Genetically Modified Children (FREE VIEWING - LIMITED TIME)
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Tuesday, October 2nd 2018 at 1:30 pm
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Stephanie Seneff
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There is a growing awareness that glyphosate is much more toxic than we have been led to believe, and I am confident that in time it will be banned worldwide, just like DDT
Introduction
Glyphosate is the active ingredient in the pervasive herbicide Roundup. Together with several colleagues, I have published extensively on the dangers of glyphosate to human health and to the ecosystem. Most people believe that glyphosate is practically non-toxic to humans, in part because governments are claiming that this is so, and people want to believe that their government is trustworthy. But there is a growing awareness that glyphosate is much more toxic than we have been led to believe, and I am confident that in time it will be banned worldwide, just like DDT.
One way to find out whether glyphosate is toxic is to get to know the people who are in the front lines: those who are exposed, environmentally or occupationally, to heavy doses of glyphosate in their daily lives. A good choice might be people who live in a small village in northern Argentina that is surrounded by fields of tobacco that have been genetically engineered to resist it. This is what two investigative journalists set out to do, and their efforts have borne fruit in the form of an informative, engaging and disturbing documentary.
Several years ago, I discovered that a genetically-engineered glyphosate-resistant tobacco seed had been developed and patented [1]; however, it is very difficult to find out what percentage of the tobacco crops are actually grown from these engineered seeds. One anecdotal answer to this question comes from the recently released documentary, Genetically Modified Children, produced and directed by Juliette Igier and Stephanie Lebrun. This movie describes the severe health issues that afflict the children of agricultural workers who live surrounded by glyphosate-resistant tobacco crops produced for sale exclusively to the tobacco company, Philip Morris. The farmers who were interviewed readily admitted that Philip Morris would most likely reject their product if they did not use glyphosate to control weeds and Bayer’s insecticide Confidor to control insects.
The documentary reveals the severe physical deformities, mental disabilities and cancers the children of these tobacco farmers are experiencing, and it offers the rather audacious hypothesis that glyphosate is inducing genetic mutations in the children of these farmers. Could they be right?
In the remainder of this article, I will first describe some of the serious health issues of the children who live near the tobacco fields, as portrayed in the documentary. After a short section about correlations between glyphosate and various diseases, I will discuss, in the following three sections, both the evidence that glyphosate causes birth defects, infertility, developmental issues, DNA damage and cancer, and the plausible biological mechanisms that could explain a link. The next section is devoted to describing the evidence that glyphosate can get into proteins by mistake in place of the coding amino acid glycine, which I now believe is its main mechanism of insidious cumulative toxicity. The rest of the article focuses on two proteins in particular that could get disrupted through glyphosate substitution, and I will explain how this disruption can lead to a runaway phosphorylation cascade resulting in cancer. I will conclude with an urgent request that we change our agricultural methods towards renewable organic solutions, in order to protect future generations from harm.
Damaged Children
The documentary included accounts of several children who were clearly very sick with disorders connected to impaired development of the neural tube and/or rare genetic diseases, as well as an unusually high incidence of cancer. Two children in this small community were born with hydrocephalus, a condition in which cerebrospinal fluid accumulates in the brain, causing an unusually large head. In infants, it is associated with a bulging fontanelle (the soft spot), irritability, seizures, vomiting and sleepiness, as well as impaired memory development. It is often linked to a rare genetic defect, and about 80-90% of newborn infants with spina bifida develop hydrocephalus. Another child suffered from another disorder directly caused by impaired neural tube development, called myelomingocele. A myelomeningocele manifests as a protrusion of meningeal membranes through an opening in the spinal column, often in the lower back region, which appears as a sac enclosing the meninges, cerebrospinal fluid, and parts of the spinal cord and nerve roots. Another child was diagnosed with congenital microcephaly, along with epilepsy, delayed motor and mental development, and multiple muscular atrophy.
Yet another child suffered from lamellar ichthyosis, a rare genetic condition often caused by mutations in the gene for the protein keratinocyte transglutaminase, which results in excessive production of keratin, causing scaly, itchy skin, along with an increased risk to hypothermia and bacterial skin infection. Children with this condition have scaly skin all over their body, and are highly susceptible to toxic exposures on the skin that readily pass the defective barrier.
There was a high rate of miscarriages among the women of the community. The rate of childhood cancer in this small community was five times as high as the rate in the general population in Argentina. Leukemia and lymphoma were prevalent among the adults as well as the children. The alarming health issues in this community, particularly neural tube defects, were documented in an interview conducted with Professor Hugo Gomez Demaio, Head of Neurosurgery at the Pediatric Hospital of Posadas, Argentina [2].
Correlations between glyphosate and various diseases
Swanson et al. published a paper in 2014 showing over 20 graphs of strong correlations in the United States over time between glyphosate usage on core crops and various diseases and conditions. Several of these were specific cancers, including pancreatic cancer (p < 4.6E-7)[1],thyroid cancer (p < 7.6E-9), bladder cancer (p < 4.7E-9), liver cancer (p < 4.6E-8), kidney cancer (p < 2.0E-8) and myeloid leukemia (p < 1.5E-6) [3]. A follow-on paper from 2015 by Hoy et al. looked specifically at diseases of newborns and found highly significant correlations between the rise in diseases of the lymph system, among children (p < 0.00043) and adults (p < 0.00023), congenital heart defects among newborns (p < 9.2E-6), newborn lung disorders (p < 3.4E-5), and newborn genitourinary disorders (p < 2.4E-5) [4], among others. Exemplary plots for pancreatic cancer and genitourinary disorders are reproduced here as Figures 1 and 2.
Figure 1: Correlation between age-adjusted thyroid cancer incidence and glyphosate applications and percentage of US corn and soy crops that are genetically engineered. Reproduced from Swanson et al. 2014 [3] .
The incidence rates for non-Hodgkin’s lymphoma, tumors of the brain and spinal cord, and liver and kidney tumors have all increased among children in the U.S. during the period from 2001 to 2014, in step with the rise in glyphosate usage on core crops [5].
Glyphosate and Spina Bifida
A myelomeningocele is the most severe form of spina bifida, which, more generally, is a defect in the maturation of the spinal column due to impaired neural tube closure at a critical time during early embryonic development. It is associated with problems in walking, problems with bladder and bowel control, and hydrocephalus. The causes of spina bifida are multifactorial, and include folate deficiency, excessive retinoic acid exposure, impaired methylation capacity, impaired transsulfation pathway, impaired glucose metabolism, and impaired nucleic acid repair systems.
Figure 2: Hospital discharge rates for newborn genitourinary disorders compared to glyphosate applications to wheat, corn and soy crops. See Hoy et al., 2015 [4] for details.
Every one of these impairments could be attributed to glyphosate, as explained in a paper I published together with colleagues linking glyphosate to anencephaly (child born with a missing cerebral cortex) [6]. Folate deficiency is probably the most well known causal factor in spina bifida, and this is what motivated the US government to require folate fortification of wheat-based products starting in 1998, just as the use of genetically engineered Roundup-Ready crops was ramping up. Glyphosate’s link to folate deficiency is easy to understand because folate is synthesized by the gut microbes for the host from products of the shikimate pathway [7]. Impaired bacterial supply of folate due to glyphosate’s interference with their shikimate pathway will lead to deficiencies in the human host. Glyphosate’s disruption of the shikimate pathway has been identified as its key mechanism of toxicity to weeds [8].
Methionine deficiency is another link to neural tube defects, because methionine supplies methyl groups that are necessary for proper development of the nervous system. Glyphosate has also been shown to deplete methionine in plants and to disrupt biological pathways in Escherichia coli (E. coli) that synthesize methionine from inorganic sulfur, as described in [6]. Methionine deficiency disrupts both the methylation pathways and the trans-sulfuration pathways. Glyphosate can also be predicted to disrupt the clearance of retinoic acid, which is metabolized through cytochrome P450 enzymes, which are inhibited by glyphosate. Details of how glyphosate disrupts these critical pathways as well as glucose metabolism and nucleic acid repair mechanisms are also discussed in the paper on anencephaly [6].
Glyphosate and the Reproductive System
Both human population studies and animal studies have shown multiple issues with the reproductive system in association with glyphosate exposure. Argentina uses 240,000 tons of glyphosate in its industrial agriculture program annually. Physicians in agricultural areas have noticed an increase in reproductive disorders in the communities they serve. A formal study published in 2018, based in the rural town of Monte Maíz, found that the rate of spontaneous abortion was three times as high as the national average, and the rate of congenital anomolies was doubled [9].
In a recent study on pregnant women in Indiana, more than 90% of them tested positive for urinary glyphosate, and higher glyphosate levels were statistically significantly associated with a shortened gestational period [10]. The US consumes more glyphosate than most other countries, and our rates of premature births have been rising in the past decade. We also have a higher rate of premature births than other Western nations [11].
An extraordinarily high rate of severe congenital malformations in piglets from a Danish farm inspired an investigation in which tissue samples from various organs of 38 deformed one-day old piglets were tested for glyphosate contamination. Glyphosate was found at levels as high as 80 micrograms/gram, with the lungs and heart showing the highest levels of contamination [12].
A new paper studying multigenerational effects of glyphosate in an animal model obtained a remarkable result that suggests that the germ cells of a fetus are especially susceptible to genetic mutation by glyphosate in utero [13]. An extraordinary feature of mammalian reproduction is that the female fetus develops its ovaries very early in gestation, long before major brain development takes place [14]. Toxic exposures to the ovaries prenatally can lead topolycystic ovary syndrome (PCOS) and premature ovarian failure (POF) [15]. More ominously, because the second generation germ cells are already present early in the gestation period, they could be subject to mutations induced by a mutagenic agent that has bridged the placental barrier.
In the experiment [13], pregnant rats were exposed to glyphosate starting at day 9 of gestation and extending beyond birth into the period when the pups were being nursed. These pups were allowed to mature and also give birth to a second generation. All three generations were evaluated for any evidence of harm by glyphosate. Two different levels of exposure were set (low and high), but both were below the daily limit set by the United States EPA.
While the mothers experienced no obvious damage from glyphosate, the second generation had a reduced litter size, suggesting impaired fertility, and the growth of their pups in utero was slow, resulting in low birth weight. However, the most remarkable result of the study was a high rate of rare mutations in the second generation pups. Three out of the 117 total second generation fetuses suffered from rare severe malformations (siamese twins and abnormally developed limbs), and these three were from three different mothers within the first generation. This suggests that exposure of germ cells in utero to glyphosate induces a high mutation rate, an idea that I will develop more fully later in this article.
Evidence that Glyphosate Causes DNA Damage and Cancer
An early step in the progression towards cancer and towards genetic mutations is DNA damage, often induced by oxidative stress -- the attack on DNA molecules by oxidizing agents such as superoxide and peroxynitrite. A common technique in the research lab for assessing the potential of a given chemical as a carcinogen is to expose cells to the chemical and then examine them under a microscope looking for evidence of damage, such as achromatic lesions (gaps in the chromosomes that are visible through a microscope) and chromatid deletions (entirely missing parts of a chromosome). These are typically caused by double strand breaks in the DNA strand that makes up the chromosome [16]. Multiple repair mechanisms exist to try to restore the proper DNA sequence following a break, but these repair mechanisms can introduce a copy error that results in a DNA mutation.
Does glyphosate induce double strand breaks in DNA? Monsanto’s own 1983 unpublished report found over twice as many achromatic lesions and chromatid deletions in rat bone marrow cells exposed to glyphosate compared to controls [17]. Several independent studies have confirmed that glyphosate causes double strand breaks and also induces the oxidative stress that usually precedes DNA damage.
A paper from Colombia showed that people living in regions surrounded by glyphosate-sprayed crops due to coca and poppy eradication efforts had significantly higher counts of cellular defects linked to DNA damage compared to people living in an area where organic coffee was grown [18]. Similarly, a study based in northern Ecuador examined DNA damage through the well-established comet assay in white blood cell samples taken from people who had been exposed to glyphosate drift from across the border (from aerial spraying in Colombia). They found that the average comet length in the exposed group was 35.5 micrometers, compared to only 25.94 micrometers in the control group [19]. The comet assay is used to detect double-strand DNA breaks.
A paper published in 2010 examined the genotoxic effects to eels of acute exposure to Roundup at realistic dosages [20]. Comet assay demonstrated that Roundup induced DNA double-strand breaks as well as chromosomal abnormalities. This paper revealed a surprising result, in that several measures of oxidative stress proved negative, implying that the DNA damage was sometimes occurring through some other mechanism besides oxidative stress.
However, a study on tropical fish exposed to sublethal doses of Roundup found evidence of an increase in the synthesis of proteins associated with antioxidant defenses, suggesting that glyphosate, or at least its formulation, Roundup, does induce reactive oxygen species, and this was associated with evidence of DNA damage in red blood cells [21]. Glyphosate exposure to a species of freshwater fish at three sublethal test concentrations caused oxidative stress in the gills and blood, as assessed through increased lipid peroxidation and increased synthesis of antioxidant defense proteins, as well as DNA damage as assessed by comet assays [22]. Damage to the gills was worse than damage to the blood.
An important study published in 2018 and conducted in response to the World Health Organization’s International Agency for Research on Cancer (IARC) classification of glyphosate as a probable carcinogen showed an increased risk to both single and double DNA strand breaks as well as oxidation of DNA nucleotides in white blood cells exposed over a 24 hour period to glyphosate, its breakdown product AMPA, or its formulation Roundup. The formulation was found to be significantly more genotoxic than the isolated chemicals. They suggested that glyphosate and AMPA cause oxidative damage by increasing production of reactive oxygen species in the cell, and this in turn induces DNA breaks [23].
DNA hypomethylation is an initiating step in the conversion of a cell to a pluripotent state, giving it stem-cell like features that are also characteristic of cancerous tissues. A 2018 study conducted in Poland showed that glyphosate exposure at a relatively low dose (0.25 millimolar) induced significant modifications in the methylation pattern on the DNA of white blood cells [24]. Specifically, globally, DNA was hypomethylated in the presence of glyphosate, whereas the promoter region of TP53, a tumor suppressor gene, was hypermethylated. Such hypermethylation has the effect of suppressing expression of this gene; i.e., enhancing the likelihood of cancer.
Many women who have gone through breast cancer treatment are aware that breast cancer cells have estrogen receptors, and the tumor will grow under estrogenic influences. This fact is what led to the sharp curtailing of hormone replacement therapy once it became clear that it was associated with an alarming increase in risk to breast cancer. Estrogen-sensitive breast cancer cells have been found to respond to minute doses of glyphosate, measured in parts per trillion, by proliferating [25]. This strongly suggests that glyphosate is an estrogenic agent.
A proteomic approach to investigate alterations in protein expression in mouse skin following topical exposure to glyphosate revealed that many proteins were either upregulated or downregulated, and the modified expression pattern was consistent with carcinogenic potential [26]. Most striking was a nearly ten-fold upregulation in a protein called calgranulin B. Calgranulin B has been shown to promote cell proliferation, migration and invasion in squamous cervical cancer [27].
Glyphosate as a Glycine Analogue
Glyphosate is a deceptively simple molecule, and it is surprising that it is so potent as an herbicide, indiscriminately killing all plants except those that are engineered to be resistant. One very unique property of glyphosate is that it is an amino acid analogue of glycine. In fact, it is a complete glycine molecule, except that extra material (namely, a methyl phosphonyl group) has been attached to the nitrogen atom. Part of its toxicity has to do with its ability to mimic glycine at glycine receptor sites, stimulating calcium entry [28, 29], and another part has to do with interfering with reactions where glycine is a substrate [30]. However, a much more insidious potential mechanism of toxicity is the possibility that it substitutes by mistake for glycine, a coding amino acid, during protein synthesis. As a consequence of my intense interest in this question, I have studied extensively the essential roles of various glycine residues in a long list of biologically important proteins, acting as enzymes, receptors, transport proteins, structural proteins, etc. Remarkably, many of the diseases whose frequency is going up alarmingly over time in step with the alarming rise in glyphosate usage on core crops can be explained through disruption of specific glycine residues in specific proteins, as has been demonstrated thus far forgout [31], amyotrophic lateral sclerosis (ALS) [32], Mesoamerican nephropathy [33], anencephaly [6], Alzheimer’s disease, diabetes, and obesity [34], and neurological diseases such as autism and multiple sclerosis [35]. Glyphosate is the only pesticide used in agriculture whose usage rate has gone up dramatically over the past two decades, in step with the dramatic rise in a long list of debilitating diseases and conditions.
One of Monsanto’s own studies from 1989 virtually proved that this is happening. The study involved exposing blue-gill sunfish to radiolabelled glyphosate and then checking for radiolabel in tissue samples. They found a remarkable discrepancy between the amount of radiolabel and the amount of glyphosate detected through a standard assay. They were able to close the gap significantly, however, by subjecting the sample to proteolysis enzymes (proteinase K), and they suggested that a plausible interpretation of their finding was that glyphosate was gettingincorporated into the protein [17]. Glyphosate assays fail to detect glyphosate when it is embedded in a peptide chain, whereas proteolysis separates the individual amino acids from the chain, freeing up the glyphosate and making it now visible to the assay.
Another huge piece of evidence comes from mutations in the enzyme in the shikimate pathway that glyphosate famously disrupts, called 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase. EPSP synthase binds electrostatically to its substrate, phosphoenol pyruvate (PEP), couched within a carefully shaped pocket. A highly conserved glycine residue helps
to form the shape of the pocket [33]. There is a lot of variability among EPSP synthases produced by different species, but essentially all of them have a glycine residue at this important spot in the peptide chain. Studies on the mechanism by which glyphosate disrupts the protein propose that it gets in the way by fitting into the pocket as a faux substrate. However, another plausible explanation is that it is swapped in in place of this especially important glycine residue along the border of the pocket, and its bulky methyl phosphonyl group protrudes into the pocket, crowding out PEP. Very compelling circumstantial evidence comes from the fact that multiple species of plants and multiple species of microbes have independently “discovered” that, if they get rid of the code for that glycine residue (changing it to the closest matching amino acid, alanine), then the enzyme fortuitously becomes completely insensitive to glyphosate exposure, even at high levels. This makes sense because the code no longer matches glyphosate, so no substitution occurs. If glyphosate were instead acting as substrate, the extra methyl group in alanine would not have had such a categorical effect on the sensitivity.
Another remarkable fact is that researchers have played around with over a thousand variants of glyphosate, various molecules that would appear to have a similar shape, but with various tweaks on the chemical structure. None of them were nearly as effective as glyphosate itself at disrupting EPSP synthase [36]. The problem is that none of these variants are amino acid analogues of glycine, and therefore they can’t work the same way glyphosate does.
Glyphosate is not unique in this ability to cause disease through misincorporation into proteins through a coding error. In fact, there are hundreds of naturally occurring amino acid analogues, and many of them have been linked to severe diseases due to substitution for coding amino acids during protein synthesis [37]. β-Methylamino-L-alanine (BMAA) is a naturally produced amino acid analogue of serine, synthesized by cyanobacteria. It has been hypothesized to misincorporate into proteins in exposed organisms, resulting in protein misfolding. Following an extremely long latency period, this can lead to an ALS-like disease that became endemic in Guam following World War II [38, 39].
Glufosinate, an herbicide that is growing in popularity with the growth in glyphosate-resistant weeds, is a naturally produced amino acid analogue of glutamate, and it is associated with impaired neurogenesis following perinatal exposure due to disruption of glutamate signaling [40]. L-azetidine-2-carboxylic acid (Aze) is yet another naturally produced non-coding amino acid analogue, found in sugar beets, and it is believed to cause multiple sclerosis in exposed humans [41]. Introduction of sugar beet leaves into the feed of lambs resulted in a swayback condition attributed to substitution of Aze for proline in myelin basic protein (MBP), a core protein of the myelin sheath in nerve fibers [42]. MBP contains a highly conserved sequence, PRTPPP, with four proline residues, which causes it to be highly susceptible to Aze toxicity through substitution for one or more of these proline residues. L-canavanine, a toxin found in the seeds of a wild potato plant in Alaska, is an amino acid analogue of L-arginine, and it is suspected to be the cause of the death of Christopher McCandless, the protagonist of the book, “Into the Wild” by Jon Krakauer [43].
AID, Nup98 and Phosphorylation Cascades
Given the growing body of evidence that suggests that glyphosate causes cancer, I have become very interested in the question of how glyphosate could cause cancer. I believe I have found an answer that starts with a triggering of epigenetic regulatory mechanisms that induce a cell to reproduce itself under conditions of stress. A breach in the barrier that protects the nucleus from attack by mutagenic agents leads to suppression of proteins that normally orchestrate a programmed cell death (apoptosis) and uncontrolled expression of proteins that induce proliferation. A regression into a stem-cell-like phenotype completes the picture that leads to rampant growth of a cancer. I find these faulty regulatory control mechanisms to be absolutely fascinating, and glyphosate’s disruption of these natural cellular processes, through widespread incorporation into proteins in place of glycine, can result in not only cancer, but also immune deficiency and autoimmune disease.
Some proteins that cells synthesize have awesome capabilities that can lead to great mischief if they get corrupted. Two of these are activation induced deaminase (AID) and nucleoprotein 98 (Nup98). Under cellular conditions of systemic extreme hyperphosphorylation, these two proteins collaborate to drive a cell into a cancerous state. Phosphorylation is an intriguing mechanism by which cells can modify their proteins and change their behavior, and it involves attaching a phosphate anion to either a serine or a threonine residue within the protein. Some proteins are profoundly affected by such a change, in some cases activated and in other cases inactivated by this small perturbation of the protein’s structure [44].
AID (not to be confused with AIDS!) was only first discovered in 1998, but the number of papers published on AID has been growing exponentially ever since. More and more of its roles in the body are being revealed over time, as well as its ability to cause harm if its expression is misregulated. AID’s specific action is to deaminate a cytosine nucleotide in a DNA molecule. This simple modification can lead to a double-strand break, which is then aggressively repaired by a suite of specialized DNA repair enzymes. However, these enzymes can make a mistake during the repair process, ending up with a DNA mutation, or even a wholesale rearrangement of a chromosome if the wrong ends get sewn together.
AID plays an essential role in the immune system, first, by launching the processes, called somatic hypermutation and class switch recombination, by which specific immunoglobulins in immature immune cells are modified within the thymus during infancy [45]. These raw immunoglobulin templates are eventually converted into an enormous number of variants that can later serve as antibodies to foreign antigens. Later on, at a time when a particular antigen, for example, from an invasive virus, presents itself, AID again becomes active to perfect the shape of the antibody so that it will be able to perfectly recognize and tag the viruses, making them readily visible to other immune cells that will then clear (phagocytose) them. If AID is dysfunctional, these processes will become imperfect, and the immune cells will not be able to optimize the shape of the antibodies. They can potentially end up tagging native proteins rather than foreign proteins, in a pathological process called molecular mimicry, leading to both autoimmune disease and immune deficiency.
AID has also been found to be expressed in some other cells besides the immune cells, most specifically, in cancer cells [46]. So-called “ectopic” expression of AID in cancer cells causes mutations in multiple proteins, which can cause activation of cancer-promoting proteins, such as the Myc oncogene [47], or suppression of proteins that protect from cancer, such as the tumor suppressor, TP53 [48]. Many cancers are associated with loss-of-function mutations in TP53, including colon cancer, breast cancer, lung cancer, lymphoma and leukemia. Over half of all human cancers carry a mutated form of TP53 that no longer functions [49].
TP53 could also be directly affected by glyphosate through a “pseudo-mutation” caused by substitution for a critical glycine residue at location 334 in the sequence [50]. A study on a lung cancer mutation, where the glycine residue at location 334 was replaced with valine, showed that this minor change induced a misfolding of the protein into amyloid fibrils and an impaired ability to form its normal tetrameric configuration [51], thus inactivating it. As mentioned earlier, glyphosate’s documented ability to hypermethylate TP53’s promoter region would also disable expression of the protein [24].
AID can also cause mutations in proteins that would normally induce apoptosis (programmed cell death), preventing them from doing so, and also in the promotor regions of proteins that induce mitosis (cell division), causing them to be permanently expressed and inducing a proliferative state that defines tumor cells. Most remarkably, AID can induce a state of pluripotency, by causing methyl groups to be removed from the cell’s DNA, a first step in reverting a cell to a stem-cell-like status [52]. Such stem-cell-like behavior is a characteristic feature of tumor cells.
Because AID can be dangerous if left unchecked in the nucleus, there are mechanisms in place to retain AID in the cytoplasm and/or to export it from the nucleus, except when it is needed to respond to an infection or other threat [53]. The nucleus is separated from the cytoplasm of the cell by the nuclear membrane. The nuclear membrane is actually riddled with open channels that are created by specialized membrane proteins. These proteins maintain a large number of nuclear pores (holes) where materials can be passaged through the membrane either to be transported from the nucleus into the cytoplasm (e.g., RNA), or to be transported from the cytoplasm into the nucleus (e.g., proteins that regulate DNA expression). These holes are normally plugged by gelled water that is maintained by nucleoporins, one of which is Nup98, which essentially keep large molecules from getting across except when escorted. Specialized transport proteins called importins and exportins can hand-carry specific molecules across the membrane through the gel, essentially by locally melting the gel while traversing the channel [54]. But, in the absence of such assistance, large molecules, such as AID, have to stay put on whichever side of the fence they’re stuck in.
Biological systems are fond of utilizing a strategy that involves minor modification of proteins, in order to change their behavior. This is part of the “epigenetic” influences that have been mentioned frequently in the media as a powerful force in nature. One of these is protein phosphorylation, which involves adding a phosphate group (PO4-2) to a protein. When Nup98 gets hyperphosphorylated (through the addition of multiple phosphates), the plug in the pore completely disintegrates, and it’s a free-for-all in terms of crossing the divide [55, 56].
AID itself uses phosphorylation of a particular serine residue to activate its ability to induce DNA double breaks. And, of course, it has to actually be on the right side of the fence (in the nucleus) in order to have access to the DNA. This is where hyperphosphorylation of Nup98 comes in. Nup98 has an “FG domain” which is a segment of the protein that is highly enriched in phenylalanine (F)-glycine (G) pairs. If multiple serine residues within this FG domain get phosphorylated, Nup98 detaches itself from the pore plug, and this induces a biophysical change that causes the entire plug to disintegrate, essentially opening the floodgates to everybody, including AID, which is normally sequestered in the cytoplasm to keep it from mutating DNA. Glyphosate substitution for glycines within this glycine-rich region would have the effect of introducing a “pseudo” phosphorylation, because, like phosphate, glyphosate carries a negative charge through its phosphonate moiety. Finally, the proteins that add phosphate groups to proteins, called kinases, are also often activated by phosphorylation, and some of these, such as calmodulin kinase II, even activate themselves by adding a phosphate group to their own threonine residue, in a positive feedback loop!
How does a phosphorylation cascade get started? Well, one common way is through excessive calcium uptake by the cell. This is something that glyphosate has been found to induce in both in vitro and in vivo experiments in multiple cell types: Sertoli cells in the testes [57], neurons [28], and cardiac muscle cells [58]. The paper showing increased calcium uptake in neurons demonstrated that glyphosate activated both NMDA receptors and voltage-dependent calcium channels, in part by acting as a glycine analogue [28]. And it also activated the serine-threonine kinase protein, calmodulin kinase II. This in turn launches a flurry of activities that ultimately results in hyperphosphorylation of a large number of proteins in the cell.
Pseudo-phosphorylation through Glyphosate Substitution
Anthony Samsel and I described, in our first paper to discuss glyphosate substitution for glycine during protein synthesis, how glyphosate substitution for critical glycine residues in kinases could be predicted to increase their activity, whereas glyphosate substitution in phosphatases (proteins that remove phosphates) would be predicted to suppress their activity [34]. These effects of course would result in systemic hyperphosphorylation. But, worse than this, glyphosate substitution for certain glycines in proteins that get phosphorylated can be expected to mimic phosphorylation, such that the modified protein acts as if it is per-manently phosphorylated! Serine/threonine kinases have a highly conserved glycine residue right next door to the serine or threonine residue that gets phosphorylated to activate them [59]. Substituting glyphosate for this glycine residue will add a methyl-phosphonyl group carrying a negative charge, which is likely a near-perfect imitation, biophysically, of a phosphate group attached to the adjacent serine residue. Multiple studies have shown that swapping out either the serine residue itself or a nearby amino acid for a negatively charged amino acid (such as aspartate, glutamate or glyphosate) creates a negative charge field that imitates permanent, irreversible phosphorylation on the serine residue [60, 61].
For example, Mayford and colleagues engineered a transgenic mouse to express a variant calmodulin kinase II where the normally present threonine residue at location 286 was replaced by the negatively charged amino acid, aspartate [60]. They showed that the effect was to increase its kinase activity level, just as would be expected to happen if the original threonine residue were phosphorylated. This modification thus produces a version of the protein that does not need calcium stimulation to be activated.
Remarkably, zebrafish have an unusual variant of the AID protein that is missing the serine residue that normally gets phosphorylated, yet it is constitutively active in inducing DNA breaks, as if it were phosphorylated [61]. Researchers hypothesized that the presence of a negatively charged amino acids, aspartate, two residues away from the place where the serine residue should be, had the same effect as serine phosphorylation, producing an enzyme that acted as if it was permanently phosphorylated. The AID protein has seven highly conserved glycine residues [62]. It is in general unpredictable what glyphosate substitution for any of these glycines might do in terms of disrupting protein behavior. However, one of them is near the phosphorylated serine residue, and its substitution by glyphosate would likely activate AID to induce double strand DNA breaks, as has been observed to occur in multiple human- and animal-based models of glyphosate exposure, as discussed previously.
Monsanto’s own studies showed that glyphosate accumulates at the highest concentrations in the bone marrow, where the stem cells that will later evolve into white blood cells reside [35]. These stem cells are precursors to cancer cells in the blood and lymph circulation in cases of leukemia and non-Hodgkin’s lymphoma. These cells naturally express AID as an essential protein involved in their maturation and the perfection of antibodies in response to antigens. It stands to reason that their response to an infection would be over-zealous in the presence of glyphosate, which would cause exuberant phosphorylation of both AID and Nup98, as well as pseudo-phosphorylation of both of these proteins through its introduction of negative charge into the protein by displacing glycine residues. This would lead to a compromised nuclear barrier and infiltration of activated AID proteins into the nucleus where they would induce DNA double strand breaks, the stripping off of methyl groups, and subsequent genetic mutations of critical proteins associated with tumor progression.
As if this weren’t enough, proteins that repair DNA breaks have multiple highly conserved glycine residues that are essential for their proper function. The DNA glycosylase enzyme, OGG1, repairs DNA lesions caused by oxidative damage to the nucleotide guanine, which is the most common defect introduced by DNA exposure to reactive oxygen species. It contains a highly conserved glycine at residue 42. If this residue is replaced by alanine, the protein becomes impaired and DNA breaks accumulate [63, 64, 34].
Another protein, directly involved with repair of double strand breaks, is called MRE11. It has a highly conserved “glycine-arginine domain” which is enriched in glycine residues and contains arginine residues that are methylated to activate the protein [65]. Substitution of glyphosate for any of these glycine residues would be expected to cause unpredictable effects, probably disrupting its ability to repair DNA damage. But hypomethylation is likely also caused by glyphosate, due to its disruption of the supply of methionine and folate, which are essential for methylation pathways.
Conclusion
As I write these words, I have just heard about the victory in the California lawsuit against Monsanto, brought on by Dewayne Lee Johnson, claiming that glyphosate caused his non-Hodgkin’s lymphoma. Johnson was awarded $289 million in a landmark case that hopefully will be the beginning of the end for glyphosate. He won despite the fact that the lawyers could not provide a clear mechanism by which glyphosate might have caused his disease. I believe that the cascade effect of insidious cumulative damage to all the proteins in the body can convincingly lead to conditions such as lymphoma, because of glyphosate’s introduction of oxidative stress, depletion of methylation capacity, and disruption of proteins such as AID and Nup98, as detailed above. AID is prominently expressed in white blood cells, the precursors to the cancer cells that ravaged Lee Johnson’s body.
In the mean time, everyone has a choice at the supermarket. It is fortunate that the United States has a regulatory process that specifies the “certified organic” label, where one strict requirement is to not use glyphosate on the crop. Eating organic can help you and your family sharply reduce the amount of glyphosate you accumulate in your tissues. By buying only certified organic, you become part of a revolution that will force the farmers to switch to organic crops through a market-driven economy. While organic food is more expensive than conventional food, it will save you money down the road on medical bills. And, as more and more people switch to organic, the price will drop due to economies of scale.
We don’t need the government to tell us not to eat toxic food. We can do this all on our own, and if enough of us do, farmers will be able to protect themselves from harm while producing a more wholesome crop that they can be proud of. If you must smoke, you can also purchase only organic cigarettes, and your lungs will thank you for this. So will the children of Argentina who are suffering so much from the toxic exposures they must endure for as long as customers continue to buy cigarettes made from the chemical-laden tobacco their parents currently produce.
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[1] i.e., the probability that this pattern could have occurred by chance is less than 0.00000046.
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