Resveratrol Attenuates Benzo(a)pyrene-Induced Dysfunctions, Oxidative Stress and Apoptosis in Pancreatic Beta-Cells

Background: Diabetes mellitus is one of the major health problems for people all over the world today. According to international diabetes federation reports, diabetes affects 382 million people worldwide. Environmental pollutants have deleterious effects on glucose metabolism and cause insulin resistance. We aimed to investigate the effects of the environmental pollutants benzo(a)pyrene, and the therapeutic potential of resveratrol. Methods: 20 μM of benzo(a)pyrene was administered after 48 h of resveratrol (80 μM) application for 24 h in INS-1 (832/13) insulinoma cells. The cells were treated with 20 μM benzo(a)pyrene for 24 hours after 48 hours initial preconditions with 10 μM resveratrol. Oxidative stress status, insulin secretion and apoptosis were analyzed by molecular techniques. Results: Though resveratrol increased the antioxidant status which was decreased by benzo(a)pyrene, interestingly, it increased the oxidative status. Resveratrol increased benzo(a)pyrenedepleted reduced glutathione levels to the control level. The mRNA expression levels of beta-cell functions associated with genes insulin-1, insulin-2 and sirtuin-1 were upregulated by resveratrol. Resveratrol treatment elevated the insulin concentration of culture medium, and the mRNA expression of forkhead box protein-1 gene. Resveratrol upregulated benzo(a)pyrene-downregulated p53 gene expression. On the other hand, benzo(a)pyrene-downregulated mRNA expression of B-cell lymphoma-2 was induced by resveratrol treatment. Conclusion: The data showed that resveratrol could reverse the oxidative alterations, functional impairments and the carcinogenetic effects of benzo(a)pyrene in pancreas beta-cells.


Introduction
Diabetes mellitus (DM) is characterized by exclusively or partially deficiency of insulin released from the pancreas. Type 2 DM (T2DM) is closely related with insulin resistance and is life threatening as significant public health problem in adults, children and adolescents [1] [2] [3]. Diabetes ranks seventh in the causes of death. In the early stages of T2DM, the beta cells produce excessive amounts of insulin to ensure the continuation of normal blood glucose levels. However, beta cells can not cope with mitochondrial dysfunction, resulting in endoplasmic reticulum stress and apoptosis [4]. Consequently, a reduction occurs in the beta cell mass [5].
The oxidative stress may develop due to the production of reactive oxygen species (ROS) in high levels and/or lack of antioxidant defense system [6]. The activities of antioxidant enzymes are increased in diabetes. It is thought that increases of hydrogen peroxide and superoxide by the events including non-enzymatic glycosylation and auto-oxidation of glucose are the basis of this phenomenon [7]. Significant decreases in plasma reduced glutathione (GSH) levels occur in diabetes [8]. The GSH is the most important bio-molecule in defense against chemicals. It also acts as a free radical scavenger and can repair cell damage caused by radicals [9].
The essential factors for the progression of the T2DM are obesity and insulin resistance. However, this results in inadequate insulin secretion from beta cells to compensate for insulin deficiency. In either case, it is regarded as the main pathway of the beta cell death, apoptosis. There are many potentially effective stimulants in the apoptosis of the beta cell. These are Fas ligand and Fas receptors as death receptors, perforins, cytokines [10] [11], ROS [12], reactive nitrogen species [13], alkylating agents [14], ceramide [15]; lack of growth factors [16].
It has been hypothesized by Longnecker and Daniels [17] that chemical contaminants may play an important role in the etiology of T2DM. This hypothesis was supported by epidemiological studies; this is an indication that the increase in insulin resistance or incidence of T2DM is a consequence of exposure to high levels of arsenic, organochlorine contaminants and air pollution [18] [19] [20] [21].
Pollution has harmfull effects on human health directly. Approximately 40% of human deaths in the world depend on environmental pollution. Even people who have never smoked in their lifetime are suffering from respiratory diseases such as lung cancer due to air pollution. Polluted waters cause many diseases. More than 1.2 million people in the world cannot find clean water for their lives. Contaminated soil carries chemicals and numerous toxins. Benzo(a)pyrene [B(a)P] is a compound with a pentacyclic polycyclic aromatic hydrocarbon structure. The most important source of atmospheric B(a)P is wood burning. It is also found in coal tar, in automobile exhaust fumes (especially in diesel engines) and in all fumes from organic materials and grilled food burning [22]. The metabolites of B(a)P are carcinogenic and cause DNA degradation. For example, Benzo(a)pyran-8-diol-9,10-epoxide forms further DNA products [23] [24].
Resveratrol (RES) is a natural phytoalex produced by many fruits and plants, including grapes. It prevents DNA damage and lipid peroxidation in cell membranes. Various research studies [25] [26] [27] have indicated that it is necessary to carry out further studies on how RES can be used in cancer treatment. Resveratrol activates sirtuin (Sirt)-1 [28] which plays an important role in many different physiological events such as cell cycle regulation, metabolism and inflammation [29]. Previous studies have demonstrated that Sirt-1 has a positive regulatory role in insulin secretion and the persistence of beta cell function [30] [31] [32].
However, to our knowledge, there is no previous report on the direct effect of environmental pollutant B(a)P on insulin levels produced by pancreatic beta cells. Therefore, the present study was designed to evaluate the possible effects of RES on oxidative stress, diysfunction and apoptosis in the pancreatic beta cells exposed to B(a)P in vitro.

Materials
All chemicals except resveratrol (Cayman Chemical, Tallin, Estonia) were purchased from Sigma-Aldrich (Germany). INS1-E pancreatic cell line was obtained from Lisa Poppe (Duke University Medical Center, USA).

Experimental Groups
INS-1 β-cells were divided into four groups: control group, resveratrol administrated group, benzo(a)pyrene treated group, and benzo(a)pyrene treated followed by resveratrol administrated group. Resveratrol was administered in an amount of 10 µM for 48 h in RES and RES + B(a)P goups [33]. After first incubation, 20 µM B(a)P [34] was added into the flasks in B(a)P and RES + B(a)P groups, and this second incubation was carried out for 24 h. After last incubation, the cells were separated from medium and used for biochemical analysis. Each assay was performed in triplicate and repeated three times.

Protein Analysis
The medium was removed, and the cells were harvested by trypsin-EDTA treatment and washed twice with PBS. The harvested cells were lysed in lysis buffer. The lysates were centrifuged at 12.000 g (4˚C, 10 min). The protein concentrations were determined in supernatants by Bradford method [35].

Measurements for Oxidative Stress-Related Parameters
The total antioxidant status (TAS) of the cell lysate was determined by the test kit from Rel Assay Diagnostics (Gaziantep, Turkey). This colorimetric method based on the bleaching of color characteristics of a more stable ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation by antioxidants [36].
The total oxidant status (TOS) levels were determined by the test kit from Rel Assay Diagnostics (Gaziantep, Turkey) using a novel automated method, which was developed by Erel [37], in the cell lysates. In this method, oxidants presented in the sample oxidized the ferrous ion-o-dianisidine complex to ferric ion. The ferric ion produced a colored complex with xylenol orange in an acidic medium.
The color intensity was measured spectrophotometrically at 530 nm wavelength.
The results were calibrated with hydrogen peroxide and expressed as µmol H 2 O 2 equiv./mg prot).
The Griess reaction was used for determining the NO levels in the cell lysates [39]. The results were expressed as μmol/mg protein.
The GSH level of samples was determined by spectrophotometer at 412 nm after the reaction between sulfhydryl groups present in supernatant and DTNB (5,5'-2-dithiobis nitrobenzoic acid) [40]. The results of the analysis were expressed as μmol/mg protein.

Total RNA Isolation and Real Time PCR Analysis
The cells were seeded in 25-cm 2 culture flasks and grown for 1 -2 days before use. Cells were collected and washed with PBS after completion of incubations given above. Total RNA was isolated by RNAeasy kit according to manufacturer's instructions (Qiagen, Valencia, CA). cDNA was generated with a First Strand  (Table 1). A melt curve analysis was performed after the last cycle, in order to investigate the specificity of the amplicon and the presence of reaction artefacts such as primer dimer, using a temperature gradient from 60˚C to 100˚C and a ramp speed of 0.5˚C·s −1 (for 10 s) and continuous fluorescence measurement.
Expression levels of the target genes were normalized to the housekeeping gene GAPDH. Gene expression values were then calculated based on the ΔΔCt method using the equation: RQ = 2 −ΔΔCt [41]. The primer sequences used in PCR reactions and PCR conditions are described in Table 1. Each assay was performed in triplicate and repeated three times.

Statistical Analysis
Since normal distribution was not observed Kruskal Wallis test was used followed by the Conover-Iman test. P < 0.05 was considered as statistically significant between the groups. The results were expressed as mean ± standard deviation. Data were analyzed with the Statistical Package for the Social Sciences (SPSS) version 20.0 for Windows.

Results
The oxidative stress-related parameters such as NO, TAS, TOS, GSH and OSI are outlined in Table 2

Discussion
Benzo(a)pyrene is known to occur after several combustion reactions [42]. It has been revealed that the air pollution was closely related to diabetes [43].
The B(a)P metabolism causes overexpression of ROS, which are effective in cancer development, by acting as a second messenger for certain cytokines and growth factors in cells [44]. It was reported that ROS could initiate the peroxidation of lipids [45].
It is well known that GSH protects the cells against oxidative damage via its thiol groups [46]. It was reported that the B(a)P application significantly reduced the levels of GSH [47]. Similarly, the level of GSH was reduced by B(a)P in our study. This could be explained by the excessive use of GSH in the process of detoxifying of peroxide radicals that were generated by lipid peroxidation. Likewise, this result was supported by the reducing effects of B(a)P on the TAS in beta cells.
A research study has reported that resveratrol causes an increase in GSH levels in the protective role against oxidative injury [48]. In our study, increase in GSH levels was detected by resveratrol treatment in both RES and B(a)P + RES groups compared to the control and B(a)P groups, respectively. In addition, it has been reported that resveratrol acts as an antioxidant function such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) [49]. This effect of resveratrol is based on its ability to protect the cell membranes against oxidative damage [50]. Resveratrol has been reported to reduce blood sugar and triglyceride levels, and also insulinemia in rats [55]. In consistent with previous studies, RES improved the insulin levels in this study. This effect of RES on insulin secretion could be related to the increased expression of Sirt-1 gene. Kong et al. [56] reported that RES improved glycemic control and insulin levels which was reduced in rats fed with high-fat diets for eight weeks period [56]. From these re- In a study used Zucker diabetic rats, Sirt-1 gene activator improved the homeostasis of glucose and positively affected the sensitivity of insulin in fat, liver and muscle tissues. It was stated in the same study that the activation of Sirt-1 in the treatment of age-related disease and T2DM was a promising new treatment strategy [57].
It was previously demonstrated that RES increasesd Sirt-1 gene expression in human endothelial cell line EA.HY926 [58]. The tumor suppressor p53, a transcription factor that is activated by various forms of cellular stress, was one of the earliest detected cancer gene [65]. Sin et al. [65] reported the increased levels of p53 protein in rats treated with the combination of RES, and sirtinol which is a Sirt-1 inhibitor. In this context, the combination consisting of RES and sirtinol can be used for preventing the harmful effects of B(a)P. In our study, although RES was used alone, expression levels of p53 in RES group and B(a)P + RES group were increased by 2.07-and by 1.60-fold compared to the control and B(a)P group, respectively. Therefore, it can be said that apoptosis is stimulated via p53 mediated mechanism reported by Advances in Bioscience and Biotechnology Soengas et al. [66], by RES in this study. The role of RES in the induction of apoptosis in various cells is controversial. The agent has been shown to induce apoptosis in cancer cells, such as skin cancer cells [67], and has been shown to play a protective role against apoptosis in human tenocytes [68], endothelial cells [69] and other cell types, particularly under oxidative stress [70]. And in this study, we confirmed the therapeutic role of RES against the reduced-apoptosis in INS-1 pancreatic cells exposure to B(a)P.
It has been reported that RES has anti-diabetic effects in different in vivo and in vitro studies [71] [72]. Xie et al. [72] showed that RES could induce the expression of several β-cell genes including insulin gene. Animal studies have shown similar beneficial effects of RES by increasing insulin secretion [73] or enhancing sensitivity to insulin in peripheral organs via activation of Sirt-1 [74].
In our study, RES increased the levels of Ins-1 and Ins-2 genes by 2.03-and 1.63-folds respectively, in RES + B(a)P group compared to the B(a)P group. In addition, insulin hormone secretion was significantly increasing in RES + B(a)P group compared with B(a)P group in this study. It has been suggested that these results are consistent with the results of previous studies mentioned above.
On the other hand, it was shown that RES increased the expression of key β-cell transcription factors such as Foxo1 and Ngn3 [72]. The increased expression of FoxO1 by resveratrol treatment in this study explains and supports the positive effect of RES on insulin levels.
The expression of the Bcl-2 gene has been reported to be significantly suppressed in the cerebral cortex of rats exposed to B(a)P. This effect of B(a)P was attributed its enhancing effect on apoptosis index [74]. In consistent with the literature, expression of Bcl-2 gene was suppressed by 1.15 fold in the B(a)P group in this study.
He et al. [75] applied RES as an activator for SIRT1 for preventing the effects of apoptosis induced by H 2 O 2 in mouse osteoblastic cells MC3T3-E1 cell lines.
With H 2 O 2 administration, p53, bax and caspase-9 were stimulated while SIRT1 and Bcl-2 were inhibited. It was determined that RES stimulated the activity of both Bcl-2 and SIRT1 genes against this effect of H 2 O 2 [75]. Similarly, RES increased the level of Bcl-2 expression in this study. This increase was also assumed to be effective in removing the reduction in Bcl-2 gene expression caused by B(a)P.

Conclusion
The harmful effects of B(a)P on beta cells of pancreas and therapeutic aspects of