Doses of glyphosate and Roundup that regulators have assumed to be safe also cause fatty liver disease and death of liver tissue.

Report by Claire Robinson and Michelle Perro, MD

At-a-glance

  • Nearly all genetically modified (GM) crops worldwide are engineered to be grown with glyphosate-based herbicides such as Roundup.
  • A 2019 study in rats provides the first definitive proof that glyphosate and Roundup can inhibit the series of biochemical reactions known as the “shikimate pathway” in the gut microbiome (bacterial population) and can cause alterations in the abundance of certain bacteria at regulatory-permitted (assumed safe) levels of exposure. The shikimate pathway is responsible for the synthesis of aromatic amino acids that are vital for the production of proteins, the building blocks of life. Studies in rats are generally accepted to be relevant to humans.
  • The health implications of these changes in the gut microbiome are unclear.
  • The study found that the inhibition of the shikimate pathway from glyphosate and Roundup exposure caused a dramatic increase in shikimic acid and 3-dehydroshikimic acid in the gut, suggesting that these substances could be used as biomarkers of exposure to these pesticides.
  • The study also showed that Roundup, and to a lesser extent glyphosate, damaged the liver and kidneys of the rats. Some of the test doses of Roundup caused an increase in lesions indicative of fatty liver disease and necrosis (death of tissue), confirming and extending previous research.
  • The new study proposes a specific mechanism through which exposure to glyphosate-based herbicides can cause cancer. Rats fed Roundup and glyphosate showed elevated levels of shikimic acid in their gut. Shikimic acid can either protect the body from oxidative stress, or it can act as a cancer promoter. The mechanism of action of glyphosate on the gut microbiome, newly identified in this study, might be of relevance to glyphosate’s ability to cause cancer.
  • It would be clinically useful to conduct surveys of human populations, such as those in the USA who are generally exposed to higher levels of glyphosate herbicides compared to other regions, to see if there is a correlation between levels of glyphosate in urine and levels of shikimic acid and 3-dehyroshikimic acid in feces, blood, and urine.

Between 85-95% of genetically modified (GM) crops worldwide are engineered to be grown with herbicides based on the active ingredient glyphosate.[1] Glyphosate-based herbicides such as Roundup are also widely used with non-GM crops and in municipal weed control programs. Therefore it is not surprising that glyphosate residues are widely found in GM and non-GM foodstuffs, as well as in the urine of the vast majority of individuals tested, demonstrating a constant daily ingestion of this pesticide.[2] The health implications of a daily intake of glyphosate-based herbicides remains controversial – but an increasing number of studies link exposure to serious illnesses such as fatty liver disease and cancer.

A new study in rats raises further concerns about the health risks of both glyphosate and glyphosate-based herbicides (e.g. Roundup). The study provides firm evidence that these pesticides disrupt the gut bacterial populations (microbiome) at doses assumed by regulators to be safe, through a particular metabolic pathway (that is, a series of chemical reactions) found in gut bacteria. The study also shows that glyphosate and glyphosate-based herbicides cause fatty liver disease and death of liver tissue at the same doses.[3]

The study, conducted by an international team of scientists based in London, France, Italy, and the Netherlands, and led by Dr Michael Antoniou of King’s College London, has been posted on the open peer-review site BioRxiv,[3] pending publication in a peer-reviewed journal.

How do glyphosate and glyphosate-based herbicides disrupt the gut microbiome?

Glyphosate herbicides kill plants by inhibiting an enzyme called EPSPS, which is part of a well-known series of biochemical reactions known as the “shikimate pathway”. The shikimate pathway is responsible for the synthesis of certain amino acids that are vital for the production of proteins, which are the building blocks of life. Thus when the synthesis of the amino acids is blocked by glyphosate’s inhibition of EPSPS, the plant dies.

Humans and other animals do not have the shikimate pathway in the cells that make up their bodies, so industry and regulators claim that glyphosate is non-toxic to humans.[4] But many strains of gut bacteria do have the shikimate pathway, leading to debate as to whether Roundup and glyphosate could affect the gut microbiome. Imbalances in gut bacteria have been linked with many diseases, including cancer, diabetes, obesity, and depression.[5]

Scientists had hypothesized that glyphosate herbicides could inhibit the EPSPS enzyme of the shikimate pathway in gut bacteria, leading to imbalance in the microbiome and thence to harmful effects on health. Some have proposed that if glyphosate herbicides do disrupt the gut microbiome, it is most likely to happen via EPSPS inhibition.

However, proof that glyphosate herbicides can inhibit the EPSPS enzyme and the shikimate pathway in gut bacteria had been lacking until now. The new study proves beyond doubt that this does indeed happen.

The study found that Roundup herbicide and its active ingredient glyphosate caused a dramatic increase in the levels of two substances, shikimic acid and 3-dehydroshikimic acid, in the gut. The levels of shikimic acid and 3-dehydroshikimic acid were undetectable in the gut of control animals not exposed to either glyphosate or Roundup. As EPSPS is responsible for converting shikimic acid and 3-dehydroshikimic acid into other compounds, the accumulation of these two substances in the gut of exposed animals shows that the EPSPS enzyme of the shikimic acid pathway was severely inhibited.

Both Roundup and glyphosate affected the microbiome at all dose levels tested, causing alterations in bacterial populations. Some changes in the abundance of certain bacteria (Eggerthella and Homeothermacea) were common to both glyphosate and Roundup treatment groups, whereas Roundup specifically caused an increase in the relative abundance of Shinella zoogleoides.

Microbiome analysis and its clinical significance is a rapidly advancing field, with dysbiosis (an unbalanced microbiome) now known to be of major importance in understanding how many diseases begin and progress.

What was the study design?

For the study, female rats (12 per group) were fed a daily dose of either glyphosate or a Roundup formulation approved in Europe, called MON 52276. Glyphosate and Roundup were administered via drinking water to give a glyphosate daily intake of 0.5 mg, 50 mg and 175 mg/kg body weight per day (mg/kg bw/day), which respectively represent the EU acceptable daily intake (ADI), the EU no-observed adverse effect level (NOAEL), and the US NOAEL.

What did the study reveal?

The study found adverse effects at all doses tested, thus disproving regulators’ assumptions that these levels have no harmful effect. Microbiome effects are not tested for in the standardized tests performed by industry for regulatory purposes.

What does the study add to previous ones?

Some previous studies also reported changes in the gut microbiome of laboratory animals exposed to glyphosate and/or Roundup. However, they did not use the in-depth molecular profiling techniques (multi-omics) used in the latest investigation, so failed to observe the inhibition of the shikimate pathway.[6]

Occurrence of oxidative stress

The researchers also saw other changes in the gut metabolome that indicated oxidative stress, a type of imbalance resulting from excessive chemically reactive oxygen that can lead to mutations in DNA, damage to cells and tissues, and diseases such as cancer. Gut bacteria respond to oxidative stress by producing substances that combat it. Marked increases in these substances (called γ-glutamylglutamine, cysteinylglycine, and valylglycine) were found in the gut of rats fed glyphosate and Roundup, showing that oxidative stress was occurring.

Occurrence of liver damage

The study also revealed that Roundup, and to a lesser extent glyphosate, damaged the liver and kidneys of the rats, even over the relatively short study period of 90 days. Histopathological (microscopic) examination of the liver showed that the study’s two higher doses of Roundup caused a statistically significant and dose-dependent increase in lesions indicative of fatty liver disease and necrosis (death of tissue).

In the glyphosate-only treatment group, there was also an increase in the incidence of the same types of liver damage but this did not reach statistical significance. In contrast, none of the control animals showed the same liver effects, so the changes in the glyphosate-fed animals may be biologically significant. As the authors state in their paper, the lack of statistical significance may have been because the numbers of animals were too low and the exposure duration too short. If the study were extended by another month or two, this may result in statistical significance for glyphosate as well as Roundup effects.

What does the study mean for people eating a glyphosate-contaminated diet?

The levels of glyphosate tested in this experiment are higher than those typically ingested daily by people, so we do not know if the same microbiome effects would be seen at typical dietary intake levels. However, a previous study also led by Dr. Antoniou, in which rats were fed a very low dose of Roundup that is well below what people could be exposed to through diet, found that even these low levels caused fatty liver disease.[7] Dr Antoniou estimates that the level of Roundup found to cause fatty liver disease in rats is at least a thousand times lower than would be ingested daily in the typical American diet. Thus, either long term exposure to a very low dose of Roundup or higher doses for a shorter time lead to the same liver toxicity. So it is likely that fatty liver disease is a concern from a typical dietary intake of glyphosate.

Medical implications of fatty liver disease findings

Americans are facing an epidemic of non-alcoholic fatty liver disease (NAFLD) – that is, fatty liver disease that is not caused by alcohol abuse. NAFLD is estimated to affect 30-40% of adult Americans.[8] One of the main issues with this disorder is that it is often clinically silent, yet it can develop into more advanced liver disease such as steatohepatitis and cirrhosis. Because it can be asymptomatic, clinicians are not routinely diagnosing this disorder. In the early stages, it is reversible. But as the disease progresses, it can cause irreversible liver damage. Worldwide, NAFLD is the major cause of chronic liver disease.[9] NAFLD and more advanced liver issues are also affecting children, especially in cases of obesity.[10]

Despite the findings of the link between glyphosate and Roundup to liver toxicity in the studies featured above[3,6], this specific causal factor is not being reported in the medical literature. Also, most medical practitioners are not exploring the link clinically. However, intoxication from “organic phosphates” (a class of chemicals to which glyphosate technically belongs) is reported as a cause of NAFLD.[11] The new study confirms that exposure to glyphosate and glyphosate-based herbicides should be specifically recognized as a hitherto overlooked risk factor for NAFLD.

Acceptable daily intake (ADI) for glyphosate is set too high

In summary, the observations of kidney and especially liver damage in the new study provide further evidence that the ADI for glyphosate currently set by regulators is likely too high and needs to be lowered to protect the public. Furthermore, the study confirms the findings of previous research[12] that commercial formulations such as Roundup

are more toxic than glyphosate alone, due to the adjuvants (added ingredients) present. This increase in toxicity needs to be taken into account by regulators when assessing the health risks of pesticides. Currently it is largely ignored.

A new mechanism for glyphosate-cancer link?

In 2015 the International Agency for Research on Cancer (IARC) classified glyphosate as a probable carcinogen. IARC identified oxidative stress, mentioned above, and genotoxicity (damage to DNA) as possible mechanisms.

The new study by Dr Antoniou and colleagues proposes a novel mechanism through which exposure to glyphosate-based herbicides can cause cancer. Rats fed Roundup and glyphosate showed elevated levels of shikimic acid in their gut. Shikimic acid can have different biological effects, including protecting the body from oxidative stress. But it has also been proposed as a cancer promoter, and a recent study found that shikimic acid can stimulate proliferation of human breast cancer cells.[13] Therefore the novel mechanism of action of glyphosate on the gut microbiome identified in the study by Dr Antoniou and colleagues might be of relevance to glyphosate’s ability to cause cancer.

First biomarker of glyphosate exposure has been identified

Crucially, this study provides the first definitive proof that glyphosate and Roundup can inhibit the shikimate pathway in the gut microbiome and can cause alterations in the abundance of certain bacteria at regulatory-permitted levels of exposure. The health implications of these changes in the gut microbiome are currently unclear. Longer term studies with larger groups of animals are needed to clarify any risks. Nevertheless, the finding that glyphosate and Roundup cause a dramatic increase in shikimic acid and 3-dehydroshikimic acid in the gut of exposed rats suggests that these two substances could be used as readily measurable biomarkers of exposure to these pesticides.

Thus, the study suggests that surveys of human populations should be conducted as matter of urgency to see if there is a correlation between levels of glyphosate in urine and levels of shikimic acid and 3-dehydroshikimic acid in feces, urine, and blood. This is especially important in regions such as the USA, where exposures are generally much higher than elsewhere (e.g. Europe). This will potentially provide insight into health risks such as NAFLD as outlined above. In the meantime, avoidance of these chemicals by favoring an organic diet and avoiding their use in the home environment is a prescription for better health.

References

1. USDA (2017). Recent trends in GE adoption. https://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-us/recent-trends-in-ge-adoption.aspx

2. Gillezeau C et al (2019) The evidence of human exposure to glyphosate: a review. Environmental Health, 18: 2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322310/

3. Mesnage R et al (2019). Shotgun metagenomics and metabolomics reveal glyphosate alters the gut microbiome of Sprague-Dawley rats by inhibiting the shikimate pathway. BioRxiv. doi: https://doi.org/10.1101/870105

4. See the Final addendum to the Renewal Assessment Report on glyphosate (https://echa.europa.eu/documents/10162/13626/renewal_assessment_report_addenda_en.pdf) (October 2015), p23. Rapporteur Member State Germany and co-rapporteur Member State Slovakia state, based on industry claims, “Action at the shikimic acid pathway is unique to glyphosate and the absence of this pathway in animals is an important factor of its low vertebrate toxicity.”

5. Zmora N et al (2019) You are what you eat: diet, health and the gut microbiota. Nat Rev Gastroenterol Hepatol. 16: 35-56. https://www.nature.com/articles/s41575-018-0061-2?draft=collection

6. Tsiaoussis J et al (2019). Effects of single and combined toxic exposures on the gut microbiome: Current knowledge and future directions. Toxicol Lett. 12: 72-79. https://www.sciencedirect.com/science/article/abs/pii/S0378427419300992

7. Mesnage R et al (2017) Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide. Sci Rep. 7: 39328. https://www.nature.com/articles/srep39328

8. Spengler EK, Loomba R (2015). Recommendations for diagnosis, referral for liver biopsy, and treatment of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Mayo Clinic Proceedings 90(9):1233–1246..
9. Kneeman JM et al (2012) Secondary causes of nonalcoholic fatty liver disease. Therap Adv Gastroenterol. 5: 199–207. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342568/

10. Temple JL et al (2016) A guide to non-alcoholic fatty liver disease in childhood and adolescence. Int J Mol Sci. 17: 947. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926480/

11. Singer C et al (2014). Non-alcoholic fatty liver disease in children. Curr Health Sci J. 40: 170–176. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340436/

12. Mesnage R, Antoniou MN (2018) Ignoring adjuvant toxicity falsifies the Safety profile of commercial pesticides. Front Public Health, 5: 361. https://www.frontiersin.org/articles/10.3389/fpubh.2017.00361/full

13. Ma X and Ning S (2019) Shikimic acid promotes estrogen receptor(ER)-positive breast cancer cells proliferation via activation of NF-κB signaling. Toxicol Lett. 312: 65-71. https://www.sciencedirect.com/science/article/abs/pii/S0378427419301201?via%3Dihub

Claire Robinson is editor, GMWatch.org. Michelle Perro, MD is a California-based pediatrician and Executive Director of GMO Science.