Reduction of the Oxidative Stress Status Using Steviol Glycosides in a Fish Model (Cyprinus carpio)

Steviol glycosides are sweetening compounds from the Stevia rebaudiana Bertoni plant. This product is considered safe for human consumption and was approved as a food additive by the Food and Drugs Administration (FDA) and European Food Safety Authority (EFSA). Its effects on the ecosystem have not been studied in depth; therefore, it is necessary to carry out ecotoxicological studies in organisms such as Cyprinus carpio. The present study aimed to evaluate the antioxidant activity by SGs on diverse tissues in C. carpio using oxidative stress (OS) biomarkers. To test the antioxidant activity, carps were exposed to four systems: (1) SGs free control, (2) CCl4 0.5 mL/kg, (3) SGs 1 g/L, and (4) CCl4 0.5 mL/kg + SGs 1 g/L at 96 h. The following biomarkers were analyzed: lipoperoxidation (LPX), hydroperoxide content (HPC), and protein carbonyl content (PCC), as well as antioxidant activity of superoxide dismutase (SOD) and catalase (CAT). It was found that both (3 and 4) systems' exposure decreases LPX, CHP, PCC, SOD, and CAT with respect to the CCl4 system. The results of this study demonstrate that the concentrations of SGs used are not capable of generating oxidative stress and, on the contrary, would appear to induce an antioxidant effect.

Due to the growing incidence of both obesity and type 2 diabetes [4] and health-related concerns such as weight gain [5,6], cancer risk [7], metabolic syndrome, hypertension, and pregnancy preterminal delivery risks [8][9][10][11] associated with the consumption of artificial high-potency sweeteners [12], much attention has been paid to low-calorie plantderived [13] sucrose substitutes. Nowadays, the food industry is increasingly interested in using natural sugars such as stevia instead of artificial sugars in order to offer a wider range of options for people who do not want or cannot eat sucrose [14].

Fish Procurement and
Maintenance. Cyprinus carpio species were obtained from a certified aquaculture facility located in Tiacaque, State of Mexico. The carps used for this experiment fit the following characteristics: 15.3 ± 0.58 cm length and 30.01 ± 4.5 g weight. Prior to toxicity studies, organisms were maintained for 30 days in tap water, at 20 ± 2 ∘ C, and exposed to natural light/dark photoperiods. Oxygen concentration was kept above 85%, pH at 7.6-7.9, total alkalinity at 17.8 ± 4.3 mg/L, and total hardness at 18.5 ± 0.4 mg/L.

Oxidative Stress Determination.
Test systems were prepared using water with the same characteristics and conditions described above in the Fish Procurement and Maintenance. The systems were static without renewal of the medium, and no food was provided to the specimens.
Previous studies were performed by determining the CCl 4 concentration that induced OS used in this study (0.15, 0.3, 0.44, and 0.62 mL/kg bw) according to Jia et al. [35]. The selected concentration of CCl 4 was 0.5 mL/kg bw.
In order to determine the concentrations of SGs used in this study, a previous experiment was performed using CCl 4 0.5 mL/kg and different concentrations of SGs (70% stevioside and 30% rebaudioside-A proportion): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1 g/L. And the oxidative stress biomarkers analyzed in this work were evaluated. The only concentration that showed effects on the biomarkers used was 1 g/L of SGs, so this was the one selected.
To test the antioxidant activity of SGs, four systems were tested: (1) SGs free control, (2) CCl 4 0.5 mL/kg, (3) SGs 1 g/L, and (4) CCl 4 0.5 mL/kg + SGs 1 g/L. Each system used 6 carps and the assays were performed in triplicate (72 fish were used in the oxidative stress evaluation). In the systems containing CCl 4 , the fish were given a caudal vein injection of CCl 4 (30% in olive oil) at a dose of 0.5 mL/kg body weight. The target concentrations used in this experiment were based on previous experiments (0.29, 0.58, 0.87, and 1.0 g/L SGs). These concentrations were determined by experimental design central composite (STATGRAPHICS Centurion XVII version). The concentration of SGs selected was 1.0 g/L. At the end of the exposure period (96 h), blood was removed by puncture of the caudal vessel, and liver, muscle, gills, and brain were removed from each specimen. Organs and tissues were placed in phosphate buffer solution (PBS) (0.138 M NaCl, 0.0027 KCl (Vetec, Sigma-Aldrich, Mexico)) at pH 7.4 and then centrifuged at 12,500 ×g and −4 ∘ C for 15 min. The following biomarkers were then evaluated: hydroperoxide content (HPC), lipoperoxidation (LPX), and protein carbonyl content (PCC), as well as the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). All bioassays were performed on the supernatant. 1.5. Determination of LPX. LPX was determined using thiobarbituric acid-reactive substances, as described by Buege and Aust's [37] method. To 100 L of supernatant, Tris-HCl buffer solution (pH 7.4) (Sigma-Aldrich, St. Louis) was added until a 1 mL volume was attained. Samples were incubated at 37 ∘ C for 30 min; 2 mL of TBA-TCA reagent [0.375% thiobarbituric acid (Fluka-Sigma-Aldrich, Toluca, Mexico) in 15% trichloroacetic acid (Sigma-Aldrich, St. Louis)] was added and samples were shaken. They were then heated up to boiling for 45 min and then allowed to cool down, and the precipitate was removed by centrifugation at 3,000 ×g for 10 min. Absorbance was read at 535 nm against a reaction blank. Malondialdehyde (MDA) content was calculated using the molar extinction coefficient (MEC) of it (1.56 × 10 5 M/cm). Results were expressed as mM MDA/mg protein.
1.6. Determination of PCC. The method of Levine et al. [38] modified by Parvez and Raisuddin [39] and Burcham [40] was used for determining PCC. Soluble proteins were obtained by centrifugation of samples at 10,500 ×g for 30 min. A test tube was filled with 100 L of supernatant and 150 L of 10 mM DNPH in 2 M HCl; the tube was incubated at room temperature for 1 h in the darkness. After the incubation time, 500 L of 20% trichloroacetic acid was added, and the solution was allowed to rest for 15 min at 4 ∘ C. The sample was centrifuged for 5 min at 11,000 ×g. Using the solution of 1 : 1 ethanol : ethyl acetate, the bud was washed three times and then dissolved in 1 mL of 6 M guanidine solution (pH 2.3) and incubated for 30 min at 37 ∘ C. Absorbance was read at 366 nm. Results were expressed as nM reactive carbonyls formed (C=O)/mg protein, using the MEC of 21,000 M/cm [41].

SOD Activity Determination. According to the Misra and
Fridovich's method [42], the activity of SOD was determined.
In a 1 cm cuvette, 40 L of the supernatant, 200 L of adrenaline (30 mM), and 260 L of carbonate buffer solution (50 mM sodium carbonate and 0.1 mM EDTA) (pH 10.2) were added. Absorbance was read at 480 nm after 30 s and 5 min. SOD activity was determined using the MEC of SOD (21 M/cm). Results were expressed as IU SOD/mg protein [41].

Hydroperoxide Content (HPC).
The amount of cumene hydroperoxide (CHP) equivalents induced by different systems is shown in Figure 1. Significant increases with respect to the control ( < 0.05) were observed in the treatment with CCl 4 in blood, gill, and brain in 892.1, 457.1, and 250.4%, respectively. In the SGs system, significant decreases were observed with respect to the control ( < 0.05) in blood (91.3%), liver (82.9%), and gill (94.4%). No significant differences were observed in the treatment with SGs + CCl 4 in any tissue. Significant decreases ( < 0.05) with respect to the CCl 4 were found in all tissues in the SGs system. The SGs + CCl 4 system showed significant decreases ( < 0.05) with respect to the CCl 4 system in blood, liver, muscle, gill, and brain in 96.0, 80.9, 83.7, 76.2, and 81.7%, respectively. Figure 2. Significant increases with respect to the control group ( < 0.05) were observed in the system with CCl 4 in blood, liver, gill, and brain. These increases were 92.6, 434.9, 99.9, and 171.5%, respectively. Significant decreases with respect to   the control group ( < 0.05) were observed in the system with SGs in blood, liver, and gill. The decreases observed were 83.7, 91.1, and 72.3%, respectively. No significant differences were observed in the treatment with SGs + CCl 4 in any tissue. Significant decreases with respect to the CCl 4 system were found in blood (91.5%), liver (91.0%), muscle (68.9%), gill (86.1%) and brain (90.6%) in the SGs system. Significant decreases with respect to the CCl 4 -induced system were found in blood, liver, muscle, gill, and brain in the SGs + CCl 4 system. The decreases observed were 69.7, 76.9, 60.0, 58.7, and 83.2%, respectively.   in blood, liver, and gill. These decreases were 91.9, 75.6, and 97.0%, respectively. No significant differences were observed in the treatment with SGs + CCl 4 in any tissue. No significant differences of the biomarker were observed in muscle in any treatment. Significant decreases with respect to the CCl 4induced system were observed in the SGs system in blood, liver, gill, and brain. Significant differences were observed in the SGs + CCl 4 system with respect to the CCl 4 system in blood (88.9%), liver (80.2%), gill (80.4%), and brain (84.0%). No significant differences were observed in muscle in any treatment. Figure 4. CCl 4 treatment showed significant increases in all tissues (blood, liver, muscle, gill, and brain) with respect to control ( < 0.05). These increases were 951. 9

Superoxide Dismutase (SOD) Activity. SOD activity is shown in
SGs 1 g/L + CCl 4 0.5 mL/kg bw mL/kg bw showed significant decreases ( < 0.05) with respect to control in blood (96.1%) and gills (97.8%). A significant increase was observed in the liver (166.3%) with the SGs treatment with respect to the control group. No significant differences were observed in the treatment with SGs + CCl 4 in any tissue. Significant decreases with respect to the CCl 4 system were observed in blood, liver, and gill in the SGs system. Decreases observed were 99.6, 65.5, and 99.4%, respectively. Significant decreases in the SGs + CCl 4 system were observed in blood (94.0%), liver (98.2%), gill (82.3%), and brain (96.9%) with respect to the CCl 4 system. Figure 5. In the CCl 4 system, significant increases were observed with respect to the control group ( < 0.05) in blood, liver, gill, and brain (203.6, 161.9, 139.8, and 1233.8%, resp.). Significant decreases with respect to the control group were found in blood (88.3%), liver (72.6%), and gill (46.2%) in the SGs system. No significant differences of the biomarker were observed in muscle in any treatment. No significant differences were observed in the treatment with SGs + CCl 4 . Significant decreases were found in the SGs system in blood (96.1%), liver (89.5%), gill (77.6%), and brain (99.1%) with respect to the CCl 4 system. Significant decreases were found in the SGs + CCl 4 system in blood, liver, gill, and brain with respect to the CCl 4 system. These decreases were 88.0, 67.8, 45.4, and 97.1%, respectively. Significant increases were observed in liver and gill with respect to the SGs system in the SGs + CCl 4 system.

Discussion
Stevia rebaudiana Bertoni, a plant species native to Northeastern Paraguay, is known to accumulate diterpene glycosides. These compounds are nontoxic, high-potency sweeteners that are used as sugar substitutes. The major SGs stevioside and rebaudioside-A constitute 60-70% and 20-30%, respectively, of the total glycosides in S. Rebaudiana. Several studies have suggested that, besides sweetness, SGs, along with related compounds, may also offer therapeutic benefits, as they have antihyperglycemic, antihypertensive, anti-inflammatory, antitumor, antidiarrheal, diuretic, and immunomodulatory effects [4]. Another important effect of SGs related to the scientific literature is their antioxidant activity. In a study conducted by Shukla et al. [45], they demonstrated that aqueous extracts also inhibited hydroxyl radicals, nitric oxide, and superoxide anions with IC 50 values of 100.86, 98.73, and 100.86 g/mL, respectively. The greater amount of phenolic compounds leads to more potent radical scavenging effects as shown by the aqueous leaf extract of S. rebaudiana.
OS is one important mechanism of toxicity, given the impact that an imbalance between reactive oxygen species (ROS) and antioxidant defenses has on vital biomolecules such as lipids, proteins, and genetic material, as well as the countless toxicants capable of inducing it (hydrocarbons, metals, pesticides, solvents, and drugs, among other compounds) [46][47][48][49][50][51]. An increase in the scientific literature suggests that diseases including cardiovascular diseases, diabetes, cancer, neural disorders, arthritis, and aging are caused by or related to the production of ROS which can result in tissue damage and cell death [52][53][54]. Kim et al. [55] reported that Stevia extracts contain high levels of compounds with ROS scavenging activity.
On the other hand, sugars are well known as ROS scavengers [56] and a number of recent findings point to a prominent role for sugars or sugar-like compounds in oxidative stress defense in plants [55][56][57]. A study conducted by Hajihashemi and Geuns [58] hypothesized that, due to the high content of sugars present in the SGs, these presented their high antioxidant capacity; however, the results showed that this did not occur.
In the same way as all aerobic organisms, fish such as Cyprinus carpio have an inherent and efficient antioxidant defense system that includes the enzymes SOD, CAT, and GPx and nonenzymatic antioxidant components such as GSH [59]. These enzymes play a preponderant role in defending the cells against free radical-mediated oxidative damage [60] decreased activities, or expressions of these enzymes may predispose tissues to free radical injury [61].
From the above, this study was carried out to investigate the antioxidant properties of SGs in CCl 4 -induced injuries in a fish model (Cyprinus carpio). The utilization of halogenated alkanes such as CCl 4 , CHCl 3 , or CHI 3 has been prohibited due to their severe toxicity; CCl 4 however continues to be used as a model substance to elucidate the mechanisms of action of hepatotoxic effects such as fatty degeneration, fibrosis, hepatocellular death, carcinogenicity, and OS. The mechanism through which CCl 4 produces its toxic effects is the formation of reactive trichloromethyl radicals ( • CCl 3 ) by CYP450 activity. In the presence of oxygen, • CCl 3 is quickly transformed into trichloromethyl peroxyl radical (CCl 3 O 2 • ).
• binds covalently to cellular proteins or lipids, which initiates the lipid peroxidation in the cellular membrane [62].
In the current work, treatment of the fish with CCl 4 at 96 h increased significantly the activities of SOD and CAT in 6 BioMed Research International all tested tissues. In addition, it increased significantly CHP, LPX, and PCC in blood, liver, gill, and brain. These results demonstrate that CCl 4 may be a good inducer of OS in common carp Cyprinus carpio.
Antioxidant defenses may be induced by diverse environmental contaminants [63]. SOD is the first mechanism of antioxidant defense and the main enzyme responsible for offsetting the toxic effects induced by the presence of ROS. This is particularly so in the case of the superoxide ion, which is a minor product of mitochondrial respiration [64] and is biotransformed by SOD to hydrogen peroxide, after which CAT and GPx take part in the capture and later dismutation of H 2 O 2 to H 2 O [31]. As can be seen in Figures 4 and 5, CCl 4 at a concentration of 0.5 mL/kg bw increases significantly the SOD and CAT activity in blood and the four tissues evaluated in this study. Also, there are increases in HPC, LPX, and PCC. Bagnyukova et al. [65] say that LPX products are apparently involved in the upregulation of certain antioxidant enzymes. Therefore, LPX increases in our study might likewise explain the increases in antioxidant enzyme activities that were found. These results would demonstrate that CCl 4 is a good inducer of oxidative stress in common carp.
SGs alone system showed an efficient decrease in the oxidative stress biomarkers used in this study with respect to control and CCl 4 systems; and to determine the effect of CCl 4 exposure on OS system and consequently potential antioxidant effects of SGs on the disturbed system by CCl 4 , a system was tested using CCl 4 0.5 mL/kg + SGs 1 g/L. The results in this system showed a significant decrease in the cellular oxidation biomarkers (CHP, LPX, and PCC) and the antioxidant enzymes (SOD and CAT) with respect to the CCl 4 systems.
These results are in agreement with those of Holvoet et al. [66]; they demonstrate that stevioside treatment of obese diabetic mice improved adipose tissue maturation and increased glucose transport, insulin signaling, and antioxidant defense in white visceral adipose tissues. Together, these increases were associated with a twofold increase of adiponectin. The adiponectin has been associated with improved insulin signaling and antioxidant defense in both the adipose tissue and the aorta of stevioside-treated mice [67].
Also, these authors showed that rebaudioside increased methionine that is directly involved in the regulation of the glutathione antioxidative system. It also increased tryptophan that is involved in the regulation of the defense system through its action as a precursor of antioxidants and its effect on the inflammatory response [68].
In addition to these findings, it was shown that SGs had a very potent • OH scavenging activity [69,70]. Several studies have shown that crude extracts of Stevia rebaudiana are responsible for antioxidant activities in murine models, neutralizing radicals such as hydroxyl radicals (OH * ), superoxide radicals (O 2 * ), and hydrogen peroxide (H 2 O 2 ).
On the other hand, when SGs were used, a decrease in HPC, LPX, and PCC (Figures 1-3) was observed with respect to the control group and the CCl 4 group at concentration of 0.5 mL/kg bw. Paradoxically, the levels of antioxidant enzymes SOD and CAT decreased significantly with respect to the control, CCl 4 at concentration of 0.5 mL/kg bw, and SGs + CCl 4 groups. The decrease in SOD and CAT of the CCl 4 and SGs + CCl 4 groups would demonstrate that the increase of ROS by tetrachloride exposure in common carp is inhibiting the antioxidant enzymes evaluated. Jira et al. [71] suggest that SOD activity may be inhibited if there is an increase in superoxide anion, H 2 O 2 , and peroxynitrite.
In the group in which only SGs were used, a significant decrease with respect to the control was observed, which would demonstrate that the glycosides of steviol are effective in reducing the ROS produced by the basal cellular activity of the carp.

Conclusion
SGs used alone are not capable of generating oxidative stress and, on the contrary, would appear to induce an antioxidant effect in the common carp Cyprinus carpio. The antioxidant properties of SGs in CCl 4 -induced injuries in Cyprinus carpio model were proven when comparing the systems with CCl 4 alone and the mixture of CCl 4 and SGs system. The concentration of SGs which showed an antioxidant activity in the model used was 1 g/L.