ISIS Report 28/03/12

Glyphosate Toxic to Mouth Cells & Damages DNA, Roundup Much Worse

Further evidence of genotoxic and cytotoxic effects – a prelude to cancer, birth defects and reproductive problems Dr Eva Sirinathsinghji

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New research finds that glyphosate causes cell and DNA damage to epithelial cells derived from the inside of the mouth and throat [1]. It raises concerns over the safety of inhaling glyphosate, one of the most common ways in which people are exposed to the herbicide.

Siegfried Knasmueller and his colleagues the Medical University of Vienna, Austria, found that Monsanto’s formulated version of glyphosate called Roundup Ultra Max caused cellular damage and DNA damage including chromosomal abnormalities and ultimately killed the cells at higher concentrations. Importantly, DNA damage occurred at concentrations below those required to induce cell damage, suggesting that the DNA damage was caused directly by glyphosate instead of being an indirect result of cell toxicity.

These are not the first findings of glyphosate-based herbicides’ cytotoxic and genotoxic effects. Numerous independent research teams have been documenting the hazards of glyphosate exposure over the last few years with in vivo, in vitro and clinical studies.

DNA damage was observed in blood samples from exposed residents in Argentina and Ecuador [2, 3]. Lab mice were found to harbour chromosomal and DNA damage in bone marrow, liver and kidney cells as well as lymphoid cells [4]. Similar effects were found in non-mammalian species, including sea urchins [5], goldfish [6, 7], eels [8], tilapia fish [9] as well as the fruitfly [10]. These experiments show that glyphosate herbicides are dangerous for humans and many other animals. Glyphosate is highly soluble in water, so impacts on aquatic wildlife may be of particular concern, especially following the recent report on the presence of glyphosate in rain water, groundwater, rivers and air [11, 12]. Its extreme toxic effects on amphibians such as frogs has already been shown (see [13] Roundup Kills Frogs, SiS 26). Cell damage has been documented in many cell types including those derived from the rat testis (see [14] Glyphosate Kills Rat Testes Cells, SiS 54), human placenta, umbilical cord, and embryo (see [15] Death by Multiple Poisoning, Glyphosate and Roundup, SiS 42), rat and carp neurones [16, 17], and liver [18, 19].  

Multiple tests all show cellular damage in response to Roundup  

To reflect occupational exposure, human buccal epithelial cells were exposed to glyphosate and Roundup for 20 minutes only at concentrations from 10 mg/L to 200 mg/L.  The Roundup formulation used for the experiments contains 450 g/L of glyphosate and should be diluted according to the manufacturer’s instructions to 1–3 % before use (final concentration 4 500–13 500 mg/l). The researchers found some significant effects with 10-20 mg/l, equivalent to a 225–1 350-fold dilution of the spraying solution.

Cell damage was assessed by the release of the membrane-bound enzyme lactose dehydrogenase into the culture medium. The integrity and viability of cells was indicated by their staining with neutral red as only healthy cells retain the dye.  Mitochondrial function was assessed by measuring the activity of the enzyme mitochondrial dehydrogenase with the substrate XXT that gives a yellow colour product.  And cell proliferation was measured by the total protein content of the cell cultures.

The results showed that the cells were much more sensitive to the Roundup formulation than glyphosate. With Roundup, a significant effect was seen at a dose level of 40 mg/L with the XXT assay, while a clear increase of the lactose dehydrogenase levels was seen already with 10 mg/L. The cell proliferation and the neutral red assays were less responsive, with significant effects detected at 80 and 100 mg/L, respectively (still well below agricultural use levels). All effects were dose-dependent.

With glyphosate, no significant effects were seen in 3 of the 4 assays, with only lactose dehydrogenase showing significant effects at over 80 mg/l. 

Multiple tests show Roundup causes DNA damage

DNA damage was analysed by two methods. The first is the Single Cell Gel Electrophoresis (SCGE) assay, which reveals single or double-stranded breaks in DNA. The second is a special comprehensive assay of chromosome instability that picks up many DNA aberrations including chromosome breakage, DNA misrepair, chromosome loss,  as well as cell death by either necrosis (cell death that results from external stressors such as toxins), apoptosis (programmed cell-death) and cell growth. Different nuclear anomalies were measured including micronuclei, a biomarker of chromosomal damage, breakage or loss; nuclear buds, a biomarker of elimination of amplified DNA and/or DNA repair complexes; and nucleoplasmic bridges reflecting the formation of dicentric chromosomes (chromosomes with 2 instead of 1 centromere) , a marker of DNA misrepair and/or end-fusions of the chromosomes.

Significant effects on DNA integrity as assessed by the SCGE assay were seen at 20 mg/l of both Roundup and glyphosate, increasing in a dose-dependent manner.

Exposure of the cells for 20 minutes also led to a significant and dose-dependent increase of nuclear anomalies including increases in the total number of micronuclei beginning at 10 mg/L of Roundup, and 15 mg/L of glyphosate. The number of nuclear buds increased with exposure concentrations, starting at 10 mg/L with both glyphosate and Roundup. In the case of the nucleoplasmic bridges, the only significant effect was obtained with the highest dose of Roundup used (20 mg/L). Apoptotic cells were observed following 20mg/L of Roundup but not glyphosate, while necrosis occurred in response to 20mg/L of both Roundup and glyphosate.

In summary, Roundup was cytotoxic at concentrations as low as 20 mg/L, while its active ingredient was not generally cytotoxic to buccal epithelial cells. Both glyphosate and Roundup elicited genotoxic effects at concentrations below the level required to induce cell damage. The different effects between the active ingredient and its commercial formulation is consistent with previous work, including experiments done on testicular, placental, embryonic and umbilical cord cells (see above).  These results may explain some of the ailments observed in people who work with this herbicide and adds yet more weight to an outright ban of the herbicide [20] Ban Glyphosate Herbicides Now, SiS 43).

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