated urinary markers of kidney disease. This was associated with lipid peroxidation and elevated levels of the inflammatory cytokine tumor necrosis factor (TNF-α) (El-Shenawy, 2009). CYP3A is constitutively expressed in human intestinal villi and plays an important role in drug metabolism (Cupp & Tracy, 1998). Celiac disease is associated with a decrease in the intestinal CYP3A (Lang et al., 1996). This defect is restored by a gluten free diet.
Impaired gallbladder bile acid production (Colombato et al., 1977) and biliary cirrhosis, an inflammatory liver disease characterized by obstruction of the bile duct (Dickey et al., 1997), have been shown to co-occur with celiac disease. CYP enzymes are crucial in the production of bile acids (Lorbek et al., 2012). An obligatory CYP enzyme in bile acid synthesis, CYP27A, has been identified as being identical to the mitochondrial vitamin D3 activating enzyme (Wikvall, 2001). In (Kemppainen et al., 1999), 64% of men and 71% of women with celiac disease were found to be vitamin D3 deficient, manifested as low spinal bone mineral density. Celiac disease is associated with impaired gall bladder function and decreased pancreatic secretions (Brown et al., 1987; Benini et al., 2012) along with recurrent pancreatitis (Patel et al., 1999). Abnormalities in bile acid secretion have been found in children suffering from celiac disease (Ejderhamn et al., 1992). Celiac patients exhibit abnormally low synthesis of cholecystokinin (Deprez et al., 2002), but it has also become apparent that the gall bladder is less responsive to stimulation of contraction by cholecystokinin (Brown et al., 1987). A reversible defect of gallbladder emptying and cholecystokinin release has been identified in association with celiac disease (Maton et al., 1985). These pathologies may be related to impaired CYP enzyme activity induced by glyphosate.
While it is clear that CYP enzymes play an important role in bile acid synthesis and in cholesterol homeostasis, the details have not yet been worked out (Lorbek et al., 2012). However, some mouse knockout experiments produce embryonically lethal effects, pointing to the importance of these enzymes to biological systems. Disruption of Cyp7A1, involved in bile acid synthesis in mice, induces elevated serum cholesterol and early death.
A link has been established between celiac disease and non-alcoholic fatty liver, which is likely due to the liver's inability to export cholesterol sulfate through the bile acids due to impaired CYP enzymes (Lorbek et al., 2012). This requires a private store of fats to house the excess cholesterol that cannot be exported in bile. This would also likely lead to insufficient sulfate supplies to the small intestine, and could result in impaired heparan sulfate synthesis in the glycosaminoglycans and subsequent pathologies. Heparan sulfate populating the glycosaminoglycans (GAGs) surrounding enterocytes is essential for the proper functioning of the small intestines. Leakage of both albumin and water in both the vasculature and tissues results when the negative charge is reduced due to insufficient sulfation of the polysaccharide units (Sunergren et al., 1987). Vascular leakage may be a consequence of degradation of sulfated GAGs due to inflammatory agents (Klein et al., 1992). A similar problem may occur in the kidneys leading to albumin loss into urine during nephrosis (Vernier et al., 1983). Intestinal protein loss in inflammatory enteropathy associated with celiac disease may also be due to a deficiency in the sulfated GAGs (Murch et al.,1993; Murch, 1995). A case study of three infants with congenital absence of enterocyte heparan sulfate demonstrated profound enteric protein loss with secretory diarrhoea and absorption failure, even though their intestines were not inflamed (Murch et al., 1996).
In (Samsel and Seneff, 2013), a hypothesis was developed that glyphosate disrupts the transport of sulfate from the gut to the liver and pancreas, due to its competition as a similarly kosmotropic solute that also increases blood viscosity. (Kosmotropes are ions that induce “structure ordering” and “salting out” of suspended particles in colloids). Insufficient sulfate supply to the liver is a simple explanation for reduced bile acid production. The problem is compounded by impaired CYP enzymatic action and impaired cycling of bile acids through defective enterocytes in the upper small intestine. The catastrophic effect of loss of bile acids to the feces due to impaired reuptake compels the liver to adopt a conservative approach of significantly reduced bile acid synthesis, which, in turn, leads to gall bladder disease.
The protein, Nuclear factor κ-lightchain-enhancer of activated B cells (NF-κB) controls DNA transcription of hundreds of genes and is a key regulator of the immune response to infection (Tieri et al., 2012). Light chains are polypeptide subunits of immunoglobulins. NF-κB responds to stimulation from bacterial and viral antigens, inflammatory cytokines like TNF-α, free radicals, oxidized LDL, DNA damage and UV light. The incidence of acute pancreatitis has been increasing in recent years (Bhatia, 2012), and it often follows billiary disease. A local inflammatory reaction at the site of injury coincides with an increase in the synthesis of hydrogen sulfide (H2S) gas. H2S regulates the inflammatory response by exciting the extracellular signal regulated (ERK) pathway, leading to production of NF-κB (Bhatia, 2012). We hypothesize that H2S, while toxic, is a source of both energy and sulfate for the pancreas, derived from sulfur-containing amino acids such as cysteine and homocysteine. Dehydroepiandrosterone (DHEA) sulfate, but not DHEA, inhibits NF-κB synthesis, suggesting that sulfate deficiency is a driver of inflammation (Iwasaki et al., 2004).
While H2S is well known as a toxic gas through its inhibition of aerobic respiration, a recent paradigm shift in the research surrounding H2S has been inspired by the realization that it is an important signaling gas in the vasculature, on par with nitric oxide (Li et al., 2011). H2S can serve as an inorganic source of energy to mammalian cells (Módis et al., 2013). 3-mercaptopyruvate sulfurtransferae (3MST) is expressed in the vascular endothelium, and it produces H2S from mercaptopyruvate, an intermediary in the breakdown of cysteine (Kimura, 2011). Endogenously produced H2S derived from 3-mercaptopyruvate stimulates additional mitochondrial H2S production, which then is oxidized to thiosulfate via at least three different pathways (Ingenbleek and Kimura,2013; Hildebrandt and Grieshaber, 2008; Goubern et al., 2007), producing ATP. The inflammatory agent superoxide can act as substrate for the oxidation of H2S to sulfite and subsequently sulfate and the activated form, PAPS (Seneff et al., 2012), but will likely induce oxidative damage in the pancreas, particularly, as we will see in section 7, if molybdenum deficiency impairs sulfite-to-sulfate synthesis.
Pancreatic beta cells express extraordinarily high levels of heparan sulfate, which is essential for their survival (Ziolkowski et al., 2012), since it protects them from ROS-induced cell death. Because sulfate transport via the hepatic portal vein is likely disrupted by glyphosate, H2S, whether derived from sulfur-containing amino acids or supplied via diffusion following its production by sulfur-reducing bacteria in the gut, can become an important source of sulfur for subsequent sulfate production locally in the pancreatic cells. Pancreatic elastase is a serine protease that is needed to assist in protein degradation, but an overabundance can lead to autolysis of tissues (Itoet al., 1998). Cholesterol sulfate inhibits pancreatic elastase (Ito et al., 1998), so a deficiency in cholesterol sulfate supply due to impaired sulfate supply to the liver and impaired CYP function should increase the risk of tissue digestion by pancreatic enzymes, contributing to the loss of villi in the upper small intestine observed in celiac disease.
In the early 1990's a newly recognized disease began to appear, characterized by eosinophil infiltration into the esophagus, which manifested as dysphagia in adults and refractory reflux symptoms in children (Lucendo & Sánchez-Cazalilla, 2012). This disease, termed eosinophilic esophagitis (EOE), is associated with a Th2 immune profile and synthesis of the cytokine IL-13, which has direct cytotoxic effects on epithelial cells. A dose-dependent induction of eosinophilia by intratracheal delivery of IL-13 confirms its association with EOE (Mishra and Rothenberg, 2003). An association has been found between EOE and celiac disease (Leslie et al., 2010). Patients with refractory celiac disease that is not corrected by dietary gluten restriction show an increased production of IL-13 in the gut (Gross et al., 2013). The incidence of EOE has increased at alarming rates in Western countries in the last three decades (Furuta et al., 2007; Liacouras et al., 2011; Prasad et al., 2009).
Glyphosate is highly corrosive to the esophageal epidermal lining, with upper GI tract injury observed in 94% of patients following glyphosate ingestion (Chang et al., 1999). In (Zouaoui et al., 2013), the most common symptoms in an acute response from glyphosate poisoning were oropharyngeal ulceration, nausea and vomiting. We hypothesize that glyphosate induces EOE via a systemic response as well as through direct contact. The pathogenesis of EOE is related to food sensitivities, but airborne exposure to chemicals in the lungs can also induce it, so it does not require physical contact to the allergen (Blanchard & Rothenberg, 2008). It is conceivable that glyphosate is responsible for the emergence of EOE.
The cytochrome P450 reductase (CPR) and cytochrome P450 (CP) enzyme system is essential for inducing nitric oxide release from organic nitrates (Li, 2006). The nitrate moiety is reduced while simultaneously oxidizing NADPH to NADP+. This system is invoked in organic nitrate drug treatment for cardiovascular therapy. The reaction depends on anaerobic, acidic conditions, a feature of venous rather than arterial blood. Since L-arginine is substrate for NO synthesis by endothelial nitric oxide synthase (eNOS) under oxidative conditions (Förstermann and Münze, 2006), it is likely that CPR and CP play an important role mainly in stimulating venous smooth muscle relaxation. Impaired venous relaxation would likely contribute to venous thrombosis, which is a well-established complication of celiac disease (Zenjari et al., 1995; Marteau et al., 1994, Grigg, 1999, Halfdanarson et al.,2007).
In summary, celiac disease is associated with multiple pathologies in the digestive system, including impaired gall bladder function, fatty liver, pancreatitis, and EOE. We have argued here that many of these problems can be traced to impaired CYP function in the liver due to glyphosate exposure, leading to insufficient flow of bile acids through the circular pathway between the liver and the gut. This results in a system-wide depletion in sulfate, which induces inflammation in multiple organs to produce sulfate locally. A potential sulfur source for sulfate synthesis could be hydrogen sulfide gas, provided in part by the local breakdown of sulfur-containing amino acids like cysteine and homocysteine and in part by diffusion of the gas produced from inorganic dietary sources by sulfur-reducing bacteria in the large intestine. Impaired CYP enzyme function may also contribute to venous thrombosis, for which celiac disease is an established risk factor.
4 Retinoic acid, celiac disease and reproductive issues
In this section, we first establish that excess retinoic acid (RA) is a risk factor for celiac disease. We then show that excess RA leads to complications in pregnancy and teratogenic effects in offspring. Glyphosate has been shown to exhibit teratogenic effects in line with known consequences of excess RA exposure to the embryo, and we propose that the mechanism for this effect may be glyphosate's known disruption of CYP enzymes (Samsel & Seneff, 2013), which are involved in RA catabolism. This then links glyphosate to increased risk to celiac disease via its direct effects on RA. And it identifies a possibly important factor in the association of celiac disease with reproductive issues. We also discuss other adverse effects of excess retinoic acid and a possible relationship to impaired sulfate supply to the gut.
In celiac disease, T cells develop antibody responses against dietary gluten, a protein present in wheat (Jabri & Sollid, 2009). RA, a metabolite of vitamin A, has been shown to play a critical role in the induction of intestinal regulatory responses (Mora et al., 2008; Coombes et al., 2007; Mucida et al., 2007). The peptide in gluten, A-gliadin p31-43, induces interleukin 15 (IL-15), a key cytokine promoting T-cell activation (Hershko & Patz,2008). RA synergizes with high levels of IL-15 to promote JNK phosphorylation (Nanda, 2011; DePaolo et al.,2011), which potentiates cellular apoptosis (Putcha et al., 2003). IL-15 is a causative factor driving the differentiation of precursor cells into anti-gluten CD4+ and CD8+ Th1 cells in the intestinal mucosa. Furthermore, in (DePaolo et al., 2011), it was discovered that RA exhibits an unanticipated co-adjuvant property to induce Th1 immunity to antigens during infection of the intestinal mucosa with pathogens. Retinoic acid has also been shown to directly suppress transglutaminase activity, another way in which it would negatively impact celiac disease (Thacheret al., 1985). Thus, it is becoming clear that excess exposure to RA would increase risk to celiac disease, and warnings have been issued regarding potential adverse effects of RA supplements on celiac disease.
It is well established that high RA levels leads to teratogenic effects both in human and experimental models. Brain abnormalities such as microcephaly, impairment of hindbrain development, mandibular and midfacial underdevelopment, and cleft palate are all implicated (Sulik et al., 1988; Clotman et al., 1998). Women with celiac disease are known to have higher rates of infertility, miscarriages, and birth defects in their offspring (Freeman, 2010; Martinelli et al., 2000; Dickey et al., 1996; Collin et al., 1996). Excess RA could be a significant factor in these complications.
A possible mechanism by which glyphosate might induce excess RA is via its interference with the CYP enzymes that metabolize RA. There are at least three known CYPs (CYP26A1, CYP26B1 and CYP26C1) that catabolize RA, and they are active in both the embryo and the adult (Taimi et al., 2004). A 1/5000 dilution of glyphosate was sufficient to induce reproducible malformations characteristic of RA exposure in frog embryos (Paganelli et al., 2010). Pathologies included shortening of the trunk, reduction in the size of the head, abnormally small eyes or the presence of only one eye (cyclopia), and other craniofacial malformations in the tadpole. Glyphosate's toxicity to tadpoles has been well demonstrated, as it killed nearly 100% of larval amphibians exposed in experimental outdoor pond mesocosms (Relyea, 2005).
According to official records, there has been a recent 4-fold increase in developmental malformations in the province of Chaco, Argentina, where glyphosate is used massively on GMO monocrops of soybeans (Carrasco,2013). In Paraguay, 52 cases of malformations were reported in the offspring of women exposed during pregnancy to agrochemicals, including anencephaly, microcephaly, facial defects, cleft palate, ear malformations, polydactily, and syndactily (Benítez-Leite et al., 2009). In in vitro studies on human cell lines, DNA strand breaks, plasma membrane damage and apoptosis were observed following exposure to glyphosate-based herbicides (Gasnier et al., 2009). Another factor in teratogenetic effects of glyphosate may be the suppression of the activity of androgen-to-estrogen conversion by aromatase, a CYP enzyme (Gasnier et al., 2009).
Ingested vitamin A, a fat-soluble vitamin, is delivered to the blood via the lymph system in chylomicrons, and excess vitamin A is taken up by the liver as retinoic acid for catabolism by CYP enzymes (Russell, 2000). Any remaining retinoic acid that is not catabolized is exported inside LDL particles, and it lingers much longer as retinyl esters in the vasculature in this form (Krasinski et al., 1990). Excess retinoic acid is more readily stored in this way in LDL particles in the elderly. Vitamin A toxicity can lead to fatty liver and liver fibrosis (Russell, 2000) as well as hypertriglyceridemia (Ellis et al., 1986). Vitamin A has a negative effect on cholesterol sulfate synthesis (Jetten et al., 1989), which might negatively impact the liver's ability to maintain adequate supplies of cholesterol sulfate for the bile acids, and therefore also interfere with the supply of cholesterol sulfate to the gastrointestinal tract.
In summary, glyphosate's disruption of the CYP enzymes responsible for RA catabolism could lead to an excess bioavailability of RA that could contribute adversely to celiac disease, as well as damaging the liver and leading to teratogenic effects in offspring of exposed individuals.
In addition to higher risk to birth defects, individuals with celiac disease have increased risk to infertility (Meloni et al., 1999; Farthing et al., 1982). Increased incidence of hypogonadism, infertility and impotence was observed in a study of 28 males with celiac disease (Farthing et al., 1982). Marked abnormalities of sperm morphology and motility were noted, and endocrine dysfunction was suggested as a probable cause. In studies conducted on Sertoli cells in prepubertal rat testis, exposure to Roundup induced oxidative stress leading to cell death (de Liz Oliveira Cavalli et al., 2013). Roundup induced the opening of L-type voltage dependent calcium channels as well as ryanodine receptors, initiating ER stress and leading to calcium overload and subsequent necrosis. Glutathione was depleted due to upregulation of several glutathione-metabolizing enzymes. This suggests that Roundup would interfere with spermatogenesis, which would impair male fertility.
5 Cobalamin deficiency
Untreated celiac disease patients often have elevated levels of homocysteine, associated with folate and/or cobalamin deficiency (Saibeni et al., 2005; Dickey et al., 2008). Species of Lactobacillus and Bifidobacterium have the capability to biosynthesize folate (Rossi et al., 2011), so their disruption by glyphosate could contribute to folate deficiency. Malabsorption in the proximal small intestine could also lead to iron and folate deficiencies. Cobalamin was originally thought to be relatively spared in celiac disease because its absorption is mostly through the terminal ileum, which is unaffected by celiac disease. However, a recent study found that cobalamin deficiency is prevalent in celiac patients. 41% of the patients studied were found to be deficient in cobalamin (<220 ng/L), and 31% of these cobalamin-deficient patients also had folate deficiency (Dahele & Ghosh, 2001). Either cobalamin or folate deficiency leads directly to impaired methionine synthesis from homocysteine, because these two vitamins are both required for the reaction to take place. This induces hyperhomocysteinemia (Refsum et al.,2001), an established risk factor in association with celiac disease (Hadithi et al., 2009). Long-term cobalamin deficiency also leads to neurodegenerative diseases (Herrmann & Obeid, 2012).
Because a deficiency in cobalamin can generate a large pool of methyl-tetrahydrofolate that is unable to undergo reactions, cobalamin deficiency will often mimic folate deficiency. Cobalamin requires cobalt, centered within its corrin ring, to function. We depend upon our gut bacteria to produce cobalamin, and impaired cobalt supply would obviously lead to reduced synthesis of this critical molecule. Glyphosate is known to chelate +2 cations such as cobalt. Glyphosate complexes with cobalt as a dimer [Co(glyphosate)2]3 in fifteen different stereoisomeric configurations, and it is facile at switching among the different stereoisomers, an unusual kinetic property compared to most Co(III) systems (Cusiel, 2005).
In fact, studies have revealed that glyphosate inhibits other cytosolic enzymes besides EPSP synthase in plants and microbes that also activate steps in the shikimate pathway (Ganson and Jensen, 1988; Bode et al., 1984). Glyphosate potently inhibits three enzymes in the shikimate pathway in yeast (Bode et al., 1984). It has been confirmed that these other enzymes depend upon cobalt as a catalyst, and glyphosate inhibition works through competitive cobalt binding and interference with cobalt supply (Ganson and Jensen, 1988). It has also been proposed that chelation by glyphosate of both cobalt and magnesium contributes to impaired synthesis of aromatic amino acids in Escherichia coli bacteria (Hoagland and Duke, 1982). Thus, it is plausible that glyphosate similarly impairs cobalamin function in humans by chelating cobalt.
6 Anemia and iron
Anemia is one of the most common manifestations of celiac disease outside of the intestinal malabsorption issues (Halfdanarson et al., 2007; Bottaro et al., 1999), and is present in up to half of diagnosed celiac patients. Celiac patients often have both cobalamin and folate deficiency, which can cause anemia, but iron deficiency may be the most important factor (Hershko & Patz, 2008). Celiac patients often don't respond well to iron treatment.
Glyphosate's chelating action can have profound effects on iron in plants (Eker et al., 2006; Bellaloui et al.,2009). Glyphosate interferes with iron assimilation in both glyphosate-resistant and glyphosate-sensitive soybean crops (Bellaloui et al., 2009). It is therefore conceivable that glyphosate's chelation of iron is responsible for the refractory iron deficiency present in celiac disease.
Erythropoietin (EPO), also called hematopoietin, is a cytokine produced by interstitial fibroblasts in the kidney that regulates red blood cell production. Low EPO levels, leading to a low turnover rate of red blood cells, is a feature of celiac disease (Bergamaschi et al., 2008; Hershko & Patz, 2008). This can lead to megaloblastic anemia, where red blood cells are large (macrocytic) and reduced in number due to impaired DNA synthesis. A recent hematological study on mice exposed to Roundup at subacute levels for just 15 days revealed an anemic syndrome in both male and female mice, with a significant reduction in the number of erythrocytes and in hemoglobin, reduced hematocrit and increased mean corpuscular volume, indicative of macrocytic anemia (Jasper et al.,2012).
7 Molybdenum deficiency
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