Abstract
Polycyclic aromatic hydrocarbons (PAHs) such as 7, 12-dimethylbenzneanthracene (DMBA), due to long-term bioaccumulation cause serious physiological processes and behavioral dysfunctions such as cancer, ageing, and hypertension. Silk sericin (SS) is instrumental in cancer applications due to presence of flavonoids and carotenoids which are natural pigments, present in the layer of sericin that has antioxidant and antityrosinase activity. It reduces oxidative stress and suppresses cancer cytokines while interacting with reactive oxygen species (ROS) to stand against lipid peroxidation. Recent research was focused to calculate the pharmacological intervention of sericin-conjugated silver nanoparticles (S-AgNO3 NPs) against DMBA-induced toxicity. For this purpose, SS protein was extracted from silkworm cocoons by degumming process and the prepared S-AgNO3 NPs via a green synthesis. In female albino mice, a total of 50 mg/kg oral administration of DMBA was used for the induction of toxicity which required almost 8 to 10 weeks approximately. After 60 days of experimentation, mice were dissected, blood samples were collected for further hematological and biochemical analysis and were euthanized via cervical dislocation. There was a significant rise in the level of red blood cells, platelets, lymphocytes, and hemoglobin at the highest applied concentration of sericin and its nanoparticles. Similarly, a reasonable decline was observed in the level of white blood cells, neutrophils, eosinophils, and monocytes as compared to the cancer-inducing group. The level of glutathione, lactate dehydrogenase, and alkaline phosphatase as well as immunoglobulins such as immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were significantly reduced in all treatment groups as compared to the DMBA-induced group. Substantial effects were demonstrated in response to S-AgNO3 NPs II (T) at the highest concentrations (200 mg/kg, BW) as follows: glutathione (2.42 ± 0.26 μmol/L), lactate dehydrogenase (493.6 ± 5.78 U/L), alkaline phosphatase (158.4 ± 6.35 U/L), IgA (4.22 ± 0.19 g/L), IgG (70 ± 1.70 g/L), and IgM (4.76 ± 0.12). The histopathological study of the liver, kidneys, and brain revealed that the DMBA-induced group showed cytotoxic effects against all selected organs of mice that were recovered by treatment of selective compounds but highly effective recovery was seen in S-AgNO3 NPs II (T). These results concluded that silk S-AgNO3 NPs showed significant pharmacological potential against cancer-inducing toxicity.
Keywords
Introduction
Polycyclic aromatic hydrocarbons (PAHs), are the derivatives of petroleum products which come from incomplete combustion of fossil foils, and oil spills, cause severe environmental pollution due to long-term bioaccumulation. One of the most important PAHs is 7,12-dimethylbenzneanthracene (DMBA), which causes a number of serious physiological anomalies and behavioral dysfunctions such as cancer, ageing, and hypertension. 1 It also generates reactive oxygen species (ROS) by increasing oxidative stress in cells during metabolism and forming DNA adducts by inducing the strand breaks and also causes oxidative modification of DNA bases which leads to mutagenic and carcinogenic effects. 1
The oxidation of DMBA is catalyzed by cytochrome P-450 monooxygenases by increasing the free radical scavengers contents and detoxification of carcinogenesis, which serve as potent in chemoprevention of PAH-induced carcinogenesis. 2 The DMBA which comes from cigarette smoke disturbed xenobiotics metabolism and potentially causes DNA damage, triggering genomic instability and inflammation, and disrupting the cross-link between DNA and proteins, a key hallmark of cancer. 3
The toxicological effects of DMBA correlate with its metabolic activation which depends on the target organ activation such as cytochrome P-450 epoxidation and peroxidative pathways. In an untreated animal model, the liver microsomal cytochrome P-450 system metabolizes DMBA to highly adrenocarticolytic agents including 7-hydroxymethyl-12-methylbenz (a) anthracene (7-OHM-12-MBA), while in induced livers, it metabolized in ring hydroxylated metabolites. These metabolites convert into dihydrodiols and phenol by animal adrenal microsomes and adrenal cells. 4 The liver plays a central role in the detoxification of carcinogen metabolism and alternations in its structure which may lead to neoplastic transformation and finally carcinogenesis. The measuring of xenobiotics metabolizing enzymes helps in evaluating the chemopreventive potential of bioactive biological compounds. 3
The silkworm Bombyx mori belongs to the family Bombycidae which constitutes mainly two different types of proteins; silk sericin (SS; 15–35%) and silk fibroin (65–85%). 5 SS contains different types of amino acids and secondary metabolites which gives it mechanical strength, good biocompatibility, low immunogenicity, and the most efficient drug delivery approaches. 6 Sericin is partially soluble in water but solubility decreases when it is converted from random coil to β sheet structure to form gel sericin at low temperature (10°C) and pH (6–7). As compared to more acidic amino acid composition than basic residue it showed the isoelectric point at around 3.5 to 4.0. Moreover, it can be used as a good emulsifying, antifrosting as well as anti-oxidant agent. 7
Sericin and its nano-micelles can be used as targeted drug delivery approaches, pharmaceutical carriers as well as in wound healing activity due to polar amino acids composition. It is also an excellent cross-linking agent due to the existence of surface functionality. The drug's cellular uptake is due to a passive mechanism or endocytosis, after that, sericin nanoparticles are degraded into smaller fragments through cellular enzymes. Sericin can be used as a potential carrier in site-specific drug delivery. 7
The green synthesis of nanocarrier is the most advanced technique to overcome drug resistance by the use of natural polymers which have drawn great attention in recent years. 8 The sericin-conjugated silver nanoparticle's (S-AgNO3 NP) existence makes these nanoparticles more stable by combining with the hydroxyl group of SS molecules. 9 Meanwhile, these nanoformulations disturb the homeostatic balance inside cancerous tissues and destroy their mitochondrial membrane and DNA machinery by bursting free radicals. Moreover, in vivo studies showed that these particles have very few systemic side effects onadjacent vital organs like the spleen, kidney, and liver. 10
The mechanism of action of sericin is due to a reaction with ROS. It protects from hydrogen peroxide (H2O2) along with ultraviolet B radiation which induces oxidative stress damage. By induction of programmed cell deaths, these proteins also prevent cancer development in different body organs. 11 SS plays a role in cancer applications due to its antioxidant properties. The presence of flavonoids and carotenoids which are natural pigments are present in the layer of sericin with antioxidant and antityrosinase activity. It reduces oxidative stress or suppresses cancer cytokines reacting with ROS to stand against lipid peroxidation. Due to its unique hydrophilic properties, it preferentially binds with DNA by surface modification and active targeting of Polyethylene glycol (PEG) and folate in cancer management. 12 The antioxidant action of SS is due to OH-related amino acid groups. In addition to cell culture media, SS avoids oxidative stress by improving cell proliferation and mitogenic effects on different cell lineages.
For diagnosis of different disorders including cancers and other immune system-related diseases, a complete blood count (CBC) test is used. CBC is considered to limit the toxicological effects of tested compounds by studying immunological and hematological parameters like red blood cells (RBCs), white blood cells (WBCs), platelets, eosinophils, neutrophils as well as hemoglobin (Hb), lymphocytes, and monocytes. 13
A present investigation is based on the use of silk protein sericin and its S-AgNO3 NPs against toxicity induced by DMBA in female Balb C mice. Therefore, we explore the relationship between hematological and biochemical parameters along with histopathological analysis of mice liver, kidneys, and brain tissues to determine the pharmacological interventions of selected treatment compounds at different concentrations and also to find out which concentration is likely to give the best preventive effects against toxicity.
Materials and methods
Ethical approval
The in vivo experiments were ethically approved by the Ethical Committee of the Zoology Department, Government College University, Lahore with reference no. GCU-IIB-1064. All animal trials were executed according to local and worldwide procedures followed by the Bureau of Animal Research Licensing, Local University committee rules, and via the Wet op de dierproeven (article 9) of Dutch law (international) as described earlier in our published articles.14–22 The animal's maintenance was done by using the “Guide for the Care and Use of Laboratory Animals” from the National Institute of Health (NIH) Publication (NRC 2011).22–24
Chemicals and reagents
Raw silk cocoons were compassionately purchased from the Sericulture sector, Forest Department, and Punjab Government Ravi Road, Lahore. Nolvadex (Tamoxifen Citrate) tablets, a nonsteroidal anti-estrogen, were purchased from Ali Imran Pharmacy, Lahore. Each tablet contained 45.6 mg of tamoxifen (Tam) citrate which is equivalent to 30 mg of Tam. From Aladdin Corporation (Shanghai, China), we acquired silver nitrate (AgNO3) (AR, 99.99%). DMBA (Sigma-Aldrich) was obtained from the Zoology Department, University of Punjab, Lahore.
Extraction and preparation of sericin powder and sericin-conjugated silver nanoparticles
In the medical toxicology laboratory, Government College University Lahore, SS solution was obtained by degumming process and it was converted into powder form by using Freeze-dried a Hero LL 3000 lyophilizer under −20°C.25–27 One gram of sericin powder was dissolved into 100 mL double distal water to make a 1% sericin stock solution. The 1 mM solution of silver nitrate (AgNO3) was added dropwise into a 1% solution of sericin and placed in the the sunlight for 36 h. 1-molar solution of sodium hydroxide (NaOH) was used to maintain the pH of the solutions between 9–11. Change of color into yellow-brown was an indication of S-AgNO3 NPs composite.
Characterization of sericin-conjugated silver nanoparticles
Different characterization techniques include ultraviolet-visible spectroscopy, scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and X-ray diffractometer were used to study the size, morphological characters, elemental composition, chemical structure, and crystalline structure of sericin nanoparticles. 28
Animals and their maintenance
Female albino mice (NMRI/Nu-Nu nude mice, 30–40 g BW, 6–8 weeks old) were purchased from the animal house of the Zoology Department, Government College University Lahore. The animal maintenance and treatment facilities were given such as 65–75 oF with 40–60% humidity, a fat-containing diet ranging from 4–11%, and 10–12 h of light/dark cycle before the start of the experiment and all such conditions followed the guidelines provided by the Ethical Committee, Government College University Lahore (Punjab, Pakistan).
Measurement of mice body weight
At the start of the experiment body weights (BW) of all individual mice were obtained with an electric weighing balance (CGolden Wall high precision Lab Scale Digital Balance) and measured the average weight in grams (g). The doses were prepared according to the average weight of every mouse measured once a week, and the same protocol was repeated during the whole experiment. The weekly weight changes were calculated in all recruited groups including cancer-inducing and all other treatment groups.
Experimental design and grouping of mice model
After the accommodation period, mice were divided into 17 groups with five replicates (n = 05) for each group and except control all treatment groups were treated with different concentrations of standard drugs such as Tam, sericin I (SI), sericin II (SII), S-AgNO3 NPs I, and S-AgNO3 NPs II as given below:
Group 1: (n = 5) The Control group received 0.4 mL of normal saline solution for 8 weeks. Group 2: Mice were given DMBA as a carcinogenic compound for induction of toxicity. 19 Group 3: (n = 5) Animals were treated with individual 0.4 mL oral administration of the standard drug Tam (100 mg/kg, BW) used as a positive control. 29 Group 4: Animals were treated with 0.4 mL oral administration of SI (100 mg/kg) during the whole 60 days of the experiment. 30 Group 5: Animals were treated with 0.4 mL orally administrated SII (200 mg/kg) till the end of the experiment. 30 Group 6: Animals were treated individually with S-AgNO3 NPs I (100 mg/kg). Group 7: Animals were administered with an individual oral dose of S-AgNO3 NPs II (200 mg/kg). Groups 8–12: After the 30th day of the experiment, the toxicity group was further divided into 4 post-treatment groups including Tam (100 mg/kg), sericin (100 and 200 mg/kg), and S-AgNO3 NPs (100 and 200 mg/kg) for the next 30 days of the experiment. Groups 13–17 included Tam (100 mg/kg) + DMBA (50 mg/kg), SI (100 mg/kg) + DMBA (50 mg/kg), SII (200 mg/kg) + DMBA (50 mg/kg), S-AgNO3 NPs I (100 mg/kg) + DMBA (50 mg/kg), and S-AgNO3 NPs II (200 mg/kg) + DMBA (50 mg/kg) were called prevention/pretreatment groups. These groups were simultaneously treated with DMBA (50 mg/kg) along with Tam (100 mg/kg), SI (100 mg/kg), SII (200 mg/kg), S-AgNO3 NPs I (100 mg/kg), and S-AgNO3 NPs II (200 mg/kg) for the end of 2 months/60 days experiment.
Induction of toxicity in animal mice model
In female albino mice, 50 mg/kg oral administration of DMBA was used for the induction of toxicity which takes almost 8 to 10 weeks approximately to induce toxicity. After 60 days of experimentation, mice were dissected, and blood samples were collected for further hematological and biochemical analysis and were euthanized via cervical dislocation. 19
Evaluation of biochemical parameters
For biochemical analysis of serum, aliquots of blood were deposited in tubes for 30 min containing coagulation activators and separator gel for clotting of blood. The aliquots were centrifuged for 5 min at 2500 rpm (Eppendorf Mini spin model Spin 1.000, Germany) for separation of the serum. Afterward, selected serum enzymes were analyzed by using different protocols. 31
Determination of glutathione
The serum samples were mixed with 1.5 mL potassium chloride solution and 3 mL of deproteinization solution (NaCl, metaphosphoric acid, Ethylene diamine tetraacetic acid (EDTA), and distilled water) and centrifuged at 1500 rpm for 5 min along with 2 mL of Na2HPO4. Afterward, 0.5 mL of supernatant and 0.5 mL Ellman reactive (5,5′-dithiobis-2-nitrobenzoic acid (DTNB), sodium citrate, and distilled water) were added. The absorbance of samples was calculated at 412 nm and compared with standard control samples. 32
Determination of alkaline phosphatase
The alkaline phosphatase (ALP) level was calculated from blood samples through the previously described High Performance Liquid Chromatography (HPLC) method in which we distinguished ALP isoenzymes and isoforms through the separation and quantitation technique. The ALP samples were separated by 0.6 M sodium acetate on an anion-exchange column (Synchropak AX300, USA), and effluents were mixed at pH 10.1 in a 0.25 M Diethanolamine (DEA) buffer along with 10.8 mM p-nitrophenyl phosphate. The ensuing reaction took place at room temperature in a packed bed postcolumn reactor and the product absorbance was calculated through the detector set at 405 nm. 33
Determination of lactate dehydrogenase
Level of lactate dehydrogenase (LDH) was measured by colorimetric method through monitoring absorbance at 430 to 550 nm. The tetrazolium salt was used for the reduction of formazan crystal which changed color from dark, deep red to orange by adding intermediate electron carriers such as NADH with phenazine methosulphate. 34
Assessment of immunoglobulins
For the assessment of immunoglobulins including immunoglobulins A (IgA), immunoglobulins M (IgM), and immunoglobulins G (IgG), the Zilva et al.’s 35 method was applied.
Histopathological study
For the histopathological study, the tissue/organ samples of dissected mice, including mammary tissues, liver, kidneys, brain, and spleen were collected after 60 days of the experiment. Afterward, they were washed with phosphate buffer saline solution to remove debris and blood, and through analytical balance (CGolden Wall high precision Lab Scale Digital Balance) weight of each organ of the mouse from each group was calculated and shown in grams (g). Thereafter, these tissues were fixed in a 10% formalin solution having a pH of 7.4 for 24 h. Then, these tissues were dehydrated in ascending grades of alcohol including 70%, 90%, and 100% and for clearance of alcohol from organs, a xylene solution was used. Further, these tissues were carried out to fix with paraffin wax at 58–60°C. The formation of blocks was done and by using a microtome (Rotary Microtome-HIS-202A), 5 mm thick sections of blocks were cut. At the end, these sections were carried out for staining protocol with the help of dyes like hematoxylin and eosin (H&E) and seen under light microscopy. 36
Statistical analysis
For appropriate statistical tests and analysis of data, GraphPad Prism (version 9.0) was applied accordingly. All the data were analyzed by mean ± standard deviation (SD) and one-way analysis of variance (ANOVA), while statistically significant values showed by P values (P < 0.01).
Results
Characterization of sericin-conjugated silver nanoparticles
Characterization analysis of the nanoparticles is reported in our previously published in vitro research data related to human breast cancer cell lines. 28
Body weight measurements
The weight (g) alterations were calculated throughout the 60 days experiment in all treatment groups, the DMBA-inducing group, and the normal untreated (UT) control group. The normal control group demonstrated a continuous increase in body weight changes, while all treatment groups illustrated a slight rise in body weight changes concerning the cancer-inducing group (Table 1).
Effects of sericin and sericin-conjugated silver nanoparticles treatment on body weight changes in experimental animals.
Abbreviation and keys: “aa” shows a significant difference in body weight of mice among 1st, and 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th week. “DMBA” stands for dimethlybenzeneanthracene (50 mg/kg), “Tam” for individual tamoxifen (100 mg/kg), “Tam (P)” for pretreatment of tamoxifen (100 mg/kg), and “Tam (T)” for post-treatment tamoxifen (100 mg/kg), “sericin II (SI)” for individual sericin (100 mg/kg), “sericin I (P)” for pretreatment of individual sericin (100 mg/kg), “sericin I (T)” for post-treatment of individual sericin (100 mg/kg), “sericin II” for individual sericin (200 mg/kg), “sericin II (P)” for pretreatment of individual sericin (200 mg/kg), and “sericin II (T)” for post-treatment of individual sericin (200 mg/kg). “Sericin-conjugated silver nanoparticles I (S-AgNO3 NPs I)” for sericin-conjugated silver nanoparticles I (100 mg/kg), “sericin-conjugated silver nanoparticles I (P)” for pretreatment of sericin-conjugated silver nanoparticles I (100 mg/kg), and “sericin-conjugated silver nanoparticles I (T)” for post-treatment sericin-conjugated silver nanoparticles I (100 mg/kg). “Sericin-conjugated silver nanoparticles II (S-AgNO3 NPs II)” for sericin-conjugated silver nanoparticles II (200 mg/kg), “sericin-conjugated silver nanoparticles II (P)” for pretreatment of sericin-conjugated silver nanoparticles II (200 mg/kg), “sericin-conjugated silver nanoparticles II (T)” for post-treatment sericin-conjugated silver nanoparticles II (200 mg/kg) (statistical icons: aa = P ≤ 0.01).
Measurement of organ index of mice
In current experimental research, after 60 days of treatment, there were significant increases in organ mass indexes (liver, kidney, brain, and spleen) of the DMBA group as in comparison with the control along with all treatment groups. In post-treatment groups, there was a significant reduction in the body mass indexes of all organs such as the kidney, liver, brain, and spleen as compared to DMBA-induced toxicity groups. In contrast to individual and prevention/pretreatment grouping, the body mass indexes were almost equal to the control group (Table 2).
Measurement of organ mass index of mice.
Abbreviation and keys: “aa” shows a significant difference in body mass indexes (liver, kidney, brain, and spleen) of the 7, 12-dimethylbenzanthracene group as compared to the control as well as all treatment groups (statistical icons: aa = P ≤ 0.01).
Analysis of hematological parameters
Level of red blood cells
In the 8th to 10th week of experimental study, the orally administrated cancer-inducing group revealed a noteworthy reduction in RBC levels. These results also showed the level of RBCs in all pretreatment/co-administration groups including Tam (P), SI (P), SII (P), S-AgNO3 NPs I (P), S-AgNO3 NPsII (P) significantly reached some extent to the normal group level as followed: Tam (P): 4.50 ± 0.06 106/µL, SI (P): 4.62 ± 0.05 106/µL, SII (P): 4.67 ± 0.03 106/µL, S-AgNO3 NPs I (P): 4.51 ± 0.03 106/µL, and S-AgNO3 NPs II (P): 4.52 ± 0.04 106/µL) when compared to DMBA (3.38 ± 0.03 106/µL). Whereas in the post-treatment groups, the level of RBCs in the blood of mice was significantly highest in Tam (T): 3.63 ± 0.02 106/µL, SI (T): 4.08 ± 0.04 106/µL, and S-AgNO3 NPs I (T): 4.07 ± 0.02 106/µL, except SII (T): 3.45 ± 0.02 106/µL and S-AgNO3 NPs II (T): 3.56 ± 0.02 106/µL as compared to cancer-inducing group (Figure 1).
Effects of sericin and S-AgNO3 NPs on RBCs, WBCs, eosinophils, and neutrophils. Abbreviation and keys: DMBA: dimethylbenzene anthracene; RBCs: red blood cells; S-AgNO3 NPs I (T): sericin-conjugated silver nanoparticles I (treatment); S-AgNO3 NPs II: sericin-conjugated silver nanoparticles II; S-AgNO3 NPs II (P): sericin-conjugated silver nanoparticles II (prevention); S-AgNO3 NPs II (T): sericin-conjugated silver nanoparticles II (treatment); SI: sericin I; SI (P): sericin I (prevention); SI (T): sericin I (treatment); SII: sericin II, SII (P): sericin II (prevention); SII (T): sericin II (treatment); S-AgNO3 NPs I: sericin-conjugated silver nanoparticles I; S-AgNO3 NPs I (P): sericin-conjugated silver nanoparticles I (prevention); Tam: tamoxifen; Tam (P): tamoxifen (prevention); Tam (T): tamoxifen (treatment); WBCs: white blood cells. “aa” shows a significant difference in the level of RBCs, WBCs, eosinophils, and neutrophils between control and dimethylbenzanthracene (50 mg/kg); “bb” depicts the difference in the level of RBCs, WBCs, eosinophils, and neutrophils between Tam, Tam (P), Tam (T) (100 mg/kg), and dimethylbenzanthracene; “cc” designates significant difference in the level of RBCs, WBCs, eosinophils, and neutrophils among SI, SI (P), SI (T) (100 mg/kg), and dimethylbenzanthracene; “dd” represents a significant difference in the level of RBCs, WBCs, eosinophils, and neutrophils between SII, SII (P), SII (T) (200 mg/kg), and dimethylbenzanthracene; “ee” represents a significant difference in the level of RBCs, WBCs, eosinophils, and neutrophils between S-AgNO3 NPs I, S-AgNO3 NPs I (P), S-AgNO3 NPs I (T), and dimethylbenzanthracene; “ff” represents a significant difference in the level of RBCs, WBCs, eosinophils, and neutrophils between S-AgNO3 NPs II, S-AgNO3 NPs II (P), S-AgNO3 NPs II (T), and dimethylbenzanthracene (statistical icons: aa, bb, cc, dd = P ≤ 0.01).
Level of white blood cells
The level of WBCs was significantly high when mice were orally administrated with a DMBA-inducing agent as compared to the control group. The level of WBCs was found to significantly low in individual-treated groups such as Tam (4.45 ± 0.02 103/µL), SI (4.18 ± 0.06 103/µL), SII (5.04 ± 0.13 103/µL), S-AgNO3 NPs I (3.81 ± 0.02 103/µL), and S-AgNO3 NPs II (4.9 ± 0.10 103/µL) as compared to cancer group (6.64 ± 0.01 103/µL). In the case of the pretreatment/ prevention study, there was also a significant reduction in all treated groups such as Tam (P), SI (P), SII (P), S-AgNO3 NPs I (P), and S-AgNO3 NPs II (P) along with toxicity group as given below: 4.64 ± 0.08 103/µL, 4.15 ± 0.03 103/µL, 5.16 ± 0.02 103/µL, 3.88 ± 0.10 103/µL, and 4.86 ± 0.11 103/µL, respectively. Similar results were obtained after post-treatment of selected compounds including Tam (T): 5.64 ± 0.02 103/µL, SI (T): 5.54 ± 0.01 103/µL, SII (T): 5.86 ± 0.13 103/µL, S-AgNO3 NPs I (T): 4.94 ± 0.01 103/µL, S-AgNO3 NPs II (T): 5.76 ± 0.06 103/µL, and there was a significant difference between all treatment groups and cancer-inducing group (Figure 1).
Level of eosinophils
The level of eosinophils in individual groups such as Tam (2.55 ± 0.01%), SI (2.54 ± 0.04%), SII (2.87 ± 0.07%), S-AgNO3 NPs I (2.66 ± 0.01%), and S-AgNO3 NPs II (2.46 ± 0.08%) was almost equal to the untreated normal group (2.61 ± 0.05%). As compared to the DMBA group (3.66 ± 0.01%), eosinophils levels between all pretreatment groups such as Tam (P), SI (P), SII (P), S-AgNO3 NPs I (P), and S-AgNO3 NPs II (P) were significantly decreased as followed: 3.07 ± 0.08%, 2.67 ± 0.03%, 2.84 ± 0.01%, 2.72 ± 0.07%, and 2.45 ± 0.01%, respectively. However, the eosinophils level in post-treatment was also significantly reduced including Tam (T): 3.35 ± 0.01%, SI (T): 3.15 ± 0.02%, S-AgNO3 NPs I (T): 2.85 ± 0.01%, and S-AgNO3 NPs II (T): 3.26 ± 0.01%, except SII (T): 3.51 ± 0.03% group in which eosinophils level decreased to some extent but no significant difference was found between SII (T) highest concentration group and DMBA-inducing group (Figure 1).
Level of neutrophils
The neutrophils level was considerably increased by toxicity-inducing chemicals, for example, DMBA-dosing group (50 mg/kg BW) in comparison with control and all treatment groups. The level of neutrophils was reduced when mice were orally treated with individual Tam (30.41 ± 0.58%), SI (32.2 ± 0.73%), SII (32.6 ± 0.91%), S-AgNO3 NPs I (25.73 ± 0.07%), and S-AgNO3 NPs II (29.2 ± 0.73%). Meanwhile, pretreatment/co-administration of Tam, sericin, and S-AgNO3 NPs along with DMBA decreased the level of neutrophils as follows: Tam (P): 35.44
Level of platelets
When DMBA-inducing chemical was orally administered to mice, there was a significant decline in the platelets level (143.2 ± 1.08 103/µL) when compared to the UT control group (247.6 ± 0.78 103/µL). The level of platelets was increased when mice were orally treated with individual Tam (237 ± 1.69 103/µL), SI (198.4 ± 3.98 103/µL), SII (184 ± 1.20 103/µL), S-AgNO3 NPs I (223.2 ± 1.32 103/µL), and S-AgNO3 NPs II (224 ± 1.20 103/µL). Meanwhile, pretreatment/co-administration of Tam, sericin, and sericin-NPs along with DMBA increased the level of platelets as follows: Tam (P): 236
Level of hemoglobin
In recent study, a 60-day experiment of orally administrated DMBA-inducing chemicals revealed a substantial reduction in Hb levels in comparison with the UT control group. The level of Hb was increased when mice were orally treated with individual Tam (13.1 ± 0.18 g/dL), SI (10.70 ± 0.19 g/dL), SII (10.5 ± 0.18 g/dL), S-AgNO3 NPs I (12.01 ± 0.02 g/dL), and S-AgNO3 NPs II (11.52 ± 0.16 g/dL). Meanwhile, pretreatment/co-administration of Tam, sericin, and S-AgNO3 NPs along with the cancer group increased the level of Hb (Tam (P): 11.93
Level of lymphocytes
The lymphocyte level was considerably decreased in the DMBA (50 mg/kg BW) dosing group in comparison to all other individuals, pretreatment, and treatment groups such as SI, SII (100 and 200 mg/kg BW), and S-AgNO3 NPs I, S-AgNO3 NPs II (100 and 200 mg/kg BW), and already used drug Tam (100 mg/kg BW). The level of lymphocytes was increased when mice were orally treated with individual Tam (74 ± 1.20%), SI (73 ± 1.20%), SII (69 ± 1.20%), S-AgNO3 NPs I (72.54 ± 0.38%), and S-AgNO3 NPs II (68.8 ± 0.90%). Meanwhile, pretreatment/co-administration of SI, SII, S-AgNO3 NPs I, S-AgNO3 NPs II along with cancer-induced group increased the level of lymphocytes as follows: Tam (P): 76.26
Level of monocytes
In recent hematological research, the monocyte level in the DMBA-inducing group was significantly higher than for the UT control group. Oral dosage of DMBA (50 mg/kg BW) for 60 days experiment caused a noteworthy increase in monocyte level (3.51 ± 0.03%) when compared to the UT control group (2.36 ± 0.02%). The level of monocytes was decreased when mice were orally treated with individual Tam (2.4 ± 0.07%), SI (2.26 ± 0.07%), SII (3.1 ± 0.08%), S-AgNO3 NPs I (2.31 ± 0.02%), and S-AgNO3 NPs II (3.04 ± 0.09%). Meanwhile, pretreatment/co-administration of Tam, sericin, and sericin-NPs along with DMBA group increased the level of monocytes as followed: Tam (P): 2.56
Analysis of biochemical parameters
Effects on glutathione
The level of glutathione (GSH) was measured by serum of mice after 60 days of the experiment which were orally administered with toxicity-inducing chemicals DMBA and selected doses of different treatment compounds divided into three basic categories including individual groups, pretreatment/co-administrated/prevention groups, and post-treatment groups. The different concentrations of selected compounds were used for treatment including two different concentrations of sericin and S-AgNO3 NPs (100 and 200 mg/kg, BW) and compared with the already used drug Tam (100 mg/kg, BW) for all biochemical parameters. The level of GSH was compared between DMBA-inducing and all individuals treated, prevention, and post-treatment groups. The level of GSH was significantly reduced in all individual treated groups such as Tam (1.74 ± 0.07 µmol/L), SII (1.8 ± 0.14 µmol/L), S-AgNO3 NPs I (1.7 ± 0.08 µmol/L), and S-AgNO3 NPs II (1.5 ± 0.07 µmol/L), except SI (2 ± 0.07 µmol/L) as compared to DMBA–cancer-inducing group (4.8 ± 0.45 µmol/L). A significant reduction in the level of GSH was also seen in all prevention groups as follows: Tam (P): 2.3 ± 0.13 µmol/L, SI (P): 2.76 ± 0.09 µmol/L, SII (P): 2.5 ± 0.04 µmol/L, S-AgNO3 NPs I (P): 2.06 ± 0.05 µmol/L, and S-AgNO3 NPs II (P): 1.82 ± 0.12 µmol/L group as compared to cancer group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 3.52 ± 0.26 µmol/L, 3.94 ± 0.07 µmol/L, 3.64 ± 0.11 µmol/L, 2.92 ± 0.08 µmol/L, and 2.42 ± 0.26 µmol/L, respectively, as shown in Figure 3. The outcomes revealed that all treatment groups were significantly different as compared to the toxicity group. The GSH level of individual treated groups including Tam (Tam), SI, SII, S-AgNO3 NPs I, and S-AgNO3 NPs II groups was almost equal to the control group except for the SI group which had an increased level of GSH than the control but significantly reduced in comparison with the DMBA group. In the case of all pretreatment/prevention groups, reduction was also detected in the GSH level, however, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). However, in the case of post-treatment groups, highest reduction in GSH level was observed in S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups at both concentrations (100 and 200 mg/kg, BW) as shown in Figure 3.
Effects on lactate dehydrogenase
The level of LDH was compared between DMBA toxicity-inducing and all individuals treated, prevention, and post-treatment groups. The level of LDH was significantly reduced in all individual treated groups such as Tam (342.2 ± 6.70 U/L), SI (353.4 ± 9.16 U/L), SII (323.4 ± 8.27 U/L), S-AgNO3 NPs I (349.4 ± 14.69 U/L), and S-AgNO3 NPs II (358.8 ± 8.95 U/L) as compared to DMBA–cancer-inducing group (993 ± 9.27 U/L). A significant reduction in the level of LDH was also seen in all prevention groups as follows: Tam (P): 450.6 ± 9.26 U/L, SI (P): 434 ± 10.71 U/L, SII (P): 451.8 ± 12.70 U/L, S-AgNO3 NPs I (P): 402.4 ± 5.61 U/L, and S-AgNO3 NPs II (P): 383.4 ± 2.60 U/L groups as compared to DMBA group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 594.8 ± 10.13 U/L, 609.8 ± 8.09 U/L, 561 ± 5.03 U/L, 518.8 ± 14.03, and 493.6 ± 5.78 U/L, respectively. The outcomes declared that there was a considerable difference between all selected treatment groups in comparison with the DMBA-inducing group. The LDH level of individual treated groups including Tam, SI, SII, S-AgNO3 NPs I, and S-AgNO3 NPs II groups was higher to some extent than the control group but significantly declined in comparison with the DMBA group. In all pretreatment/prevention groups, decline was also detected in the LDH level, however, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). However, in the case of post-treatment groups, highest reduction in LDH level was observed in the S-AgNO3 NPs II (T) group as shown in Figure 3.
Effects on alkaline phosphatase
The level of ALP was compared between DMBA-inducing and all individuals treated, prevention, and post-treatment groups. The level of ALP was significantly reduced in all individual treated groups such as Tam (139.6 ± 3.83 U/L), SI (143.6 ± 3.88 U/L), SII (131.2 ± 3.26 U/L), S-AgNO3 NPs I (117.4 ± 7.33 U/L), and S-AgNO3 NPs II (103.6 ± 4.83 U/L) as compared to DMBA–cancer-inducing group (353.4 ± 15.25 U/L). A significant reduction in the level of ALP was also seen in all prevention groups as follows: Tam (P):183.4 ± 9.95 U/L, SI (P): 197.4 ± 5.27 U/L, SII (P):172 ± 3.49 U/L, S-AgNO3 NPs I (P): 156.8 ± 4.24 U/L, and S-AgNO3 NPs II (P): 142.4 ± 5.71 U/L groups as compared to DMBA group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 227.6 ± 6.15 U/L, 243.4 ± 9.12 U/L, 205.4 ± 7.47 U/L, 194.8 ± 19.46 U/L, and 158.4 ± 6.35 U/L, respectively. The outcomes declared that there was a considerable difference between all selected treatment groups in comparison with the DMBA-inducing group. The ALP level of individual treated groups including Tam, SI, SII, S-AgNO3 NPs I, and S-AgNO3 NPs II groups was almost equal to the control group but significant decrease in comparison with the cancer-inducing group. In all pretreatment/prevention groups, reduction was also detected in the ALP level. However, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). Meanwhile in the case of post-treatment groups, highest reduction in ALP level was observed in S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups at both lower and highest concentrations (100 and 200 mg/kg, BW) as shown in Figure 3.
Effects of sericin and its sericin-conjugated silver nanoparticles on immunoglobulins
In curent research, the effects of sericin and their S-AgNO3 NPs on immunoglobulins levels such as on IgA, on IgG, and IgM were investigated. These immunoglobulins are the most important part of the immunosystem which interacts with any foreign compounds resulting in an immune cascade and ultimately leading to the clearance of these compounds from the organisms. The immunoglobulin levels were increased in toxicity-inducing DMBA mice group in comparison with the UT control group.
Effects on immunoglobulin A level
The level of IgA was compared between DMBA-inducing and all individuals, prevention, and post-treatment groups. The level of IgA was significantly reduced in all individual treated groups such as Tam (3.84 ± 0.13 g/L), SI (4.24 ± 0.18 g/L), SII (3.8 ± 0.14 g/L), S-AgNO3 NPs I (3.78 ± 0.12 g/L), and S-AgNO3 NPs II (3.5 ± 0.07 g/L) as compared to DMBA–cancer-inducing groups (7.6 ± 0.56 g/L). A significant reduction in the level of IgA was also seen in all prevention groups as follows: Tam (P): 4.28 ± 0.12 g/L, SI (P): 4.78 ± 0.11 g/L, SII (P): 4.6 ± 0.07 g/L, S-AgNO3 NPs I (P): 3.78 ± 0.12 g/L, and S-AgNO3 NPs II (P): 3.38 ± 0.38 g/L groups as compared to cancer group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 5.52 ± 0.26 g/L, 6 ± 0.07 g/L, 5.62 ± 0.12 g/L, 4.92 ± 0.11 g/L, and 4.22 ± 0.19 g/L, respectively. The outcomes declared that they were significantly different among all treated groups in comparison with the toxicity-induced group. The level of IgA of individual treated groups including Tam, SI (SI), SII (SII), S-AgNO3 NPs I, and S-AgNO3 NPs II groups was almost equal to the control group but considerably decreased in comparison with the toxicity-inducing group. In all pretreatment/prevention groups, a significant decline was also detected in the IgA level. However, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). Meanwhile, in the case of post-treatment groups, highest reduction in IgA level was observed in S-AgNO3 NPs I (T) and S-AgNO3 NPs II (T) groups at both concentrations (100 and 200 mg/kg, BW) as shown in Figure 4.
Effects on immunoglobulin G level
The level of IgG was significantly reduced in all individual treated groups such as Tam (38.4 ± 1.50 g/L), SI (45.6 ± 1.50 g/L), SII (38.2 ± 2.56 g/L), S-AgNO3 NPs I (42.2 ± 1.71 g/L), and S-AgNO3 NPs II (47.6 ± 1.36 g/L) as compared to cancer-inducing group (121.6 ± 2.99 g/L). A significant reduction in the level of IgG was also seen in all prevention groups as follows: Tam (P): 66.2 ± 1.59 g/L, SI (P): 74 ± 2.10 g/L, SII (P): 60.8 ± 1.59 g/L, S-AgNO3 NPs I (P): 56.6 ± 2.38 g/L, and S-AgNO3 NPs II (P): 51.4 ± 1.21 g/L groups as compared to cancer group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 89.6 ± 3.37 g/L, 101.8 ± 1.77 g/L, 92.4 ± 1.44 g/L, 79.2 ± 2.15 g/L, and 70 ± 1.70 g/L, respectively. The IgG level of individual treated groups including Tam, SI, SII, S-AgNO3 NPs I, and S-AgNO3 NPs II groups was almost equal to the control group but considerably decreased in comparison with the DMBA-inducing group. In all prevention groups, a significant decline was also detected in the IgG level, however, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). Meanwhile, in the case of post-treatment groups, highest reduction in IgG level was observed in S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups at both concentrations (100 and 200 mg/kg, BW) as shown in Figure 4.
Effects on immunoglobulin M level
The level of IgM was compared between DMBA toxicity-inducing and all individuals, prevention, and post-treatment groups. The level of IgM was significantly reduced in all individual treated groups such as Tam (4.8 ± 0.10 g/L), SI (5.02 ± 0.07 g/L), SII (5.04 ± 0.11 g/L), S-AgNO3 NPs I (4.74 ± 0.09 g/L), and S-AgNO3 NPs II (4.5 ± 0.07 g/L) as compared to cancer-inducing group (8.96 ± 0.36 g/L). A significant reduction in the level of IgM was also seen in all prevention groups as follows: Tam (P): 5.3 ± 0.13 g/L, SI (P): 5.78 ± 0.11 g/L, SII (P): 5.5 ± 0.04 g/L, S-AgNO3 NPs I (P): 5.16 ± 0.08 g/L, and S-AgNO3 NPs II (P): 4.8 ± 0.14 g/L groups as compared to cancer group. Similar results were observed in post-treatment groups such as Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) groups: 6.3 ± 0.35 g/L, 6.94 ± 0.07 g/L, 6.64 ± 0.11 g/L, 5.94 ± 0.09 g/L, and 4.76 ± 0.12 g/L, respectively. The outcomes declared that there was a significant difference among all treated groups in comparison with the DMBA-inducing group. The IgM level of individual treated groups including Tam (Tam), SI (SI), SII (SII), S-AgNO3 NPs I, and S-AgNO3 NPs II groups was almost equal to the control group but considerably declined in comparison with the cancer-inducing group. In all pretreated/prevention groups decline was also detected in the IgM level, however, the highest reduction was seen in S-AgNO3 NPs I (P) and S-AgNO3 NPs II (P) groups at both concentrations (100 and 200 mg/kg BW). Meanwhile, in the case of post-treatment groups, highest reduction in IgM level was observed in S-AgNO3 NPs I (T) and S-AgNO3 NPs II (T) groups at both concentrations (100 and 200 mg/kg, BW) as shown in Figure 4.
Histopathological study
The mice organs such as the kidney, liver, and brain were washed with ice-chilled physiological saline and stored in 10% formalin for 24 h, afterwards, they were transferred into 75% alcohol for histopathological examination.
Histology of liver
The liver is the most important and biggest organ in the body and performs multiple functions including maintaining normal glucose levels, removal of toxic substances from the blood, regulating blood clotting, and also being involved in protein synthesis as well as storage of vitamins and minerals. Histopathological characteristics in the liver of DMBA-inducing mice treated with silk protein sericin and sericin-silver NPs were illustrated. DMBA-induced toxic mice group showed morphological changes in the liver such as hepatocytes swollen, Kupffer cell proliferation, cytoplasmic degeneration, accumulation of necrotic foci, binucleated cells indicating the process of regeneration, hemorrhage, vacuolation, infiltration of inflammatory cells, congestion and few hepatic cells along with large intercellular spaces. The UT control group showed normal liver structure with well-organized hepatocytes, normal nuclei, intact central vein structure, a large number of hepatocyte cells with few intercellular spaces, and performed function normally. The individually treated groups such as Tam, SI (SI), SII (SII), S-AgNO3 NPs I, and S-AgNO3 NPs II groups did not show any adverse effects on the liver structure when compared with the control group. In prevention groups including Tam (P), SI (P), SII (P), S-AgNO3 NPs I (P), and II (P), there were no significant adverse effects in the liver structure as compared to the control group, and all pretreatment groups shown histology of liver almost near to the control group. Whereas post-treatment groups like Tam (T), SI (T), SII (T), S-AgNO3 NPs I (T), and S-AgNO3 NPs II (T) reversed the changes caused by the DMBA-induced group to some extent and all these post-treatment groups showed positive significant effects on the histopathology of the liver as shown in Figure 5.
Histology of kidney
The kidneys are the reddish-brown bean-shaped organs having the main structural and functional unit called the nephron span the outer renal cortex and inner renal medulla. The most important functions of kidneys include removal of waste products along with drugs from the body, balanced electrolytes, regulation of blood pressure by liberation of hormones, and control of the production of red blood corpuscles. The renal histopathology of kidneys showed the normal morphological appearance of the kidney structure as compared to the DMBA-induced group which caused damage in normal kidneys such as mild narcosis, widening the glomeruli capsular spaces, destruction of Bowman's capsule epithelial flat-lining, vacuoles in epithelial cells, damage of epithelial cells in proximal convoluted tubules, inflammatory cells infiltration, diminished and distorted glomeruli, edema exudates, and dilation in distal convoluted tubules along with vacuoles formation in epithelial cells. Prevention and treatment with sericin and its S-AgNO3 NPs showed the renal protective effect by improving the renal injury caused by the cancer group. Treatment with Tam showed no remarkable recovery in the renal injury as compared to the cancer group. These results showed that sericin and S-AgNO3 NPs could better defend against DMBA-induced kidney damage in mice than the standard drug Tam as shown in Figure 6.
Histology of brain
The normal brain contains blood vessels and neuroglial cells, meningeal cells, and vascular epithelium, and together with the spinal cord forms the central nervous system. The brain performs various functions including controlling and regulating body temperature, hunger, pain, vision, motor skills, emotions, and most importantly breathing. The histological section of the brain showed that in the toxic group, massive lacerations were seen in the cerebrum, nuclear pleiomorphism, increased cell density, binucleation, dead neurons, cells without a nucleus, and necrosis with pseudo-palisading. It has also been found that the hippocampus area of the DMBA group revealed a reduction in the number of pyramidal cells with the presence of various shrunken degenerated cells. However, in the control group, normal histology of the brain showed different neurons in the cerebral cortex, intact neurons, without any loss in neuron structure, striatum, and hippocampus. The different types of neuroglial cells and well-organized pyramidal cells were scattered inside the neuropil matrix. The individual treated groups showed a similar structure of the brain when compared with the control group and no significant cytotoxic effects of SI, and S-AgNO3 NPs I except SII (SII) and S-AgNO3 NPs II which showed some structural changes in brain structure as compared to normal control groups such as binucleation, dead neurons, and cells without a nucleus. Prevention with sericin and S-AgNO3 NPs showed a condensed layer of pyramidal cells with vesicular nuclei, intact neurons, and normal physical appearance of neuron structure as compared to DMBA except at higher concentrations of sericin and S-AgNO3 NPs which caused damage to normal neurons structure with binucleated and dead neurons. Post-treatment with sericin and S-AgNO3 NPs also overcame the loss of neurons in the DMBA-treated group but this alleviation was less than in the prevention groups as the brain section did not closely resemble the control group when compared to the prevention group. The outcomes showed that sericin and S-AgNO3 NPs may have the capability to as neuroprotective agents against cancer-induced mice at lower concentrations (100 mg/kg, BW) (Figure 7).
Discussion
Numerous experiments have been performed to demonstrate the potential of sericin and its different types of nanoparticles on blood parameters. Khan et al. 19 demonstrated the in vitro and in vivo effect of N-(benzylidene)-2-(2-hydroxynapthalen-1-yl) diazenyl) benzohydrazides (1–2) (NCHDH and NTHDH) on DMBA-inducing tumor models. They declared that selective treatment compounds enhanced the serological and hematological considerations following the DMBA-inducing model animal group. Our study also declared the same results in which sericin and its conjugated nanoparticles treatment groups improved their hematological effects as compared to the cancer-induced group. Sumaiah et al. 37 worked on the effects of gold nanoparticles (GNPs) for the treatment of human adenocarcinomas with hematological parameters. The results declared that blood parameters in all treatment groups as well positive group were less than the normal range which is due to the effect of toxic reaction. 13 The results of current study are similar to these previous studies in which all treatment group values were almost equal to the control group.
The study was conducted to evaluate the in vitro and in vivo inhibition or stimulation of human Wharton's jelly mesenchymal stem cells-derived secretomes (hWMSCs). The results showed that these secretomes could improve hematological indices and prolonged survival rates in tumor-bearing mice and inhibit growth during in vitro cell line experiments. 38 Similar results were evaluated from a recent study in which all treatment groups improved blood group indices as compared to DMBA-inducing mice. Yu et al. 39 improved the sustainable release and bio-availability of carbon nanotube (CNT)–drug complex in human breast cancer cells (HBCCs). The results evaluated that CNTs carried inhibitory effects against HBCCs and reduced platelet counts along with increasing aspartate aminotransferase levels during blood biochemical testing. 39 The results of our treatment groups also declared similar effects on platelet count.
Pateliya et al. 40 evaluated the effects of metformin and naringenin in combination with doxorubicin chemotherapy in methyl nitrosourea-induced experimental breast carcinomas. They concluded that both these selected compounds along with doxorubicin proved a noteworthy decline in cancer weight as well as its volume in the 4T1-induced orthotopic mouse model, suggesting a combination treatment to enhance anticancer action against in vivo experiments. Furthermore, they also suggested that histology of tumor biopsies showed that combination therapy enhanced antibreast cancer action through rising cancer necrosis. Hematological parameters, body weight, and survival rates offered that adjunct treatment is remarkably safe at lower dose levels. 40 Our experimental results also suggested that in some blood parameters SI and S-Ag NO3 NPs I showed the highest significant activity as compared to SII and S-Ag NO3 NPs II treatment groups. So, they are also effective at lower doses as compared to higher doses. The present research was performed to check the in vitro and in vivo anticancer potential of Jasminum sambac on Dalton's ascites lymphoma (DAL)-induced Swiss albino mice. The results presented that at an oral dose of methanolic extract, 100 mg/kg (BW), of mice displayed a considerable modification in blood-related parameters level between treated and cancer-inducing mice. Furthermore, it could be accomplished that the methanolic extract of J. sambac showed considerable anticancer effects. 24 The results of our treatment groups also declared similar effects on blood count.
Antony et al. 41 demonstrated the anticancer action of green synthesized silver nanoparticles (AgNO3 NPs) utilizing Ficus religiosa for the treatment of DAL in an animal mice model. Hematological, biochemical, and antioxidant assay results proved that silver NPs along with F. religiosa were useful for the treatment of the DAL-inducing mice experiment. Furthermore, after treatment of the DAL-induced group, the values of blood parameters significantly reached their normal control group. Similar results were also yielded from our study. 41
GSH is an antioxidant agent normally involved in different cellular processes including cell proliferation, differentiation, and cell division, and reacts with ROS to prevent apoptosis and detoxify harmful substances. In the case of cancer cells at moderate ROS level, it supports the dual role in the progression, removal, and detoxification of cancerous substances by activating the signaling pathway mechanism. But at a high ROS level, GSH promotes cell metastasis to distant organs and also causes several chemotherapeutic drug resistance in different cancer types such as liver, breast, colon, and lung cancer. 42 In our recent study, an increased level of GSH in the cancer-induced group has been found in comparison with the control group. There was a significant difference between the DMBA-induced group and all treatment groups. In individuals treated as well as prevention/pretreatment groups, the level of GSH was nearly equal to control groups which showed that sericin and its S-AgNO3 NPs can prevent elevation of the GSH group due to the presence of flavonoids contents that react with ROS species. The post-treatment groups of sericin and S-AgNO3 NPs also decreased the level of GSH compared to the DMBA-induced group, but their recovery was lesser than the prevention group.
Zhang et al. 43 worked on the involvement of the GSH enzyme in the occurrence and involvement of breast cancer. GSH enzyme balances the hemostasis level by reducing ROS and catalyzing the reduction of hydrogen peroxide (H2O2) to convert into alcoholic compounds. ROS plays different role at the initial stage of cancer such as causing DNA damage, acting with carcinogenic compounds, and interfering with the response to DNA damage. However, an imbalance in the homeostasis level of ROS due to the abnormal production of GSH significantly increased the resistance of breast cancer cells to different therapeutic agents such as doxorubicin. 43 In a recent study, toxicity was chemically induced by DMBA in female Swiss albino mice, and from the serum of mice, GSH level was measured. The DMBA group showed an elevated level of GSH in comparison with the control and all treated groups. In individuals treated as well as prevention/pretreatment groups, the level of GSH was nearly equal to control groups which showed that sericin and it is S-AgNO3 NPs can prevent elevation of the GSH group due to the presence of flavonoid contents that react with ROS species. The post-treatment groups of sericin and S-AgNO3 NPs also decreased the level of GSH compared to the cancer-induced group, but their recovery was lesser than the prevention group.
In current study, we analyzed LDH, gamma-glutamyl transferase (GGT), and ALP from the serum of cancer-induced and treated animal mice models. The results declared that in the serum of mice group with cancer-induced toxicity, the level of LDH, GGT, and ALP was considerably higher in comparison with the UT control group. The treatment compounds such as sericin along with its S-AgNO3 NPs significantly declined the LDH, GGT, and ALP levels in comparison with the DMBA-induced group. The values of treatment groups demonstrated a decrease in enzyme levels in the cancerous group due to the presence of ROS and the secondary amino acid composition of SS which reduced silver ions into their metallic form and prevented cell proliferation and apoptosis of cells. Alipanah et al. 44 studied the inhibitory effects of the natural herb Viola odorata extract (VOE) on tumors and their metastasis in the 4T1 breast cancer rat model. They treated 4T1 breast cancer rat models with different concentrations (50, 150, and 250 mg/kg, BW) of Viola odorata for 21 days experiment. They concluded that at 250 mg/kg, the body weight concentration of the natural herb reduced the enzymatic level in comparison with the control group. The immunohistopathological analysis of spleen, liver, and lung tissues showed metastasis of tumors in rats. The VOE250 significantly reduced the metastasis in the liver and lungs of rats as compared to other selected doses of VOE. 44
In the present study, elevated levels of ALP, GSH, GGT, and Hb were observed in DMBA-inducing mice in comparison with the control group. The treatment with selected concentrations of sericin and its S-AgNO3 NPs showed a significant decline in these parameters in comparison with the cancer-inducing group, which indicated that the natural silk protein sericin and S-AgNO3 NPs have pharmacological potential without any side effects on normal body tissues. Mishra et al. 45 reported that significant increase in the level of ferritin, GTT, ALP, GSH, and Hb in patients with cancer without metastasis in comparison with the control group. Meanwhile, in the case of metastasis, further elevated levels of these parameters were observed as compared to those without metastasis. Mastectomy, in both cases, led to no significant reduction in all biochemical parameters signifying that longer follow up could perform a post-surgery decline in biochemical parameters. 45 Similar outcomes were observed in this study where mice groups experiencing toxicity had elevated levels of these biochemical parameters that were decreased and settled down when treated with selected concentrations of sericin and S-AgNO3 NPs.
In a previous study, a mouse model of breast cancer metastasis was developed in which breast cancer cells and osteotropic metastatic bone targets both had a human origin. The engrafted human bone was functional based on finding human IgG in the mice's bloodstream, with normal bone histology, and human B cells in the spleen of the mouse. The results showed that in the case of bone metastasis of cancer, human cells were implanted into the mouse skeleton, which indicated the species-specific tropism. The model also replicates the events observed in breast cancer patients’ skeletal metastasis and also serves as a relevant and beneficial model for studying the disease. 46 Another study worked on antibodies to mouse mammary tumor virus-related antigens in the serum of patients with breast cancer. They showed a correlation between patients with breast carcinoma with age and the probability of detection of these antibodies. Immunological and histopathological analysis indicated that immunoglobins such as IgG, and IgM activities were present more often in patients with filtrating ductal carcinoma as compared to other breast cancer tumor types, and no correlation was found between the incidence rate of antibodies and disease stage. 47 In the current study, we also determined the level of immunoglobins (IgA, IgG, and IgM) in the serum of DMBA-induced mice and selected concentrations of sericin and S-AgNO3 NPs in an animal mice model. The cancer-inducing group showed a higher level of immunoglobins in comparison with the control group. Meanwhile, treatment with sericin and S-AgNO3 NPs indicated a significant reduction in the immunoglobins level in comparison with the cancer-induced group.
We conducted research and worked on biochemical and histopathological evaluation of an in vivo animal mice model. The DMBA chemical was used for inducing toxicity and results revealed a significant difference in oxidative stress biochemical parameters between cancer-treated groups and control groups and these changes were dose-dependent. The histopathological studies indicated that triple-negative mammary tumors developed in mice. 48 Similar findings were calculated from the present research study, in which we induced toxicity by DMBA and treated with different concentrations of sericin and S-AgNO3 NPs. The biochemical and histopathological findings also revealed that both selected concentrations of silk protein sericin showed a significant difference among the treated groups and DMBA group along with a comparison to the UT control group. No significant difference was found between individual, and pretreatment groups of sericin and S-AgNO3 NPs in comparison with the control group. The results declared that there were no side effects of selected compounds on normal cells of animals, except the highest concentration of sericin and its nanoparticles (200 mg/kg BW) had some side effects on histopathological analysis.
Santos et al. 49 worked on histopathology, oxidative stress, and bioenergetics in mice livers exposed to N-diethylnitrosamine (DEN) hepatocellular carcinomas. They showed that histopathology of the DEN group identified a preneoplastic lesion along with induced bioenergetics and antioxidant changes in the liver of mice. 49 Another study on the histopathology of the kidney, spleen, and liver of mice with different dosages of GNPs. They worked on the effects of single-dose as well as priming doses of 5, 20, and 50 nm diameter GNPs on the structural changes in the spleen, liver, and kidney of mice along with some biochemical parameters. The outcomes showed that a single dose of small GNPs (5 nm) showed reasonable adverse changes on the second day in the structure of the liver that progressively decreased after 8 days. Similarly, large-dose and medium nanoparticles (50 and 20 nm) also showed significant changes on day 2 in the mice spleen that continued on day 8 as well. There were no pathological or significant adverse effects in the kidneys of mice irrespective of the GNP size. While the primed animals could not show any pathological changes on pre-exposure to the second dose of GNPs. 50 The present study also declared the same pathological changes in the liver, kidney, and spleen of mice exposed to DMBA-induced mice that were gradually reduced with different concentrations of silk sericin (SI and SII) and their S-AgNO3 NPs (S-AgNO3 NPs I and S-AgNO3 NPs II).
In the current study, we measured the histopathological effects in the DMBA-induced brain of mice. These changes were included as massive lesions were seen in the cerebrum, nuclear pleiomorphism, increased cell density, and necrosis with pseudopalisading as well as a decline in the number of pyramidal cells in the hippocampus area of brain cells. The damage in the brain cells of mice could be treated with select doses of SI, SII, S-AgNO3 NPs I, and S-AgNO3 NPs II. The treatment groups showed significant recovery in the histopathological damage in the brain cells of mice. The recovery showed that SS and their S-AgNO3 NPs have histopathological potential against DMBA-induced mice. Tamase et al. 51 identified the tumor-initiating cells in a highly aggressive solid brain tumor by using promoter activity of nucleostemin drives Green fluorescent protein (GFP) expression Nucleostemin gene-Green fluorescent protein (NS-GFP). By using NF-GFP expression, they were monitoring the differentiation processes of normal neural stem cells by analyzing GFP fluorescence intensity. They revealed by detailed analyses of the NS-GFP+ brain tumor cells that tumor-initiating cells showed the activation of tyrosine kinase c-Met receptors which involved tumor invasiveness. They concluded that the NF-GFP system provides a powerful tool to elucidate stem cell biology of normal as well as malignant tissues. 51 Another study on mouse brain tumors and their application in preclinical trials suggested that major genetic alterations of human brain tumors such as gliomas and medulloblastomas occurred due to gene-encoding proteins involved in signal transduction or cell cycle regulation. They used a genetically engineered mice model, to study the correct recapitulation of these genetic alterations with etiology, biology, and histopathology. They declared that the animal mice model increased our understanding of brain tumor initiation, progression, formation, and metastasis for the discovery of novel therapeutic test agents as well as their targets. 52 The majority of the findings of the current study are promising and can pave the way for further research. However, power analysis was not performed before the selection of the sample size which could be the limitation of the current study. Keeping in view the importance of the power analysis of sample size, upcoming studies’ sample size/replicates will be designed using power analysis. By applying the power analysis for sample size, the probability could be better for even positive outcomes.
Conclusion
Both PAHs: DMBA causes numbers of serious physiological anomalies and behavioral dysfunctions such as cancer, ageing, and hypertension. Sericin and its conjugated silver nanoparticles were successfully designed to develop therapeutic efficiency against cancer-induced toxicity. The consequences suggested that the use of different concentrations of sericin and its S-AgNO3 NPs significantly improved blood parameters as well as biochemical parameters index between treatment groups and the DMBA-inducing group. Moreover, in vivo studies showed that these particles have very little immuno-toxicity profile against vital organs like the kidney, liver, and brain tissues. However, sericin applications are so far at the laboratory scale and additional investigations are needed to use them at industrial scale as well as more experimentation is mandatory to reveal the comprehensive mechanism.
Footnotes
Acknowledgements
We are obliged to the Department of Zoology, Government College University Lahore, and University of Health Sciences Lahore for providing the necessary equipment and environment for the completion of current research work.
Authors contributions
SM, SA, AP, UAA, LK, and TAM wrote the main manuscript. SM, SA, and TN prepared figures. HMT, MS, and SM analyzed the data. All authors reviewed the manuscript.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Ethical approval
The in vivo experiment was ethically approved by the Ethical Committee of Zoology Department, Government College University, Lahore with reference No. GCU-IIB-1064. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Author biographies
Samaira Mumtaz has completed her PhD in zoology with specialization in cancer biology/medical toxicology from Government College University Lahore, Pakistan. She has 1 year of teaching experience as a Visiting Lecturer in Women University Bagh Azad Jammu & Kashmir, Pakistan, and 6 months of experience as a Visiting Lecturer in the University of Education, Lahore, Pakistan. She has published 10 research articles and 6 review articles.
Shaukat Ali is working as Associate Professor at the Department of Zoology, Government College University Lahore. He has obtained a PhD degree in medical toxicology from Leiden University, The Netherlands. He is working on the induction of human diseases in animal models to understand the underlying mechanisms of these diseases and the development of therapeutic agents. He has published 190 research articles and 8 book chapters in international peer-reviewed journals. He has won 5 competitive research grants during his scientific carrier. Furthermore, he has supervised 8 PhD, 45 post-graduate and 50 graduate students for their thesis-oriented research.
Asim Pervaiz obtained his PhD degree in translational biology and cancer therapeutics from German Cancer Research Centre (DKFZ), Heidelberg, Germany. During his PhD tenure, he learned and experienced a broad range of in vitro and in vivo techniques. Later on, he joined the Institute of Biomedical and Allied Health Sciences, University of Health Sciences, Lahore, Pakistan, as Assistant Professor. His professional experience as a teacher has also polished his skills overtime to apply the learned skills, while training the next-generation learners to be better leaders of the future. So far, he has published 16 research articles, 5 abstracts and 1 book chapter in international peer-reviewed journals. In addition, he has been awarded with eight national/international awards and three competitive research grants during his scientific carrier. Furthermore, he has supervised 4 PhD and 27 post-graduate students for their thesis-oriented research.
Uzma Azeem Awan has completed her PhD in biotechnology with specialization in nanobiotechnology/nanomedicine from the University of Azad Jammu and Kashmir, Pakistan. She has completed her research project from Georgia Institute of Technology, Atlanta, USA. Currently, she is working as Assistant professor at National University of Medical Sciences (NUMS), Rawalpindi, Pakistan. With over 5 years of experience, she has published 16 research articles, 6 review articles and 6 book chapters. She has been awarded with three national research grants as Principal Investigator and three national research grants as Co-Principal Investigator. She has actively participated in various national and international scientific conferences/workshops. Furthermore, she is a member of American Society of Chemistry and British Society of Nanomedicine.
Tooba Nauroze has completed her PhD in zoology with specialization in nanobiotechnology/medical toxicology from Government College University Lahore, Lahore, Pakistan. Currently, she is working as Lecturer at University of Education, Lahore, Pakistan. With over 5 years of experience, she has published seven research articles. She has actively participated in various national and international scientific conferences/workshops.
Lubna Kanwal is currently working as Lecturer in the Department of Zoology, University of Okara, Pakistan. Kanwal earned her PhD degree under the supervision of Shaukat Ali, from Government College University Lahore, Pakistan in 2023. The PhD Research title was “Targeting Glucose-6-phosphate dehydrogenase by phytochemicals for the treatment of Hepatocellular carcinoma.” During the last 15 years of teaching and research experience she has also supervised more than 20 research students of BS, MSc and MPhil level. Kanwal is carrying out research activities in molecular biology, biochemistry, cancer biology. Kanwal has published more than 10 articles in peer-reviewed international SCI scientific journals. Kanwal is also a co-author of the book chapter “Alkaloids in the Treatment of Gastrointestinal Cancer.”
Muhammad Summer is working as a Lecturer in the Department of Zoology, Government College University Lahore, Pakistan. His area of interest includes nanotechnology, microbiology, and toxicology. He has been publishing research and review articles in the above-said fields from 2020. He has published more than 25 research articles.
Shumaila Mumtaz has earned her PhD in zoology with a specialization in nano-biotechnology (nanomedicine) from Government College University Lahore, Pakistan. Currently, she holds the position of Assistant Professor at the University of Poonch, Rawalakot, Azad Jammu and Kashmir, Pakistan. With 2.5 years of experience, she has authored 30 research articles and 10 review articles.
Tafail Akbar Mughal has earned his PhD in zoology with a specialization in nano-biotechnology (nanomedicine) from Government College University Lahore, Pakistan. Currently, he holds the position of Lecturer at the Women University of Azad Jammu and Kashmir Bagh, Pakistan. With 2.5 years of experience, he has authored 22 research articles and 3 review articles.
Hafiz Muhammad Tahir has obtained his PhD from the University of the Punjab, Lahore, Pakistan. He has completed his postdoctoral research from the USA. He is working as Director of Dr. Nazir Ahmad Institute of Biological Sciences, Government College University Lahore. His research areas are applied entomology and biomaterial research. He has published over 200 research articles in various fields biological sciences in well reputed journals and supervised 10 PhD and over 100 MPhil and BS students.