Abstract
The effect of Roundup® on adrenal gland steroidogenesis and signaling pathway associated with steroid production was investigated. Doses of 10, 50, 100 and 250 mg/kg bw/d Roundup® were administered for two weeks to adult male rats. The 10 mg/kg bw/d dose which reduced circulatory corticosterone levels, but did not change food consumption and body weight, was selected for further study. The expression of cholesterol receptor (low density lipoprotein receptor), de novo cholesterol synthesis enzyme (3-hydroxy-3-methylglutaryl-Coenzyme A synthase), hormone-sensitive lipase, steroidogenic acute regulatory protein (StAR) mRNA and phosphorylated form was decreased. Adrenocorticotropic hormone receptor (ACTH), melanocortin-2 receptor, expression was not changed but circulatory ACTH levels and adrenal cortex protein kinase A (PKA) activity were reduced. Surprisingly, exogenous ACTH treatment rescued steroidogenesis in Roundup®-treated animals. Apoptosis was evident at 250 mg/kg bw/d, but not at 10 mg/kg bw/d dose. These results suggest that Roundup® may be inhibitory to hypothalamic-pituitary axis leading to reduction in cyclic adenosine monophosphate (cAMP)/PKA pathway, StAR phosphorylation and corticosterone synthesis in the adrenal tissue.
Abbreviations
- EDC, Endocrine disrupting chemical;
- LD50, Lethal dose, 50%;
- Ldlr, Low density lipoprotein receptor;
- Sr-b1, Scavenger receptor class B member 1;
- Hmgcs, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase;
- Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase;
- Hsl, Hormone-sensitive lipase;
- StAR, Steroidogenic acute regulatory protein;
- Creb, cAMP response element-binding protein;
- ACTH, Adrenocorticotropic hormone;
- Mc2r, Melanocortin-2 receptor;
- PKA, Protein kinase A;
- cAMP, Cyclic adenosine monophosphate;
- L:D cycle, Light Dark cycle;
- RIA, Radioimmunoassay;
- ELISA, Enzyme-linked immunosorbent assay;
- EIA, Enzyme Immunoassay;
- qPCR,Quantitative real-time PCR;
- DPX, Distrene, Plasticiser, Xylene;
- DAPI, 4',6-diamidino-2-phenylindole;
- RIPA buffer, Radioimmunoprecipitation assay buffer;
- SDS PAGE,Sodium dodecyl sulfate polyacrylamide gel electrophoresis;
- EDTA,Ethylenediaminetetraacetate;
- EGTA, Ethylene glycol tetraacetate;
- β ME, Beta Mercaptoethanol;
- PBS, Phosphate buffer saline;
- TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling;
- TdT, Terminal deoxynucleotidyl transferase;
- SD,Standard deviation
Keywords
- Endocrine disruptor;
- Glyphosate;
- Steroidogenesis;
- StAR;
- Adrenal gland;
- Rat
1. Introduction
Agricultural advancements have increased production and correspondingly increased the usage and release of herbicides into the environment. Among the herbicides, the glyphosate-based herbicide Roundup® is most extensively used world over [14]. Roundup® is non-selective and broad spectrum herbicide utilized in agricultural fields, gardens, play grounds, road sides etc. [10]. The half life of Roundup® is ∼47 days in soil and up to 90 days in water with low microbial metabolism and disintegration [18],[37] and [39]. In rats, Roundup® whole body pharmacokinetics is biphasic for single 10 mg/kg bw dose with half-life of the alpha phase is 6 h and 79 to 106 h for beta phase [43]. Roundup® and its metabolite, aminomethyl-phosphonic acid, have been detected in water and crops [11], [25], [26] and [35]. Therefore, there is increased probability of Roundup® exposure to animals and human, and it becomes of interest to study its toxic effects, if any.
Glyphosate or its formulation Roundup® acts via specific inhibition of plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase which is essential for synthesis of aromatic amino acids [19], [34] and [36] and thus, considered non toxic to animals. However in recent past, several studies have suggested glyphosate toxic effects such as carcinogen[28], [38] and [40], teratogen [12] and [13] and as an endocrine disruptor (ED). The endocrine disrupting chemicals (EDC) represent a broad class of exogenous substances that adversely affect the endocrine system by interfering with hormone biosynthesis, metabolism or action [23]. As an ED, glyphosate and its formulation, Roundup® was reported to decrease testosterone hormone levels in adult rats [9]. The prenatal exposure of glyphosate disrupted the masculinization process and caused endocrine dysfunction in reproductive parameters of male offspring [32]. Moreover, various formulations of glyphosate including Roundup® were reported to disrupt aromatase activity, enzyme required for estrogen synthesis, in human liver HepG2 cells [15]. The human placental JEG3 cells treated with Roundup® altered aromatase mRNA levels and enzymatic activity by interacting with the active site of the purified enzyme [31]. A study involving MA-10 Leydig tumor cell line reported down regulation of StAR mRNA levels, a key regulatory steroidogenic gene, and dibutyryl cAMP-stimulated progesterone production upon treatment [42]. It has been observed that the commercial formulation had more adverse effects than the active ingredient i.e. glyphosate [3]. The EDC effects of Roundup® in male reproductive system have been described; however, studies detailing EDC effect on the adrenal gland steroidogenesis have not been reported in animals.
In the present study, experiments have been conducted to examine effects of Roundup® on adrenal steroidogenesis at systemic as well as at the tissue level. Different doses of Roundup® were orally administered to adult male rats for 14 days daily although, only the lowest dose required for disrupting the major adrenal gland steroid hormone i.e. corticosterone level was selected for further detailed study. The EDC effect of Roundup® was examined on important regulatory genes for steroidogenesis; StAR and p450scc, the steroid precursor, cholesterol levels, and cholesterol homeostasis genes and the signaling involved. An ACTH challenge experiment was also performed to evaluate Roundup® action to be via hypothalamic pituitary axis or directly upon the gland.
