Cell damaging effects of glyphosate – Are we ready for Roundup?

Table 1: Characterization of tested nutritional supplements

Reagent preparation and Assays. All nutritional supplements were treated identically in accordance with the protocol recommended by the Unites States Pharmacopeia⁸. Three recommended daily doses of each supplement were powdered (tablets were crushed using ceramic pestle and mortar, capsules were cut open and powder poured out), placed into a glass container with 900 ml of 0.1N hydrochloric acid and incubated for 1 hour at 37°C in a shaking incubator set with rotation speed of 75 rpm.

Resulting solutions were filter-sterilized using 0.2 micrometer pore size filters, aliquoted and kept frozen at -20°C until analyses. Amounts of samples taken for analysis are expressed as number of millionth parts of recommended daily dose of the respective supplement.

Cell viability assay. The assay was performed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) supplied by Promega, Madison, WI, USA. This is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes in viable cells reduce the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan. The concentration of formazan produced is measured by optical density at 490 nm and it is directly proportional to the number of live cells in culture.

Protein Biomass Assay- Sulforhodamine B based. After appropriate treatments, dead cells were washed away. Cell proteins were fixed, and stained with Sulforhodamine B dye. The incorporated dye was then liberated from the cells by a Tris base solution and quantified by optical density at 565 nm. An increase or decrease in the number of cells (total biomass) defines the amount of dye incorporated by the living cells in the culture as the dead cells are washed away. The optical density therefore determines total protein biomass.

Treatment of Human Dermal Fibroblasts. Normal Human Dermal Fibroblasts (NHDF) were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA) and maintained in Dulbecco’s  Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin. In the experiments, the cells were grown to confluency in a 48 well plate.

NHDF’s Cells were treated with various supplements for 24 hours, all at 0.5 millionth part daily dose in DMEM supplemented with 5% FBS, except for the “No additions” and “No protection” controls which only received fresh media supplemented with 10% FBS.

After 24 hours, the media was aspirated away, and cells were exposed to various concentrations of glyphosate in a fresh solution containing respective supplements at 0.5 millionth part daily dose in DMEM supplemented with 5% FBS for 24 hours. The “No additions” wells were incubated with fresh DMEM supplemented with 5% FBS and the “No protection” wells were treated with various concentrations of glyphosate in media without supplements.

After 24 hours all treatments were aspirated away, cells were washed 3 times with PBS and cell viability assay was performed or their pictures taken after staining with Hematoxylin and Eosin.

Treatment of Human Kidney Cells. Human Kidney Cells (HEK-293) were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA) Catalog No. CRL-1573 and maintained in DMEM and supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were grown to 50% confluency in a 96 well plate.

Glyphosate mixed with each tested nutrient mixture was added to the cells in DMEM supplemented with 5% FBS and incubated for 24 hours. After 24 hours, treatments were removed and cells were incubated in fresh DMEM supplemented with 10% FBS for 48 hours for a recovery phase. After 48 hours of recovery period, protein biomass assay was performed as described.

The toxicity of a generic, easily available glyphosate concentrate solution was tested at a range of concentrations from 0.025 to 0.05% against human dermal fibroblasts (derived from the lower skin layers) and human kidney cells.