Ovarian mitochondrial and oxidative stress proteins are altered by glyphosate exposure in mice

https://doi.org/10.1016/j.taap.2020.115116Get rights and content

Highlights

  • A dose-dependent altered ovarian proteome resulted from glyphosate exposure.

  • The highest glyphosate dose increased ovarian weight and follicle number.

  • Glyphosate did not impact ovarian steroid hormone production.

Abstract

Glyphosate (GLY) usage for weed control is extensive. To investigate ovarian impacts of chronic GLY exposure, female C57BL6 mice were orally administered saline as vehicle control (CT) or GLY at 0.25 (G0.25), 0.5 (G0.5), 1.0 (G1.0), 1.5 (G1.5), or 2 (G2.0) mg/kg for five days per wk. for 20 wks. Feed intake increased (P < .05) in G1.5 and G2.0 mice and body weight increased (P < .05) in G1.0 mice. There was no impact of GLY on estrous cyclicity, nor did GLY affect circulating levels of 17β-estradiol or progesterone. Exposure to GLY did not impact heart, liver, spleen, kidney or uterus weight. Both ovarian weight and follicle number were increased (P < .05) by G2.0 but not affected at lower GLY concentrations. There were no detectable effects of GLY on ovarian protein abundance of pAKT, AKT, pAKT:AKT, γH2AX, STAR, CYP11A1, HSD3B, CYP19A, ERA or ERB. Increased (P < .05) abundance of ATM protein was observed at G0.25 but not higher GLY doses. A dose-dependent effect (P < .10) of GLY exposure on ovarian protein abundance as quantified by LC-MS/MS was observed (G0.25–4 increased, 19 decreased; G0.5–5 increased, 25 decreased; G1.0–65 increased, 7 decreased; G1.5–145 increased, 2 decreased; G2.0–159 increased, 4 decreased). Pathway analysis was performed using DAVID and identified glutathione metabolism, metabolic and proteasome pathways as GLY exposure targets. These data indicate that chronic low-level exposure to GLY alters the ovarian proteome and may ultimately impact ovarian function.

Introduction

Proper ovarian function is important for reproductive and overall female health (Hoyer, 2005; Hoyer, 2002; Hoyer and Keating, 2014). Impacts of chemical exposures on ovarian function range from temporary (altered cyclicity and ovulation) to permanent (complete depletion of ovarian follicular structures; (Hoyer and Keating, 2014; Keating, and C JM, Sen N, Sipes IG, Hoyer PB., 2009)). In addition, endocrine disrupting chemicals can target the ovary, which can interfere with steroid hormone production; a scenario which can negatively impact female fertility (Gore et al., 2015; Patel et al., 2015; Rattan et al., 2017). Loss of ovarian function and cessation of associated uterine functionality result in menopause, a timeframe at which women are at heightened risk for development of a number of diseases and health disorders (De Vos et al., 2010; Hoyer and Sipes, 1996; Senapati, 2018). Thus, premature complete or partial loss of ovarian function is detrimental for female health.

Glyphosate (GLY) is a widely applied non-selective herbicide and has been used for approximately 3.5 decades (Williams et al., 2000). With the introduction of GLY-resistant crops, GLY became one of the most predominant utilized herbicides in the U.S. (Benbrook, 2016). Detection of GLY residues in food stuffs (Zoller et al., 2018) and in human urine (Curwin et al., 2007a, Curwin et al., 2007b; Knudsen et al., 2017; Mills et al., 2017; Soukup et al., 2020) has placed potential health effects of GLY exposure under scrutiny. Urinary presence of GLY does not automatically indicate a health risk – in fact, it has been recognized that GLY is readily excreted, through feces and urine (Williams et al., 2000). Furthermore, although urinary exposure has been detected, there is no difference in urine levels in rural agricultural (presumably a higher exposure demographic) versus non-agricultural individuals (Curwin et al., 2007a, Curwin et al., 2007b).

There are conflicting reports as to whether GLY should be considered a risk factor for female reproductive health. The Ontario Farm Family Health study correlated GLY exposure during late pregnancy with spontaneous abortion (Arbuckle et al., 2001), and association between GLY exposure and a shortened gestational length was reported in a birth cohort from Indiana (Parvez et al., 2018). Exposure in vivo for 60 days to GLY (126 or 315 mg/Kg/d) increased atretic follicle number, reduced antral follicle surface area in vivo in rats and decreased 17β-estradiol (E2) production (Hamdaoui et al., 2019). Another in vitro study determined no impact of GLY (1–300 μg/ml) on cell number, E2 or progesterone (P4) production in granulosa cells, but did note impacts of a GLY-based herbicide (GBH) exposure on steroid hormone concentration in media (Perego et al., 2017). Postnatal exposure of lambs to a GBH; 2 mg/Kg/day) did not alter ovarian weight but increased proliferation of granulosa and theca cells as evidenced by increased abundance of a proliferation marker and decreased mRNA encoding follicle stimulating hormone receptor and growth and differentiation factor 9 (Alarcon et al., 2019). GLY-based herbicide exposure (2 mg/Kg/day) in neonatal rats increased uterine luminal hyperplasia, estrogen receptor alpha, and P4 receptor (Ingaramo et al., 2019). In contrast, no effect of GLY on E2 production using a standardized H295R steroidogenic assay was observed (Hecker et al., 2011), nor was there any impact of GLY on gross phenotypic reproductive measures in acute and sub-chronic exposure studies (Williams et al., 2000), illustrating conflicting data in the literature.

Additional reasons for inconsistency between the aforementioned studies is the formulation used: GLY vs. a GBH mixture. Additionally, as alluded to above the doses used in vivo are often extremely high and at non-relevant human exposure levels. The purpose of the current study was to explore the hypothesis that chronic GLY exposure at levels relevant to human exposure would impact ovarian function in ways that could contribute to female infertility.

Section snippets

Materials

Glyphosate (CAS # 1071-83-6), 2-β-mercaptoethanol, Tris base, Tris HCL, Sodium chloride, Sucrose, EDTA, Paraformaldehyde and Tween-20 were purchased from Sigma-Aldrich Inc. (St Louis, MO). Glycerol, Sodium citrate, Citric acid and Pierce BCA protein assay kit were from Thermo Fisher Scientific. 4–15% mini-PROTEAN TGX™ precast protein gels were obtained from BioRad, USA. 4–20% TGX stain free precast protein gels were purchased from Criterion. iBlot 2NC regular stacks were from Invitrogen.

Effect of glyphosate on body weight and feed intake

As anticipated, body weight increased over the duration of the dosing period (Fig. 1A). There was increased (P < .05) body weight observed in the mice that received G1.0, but no other dosage groups differed from CT. Relative to CT, G2.0 and G1.5 treated mice had increased (P < .05) feed intake (Fig. 1B) but there were no other GLY-induced impacts on feed intake.

Impact of GLY exposure on the estrous cycle

Daily vaginal cytology was performed over 21 weeks and the percentage time spent at stages of the estrous cycle determined as a

Ovarian global proteome impacts of GLY exposure

LC-MS/MS was performed on ovarian protein homogenates and bioinformatic comparison between each dose with CT-treated mouse ovaries performed. There was a dose-dependent effect (P < .1) of GLY exposure on ovarian protein abundance. (See Fig. 9.)

Exposure to G0.25 increased four and decreased 19 proteins relative to CT (Supplemental Table 1). Ovaries from mice that received G0.5 had five increased and 25 decreased proteins. (Supplemental Table 5). G1.0 exposure increased abundance of 65 and

Discussion

Glyphosate is an herbicide used for weed control since 1974 and introduction of GLY resistant crops in 1996 has dramatically increased usage (Duke and Powles, 2008). A dose of GLY of 1 mg/kg/day from chronic dietary exposure has been deemed safe (Agency, 2017) and a NOAEL for reproductive toxicity has been identified as 2132 mg/kg/day (Williams et al., 2000). While many other studies have investigated dramatically higher GLY exposure levels, the current study sought to determine ovarian impacts

Funding

Supported by R21ES026282 from the National Institute of Environmental Health Sciences to AFK.

CRediT author statement

Shanthi Ganesan: Animal exposures and all Laboratory analysis; Original paper draft preparation. Aileen F. Keating: Conceptualization; Supervision; Writing - Reviewing and Editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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