Protective effects of resveratrol against fumonisin B1-induced liver toxicity in mice

Abstract

The aim of this study was to investigate the effects of resveratrol against fumonisin B₁ (FB₁)-induced liver toxicity, as, to the best of our knowledge, these effects have not been investigated yet, even though the toxic effects and mechanisms of FB₁ and the antioxidative effects of resveratrol are well known. 40 BALB/c mice were divided into control, FB₁, resveratrol, and FB₁+resveratrol groups. Control received saline for 14 days. The FB₁ group received 2.25 mg/kg FB₁ every other day for 14 days. The resveratrol group received 10 mg/kg resveratrol for 14 days. The FB₁+resveratrol group received 2.25 mg/kg FB₁ every other day and 10 mg/kg resveratrol every day for 14 days. All administrations were peritoneal. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total sialic acid (TSA) levels were analysed in serum samples, while total antioxidant status (TAS) and total oxidant status (TOS) were measured in the liver. Additionally, the liver tissue was examined for histopathological changes. AST, ALT, and TSA were significantly higher in the FB₁ group than control. Resveratrol countered FB₁ effects for all parameters, including TOS and TAS. Liver histology showed FB₁-induced hyperaemia, infiltrations, and megalokaryosis in some hepatocytes. No pathological findings were detected in the control, resveratrol, or FB₁+resveratrol group. Our findings confirm resveratrol’s protective effect against liver damage and oxidative stress caused by FB₁. In addition, they suggest that increased serum TSA levels can be used as a biomarker of FB₁-induced hepatotoxicity.

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.

MATERIALS AND METHODS

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).

AST and ALT measurements

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

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.

Analysis of total oxidant status

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.

Total sialic acid measurement

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 g for 4 min. The obtained supernatants (1 mL) were transferred to clean test tubes, which were added 0.2 mL of Ehrlich reagent, and boiled in a water bath at 100 °C for 15 min. After boiling, the tubes were cooled and 1 mL of distilled water added for spectrophotometry. Optical densities (OD) were read at 525 nm and serum TSA levels determined by means of the standard curve obtained by serial dilution of n-acetyl neurominic acid (NANA) (Sigma-Aldrich, St. Louis, MO, USA).

Histopathological examination

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).

Statistical analysis

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.

RESULTS

Serum AST and ALT levels

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.

Table 1.

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)	623.62±150.52b	368.00±119.16b
FB1 +resveratrol	207.99±34.93a	121.11±23.50a

^{a, b}– 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

Liver TAS and TOS and serum TSA levels

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.

Table 2.

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)	1.53±0.07a
Resveratrol (10 mg/kg)	2.69±0.25b
FB 1+resveratrol	2.15±0.20ab

^{a, b}– 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.

Table 3.

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)	49.77±2.50b
Resveratrol (10 mg/kg)	34.92±2.88a
FB1+Resveratrol	40.86±2.71ab

^{a, b}– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. TAS – total oxidant status; qad – every other day

Table 4.

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)	1594.99±33.65b
Resveratrol (10 mg/kg)	1411.41±37.87a
FB1 + Resveratrol	1513.99±25.95ab

^{a, b}– statistically significant (P<0.05) differences between the groups are marked with different letters in superscript. TSA – total sialic acid; qad – every other day

Histopathological findings

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

DISCUSSION

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.

Funding Statement

This research was supported by the Burdur Mehmet Akif Ersoy University Scientific Research Projects Coordinatorship (project No, 0483-YL-17).