Ovarian mitochondrial and oxidative stress proteins are altered by glyphosate exposure in mice
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.
References (113)
- et al.
Neonatal exposure to a glyphosate-based herbicide alters the histofunctional differentiation of the ovaries and uterus in lambs
Mol. Cell. Endocrinol.
(2019) - et al.
Chronic exposure to a glyphosate-containing pesticide leads to mitochondrial dysfunction and increased reactive oxygen species production in caenorhabditis elegans
Environ. Toxicol. Pharmacol.
(2018) - et al.
Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol
Toxicol. Appl. Pharmacol.
(2016) - et al.
The effects of in utero bisphenol a exposure on the ovaries in multiple generations of mice
Reproductive Toxicol. (Elmsford, NY)
(2016) - et al.
Protective role for ovarian glutathione s-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity
Toxicol. Appl. Pharmacol.
(2012) - et al.
Glutathione s-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during vcd-induced ovotoxicity in neonatal rat ovaries
Toxicol. Appl. Pharmacol.
(2013) - et al.
Atm phosphorylates histone h2ax in response to DNA double-strand breaks
J. Biol. Chem.
(2001) - et al.
Glutathione s-transferase mu modulates the stress-activated signals by suppressing apoptosis signal-regulating kinase 1
J. Biol. Chem.
(2001) - et al.
Transport of glutathione and glutathione conjugates by mrp1
Trends Pharmacol. Sci.
(2006) - et al.
Pesticide dose estimates for children of Iowa farmers and non-farmers
Environ. Res.
(2007)
Primary ovarian insufficiency
Lancet.
Destruction of preantral follicles in adult rats by 4-vinyl-1-cyclohexene diepoxide
Reproductive toxicology (Elmsford, NY).
Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage
Toxicol. Appl. Pharmacol.
Protein phosphorylation and its role in the regulation of annexin a2 function
Biochim. Biophys. Acta, Gen. Subj.
Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary
Toxicol. Appl. Pharmacol.
Di(2-ethylhexyl) phthalate inhibits antral follicle growth, induces atresia, and inhibits steroid hormone production in cultured mouse antral follicles
Toxicol. Appl. Pharmacol.
Metabolism disrupting chemicals and metabolic disorders
Reproductive toxicology (Elmsford, NY)
Assessment of the oxidative and genotoxic effects of the glyphosate-based herbicide roundup on the freshwater shrimp, macrobrachium nipponensis
Chemosphere.
Acute uterine effects and long-term reproductive alterations in postnatally exposed female rats to a mixture of commercial formulations of endosulfan and glyphosate
Food Chem. Toxicol.
Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries
Toxicol. Appl. Pharmacol.
Dual protective role for glutathione s-transferase class pi against vcd-induced ovotoxicity in the rat ovary
Toxicol. Appl. Pharmacol.
Control of mammalian oocyte growth and early follicular development by the oocyte pi3 kinase pathway: new roles for an old timer
Dev. Biol.
Toxicity induced by glyphosate and glyphosate-based herbicides in the zebrafish hepatocyte cell line (zf-l)
Ecotoxicol. Environ. Saf.
Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity
Toxicol. Appl. Pharmacol.
Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents
Food Chem. Toxicol.
Prepubertal subchronic exposure to soy milk and glyphosate leads to endocrine disruption
Food Chem. Toxicol.
A critical role for histone h2ax in recruitment of repair factors to nuclear foci after DNA damage
Curr. Biol. : CB.
Influence of a roundup formulation on glyphosate effects on steroidogenesis and proliferation of bovine granulosa cells in vitro
Chemosphere.
Low-concentration exposure to glyphosate-based herbicide modulates the complexes of the mitochondrial respiratory chain and induces mitochondrial hyperpolarization in the danio rerio brain
Chemosphere.
Activation of akt (pkb) and suppression of fkhrl1 in mouse and rat oocytes by stem cell factor during follicular activation and development
Dev. Biol.
DNA double-stranded breaks induce histone h2ax phosphorylation on serine 139
J. Biol. Chem.
Noncanonical function of glutamyl-prolyl-trna synthetase: gene-specific silencing of translation
Cell.
Infertility: a marker of future health risk in women?
Fertil. Steril.
Mechanism of elimination of phosphorylated histone h2ax from chromatin after repair of DNA double-strand breaks
Mutat. Res.
Glyphosate induces human breast cancer cells growth via estrogen receptors
Food Chem. Toxicol.
Image processing with imagej
Biophoton. Int.
Regulation of jnk signaling by gstp
EMBO J.
Glyphosate
Glyphosate. Dietary exposure analysis in support of registration review
The in vitro impact of the herbicide roundup on human sperm motility and sperm mitochondria
Toxics.
The effect of glyphosate on human sperm motility and sperm DNA fragmentation
Int. J. Environ. Res. Public Health
An exploratory analysis of the effect of pesticide exposure on the risk of spontaneous abortion in an Ontario farm population
Environ. Health Perspect.
Ameliorative effect of resveratrol on testicular oxidative stress, spermatological parameters and DNA damage in glyphosate-based herbicide-exposed rats
Andrologia
Physiological and pharmacological significance of glutathione-conjugate transport
J Toxicol Environ Health B Crit Rev.
Trends in glyphosate herbicide use in the United States and globally
Environ. Sci. Eur.
Metabolome disruption of pregnant rats and their offspring resulting from repeated exposure to a pesticide mixture representative of environmental contamination in brittany
PLoS One
Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse
Toxicol. Sci.
Subfertility caused by altered follicular development and oocyte growth in female mice lacking pkb alpha/akt1
Biol. Reprod.
Defective pitrm1 mitochondrial peptidase is associated with abeta amyloidotic neurodegeneration
EMBO molecular medicine.
Acute exposure to a glyphosate-containing herbicide formulation inhibits complex ii and increases hydrogen peroxide in the model organism caenorhabditis elegans
Environ. Toxicol. Pharmacol.
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2022, Environmental ResearchCitation Excerpt :The oxidative stress and mitochondrial dysfunction explain the cytotoxicity, biochemical changes, immune suppression, and at least part of the endocrine dysfunction. While oxidative stress has been reported to harm both sperm (Nerozzi et al., 2020) and ovaries (Ganesan and Keating, 2020), others have reported direct actions on both androgen and estrogen receptors, which lead to gene induction (Gasnier et al., 2009). Thus, the endocrine disruption is a result of two independent mechanisms.