Comparative cyto- and genotoxicity assessment of glyphosate and glyphosate-based herbicides in human peripheral white blood cells

https://doi.org/10.1016/j.envres.2019.108851Get rights and content

Highlights

  • Glyphosate alone did not induce significant cytotoxicity and genotoxicity.

  • Glyphosate based herbicide formulations substantially decreased cell viability and induced DNA damage.

  • The observed DNA damage can be due to the cell death exerted by the formulations.

  • Metabolic activation increased the genotoxicity of the Glyfos formulation.

Abstract

Glyphosate is the most heavily applied active compound of agricultural pesticides. It is solely used in more than 750 different glyphosate-based herbicide formulations (GBHs) that also contain other substances, mostly presumed as inert by regulatory agencies. The toxicity of formulations is currently assessed substance by substance, neglecting possible combined effects in mixtures and many of the findings regarding the toxic effects of glyphosate and GBHs to human cells are inconsistent. This is the first study to investigate and compare the cyto- and genotoxic potential of the active ingredient glyphosate and GBHs in human mononuclear white blood (HMWB) cells.

HMWB cells were treated for 4 h at 37 °C with increasing concentrations (1–1000 μM) of glyphosate alone and in three GBHs (Roundup Mega, Fozat 480 and Glyfos) to test cytotoxic effect with fluorescent colabelling and genotoxic effect with comet assay. In addition, each concentration was tested with and without metabolic activation using human liver S9 fraction.

We found that glyphosate alone does not induce significant cytotoxicity and genotoxicity over the tested concentration range. Contrarily, GBHs induced statistically significant cell death from 250 μM (Roundup Mega and Glyfos) and 500 μM (Fozat 480), as well as statistically significant increase of DNA damage from 500 μM (Roundup Mega and Glyfos) and 750 μM (Fozat 480); however, the latter observation may not be explained by direct DNA injuries, rather due to the high level of cell death (>70%) exerted by the formulations. Metabolic activation significantly increased the DNA damage levels induced by Glyfos, but not of the other GBHs and of glyphosate.

The differences observed in the toxic pattern of formulations and the active principle may be attributed to the higher cytotoxic activity of other ingredients in the formulations or to the interaction of them with the active ingredient glyphosate. Hence, further investigation of formulations is crucial for assessing the true health risks of occupational and environmental exposures.

Introduction

Glyphosate, as an expectant herbicide, was patented by the American Monsanto Company in 1971. The developed product was called “Roundup”. Glyphosate has become one of the market-leading active ingredients in today's plant protection products. This situation is intensified with the spread of glyphosate-tolerant genetically modified plants beyond Europe (Perry et al., 2016). The estimated usage in 1994 was 56 thousand tons, increasing almost 15-fold, to roughly 826 thousand tons by 2014 (Benbrook, 2016). Glyphosate is quite persistent in soil due to the presence of a chemically inert C–P bond in its chemical structure (Chekan et al., 2016). The widespread and large-scale application of glyphosate has led to its accumulation in the environment and in edible products (Bai and Ogbourne, 2016). The consequent human exposure, however, has not been estimated sufficiently (Gillezeau et al., 2019).

In recent years, genotoxicity and carcinogenicity of glyphosate has been under vigorous – and so far inconclusive – review and debate by scientific and regulatory agencies. The International Agency for Research on Cancer (IARC) classified glyphosate as “probably carcinogenic to humans” (Group 2A) in 2015 based on limited epidemiological evidence in humans (for non-Hodgkin lymphoma), sufficient evidence in experimental animals with strong evidence for two carcinogenic mechanism, genotoxicity and oxidative stress (IARC, 2015; Portier et al., 2016). In the same year, the European Food Safety Authority (EFSA) concluded that “glyphosate is unlikely to be genotoxic (i.e. damaging to DNA) or to pose a carcinogenic threat to humans” because its experts did not find evidence from neither epidemiological nor animal studies on the causality between exposure to glyphosate and the development of cancer in humans (EFSA, 2015a). However, in agreement with the IARC evaluation, EFSA has confirmed that glyphosate induces oxidative stress, nevertheless emphasized that the ability of inducing oxidative stress alone is not sufficient to classify glyphosate as carcinogenic (Portier et al., 2016). EFSA also criticized the assessment from IARC for not including some studies in the evaluation process which was one of the reasons for reaching different conclusions. In 2016, the U.S. Environmental Protection Agency (US EPA) also declared that “glyphosate is not likely to be carcinogenic to humans” as a result of a comprehensive review of dietary, residential/non-occupational, aggregate, and occupational human exposures, as well as an in-depth review of the glyphosate cancer database, including data from epidemiological, animal carcinogenicity, and genotoxicity studies (US EPA, 2016). The controversy over the classification of glyphosate's genotoxicity and carcinogenicity is likely to be explained by the substantially different evaluation methodologies of the authorities and makes it difficult to bring us closer to a final conclusion on the DNA damaging or cancer-causing ability of this herbicide.

Glyphosate is often marketed in aqueous solution as isopropylamine, dimethylamine, trimesium, and potassium salts to enhance its solubility. Besides, other ingredients labelled as “inerts”, “adjuvants“ or “co-formulants” are also added to the final formulations in order to improve solution, stability, absorption and thus the herbicidal action of glyphosate (Cox and Surgan, 2006). The identity of supposedly “inert” adjuvants is mostly kept confidential by the manufacturing companies; the composition of glyphosate-based herbicides (GBHs) is usually unknown and the knowledge about the hazards posed by the combined effects of different ingredients in the GBHs remains limited. Although adjuvants added to GBHs vary among countries and manufacturers, most marketed GBHs contain surfactants, such as polyethoxylated tallow amines (POEAs). It is recognized that POEAs or GBHs containing POEAs are more toxic in acute and chronic toxicity tests than glyphosate alone, therefore GBHs containing these surfactants are gradually being withdrawn from the market in Europe and substituted by a new generation of surfactants. The acute toxicity of newly introduced GBHs is considered to be lower than that of POEAs (Mesnage et al., 2019); nevertheless, the chronic adverse effects potentially posed by the exposure to commercially available GBHs with still highly variable composition should be assessed in a more systematic way to allow for effective protection of human health.

The genotoxic and related effects of glyphosate and GBHs have been extensively studied in human cells in vitro by several researchers using a variety of techniques over the past few decades. Glyphosate as an active principle induced significant DNA injury (as measured by the comet assay) in Hep-2 cells from the concentration of 3 mM (Manas et al., 2009b), in fibrosarcoma (HT1080) cells from the concentration of 4 mM (Monroy et al., 2005), in buccal epithelial carcinoma cells from the concentration of 20 μg/ml (118.3 μM) (Koller et al., 2012), and in lymphocytes from the concentrations of 3.5 μg/ml (20.7 μM) (Mladinic et al., 2009a) and 0.12 μg/ml (0.7 μM) (Alvarez-Moya et al., 2014). A more recent research exposing Burkitt's lymphoma (Raji) cells to glyphosate in the concentration range of 0.1 μM–15 mM suggested that glyphosate was lethal to cells at concentrations above 10 mM but has no cytotoxic effect at physiologically relevant (≤100 μM) concentrations. Although concentrations of 1 mM and 5 mM induced statistically significant DNA damage of Raji cells following 30–60 min of treatment, the cells show a slowed recovery from initial damage and cell viability is unaffected after 2 h (Townsend et al., 2017). Kwiatkowska and colleagues investigated the glyphosate-induced DNA damage and DNA methylation in peripheral blood mononuclear cells (PBMCs) and found increased DNA damage at concentrations of 0.5–10 mM as well as increased p53 gene promoter methylation following 0.25 mM and 0.5 mM glyphosate treatment (Kwiatkowska et al., 2017). Chromosomal damage in lymphocytes was noted as a result at ≥1000 μg/ml (sister-chromatid exchange) (Bolognesi et al., 1997) but not at ≤6 mM (chromosomal aberrations) (Manas et al., 2009b) and ≤580 μg/ml (3.4 mM) (micronucleus formation) (Mladinic et al., 2009b) glyphosate exposure. GBHs induced DNA strand breaks of HepG2 cells from 5 ppm (29.6 μM) (Gasnier et al., 2009) and of buccal epithelial carcinoma cells from 20 μg/ml (118.3 μM) (Koller et al., 2012) measured by the comet assay. Recently, Woźniak and colleagues assessed the cyto- and genotoxic effects of a GBH (Roundup 360 PLUS) and its active ingredient glyphosate by means of the comet assay in the concentration range from 1 to 1000 μM in PBMCs with the conclusion that Roundup 360 PLUS caused damage to DNA from 5 μM, while glyphosate induced DNA lesions from the concentration of 500 μM (Wozniak et al., 2018). Sister-chromatid exchanges in lymphocytes were also reported as a results of ≥250 μg/ml (1.5 mM) (Vigfusson and Vyse, 1980) and 100 μg/ml (0.6 mM) (Bolognesi et al., 1997) GBH exposure. The European Commission, being aware of these findings, has recently re-evaluated the exposure limit values for glyphosate, and has kept its Acceptable Daily Intake (ADI) at 0.5 mg/kg bw per day (European Comission, 2017).

Keeping in mind the knowledge gaps on the possible complex interactions between ingredients in GBHs, and also the lack of studies comparing the effects of glyphosate with its formulated products, the present research aimed to first compare the cyto- and genotoxic effects of three marketed GBHs with different composition to the declared active ingredient glyphosate as well as to each other, in order to clarify whether the adjuvant content of the formulations may influence the toxic effects.

Section snippets

Chemicals

Analytical-grade glyphosate (N-(phosphonomethyl) glycine, CAS No: 1071-83-6) was purchased from VWR International Kft (Debrecen, Hungary) and samples of three GBHs, i.e.

  • Roundup Mega containing 551 g/L or 42% (w/w) potassium salt of glyphosate (CAS No: 70901-12-1; equivalent to 450 g/L glyphosate) and 7% (w/w) ethoxylated etheralkylamine (CAS No: 68478-96-6);

  • Fozat 480 containing 480 g/L or 41% (w/w) isopropylammonium salt of glyphosate (CAS No: 38641-94-0; equivalent to 360 g/L glyphosate) and

Cytotoxicity

The viability of HMWB cells treated with glyphosate alone was found to be over 86% in the absence and presence of S9 over the entire concentration range (Fig. 1). Regardless of metabolic activation, GBHs induced a significant decrease in the proportion of living cells from 250 μM of Round Mega and Glyfos whereas from 500 μM of Fozat 480.

DNA damage

Exposure of HMWB cells to glyphosate in the 0–1000 μM concentration range did not result in dose-dependent increase of DNA damage measured by the comet assay

Discussion

The use of glyphosate containing products both for agricultural and residential purposes continues to rise since their first introduction to the market in 1974 (Benbrook, 2016). Today, uncountable GBHs are registered in more than 130 countries worldwide (Bayer, 2019). The unrestricted application of GBHs in the past few decades has resulted in the accumulation of glyphosate residues in environmental media (e.g. water, soil, food) (Bai and Ogbourne, 2016) exposing – and also impairing the health

Conclusion

This is the first study that compared toxic effects of various glyphosate-based herbicide formulations to each other and with the declared active ingredient glyphosate in isolated human mononuclear white blood cells. GBHs caused much stronger cytotoxic effect on HMWB cells in comparison to glyphosate that may be attributed to the effect of various surfactants added to the formulations or their interaction with the active ingredient glyphosate and/or with other components of GBHs. Therefore, the

Funding sources and ethical statement

The study was carried out with the support of the Diagnosis, Monitoring and Prevention of Exposure-Related Noncommunicable Diseases (DiMoPEx) COST Action project (CA15129) and the Stipendium Hungaricum Programme of the Tempus Public Foundation, and was approved by the Hungarian Ethical Committee for Medical Research (document 147-5/2019/EÜIG).

Declaration of competing interest

None.

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