Moderate levels of glyphosate and its formulations vary in their cytotoxicity and genotoxicity in a whole blood model and in human cell lines with different estrogen receptor status

Abstract

In vitro studies were conducted to determine the short-term cytotoxic and genotoxic effects of pure glyphosate and two glyphosate formulations (Roundup^® and Wipeout^®) at concentrations relevant to human exposure using whole blood (cytotoxicity) and various cancer cell lines (cytotoxicity and genotoxicity). Pure glyphosate (pure glyph) and Roundup^® (Ro) showed similar non-monotonic toxicological profiles at low dose exposure (from 10 µg/ml), whereas Wipeout^® (Wo) demonstrated a monotonic reduction in cell viability from a threshold concentration of 50 µg/ml, when tested in whole blood. We evaluated whether using various cancer cells (the estrogen-E2-responsive HEC1A, MCF7 and the estrogen-insensitive MDA-MB-231) exposed to moderate doses (75-500 µg/ml) would indicate varied toxicity and results indicated significant effects in the HEC1A cancer cells. A non-monotonic reduction in cell viability was observed in HEC1A exposed to pure glyph (75-500 µg/ml) and proliferative effects were observed after exposure to Wo (75, 125 and 250 µg/ml). Genotoxicity assessment (test concentration 500 µg/ml) demonstrated DNA damage in the HEC1A and MDA-MB-231 cells. Adjuvants and/or glyphosate impurities were potential contributing factors of toxicity based on the differential toxicities displayed by Ro and Wo in human whole blood and the HEC1A cells. This study contributes to the existing knowledge about in vitro exposure to moderate concentrations of glyphosate or glyphosate formulations at cytotoxic and genotoxic levels. In addition, a suggestion on the relevance of the estrogen receptor status of the cell lines used is provided, leading to the need to further investigate a potential endocrine disruptive role.

Keywords: Cytotoxicity; Genotoxicity; Glyphosate; Roundup®; Wipeout®.

Fig. 1

The effect of a1 pure glyph (99.5%) and its formulations, Ro (b1) and Wo (c1) on the cell viability in human whole blood. The cells were exposed over an 18-h period at varying herbicide concentrations (0–500 µg/ml). Lipolysaccharide (LPS, 5 µg/ml) was used as a positive control in this study. Data points represent the means of five replicates (± SEM). ANOVA single-factor analysis was used to determine significant differences from the untreated control (not shown) *P ≤ 0.05, ^#P ≤ 0.01. Non-linear regression analysis using Graphpad Prism 6 software was used to describe the effect of pure glyph (a2), Ro (b2) and Wo (c2) in human whole blood. a2 Bell-shaped curve (biphasic curve): Ymin + (Ymax − Ymin)/1 + $\text{1}{0}^{(\text{logEC5}{0}_1-\text{log}[x]\ast n\text{H1}}$ + Ymax₂ − Ymin/1 + $\text{1}{0}^{\left(\text{log[}x\text{]}-\text{logEC5}{0}_{\text{2}}\right)\ast n\text{H2}}$, b2 bell-shaped curve (biphasic curve): Ymin + (Ymax − Ymin)/1 + $\text{1}{0}^{(\text{logEC5}{0}_1-\text{log}[x]\ast n\text{H1}}$ + Ymax₂ − Ymin/1 + $\text{1}{0}^{\left(\text{log[}x\text{]}-\text{logEC5}{0}_{\text{2}}\right)\ast n\text{H2}}$, c2 linear curve: Y = m*log(x) + c. Goodness of fit was assessed by R², R²_adjusted values and upper and lower 95% confidence intervals around the fit (dash lines)

Fig. 2

The effect of a pure glyph (99.5%) and its formulations, Ro (b) Wo (c) on the cell viability of the cancer cell lines (MCF-7, HEC1A and MDA-MB-231). The cancer cell lines were exposed over a 24-h period at varying herbicide concentrations (0–500 µg/ml). Camptothecin (100 µM) was used as a positive control in this study. Data points represent the means of three replicates (± SEM). ANOVA single-factor analysis was used to determine significant differences from the untreated control (not shown) *P ≤ 0.05, ^#P ≤ 0.01

Fig. 3

A representation of (HEC1A endometrial carcinoma cancer cell line) images developed in the comet assay (fluorescence microscope), a exposure to 500 µg/ml pure glyph b exposure to 500 µg/ml Ro c exposure to 500 µg/ml Wo d untreated control. Cells were exposed for a period of 4 h at 37 °C. FITC filter, magnification bar = 50 µm

Fig. 4

DNA damage in HEC1A, MCF7 and MDA-MB-231 cancer cell lines presented as TL (µm): a pure glyph c Ro and e Wo and TM, b pure glyph, d Ro and f Wo. Cells were exposed for 4 h at 37 °C at test and reference herbicidal concentrations. Camptothecin (35 µg/ml) was the positive control in this study and data points represent the means of 50 cells analyzed per experimental treatment (± SEM). ANOVA single-factor analysis was used to determine significant differences from the untreated control. ^*P ≤ 0.05, ^#P ≤ 0.01