Anticancer and Hepatoprotective Role of Selenium Nanoparticles against Liver Carcinogen Acrylamide Induced Toxicity: In vitro and in vivo Studies

Background and Objectives: Selenium is considered one of the essential components of many enzymes, particularly the antioxidant enzymes such as thioredoxin reductase. Vegetables, among other food sources, are common sources of selenium. In the present study, the green synthesis of Selenium nanoparticles (SeNPs) from Ocimum basilicum’s aerial part was carried out, as were the anticancer and hepatoprotective roles of selenium nanoparticles against liver carcinogen acrylamide-induced toxicity. Materials and Methods: The classification of SeNPs was done by different spectroscopic methods, including UV-spectroscopy, FT-IR, XRD, and HR-TEM. The synthesized SeNPs were tested for their anticancer properties using MTT assays (the normal human liver fibroblast cell line (WISH) and Human liver cancer (HepG2), respectively). Results: The histopathological studies indicated that the liver acrylamide induced in mice showed necrosis and damage in liver cells, but the treatment of mice with SeNPs, reduced the effect of acrylamide significantly. Conclusion: The results indicated the protective effect of SeNPs on acrylamide-induced cells both in vitro and in vivo .


INTRODUCTION
The Lamiaceae family includes an annual, herbaceous, 20-60 cm tall, white, and purple flowering plant, Ocimum basilicum.According to Chalchat and Ӧzcan, the plant was first found in Iran and India. 1,2Ocimum plant is considered an excellent source of antioxidant potential due to the presence of large quantities of secondary metabolites such as polyphenolic compounds. 3ccording to pharmacological surveys, 4 a wide variety of terpenoids and phenolic chemicals were stored in glandular trichomes (specialized glands) present on the surface of their leaves.][8] Since Selenium (Se) is an essential component of the antioxidant enzymes (glutathione peroxidase and thioredoxin reductase), it has recently gained prominence as one of the most essential dietary supplements.Due to their function as antioxidants that scavenge Reactive Oxygen Species (ROS), these enzymes are extremely valuable. 9,10Oxidative stress and ROS play a substantial part in cancer, and adequate selenium may also play a major role in normal cell growth 11 Inorganic versions of some Se supplements, in particular, have documented toxicity at larger doses.As a result, this trace element's Nanoparticles (NPs) were created to reduce their toxicity and improve their biological activities.In this study, we studied Ocimum basilicum's potential for green SeNP Faridi, et al.: Anticancer and Hepatoprotective Properties of Selenium Nanoparticles production.Then, the cytotoxic and hepatoprotective properties of the improved green SeNPs were examined.

Chemicals
The chemicals used in the study were purchased from S (USA).For qualitative and quantitative evaluations, the following devices and frameworks were used: The WARING Lab Blender (Dynamics Corp., USA), the Hei-VAP Rota vapor (Germany), the LC: 1260 Infinity II (Agilent, USA), the 150/vt Ultrasonic homogenizer (Biologics, North Carolina), the PANalytical X'Pert PRO X-ray Diffraction System (Netherlands), the JEOL JEM-2100 High-Resolution Transmission Electron Microscope (Ja: Shenzhen Mindray Bio-Medical Electronics, China).

Plant Collection
Aerial parts of O. basilicum were collected in Tanta, Tanta Governorate, Egypt, and identified in the faculty of science's plant taxonomy department.

Plant Extraction
The collected plant samples were thoroughly cleaned with distilled water to remove any pollutants before being dried at 25ºC in the dark room.After shade drying, the aerial part of the plant sample was finely ground and used for further studies.The aqueous extract was made using the cold maceration method. 12riefly 250 g of the plant powder was thoroughly mixed in a shaker for continuous agitation at 100 rpm after being soaked in 4L of distilled water for 24 hr at 20ºC.The extract was then filtered and kept at -4ºC for further analysis.

Green Synthesis of Selenium Nanoparticles
Using a magnetic stirrer, 50 mL of double-distilled water (heated to 60-80ºC) was used to dilute the crude aerial portions of O. basilicum extract (approximately 30 mL), followed by the addition of 30 mL of a 40 mM selenium acid solution (H 2 SeO 4 ), and then at 27ºC, the mixed solution was agitated for 24 hr.until the color changed from pale yellow to crimson.This combination was kept at 27ºC in a hot air oven for 24 hr.The obtained SeNP product was centrifuged at 20,000 rpm for 20 min with Double-Distilled Water (DDW) to eliminate contaminants.The resulting crimson pellets were employed for further research after being dried in a freeze-dryer for two days in an airtight container. 14

Characterization of SeNPs
The synthesized SeNPs were principally examined by Rigol Ultra-3660 UV-vis spectroscopy [200-800 nm].The functional groups and different phytochemicals involved in the reduction process and stability of the produced SeNPs at 4000-400 cm -1 were then identified by using FTIR in the Attenuated Total Reflectance (ATR) mode.The size and crystallite of SeNPs were examined by using CuK1-X Ray diffractometer radiation (=1.5406Aº) running at 40 kV and 30 mA with 2 varying from 30º-140º, SeNP pellets were coated on a copper grid after being suspended in ethanol, sonicated, and allowed to dry before being investigated with a JEOL-2100 HR-TEM and FE-SEM with EDS mapping analysis (JEOL 7401 F).

Cell lines
Human liver carcinoma (HepG2) and the liver of a normal human fibroblast cell line (WISH) were acquired from a tissue culturelLab at VACSERA Institute, Agoza, Egypt.

Cell culture and MTT assay
To evaluate the cytotoxicity of SeNP, the MTT (3-(4, 5dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide) colorimetric assay was performed in 96-well plates. 15The entire procedure was kept under sterile conditions by using a laminar air-flow cabinet, following culturing and sub-culturing techniques adopted by Thabrew, et al. 16 In vivo anticancer Activity 18 mature male albino rats were used for the in vivo studies.Separate cages with a humidity level of between 55 and 65% were used to keep the animals.There were three groups of six animals each.In the animals of groups B and C, hepatocellular carcinoma was induced using Diethyl Nitrosamine (DEN) (50 mg/kg) and acrylamide (2 mg/Kg), which led to oxidative stress as evidenced by an increase in Malondialdehyde (MDA) and a super-decrease in Glutathione (GSH) content.Group (A) served as normal control.Histopathological evidence confirmed the hepatocellular carcinoma's progression.After four weeks, rats were intraperitoneally injected with a single dosage of acrylamide.Group (C) received corresponding treatments with Selenium Nanoparticles (SeNPs).

Sample collection and/or in vivo liver function analysis
The animals were sacrificed by the method of decapitation after eight weeks of treatment.On days 15 and 30 of the treatment period, the cardiac puncture was utilized to collect 5 mL of blood in gel tubes.In a 14-k Humax centrifuge, samples were spun at 3000 rpm for 15 min.Using standard reagents provided by Human Germany, levels of blood glucose were examined on a Humalyzer 3000.Sections of the liver's lobes were preserved in a 10% formalin solution for histological analysis of any cancerous lesions.At the start and end of the experiment, blood (5 mL) was drawn from the vein, and liver function tests, including AST, ALT, and ALP, were performed (Table 1).

Statistical analysis
SPSS version 16 was used to perform the statistical analysis.All values were expressed as mean±standard error to the mean and were compared with the values of control by one-way ANOVA.p values less than 0.05 were considered statistically significant, and values less than 0.01 were considered highly significant.

HPLC Analysis
Several polyphenols, including catechin, caffeine, caffeic acid, and quercetin, were found in the samples in the HPLC analysis (Figure 1).Table 2 displays the concentrations and structures of polyphenols.The aerial portions of O. basilicum contain significant amounts of chlorogenic acid (962.12g/g), catechin (904.19), and quercetin (922.23 g/g).There were some unidentified peaks, which could not be identified due to the low concentration.These polyphenolic chemicals are crucial for the synthesis of selenium nanoparticles.Additionally, O. basilicum's 4'.7-dihydroxy isoflavone, a polyphenolic molecule, is what gives selenium nanoparticles their ability to last for 18 months without aggregating.Propyl gallate and naringenin were detected in high concentrations in the majority of phenolic compounds.It has been discovered that phenolics are efficient H-donors, (due to the presence of a hydroxyl function group), which accounts for a wide range of biological activities.

Green synthesis of SeNPs
The conversion or reduction of selenic acid to Selenium dioxide nanoparticles (SeO 2 ) was indicated by the change in color of the solution and the formation of a red pellet precipitate.

U.V. Spectrophotometric Analysis and FTIR Spectroscopic analysis
Metal nanoparticles are elucidated due to the differences in solution color and peak locations.UV-vis absorbance spectroscopy has proven to be a very important technique for the investigation of MNPs because of its sensitivity to the size of nanoparticles.The formation of SeNPs was confirmed by the UV-vis spectrum (200-800 nm).U.V. spectrum of SeNPs prepared by green synthesis exhibited a characteristic peak at 282 nm.The calculated direct band gap (Eg) was found to be 3.32 eV (Figure 2A).
The FTIR technique is usually used to confirm the formation of nanoparticles, and it offers an impression of the vibrational and rotational modes of the existing molecules.FTIR analysis was used to detect the functional groups involved in the reduction and stabilization of SeNPs. Figure 2B depicts the FTIR spectrum of SeNPs, which showed a broad vibration band at 3406 cm -1 corresponding to the O-H stretch of alcoholic and phenolic groups.A characteristic band at 2330 cm -1 corresponds to nitro compounds (N-O asymmetric stretch) present in the compounds, and the absorption band at 2966 and 2897 cm -1 represent the C-H stretch of alkynes groups.The strong bands at 1539 and 1489 cm -1 are due to the aromatics and alkanes ring (C-C and C-H stretching).Another characteristic band at 1384-1219 cm -1 corresponds to the bending O-H stretches of carboxylic groups,  therefore, these functional groups indicate and confirm the involvement of different reducing and stabilizing agents in the synthesis of SeNPs. 17,18In the FTIR spectrum, the presence of other bands may indicate the existence of enzymes, proteins, and metabolites such as flavonoids, polyphenols, and carboxylic acid, which remained bound to SeNPs and assisted in the reduction of zinc ions to ZnO.NPs.

HR-TEM Analysis
The spectrum of HR-TEM was used to explain the crystalline characteristics and size of the synthesized SeNPs.The HR-TEM analysis was conducted using JEOL-2100, and the images at different magnifications (50 and 100 nm) are shown in Figure 4. TEM images showed individual and spherical nanostructures as the major series of particles that were between 18.96 and 36.28 nm.

In vitro assay
A MTT assay was carried out to investigate the cytotoxicity of SeNPs on normal and liver cancer cell lines.Doxorubicin was used as the positive control.The IC 50 inhibitory concentration (50%) was 19.1 µg/mL for HepG2 and 68.3 µg/mL for WISH cell lines.The cytotoxicity results clearly indicated that the SeNPs have selective toxicity towards the liver cancer cell-line (HepG2) in comparison with the normal cell-line WISH.The IC 50 value of doxorubicin was 13.7 against hepG2 cell lines, which is almost similar to the IC 50 of SeNPs, which indicates that SeNPs can be one of the important anticancer drug candidates against liver cancer (Table 3).

Biochemical analysis
The selenium nanoparticles' in vivo anticancer potential was assessed against human liver cancer cell lines.The findings of this investigation demonstrated that synthetic SeNPs have powerful and selective anti-liver cancer cell line action (HepG2).SeNPs were therefore examined in vivo against the same cell line.The studies on liver enzymes were performed, and the levels of liver enzymes were monitored.The levels of AST, ALT, and ALP were 69.02±0.

Histopathological examination
Hepatocellular carcinoma-affected liversdisplayed enlargement, granular, and vacuolar degeneration of the hepatic cells, along with the presence of numerous necrotic cells.The necrotic cells either had pyknotic nuclei or seemed homogeneous in structure without any nuclei.The cells had multiple areas of vacuolar degeneration, and their nuclei were atypical.Rats from Group 1 showed normal hepatic lobule histological structure under a microscope.SeNPs treatment demonstrated moderate granular and vacuolar degeneration, necrosis of hepatic cells with some nuclear pyknosis and activated Kupffer cells, as well as very good repair of the hepatic parenchymal cells, which appeared close to normal with a few scattered necrotic cells.The histopathological studies validated the results of the liver function test and cytotoxicity test.In all the above studies, SeNPs were shown to have significant anticancer as well as hepatoprotective effects both in vitro and in vivo (Figure 5).

CONCLUSION
Selenium is one of the most important micronutrients and is an essential part of various enzymes.A deficiency of selenium causes susceptibility to oxidative stress in animals and humans. 20elenium nanoparticles showed anticancer activity, which could be due to their presence in antioxidative enzymes.Oxidative stress decreases drastically, which is one of the most common causes of carcinogenesis. 21The present study clearly indicated that the green synthesized SeNPs exhibit both in vitro and in vivo anticancer and hepatoprotective effects.

Faridi,
et al.: Anticancer and Hepatoprotective Properties of Selenium Nanoparticles

Figure 5 :
Figure 5: Effect of SeNPs on liver sections of different groups: A) of control rats revealed normal histological; B) induced control group (acrylamide); C); experimental group treated with SeNPs.

Table 2 : Concentrations and structures of components of aerial parts of O. basilicum.
19ridi, et al.:Anticancer and Hepatoprotective Properties of Selenium Nanoparticles planes, respectively.The crystalline nature of SeNPs is evidenced by this reflection, which is in agreement with the literature.19