Glyphosate and the Gut Microbiome: Another Bad Argument Annihilated

Introduction:

Glyphosate is a broad spectrum herbicide that was first introduced by the Monsanto company in the 1970s under the brand name Roundup. The already popular product grew even more popular among farmers upon the introduction of various commodity crops which were genetically engineered to resist the herbicide while it killed the surrounding weeds with which the crops would otherwise compete for water and nutrients. Glyphosate went off patent back in the year 2000, and since then many manufacturers have cashed in on its popularity [1]. Although it is of unusually low toxicity, glyphosate receives a level of scrutiny and vehemence of criticism that is disproportionate to its actual established risks [2],[3],[4]. This is attributable in part to its ubiquity in modern conventional farming, but it’s likely even more attributable to its association with Monsanto, against which a large and well-organized counter-movement has emerged [5].

Consequently, many different arguments have been formulated and circulated among this counter-movement and beyond. The purpose of this piece is address one of those arguments in particular. More specifically, on numerous occasions I have heard glyphosate critics argue that glyphosate should be opposed because it might alter the microbiome in humans. In a post on his facebook page, The Mad Virologist discussed a recently published study on the effects of glyphosate on gut microorganisms, and inspired me to unpack the microbiome argument against glyphosate and explain what’s wrong with it.

Background

Glyphosate binds to and inhibits the action of an enzyme known as EPSP synthase, which plants need in order to make three important aromatic amino acids: phenylalanine, tyrosine, and tryptophan via what’s known as the shikimic acid pathway, which occurs in plants, bacteria, fungi, algae and some protozoan parasites [6],[7]

Image c/o Zucko et al 2010 [37].

Glyphosate does this by acting as what’s called an uncompetitive inhibitor. That means that it can only bind to the enzyme-substrate complex – the substrate being shikimate-3-phosphate in this case – and cannot bind the enzyme when the substrate is unbound [8],[9]. Upon binding to the enzyme-substrate complex, glyphosate prevents the complex from forming its product, 5-enopyruvylshikimate-3-phosphate (EPSP). Normally the complex would form EPSP by reacting with another molecule called phosphoenol pyruvate (PEP), but sufficient concentrations of glyphosate reduces the number of units of the enzyme-substrate complex available to form their product. The shikimic acid pathway doesn’t exist in us. Humans and other mammals, for example, can’t make those amino acids at all to begin with, so we get them directly from our food. Plants need those amino acids in order to grow and to make proteins, so if they are unable to synthesize them, they can’t grow, and therefore they die.

Additionally, mammals such as ourselves have lived in co-evolutionary association with myriad microorganisms whose aggregate is referred to as the microbiome. The roles of the microbiome in human health and the effects resulting from changes in its composition are active areas of scientific investigation [33].

Image: retrieved from UmassMed.edu

However, our collective knowledge of the relationship between the microbiome and human health is still in its infancy. Consequently, the topic is an easy target for exploitation by proponents of pseudoscience who would leverage it as a promotional tool for their own agendas, and/or extrapolate to claims which overstep what the current body of scientific literature actually supports [34],[35],[36].

The Gut Microbiome Argument Against Glyphosate

Keeping that in mind, the reasoning underlying the gut microbiome argument against glyphosate can be summarized as follows:

1. The makeup of a person’s gut microbiome is relevant to human health in ways which are only recently starting to be elucidated.

2. Bacteria possess the shikimic acid pathway and can use it to synthesize aromatic amino acids.

3. Glyphosate inhibits a key enzyme used in the shikimic acid pathway.

4. Therefore, glyphosate might be altering people’s microbiome in detrimental ways.

Simple enough?

Good.

Is This Argument Biologically Plausible?

However, the problem is that this argument flies in the face of one of the basic principles of microbiology: that microbes grow in the presence of abundant nutrients. As I’ve explained on several occasions when this has come up in my facebook comment sections, bacteria shouldn’t need to synthesize aromatic amino acids when they are literally bathing in them in the gut, therefore this argument against glyphosate is grasping at straws and not plausible. A recent study tested this more formally (in vivo) [10].

How did I know in advance that this was extremely unlikely to be a major issue before this research? It was because I knew that gut bacteria live in… Wait for it… the GUT!!! Where aromatic amino acids are abundant. That means they will continue to grow if the final product of a given biosynthetic pathway is supplemented to them – which is what we are doing by supplementing them with aromatic amino acids through the food we eat – even in the presence of something that inhibits that specific pathway.

This principle is the basis for experiments that allow scientists to functionally characterize which genes’ enzymes act on which substrates in a given biochemical pathway (called functional complementation analysis), and has been in common use for the last century or so as a method for ascertaining the specific steps of varous metabolic pathways, and/or the genes which code for the enzymes which catalyze each reaction [11],[12],[13],[14].

For an example of how this immersion technique has been used, consider the elucidation of the arginine synthesis pathway in N. crassa fungi by Srb et al 1944 [15]. The authors used radiation to induce mutations in the cells, and then performed a genetic screen to isolate those with mutations relevant to the arginine synthesis pathway. This was accomplished by growing colonies of mutants in a medium which included arginine, and then in one which lacked arginine. Cells which grew in an arginine-containing medium but not without it were deemed incapable of synthesizing their own arginine, and were subsequently grown under four different conditions:

  1. In a medium lacking ornithine, citrulline, and arginine.
  2. The same medium as 1, except supplemented with ornithine only (no citrulline or arginine).
  3. The same medium as 1, except supplemented with citrulline only (no ornithine or arginine).
  4. The same medium as 1, except supplemented with arginine only (no ornithine or citrulline).

The results were as follows: 

Image c/o Biological Science 4th ed [16].

This implied that there were three types of mutants. Some had mutations preventing them from producing functional copies of the enzyme responsible for catalyzing the reaction to produce ornithine from its precursor, some for the enzyme responsible for catalyzing the reaction to produce citrulline from ornithine, and some for the enzyme responsible for catalyzing the reaction to produce arginine from citrulline. This is a simple textbook example, but the point here is that supplementing cells with the end product of a metabolic pathway negates the need for the cell to synthesize it itself through that pathway. This particular example used bread mold, but the same principle applies to bacteria.

Moreover, the Shikimic acid pathway is also metabolically expensive, so it’s not likely that the bacteria are actively using this pathway in the presence of abundant aromatic amino acids (i.e. phenylalanine, tyrosine, and tryptophan), especially when they are in competition with other microbes [17]. So, unless the person (the host) is literally starving to death, then it is far more likely gut bacteria are taking them in the easy way by just absorbing them from their environment.

If the host actually is literally starving to death or suffering from severe malnutrition, then they have far bigger and more urgent problems to worry about than their gut microbiome. Starvation and severe malnutrition themselves cause harm [18]. Consequently, parsing out and identifying harm to the host attributable to malnutrition and distinguishing it from harm to the host due to glyphosate-induced alterations to the microbiome would be problematic, especially considering that any hypothetical problems caused by the latter would be avoided by mitigating or preventing the former.

None of this is new or controversial, which is part of the reason why researchers never bothered with a full blown in vivo experiment until recently on the effects of glyphosate on the microbiome. It is also the reason why the results of the recent study should not be surprising.

Earlier Studies

Earlier studies on glyphosate’s effects on bacteria were either full of methdological problems, and/or not setup in such a way as to test the question of how it affects the microbiome in vivo, where aromatic amino acids are abundant. I’ll start with the lowest hanging fruit before dealing with more credible studies, for which the strengths and limitations are more subtle.

Samsel and Seneff

Computer scientist Stephanie Seneff is an anti-vaccine, anti-GMO, and anti-glyphosate activist who claims that GMO foods cause concussions and suggests that glyphosate in vaccines have contributed to school shootings and the Boston Bombing [19],[20]. Seriously, you can’t even make this shit up, but I digress. She and her co-author, a retired consultant by the name of Anthony Samsel, published a series of papers in a predatory pay-to-play journal (entropy) implicating glyphosate in a whole host of conditions (including celiac disease, MS, Parkinson’s, cancer, and autism), many of which involved convoluted non-sequitur arguments based on glyphosate’s alleged effects on the microbiome [21],[22]. Eric from Skeptoid has meticulously broken down the plethora of flaws and red flags in that paper, which would take way too long to reiterate here [23]. To get an idea of just how terrible that paper is, Thoughtscapism points out that it has actually been used as an example of how to spot bogus science journals: a little factoid I found far too hilarious to omit [24],[22].

Other Earlier Studies

This 1986 study showed significant growth inhibition, but only at glyphosate concentrations on the order of a millimolar or more, which is thousands of times the amounts realistically occurring in the gut from food [25]. To put this into perspective, legumes are the food crop with the highest allowed pesticide residue limit in the US (5.0 ppm) [26]. 5.0 ppm = 5.0 mg of glyph/kg of legumes, and glyphosate has a molar mass of 169.07 g/mol.

So, if we estimate that an average full stomach is roughly 1 L in volume while assuming homogeneous distribution, then we get that millimolar concentrations in the gut would involve (1 L)*(10^-3 mol of glyph/L)*(169.07 g of glyph/mol of glyph)*(10^3 mg/g) = 169.07 mg of glyphosate.

If we then assume the maximum permissible amount of glyphosate on the food crop with the highest maximum allowable glyphosate residue limit, we can calculate that millimolar concentrations in the gut by dividing the mass of glyphosate required to achieve millimolar concentrations by the mass of glyphosate per unit of mass of legumes at the maximum allowable residue limits.

When we do that, we find that it would require ingesting about 33.8 kg of legumes (or about 74.5 lbs).

i.e. (169.07 mg glyph)/(5.0 mg of glyph/kg of legumes) = 33.8 kg of legumes.

This of course assumes 100% absorption, which, as neuroscientist/geneticist/toxicologist, Alison Bernstein (aka Mommy, PhD) explains here, is actually not the case. So, the actual amount of legumes required to reach such concentrations in the gut may actually be many times higher than my sample estimate.

As Thoughtscapism points out, even at those extreme doses, the bacteria were not killed, but rather grew at a slower rate, and even that effect was partially mitigated when the researchers supplemented the bacteria with aromatic amino acids to simulate conditions likely to occur in the gut [27]. This 2010 study suffered from similar limitations [28].

Similarly, the following study showed a significant reduction in colony forming units (CFU) in vitro, but the concentrations were again on the order of a millimolar (and up to 29.5 mM), and no aromatic amino acids were supplemented to any of the test groups, which again means that it cannot be extrapolated to the gut microbiome where aromatic amino acids are abundant [29].

The Danish Study

In the new study, researchers from Denmark mapped the microbiome of Sprague Dawley rats using next generation sequencing techniques both before and after exposure both to high doses glyphosate and a commercial glyphosate formulation [10]. The researchers found that even doses 50 times that European Acceptable Daily Intake value (ADI = 0.5 mg/kg of body mass) had limited effects on microbiome composition over the course of two weeks, and that glyphosate’s effects on prototrophic bacteria growth was highly dependent on the availability of aromatic amino acids in the intestinal environment. If you are thinking that two weeks isn’t very long, you have to consider the fact that the average generational time for bacteria is roughly on the order of about 20-30 minutes (or often even less). That means that two weeks represents something on the order of (2 wks)*(7 days/wk)*(24 hrs/day)*(2-3 generations/hr) = 672–1,008 generations. Given the life expectancy of Sprague Dawley rats relative to humans, this duration is also comparable to roughly a year and a half in the life of a human [30].

What this means is that anyone continuing to promote the wrongheaded argument that glyphosate can affect health by altering the composition of the microbiome will have to hypothesize a completely new mechanism by which this is supposed to occur (preferably a biologically plausible one). This is because the reasoning behind this argument is based on the premise that glyphosate-induced inhibition of the shikimic acid pathway in gut microorganisms should prevent them from growing due to their (wrongly) assumed dependence on it for the synthesis of aromatic amino acids. This hypothesis predicts that hundreds of generations of bacteria should not be permitted to grow normally if this effect is occurring to any meaningful degree. The evidence falsifies this prediction.

Conclusion

The claim that glyphosate harms human health via disruption of the microbiome was never a biologically plausible one, because it only makes sense when the system is not being viewed as a whole. Ironically, glyphosate and GE food opponents like to say that they take a holistic approach, but this is not a holistic argument, because it ignores the environment in which the microbiome exists.

We know that organisms don’t bother synthesizing compounds they can already get from their environment. Knocking out one step of a biochemical pathway and growing microorganisms on different media with various substrates is a tried and true classical method for identifying which substrates are involved in a given pathway and/or the enzymes which catalyze their reactions. We also know that the human gut contains abundant aromatic amino acids alleviating the need for resident microorganisms to synthesize them. Running out of them is not a concern because they are replenished multiple times per day. The exception to this would be cases of starvation or malnutrition, in which case malnutrition would be the problem to address: not glyphosate. Despite this, in vivo research has been done, and reaffirms exactly what theoretical predictions would imply. Gut microorganisms grew and replicated for hundreds of generations, thus contradicting the predictions of the hypothesis under discussion.

In order to continue to argue that glyphosate had some other negative effect on the microbiome which would be undetectable within the first several hundred or more generations, a contrarian would have to either postulate a different mechanism by which this could be rendered into a testable scientific hypothesis, or appeal to vague and unspecified unknowns.

In the former case, this would constitute an abandonment of the original argument in place of a new hypothesis leading to predictions distinct from those of the hypothesis under discussion. Essentially, this would mean conceding (either explicitly or implicitly) that the original claim was false (or at least not supported), and then moving the goalpost to a new claim based on a different mechanism.

In the latter case, such vague and half-baked speculation could be applied just as easily to virtually anything. It makes no specific postulates and thus makes no testable predictions, and is therefore unscientific. It is what we sometimes refer to as “not even wrong” [31].

– Cred Hulk

For more on glyphosate and common myths about it, Thoughtscapism has put together the most comprehensive piece I’ve ever seen on the subject for a general audience [32].

References

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[2] (2017). Www3.epa.gov. Retrieved 10 December 2017, from https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-103601_20-Feb-02_a.pdf

[3] Hulk, C. (2015). Glyphosate toxicity: Looking past the hyperbole, and sorting through the facts. By Credible HulkThe Credible Hulk. Retrieved 10 December 2017, from http://www.crediblehulk.org/index.php/2015/06/02/glyphosate-toxicity-looking-past-the-hyperbole-and-sorting-through-the-facts-by-credible-hulk/

[4] Scientific evidence that Roundup is dangerous has been mounting.. (2017). Greenpeace International. Retrieved 10 December 2017, from http://act.greenpeace.org/ea-action/action?ea.client.id=1844&ea.campaign.id=37624

[5] Millions march against GM crops. (2013). the Guardian. Retrieved 10 December 2017, from https://www.theguardian.com/environment/2013/may/26/millions-march-against-monsanto?INTCMP=SRCH

[6] Glyphosate | Glyphosate: mechanism of action. (2017). Glyphosate.eu. Retrieved 10 December 2017, from http://www.glyphosate.eu/glyphosate-mechanism-action

[7] Starcevic, A., Akthar, S., Dunlap, W. C., Shick, J. M., Hranueli, D., Cullum, J., & Long, P. F. (2008). Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins. Proceedings of the National Academy of Sciences105(7), 2533-2537.

[8] Sammons, R. D., Gruys, K. J., Anderson, K. S., Johnson, K. A., & Sikorski, J. A. (1995). Reevaluating glyphosate as a transition-state inhibitor of EPSP synthase: Identification of an EPSP synthase. cntdot. EPSP. cntdot. glyphosate ternary complex. Biochemistry34(19), 6433-6440.

[9] Alibhai, M. F., & Stallings, W. C. (2001). Closing down on glyphosate inhibition—with a new structure for drug discovery. Proceedings of the National Academy of Sciences98(6), 2944-2946.

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[13] Smits, T. H., Balada, S. B., Witholt, B., & van Beilen, J. B. (2002). Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria. Journal of bacteriology184(6), 1733-1742.

[14] Salcedo, E., Cortese, J. F., Plowe, C. V., Sims, P. F., & Hyde, J. E. (2001). A bifunctional dihydrofolate synthetase–folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria. Molecular and biochemical parasitology112(2), 239-252.

[15] Srb, A., & Horowitz, N. H. (1944). The ornithine cycle in Neurospora and its genetic control. Journal of Biological Chemistry154(1), 129-139.

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[20] Who is Stephanie Seneff?. (2017). VAXOPEDIA. Retrieved 10 December 2017, from https://vaxopedia.org/2017/07/28/who-is-stephanie-seneff/

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[26] (2017). Gpo.gov. Retrieved 10 December 2017, from https://www.gpo.gov/fdsys/pkg/FR-2013-05-01/pdf/2013-10316.pdf

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[29] Shehata, A. A., Schrödl, W., Aldin, A. A., Hafez, H. M., & Krüger, M. (2013). The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. Current microbiology66(4), 350-358.

[30] Andreollo, N. A., Santos, E. F. D., Araújo, M. R., & Lopes, L. R. (2012). Rat’s age versus human’s age: what is the relationship?. ABCD. Arquivos Brasileiros de Cirurgia Digestiva (São Paulo)25(1), 49-51.

[31] Burkeman, O. (2005). Briefing: Not even wrongthe Guardian. Retrieved 10 December 2017, from https://www.theguardian.com/science/2005/sep/19/ideas.g2

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One Comment

  1. Great article. Thanks for all the work you put in.
    May I suggest a topic for another article?
    Another major problem with the ‘glyphosate kills gut bacteria’ argument is that it totally ignores the mutations that occur spontaneously in the bacterial population. Even if glyphosate were inhibitory, and gut bacteria were unable to take up the aromatic amino acids from their environment, the presence of glyphosate would select for those bacteria that had a workaround for the problem. It could be through alteration in the shikimate pathway, a new ability to transport in the amino acids, or even the ability to break down glyphosate. After all, glyphosate-tolerant crops were developed using bacterial genes that had mutated and been selected for in the presence of glyphosate.

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