Protective effects of chlorogenic acid against glyphosate-induced organ and blood toxicity in Wistar rats

We used a Knock-out^® glyphosate formulation marketed in Turkey by its producer Hektaş (Gebze, Turkey). The active ingredient in this herbicide is the 48 % isopropylamine salt of glyphosate. We selected this brand due to its popularity in controlling weeds resistant to other high glyphosate herbicides.

CGA (Cat. No. C3878, purity ≥95 %) was acquired from Sigma (Sigma-Aldrich, Shanghai, China).

For 8-hydroxy-2′-deoxyguanosine (8-OHdG) determination we used the ELISA kit from the Shanghai Sunred Biological Technology (Shanghai, China, Cat. No. 201-11-0032).

All other reagents and chemicals mentioned below were of analytical grade. GBH and CGA were diluted in distilled water.

For the experiment we used 42 adult (10–12 weeks old) male Wistar rats, weighing 200–250 g, acquired from the Experimental Research and Application Centre at Afyon Kocatepe University in Afyonkarahisar, Turkey. The animals were housed in polypropylene cages under standard laboratory conditions of 55±5 % relative humidity, 22±2 °C room temperature, and 12-hour light/dark cycle, with free access to food pellets and tap water.

The animals were randomly divided into six groups of seven rats to receive distilled water or the following daily doses of CGA, GBH, or their combination by gavage for 49 days in a row. The control group received 0.5 mL of distilled water (otherwise used as solvent for CGA). The CGA50 group received 50 mg/kg of CGA dissolved in 0.5 mL of distilled water. The GBH group received 800 mg/kg of GBH (one tenth of their LD₅₀) dissolved in 0.5 mL of distilled water. The CGA12.5+GBH group received 12.5 mg/kg CGA one hour before receiving 800 mg/kg of GBH. The CGA25+GBH group received 25 mg/kg of CGA as described for the previous group. The CGA50+GBH group received 50 mg/kg of CGA as described for the previous group.

The dosage of GBH administered to the animals was determined by Turkmen and Dogan (13), whereas the dosage of CGA was taken from Qi et al. (14).

The study protocol was approved by the Ethics Committee for Animal Experiments of the Afyon Kocatepe University (approval No. 2018-49533702/65).

On day 50, we took blood from the animals under mild sevoflurane anaesthesia (1–3 % concentration in oxygen) by cardiopuncture into heparinised and non-heparinised tubes. Blood was centrifuged at 600 g and 4 °C for 15 min within 30 min of blood collection to precipitate the erythrocytes and extract plasma and serum. Serum was used for biochemical measurements of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, creatinine, malondialdehyde (MDA), and reduced glutathione (GSH), while plasma was used to measure 8-OHdG.

After separating the buffy coat, we washed erythrocytes three times in isotonic saline, placed them into vials with an equal volume of isotonic saline, and stored at approximately −20 °C for 3 days. At the end of the storage period, we obtained the erythrocyte lysate for analysis by inducing haemolysis. To ensure complete cell lysis, a repeated freeze-thaw process was applied. This approach allowed for the preparation of a homogeneous lysate for subsequent analyses. To prevent the erythrocyte suspensions from being disrupted due to osmotic pressure, we added five times the volume of cold deionised water. This ratio ensured controlled haemolysis of the cells, allowing for the preparation of a homogeneous lysate suitable for enzymatic analyses.

The erythrocyte lysate was then stored at 4 °C for 3 days before measuring SOD and CAT activity. This storage period was chosen based on previous studies (15), which found it sufficient to maintain enzyme stability and ensure accurate activity measurements.

The brain, heart, liver, and kidney were promptly dissected, rinsed with ice-cold 0.9 % NaCl solution, and homogenised in a homogeniser (VWR International GmbH, Darmstadt, Germany) using 1.15 % potassium chloride buffer to obtain a 1:10 (w/v) homogenate. Homogenates were then centrifuged at 600 g and 4 °C for 15 min for the analysis of MDA, SOD, and CAT. To quantify GSH concentration in homogenates, the samples were centrifuged at 4900 g and 4 °C for 20 min.

Tissue specimens were preserved in 10 % neutral buffered formalin, dehydrated using a graded alcohol series, and embedded in paraffin. Sections were 5 µm thick, stained with haematoxylin and eosin (H&E), and analysed under a light microscope (Olympus BX51 with DP20 Microscopic Digital Image Analysis System, Tokyo, Japan) by a blinded pathologist.

Semi-quantitative scoring of brain, heart, liver, and kidney lesions followed the methodology outlined by Gibson-Corley et al. (16).

Serum levels of AST, ALT, ALP, urea, and creatinine were determined with the COBAS test kits (Roche Diagnostics Systems, Istanbul, Turkey) following the manufacturer’s instructions (17).

Serum MDA was determined using the method described by Yoshioka et al. (18), while tissue homogenate MDA was determined using the method described by Ohkawa et al. (19). Both methods rely on the spectrophotometry of the colour produced by the reaction between MDA and thiobarbituric acid (TBA) at 532 nm on a Shimadzu 1601 UV-VIS spectrophotometer (Shimadzu, Tokyo, Japan). MDA concentration was determined using the absorbance coefficient of the MDA-TBA complex and the results are reported in nmol/mL of blood or nmol/g of tissue.

GSH was quantified in serum and tissue homogenates using the method described by Sedlak and Lindsay (20). Optical density was measured spectrophotometrically (Shimadzu 1601 UV-VIS) at 412 nm and the results are reported as nmol/mL of blood or nmol/g of tissue.

SOD in erythrocyte lysate and tissue homogenates was measured as described by Sun et al. (21). The absorbance of blue formazan obtained from the reduction of nitro blue tetrazolium (NBT) by superoxide radicals was determined spectrophotometrically (Shimadzu 1601 UV-VIS) at 560 nm. SOD activity is expressed as U/g of haemoglobin (Hb) for erythrocytes or U/g of protein for tissue.

CAT activity in erythrocyte lysates and tissue homogenates was determined as described by Aebi (22). One unit of CAT activity corresponds to the amount of enzyme that decomposes 1 µmol of H₂O₂ per minute at pH 4.5 and 25 °C. CAT activity (k; nmol/min) was expressed as catalase/g of Hb for erythrocytes or catalase/g of protein for tissue.

Plasma 8-OHdG levels were measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit and a Multiskan Go microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA), in accordance with the Sunred ELISA kit protocols for rat 8-OHdG (Cat. No. 201-11-0032) assay. The absorbance was measured at 450 nm and the 8-OHdG concentration quantified using a standard curve and reported in ng/mL.

Hb was quantified in erythrocyte lysates using the cyanmethaemoglobin method as described by Drabkin and Austin (23). The protein concentration of the sample in tissue homogenates was evaluated using the colourimetric method established by Lowry et al. (24).

The sample size calculated with a power test using G*Power 3.1 software (Heinrich Heine University, Düsseldorf, Germany) to meet one-way ANOVA or Kruskal-Wallis test requirements for six independent groups. Based on the calculation, the required sample size was 114 animals, assuming an effect size of 0.4, an error probability (alpha) of 0.05, and a power (1-beta) of 0.9. However, due to practical constraints, including limitations in time, resources, and animal availability, a smaller sample size of 42 animals was used in this study. It is recommended to use the smallest number of animals possible in studies, in accordance with ethical principles outlined by the guidelines for animal research, such as those provided by the National Institutes of Health (NIH) and local ethical review boards. Since the sample size obtained with the power test was very large, similar studies were used to determine the sample size (25, 26). Despite the reduced sample size, statistical analyses were conducted with careful consideration of the limitations, and we believe that the findings are still meaningful and provide useful insights under the given conditions.

For statistical analysis of the data we used the SPSS version 20.0 (SPSS, Chicago, IL, USA) software package. The normality of distribution was established for all data with the Shapiro-Wilk test, and one-way ANOVA used for further analysis. In cases where differences were statistically significant (p<0.05), we used the post-hoc Duncan test to determine which groups were different. The results are presented as means ± standard deviations (SD). The level of significance was set at p<0.05.

AST, ALT, ALP, urea, and creatinine were higher in the GBH group than control (p<0.05), but CGA pre-treatment lowered them in a dose-dependent manner. There were no significant changes in the CGA alone group (Figure 1A–E).

Figure 1

The effects of GBH and CGA treatments on AST (A), ALT (B), ALP (C), urea (D), and creatinine (E) levels in the serum of Wistar rats. Values are expressed as mean ± SD of seven rats per group. Different superscripts (a–c) above the bar indicate significant difference between groups (p<0.05)

GBH significantly increased MDA levels in the serum, brain, heart, liver, and kidney tissues relative to the control and CGA50 group (p<0.05), but CGA pre-treatment lowered them in a dose-dependent manner, restoring them to normal (control) with the highest, 50 mg/kg dose (Table 1).

A similar pattern was observed with the antioxidant enzyme activities. GBH alone significantly lowered them (p<0.05), whereas CGA pre-treatment increased them in a dose-dependent manner. No significant change was observed in the CGA50 group compared to the control (Tables 2–4).

8-OHdG levels significantly increased in rats receiving GBH alone compared to control (p<0.05), but CGA pre-treatment lowered them, although never restoring them to control values. CGA alone treatment did not change them significantly compared to control (Figure 2).

Figure 2

The effects of GBH and CGA treatments on 8-OHdG levels in the plasma of Wistar rats. Values are expressed as mean ± SD of seven rats per group. Different superscripts (a–d) above the bar indicate a significant difference between groups (p<0.05)

Figure 3A–D shows histopathological changes in the brain, heart, liver, and kidney tissues. In the control and CGA50 groups their structure is normal, whereas the GBH group shows neuronal degeneration and vacuolation, vessel hyperaemia, and focal gliosis in the brain (Figure 3A3), myocardial hyaline degeneration and focal haemorrhage in the heart(Figure 3B3), expansion of sinusoids, binucleated hepatocytes in periportal areas, and infiltration by mononuclear cells in the liver (Figure 3C3), and hyaline cylinder formations within the lumens of the tubules in the kidney, accompanied by degenerative and necrobiotic changes in tubular epithelial cells (Figure 3D3).

Figure 3

H&E-stained slides showing the protective effects of CGA against GBH-induced damage in the brain (A), heart (B), liver (C), and kidney (D) of Wistar rats(20× magnification; scale bar 200 µm). 1 – control; 2 – CGA50; 3 – GBH; 4 – CGA12.5+GBH; 5 – CGA25+GBH; 6 – CGA50+GBH. Slides A3–A6: arrows indicate neuronal degeneration and focal gliosis; arrowheads indicate hyperaemia in the vessels and curved arrows indicate neuronal vacuolation in the brain. Slides B3–B6: arrows indicate myocardial hyaline degeneration; arrowheads indicate focal haemorrhage in the heart. Slides C3–C6: arrows indicate mononuclear cells infiltration in periportal areas; arrowheads indicate sinusoidal dilatation and curved arrows indicate binucleated hepatocytes in periportal areas in the liver. Slides D3–D6: arrows indicate degenerative and necrobiotic changes in tubular epithelial cells; arrowheads indicate hyaline cylinder formations within the lumens of tubules in kidney tissue

In the CGA pre-treated groups (particularly CGA50+GBH), only slight histopathological changes were observed in the brain, heart, liver, and kidney compared to the GBH group (Figures 3A4–6, 3B4–6, 3C4–6, and 3D4–6, respectively).

Semi-quantitative scoring of tissue lesions (0 – no, 1 – mild, 2 – moderate, and 3 – severe) in Table 5 reflects the patterns reported above, with no scores in the control and CGA50 alone groups and the highest scores in the GBH alone group.