Introduction

Zearalenone (ZEN) or the F-2 or RAL (resorcylic acid lactone) is a mycotoxin primarily produced by the fungus Fusarium graminearum (Zhang et al. 2016; Zinedine et al. 2007). ZEN contaminates agricultural products worldwide (Darwish et al. 2014; Wang et al. 2020). Its exposure results in harmful effects on humans and animals’ health (Aiko and Mehta 2015; Bennett and Klich 2003; Binder et al. 2017). ZEN is commonly found in the moderate climate zone, and exhibits high stability during storage and does not degrade when exposed to high temperature (Appell et al. 2017; Bai et al. 2018).

Studies have shown that exposure to ZEN is hepatotoxic (Bai et al. 2018), alters immune and hematologic function (Ben Salem et al. 2015; Wang et al. 2018b) and is systemically toxic (Rai et al. 2019). Thus elimination of ZEN from contaminated produce is of great importance (Wang et al. 2018c), as ZEN can be absorbed into the body, metabolised via hydroxylation to form either toxic α- and β-Zearalenol (α- and β-ZEL) (Olsen et al. 1987; Rai et al. 2019) in the liver (Malekinejad et al. 2006), and the gastrointestinal tract (Biehl et al. 1993). α-ZEL happens to be the more toxic of the two metabolites. (Brodehl et al. 2014) After absorption, ZEN is highly distributed in the liver (Rai et al. 2020). It also gets distributed to the kidney, intestine, fatty tissues, reproductive organs like the uterus, testes, and ovaries (Jiang et al. 2012; Koraichi et al. 2012). Plants and fungi can also metabolize ZEN by attaching polar residues like glucose and sulphates to mask the mycotoxin (Berthiller et al. 2013; Binder et al. 2017). Primarily, ZEN and its metabolites are glucuronidated in the liver and intestine of humans and animals at the sterically unobstructed phenolic 14-hydroxy group to form ZEN-16-GlcA (Pfeiffer et al. 2010). Glucuronic acid conjugation of α-ZEL mediates its final metabolic fate and excretion (Videmann et al. 2008). Recent studies revealed that α- and β-Zearalenol mediate their toxicities in the liver, kidney and other cells of the body by upregulating the activities of the glucose-regulated protein (GRP78) and, Growth arrest and DNA-damage induced protein (GADD34), resulting in endoplasmic reticulum stress (ERS)(Ben Salem et al. 2016). The induction of ERS is characterized by increased reactive oxygen species (ROS) generation (Marin et al. 2013), inflammatory responses (Jia et al. 2014), lipid peroxidation (Ben Salem et al. 2016; Wang et al. 2019), thereby decreasing endogenous antioxidant activities.

Considering the adverse impacts that ZEN and other mycotoxins have on human and livestock well-being, it is of immense public health significance to create plausible stratagems for neutralizing mycotoxins (Kowalska et al. 2016). Although the most effective method is prevention, it is relatively impractical to avoid mycotoxin contamination of crops.

Several strategies have been devised to reduce human and livestock exposure to ZEN. Among these strategies is microbial degradation of ZEN in situ (Wang et al. 2018c). Adding Bacillus velezensis A2 as an additive could effectively remove ZEN in feed and protect mice against ZEN toxic damage from the contaminated diet (Wang et al. 2018a) and wheat treatment with manganese to protect Zearalenone infection and its derivatives (Gzyl-Malcher et al. 2019). Supplementation with the carotenoid Crocin has been demonstrated to help mitigate ZEN-induced oxidative stress in the liver and kidney (Ben Salem et al. 2015) of mice. Furthermore, in vitro studies revealed that Kefir protect against ZEN toxicity and oxidative damage in cultured HCT-116 cell line (El Golli-Bennour et al. 2019) and opuntia ficus-indica protected Balb/C mice against oxidative damage induced by Zearalenone (Zourgui et al. 2008). Polyphenolic compounds, which can confer beneficial effects, and serve as chemoprotective pharmacological agents in preventing ZEN toxicity, have been investigated in in vitro and systemically. Gallic acid (GA, 3,4,5-trihydroxybenzoic acid) is a polyphenolic compound naturally present in some herbal formulation used in phytomedicine; with various biological properties which aid its ability to protect tissues and organs against the injuries induced by toxic compounds(Constant 1997; Mansouri et al. 2013; Owumi et al. 2020a). GA is readily absorbed in the gastrointestinal tract compared to several other polyphenolic acids, although its bioavailability is limited due to rapid metabolism and high elimination rate (Shahrzad et al. 2001). GA has been documented to exhibit antioxidant and anticancer activities (Badhani et al. 2015), substantial anti-inflammatory property (Banerjee et al. 2015), and a beneficial modulator of the immune system fending off microbial infection (Choubey et al. 2018). GA is recognized as safe by the Food and Drug Administration and has been used as an antioxidant in food, cosmetics, and pharmaceuticals (Shahrzad et al. 2001). GA has been reported to elicit protective effects against other mycotoxins – aflatoxin B1- which induces hepatorenal damage and oxido-inflammatory in rats’ stress (Owumi et al. 2020b). However, the shortage of data validating the protective effects of GA against ZEN-induced toxicity in relevant animal models necessitated this study.

Herein, we report in vivo findings on ZEN and GA’s effects on hepatorenal function in albino Wistar rats. We observed that GA ameliorated ZEN-induced hepatorenal dysfunction by decreasing oxidative stress, lipid peroxidation and inflammation in rats. Furthermore, GA protected against histopathological changes in the liver and kidney of rats exposed to ZEN for 28 consecutive days. To our knowledge, there is no reported study in the literature describing the protective in vivo effects of GA on ZEN-induced hepatorenal toxicity and oxidative damage, inflammatory responses and induction of apoptosis. This study is of significance, giving GA and gallate esters everyday use as antioxidants in cosmetics, food, and pharmaceutical industries (Monteiro et al. 2017; Ow and Stupans 2003).

Materials and methods

Chemicals

Zearalenone (≥ 99 %) and gallic acid were purchased from AKSci Scientific (San Mateo, CA, USA) and Sigma-Aldrich Chemical Company (St. Louis, Mo), respectively. 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), hydrogen peroxide (H2O2), 5’, 5’-dithiobis-2-nitrobenzoic acid (DTNB), epinephrine and glutathione (GSH) were procured from Sigma Aldrich Chemical Co. (MO, USA). Interleukin-1β (IL-1β), interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α), Enzyme-Linked Immunosorbent Assay (ELISA) kits were purchased from E-labscience Biotechnology (Beijing, China). Other chemicals used for these experiments were pure analytical grade and purchased from British Drug Houses (Poole, Dorset, U.K.).

Animal model and care for experimental animals

Adult Wistar rat (sex: male; ages: 10 weeks old; weight: 150 ± 30 g; n = 50) were procured from the Primate Colony Animal House Facility, College of Medicine, University of Ibadan and housed in polycarbonate cages in a well-ventilated animal house with free access to standard rat pellets (Ladokun™ Feeds, Ibadan, Nigeria) and water. Rats were allowed to adapt (one week) to their new environment preceding experimentation for a photoperiod of 12-hr light: 12-hr dark cycle and adequately cared for as specified by ‘Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. All experiment was performed following the approved procedures by the University of Ibadan Ethical Committee and the guidelines of the United States National Academy of Sciences.

Experimental protocol

Experimental rats were assigned randomly to five groups consisting of ten rats each post acclimatization. They were dosed per os (p.o.) either with ZEN:100 µg/kg or GA: 20 and 40 mg/kg body weight and in combination in co-treated groups as required for four consecutive weeks as:

  • Control: treated with corn oil alone (2 ml/kg; p.o.).

  • Zearalenone (ZEN) alone: treated with ZEN (100 µg /kg; p.o).

  • Gallic Acid (GA) alone: treated with GA (40 mg/kg; p.o).

  • Zearalenone + GA1: treated with ZEN + GA (100 µg /kg + 20 mg/kg; p.o).

  • Zearalenone + GA2: treated with ZEN + GA (100 µg /kg + 40 mg/kg; p. o).

The doses of ZEN (100 µg/kg) (Cressey and Thomson 2006; Shin et al. 2009) and GA (20 and 40 mg/kg) (Garud and Kulkarni 2018; Owumi et al. 2020b) bodyweights used for this study were based on earlier published reviews. On day 29, after the last treatment, terminal body weights of experimental animals were recorded before exsanguination via the retro-orbital venous plexus to collect blood samples into pre-labelled plain tubes. Subsequently, rats were sacrificed by dislocation of the cervical vertebrae light ether anaesthesia. Serum was obtained by centrifugation (4000 g; 10 min; 40 C) of blood allowed to clot at room temperature. Subsequently, serum samples were preserved at -20 °C pending routine analysis of hepato-renal function biomarkers.

Evaluation of biomarkers of hepato-renal function

Serum levels of biomarkers of hepato-renal function such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), urea and creatinine were determined using commercial kits (Randox™ Laboratories Limited, Crumlin, U.K.).

Preparation of tissue homogenates and evaluation of biomarkers of hepato-renal antioxidant status and lipid peroxidation

Homogenization of the rat liver and kidney samples were performed in Tris–HCl buffer (50 mM; pH 7.4), the tissue homogenates were centrifuged (10,000 g; 15 min; 4 °C) to obtain the supernatants utilized in examining for the biomarkers of apoptosis, inflammation, and oxidative stress. Hepato-renal protein concentration was determined according to Lowry’s method (Lowry et al. 1951). Superoxide dismutase (SOD) activity was evaluated using the method described by Misra and Fridovich (Misra and Fridovich 1972), while catalase (CAT) enzyme activity was assessed by Claiborne’s technique (Clairborne 1995; Owumi et al. 2019b). Glutathione-S-transferase (GST) and glutathione peroxidase (GPx) enzyme activities were evaluated using Habig’s (Habig et al. 1974; Owumi et al. 2019b) and Rotruck’s (Owumi et al. 2020a; Rotruck et al. 1973) protocols, respectively. Furthermore, Glutathione (GSH) and Lipid peroxidation – by measuring malondialdehyde (MDA)- levels were assayed for, using the methods of Ellman (Ellman 1959) and Okhawa (Ohkawa et al. 1979), respectively.

Evaluation of reactive oxygen and nitrogen species (RONS) level

RONS generated in the hepatic and renal tissues was estimated with an established protocol that depends on RONS-dependent oxidation of 2’,7’-dichlorodihydrofluorescin diacetate (DCFH-DA) to 2’,7’-dichlorofluorescin (DCF) (Owumi and Dim 2019; Perez-Severiano et al. 2004). Quickly, the reaction mixture {sample + potassium phosphate buffer (0.1 M; pH 7.4) + DI water + DCFH-DA} was prepared with minimal exposure to air. Fluorescence emission of DCF arising from DCFHDA oxidation was evaluated for 10 min at an emission and excitation wavelengths of 525 and 488nm. The readings were obtained with M384 SpectraMax™ Multi-modal plate reader (Molecular Devices, San Jose, CA, USA). The proportion of DCF formation was expressed as a percentage fold over the control group.

Estimation of inflammatory biomarkers

In the hepato-renal samples, Nitric oxide (NO) levels were determined by the methods of Green (Green et al. 1982). The reaction mixture containing equal volume (sample + Griess reagent) was incubated for 15 min, and absorbance readings recorded at 540 nm. NO levels were extrapolated from a generated standard curve and expressed as Units/mg protein. Myeloperoxidase (MPO) activity was assayed following the previously described method by Granell (Granell et al. 2003; Owumi et al. 2020c).

Furthermore, hepatic and renal concentrations of IL-10 and IL-1β were evaluated using ELISA Kits from E-labscience (Beijing, China) following the manufacturer’s protocol. All readings were obtained with M384 SpectraMax™ Multi-modal plate reader (Molecular Devices, San Jose, CA, USA).

Histopathological examination of liver and kidney

Liver and kidney samples that were earlier fixed in phosphate-buffered formalin (10 %; for three days) were randomly selected for histological analysis. The samples were embedded in paraffin following sequential dehydration processes. Subsequently, microtome sliced tissue Secs. (4–5 μm) were fixed on charged microscopic glass slides before hematoxylin and eosin (H & E) staining (Bancroft and Gamble 2008; Owumi et al. 2019a). Histopathologic examination of hepatic and renal tissue was conducted with the aid of a light microscope (Leica DM 500, Germany), pathological abnormalities were scored by a pathologist blinded to experimental slides. Images were captured using a digital camera (Leica ICC50 E, Germany) attached to the microscope.

Statistical analysis

Experimental data were expressed as mean ±S. D, n = 10 per group. The values obtained were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons tests, performed using GraphPad Prism version 8.3.0 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com; Statistically significant values were set at p < 0.05.

Results

GA caused a reduction in terminal body weight and relieved hepatic and renal toxicity in ZEN treated rats

The bodyweight of rat treated with ZEN alone increased by 7.33 % compared to the untreated control. ZEN + GA1 and ZEN + GA2 co-treated groups also differed by 4.89 and 3.30 %, respectively, compared to the ZEN alone treated group Table 1. A 4.54 % increase in the body weights of rats treated with GA alone relative to the untreated control was also observed. ZEN alone treated exhibited almost no treatment-related body and relative organ weight gains. The effect of GA on ZEN-induced hepatorenal toxicity was evaluated by creatinine and urea and hepatic transaminases (ALT, AST, ALP and GGT) level in the serum. Figure 1 describes GA’s influence on biomarkers of hepatic and renal toxicity in ZEN-treated rats. Exposure to ZEN (100 µg/kg) increased significantly (p < 0.05) biomarkers of hepatic and renal injury in rat serum compared to the control rats. The AST, ALT, ALP, and GGT increased by 135.4 %, 222.1 %, 110.9 %, and 111.1 %, respectively, compared to the control group. In rats co-treated with GA (20 and 40 mg/kg) and ZEN, GA reduced ZEN-mediated increases in creatinine and urea levels, in addition to ALT, AST, ALP and GGT activities in rat serum compared to ZEN alone treated rats Fig. 1. Treatment with GA (40 mg/kg) alone caused almost no treatment-related changes in hepatorenal biomarkers of toxicity. Treatment with GA alone at 40 mg/kg for 28 successive days caused almost no treatment-related effect on these biomarkers.

Table 1 Effect of Zearalenone (ZEN) and gallic acid (GA) on the relative organ weight in treated rats
Full size table
Fig. 1
figure 1

Effect of gallic acid (GA) on hepatorenal functional enzymes in Zearalenone (ZEN)-treated rats. Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance. AST: aspartate aminotransferase; ALT: alanine aminotransferase; GGT: gamma-glutamyl transferase; ALP: alanine aminotransferase; Creatinine and Urea

Full size image

GA treatment restored ZEN-induced reduction in hepatic and renal antioxidant enzymes activities, GSH, and total thiol levels in rats

The influence of GA on the oxidative stress indices in ZEN-treated rats was evaluated and the results presented in Figs. 2 and 3. Results showed a decrease (p < 0.05) in the activities of SOD CAT and total thiols (TSH) in the liver of ZEN treated rats by 90.18 %, 71.37 and 45.01 %, respectively and in the kidney by 94.2 %, 29.7 %, and 29 % respectively compared to the control Fig. 2. There was also a decrease (p < 0.05) in the activities of GST, GPx, and GSH in the liver of ZEN alone treated rats by 83.78 %, 47.25 %, and 48.65 %, respectively, and in the kidney of ZEN alone treated rats by 54.4 %, 32.69 %, and 56.4 % respectively when compared to the control groups Fig. 3. Co-treatment with GA at 20 and 40 mg/kg decreased (p < 0.05) the reduction in the activities of these antioxidant enzymes (SOD, CAT, GST and GPx) and significantly restored their hepatic and renal activities similar to control levels in ZEN + GA2 (40 mg/kg) -treated rats. Treatment with GA alone significantly increased hepatorenal GSH and TSH level relative to both control cohorts - untreated controls and ZEN alone.

Fig. 2
figure 2

Effect of gallic acid (GA) on SOD, CAT activities, and TSH levels in the liver and kidney of rats treated with Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance. SOD: superoxide dismutase; CAT: catalase, and TSH: total thiol group

Full size image
Fig. 3
figure 3

Effect of gallic acid (GA) on GST, GPx activities, and GSH levels in the liver and kidney of rats treated with Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D.; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance. GST: glutathione-S-transferase; GPx: glutathione peroxidase, and GSH: glutathione

Full size image

GA attenuates ZEN-induced hepatorenal RONS generation, xanthine oxidase (XO) activity and induction of lipid peroxidation (LPO) in rats

The effect of GA on RONS generation, xanthine oxidase (XO) activity and subsequent lipid peroxidation (LP) in experimental rats were evaluated, and the results are presented in Fig. 4. Treatment with ZEN alone evidently increased (p < 0.05) RONS levels in the liver (40.79 %), kidney (56.2 %) relative to the untreated control group. Also, LPO levels in the kidney and liver of ZEN alone treated rats were also increased (p < 0.05) by 125.6 and 50 %, respectively, compared to untreated control. Co-treatment with GA protected against renal and hepatic lipid peroxidative damage evidenced by decreases (p < 0.05) in RONS and LPO levels relative to rats treated with ZEN alone. Reduction in RONS levels in groups co-treated with ZEN + GA1 and ZEN + GA2 was 23.14 and 34.43 %, respectively, compared to the ZEN alone group in the liver and 22.4 and 34.6 % respectively compared to ZEN alone group in the kidney. LPO levels in groups co-treated with ZEN + GA1 and ZEN + GA2 were reduced (p < 0.05) by 20.37 and 42.59 % in the liver, 24.4 and 65.6 % in the kidney relative to ZEN alone treated rats. Furthermore, XO activity was decreased (p < 0.05) in the liver and kidney of ZEN + GA treated rats dose-dependently by GA1: 16.5 and 22.8 %; and GA2: 55.7 and 42.3 %, respectively. ZEN alone treated rats exhibited high levels of XO activity in the liver (169.44 %) and Kidney (105.1 %) Fig. 4.

Fig. 4
figure 4

Effect of gallic acid (GA) on RONS, LPO levels, and XO activities in the liver and kidney of rats treated with Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D.; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance. RONS: reactive oxygen and nitrogen species; LPO: lipid peroxidation, and XO: xanthine oxidase

Full size image

GA suppresses ZEN mediated increase in biomarkers of inflammation in rats

The influence of GA on the biomarkers of inflammation was assessed in ZEN-treated rats’ liver and kidney. Figures 5 and 6 depict the impact of GA on biomarkers of inflammation, administration of ZEN alone increased (p < 0.05) the hepatic and renal MPO activity, and levels of NO, and IL-1β, and suppressed IL-10 levels relative to the untreated control group. In the liver, NO level and MPO activity in ZEN alone treated rats increased by 81.28 and 182.4 %, respectively, compared to the untreated control Fig. 5. NO and MPO in the kidney also increased by 80 and 93.2 % compared to the untreated control. In ZEN alone, treated rats level of IL-1β increased in the liver (361.5 %) and kidney (81.9 %), while IL-10 levels decreased in the liver (55 %) and kidney (68.57 %) relative to untreated control Fig. 6. Co-treatment with GA alleviated ZEN-mediated increases in the preceding biomarkers of inflammation. Group co-treated with ZEN + GA2 dose-dependently decreased (p < 0.05) IL-1β level and increased IL-10 levels relative to group co-treated with ZEN + GA1. The levels of IL-1β and IL-10 in the liver and kidneys of rats treated with GA only did not differ (p < 0.05) compared to the untreated control rats.

Fig. 5
figure 5

Effect of gallic acid (GA) on NO level, and MPO activity in the liver and kidney of rats treated with Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D.; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance. NO: nitric oxide; MPO: myeloperoxidase

Full size image
Fig. 6
figure 6

Effect of gallic acid (GA) on IL-1β, and IL-10 levels in the liver and kidney of rats treated with Zearalenone (ZEN: 100 µg/Kg); GA1: 20 mg/kg; GA2: 40 mg/kg. Data are shown as mean ± S.D.; (n = 10) per group. Significance markers indicate groups compared to one another (p < 0.05). * to ****: indicates the level of significance—IL-1β: Interleukin-1beta, and IL-10: Interleukin-10

Full size image

GA reversed ZEN-induced hepatorenal lesions in exposed rats

The representative photomicrographs depicting GA’s influence on ZEN-induced histological damage in the liver and kidney of experimental rats are shown in Figs. 7 and 8. The kidney and liver of control and rats treated with GA alone appeared normal with typical, well-preserved histological architecture. Administration of ZEN alone caused disseminated congestion, infiltration of inflammatory cells and glomerular dystrophy with disaggregated podocytes, whereas the liver showed significant congestion and focal periportal infiltration by inflammatory cells. However, rats treated with GA1 and GA2 combined with ZEN presented typical liver and kidney histological features and were similar to control.

Fig. 7
figure 7

Representative photomicrographs of the liver from control, ZEN only, GA only, co-exposure groups. Control and gallic acid (GA) only-treated groups showed typical hepatic architecture. Zearalenone (ZEN) only-treated rats showed marked disseminated portal fibrosis and congestion (black arrow), infiltration by inflammatory cells (red arrow) and necrosis (green arrow). ZEN + GA1 and ZEN + GA2 appeared comparable to the control. H and E stained, Magnification: x400

Full size image
Fig. 8
figure 8

Representative photomicrographs of the kidney section from control, ZEN only, GA only, co-exposure groups. Control and gallic acid (GA) only-treated groups showed typical kidney architecture. Zearalenone (ZEN) only-treated rats showed severe disseminated tubular necrosis (black arrow), infiltration by inflammatory cells (red arrow) and disseminated segmental glomerular necrosis (green arrow). The kidney of rats treated with ZEN and GA appeared comparable to the control. H and E stained, Magnification: x400

Full size image

Discussion

ZEN is a mycotoxin that contaminates feedstuff and crops worldwide, resulting in toxic effects on human and animal health (Aiko and Mehta 2015; Bennett and Klich 2003; Binder et al. 2017; Darwish et al. 2014; Wang et al. 2020) exposed to such contaminated product. Here we investigated the protective role of GA against ZEN-induced oxidative, inflammatory and pathological changes in rats. After exposure to ZEN for 28-days, rats showed an attenuated gain in body weight relative to the untreated control (p < 0.05) (Table 1). This reduction in weight gain may be attributed to loss of appetite (Joslyn and Glick 1969; Roberts et al. 2007), decreased nutrient digestibility, or reduced growth (Wang et al. 2012). Co-treatment with GA reversed ZEN-induced reduction in weight gain dose-dependently. This result is indicative of a beneficial role of GA in enhancing digestibility (Banerjee et al. 2005). However, the peculiar drop in body weight gain, especially in rats co-treated with ZEN + GA1, can perhaps be attributed to GA’s ability to cause a reduction in body weight (Totani et al. 2011). Previously reported data indicates that GA decreased bodyweight when GA was administered intraperitoneally to experimental rats (Roberts et al. 2007). Thus, the mechanism by which GA reduces food intake involves more than taste aversion or gastrointestinal factors. The reason could also be related to ZEN metabolites’ fate and kinetics, which was administered to the animals and competitive binding to the metabolic site.

Increases in hepatic transaminases in serum indicate hepatobiliary system dysfunction (Kaplan 1993; Ramaiah 2007) and subsequent seeping of hepatic specific transaminases -AST, GGT, ALT ALP- into circulation that are easily quantified as biomarkers of hepatobiliary-toxicity. ZEN treatment resulted in increases (p < 0.05) of hepatic transaminases -AST, ALT, and ALP- activities when compared to the control, a reflection of the hepatotoxicity of ZEN as previously reported (Gao et al. 2018; Zhou et al. 2015). GA co-treatment ameliorated the damage induced by ZEN by decreasing (p < 0.05) serum hepatic transaminases in the GA co-treated groups dose-dependently.

The kidneys are key players in the excretion of waste products and toxins such as urea and creatinine generated from the degradation of proteins and waste produced from muscle breakdowns (Katari et al. 2017; Rubenstein et al. 2012). Urea and creatinine clearance reflect kidney function, and increases in serum urea and creatinine levels indicate a dysfunctional kidney (Bidani and Churchill 1989). ZEN treatment caused significantly high serum creatinine and urea levels in experimental rats; these increases could suggest impaired renal function/renal damage, dehydration, and protein catabolism (Hejazy and Koohi 2017) caused by ZEN exposure. GA dose-dependently reduced serum urea and creatinine levels, further underscoring GA’s ability to protect against ZEN toxicity.

ZEN has been reported to increase oxidative stress (Wang et al. 2012), supporting our current finding, where ZEN treatment resulted in increased generation of RONS, depleting GSH / total thiol (TSH) levels and upregulated endogenous antioxidant enzyme (GPx, GST, SOD, CAT) activities primarily involved in mitigating oxidative stress in the experimental animal. GA’s antioxidative and –inflammatory activities (Badhani et al. 2015; Banerjee et al. 2015; Choubey et al. 2018; Usha et al. 2014), without manifesting toxicity or any adverse clinical indications, have been well demonstrated in the literature. GA enhanced antioxidant levels by increasing GSH and TSH levels in the liver and kidney of treated rats. TSH status, especially thiol (-SH) groups present on protein, are considered significant plasma antioxidants in vivo, and most of them are present in albumin (Prakash et al. 2004). These -SHs are the major reducing groups present in the body fluids (Monod et al. 1965). Oxidative damage is mainly caused by the generation of many RONS and free radicals. RONS generated in the tissues can stimulate oxidative tissue damage due to the inability to detoxify these toxic radicals. Exposure to ZEN alone caused an increase in RONS levels in the liver and kidney tissues, and such exposure is implicated in LPO formation. As expected, we also observed an increase in LPO levels in the liver and kidneys of ZEN treated rats, indicated by the significant rise in Malondialdehyde (MDA) content compared to the control (Ben Salah-Abbes et al. 2009; Zourgui et al. 2008). GA co-treatment significantly reduced RONS and LPO levels in rats’ liver and kidney, attributable to GA’s reported free radical scavenging ability (Choubey et al. 2018).

XO is a valuable marker for the assessment of liver function (Battelli et al. 2001). We observed that XO activities increased in liver and kidney of ZEN treated rats while co-treatment with GA showed a dose-dependent decrease in XO activity compared to ZEN alone. NO is a signalling molecule involved in the onset of inflammation (Sharma et al. 2006), and it is a marker of inflammation. NO toxicity is attributable to its ability to act in conjunction with superoxide anion and disrupt the structure of macromolecules such as proteins, thus inhibiting their function (Carr et al. 2000), triggering an inflammatory response. Increased NO levels in the liver and kidney of rats treated alone with ZEN indicate an inflammatory response. GA resisted the enhanced production of NO exemplified by decreases in NO levels in the co-treated groups, in agreement with earlier reports of GA’s anti-inflammatory properties (Choubey et al. 2018).

MPO is a hemoprotein expressed in neutrophils and monocytes (Valko et al. 2006). An increase in MPO activity is associated with leukocyte infiltration, a significant inflammatory response player (Badhani et al. 2015; Usha et al. 2014). We observed that ZEN treatment significantly increased MPO activity in liver and kidney rats. In contrast, GA co-treatment reversed this observed increase in MPO activity, indicative of inhibition of leukocytes infiltration. IL-1β is a known pro-inflammatory cytokine involved in the mediation of inflammatory response (Valko et al. 2006).

Conversely, IL-10 is an anti-inflammatory cytokine capable of enhancing antibody production and β-cell survival (Gutierrez-Murgas et al. 2016; Steen et al. 2020). ZEN treated rats showed a significant increase in IL-1β in liver and kidney tissues than the control group. Co-treatment with GA caused a concomitant decrease in IL-1β and increased IL-10 (p < 0.05) levels in test rats’ liver and kidney compared to the controls. Other parameters evaluated were the histopathological changes in the test rats’ hepatorenal system. While control rats presented with typical histoarchitecture of the liver and kidney, ZEN treatment resulted in moderate to marked disseminated periportal infiltration by inflammatory cells and focal congestion, in line with previous findings reported Jia et al. (2014). Co-treatment with GA alleviated histological damages induced by ZEN alone with moderate disseminated congestion and mild infiltration by inflammatory cells.

On the other hand, the kidney of rats treated with ZEN alone shows a focal area of congestion and mild disseminated glomerular congestion/hypercellularity. These were reserved by GA co-treatment, where the kidney appears to be similar to the control rat kidney—taken together, dosing rats adversely altered all parameters measured with ZEN alone. They were mitigated in the presence of GA, indicating that GA can serve as an antidote to diminish ZEN-mediated toxic biochemical alteration and oxido-inflammatory responses, as shown in our proposed mechanism of GA action Fig. 9. Collectively, GA could serve as a protective biomolecule against ZEN inadvertently present in the food supply.

Fig. 9
figure 9

Structures of naturally occurring Zearalenone (ZEN), illustrating the metabolic pathways involved in ZEN metabolism by 3α and 3β -hydroxysteroid dehydrogenase (HSDs) and cytochrome P450 s(CYP450s)-mediated activation of ZEN. We focused on the formation and fates of α and β-Zearalenol that result in the cyto-, hepato- and genotoxicity of ZEN. The plausible mechanism(s) of GA-mediated phytochemical based prevention of ZEN-induced injury in rats’ liver and kidney. Note that uridylyl-glucuronic transferase (UGT) is known to detoxify α and β-Zearalenol further, here was used as a model to illustrate the consequence of induction of Phase II drug-metabolizing enzymes by GA. Red arrow indicates upregulation, black arrow indicates downregulation, and blue T-arrow indicates suppression

Full size image

Conclusions

The free radical scavenging activity of GA as a result of its polyphenolic content makes GA not only valuable for the food industry, especially in baked foods where it helps prevent rancidity, but also in cosmetics pharmaceutical industries. The present study showed GA’s ability to reverse oxidative, inflammatory and pathological damages induced by ZEN in the liver and kidney of male Wistar rats due to its antioxidative, anti-inflammatory capacity. These results suggest that GA can protect the hepatorenal system of rats exposed to ZEN from damages by enhancing antioxidant activities, decreasing hepatorenal toxicity, and reducing some inflammatory biomarkers levels and activities in the liver and kidney of rats in the co-treated groups. Due to the beneficial and pivotal role played by GA in mitigating hepatorenal alterations induced by ZEN in rats, GA could be considered as an additive in the rat diet to prevent damages caused by accidental exposure to ZEN, as well as other probable mycotoxins exposure.