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. 2019 Jan 7;8(2):238–245. doi: 10.1039/c8tx00287h

Protective effects of resveratrol on biomarkers of oxidative stress, biochemical and histopathological changes induced by sub-chronic oral glyphosate-based herbicide in rats

Ruhi Turkmen a,✉, Yavuz Osman Birdane a, Hasan Huseyin Demirel b, Mustafa Kabu c, Sinan Ince a
PMCID: PMC6417488  PMID: 30997023

graphic file with name c8tx00287h-ga.jpgThe aim of this sub-chronic toxicity study is to determine the protective effects of Resveratrol (Res) on oxidative stress, biochemical and histopathological changes induced by glyphosate-based herbicide (GBH) in the blood, brain, heart, liver and renal tissues.

Abstract

The aim of this sub-chronic toxicity study is to determine the protective effects of Resveratrol (Res) on oxidative stress, biochemical and histopathological changes induced by glyphosate-based herbicide (GBH) in the blood, brain, heart, liver and renal tissues. A total of 28 male Wistar rats were equally divided into 4 groups so that each group included 7 rats. In the study, Group I (control group) was given normal rodent feed and tap water ad libitum. Group II (Res group) was given Res 20 mg kg–1, Group III (GBH group) was given GBH of 375 mg kg–1 to achieve 1/10 of Lethal Dose 50% (LD50), and Group IV (Res + GBH) was given Res 20 mg kg–1 and GBH 375 mg kg–1 with oral gavage once a day for 8 weeks. While GBH decreased the glutathione (GSH) levels in the blood, brain, heart, liver and renal tissues, it significantly increased malondialdehyde (MDA) levels. In contrast, the aforementioned parameters were seen to recover in the group to which Res was administered. Moreover, it was observed that Res improved the histopathological changes induced by GBH in rat tissues. In conclusion, Res prevents oxidative stress caused by GBH by preventing lipid peroxidation (LPO) and boosting the antioxidant defense system and decreases the damage in the brain, heart, liver and renal tissues of Wistar rats.

Introduction

Glyphosate (N-(phosphonomethyl) glycine)- and glyphosate-based herbicides (GBHs) are the world's leading systemic organophosphonate, broad-spectrum and non-selective herbicides which are used to eliminate weeds.1 Glyphosate has a relatively low solubility in water (12 g L–1 at 25 °C and 60 g L–1 at 100 °C). It does not dissolve in common organic solvents like acetone, methanol and chloroform. Therefore, commercial formulations of glyphosate are generally in the form of salts for increasing the herbicidal effectiveness of the parent compound and providing higher solubility.2 Glyphosate isopropylamine salt that was used for this study is the most commonly used commercial product in Turkey. Especially with the increase in the use of glyphosate-resistant plants in agriculture, it is inevitable that these chemicals will contaminate air, water and foods. As a result of this, non-target organisms such as humans and animals are exposed to GBH residues more, and this may cause different health problems in these living beings.3,4 GBHs, classified as class E in 1991, are believed to be safe and non-toxic compounds for both humans and animals.5,6 However, a previous study revealed that GBHs, which are commonly used around the world, may have toxic effects even below the acceptable limit values.1 It was concluded in studies on frogs,7 rats8 and human cell cultures9,10 that commercial formulations of glyphosate are only more toxic than glyphosate active substances. Especially, Lipok et al. (2010) found that isopropylamine alone was more toxic than glyphosate on aquatic phytoplankton such as cyanobacterial strains.11

Glyphosate is an herbicide which inhibits the synthesis of aromatic amino acids in plants such as tryptophan, tyrosine and phenylalanine. 5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is inhibited by the glyphosate in plants. EPSPS is primarily present in plastids, and its inhibition causes shikimate-3-phosphate accumulation. Thus, the production of aromatic amino acids is hindered, and this means prevention of protein synthesis.12 Since only plants and some micro-organisms have the shikimate aromatic synthesis pathway, the toxic effects it is thought to have on mammals and other animals are caused by different reasons. Although the toxicity mechanism of GBH in animals has not been clarified yet, it has been reported in many studies that formation of reactive oxygen species (ROS) or oxidative stress, known as deterioration of the antioxidant defense mechanism, may have a role.13 A sub-chronic toxicity study was carried out to induce oxidative stress and histopathological changes as a result of oral administration of GBH in rats for 8 weeks at a dose of 375 mg kg–1.14 Therefore, in this sub-chronic study, we used 375 mg kg–1 GBH dose of the previous study.14 As a result of a treatment administered with various antioxidants, it was reported that LPO- and intoxication-related oxidative stress caused by GBH may emerge.14–17

A promising diet phytoalexin, Res (trans-3,5,4′-trihidroxystilbene) is a strong antioxidant and polyphenolic compound which is produced by photosynthetic microorganisms18 and many plants against drought, cold air, stress, injury, UV rays and fungal infections.19,20 The biological characteristics of Res such as anti-inflammatory activity,21 antiviral activity,22 antiplatelet aggregation activity23 and especially antioxidant effectiveness24,25 have become the focus of researchers in many studies. Another study showed promising results of Res usage as secondary prophylaxis for stroke.18 Furthermore, it was reported that Res had the ability to catch both superoxide and hydroxyl radicals.26 Moreover, Akbel et al. (2018) stated that Res may have a protective role against LPO and oxidative stress induced by malathion, an organophosphorus pesticide.27 Although there are several studies in the literature which revealed the antioxidant effectiveness of Res, to the best of our knowledge, there has so far been no study which investigated the potential antioxidant effectiveness of Res against toxicity induced by GBH, which is used commonly. Therefore, this study is intended to biochemically and histopathologically examine the potential antioxidant effects of Res on blood and tissues against LPO and oxidative damage induced by sub-chronic GBH exposure.

Materials and methods

Materials and experimental protocol

Knockdown 48 SL (Safa Agriculture Corp., Turkey), which contained 480 g L–1 a glyphosate isopropylamine salt, was used to induce GBH toxicity in this study. Res, administered to the treatment groups, was purchased from Terraternal (Santa Clara, CA, USA). The chemicals which would be used to determine the parameters to be analyzed were procured from the relevant firms. 28 3-month-old (200–300 g) male Wistar rats that were used in this study were procured from the Laboratory of Experimental Animals Research and Application Center (Afyon/Turkey). All experimental procedures were performed in compliance with the ARRIVE guidelines for the ethical treatment of experimental animals. Afyon Kocatepe University Experimental Animals Ethics Committee approved this study with the protocol no 2016/54. The care of the rats was carried out at Afyon Kocatepe University Experimental Animals Research and Application Center. Feeding and care of rats were facilitated under the conditions of 21 ± 2 °C room temperature, 55–60% moisture and a 12 : 12-hour light–dark cycle throughout the study. The rats in the sample were given standard rat feed and clean potable water ad libitum.

The rats were divided into 4 groups so that each group included 7 rats. Distilled water (0.5 ml) was provided to the control group. GBH (375 mg kg–1 was dissolved in distilled water) was given to the GBH group. Res (20 mg kg–1 was dissolved in distilled water) was given to the Res group. Res and GBH were given to the other group (Res + GBH) in the same amounts. Administrations of the substances were performed with a gastric gavage once a day for 56 days. Furthermore, Res was given to the rats 1 hour before the administration of GBH. Res and GBH doses in this study were selected based on previous studies.27,28

Blood collection and preparation of erythrocytes

Blood samples were collected from each group by cardiopuncture into heparinized and non-heparinized tubes under light ether anesthesia at the end of 56 days. Within 30 min of blood collection, centrifugation was used for precipitating the erythrocytes at 600g for 15 min at 4 °C, and the plasma and serum were removed. The erythrocytes were washed three times by isotonic saline, and the puffy coat was separated. Later, the same volumes of isotonic saline and erythrocyte were added into vials and kept at –20 °C in a deep freezer. When the osmotic pressure that was used destroyed erythrocyte suspensions, five times in volume of cold deionized water was used. The erythrocyte lysate was kept at 4 °C until measurements within 3 days.29

Homogenate preparation

Cervical dislocation was utilized to euthanize the rats, and the brain, heart, liver and renal tissues were washed immediately with ice-cold 0.9% NaCl. Each tissue was trimmed free of extraneous tissue rinsed in a 0.15 M chilled Tris–HCl buffer (pH 7.4). The aforementioned tissues were blotted dry and homogenized in a 0.15 M Tris–HCl buffer (pH 7.4) to yield a homogenate of 10% (w/v). Later, they were centrifuged at 2100g for 10 min at 4 °C. The nuclear fraction was symbolized by the pellets, and the supernatants were subjected to centrifugation at 18 600g for 20 min at 4 °C. The mitochondrial fraction and the cytosolic (including microsomal fraction) fraction were symbolized by the emerging pellets and the supernatants, respectively. Formation of reactive oxygen species was seen in all the fractions, as well as the entire homogenate.

Preparation of tissues for histopathological analysis

After necropsy, the brain, heart, liver and renal tissues of each rat were set in a formalin solution of 10% and fixed in paraffin blocks after processing. After they were sliced into 5–6 μm sections, they were stained with hematoxylin&eosin (H&E). In conclusion, each section was examined under a light microscope (Olympus BX51 and DP20 added Microscopic Digital Image Analysis System, Tokyo, Japan). The scoring of the findings and histopathological changes in the tissues is shown as follows: –: no lesion; +: mild; ++: moderate; +++: severe.

Measuring LPO and GSH in whole blood and tissue homogenates

As a marker for LPO, MDA was detected with the methods reported by Draper and Hadley30 in the whole blood and Ohkawa et al.31 in the tissue homogenates. The spectrophotometric measurement of the colors generated during the reaction of thiobarbituric acid with MDA required that the principle of the methods and spectrophotometric measurement of its absorbance utilized a wavelength of 532 nm. The concentration of MDA is stated in nmol ml–1 blood and nmol g–1 protein. The method described by Beutler et al.32 was used to measure the GSH concentration in the whole blood and tissue homogenates. The measurement of optical density was carried out at 412 nm in the spectrophotometer. The results are explained as nmol ml–1 blood and nmol g–1 protein. A Shimadzu 1601 UV–VIS spectrophotometer (Tokyo, Japan) was used to carry out the spectrophotometric measurements.

Measuring superoxide dismutase (SOD) and catalase (CAT) activities in erythrocyte lysate and tissue homogenates

The measurement of the antioxidant enzyme activity of SOD in erythrocyte lysate and tissue homogenate was performed based on the method reported by Sun et al.33 SOD is measured depending on the principle where xanthine reacts with xanthine oxidase as a source of substrate (superoxide) and reduced nitro blue tetrazolium (NBT) as a superoxide indicator. A superoxide flux was formed using xantine-xantine oxidase in this method. The absorbance provided by NBT's reduction into blue formazan by superoxide was determined spectrophotometrically at 560 nm. SOD activity is explained as U gHb–1 erythrocyte and U μg–1 protein tissue. CAT activities in erythrocyte lysate and tissue homogenate were identified based on the methods by Luck34 and Aebi,35 respectively. The decomposition of H2O2 by the catalase was the basis of the method. A 50 mM phosphate buffer of pH 7.0, 10 mM H2O2 and the sample formed the reaction mixture. The reduction rate of H2O2 was pursued at 240 nm for 45 s at room temperature. One unit of catalase is the amount of catalase that decomposes 1.0 μmol of H2O2 per min at pH 4.5 at 25 °C, and the catalase activity (k; nmol min–1) was explained in k gHb–1 erythrocyte and k μg–1 protein tissue.

Measurement of serum biochemical parameters

COBAS test kits (Roche Diagnostics Systems, Istanbul, Turkey) were used to determine the serum total protein (Tp), aspartate aminotransferase (AST), aspartate alanine transferase (ALT), alkaline phosphatase (ALP), albumin, urea, creatinine and creatine kinase-MB (CK-MB) levels by considering the manufacturers’ instructions at the Molecular Biology and Genetics Laboratory, Faculty of Veterinary Medicine, University of Afyon Kocatepe (Turkey). Moreover, the levels of serum rat cardiac troponin I (cTn-I) were determined using diagnostic commercial kits (Catalog Number: CSB-E08594r) as described by the manufacturer (Cusabio Biotech Co., Ltd) by an ELx800 Absorbance Microplate Reader (BIOTEK, Bad Friedrichshall, Germany).

Measurement of hemoglobin (Hb) and protein concentrations

Hemoglobin was determined using colorimetric the cyanmethemoglobin method according based on the study by Drabkin and Austin,36 and the analysis of tissue protein content was conducted based on the colorimetric method by Lowry et al.37

Statistical analyses

The data that were obtained from the experimental animals are expressed as means and standard deviations (±SD) and analyzed using one-way analysis of variance (ANOVA), followed by Duncan post-hoc tests on the SPSS (20) software. A difference in the mean values of by p < 0.05 was considered to be significant.

Results

Effects on LPO and GSH levels

A more significant increase was observed in the MDA levels in the whole blood, brain, heart, liver and renal tissues (p < 0.05) of the rats to which GBH was administered, than those in the control group. However, a more significant decrease occurred in the MDA levels in the whole blood, brain, heart, liver and renal tissues of the Res + GBH group than those in the group to which GBH was administered (Table 1). A more significant decrease was observed in the GSH levels in the whole blood, brain, heart, liver and renal tissues (p < 0.05) of the rats to which GBH was administered than those in the control group. However, the GSH levels in the whole blood, brain, heart, liver and renal tissues increased more significantly in the Res + GBH group than the group to which GBH was administered (Table 2).

Table 1. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the malondialdehyde (MDA) levels in the whole blood, liver, kidney, heart and brain homogenates of the rats.

Groups Blood (nmol ml–1) Liver (nmol g–1 tissue) Kidney (nmol g–1 tissue) Heart (nmol g–1 tissue) Brain (nmol g–1 tissue)
Control 4.53 ± 0.82b 3.53 ± 0.25b 4.38 ± 0.25b 3.56 ± 0.35b 3.38 ± 0.23b
RES 4.49 ± 0.66b 3.35 ± 0.18b 3.75 ± 0.41c 3.27 ± 0.31b 3.17 ± 0.18b
GBH 8.86 ± 2.13a 5.02 ± 1.19a 5.94 ± 0.97a 4.39 ± 0.69a 4.84 ± 0.65a
RES + GBH 5.00 ± 0.74b 3.80 ± 0.23b 4.47 ± 0.26b 3.55 ± 0.48b 3.52 ± 0.25b
P value 0.000 0.000 0.000 0.002 0.000
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Table 2. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the glutathione (GSH) levels in the whole blood, liver, kidney, heart, and brain homogenates of the rats.

Groups Blood (nmol ml–1) Liver (nmol g–1 tissue) Kidney (nmol g–1 tissue) Heart (nmol g–1 tissue) Brain (nmol g–1 tissue)
Control 24.51 ± 2.29ab 5.86 ± 0.47b 6.03 ± 0.65b 4.31 ± 0.14a 4.70 ± 0.30a
RES 26.58 ± 2.48a 6.74 ± 0.52a 6.62 ± 0.35a 4.48 ± 0.36a 4.93 ± 0.91a
GBH 14.75 ± 0.85c 4.80 ± 0.23c 4.93 ± 0.22c 3.85 ± 0.14b 3.55 ± 0.25b
RES + GBH 23.74 ± 3.22b 5.67 ± 0.34b 5.78 ± 0.33b 4.22 ± 0.29a 4.61 ± 0.35a
P value 0.000 0.000 0.000 0.001 0.000
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Effects on antioxidant enzymes

Among antioxidant enzymes, SOD and CAT activities were determined as indicated respectively in Tables 3 and 4 in the rats’ erythrocytes and brain, heart, liver and renal tissues. A significant decrease occurred in the erythrocyte SOD activity (p < 0.05) of the rats to which GBH was administered, and no significant change occurred in their brain, heart, liver, and renal tissues (p > 0.05). Similarly, it was observed that a significant decrease occurred in the erythrocyte CAT activity (p < 0.05) of the rats to which GBH was administered, but no significant change occurred in the brain, heart, liver and renal tissues (p > 0.05) of the rats. Moreover, in the Res + GBH group, the Res that was administered improved the changes of erythrocyte SOD and CAT activities caused by GBH.

Table 3. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the superoxide dismutase activity (SOD) in the erythrocyte, liver, kidney, heart and brain homogenates of the rats.

Groups Erythrocyte (U gHb–1) Liver (U μg–1 protein) Kidney (U μg–1 protein) Heart (U μg–1 protein) Brain (U μg–1 protein)
Control 36.34 ± 7.20b 1.09 ± 0.18 2.70 ± 0.27 1.84 ± 0.57 5.28 ± 0.89
RES 30.96 ± 5.33b 1.08 ± 0.03 2.69 ± 0.31 2.00 ± 0.38 5.24 ± 0.92
GBH 45.07 ± 4.50a 1.21 ± 0.14 2.80 ± 0.30 1.79 ± 0.74 5.12 ± 0.91
RES + GBH 36.43 ± 4.57b 1.16 ± 0.19 2.76 ± 0.34 1.89 ± 0.44 5.21 ± 0.83
P value 0.001 0.314 0.899 0.906 0.989
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Table 4. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the catalase activity (CAT) in the erythrocyte, liver, kidney, heart and brain homogenates of the rats.

Groups Erythrocyte (k gHb–1) Liver (k μg–1 protein) Kidney (k μg–1 protein) Heart (k μg–1 protein) Brain (k μg–1 protein)
Control 18.71 ± 1.79a 0.72 ± 0.27 0.81 ± 0.10 0.15 ± 0.07 0.03 ± 0.00
RES 17.90 ± 1.00ab 0.85 ± 0.25 0.83 ± 0.28 0.09 ± 0.02 0.02 ± 0.00
GBH 12.30 ± 1.28c 1.17 ± 0.41 0.93 ± 0.44 0.09 ± 0.03 0.02 ± 0.01
RES + GBH 16.56 ± 1.07b 1.00 ± 0.53 0.84 ± 0.18 0.16 ± 0.07 0.02 ± 0.00
P value 0.000 0.185 0.857 0.650 0.233
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Effects on serum biochemical parameters

Glyphosate-based herbicide (375 mg kg–1, with oral gavage) administrations significantly increased the Tp, AST, ALT, ALP, Albumin, Urea, Creatinine, CK-MB and cTn-I levels (p < 0.05). On the contrary, treatment with Res improved the changes in the serum biochemical parameters caused by GBH in the male Wistar rats (Table 5).

Table 5. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the serum biochemical parameters as serum levels of total protein (Tp), aspartate aminotransferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), albumin, urea, creatinine, creatine kinase-MB (CK-MB) and cardiac troponin-I (cTn-I) of the rats.

Groups and parameters Control RES GBH RES + GBH P value
Tp (g dL–1) 65.82 ± 1.96b 65.01 ± 1.20b 89.47 ± 2.28a 64.60 ± 2.05b 0.000
AST (U L–1) 30.87 ± 3.99d 38.47 ± 2.87c 60.91 ± 1.67a 49.01 ± 7.89b 0.000
ALT (U L–1) 40.88 ± 4.28c 48.88 ± 1.30b 71.91 ± 4.18a 50.81 ± 10.69b 0.000
ALP (U L–1) 80.50 ± 2.53c 89.13 ± 2.48b 125.21 ± 8.02a 92.91 ± 7.21b 0.000
Albumin (g dL–1) 42.26 ± 2.00b 39.29 ± 3.84b 55.16 ± 3.45a 40.51 ± 2.10b 0.000
Urea (mg dL–1) 56.18 ± 3.32b 45.38 ± 3.06c 66.08 ± 6.39a 53.75 ± 2.47b 0.000
Creatinine (mg dL–1) 0.19 ± 0.03c 0.16 ± 0.03c 0.29 ± 0.04a 0.23 ± 0.03b 0.000
CK-MB (IU L–1) 165.92 ± 25.22c 159.13 ± 10.59c 389.99 ± 23.98a 211.04 ± 38.24b 0.000
cTn-I (pg ml–1) 0.47 ± 0.12d 2.58 ± 0.25c 6.21 ± 0.47a 3.90 ± 0.22b 0.000
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Histopathological examination

The histopathological changes in the organs of the rats in the groups are identified in detail and shown in Fig. 1. Degenerative changes and focal gliosis (Fig. 1A3) were encountered in the brain tissues in the GBH group. Apoptotic heart muscle cells and hemorrhage areas (Fig. 1B3) were seen in the heart tissues. Dilatation of sinusoids and hyperemia and mononuclear cell infiltration (Fig. 1C3) were observed in the liver tissues of the rats. In the renal tissue of rats, degeneration in the tubular epithelial cells and dilatation and vacuolar degeneration in the glomerulus Bowman's capsules (Fig. 1D3) were found.

Fig. 1. The effect of resveratrol (Res) on damage induced by glyphosate-based herbicide in the brain (A), heart (B), liver (C) and kidney (D) of the rats. Representative figures were stained with H&E. The original magnification was ×20 and the scale bars represent 100 μm. Arrows and arrow heads indicate focal gliosis and neuronal degenerations (A3) and focal gliosis (A4) in the brain, apoptotic heart muscle cells and hemorrhage areas (B3) and hyaline degenerations in the heart (B4), sinusoidal dilatation and hyperemia (C3), mononuclear cell infiltration in the liver (C4), and degenerations in the tubular epithelial cells and dilatation and vacuolar degeneration in the glomerulus Bowman's capsule in the kidney (D3) of rats. (1) Control group, (2) Res group, (3) animals treated with 375 mg kg–1 day–1: GBH group, (4) animals treated with 20 mg kg–1 day–1 Res and 375 mg kg–1 day–1 GBH: Res + GBH group.

Fig. 1

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In the GBH group, to which Res was administered, very few histopathological changes were observed in the brain and heart tissues (Fig. 1A4 and B4, respectively). In the control group (Fig. 1A1, B1, C1 and D1) and the group to which Res was administered (Fig. 1A2, B2, C2 and D2), histopathological changes were observed to be normal in the brain, heart, liver and renal tissues, respectively to the figures.

Discussion

Herbicides form a significant class of pesticides, and they are frequently used for weed control both in Turkey, which is an agricultural country, and in the world in general. The effects of Res on oxidative stress caused by pesticides were investigated in different studies.27,38 However, to the best of our knowledge, this is the first experimental study to report the antioxidant effects of Res on oxidative stress induced by glyphosate-based herbicide.

Studies showed that pesticides may disturb the balance between prooxidants and antioxidants and cause membrane damage in the body as a result of LPO.39 MDA is one of the primary oxidation products of the peroxidation of poly-unsaturated fatty acids, and an increase in MDA levels is the most important indicator of LPO. GSH takes on important tasks as a non-enzymatic antioxidant in the antioxidant defense system of the body. GPx not only is the co-substrate of different antioxidant enzymes such as glutathione-S-transferase (GST) but also directly plays a role in scavenging free radicals.40 They generate conjugate forms by reacting with xenobiotics such as pesticides. Therefore, the decrease in GSH levels after exposure to glyphosate-based herbicides may be based on the consumption of GSH in a conjugation reaction or a decrease in its biosynthesis. A previous study revealed that MDA levels increased in case of oxidative stress caused by GBH, it caused a significant decrease in GSH levels, MDA levels decreased, and GSH levels increased with the application of Ginkgo biloba.17 Jasper et al.41 reported that MDA levels increased in both doses when they administered it to rats orally in doses of 50 and 500 mg kg–1 for 15 days. In our findings, similar to previous studies, an increase in blood and tissue MDA levels and a decrease in GSH levels were observed as a result of sub-chronic administration of glyphosate-based herbicide to rats for 8 weeks. This may be a result of the fact that glyphosate-based herbicide causes an increase of LPO in the cell membrane and consumes GSH sources. MDA and GSH were on the normal levels in the GBH group to which Res was administered, and this may be a result of Res decreasing the peroxidative activity among the cells.

The first line of defense against oxidative stress is formed by antioxidant enzymes such as SOD and CAT which transform superoxide anions into hydrogen peroxide and water. These enzymes move in coordination, and cells may be pushed into oxidative stress if any change occurs in the level of these enzymes.42 Moreover, few studies were carried out using mammals, and most previous studies were carried out using herbicide-sensitive aquatic organisms.43–45 Therefore, investigating the effects of glyphosate on mammals is important to form the relevant toxicity parameters and determine potential treatments in case of occupational or accidental intoxication. In this study, where the effects of glyphosate on antioxidant enzymes in mammalian organisms were investigated, GBH was orally administered to rats for 56 days, and a significant decrease was observed in the rats’ blood SOD and CAT activities. The decrease in the activity of these enzymes in the blood after administration of GBH may be a result of the increase in the superoxide anions in the blood. On the other hand, it was found that the activities of these enzymes were regained significantly when Res was administered with GBH. These results indicated that free radicals were scavenged by Res, a strong antioxidant.

Apart from oxidative stress markers, the markers of liver, renal and heart functions are important for bio-monitorization of exposure to environmental pollutants. We showed that, in comparison to the rats in the control group, the serum Tp, AST, ALT, ALP, albumin, urea, creatinine, CK-MB and cTn-I levels increased with administration of GBH and were improved by oral administration of Res. As far as we know, this study is the first report on the effectiveness of Res on the biochemical marker levels of rats under oxidative stress caused by GBH. A decrease in serum albumin and Tp levels may contribute to inhibition of the oxidative phosphorylation process, and this may cause a decrease in protein absorption, a decrease in protein synthesis and an increase in the catabolic process.46 In this study, an increase was observed in the serum albumin and Tp levels, as well as in the markers of liver injury (AST, ALT and ALP), a reflex of hepatocyte damage induced by GBH. These results were in harmony with the findings obtained in other studies carried out on rats, on which toxicity was induced with glyphosate.16,17,41 Serum urea and creatinine are useful bioindicators in evaluating renal functions in both in vivo and in vitro studies. In this study, in comparison to the control group, the serum urea and creatinine levels were observed to increase in the group to which GBH was administered. Similarly, Çavuşoğlu et al.17 also reported that serum urea and creatinine levels increased in rats to which glyphosate was administered intraperitoneally at a dose of 50 mg kg–1. CK-MB and cTn-I are regarded as the most typical markers of myocardial damage. Although there are many in vivo,47 ex vivo48 and in vitro49,50 studies which have investigated the effects of glyphosate on the heart and the development of the heart until now, no study on CK-MB and cTn-I levels in rats has been encountered yet. In this study, in comparison to the control group, the serum CK-MB and cTn-I levels were found to be very high in the group to which GBH was administered. On the contrary, in the group to which Res was administered, it was seen that Res decreased these increasing values depending on the cardiotoxicity caused by GBH's reducing effect.

Glyphosate-based herbicide caused significant histopathological changes in the brain, heart, liver and kidney (Fig. 1 and Table 6). Neuronal changes and focal gliosis were observed in the brains of rats in the group to which only GBH was administered; these included apoptosis of heart muscle cells and hyaline degeneration and cardiac hemorrhage in the heart, dilatation of the sinusoids, hyperemia, degeneration of hepatocytes and Kupfer cell activation in the liver and expansion of the Bowman's capsule and vacuolar degeneration in the kidney. Hamdaoui et al.51 showed that, as a result of oral administration of Kalach 360 SL, which is a commercial product of GBH, to rats for 60 days, the substance caused peritubular inflammatory reaction, nephrosis, fragmented glomerulus, necrotic epithelial cells and tubular expansion in the kidney. Moreover, Alp et al.15 stated in a study they carried out that oral administration of GBH at a dose of 4 mg kg–1 day–1 to rats caused liver and renal damage, and this damage was reduced with CAPE, a flavonoid antioxidant. On the contrary, the group to which only GBH was administered, since Res has strong antioxidant and anti-inflammatory characteristics,52 kept cellular damage caused by GBH in the liver, renal, heart and brain tissues.

Table 6. Effects of sub-chronic exposure to glyphosate-based herbicide (GBH-375 mg kg–1), resveratrol (Res-20 mg kg–1) and Res (20 mg kg–1) + GBH (375 mg kg–1) on the histopathological changes in the brain, heart, liver and kidney of the rats.

Tissue Histopathological changes Control Res GBH Res + GBH
Brain Neuronal degeneration and focal gliosis –(7/7) –(7/7) +(3/7); ++(3/7); +++(1/7) –(4/7); +(3/7)
Heart Apoptosis of cardiac muscle cells and hyaline degeneration –(7/7) –(7/7) +(5/7); ++(2/7) –(4/7); +(3/7)
Cardiac hemorrhage –(7/7) –(7/7) +(3/7); ++(4/7) –(4/7); +(3/7)
Liver Dilatation of sinusoids, hyperemia, degeneration of hepatocytes –(7/7) –(7/7) +(5/7); ++(2/7) –(4/7); +(3/7)
Kupffer cell activation –(7/7) –(7/7) +(4/7); ++(3/7) –(3/7); +(4/7)
Kidney Dilatation of Bowman's space and vacuolar degeneration –(7/7) –(7/7) –(1/7); +(4/7); ++(2/7) –(5/7); +(2/7)
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Conclusion

In conclusion, we are reporting, for the first time, the protective effects of Res against oxidative damage which emerges depending on sub-chronic GBH exposure in rats. In the light of the findings in this study, we may state that Res prevents LPO with its strong antioxidant effects, and it has the ability of decreasing sub-chronic toxicity induced by GBH by supporting the antioxidant defense system. Therefore, Res is likely to be used as promoter for routine treatment by decreasing tissue damage induced by GBH.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgments

This study was supported by Afyon Kocatepe University Scientific Research Project Coordination Unit (16.VF.12). The study was also presented at the 5th International Multidisciplinary Congress of Eurasia, 24-26 July, 2018, in Barcelona, Spain.

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