pCramoll and rCramoll as New Preventive Agents against the Oxidative Dysfunction Induced by Hydrogen Peroxide

Oxidative stress plays an important role in the induction of cell death and is associated with various pathologic disorders; therefore, the search for natural products that attenuate the effects produced by oxidant agents is greatly increased. Here, the protective effects of native lectin from Cratylia mollis seeds (pCramoll) and recombinant Cramoll 1 (rCramoll) against H2O2-induced oxidative stress in Vero cells were evaluated. Both lectins significantly attenuated the H2O2-induced cytotoxicity in a concentration-dependent way. The maximum protective effects were 96.85 ± 15.59% (rCramoll) and 59.48 ± 23.44% (pCramoll). The Live/Dead analysis showed a reduction in the percentage of dead cells from 65.04 ± 3.29% (H2O2) to 39.77 ± 2.93% (pCramoll) and 13.90 ± 9.01% (rCramoll). The deleterious effects of H2O2 on cell proliferation were reduced to 10.83% (pCramoll) and 24.17% (rCramoll). Lectins treatment attenuated the excessive superoxide production, the collapse of the mitochondrial membrane potential, and the lysosomal and DNA damage in H2O2-treated cells. In conclusion, our results suggest that pCramoll and rCramoll blocked H2O2-induced cytotoxicity through decreasing reactive oxygen species, restoring the mitochondrial potential, preventing the lysosomal damage and DNA fragmentation, and thus promoting cell survival and proliferation.


Introduction
Oxidative stress is characterized by an imbalance in the redox status of the cell and has been implicated in a range of age-associated and neurodegenerative diseases, such as aging, cancer, diabetes, Alzheimer's disease, and Parkinson's disease [1]. The reactive oxygen species (ROS) are oxygen-containing molecules that are constitutively produced in cells as a result 2 Oxidative Medicine and Cellular Longevity of normal metabolic processes. They include superoxide anions (O 2 − ), hydroxyl radicals (OH • ) and hydrogen peroxide (H 2 O 2 ; nonradical derivative of oxygen). ROS are known to be responsible for cell toxicity when the generation of ROS exceeds the clearance capacity of the cellular antioxidant systems [2]. H 2 O 2 is thought to be the major precursor of highly reactive free radicals, such as hydroxyl radicals via Fenton's reaction [3]. ROS may damage relevant classes of biological macromolecules in the cells through direct oxidation of lipids, proteins, and nucleic acids, thereby disrupting cellular function and integrity, which leads to cell death [1,3]. Nowadays, the search for natural products that attenuate the effects produced by oxidant agents is greatly increased [4,5].
Lectins are a heterogeneous group of nonimmune proteins and glycoproteins that specifically and reversibly bind with high affinity to carbohydrates without altering the covalent structure of any of their recognized ligands. Lectins can agglutinate cells through binding to cell surface glycoconjugates. They are distributed in plants, animals, and microorganisms [6,7].
Cratylia mollis Mart (Fabaceae family) is a native leguminous forage from the semiarid region of the Northeast of Brazil (Caatinga biome), popularly known as Camaratu bean. Four multiple molecular forms of lectin have been purified from this plant: Cramoll-1, Cramoll-2, Cramoll-3, Cramoll-4; which exhibit different carbohydrate specificities. The isoforms 1, 2, and 4 are nonglycosylated and glucose/mannose specific proteins; and Cramoll 3 is a galactose specific glycoprotein [8,9]. Cramoll 1,4 (preparation containing isolectins 1 and 4; pCramoll) is isolated in a similar way to concanavalin A (Con A), a well-known lectin from Canavalia ensiformis seeds [8]. This preparation has shown interesting biological activities such as immunomodulatory, antitumoral, antiparasitic, and healing agent [9]. Biotechnological applications of Cramoll also involve the characterization of human malignant tissues, affinity matrix for protein purification, and the development of sensors for microbial detection [9]. Cramoll 1 (major isolectin in this preparation) consists of 236 residues with 82% identity with Con A. Cramoll 1 tertiary structure was determined by X-ray crystallography at 1.77Å and revealed three -sheets connected by loops, known as the jellyroll domain (this topological architecture is essentially identical to Con A) [9]. Recently, the expression of soluble, functional recombinant Cramoll 1 in Escherichia coli was reported by our group: rCramoll, which shares the molecular mass, charge density, sugar recognition, and secondary and tertiary structures of pCramoll [10,11].
In this study the cytoprotective effects of pCramoll and rCramoll against H 2 O 2 -induced oxidative damage in Vero cells were investigated. We found that the cytoprotective effects of these lectins are mediated by the decrease of superoxide species production that prevent the mitochondrial and lysosomal dysfunctions and the DNA damage.

Cell
Culture. The monkey kidney fibroblast line (Vero) was maintained at 37 ∘ C in an incubator with humidified atmosphere of 5% CO 2 . Cells were cultured in RPMI medium containing 10% heat-inactivated fetal calf serum, penicillin, and streptomycin (100 U/mL), all from Sigma-Aldrich.

MTT Assay.
Cell viability was evaluated using the MTT assay, which measures the metabolic conversion of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) salt to the colored formazan dye. Vero cells (1 × 10 5 /mL) were incubated in a 96-well plate in quadruplicate for 24 h (37 ∘ C and 5% CO 2 ), treated with lectins (0.625-10 M) for 30 min and subsequently with H 2 O 2 (1 mM) for 24 h. At the end of the incubation time, the medium was removed and a MTT solution (5 mg/mL in RPMI) was added to the culture and the cells were incubated for additional 3 h (37 ∘ C and 5% CO 2 ). Afterwards, the medium was removed again and the intracellular formed formazan product was dissolved in DMSO. The optical density (OD) was measured at 595 nm in a microplate reader (Benchmark Plus, Bio-Rad, California, EUA). Cell viability (CV) was calculated in comparison to the OD obtained by control cell, considered as 100%. The protective effect was calculated following this formula: (1 mL/5 M) was added and the samples were incubated for 10 minutes at 37 ∘ C, protected from light. The cells were washed with warm buffer (three times) and analyzed by flow cytometry (FACSCalibur-BD, San Jose, CA) using the software CELLQuestPro (Becton Dickinson Co., San Jose, CA) for acquisition and analysis of data.

Lysosomal Membrane
Stability. An experiment using the lysosomotropic base acridine orange was carried out to measure severe or late lysosomal membrane stability by assessing change in red fluorescence (AO uptake method). The cells were prepared as described above (2.5) and incubated at 37 ∘ C in the presence of 1 mM acridine orange (Sigma-Aldrich) and washed in PBS (2X) after 30 min. The pellet containing the cells was resuspended in PBS and analyzed by flow cytometry (FACSCalibur-BD, San Jose, CA; FL3 channel) using the software CELLQuestPro (BD Bioscience, San Jose, CA) for acquisition and analysis of data.

Determination of Mitochondrial Membrane Potential (ΔΨm).
The uptake and retention of the cationic fluorescent dye rhodamine123 was used to evaluate the mitochondrial membrane potential (ΔΨm). Treated cells were trypsinized, washed twice with PBS, and centrifuged at 300 ×g for 10 min. The cell pellet was then resuspended in 1 mL of fresh medium containing 2 M rhodamine123 and incubated at 37 ∘ C in a thermostatic bath for 20 min with gentle shaking. The stained Vero cells were washed and then resuspended in 1 mL of PBS. The variation index (VI) was calculated following this formula: where FI c is the mean of fluorescent intensity of control and FI s the mean of treated cells.

CFSE Proliferation Assay.
To evaluate the cytoprotective effect of Cramoll on Vero cells the CFSE (carboxyfluorescein diacetate succinimidyl ester) proliferation assay was used. The Vero cells (1 × 10 5 /mL) were stained with CFSE (2.5 M) and incubated at 37 ∘ C for 10 min. The reaction was stopped by the addition of cold RPMI. The cells were centrifuged (300 g for 10 min) and the cell pellet was washed with PBS in the same conditions. Afterwards, the cells were resuspended in RPMI and pretreated with lectins, following the addition of H 2 O 2 as described (Section 2.5). The proliferation indexes were determined after 72 h by flow cytometry (FACSCalibur-BD, San Jose, CA) using the software CELLQuestPro (BD Bioscience, San Jose, CA) for acquisition and the FlowJo software (Tree Star, Ashland OR) for analysis of data. The proliferation index of control cells was considered as 100%.

Terminal Deoxynucleotidyl Transferase dUTP Nick End
Labeling (TUNEL) Click-iT Assay. TUNEL assay was performed using the Click-iT TUNEL Alexa Fluor 488 (Invitrogen) following the manufacturer's protocol. Briefly, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized (0.25% Triton X-100 in PBS) for 20 minutes at room temperature. Then they were washed twice with deionized water. The TdT reaction cocktail was added and incubated for 1 h, followed by 30 min incubation with the Click-iT reaction solution. The stained Vero cells were washed and resuspended and the fluorescence intensity was analyzed by flow cytometry (FACSCalibur-BD, San Jose, CA) using the software CELLQuestPro (BD Bioscience, San Jose, CA) for acquisition and analysis of data.

Spectroscopic
Measurements. The effects of H 2 O 2 treatment on lectin tertiary structure were evaluated by intrinsic fluorescence using a Jasco FP-6300 spectrofluorometer (Jasco, Tokyo, Japan). Both lectins (10 M) were incubated with H 2 O 2 (1 mM). The fluorescence emission of tryptophan was measured at 25 ∘ C in a rectangular quartz cuvette with a 1 cm path length, the excitation was at 295 nm, and emission was recorded from 305 to 450 nm using 5 nm band pass filters (for both).

Statistical Analysis.
Data is analyzed by one-way analysis of variance (ANOVA) and Turkey test to determine the statistical significance. A value of <0.05 was considered to be statistically significant.

pCramoll and rCramoll Attenuated the H 2 O 2 -Induced
Cytotoxicity. The cytoprotective effects of lectins against H 2 O 2 -induced cell death were evaluated by MTT assay, which measures the loss of metabolic activity of cells and it is an early indicator for cell death. Both lectins inhibited the cytotoxicity in a concentration-dependent way ( Figure 1). pCramoll showed maximum protective effects at 5 M (48.36 ± 8.12%) and 10 M (59.48 ± 23.44%), with no statistically significant differences ( > 0.05) between these concentrations. rCramoll induced higher/maximum protection at 10 M (96.85 ± 15.59%) ( < 0.05). It is important to note that rCramoll was more effective than the pCramoll at 5 and 10 M ( < 0.05). Because the best activity of both lectins was achieved at 10 M we chose this concentration to further investigate the subcellular effects involved in the Cramoll-mediated cytoprotective effects. The cytoprotective action of both lectins was confirmed by Live/Dead assay, a flow cytometry analysis that uses the hydrolysis of calcein AM by intracellular esterases of live cells and the ethidium heterodimer (EthD-1) that enter nucleus of cells with damage membranes to discriminate viable from unviable cell, respectively. Figure 2 shows an increase of cell death when Vero cells were treated with H 2 O 2 alone (65.04 ± 3.29%) compared to control cells. On the other hand, both lectins reduced the rates of cell death to 39.77 ± 2.93% for pCramoll and 13.90 ± 9.01% for rCramoll.

pCramoll and rCramoll Inhibited the Deleterious Effects of H 2 O 2 on Cell Proliferation.
To evaluate the cell proliferation a FACS assay using CFSE staining followed by FlowJo analysis was performed. CFSE proliferation assay is based on the ability of CFSE probes to bind to lipids within the cell membrane. After each mitosis, the fluorescence intensity decreases to approximately the half. The H 2 O 2 -treated cells showed, after 72 h, a decrease of proliferation index of 34.71% compared to the control cells. The lectins were able to enhance the proliferation index in the presence of H 2 O 2 (78.84 ± 4.04% for pCramoll and 84.20 ± 1.16% for rCramoll) (Figure 3). Figure 4, after H 2 O 2 exposition ROS generation increased more than 52-fold as compared to control. Pretreatment of the cells with 10 M of lectins for 30 minutes induced significantly the attenuation of mitochondrial ROS production. The reduction rates (in relation to H 2 O 2 ) were 20.31 ± 7.82% and 39.84 ± 2.36%, for pCramoll and rCramoll, respectively ( < 0.05).  (Figure 6).

pCramoll and rCramoll Prevent the Fragmentation of
Nuclear DNA. The formation of DNA ladders is a consequence of a specific nucleosomal-sized fragmentation and is a conventional event in apoptotic process. The TUNEL assay was carried out to detect the extension of DNA degradation in apoptotic cells. DNA damage was quantified in relation to control cells. As expected, no significant DNA injury could be detected in the control cells. On the other hand, H 2 O 2treated cells presented an increase in the TUNEL fluorescence intensity, indicative of intense DNA fragmentation (this fluorescence was 170.41% higher than control cell). Pretreatment with both lectins leads to decrease in TUNEL staining in about 2-fold (61.62 ± 1.44% for pCramoll and 55.53 ± 5.86% for rCramoll) (Figure 7).

Discussion
In this work we reported the protective effects of native and recombinant Cramoll against H 2 O 2 -induced cell death. The induction of cell death by H 2 O 2 has been already reported in the literature using different cell types [5,[12][13][14]. The severe damage caused by H 2 O 2 is related to its capacity to cross the cellular membranes and react with intracellular metal ions, yielding highly toxic hydroxyl radicals, which are able to cause serious damage to macromolecules, including DNA, proteins, and lipids, and ultimately lead to the cell death [2]. In fact, several diseases had the oxidative stress as the main trigger (cancer, neurodegenerative, and cardiovascular disorders) [1,15]. In the present work, H 2 O 2 induced significant toxicity, reducing the cell viability to 34.96% (Live/Dead kit) and suppressing the cell proliferation. The pretreatment with different concentrations of both rCramoll and pCramoll lectins had a significant cytoprotective effect reestablishing the cell viability and proliferation to the rates near to those found in the control cells.
The secondary production of other mitochondrial ROS (such as superoxide) induced by H 2 O 2 is one of potential mechanisms of cell damage [16]. In this study, we measured the superoxide generation using MitoSOX probe. The treatment of cells with H 2 O 2 increased significantly the superoxide production by the mitochondria ( < 0.05). This effect was attenuated in both lectins-pretreated groups. These results indicate that the anticytotoxic effects of both lectins were related to the inhibition of mitochondrial ROS production.
Mitochondria play a critical role in maintaining the physiology of the cell and its dysfunction is an important pathway in the cell death [17]. It is well known that several proapoptotic mediators (cytochrome c, AIF, Smac/DIABLO, and endoG) are located in the mitochondria. These factors are released into the cytosol where they activate diverse enzymatic reactions that lead to the specific degradation of proteins and DNA during apoptosis [18]. The conservation of ΔΨm is essential for mitochondrial integrity and functions (#) Significant differences between lectins. [19]. H 2 O 2 induces mitochondrial dysfunction by the loss of ΔΨm [20]. Our results showed that native and recombinant Cramoll are able to inhibit the deleterious effects of H 2 O 2 on the ΔΨm. Although studies on oxidative-stress-induced cell dysfunction are focused on the alterations in the mitochondrial bioenergetic and proapoptotic compounds release, lysosomes are also susceptible to the oxidative stress and have been associated with necrotic, autophagic, and apoptotic cell death [21,22]. Different kinds of hydrolytic enzymes (proteases, lipases, nucleases, glycosidases, phospholipases, phosphatases, and sulfatases) are present in this organelle and damage in this structure caused by toxic agents as H 2 O 2 can lead to the leakage of these enzymes in the cytoplasm resulting in the vanishing of the AO red labeling, as observed in our study [23][24][25]. The lysosomal proteases such as cathepsins family (A, B, D, and L) are potent activators of apoptotic effectors [26]. The lectins tested in this study were able to prevent lysosomal damage induced by H 2 O 2 and promoting the cell survival.
Furthermore, the activation of endonucleases leading to the genomic DNA fragmentation is one of the most representative events during apoptosis [27]. The protective actions of the tested lectins against DNA ladder fragmentation induced by H 2 O 2 in Vero cells were confirmed by TUNEL analysis.  pCramoll and rCramoll share several biophysical properties, the ability to recognize Glc/Man moieties, and the same pH dependent dimer-tetramer equilibrium. However, the tetramers of rCramoll are composed of intact monomers due to the absence of natural fragmentation process, and this characteristic is related to the little enhancement in its stability when probed with acidification, high temperatures, or hydrostatic pressure [10,11]. As different degrees of protective effects were observed in pCramoll and rCramoll (these differences could be also observed in other biological activities of these proteins [10,28]), we decided to examine the effects of H 2 O 2 on their structures using intrinsic fluorescence emission. Cramoll has four tryptophan residues and two of them (40 and 109) are located in the protein core [9]. Tryptophan is easily oxidized by hydrogen peroxide by photooxidation in the presence of oxygen [29,30]. Oxidative damage distorts the hydrophobic surface and the hydrophobic core of proteins, and partial aromatic amino acid substitution, caused by hydrogen abstraction, results in a decrease in the fluorescence emission. This work showed a decrease in the intrinsic fluorescence emission for both proteins when treated with H 2 O 2 at 1 mM, suggesting that the different effects of each lectin could be related to other factors, such as sugar affinity.
In summary, pCramoll and rCramoll ameliorate the H 2 O 2 -induced oxidative stress and cell death. The protective effects were related to the inhibition of mitochondrial superoxide generation, the reestablishment of ΔΨm, and the blocking of the deleterious effects of oxidative stress on lysosomal integrity and DNA fragmentation, promoting cell survival and proliferation. Although the molecular mechanisms responsible for the antioxidant properties of pCramoll and rCramoll remain to be elucidated, some studies carried out by our and other research groups have demonstrated that some lectins act directly as scavengers of ROS [31][32][33]. However, we cannot rule out the possibility that pCramoll and rCramoll act indirectly on the antioxidant enzymes triggering detoxification mechanisms that protect the cells against oxidative stress induced by H 2 O 2 treatment. Further studies are needed to investigate in more detail the molecular mechanisms involved in the protective role of lectins against the damage caused by the oxidative stress ( Figure 8).