Curative Role of Dithiophenolato Titanium (IV)-Chitosan Nanocomposite Complex Against Carbon Tetrachloride-Induced Liver Injuries

Abstract


Introduction
The liver is the largest tough organ in the body and is requisite for survival.The liver has numerous functions including the synthesis of proteins, glucose, bile, and clotting factors, besides breaking down hormones, certain drugs, and xenobiotics [1,2].Hepatic metabolism of medicinal agents and toxins as carbon tetrachloride (CCl 4 ) involves disturbed hepatic cell biochemistry with the augmented generation of free radicals and reactive oxygen species (ROS) and redox imbalance with secondary deterioration to proteins, lipids, carbohydrates, and nucleic acids [3,4].The xenobiotic-induced hepatotoxicity ranging from a subclinical anicteric state to severe necroin ammatory hepatitis (acute, recurrent, or chronic) and cirrhosis, relies on the dosage, nature, and period of exposure to the xenobiotics, the antioxidant defense, and attendant exposure to another disease or xenobiotics [1,2].ROS and reactive nitrogen species (RNS) are well known for playing a double function as both mischievous and bene cial species.The "two-faced" of ROS is adduced by a growing body of proof that ROS within cells act as secondary messengers in intracellular signaling cascades, which stimulate and preserve the oncogenic phenotype of cancer cells, while, ROS can also prompt cellular senescence and apoptosis and can thus function as antitumorigenic species [5,6].The cumulative production of ROS/RNS through either endogenous or exogenous insults is titled oxidative stress (OS) and is popular for numerous types of cancer cells that are connected with altered redox organization of cellular signaling paths.OS prompts a cellular redox imbalance that has been found to be present in different cancer cells as compared with normal cells; the redox imbalance thus may be linked to oncogenic inducement [7].
CCl 4 is an industrial chemical found in refrigerants and solvents for waxes, varnishes and other materials.CCl 4 is one of the most potent hepatotoxins, so it is widely used in scienti c research to evaluate hepatoprotective agents [8].Chronic exposure to CCl 4 can cause liver and kidney damage and could result in cancer [5,6].In liver, CCl 4 is metabolized by cytochrome P450 into trichloromethyl radical ( • CCl 3 ) which converts to trichloromethylperoxy radicals (CCl 3 OO • ) [5,6].
Liver diseases represent a major cause of serious and public health problems leading to morbidity and mortality in worldwide and Egypt [8,9].Therefore, scientists are working on discovering new treatments for liver diseases that are effective and do not have side effects or have minor side effects.During the last 100 years, metals have been used in therapy and other medical applications in a rational manner.A wide variety of metallodrugs have been investigated which showed excellent anticancer results [10][11][12][13].Gallium, titanium, platinum, ruthenium, tin, gold and cobalt have shown effective anticancer activities [10,13].
Titanium Ti(IV) as a biomaterial is generally used for orthopedic implants such as the substitution of teeth and bone [14].The advantage of titanium compounds lies in their elevated effectiveness and low toxicity.In biological mediums, hydrolysis of Ti(IV) complexes leads to safe and inert titanium oxide (TiO 2 ) [15].The next generation of phenolato-based complexes showed markedly improved stability and high activity.Ti(IV) is chie y accumulated in the cellular nucleic acid-rich areas, mainly in the nucleic chromatin of tumor cells [16] and interacts with the oxygen (hard base) of the phosphoesters and with the nitrogen of the nucleobase in DNA that leads to cell cycle inhibition.Phenolato Ti complexes are considered a novel and e cient anticancer drug with nontoxic effect at extents of the effective doses [17,18].In 2012, we synthesized the dithiophenolate titanium (IV)-complex [{Ti(N 2 H 2 S 2 ) (O i Pr)}c 2 ] (DBT: Fig. 1) that contains the thiophenolate and amine donors instead of phenolate donors [19].DBT complex is stable under physiological conditions, but, it is characterized by low solubility.Therefore, we prepared DBT-CS nanoparticles (DBT-CSNPs) by loading DBT on chitosan nanoparticles to improve bioavailability and the therapeutic e cacy of DBT [20].Chitosan, a natural biomaterial, is a modi ed biopolymer, derived by partial deacetylation of chitin.It has been investigated for numerous biomedical applications due to its biodegradability, biocompatibility, antimicrobial properties, and functionality [21].Further, chitosan nanoparticles (CSNPs) have the advantages of chitosan and the properties of nanoparticles such as surface and interface effect, small size and quantum size effects.Therefore, chitosan nanoparticles are greatly studied as nanocarriers for drug, protein, and gene delivery systems [22,23].Our previous studies revealed that DBT and DBT-CSNPs have antibacterial and cytotoxic activities and can bind with the DNA helix via groove binding [20].
Therefore, in this study, and for the rst time, the LD50 values of DBT and DBT-CSNPs were determined as well as their therapeutic effects were evaluated against CCl 4 -induced liver injuries.The study focused on the determination of the markers of OS, apoptosis, liver functions, lipid pro le, and kidney functions besides liver histopathology were determined.Additionally, the effect of DBT and DBT-CSNPs on cell cycle analysis in normal liver (THLE2) and human liver cancer (HepG2) cell lines were investigated to recognize if these compounds have antitumor activity.

Results And Discussion
The results showed that the prepared DBT-CSNPs have a spherical morphology with an average particle size of ~ 85 ± 2 nm.The presence of DBT in the DBT-CSNPs was found to increase the thermal stability of the composite material in comparison to DBT [20].

LD50 of DBT and DBT-CS NPs
The current results showed that LD50 values of DBT and DBT-CSNPs were about 1350 mg/Kg and 1800 mg/Kg, respectively (Table 2).The previous results revealed that the LD50 values of oral titanium tetrachloride and oral titanium oxide nanoparticles (TiO 2 NPs) are 1780 mg/kg and greater than12 g/kg, respectively [24].

Effect of different studied compounds on OS
The present results revealed that CCl 4 injection caused a signi cant elevation (P < 0. 05) in the malondialdehyde (MDA) level and GR activity, with a signi cant decline (P < 0. 05) in the level of reduced glutathione (GSH) and the activities of glutathione-S-transferase (GST), total glutathione peroxidase (GPx) and superoxide dismutase (SOD) as compared with the control group (Fig. 3).MDA is the main product of peroxidation of polyunsaturated fatty acids and elevated MDA content is a signi cant indicator for the lipid peroxidation [25].The formation of lipid peroxides in the liver tissues may be related to the effect of reactive free radicals of CCl 4 metabolites, • CCl 3 and CCl 3 OO • , that accelerated the peroxidation of native membrane lipids.The elevation in the peroxidation of the mitochondrial membrane increased membrane permeability and altered calcium homeostasis, resulted in the loss of cell integrity that contributed to cell death [5,6].On the other hand, free radical scavengers are nonenzymatic as GSH and enzymatic such as GPx, glutathione reductase (GR), GST and SOD could protect the biological systems from the deleterious effects of the free radicals [26].Where, GSH plays an important role against CCl 4 -induced lipid peroxidation by covalently binding to • CCl 3 and CCl 3 OO •• radicals [27].Additionally, GSH acts as a cofactor for GPx and as a nucleophilic scavenger of numerous compounds [26].The depletion in GSH level after CCl 4 administration could contribute to the stimulation of lipid peroxidation [1,27].Otherwise, GR plays a key role in cellular defense against OS by preventing the accumulation of oxidized glutathione (GSSG) and thus maintaining the redox state.The increase in GR activity after CCl 4 administration possibly re ects an adaptation to oxidative condition and this agreed with previous studies [1,2,28].Additionally, SOD is the rst line of protection in the body against superoxide radicals [25].SOD catalyzes the dismutation of the superoxide radical into ordinary molecular oxygen and H 2 O 2 .GPx, a selenium-containing enzyme, is the second line of protection against hydroperoxides through catalyzing the reduction of H 2 O 2 and lipid peroxides, in the presence of GSH, to water and lipid alcohols, respectively, while GSH is transformed into GSSG [1,27].Otherwise, GSTs are major phase II detoxi cation enzymes found mainly in the cytosol.They catalyze the conjugation of GSH to a wide range of electrophilic substrates, usually resulting in detoxi cation.Subsequently, the reduction in the activities of antioxidant enzymes may be related to their inhibition by CCl 4 and its reactive metabolites through direct interaction with the enzyme molecules.Also the inhibition of GPx and GST activities probably due to GSH depletion.SOD inhibition may be concerned with the oxidation of cysteine residues in the enzyme molecules by superoxide radicals and H 2 O 2 [3,29].
On the other hand, treatment with DBT and DBT-CSNPs after CCl 4 injection increased MDA level (non-signi cantly) and GR activity (signi cantly: P < 0.05) but decreased signi cantly the antioxidants (GSH level, GPx, GST and SOD activities) as compared to the CCl 4 group (Fig. 2).This indicates that DBT and DBT-CSNPs increased non-signi cantly lipid peroxidation in liver tissues, in spite of, they improved the malfunctioning liver and kidney functions, and lipid pro le as mentioned in (Table 3).The results clarify that DBT and DBT-CSNPs deteriorated the unwanted hepatocytes via non signi cant oxidative stress.The induction of OS may be related to the effect of DBT and DBT-CSNPs and their metabolites, TiO 2 and TiO 2 NPs, respectively.TiO 2 induces the generation of ROS such as O 2 -• and H 2 O 2 , resulting in increasing oxidative stress, lipid peroxidation, membrane damage, and increases the apoptosis of the unwanted cells [30].Also, the thiol groups in the DBT and DBT-CSNPs are able to oxidize in the presence of H 2 O 2 , forming O-S-O [31] resulting in the reduction of H 2 O 2 and this leads to the non-signi cant elevation in the MDA level compared with CCl 4 .Moreover, the reduction in the antioxidant enzymes may be related to the fact that SOD and GPx are Zn-and selenium-dependent enzymes, respectively.Therefore their inhibition may be due to the replacement of zinc and selenium by Ti.Also, the reduction of GSH level may be due to the binding of Ti with the sulfhydryl group and accordingly decreasing GPx and GST activities [32].These results indicate that DBT and DBT-CSNPs may promote its antineoplastic effect, though the induction of the oxidative stress, which in consistence with the action of antineoplastic drugs, including doxorubicin and cyclophosphamide [33,34].
The results showed that the treatment with CSNPs after CCl 4 injection increased non-signi cantly MDA level and GR activity (Fig. 3).While this treatment decreased signi cantly the antioxidant parameters as compared to the CCl 4 group (Fig. 3).Also, Fig. 3 shows that the administration of CSNPs, DBT-CSNPs and DBT to healthy rats for 14 days caused non-signi cant changes (increases or decreases) in the levels of markers of lipid peroxidation and the antioxidant markers compared with the control group.This led to nonsigni cant changes in the markers of lipid pro le, liver and kidney functions.In general, the results revealed that DBT-CSNPs had a greater effect than DBT and this may be due to the physicochemical properties of the nanoparticles which is distinguished by nano-size, raised surface area/unit mass and surface property effects that facilitated its passage through the cellular membranes, hydrolyzed into TiO 2 NPs and interrupted the biological systems [35][36][37][38].Our results are in agreement with the studies which showed that the nanoparticles as CSNPs induce OS and this process is in a dose-dependent manner [39,40].Therefore, DBT and DBT-CSNPs must not be taken without medical indications and must be taken for a short period.
Otherwise, Fig. 3 reveals that treatment with cisplatin (for 4 days) after CCl 4 injection signi cantly increased OS as shown from the elevation of lipid peroxidation and reduction of antioxidant parameters leading to liver damage resulting in the elevation of serum liver enzymes and changing the lipid pro le [41][42][43].

Effect of different studied compounds on apoptosis
Apoptosis is a basic biochemical process for a selective and controlled removal of cells within multicellular organisms and a key mechanism for the elimination of injured, infected, or mutated cells [44][45][46].Apoptosis is tightly regulated by speci c genes, including several pro-and anti-apoptotic proteins [44,45].Proteins of the Bcl-2 family exert different effects; for example, Bcl-2 and Mcl-1 are anti-apoptotic proteins, while white B-cell lymphoma X protein (bax, Bad and Bak) exhibit pro-apoptotic effects.Dysfunction of apoptosis renders the cancer cell resistant to treatment as well as promotes the tumorigenesis [45,47].Bax activation elicits cytochrome-c release, procaspase-3 activation and Poly (ADP-ribose) polymerase cleavage through the stimulation of Apaf-1 (apoptosis protease activating factor-1) which leads to the induction of the apoptotic response and cell death [48].Caspase-8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway.Activation of caspase-8 induces apoptosis through the direct activation of caspase-3 or activation of Bax which in turn activates caspase-3.These activations led to the cleavage of essential substrates for cell viability, inducing cell death [48,49].Additionally, cleavage of chromosomal DNA into oligonucleosomal size fragments is an essential part of apoptosis [44][45][46].
The current results revealed that CCl 4 administration showed a signi cant (P < 0.05) down-regulation of Bcl2 expression with a signi cant (P < 0.05) upregulation of Bax and caspase-8 expressions as well as reduction in DNAF level compared to the control group (Fig. 4a, b and c "lane 1").Furthermore, a signi cant (P < 0.05) elevation was observed in the ratio of Bax/Bcl-2.This indicates that CCl 4 administration induced apoptosis where the elevation in Bax/Bcl-2 ratio caused a change in the integrity of the mitochondrial membrane leading to cytochrome C release to the cytoplasm.Where cytochrome C binds to the Apaf-1 resulting in the activation of procaspase 9 which in turn activate procaspases-3 and 7 leading to apoptosis and the cell death [4,50].Moreover, as shown from the current results, the elevation in caspase-8 after CCl 4 administration induced apoptosis.Where caspase-8 activates caspase-3 (direct) or activates Bax, which in turn activates caspase-3 (indirect pathway) leading to the cleavage of the fundamental substrates for cell viability, inducing cell death [4,50,51].Otherwise, as shown from our results, CCl 4 increases the ROS, which plays an important role in the activation of apoptosis through intrinsic and extrinsic pathways.The elevation in ROS, leads to the increase of p53 signaling which in turn stimulates the Bax expression, but inhibits Bcl-2 expression [33,34].Moreover, DNAF and DNA damage may be related to the reaction of CCl 4 metabolites ( • CCl 3 and CCl 3 OO • ) and other lipid peroxidation products with DNA forming adducts [1,2] that activated pro-apoptotic factors, promoting apoptosis and cell death [52].Our results are in agreement with the previous studies which demonstrated that CCl 4 induces rat liver brosis through the activation of apoptosis [4,50,51].
In contrast, treatment with DBT and DBT-CSNPs, and cisplatin after CCl 4 injection caused a signi cant (P < 0.05) elevation in the Bcl-2 expression level associated with a signi cant (P < 0.05) decline in the expression levels of Bax and caspase-8 as well as reduction in the Bax/Bcl-2 ratio and DNAF level compared to the CCl 4 group (Fig. 4a-c).Also, treatment with CSNPs after CCl 4 injection caused a nonsigni cant elevation in all markers of apoptosis when compared with the CCl 4 group.This indicates that the treatment with DBT-CSNPs after CCl 4 was related to the effect of DBT not to that of CSNPs.DBT, DBT-CSNPs, and their metabolites as well as cisplatin reduced the apoptosis induced by CCl 4 .A possible mechanism for such effect is the reduction of Bax/Bcl2 ratio led to the stabilization of the mitochondrial membrane and prevented the releasing of cytochrome C. Interestingly, treatment with DBT-CSNPs showed a greater effect than DBT and this may be due to the differences in the physicochemical properties of these compounds and their metabolites as mentioned before.These results agree with the previous studies which showed that TiO 2 NPs have anti-apoptotic action greater than TiO 2 [53].Also, the studies showed that nanoparticles act as anti-apoptotic effect depending on their doses and period as well as cellular types [50].
Otherwise, treatment with DBT-CSNPs and DBT gave better results than cisplatin treatment.Where treatment with cisplatin (for 4 days) after CCl 4 injection signi cantly increased OS and lipid peroxidation leading to liver damage resulting in the elevation of serum liver enzymes and changing the lipid pro le (Table 3) which in accordance with Silici [54] and Ateyya [55].
On the other hand, the administration of each DBT, DBT-CSNPs, or CSNPs for the healthy rats caused non-signi cant changes in all markers of apoptosis as compared with the control group (Figs. 3 and 4).This indicates that DBT-CSNPs and DBT have no effect on the apoptotic markers in normal animals.

Liver and kidney functions and lipid pro le
The results showed that CCl 4 administration caused signi cant (P < 0.05) elevation in the activities of serum ALT, AST and ALP with signi cant reductions in serum albumin and TP and liver TP levels compared to the control group (Table 3).Also, there were signi cant increases (P < 0.05) in serum cholesterol, TG and LDL-cholesterol with a signi cant decrease in serum HDL-cholesterol level (Table 3).This may be due to liver damage induced by CCl 4 which led to the leakage of liver enzymes into the blood circulation and decreased protein biosynthesis [56].Additionally, blood urea and serum creatinine levels in rats injected with CCl 4 were signi cantly increased (P < 0.05, Table 3) indicating that CCl 4 induced nephrotoxicity and these results are in accordance with the results of Safhi [57].
Otherwise, treatment with DBT and DBT-CSNPs after CCl 4 administration improved liver functions, lipid pro le and kidney functions with different degrees as shown from the levels of all parameters (Table 3).This indicates that treatment with DBT and DBT-CSNPs reduced the hepato-and nephrotoxicity induced by CCl 4 .Also, treatment with DBT-CSNPs showed a better effect than DBT.Treatment with CSNPs after CCl 4 administration caused nonsigni cant changes in all markers of liver and kidney functions and lipid pro le.
Treatment with cisplatin after CCl 4 administration improved liver function and lipid pro le, but to a lesser degree than those treated with DBT and DBT-CSNPs (Table 3).Furthermore, cisplatin treatment caused non-signi cant improvement in kidney functions compared to the CCl 4 group.These results agree with the previous studies which showed that treatment of different cancers with cisplatin causes nephro-, neuro-, cardio-and hepatotoxicity, due to its accumulation in the liver, kidney and other organs leading to production of ROS and induces OS resulting in kidney failure and liver damage [71].
On the other hand, Table 3 shows that DBT and DBT-CSNPs administration for the healthy rats for 14 days caused signi cant changes (P < 0.05) in some markers of liver and kidney functions and lipid pro le as compared to the control group.These effects on healthy rats may be related to the long period (14 days) of the administration of DBT and DBT-CSNPs and the accumulation of their metabolites.The results showed that CSNPs administration to healthy rats for 14 days caused nonsigni cant changes in liver and kidney functions and lipid pro le as compared to the control group.

Histopathological analysis
The histopathological characteristics of liver tissues from the different studied groups are shown in Fig. 5 (A-K).Administration of the healthy rats with each CSNPs, DBT and DBT-CSNPs, separately had no effect on normal liver architecture.While, treatment with DBT and DBT-CSNPs after CCl4 administration improved liver histopathology induced by CCl4 indicating the therapeutic roles of DBT and DBT-CSNPs that con rm the biochemical analysis.

BT and DBT-CSNPs toxicity and cell cycle arrest
The viability of the HepG2 and THLE2 cells was measured under the effect of different concentrations of each DBT and DBT-CSNP for 24 h (Fig. 6a).The results showed that the cell viability of HepG2 and THLE2 cells was decreased with increasing the concentration of these compounds.Also, both DBT and DBT-CSNP showed selective cytotoxic, where DBT-CSNP exhibits greater cytotoxicity against HepG2 cells than DBT cytotoxicity (their IC50 values were 44.04 and 31.76 µg/ml, respectively).While, DBT-CSNP exhibits lower cytotoxicity towards the normal THLE2 cells than DBT cytotoxicity (their IC50 values were 1915 and 2094 µg/ml, respectively).
Cell growth and proliferation are controlled by the cell cycle regulation, and an imbalance between them causes apoptosis that is implicated in the growth and progression of most tumors [58].Therefore, anticancer agents that interfere with the cell division and halt the uncontrolled proliferation of cancer cells are considered an important treatment strategy [58,59].The current results showed that treatment with DBT and DBT-CSNPs caused a signi cant decrease in the population of HepG2 cells in G0/G1 and S phases compared to the normal cells (Fig. 6b).High populations of HepG2 cells were arrested at G2/M checkpoint as compared to untreated cells.Also, the results showed that the cells treated with DBT-CSNPs showed the lowest levels of G0/G1 and S phases and the highest level of G2/M phase (Fig. 6b).Cell cycle arrest in the G2/M phase leads to disruption of the tubulin-microtubule equilibrium and allows the time for the repair of DNA damage [60].This means that these complexes have anticancer activities and this may be related to the effect of the DBT and DBT-CSNPs and their hydrolyzates; (TiO 2 and TiO 2 NPs), respectively.It has been reported that TiO 2 NPs may induce OS in HepG2 cellscausing DNAF and p53 activation which lead to apoptosis [30,37].The results also indicate that each DBT and DBT-CSNPs inhibited cell growth in S-and G2/M-phases with different degrees and DBT-CSNPs has a potent effect than DBT, this may be due to the simplicity of these NPs which can pass throughout the cellular membranes and disturb the biological systems.
In general, our results revealed that DBT-CSNPs and DBT treatments decreased rat liver apoptosis induced by CCl 4 resulting in the improvement of liver architecture and functions.
In contrast, the results showed that DBT-CSNPs and DBT prevented the proliferation of the HepG2 cell line, where they arrested the cell cycle in the G2/M phase.Also, these compounds showed pro-apoptotic effects in HepG2 cells.These results indicate that both complexes are characterized by selectivity, i.e. they have an ability to differentiate between liver injuries and cancer cell lines.These results are in agreement with the previous studies which reported that the effect of some drugs and xenobiotics are not matched in vivo and in cell lines [67, 68].These results are in agreement with the previous studies which reported that the effect of some drugs and xenobiotics in vivo and in cell lines are unlike each other [61,62].Where, these studies revealed that tumor cell lines have important limits, and there are some variations between cell lines and tumor tissues in vivo including gene expression.Also, Cell lines lack interactions with the other cell types, their growth is not affected by cytokines and other cell signaling molecules, and the native tissue architecture is lost [61, 62].

Conclusion
LD50 values of DBT and DBT-CSNPs are 1350 mg and 1800 mg, respectively.DBT and DBT-CSNPs have a therapeutic effect against CCl 4 -induced liver injuries by reducing apoptosis through the reduction of DNAF, caspase 8, and the Bax/Bcl2 ratio as well as remediation of the antioxidant parameters.DBT-CSNPs "due to their structure" have a greater effect than DBT and both compounds have greater effects than cisplatin.The administration of healthy rats with low doses of DBT and DBT-CSNPs for 14 days has no effect on apoptotic markers and non-signi cant effect on the oxidative stress.Otherwise, DBT and DBT-CSNPs have antineoplastic activities against the HepG2 cell line, but DBT-CSNPs has a greater effect due to their nanostructure.Therefore, the pharmacokinetics of DBT-CSNPs and DBT should be evaluated to know their pharmacological roles.

Chemicals
Adherent HepG2 were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia, USA).GIBCO® Minimum Essential Medium (MEM), was purchased from GIBCO, USA.Dulbecco's Phosphate Buffered Saline medium and L-glutamine were obtained from Invitrogen, USA.Trypsin-EDTA, penicillin and streptomycin were purchased from Thermo Fisher Scienti c, USA.Cisplatin, CCl 4 , dimethylsulfoxide (DMSO) and other analytical grade chemicals were purchased from Sigma Aldrich, USA.MTT assay kit and RNA extraction kit were obtained from Eugene, Oregon, USA and Thermo Scienti c, Fermentas, respectively.All other analytical grade chemicals were obtained from Sigma (Germany) and Merck (Germany).All dilutions were made with high purity deionized water, obtained from a Milli-Q® system (Merck Chemicals GmbH, Darmstadt, Germany).

Animals
One hundred and twenty Sprague Dawley rats weighing 100-150 g were obtained from Faculty of Medicine, Alexandria University.The animals were housed in stainless cages under standard laboratory conditions of 12 h light/dark cycle, 55 ± 5% air humidity at room temperature of 22±3 °C and received a standard laboratory diet and tap drinking water for 2 weeks, as an adaptation period.All animal methodology was accomplished following the Institutional Animal Care and Use Committee (IACUC) and approved via the Committee of the Animal Care and Use in Alexandria University (Ethical approval reference number: AU 04 20 06 20 2 02).

Biological effects of DBT, DBT-CSNPs and cisplatin on CCl 4 -induced hepatotoxicity
The doses of DBT and DBT-CSNPs were chosen to be safe, away from their LD50 values where these doses approach that of cisplatin.DBT and DBT-CSNPs and CSNPs were dissolved in 2% DMSO [64,65].Seventy two Sprague Dawley rats were divided into nine groups (8 animals/group).Fig. 2 shows the experimental design, since the period of the experiment was 12 weeks comprehensive 2 weeks the adaptation period.Control group (C): the rats were treated with 0.5 ml DMSO (2%) for 14 days (at 9th and 10th weeks), CSNPs group: the rats were treated (i.p) with 3.0 mg CSNPs / kg BW/day for 14 days (at 9th and 10th weeks), DBT group: the rats were treated (i.p) with DBT (4.5 mg/kg BW/day for 14 days (at 9th and 10th weeks), DBT-CSNPs group: the rats were treated (i.p) with DBT-CSNPs (3.0 mg/ kg BW/day for 14 days (at 9th and 10th weeks), CCl 4 group: the rats were injected (i.p) with 0.5 ml of 99.9% CCl 4 /kg BW, with equal amount of olive oil, day after day for 6 weeks (at 3 rd and 8 th weeks) [66].CCl 4 -CSNPs, CCl 4 -DBT, CCl 4 -DBT-CSNPs groups: the rats were injected with CCl 4 for 6 weeks, then they treated with the same doses and periods of CSNPs, DBT and DBT-CSNPs, respectively.CCl 4 -Cisplatin: the rats were injected with CCl 4 for 6 weeks then they treated (i.p.) with 4 mg of cisplatin/kg BW/day for 5 consecutive days [67].At the end of the experimental period, the rats were fasted overnight and sacri ced after anesthetized by carbon dioxide.Blood was collected from caudal vena cava, kept at room temperature for 15 min, then centrifuged at 3000 rpm for 10 min and serum was stored at -20 o C until used.The livers were removed immediately where small portions were taken and xed in 10% formalin for the histopathological examination.The remaining livers were washed with cold saline solution (0.9% NaCl), divided into two parts and kept at -80 o C. The rst part was used for determination of the expression levels of caspase-8, Bcl-2, Bax and DNA fragmentation (DNAF).The second part was homogenized in 9 volumes of cold 0.1 M sodium phosphate buffer (pH 7.4) containing 0.9% NaCl, using a glass-Te on Homogenizer and the homogenate was centrifuged at 4000 rpm for 15 min at 4°C.The supernatant was kept, at -80°C till used for determination of the markers of OS [malondialdehyde (MDA) and reduced glutathione (GSH) levels, and the activities of glutathione-S-transferase (GST), total glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD)].

Determination of DNAF
It was determined using agarose gel electrophoresis technique [74].Liver tissues were homogenized in ve volumes of 50 mM Tris-HCl buffer containing 50 mM EDTA and 20% sucrose, pH 7.6.The genomic DNA was separated using the DNA puri cation kit (G-spin TM Total, Cat.No.17045, Korea).Then 15 µg of DNA/lane was loaded and separated by electrophoresis for 2h on 1% agarose gel containing ethidium bromide (10 µg/ml).Finally, the DNA bands were visualized using trans-illumination with ultraviolet light (Consort, Turnhout, Belgium).

Liver histopathological analysis
Liver tissues were xed, processed and embedded in para n wax.Sections of 5 µm in thickness were cut and stained with hematoxylin and eosin (H & E) for examination using light microscope [75].wells.After incubation, 10 μL of MTT solution (5 mg/ml) in phosphate-buffered saline (PBS) was added to each well and incubated for another 4 h at 37 °C.After the medium was discarded, 100 μL of DMSO was added to each well to dissolve the purple formazan crystal formed at the bottom of the wells.
Absorbance at 490 nm was measured in a BioTek Instruments Elx800 Universal Microplate Reader (USA) and the percentage of cell inhibition was determined.

Cell cycle analysis
The distribution of HepG2 cells in the different cell cycle phases (G0/G1, S and G2/M) were     Moreover, a periportal in ammatory reaction with degenerated hepatic cord and disrupts cell plates were observed in tissue of the cisplatin treated group, Figure 5 J.These histopathological results revealed the hepatoprotective effects of DBT and DBT-CSNPs that con rm the biochemical analysis.
left at 25 o C for 12 hours, DBT was added, left for 40 min and the solvent was removed at 40 o C. The characterization of DBT-CSNPs was examined by High Resolution Transmission Electron Microscope (HR-TEM), Scanning Electron Microscope (SEM) with EDX detector, X-Ray Diffraction (XRD), Fourier transforms infrared (FT-IR) and thermographymetric analysis (TGA) [20].4.4 Determination of LD50 values of DBT and DBT-CSNPs For determination of LD50 of DBT or DBT-CSNPs, 48 rats were used.The animals were divided into 12 groups and six doses of each DBT or DBT-CSNPs dissolved in 2% DMSO (200, 400, 800, 1200, 2000 and 3000 mg/kg) were used for the administration once, intraperitoneally (i.p).The animals were examined for any abnormal clinical signs and behavioral changes for 24 h.The number of dead rats in each group was recorded (% dead) and LD 50 was calculated by the arithmetic method of Kärber [63].LD100 = Lethal dose causing the 100% mortality.n = Group population: total number of animals/ group.a = Dose difference: the difference between two successive doses of the administered substance.b = Mean mortality: the average number of dead animals in two successive doses.

4. 6
Disclosure of the antitumor activities of DBT and DBT-CSNPS4.6.1 Cell viabilityThe anti-proliferative activity of the DBT and DBT-CS nanocomposite was studied against HepG2 and THLE2 cell lines using methyl thiazolyl tetrazolium (MTT) assay.MTT assay isused to quantify the number of viable cells, depends on the reduction of the yellow tetrazolium salt MTT to a purple MTT-formazan crystal by viable cells with active metabolism[76].HepG2 and THLE2 Cells were plated in 96-well plates at 10 4 cells/well and treated after 24 h with serial dilutions (0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 µg/ml DMSO (0.1%) of DBT and DBT-CS nanocomposite separately.Each concentration was repeated in 4

Figure 2 The experimental design Figure 3
Figure 2The experimental design

Figure 5 Effect
Figure 5 Effect of different compounds on the liver histopathology.H & E stained sections of liver tissue from the different examined groups.A: control, B: DBT, C: DBT-CSNPs, D-G: CCl4, H: CCl4-DBT, I: CCl4-DBT-CSNPs and J: CCl4-cisplatin (X200).Liver tissues of the control group showed normal lobular architecture, individual hepatocytes disclosed no pathology and the portal triad was unremarkable.Liver tissues of DBT and DBT-CSNPs groups exhibited normal hepatocytes around the central vein (Figure 5 B and C).Tissue sample of CCl4 intoxicated group indicated cellular in ltration, congestion of central vein, mild portal in ammation, hemorrhage as well as centrilobular hepatic necrosis and focus of lytic necrosis & dispersed apoptotic bodies both intra & extracellular in location (Figure 5 D-G).The specimen of DBT treated rats con rmed a mild degree of hepatic degeneration represented by cell swelling (arrow), Figure 5 H. On the other hand, liver tissue of DBT-CSNPs treated rats revealed a marked decrease of hepatic degeneration unless single cells degeneration (arrow), Figure 5 I.

Table 1 .
Primers used in the RT-PCR estimated [77].Brie y, HepG2 cells were untreated (control, reseived only DMSO, 0.1%) and treated with 40 μg/ml of DBT and DBT-CSNPs separately for 24 h, then cells were harvested and xed in ice cold 70% ethanol Tables

Table 2 :
Karber's method for determination of LD50 of DBT and DBT-CSNPs rats