Therapeutic effect of dithiophenolato chitosan nanocomposites against carbon tetrachloride-induced hepatotoxicity in rats

Our previous study showed that dithiophenolate (DTP) and its chitosan nanoparticles (DTP-CSNPs) have abilities to bind with DNA helixes. So in this study, their lethal doses (LD50) and therapeutic roles against rat liver injuries induced by carbon tetrachloride (CCl4) were evaluated. The study focused on the determination of the markers of oxidative stress (OS) and apoptosis and compare the results with those of cisplatin treatment. The results revealed that LD50 values of DTP and DTP-CSNPs are 2187.5 and 1462.5 mg/kg, respectively. Treatment with DPT and DPT-CSNPs after CCl4 administration reduced liver injuries, induced by CCl4, and improved liver functions and architecture through the reduction of OS and apoptosis. Where the oxidant marker was decreased with elevations of antioxidant markers. Also, there was an elevation in Bcl-2 value, with decreases in caspase-8, Bax, and Bax/Bcl-2 ratio. DPT-CSNPs treatment gave preferable results than those treated with DPT. Moreover, DTP and DPT-CSNPs treatment gave better results than cisplatin treatment. The administration of healthy rats with low doses of DTP and DTP-CSNPs for 14 days had no effect. Otherwise, the study on HepG2 cell line showed that DTP and DPT-CSNPs inhibited cell growth by arresting cells in the G2/M phase and inducing cell death. In conclusion, DTP and DTP-CSNPs have antiapoptotic and anti-oxidative stress toward hepatotoxicity induced by CCl4. Moreover, DTP and DTP-CSNPs have anticancer activity against the HepG2 cell line. Generally, DTP-CSNPs are more effective than DTP. So, they can be used in the pharmacological fields, especially DTP-CSNPs.


Introduction
The liver is a crucial organ of the metabolism and elimination of foreign substances. Therefore, the liver is a preferred target for xenobiotics toxicity (Shaban et al. 2014). Liver injury induced by xenobiotics can impersonate all forms of acute or chronic liver disease. The pathogenesis of xenobioticsinduced liver diseases involves cell stress, mitochondrial feebleness, and definite immune reactions. Mitochondrial feebleness leads to apoptosis and/or necrotic cell death. Apoptosis, as well-referred to as programmed cell death, is an organized form of cell death that comprises distinct biochemical and morphological changes (Youle and Strasser 2008). Apoptosis can be triggered by both internal and external factors (Kvansakul and Hinds 2015;Strasser and Vaux 2018). The internal factors involve misfolded proteins and deregulated signaling while the external factors include the loss of nutrients, heat, radiation, stimulation of the cell surface receptors, and xenobiotics such as carbon tetrachloride (CCl 4 ) (Suraweera et al. 2021), paracetamol (Gokkaya et al. 2021), diethylnitrosamine (Shaban et al. 2013), thioacetamide , and phenobarbital (Shaban et al. 2014).
Cellular reactive oxygen species (ROS) are produced endogenously such as in the process of mitochondrial oxidative phosphorylation or they may be produced from the interactions with exogenous sources as xenobiotic compounds. Oxidative stress (OS) occurs when ROS overcome the cellular antioxidant defense system whether via an increase in ROS levels or a decrease in the cellular antioxidant ability. The OS performs in direct or indirect ROS-mediated damage of proteins, nucleic acids, and lipids, and has been implicated in diabetes, atherosclerosis, neurodegeneration, and carcinogenesis (Ray et al. 2012). The molecular mechanism of hepatotoxicity induced by CCl 4 has been well reported in rats (Saygili et al. 2017). CCl 4 is biotransformed by liver cytochrome P450 enzymes to form the trichloromethyl free radical (CCl 3 ) that are reacting rapidly with molecular oxygen to produce the trichloromethyl peroxyl radical (CCl 3 O 2 ). These toxic radicals are accountable for the abstraction of hydrogen atoms from unsaturated fatty acids of phospholipids existing in the cell membrane, inducing lipid peroxidation in the hepatocytes and others. It has been authenticated that lipid peroxidation was the master mechanism in the pathogenesis of liver injuries induced by CCl 4 (Shaban et al. 2013).
Chemotherapy is still considered to have a remarkable curative effect with great success in clinical practice. In this regard, there is a significant requirement for novel pharmaceutical agents (Saygili et al. 2017). Nanotechnology is presently involved vastly in human lives with a lot of implementations, especially in sciences, food industry, paints, biology, medicine, drug delivery, etc. Nanomedicines (or nanotherapeutics) are usually intended as miniaturized delivery systems which aim to improve the therapeutic efficiency of currently available chemotherapeutic agents, combining them with a nanoscale delivery component (Baek et al. 2015;Chan et al. 2013).
Natural products exemplify a considerable family of assorted chemical entities with a broad diversity of biological activities that have found various uses, especially in agriculture and in human and veterinary medicine (Katz and Baltz 2016). They originate from plant, marine animal, bacterial, and fungal sources. Where the bacterial and fungal natural product molecules are the products of secondary metabolism. However, the natural macromolecules (protein, DNA, and RNA), their precursors and building blocks, besides intermediates of primary metabolism, are excluded from the working determination of natural products (Katz and Baltz 2016). Lignin is the second most considerable component of plant material and has been confirmed to be a hopeful natural resource for adhesive and coating applications (Mu et al. 2021). Otherwise, huge quantities of natural polysaccharides, as chitosan (CS), chitin, and chitin-glucan copolymers are easily produced, and existent unique characteristics are frequently not found for synthetic polymers (Shaban et al. 2020). CS is isolated from marine natural sources such as crustacean shells. The significant properties of CS, such as biocompatibility, biodegradation, cellular toxicity, and antimicrobial activity, encouraged researchers to make it more suitable and ameliorate its physical and chemical properties. Additionally, CS is used in further applications such as production and modulation of CS-based membrane (Sun et al. 2020b), potential embolization , and removing the hexavalent chromium Cr(VI) from the wastewater system (Gu et al. 2018).
Nanocomposites are the best nominee that improves the microscopic properties of the products significantly. They have much better mechanical properties due to their very high surface-to-volume ratio. Some polymeric nanocomposites are used for biomedical applications such as drug delivery, tissue engineering, and cellular therapies. Because of the unique interactions between the polymer and nanoparticles, a range of possession combinations can be engineered to imitate native tissue structure and properties. Also, nanocomposites are used for enhancing each of the following: the energy storage (Zhou et al. 2020b), the service life and stability of electronic devices (Zhou et al. 2020a), the morphology and dielectric properties of materials (Zhou et al. 2021), anticorrosion properties (Liu et al. 2020), the antiflammable material (Das et al. 2020), improving dielectric performances of topologicalstructured polymer (Sun et al. 2020a), and the application of hot embossing in polymer processing (Sun et al. 2019). Additionally, nanoparticles have a role in the medical field, such as therapeutic, diagnostic, immunization, and vaccine production (Ahmad et al. 2020;El-Sayed and Kamel 2020).
Chemotherapy induces many problems which may be related to drug nonspecificity resulting from poor drug delivery systems. At present, these problems are overcome using nanomedicine by using nanoparticles such as drug delivery systems or nanocarriers (Bhattacharya et al. 2021). Drug delivery systems can be carried out using various methods such as nanoparticles (Hu et al. 2018), electrospinning for the encapsulation of the probiotics (Zhang 2020), microencapsulation , hydrogel polymer systems , and a promising emulsion system to encapsulate astaxanthin-enriched camelina oil (Xie et al. 2020). Furthermore, natural protein-based nanoparticles for example lipoprotein, lectins, and ferritin from the natural origins have acquired wide importance of scientific society level as nanovehicle for efficient drug delivery and picture vocal labeling to replace many synthetic nanocarriers which have demonstrated bounded therapeutic results (Bhattacharya et al. 2021).
CSNPs are perfect drug carriers because of their good biocompatibility and biodegradability and can be easily modified (Saneja et al. 2016). CSNPs have attractive physiochemical properties like size and surface potential making them a perfect candidate for anticancer effects but the major limitation was the low solubility at physiological pH (Shaban et al. 2020). They have attracted rising attention for their broad applications such as loading protein drugs, anticancer chemical drugs, and gene drugs, where they are given via different routes involving oral, ocular, nasal, and intravenous (Rosiere et al. 2018;Shah and Rajput 2018;Wang 2011). In addition, CSNPs are used as building blocks to construct electromagnetic composites Xie et al. 2021).
Previous studies revealed that the exposure of chitin and chitosan-based multifunctional nanomaterial composites for hopeful applications in the field of biomedical science build, synthesis besides possibility usage from a colossal angle. Both chitin and chitosan-based nanomaterials were elaborated crucially with their potential application toward biomedical science. For various biomedical implementations, it utilizes in the form of a scaffold, nanoparticles, aerogels, microsphere, and the form hydrogels. Therefore, it had been mixed with various polymers such as cellulose, carboxymethyl cellulose, starch, lipid, hyaluronic acid, alginate, polyvinyl alcohol, and curcumin (Ahmad et al. 2020;Kabir et al. 2021).
In our previous studies, dithiophenolato ligand N2H2S2\H2 (DTP) (Fig. 1) was prepared (Shaban et al. 2012), as well as DTP-CS nanocomposite (DTP-CSNPs), by loading DTP on CS-tripolyphosphate nanocomposite (Shaban et al. 2020). Also, their characterization was studied where the study showed that DTP-CSNPs bind strongly to DNA as compared to DTP, and both bind via a static quenching mechanism. Therefore, in this study, DTP and DTP-CSNPs were prepared (Shaban et al. 2020) and their lethal doses (LD 50 : the amount of a material, given all at once, which causes the death of 50% of a group of test animals) were determined. Then the therapeutic roles of both compounds against CCl 4induced hepatotoxicity in male rats were studied and the results were compared with those that resulted from cisplatin treatment (as a standard drug, but has an adverse effect). Where the study focused on the determination of the markers of oxidative stress, apoptosis, lipid profile, liver, and kidney functions besides liver histopathological examination. Additionally, the antitumor efficacies of DTP and DTP-CSNPs against human liver cancer (HepG2) cell lines via cell cycle analysis were evaluated.

Animals
One hundred and twenty Sprague-Dawley rats weighing 100-150 g were obtained from the 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.

Preparation of DTP-CSNPs
DTP-CSNPs were prepared (from DTP , sodi um tripolyphosphate (TPP), and CS) and characterized as mentioned in our previous paper (Shaban et al. 2020). In brief, TPP solution was added to CS solution, left at 25°C for 12 h; DTP was added, left for 40 min and the solvent was removed at 40°C. The characterization of DTP-CSNPs was examined by high-resolution transmission electron microscopy (HRTEM), scanning electron microscope with EDX detector, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA) (Shaban et al. 2020).

Determination of LD 50 values of DTP and DTP-CSNPs
To facilitate the determination of LD 50 in vivo, the LD 50 values of DTP or DTP-CSNPs were estimated first using the values of IC 50 (μg/ml) according to the regression formula obtained from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) log LD 50 (mg/kg) = 0.372log IC 50 (μg/ml) + 2.024 (Wong et al. 2017).
For the determination of LD 50 of DTP or DTP-CSNPs, 48 rats were used after the acclimation period. The rats were divided into 12 groups, six groups for each compound, where six doses of DTP and DTP-CSNPs (200, 500, 1000, 1500, 2500, 3500 mg/kg body weight (BW)) were used for the administration once, intraperitoneally (IP). Any abnormal clinical signs and behavioral changes on the animals were observed for 24 h. In each group, the number of dead rats was recorded (% dead) and LD 50 was calculated by the arithmetic method of (Kärber 1931).
ð Þ=n LD 100 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 the mean mortality: the average number of dead animals in two successive doses The biological effects of DTP, DTP-CSNPs, and cisplatin on hepatotoxicity DTP and DTP-CSNPs were dissolved in 2% DMSO and their doses were chosen to be safe away from the values of LD 50 where these doses were around to that of cisplatin as a standard drug. Seventy-two Sprague Dawley rats were divided after the adaptation into nine groups (8 animals/group) (Fig. 2). Control group (C): the rats were treated with 2% DMSO (0.5 ml/kg BW) and olive oil (0.5 ml/kg BW) day after day, IP for 8 weeks (the experimental period); CSNPs group: rats were treated (IP) with 4.41 mg of CS (dissolved in 1 ml of 2% DMSO) per kg BW/day for 2 weeks (at 7th and 8th weeks); DTP group: the rats were treated (IP) with 6.71 mg of DTP dissolved in 1 ml DMSO per kg BW/day for 2 weeks (at 7th and 8th weeks); DTP-CSNPs group: rats were treated (IP) with 4.41 mg of DTP-CSNPs dissolved in 1 ml DMSO per kg BW/day for 2 weeks (at 7th and 8th weeks); CCl 4 group: the rats were injected (IP) with 0.5 ml of 95% CCl 4 dissolved in 0.5 ml olive oil/kg BW, day after day for the first 6 weeks (Karaca et al. 2017). The groups CCl 4 -CSNPs, CCl 4 -DTP, and CCl 4 -DTP-CSNPs: the rats were injected with CCl 4 for the first 6 weeks as in the CCl 4 group, then at the 7th and 8th weeks, they were treated with the same doses and periods of CSNPs, DTP, and DTP-CSNPs, respectively. CCl 4 -cisplatin: the rats were injected with CCl 4 as in the CCl 4 group, then at the beginning of the 7th week, they were treated (IP) with 4 mg of cisplatin dissolved in 0.5 ml olive oil/kg BW/day for 5 consecutive days (Wang et al. 2010). Figure 2 shows this experimental design. At the end of the experimental period, the rats were fasted overnight and sacrificed after being 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°C until used. Livers were removed immediately and small portions were fixed in 10% formalin for histopathological examination. The remaining liver was washed with cold saline solution (0.9% NaCl), divided into two parts, and kept at −80°C. The first part was used for determining the expression levels of caspase-8, Bcl-2, and Bax. 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 Teflon Homogenizer and the homogenate was centrifuged at 4000 rpm for 15 min at 4°C . The supernatant was kept at −80°C until used for the determination of malondialdehyde (MDA), SOD activities, GSH levels, glutathione-S-transferase (GST), total glutathione peroxidase (GPx), and glutathione reductase (GR).
Effect of the studied compounds on apoptotic markers (Bcl-2, Bax, and caspase-8) RNA isolation and quantitative real-time PCR analyses Total RNA was isolated from liver tissues via the RNA extraction kit (Thermo Fisher Scientific, Fermentas, #K0731). Total RNA was quantified using a NanoDrop™ Q5000 (UV-Vis spectrophotometer Q5000, USA). The complementary DNA was produced using reverse transcription kits (Thermo Fisher Scientific, Fermentas, #EP0451). The synthesized cDNA was amplified using 2X Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, USA, #K0221). The primers for Bcl-2, Bax, caspase-8, and β-actin are listed in (Table 1). RT-PCR cycle parameters included 10 min at 95°C followed by 40 cycles involving the denaturation for 15 s at 95°C, annealing for 30 s at 60°C and elongation for 30 s at 72°C , then final elongation at 72°C for 5 min. qRT-PCR was operated using StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, USA). A division curve program was one after each reaction to confirm the purity of the PCR products. The quantities cycle threshold (Ct) of the target gene was normalized with quantities (Ct) of the housekeeping gene (β-actin) by using the 2 −ΔΔCt method to calculate the fold change in the target gene.

Liver histopathological analysis
Liver tissues were fixed, processed, and immersed in paraffin wax. Sections of 5 μm in thickness were obtained and stained with hematoxylin and eosin (H&E) for investigation via a light microscope (Suzuki and Suzuki 1998).

Cell cycle analysis
The distribution of HepG2 cells in the dissimilar cell cycle p h a s e s ( G 0 / G 1 , S , a n d G 2 / M ) w e r e m e a s u r e d (Shanmugasundaram et al. 2017). In brief, HepG2 cells were untreated and treated with 40 μg/ml of DTP and DTP-CSNPs separately for 24 h, then cells were harvested and fixed in icecold 70% ethanol for 12 h at 4°C and centrifuged for 5 min at 1000 rpm. The pellets were resuspended in propidium iodide (0.05 mg/ml) and RNase (100 U/ml) in phosphate buffer (pH 7.4), incubated at 37°C for 30 min, and DNA was analyzed by a flow cytometer (Attune® Acoustic Focusing Flow Cytometer, Thermo Fisher Scientific, USA). Finally, the cell cycle data were analyzed using CellQuest software.

Statistical analysis
The data were expressed as means ± SD (standard deviation). One-way analysis of variance (ANOVA) via SPSS 18.0 Software and the individual comparisons were acquired by Duncan's multiple range test (DMRT). Values were considered statistically significant (P ≤ 0.05).

Results
The results investigated that the prepared DTP-CSNPs have a spherical morphology with an average particle size of~150 ± 5 nm. The conversion of DTP into nanoparticles forms (DTP-CSNPs) was found to increase the thermal stability of the composite material in comparison to DTP (Shaban et al. 2020).

LD 50 of DTP and DTP-CSNPs
Karber's method for the determination of LD 50 showed that LD 50 values of DTP and DTP-CSNPs were about 2187.5 and 1462.5 mg/kg, respectively ( Table 2).

Effect of different treatments on the oxidative stress
Administration of CCl 4 caused a significant (P ≤ 0.05) increase in MDA level and GR activity with significant (P ≤ 0.05) decreases in the level of GSH and the activities of GPx, GST, and SOD when compared with the control group. However, treatment with DTP and DTP-CSNPs after CCl 4 administration decreased significantly (P ≤ 0.05) MDA levels and GR activity, while GSH level and GPx, GST, and SOD activities were increased significantly (P ≤ 0.05) when compared with the CCl 4 group (Fig. 3), whereas the animals treated with CSNPs only after CCl 4 administration showed nonsignificant effects on the oxidative stress parameters when compared with the CCl 4 group. In contrast, treatment with cisplatin after CCl 4 administration significantly increased (P ≤ 0.05) MDA level and GR activity as compared to the CCl 4  Values represent the values mean ± SD of 8 rats. One-way ANOVA followed by Tukey's test was used (a P ≤ 0.05 vs. control group, b P ≤ 0.05 vs. CCl 4 group, and c P ≤ 0.05 vs. DTP-CSNPs treated group) group but decreased significantly (P ≤ 0.05) GPx, GST, and SOD activities and GSH level when compared to the CCl 4 group (Fig. 3).
On the other hand, the administration of CSNPs, DTP, and DTP-CSNPs to healthy rats caused nonsignificant changes (increased or decreased) in levels of MDA, GSH, and the activities of GR, GPx, GST, and SOD compared to the control group (Fig. 3).

Effect of different treatments on the apoptotic markers
Administration of CCl 4 investigated a significant (P ≤ 0.05) downregulation of the Bcl-2 gene expression with a significant upregulation of Bax and caspase-8 gene expressions as compared with the control group (Fig. 4a). Treatment with DTP, DTP-CSNPs, and cisplatin after CCl 4 injection showed significant downregulation in the gene expression levels of Bax and caspase-8 with a significant (P ≤ 0.05) upregulation in Bcl-2 gene expression when compared with the CCl 4 group (Fig. 4a), while the animals treated with CSNPs only after CCl 4 administration showed nonsignificant impact on the apoptotic markers as compared with the CCl 4 group. Also, there were nonsignificant changes in all parameters in the healthy rats after administration with CSNPs, DTP, or DTP-CSNPs when compared with the control group (Fig. 4a). Figure 4b shows that CCl 4 injection increased Bax/Bcl-2 ratio, while treatment with DTP, DTP-CSNPs, and cisplatin after CCl 4 injection reduced this ratio by different degrees.

Effect of different compounds on lipid profile and liver and kidney functions
Administration of CCl 4 significantly increased (P ≤ 0.05) serum cholesterol, TG, and LDL cholesterol levels but significantly decreased (P ≤ 0.05) HDL cholesterol levels as compared with the control group (Fig. 5a). However, their levels were significantly (P ≤ 0.05) improved in rats treated with DTP-CSNPs over DTP and cisplatin as compared to the CCl 4 group, while the animals treated with CSNPs after CCl 4 administration showed nonsignificant changes in lipid profile as compared with the CCl 4 group (Fig. 5a).
CCl 4 administration resulted in significant (P ≤ 0.05) increases in the activities of ALT, AST, and ALP with a significant decrease in the levels of STP and LTP compared to the control group (Fig. 5b-d). In contrast, their levels were significantly (P ≤ 0.05) improved in rats treated with DTP-CSNPs,  DTP, and cisplatin after CCl 4 injection as compared to the CCl 4 group, where DTP-CSNPs gave better results than DTP and cisplatin. We noticed that cisplatin treatment decreased STP and LTP as compared with the CCl 4 group. Urea and creatinine levels were increased after CCl 4 injection as compared to the control group, while their levels were improved after treatment with DTP-CSNPs over DTP and cisplatin as compared to the CCl 4 group. However, the animals injected with CSNPs after CCl 4 administration revealed nonsignificant changes in the markers of liver functions, lipid profile, and kidney functions as compared with the CCl 4 group (Fig. 5). Administration of CSNPs, DTP, and DTP-CSNPs to the healthy rats caused nonsignificant changes in the levels of liver functions, lipid profile, and kidney functions when compared to the control group (Fig. 5).

Histopathological analysis
The histopathological characteristics of liver tissues of the different examined groups are shown in Fig. 6. The histopathological results revealed that treatment with DTP and DTP-CSNPs after CCl 4 administration improved liver histopathology induced by CCl 4 indicating their therapeutic roles which confirm with the biochemical analysis. The results revealed no changes in liver histology of rats administered with CSNPs, DTP, and DTP-CSNPs when compared with the control group.

Induction of cell cycle arrest by DTP and DTP-CSNPs
Treatment with DTP and DTP-CSNPs resulted in a significant decrease in the population of HepG2 cells in G0/G1 and S phases as compared to the control cells (Fig. 7). Moreover, high populations of HepG2 cells were arrested at the G2/M checkpoint as compared with untreated cells. Cells treated with DTP-CSNPs showed the lowest levels of both phases, G0/G1 and S with the highest level in the G2/M phase when compared with that treated with DPT (Fig. 7).

DTP and DTP-CSNPs treatment diminished liver injuries induced by CCl 4
The present results showed that LD 50 values of DTP and DTP-CSNPs were about 2187.5 and 1462.5 mg/kg, respectively, ( Table 2). So in this study, the rat hepatotoxicity induced by CCl 4 administration was treated with safe doses of DTP and DTP-CSNPs (6.71 and 4.41 mg/kg BW, respectively).
Our results showed that CCl 4 administration caused hepatotoxicity leading to severe liver injuries as shown from the histopathological results, which were confirmed by the biochemical results involving the markers of liver functions, lipid profile, oxidative stress, and apoptosis in liver tissues. The mechanism of hepatotoxicity by CCl 4 may be related to the deleterious effect of CCl 4 and its highly reactive metabolites (CCl 3 * and CCl 3 O 2 *). Since these free radicals caused oxidative stress as shown from the elevation of MDA level (the end product of lipid peroxidation) and GR activity, with the reduction in the levels of antioxidants, GSH, GPx, GST, and SOD. The lipid peroxidation of polyunsaturated fatty acids of the membrane of the hepatocytes led to the disturbance in Ca 2+ homeostasis resulting in the destruction of the cells and their intracellular organelles and also protein content leading to hepatocellular injuries. The elevation in GR activity may be related to the adaptation to the elevation in lipid peroxidation. Free radical scavengers may protect biological systems from the deleterious effects of the free radicals induced by xenobiotics including CCl 4 (Kurutas 2016). GSH plays an important role against CCl 4 -induced lipid peroxidation by covalently binding to CCl 3 · radicals and enhancing the activities of GR (Shah et al. 2017). Also, GSH acts as a cofactor to GPx besides its action as a nucleophilic scavenger of numerous compounds via enzymatic and chemical mechanisms (Kurutas 2016). The reduction in GSH levels after CCl 4 administration may be regarded to the reactions through oxidation and/or conjugation, leading to the elimination of the products of lipid peroxidation, peroxides, and aldehydes. GSH depletion may lead to the inhibition of GPx activity and the elevation of lipid peroxidation (Shaban et al. 2014;Shah et al. 2017). Furthermore, SOD is considered the most effective antioxidant in the body against superoxide radicals (Alkreathy et al. 2014) and its inhibition may be owed to the action of superoxide radicals or after their conversion to H 2 O 2 by oxidation of the cysteine in the enzyme (Ighodaro and Akinloye 2018).
Apoptosis is a form of cell death in which a programmed sequence of proceedings leads to the removal of unnecessary cells without releasing harmful substances into the surrounding area. Apoptosis is regulated by specific genes, including Fig. 6 Histopathological analysis of liver tissue samples from the different examined groups H&E, ×200. Liver tissues in rats after CCl 4 administration showed cellular infiltration congestion of hepatic sinusoids and portal vein, multiple hemorrhages, and center lobular hepatic necrosis. While, after treatment with DTP confirmed a marked decrease of hepatic degeneration with granular hepatic vacuolation (arrow), and also after DTP-CSNPs treatment, liver tissues revealed a marked decrease of hepatic degeneration with mild to moderate hepatic vacuolation (arrow). CSNPs are a portal tract infiltrated by inflammatory cells, mainly lymphocytes. The infiltrate extends into the adjacent parenchyma. Some hepatocytes exhibit ballooning. Otherwise, after treatment with cisplatin, a periportal inflammatory reaction with degenerated hepatic cord and disrupts cell plates were observed in liver tissues. Liver tissues in healthy rats after administration of each CSNPs, DTP, and DTP-CSNPs exhibited normal hepatocytes (arrow) around the central vein many pro and antiapoptotic proteins. Additionally, caspase-8 is a member of the cysteine proteases, which are involved in apoptosis and cytokine processing. Caspase-8 is synthesized as an inactive enzyme and is activated by proteolytic cleavage.
Our results revealed that CCl 4 administration caused upregulation of proapoptotic proteins Bax and caspase-8, with downregulation of antiapoptotic proteins Bcl-2. The changes in Bax and Bcl-2 levels led to the disturbance in the ratio of Bax/Bcl-

Control group (C)
CSNPs group

CCl4-DPT group
CCl4-Cisplatin group 2 which became much greater than that of the control group. The elevation in Bax/Bcl-2 ratio causes permeabilization of the outer membrane of the mitochondria and releases cytochrome c into the cytoplasm where it binds with the Apaf-1 (apoptotic protease activating factor-1) leading to the activation of procaspase-9 which in turn activates procaspase-3 leading to the induction of apoptosis and cell death (Campbell and Tait 2018;Pistritto et al. 2016). Moreover, CCl 4 caused apoptosis through the activation of caspase-8 where caspase-8 activates caspase-3 directly or indirectly via activation of Bax, which in turn activates caspase-3 resulting in the cleavage of the essential substrates for cell viability inducing apoptosis and cell death (Kalkavan and Green 2018;Pistritto et al. 2016). In addition, the rise in ROS after CCl 4 administration leads to an increase of p53 signaling which in turn activates Bax expression but inhibits Bcl-2 expression (i.e., the ratio of Bax/Bcl-2 was elevated) leading to apoptosis (Eltahir and Nazmy 2018). Also, the previous studies revealed that CCl 4 induced apoptosis via induction of DNA fragmentation (Eidangbe et al. 2021). Generally, our biochemical results showed that CCl 4 administration induced oxidative stress and apoptosis, and this led to severe liver injuries where these results are in harmony with the histopathological results. Also, liver injuries after CCl 4 administration were confirmed by the elevation of the activities of liver enzymes (ALT, AST, and ALP), lipid profile (TC, TG, and LDL cholesterol) in serum with a decline of albumin, STP, LTP, and HDL cholesterol, where the liver damage led to the leakage of liver enzymes into the blood circulation and decreased the protein biosynthesis. In addition, CCl 4 administration induced nephrotoxicity as shown from the elevation of creatinine and urea levels in serum. These results agree with the previous studies (Krithika and Verma 2019;Safhi 2018).
On the other hand, the current results showed that LD 50 values of DTP and DTP-CSNPs were about 2187.5 and 1462.5 mg/kg, respectively ( Table 2). The results showed an improvement of the hepatic histopathology in rats treated with DTP and DTP-CSNPs after CCl 4 administration as shown  (Fig. 6). These results were confirmed by the improvement of liver functions and lipid profiles. AST, ALT, and ALP levels, as well as total cholesterol, TG, and LDL cholesterol became lower than those of the CCl 4 group, while STP, LTP, and HDL cholesterol became greater. Also, the renal functions were improved where the levels of urea and creatinine became lower than the CCl 4 group. All these positive results may be related to the therapeutic effects of DTP and DTP-CSNPs where they reduced liver injuries induced by CCl 4 through the reduction of both oxidative stress and apoptosis, induced by CCl 4 . Firstly, these treatments reduced the oxidative stress and lipid peroxidation induced by CCl 4 as MDA levels and GR activities became lower than those in the CCl 4 group. However, the levels of antioxidants (GSH, GPx, GST, and SOD) became greater than those of the CCl 4 group. This indicates that DTP and DTP-CSNPs have antioxidant activities against the oxidative damage induced by CCl 4 . This antioxidant activity may be related to the effect of dithiol groups (2 SH) in both compounds which may be oxidized forming an S-S bond (Fig. 1). This means that the presence of the dithiol groups makes these compounds similar to cysteine and glutathione. Additionally, the previous studies showed that the metabolism of sulfur-containing xenobiotics can produce bisulfite (HSO 3− ) and sulfite (SO 3 2− ) (Shaban et al. 2010). The bisulfite can be oxidized by both one-and two-electron-forming sulfur trioxide radical anion (SO 3 ) (Shaban et al. 2010). The results in Fig. 3 show that the toxic effects of the reactive metabolites are lower than the useful effects of the antioxidants present in the dithiol groups of DPT and DTP-CSNPs. Secondly, the reduction of apoptosis is obviously from the reduction of proapoptotic marker levels, Bax, and caspase-8, and elevation of antiapoptotic marker (Bcl-2) which in turn changed the ratio of Bax/Bcl-2 and became lower than that of the CCl 4 group. In addition, treatment with DTP and DTP-CSNPs reduced the oxidative stress induced by CCl 4 and this led to the reduction of apoptosis. This indicates that DTP and DTP-CSNPs act as an antiapoptotic action against the apoptosis induced by CCl 4 . A possible mechanism for such effect is by elevation of the antiapoptotic proteins and the reduction of proapoptotic proteins causing the stabilization of the mitochondrial membrane which in turn preventing the leakage of cytochrome c into the cytoplasm resulting in preventing the activation of caspase-3 leading to the prohibition of apoptosis and cell death. Additionally, the reduction in oxidative stress via treatment with DTP and DTP-CSNPs leads to a reduction of apoptosis induced by CCl 4 . Nevertheless, treatment with DTP-CSNPs gave preferable results than DTP treatment and this may be owing to the simplicity of the DTP-CSNPs which can pass via the cellular membranes. Additionally, the large number of DTP-CSNPs leads to an increase in their active surface area which may raise the absorption rate resulting in troubles in the biological systems (Mohammed et al. 2017).
Otherwise, treatment with cisplatin for 4 days, after CCl 4 administration increased oxidative stress as shown from the markers of MDA and antioxidants, so the MDA level and GR activity became greater than those of CCl 4 , while the antioxidants became lower. However, cisplatin treatment reduced apoptosis with a degree lower than those induced by treatment with DTP and DTP-CSNPs. Therefore, cisplatin treatment improved liver and kidney functions and lipid profile with a degree lower than those resulted from DTP-CSNPs and DTP.
On the other hand, the results showed that administration of DTP and DTP-CSNPs for healthy rats for 14 days had no effect on the studied apoptotic markers as compared with the control group. However, they caused nonsignificant changes in some oxidative stress markers (MDA, GR, GST, and SOD) (Fig. 3), and this may be related to the effects of their reactive metabolites (O 2 •− and SO 5

•−
) (Shaban et al. 2010) which may be accumulated in the liver. Therefore, we noticed some changes (increase or decrease) in the markers of lipid profile and liver and functions, but there were no changes in liver histopathology when compared with the control.

DTP and DTP-CSNPs have antitumor activities against HepG2 cells
Cell growth and proliferation are controlled by the cell cycle management, so its interruption causes the development and progression of most tumors due to an imbalance between proliferation and apoptosis (Hong et al. 2019). Our previous study showed that the DTP and DTP-CSNPs have the ability to bind to DNA and they also have cytotoxic effects, so, in this study, we examined their antiproliferative effect against HepG2 cell line via cell cycle analysis to elucidate if these compounds have antitumor activities. Accordingly, the current results showed that the treatment of HepG2 cells with DTP and DTP-CSNPs resulted in a significant reduction in the population of HepG2 cells in G0/G1 and S phases as compared with the control cells. This indicates that these compounds interfered with DNA synthesis and disrupted the progression of the cell cycle, leading to apoptosis (Yuan et al. 2015). Furthermore, both DTP and DTP-CSNPs arrested high populations of HepG2 cells at the G2/M checkpoint as compared with untreated cells, indicating that the cell cycle arrest led to disruption of the tubulin-microtubule equilibrium and allowed the time for the repair of DNA damage or allowed cells to survive with persistent DNA damage (Sosnowska et al. 2019;Yuan et al. 2015). In general, DTP and DTP-CSNPs have anticancer activities through interfering with the cell division and arresting the uncontrolled proliferation of cancer cells, initiating apoptosis and this is considered to be an important strategy. These results agree with our previous studies which demonstrated that DTP and DTP-CSNPs have a strong ability to interact with DNA helix (Fouad et al. 2016). The antiproliferative and apoptotic activities of DTP and DTP-CSNPs probably may be due to the cytotoxicity of these compounds and their metabolites (bisulfite (HSO 3− ) and sulfite (SO 3 2− ) (Fouad et al. 2016;Shaban et al. 2010). These results agree with the behavior of numerous anticancer agents such as vinblastine and Taxol (Hong et al. 2019;Li et al. 2007). Additionally, treatment with DTP-CSNPs gave better results than that of DTP and this may be due to the naturalness of the DTP-CSNPs as we mentioned before. In addition, the previous studies revealed that the nanoparticles induce apoptosis (Mohammed et al. 2017). Also, previous studies revealed that a drug delivery system reinforces clinical consequences by enhancing permeability and its rate, improving drug internalization, enabling targeted delivery, and prolonging circulation and easy distribution of the drug resulting in improving the therapeutic efficacy of several drugs (Bhattacharya et al. 2021).
In general, in vivo treatment with DTP-CSNPs and DTP reduced rat liver injuries induced by CCl 4 through diminishing apoptosis and oxidative stress induced by CCl 4 resulting in the improvement of liver architecture and function as well as decreasing the nephrotoxicity. Moreover, DTP-CSNPs and DTP showed anticancer activities against HepG2 cell lines where they prevented the proliferation of HepG2 cell line by increasing apoptosis via arresting cell cycle in the G2/M phase. These results designate that these complexes are characterized by selectivity (i.e., they have an ability to differentiate between liver injuries and cancer cell lines). These results agree with the previous studies which showed that the effect of the drug (or xenobiotics) are not matched in vivo and in vitro (Mirabelli et al. 2019;Weinstein 2012).

Conclusion
DTP-CSNPs and DTP exposed their therapeutic effect against liver injuries induced by CCl 4 by reducing both oxidative stress and apoptosis, where LD 50 values of DTP and DTP-CSNPs are 2187.5 and 1462.5 mg/kg, respectively. DTP-CSNPs have a greater effect than DTP and both compounds have a greater effect than cisplatin. The administration of healthy rats with low doses of DTP and DTP-CSNPs for 14 days has no effect on apoptotic markers and causes nonsignificant changes in oxidative stress markers. Furthermore, DTP-CSNPs and DTP showed anticancer activities against HepG2 cell lines. So, the pharmacokinetics of DTP-CSNPs and DTP must be studied to evaluate their clinical applications.
Acknowledgements The authors thank Dr. Kamel R. Shoueir (Institute of Nanoscience & Nanotechnology, Kafrelsheikh University, Egypt) for his participation in the preparation of the studied complexes in the form of nanoparticles. Also, the authors thank Dr. Ahmed Alaa Abdelazis (Alexandria University, Faculty of Medicine) for his participation in the histological examination of the liver.
Author contribution Nadia Z. Shaban suggested this study, designed and organized and participated in the sequence arrangement, wrote and reviewed and approved the manuscript, and agreed to be accountable for all aspects of the work by ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ahmed M. Aboelsaad participated in the suggestion of this study, participated in its design, carried out the experimental part in vitro and in vivo, performed the statistical analysis and drew the figures, and was a major contributor in writing and reviewing the manuscript. Doaa Awad participated in the statistical analysis. Shaymaa A.
Abdulmalek participated in the supervision of the experiments in vitro. Shaban Y. Shaban participated in the supervision of the preparation of the studied complexes, read, and approved the final manuscript.
Availability of data and materials All data generated or analyzed during this study are included in this published article.

Declarations
Ethics approval and consent to participate HepG2 cell lines and all animal methodology were done following the Institutional Animal Care and Use Committee (IACUC) and approved via the Committee for Animal Care and Use in Alexandria University (ethical approval reference number: AU 042008 15 3 01).

Consent for publication Not applicable.
Competing interests The authors declare no competing interests.