Abstract

INTRODUCTION

Glyphosate is the active ingredient in Roundup®, the most widely used herbicide on the planet.[314] Glyphosate enjoys widespread usage on core food crops, in large part because of its perceived nontoxicity to humans. The adoption of genetically engineered “Roundup^®-Ready” corn, soy, canola, cotton, alfalfa, and sugar beets has made it relatively easy to control weeds without killing the crop plant, but this means that glyphosate will be present as a residue in derived foods. Unfortunately, weeds among GM Roundup^®-Ready crops are developing ever-increasing resistance to Roundup^®,[107,221] which requires an increased rate of herbicide application.[26] In 1987, glyphosate was the 17^th most commonly used herbicide in the United States, but, in large part due to the introduction of glyphosate-resistant core crops, it became the number one herbicide by 2001.[146] Its usage has increased steadily since then, in step with the rise in autism rates. Glyphosate’s perceived nontoxicity is predicated on the assumption that our cells do not possess the shikimate pathway, the biological pathway in plants, which is disrupted by glyphosate, and whose disruption is believed to be the most important factor in its toxicity.

It may seem implausible that glyphosate could be toxic to humans, given the fact that government regulators appear nonchalant about steadily increasing residue limits, and that the levels in food and water are rarely monitored by government agencies, presumably due to lack of concern. However, a paper by Antoniou et al.[12] provided a scathing indictment of the European regulatory process regarding glyphosate’s toxicity, focusing on potential teratogenic effects. They identified several key factors leading to a tendency to overlook potential toxic effects. These include using animal studies that are too short or have too few animals to achieve statistical significance, disregarding in vitro studies or studies with exposures that are higher than what is expected to be realistically present in food, and discarding studies that examine the effects of glyphosate formulations rather than pure glyphosate, even though formulations are a more realistic model of the natural setting and are often orders of magnitude more toxic than the active ingredient in pesticides.[189] Regulators also seemed unaware that chemicals that act as endocrine disruptors (such as glyphosate[108]) often have an inverted dose–response relationship, wherein very low doses can have more acute effects than higher doses. Teratogenic effects have been demonstrated in human cell lines.[212] An in vitro study showed that glyphosate in parts per trillion can induce human breast cancer cell proliferation.[289]

Adjuvants in pesticides are synergistically toxic with the active ingredient. Mesnage et al.[189] showed that Roundup^® was 125 times more toxic than glyphosate by itself. These authors wrote: “Despite its relatively benign reputation, Roundup^® was among the most toxic herbicides and insecticides tested.”[189]

The industry dictates that 3 months is a sufficiently long time to test for toxicity in rodent studies, and as a consequence none of the industry studies have run for longer than 3 months. The only study we are aware of that was a realistic assessment of the long-term effects of GM Roundup^®-Ready corn and soy feed on mammals was the study by Séralini et al. that examined the effects on rats fed these foods for their entire life span.[261] This study showed increased risk to mammary tumors in females, as well as kidney and liver damage in the males, and a shortened lifespan in both females and males. These effects occurred both in response to Roundup and to the GM food alone. These effects only began to be apparent after 4 months.

There are multiple pathways by which glyphosate could lead to pathology.[248] A major consideration is that our gut bacteria do have the shikimate pathway, and that we depend upon this pathway in our gut bacteria as well as in plants to supply us with the essential aromatic amino acids, tryptophan, tyrosine, and phenylalanine. Methionine, an essential sulfur-containing amino acid, and glycine, are also negatively impacted by glyphosate. Furthermore, many other biologically active molecules, including serotonin, melatonin, melanin, epinephrine, dopamine, thyroid hormone, folate, coenzyme Q10, vitamin K, and vitamin E, depend on the shikimate pathway metabolites as precursors. Gut bacteria and plants use exclusively the shikimate pathway to produce these amino acids. In part because of shikimate pathway disruption, our gut bacteria are harmed by glyphosate, as evidenced by the fact that it has been patented as an antimicrobial agent.[298]

Metal chelation and inactivation of cytochrome P450 (CYP) enzymes (which contain heme) play important roles in the adverse effects of glyphosate on humans. A recent study on rats showed that both males and females exposed to Roundup^® had 50% reduction in hepatic CYP enzyme levels compared with controls.[156] CYP enzyme dysfunction impairs the liver’s ability to detoxify xenobiotics. A large number of chemicals have been identified as being porphyrinogenic.[77] Rossignol et al.[242] have reviewed the evidence for environmental toxicant exposure as a causative factor in autism, and they referenced several studies showing that urinary excretion of porphyrin precursors to heme is found in association with autism, suggesting impaired heme synthesis. Impaired biliary excretion leads to increased excretion of heme precursors in the urine, a biomarker of multiple chemical sensitivity syndrome.[77] We later discuss the ability of glyphosate to disrupt bile homeostasis, which we believe is a major source of its toxic effects on humans.

Glyphosate is a likely cause of the recent epidemic in celiac disease.[249] Glyphosate residues are found in wheat due to the increasingly widespread practice of staging and desiccation of wheat right before harvest. Many of the pathologies associated with celiac disease can be explained by disruption of CYP enzymes.[156] Celiac patients have a shortened life span, mainly due to an increased risk to cancer, most especially non-Hodgkin’s lymphoma, which has also been linked to glyphosate.[85,253] Celiac disease trends over time match well with the increase in glyphosate usage on wheat crops.

Glyphosate is also neurotoxic.[59] Its mammalian metabolism yields two products: Aminomethylphosphonic acid (AMPA) and glyoxylate, with AMPA being at least as toxic as glyphosate. Glyoxylate is a highly reactive glycating agent, which will disrupt the function of multiple proteins in cells that are exposed.[90] Glycation has been directly implicated in Parkinson’s disease (PD).[57] Glyphosate has been detected in the brains of malformed piglets.[155] In a report produced by the Environmental Protection Agency (EPA), over 36% of 271 incidences involving acute glyphosate poisoning involved neurological symptoms, indicative of glyphosate toxicity in the brain and nervous system.[122]

In the remainder of this paper, we first introduce the link between glyphosate and manganese (Mn) dysbiosis, and briefly describe the main biological roles of Mn. We then describe how glyphosate’s disruption of gut bacteria may be a major player in the recent epidemic in antibiotic resistance. We then explain how glyphosate can influence the uptake of arsenic and aluminum, and propose similar mechanisms at work with Mn. In the next section, we describe how Mn deficiency can lead to a reduction in Lactobacillus in the gut, and we link this to anxiety disorder. We follow with a discussion on mitochondrial dysfunction associated with suppressed Mn superoxide dismutase (Mn-SOD), and then a section on implications of Mn deficiency for oxalate metabolism. The following section explains how Mn deficiency can lead to the overexpression of ammonia and glutamate in many neurological diseases. The next two sections show how Mn accumulation in the liver is linked to cholestasis and high serum low density lipoprotein (LDL), and how this can also induce increased susceptibility to Salmonella poisoning. We then identify a role for Mn in chondroitin sulfate synthesis, and the implications for osteomalacia. The next two sections explain how glyphosate exposure can lead to Mn toxicity in the brain, and discuss two neurological diseases that are associated with excess Mn, PD and prion diseases. After a section on the link between male infertility and Mn deficiency in the testes, we discuss evidence of exposure to glyphosate and end with a short summary of our findings.

SUPPORTIVE EVIDENCE OF MANGANESE DYSBIOSIS DUE TO GLYPHOSATE

Glyphosate’s disruption of the shikimate pathway is due in part to its chelation of Mn, which is a catalyst for enolpyruvylshikimate phosphate synthase (EPSPS), a critical early enzyme in the pathway.[63] A recent study on Danish dairy cattle investigated mineral composition in serum of cattle fed Roundup^®-Ready feed.[154] The study identified a marked deficiency in two minerals: Serum cobalt and serum Mn. All of the cattle on eight different farms had severe Mn deficiency, along with measurable amounts of glyphosate in their urine. In Australia, following two seasons of high levels of stillbirths in cattle, it was found that all dead calves were Mn deficient.[184] Furthermore, 63% of newborns with birth defects were found to be deficient in Mn.

Mn, named after the Greek word for “magic,” is one of 14 essential trace elements. Mn plays essential roles in antioxidant protection, glutamine synthesis, bone development, and sperm motility, among other things. Although Mn is essential, it is only required in trace amounts. And an excess of Mn can be neurotoxic.

Remarkably, Mn deficiency can explain many of the pathologies associated with autism and Alzheimer’s disease (AD). The incidence of both of these conditions has been increasing at an alarming rate in the past two decades, in step with the increased usage of glyphosate on corn and soy crops in the United States, as shown in [Figures ​[Figures11 and ​and2].2]. Although correlation does not necessarily mean causation, from 1995 to 2010, the autism rates in first grade in the public school correlates almost perfectly (P = 0.997) with total glyphosate application on corn and soy crops over the previous 4 years (from age 2 to 6 for each child) [Figure 1]. Such remarkable correlation necessitates further experimental investigation. These neurological disorders are associated with mitochondrial impairment[197,241,243,281,316] and with excess glutamate and ammonia in the brain,[2,109,265] leading to a chronic low-grade encephalopathy.[256,260] As we will show later, Mn deficiency is critically associated with these pathologies.

Thyroid dysfunction can be predicted as well, and low maternal thyroid function predicts autism in the fetus.[238] Furthermore, increases in bone fractures in both children and the elderly can also be explained by Mn deficiency, due to its critical role in bone development.[276] Osteoporosis, which is a serious problem among the elderly today, is also likely promoted by Mn deficiency,[247] and osteoporosis leads to increased risk to fractures.[98,139,140]

Sprague-Dawley rats fed a Mn-deficient diet had significantly reduced concentrations of Mn in liver, kidney, heart, and pancreas, compared with controls.[18] Furthermore, pancreatic insulin content was only 63% of control levels, and insulin release following glucose administration was also reduced. Mn deficiency not only impairs insulin secretion in Sprague-Dawley rats, but it also causes reduced glucose uptake in adipose tissue,[19] so Mn deficiency could contribute to impaired glucose metabolism in both type 1 and type 2 diabetes, which are a growing problem worldwide.[199] Type 1 diabetes in children is associated with a decrease in Lactobacillus and Bifidobacterium, and an increase in Clostridium, in the gut.[195] These same pathologies are also found in gut bacteria from poultry fed Roundup^®-Ready feed.[263] The increased incidence of diabetes in the US is strongly correlated with glyphosate usage on corn and soy, as shown in [Figure 3].

Much remains elusive about Mn’s roles in cellular metabolism, but it is clear that it is very important. For instance, Target of Rapamycin Complex 1 (TORC1) accelerates the aging process in cells from yeast to mammals,[231] and Mn inhibits TORC1, but only if it is present in the Golgi.[86] Zinc (Zn) is essential for DNA and RNA replication and cell division. Zn deficiency leads to greatly enhanced Mn uptake by cells, and this induces modifications to messenger RNA such that the ratio of guanine and cytosine nucleotides (C + G) to adenine and thymine (A + T) is sharply increased.[100] Clearly, more research is needed to explain the significance of these phenomena.

We infer, paradoxically, that both Mn deficiency and Mn toxicity, attributable to glyphosate, can occur simultaneously. Because of glyphosate’s disruption of CYP enzymes, the liver becomes impaired in its ability to dispose of Mn via the bile acids, and instead it transports the Mn via the vagus nerve to brainstem nuclei, where excess Mn leads to PD. Recently, PD has also increased dramatically, in step with glyphosate usage on corn and soy [Figure 4].

Ironically, while the brainstem suffers from excess Mn, the rest of the brain incurs Mn deficiency due to the depressed serum levels of Mn. Mn is particularly important in the hippocampus, and deficiency there can lead to seizures. A high incidence of seizures is found in children with autism.[302] Seizures are also associated with reduced serum Mn,[54,88,269] and this is consistent with the liver’s inability to distribute Mn to the body via the bile acids. Antibiotics have been found to induce seizures.[132]

Mn uptake in the brain is normally enhanced during the neonatal period in rats, and proper development of the hippocampus depends on Mn.[284] Soy formula increases the risk of seizures in autism,[310] hardly surprising when one considers that soybean crops are now 90% Roundup^®-Ready. A recent paper has confirmed that alarmingly high glyphosate residues appear in Roundup^®-Ready soy.[35] The US Department of Agriculture analyzed glyphosate residues in soy in 2011, and reported that 91% of the 300 samples tested were positive for glyphosate, with 96% being positive for AMPA, an equally toxic by-product of glyphosate breakdown.[297] Our own analysis confirms that glyphosate is present in infant formula. Out of several soy-based baby formulas we tested, only one contained glyphosate residues. We found levels of 170 ppb in Enfamil ProSobee liquid concentrate. Further testing is underway. Soybean product sourcing and residue testing should be required prior to product manufacturing and is necessary to prevent inadvertent infant exposure.

Another mechanism by which glyphosate in soy formula could cause seizures is through bilirubin production. Serum concentrations of bilirubin were elevated in catfish exposed to sublethal doses of Roundup®, in a dose-dependent relationship.[208] Neonates, due to an immature digestive system, are unable to metabolize bilirubin in the gut, and it can therefore build up in the blood and even penetrate their immature blood-brain barrier to cause seizures.[308]

GLYPHOSATE AND MICROBIAL ANTIBIOTIC INTOLERANCE

Microbial antibiotic tolerance and resistance are a growing problem worldwide, likely fueled by horizontal gene transfer among different bacterial species.[106,121] Multiple-drug resistant commensal bacteria in the guts of both animals and humans form a reservoir of resistance genes that can spread to pathogenic species. Methicillin-resistant Staphylococcus aureus (MRSA),[119] Clostridium difficile,[183] and Pseudomonas aeruguinosa[169] are all becoming major threats, especially in the hospital environment. A generic mechanism of upregulated efflux through membrane pores offers broad-domain resistance to multiple antibiotics.[169] Exposure to antibiotics early in life can even lead to obesity as a direct consequence of the resulting imbalance in gut bacteria.[73]

Studies have shown that increased mutation rates due to chronic low level exposure to one antibiotic can induce an accelerated rate of development of resistance to diverse other antibiotics.[151] Glyphosate, patented as an antimicrobial agent,[298] is present in steadily increasing amounts in the GM Roundup-Ready corn and soy feed of cows, pigs, chickens, farmed shrimp, and fish, and it is ubiquitous in the Western diet of humans. Pseudomonas aeruginosa can use glyphosate as a sole source of phosphorus,[192] and it is one of a small number of resistant bacterial species with the ability to metabolize glyphosate, a feature that might be exploited for soil remediation.[1] However, DNA mutations due to exposure would enhance tolerance to glyphosate and other antibiotics, perhaps explaining the current epidemic in multiple antibiotic resistant P. aeruginosa infections, which have a 20% mortality rate.[190] Antibiotic resistance sequences engineered into GM crops may also play a role in the current crisis concerning antibiotic resistant pathogens.

Glyphosate has also been demonstrated as a remarkable antimicrobial synergist. It greatly increases the cidal effects of other antimicrobials, particularly when combined as salts of glyphosate. A concentration dependent synergy index (SI) ranging from 0.34 to 5.13 has been recorded for the Zn salt of glyphosate.[299] This has serious implications for glyphosate ingested with pharmaceuticals or residues of other widely used agricultural chemicals, such as the herbicides Diquat, Paraquat, 2,4 D and Glufosinate, the fungicide Chlorothalonil and the systemic neonicotinoid insecticides Acetamiprid, Imidacloprid, Thiacloprid, Thiamethoxam, and Clothianidin.

Glyphosate acts as a catalyst for the development of antibiotic resistance genes in pathogens. Since both poultry and cow manure are used as natural fertilizers in crops, it can be expected that a vector for microbial resistance to multiple drugs is through contamination of fruits and vegetables. Indeed, multiple resistance genes have been identified from diverse phyla found in cow manure, including Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, that is, in phylogenetically diverse organisms.[311]

One of the ways in which glyphosate is toxic to plants is through disruption of chlorophyll synthesis, due to suppression of the activity of the first enzyme in pyrrole synthesis.[61,69,143,319] Pyrrole is the core building block of both chlorophyll and the porphyrin rings, including corrin in cobalamin and heme in hemoglobin and cytochrome enzymes. Several cofactors containing a structurally complex tetrapyrrole-derived framework chelating a metal ion (cobalt (Co), magnesium (Mg), iron (Fe), or nickel (Ni)) are synthesized by gut bacteria and supplied to the host organism, including heme and corrin.[236]

Thus, glyphosate can be expected to disrupt synthesis of these biologically essential molecules. Pseudomonas normally thrives in the small bowel and produces abundant cobalamin that may be a significant source for the human host.[6] P. aeruginosa‘s successful colonization may be due in part to its ability to produce cobalamin despite the presence of glyphosate. Only recently has it also been recognized that a Mn–porphyrin complex can protect from mitochondrial overproduction of hydrogen peroxide (H₂O₂) in response to ionizing radiation.[274] It can be predicted that homeostasis of all of these minerals in the gut (Co, Mn, Fe, Ni, and Mg) is impaired in the presence of glyphosate, and this will have serious consequences not only to the gut bacteria but also to the impaired regulation of these minerals. The implications of impaired heme and cobalamin synthesis will be further addressed in a future paper.

A key component of glyphosate’s action is its ability to chelate minerals, particularly transition metals such as Mn. Glyphosate forms strong complexes with the transition metals via the amino, the carboxylic, and the phosphonic moieties in the molecule. Each of these can coordinate separately to metal ions or in combination as bidentate or tridentate ligands.[194,296]

ANALOGY WITH ARSENIC AND ALUMINUM

Chronic kidney disease is clearly associated with multiple environmental toxicants.[268] There has been an epidemic in recent years in kidney failure among young agricultural workers in Central America, India, and Sri Lanka, particularly those working in the sugar cane fields.[249] A recent paper reached the unmistakable conclusion that glyphosate plays a critical role in this epidemic.[133] A growing practice of spraying sugar cane with glyphosate as a ripener and desiccant right before the harvest has led to much greater exposure to the workers in the fields. The authors, who focused their studies on affected workers in rice paddies in Sri Lanka, identified a synergistic effect of arsenic, which contaminated the soil in the affected regions. This paper is highly significant, because it proposes a mechanism whereby glyphosate greatly increases the toxicity of arsenic through chelation, which promotes uptake by the gut. Glyphosate also depletes glutathione (GSH)[60,128] and glutathione S transferase (GST) is a critical enzyme for liver detoxification of arsenic.[295] As a consequence, excess arsenic in the kidney causes acute kidney failure, without evidence of other symptoms such as diabetes usually preceding kidney failure.

Arsenic is normally disposed of by the liver through biliary excretion. In rats exposed to arsenic, large amounts of GSH appeared in the bile simultaneously with biliary excretion of arsenic.[113] It was first hypothesized, and later confirmed, that arsenic is transported in bile acids in the form of unstable GSH complexes (monomethylarsonous acid), which release GSH upon decomposing. Since glyphosate disrupts CYP enzymes necessary for bile acid formation,[248,249] as well as depleting GSH,[60,128] it can be expected that glyphosate would disrupt the process of biliary excretion of arsenic, thus forcing arsenic to be redirected toward urinary excretion, leading ultimately to kidney failure.

Glyphosate also chelates aluminum,[230] and it has been reasoned that this enables aluminum to get past the gut barrier more readily through direct analogy with the situation with arsenic, which is also a 3+ cation.[193]

However, it has been demonstrated through experimentation that glyphosate prefers divalent cations. Thus, aluminum would enter the bloodstream via the digestive tract’s portal vein to the organs traveling with albumin, which is known to attach and transport many xenobiotics. It is well established that citrate also binds aluminum and promotes its uptake past the gut barrier through a mechanism that parallels glyphosate’s binding to aluminum.[68,148] Both are small molecules that easily pass through a leaky gut barrier.

Considering these observations regarding aluminum and arsenic, it is reasonable to expect that something similar might happen with Mn. Unlike these other two, however, Mn plays many essential roles in the body, and so its chelation by glyphosate would interfere with its bioavailability in the general circulation. Just as for arsenic, bile acids play a critical role in Mn homeostasis. Bile is the major excretory route of injected Mn.[17] Malecki et al. wrote: “Biliary excretion may be a major homeostatic mechanism for preventing both deficiency and toxicity of Mn.”[179, p. 489]