Protective effects of resveratrol against fumonisin B1-induced liver toxicity in mice
Article Category: Original article
Pubblicato online: 26 giu 2023
Pagine: 115 - 119
Ricevuto: 01 apr 2022
Accettato: 01 mag 2023
DOI: https://doi.org/10.2478/aiht-2023-74-3648
© 2023 Rıza Yalçın et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Much is already known about mycotoxins and fumonisins in particular (1–3). Fumonisin B1 (FB1) accounts for 70–80 % of all fumonisins in food and feed products and can be found in urine, serum, and hair (1, 4). According to the International Agency for Research on Cancer (IARC), FB1 is a Group 2B possible human carcinogen (5), and the high concentrations found in Chinese maize have been associated with human oesophageal and liver cancer in China (6, 7).
Among many harmful effects and mechanisms of fumonisin toxic action (8–11), oxidative stress stands out as it causes damage to nucleic acids, proteins, and lipids (12, 13).
The aim of our study was to see if the well-known antioxidant, resveratrol, would counter the effects of FB1, because, to the best of our knowledge, its protective action has never been tested against this mycotoxin, even though its effects have been largely evidenced (14–18). To do that, we focused our study in mice on the protection against FB1-induced liver injury by evaluating serum AST and ALT levels, total sialic acid level, total antioxidant, total oxidant status, and histopathological changes.
The study included 40 (20 male and 20 female) 8–10-week-old BALB/c mice weighing 25–35 g on average, kept in cages in a 12:12-hour light/dark cycle with free access to standard pellet and water.
The mice were randomly divided into four groups of 10 (five male and five female). The first, control group, was receiving saline (10 mL/kg body weight, bw) intraperitoneally (IP) every other day for 14 days. The second, FB1-only group was receiving an IP dose of 2.25 mg/kg bw FB1 (Adoq Bioscience, Irvine, CA, USA) every other day for 14 days. The third, resveratrol-only group, was receiving a daily IP dose of 10 mg/kg bw resveratrol (Cayman Chemical, Ann Arbor, MI, USA) for 14 days. The fourth, FB1+resveratrol group, received a combination of FB1 and resveratrol as described above.
On day 15, the animals were taken blood samples by intracardiac puncture under inhalation anaesthesia (Isoflurane, Adeka Pharmaceuticals Industry and Trade Inc., Istanbul, Turkey). Later, the mice were euthanised by cervical dislocation and the liver samples were taken.
The study was approved by the Burdur Mehmet Akif Ersoy University animal experiments ethics committee (approval No. 328 of 13 September 2017).
Serum AST and ALT were analysed spectrophotometrically on a Randox Monaco autoanalyser (Randox Laboratories, Monaco, UK) with corresponding chemical kits (AS8306 and AL8304, respectively).
Total antioxidant status (TAS) was measured in the liver with a commercial TAS kit (Rel Assay Diagnostic, Gaziantep, Turkey) (19). The method is based on the formation of the colored 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical in a colourless reduced form by the antioxidants present in the sample. This change in colour was measured spectrophotometrically at 660 nm. The method was calibrated with Trolox and data expressed as mmol Trolox eq/L.
Total oxidant status (TOS) was also measured in the liver with a commercial TOS kit (Rel Assay Diagnostic) (20). The oxidants in the sample oxidise ferric iron ions bound to the chelator. In the acidic medium, these ferric ions form a coloured complex with chromogen. Colour intensity is measured spectrophotometrically at 530 nm. The assay was calibrated with hydrogen peroxide (H2O2) and results are expressed as μmol H2O2 eq./L.
As an indicator of liver damage (21, 22), total sialic acid (TSA) was determined in serum according to the method described by Sydow (23). Briefly, 0.2 mL of serum was taken into test tubes and then 1.5 mL of perchloric acid solution was added. The tubes were boiled in a water bath at 100 °C for 5 min, cooled to 4 °C, and centrifuged at 2500
Liver tissue samples collected during necropsy were fixed in a 10 % buffered formaldehyde solution and paraffin-blocked using a Leica ASP300S autotechnicon (Leica Microsystems, Wetzlar, Germany). After 4–5 hours of cooling, we cut them into 5 µm thick serial slices with a rotary microtome (Leica 2155), stained with haematoxylin-eosin (Surgipath, Deer Park, IL, USA), and covered with entellan for examination under a light microscope (Olympus CX21, Olympus Co., Tokyo, Japan). Images were taken with a digital camera (Olympus DP74) and processed using Cell Sens imaging software (Olympus Co., Tokyo, Japan).
For statistical analysis we used the Statistical Package for Social Sciences (SPSS) 16.0 (SPSS Inc., Chicago, IL, USA). Initially, the data were analysed for normality of distribution with the Shapiro-Wilk test. Since the data showed a normal distribution (P>0.05), comparisons between the groups were made with one-way analysis of variance (ANOVA). Pairwise differences between the groups were determined with the post hoc Tukey test. The data are shown as means ± standard error, and the significance was set to P<0.05.
Table 1 shows AST and ALT findings. Serum AST levels were significantly higher in the FB1 than other groups (P<0.05). The other groups did not differ significantly from each other. The same is true for serum ALT levels.
Mean (±standard error) serum AST and ALT levels in the control, resveratrol, FB1, and FB1 +resveratrol groups receiving IP treatment for 14 days
| Groups | AST (U/L) | ALT (mg/dL) |
|---|---|---|
| Control (saline 14 days) | 89.51±7.87a | 51.62±8.99a |
| Resveratrol (10 mg/kg) | 88.30±7.75a | 40.66±3.22a |
| FB1 (2.25 mg/kg, qad, 14 days) | ||
| FB1 +resveratrol | 207.99±34.93a | 121.11±23.50a |
– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. ALT – alanine aminotransferase; AST – aspartate aminotransferase; qad – every other day
Table 2 shows that liver TAS levels were significantly lower in the FB1 than control and resveratrol group (P<0.05), but not between the FB1+resveratrol group and the rest.
Mean (±standard error) liver TAS in the control, resveratrol, FB1, and FB1 +resveratrol groups receiving IP treatment for 14 days
| Groups | TAS (mmol Trolox eq./L) |
|---|---|
| Control (saline 14 days) | 2.58±0.33b |
| FB1 (2.25 mg/kg qad, 14 days) | |
| Resveratrol (10 mg/kg) | 2.69±0.25b |
| FB 1+resveratrol | 2.15±0.20ab |
– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. TAS – total antioxidant status; qad – every other day
TOS and TSA levels show the same pattern (Tables 3 and 4, respectively) in the sense that the FB1 group differs significantly from the control and resveratrol groups, whereas the FB1+resveratrol differs from none of the three, which points to attenuating effects of resveratrol against FB1.
Mean (±standard error) liver TOS in the control, resveratrol, FB1, and FB1 +resveratrol groups receiving IP treatment for 14 days
| Groups | TOS (μmol H2O2 eq./L) |
|---|---|
| Control (saline 14 days) | 38.40±1.20a |
| FB1 (2.25 mg/kg qad, 14 days) | |
| Resveratrol (10 mg/kg) | 34.92±2.88a |
| FB1+Resveratrol | 40.86±2.71ab |
– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. TAS – total oxidant status; qad – every other day
Mean (±standard error) serum TSA levels in the control, resveratrol, FB1 and FB1 +resveratrol groups receiving IP treatment for 14 days
| Groups | Sialic acid (mg/dL) |
|---|---|
| Control (saline 14 days) | 1358.37±76.38a |
| FB1 (2.25 mg/kg qad, 14 days) | |
| Resveratrol (10 mg/kg) | 1411.41±37.87a |
| FB1 + Resveratrol | 1513.99±25.95ab |
– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. TSA – total sialic acid; qad – every other day
Liver samples of mice treated with FB1 alone displayed hyperaemia, infiltrations, and large nuclei (megalokaryosis) in some hepatocytes, whereas no pathological findings were detected in the other groups (Figure 1A–D).
Figure 1
Comparison of liver histologies between the study groups. (A) normal liver histology in a mouse from the control group; (B) severe hyperaemia (white arrows), inflammatory cell infiltrations (black arrow), and megalokaryosis (arrow head) in a mouse from the FB1 group; (C) normal liver histology in a mouse from the resveratrol group; (D) normal liver histology in a mouse from the FB1+resveratrol group. Scale bar=50 µm
Our findings confirm reports of increased liver enzyme levels by the same FB1 dose (2.25 mg/kg bw) (24–26). Administration of resveratrol in our study attenuated these effects to the levels no longer significantly higher than control. Similar protective effects of resveratrol were reported by Sehirli et al. (27) against naphthalene in BALB/c mice.
The same is true for oxidative stress parameters TAS and TOS in the liver of our mice, as resveratrol attenuated the adverse effects of FB1. These findings confirm the reports of resveratrol protecting against fluoride-induced oxidative stress in rats by increasing TAS and decreasing TOS levels (28) or by inhibiting aflatoxin B1-induced oxidative stress in bovine mammary epithelial cells (29). Rašić et al. (30), however, have pointed to a limited resveratrol protection against oxidative stress induced by a combination of ochratoxin A and citrinin, in the sense that resveratrol was capable of restoring glutathione levels in all tissues but had no protective effect against increased malondialdehyde levels and DNA damage. Although resveratrol is generally considered to be safe, it can also be toxic, depending on the dose and duration of exposure. At high doses it may induce oxidative stress by decreasing catalase, glutathione, and superoxide dismutase activity and increasing reactive oxygen species (ROS) and lipid peroxidation (31). In addition, Karaica et al. (32) reported that resveratrol in combination with ochratoxin A and citrinin reduced the expression of renal organic anion transporters (OATs), which led to the accumulation of OTA.
As for TSA, resveratrol lowered the FB1-induced increase in serum sialic acid to levels not significantly higher than control, yet not significantly lower than those in the FB1 group. Total sialic acid levels have been reported to reflect liver-related pathological conditions (21, 22) and that its levels increase with ALT/AST and liver damage (33–35). Similar was observed in the FB1 group in our study, which supports that TSA levels can be used as a biomarker of FB1-induced liver injury.
Our liver histology findings further support biochemistry analyses with hyperaemia and inflammatory cell infiltrations in the liver, as well as megalokaryosis in many hepatocytes of mice in the FB1 group and no pathological changes in the remaining three groups, the FB1+resveratrol in particular. They are in line with an earlier study by Sehirli et al. (27), who reported that 10 mg/kg resveratrol reduced the naphthalene-induced pathological changes in the lung, liver and kidney tissues. The authors also reported that resveratrol helped tissue regeneration.
In conclusion, our biochemical and histopathological findings evidence that resveratrol has a protective effect against oxidative stress and liver damage caused by FB1. It also confirms that total sialic acid levels in the serum can serve as a biomarker of FB1 toxicity and liver damage.