الفهرس 
Introduction and research objectivesOnly 14 pages are availabe for public view
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Abstract
The present study was conducted to evaluate the targeted antitumor effect of AgNPs in a rat model of hepatocarcinogenesis induced by NDEA and CCL4. The study also investigated the role of AgNPs targeted therapy on modulating the incured biochemical indices compared to those of the carcinogen control group.
To fulfill our goals, we were devoted to green synthesis of AgNPs employing the biomolecules of G. glabra root extract which exhibit stabilizing and targeted hepatic action. Thereafter, characterization of the obtained particles was done to confirm their reliable nanosize (9.18-23.4 nm), morphology and crystalline nature. FTIR spectra was analysed for emphasizing the presence of different functional groups of G. glabra involved in capping the silver nanoparticles. The stability of biosynthesized AgNPs was tested in the presence of sodium chloride and rat serum to simulate the in vivo condition and cofirm the steric stabilization of the nanoparticles created by the biomolecules of G. glabra which coat their surfaces. After that, we optimized the therapeutic dose of AgNPs (0.5 mg/kg b. wt.) that should be given to the assigned experimental group exhibiting minimal hepatotoxic effects.
Afterwards, 91 male albino rats weighting about 140-160 gms were used for this study. We induced the hepatocarcinogenesis in 60 rats which received single intraperitoneal (i.p.) injection of NDEA (200 mg/kg body weight) in normal saline (0.9%). One week after NDEA challenging, rats received weekly subcutaneous injections of carbon tetrachloride solution (CCL4/olive oil; 1:1; 3ml/kg body weight) for 6 consecutive weeks.
After the induction of the hepatocarcinogenesis process, we aimed to monitor and confirm the evidence of preneoplastic hepatic lesions as detrminent criteria for commencing an effective AgNPs therapy. The monitoring process was accomplished via biochemical screening the liver enzyme seromarkers (ALT&AST) in serum samples obtained from 30 rats randomly selected at 8, 12, 16, 20, 24 and 28 weeks post NDEA administration. The serum levels of ALT and AST showed significant upregulation at weeks 24 and 28 post NDEA dosing in comparison with normal control rats. The confirmation of the hepatic preneoplastic lesions was done by sacrificing 5 rats at each week and submitted to histopathological examination. At week 24, microscopic examination of H&E stained hepatic sections revealed revealed early preneoplastic lesions in all 5 examined rats. These lesions were observed as mild nuclear atypia which expressed by evidence of meganuclei with dotted and condensed chromatic and others with prominent nucleoli as wll as condensed nuclear membranes. At week 28, all 5 sacrificed rats demonstated progressive preneoplastic lesions in the form of foci of cellular alteration which could be classified according to their cell size and cytoplasmic tinctorial appearance into; eosinophilic, tigroid basophilic, clear cell, vacuolated, amphophilic and mixed cell foci.
Following the confirmation of hepatic preneoplastic changes, the remaining 50 rats were randomized into two groups at the beginning of week 31 post NDEA administration including carcinogen control group and silver nanoparticles-treated group.
In the first group (carcinogen control group), 25 rats received only NDEA and CCL4 at the biggening of the experiment and kept without treatment. Five rats were sacrificed after 32, 34, 36, 38 and 40 weeks post NDEA dosing in 5 sacrifices representing different subgroups. The carcinogenic effect of NDEA and CCL4 was assessed grossly and histopathologically as well as via immunohistochemical phenotyping of the developed neoplastic lesions.
The gross findings were expressed by hepatomegaly (52%), minute whitish foci (12%), whitish-gray discrete nodular lesions projected above the surface (52%), haemorrhagic nodular lesions (8%), in addition to combined focal and nodular whitish lesions (20%).
The histopathological results revealed different proliferative and neoplastic lesions involving the hepatocytes and intrahepatic bile ducts. The hepatocellular proliferative and neoplastic lesions included foci of cellular alteration (100%), Hepatocellular adenomas (28%) and hepatocellular carcinomas (96%). Based on the histological arrangement of malignant cells, HCCs were observed in 7 variable growth patterns, including trabecular (36%), pseudoglandular (8%), solid (20%), steatotic (8%), clear cell (8%), fibrolamellar (8%) and mixed pattern (8%) HCCs.
The cholangiocellular proliferative and neoplastic lesions were expressed by bile duct hyperplasia (80%), biliary cysts (16%), oval cell hyperplasia (100%), cholangiofibrosis (36%), cholangiocellular adenoma (8%), cholangiocellular carcinoma (8%) and combined hepatocellular cholangiocarcinoma (4%).
Metastatic evidence of hepatocellular carcinomas was detected both intrahepatic and exrtahepatic (pulmonary) and were evaluated histopathologically and immunohistochemically. The intrahepatic metastatic lesions were seen in (48%) of the carcinogen control rats and found as multiple focal satellite lesions within the hepatic parenchyma. Immunohistochemically, intrahepatic metastatic lesions showed similar immunophenotypes to the primary liver carcinomas. The extrahepatic metastasis was identified in lung of (12%) of the carcinogen control rats. Microscopically, these metastatic lesions were detected either as metastatic emboli within pulmonary blood and lymph vessels or within lung tissues. The neoplastic cells showed pronounced cytological and nuclear atypia. Immunostaining of the pulmonary metastatic lesions with hepatocytes specific monoclonal antibody (HepPar-1) showed strong cytoplasmic granular immunoreactivity confirming the hepatocytic origin of these lesions.
Immunohistochemically, a panel of monoclonal antibodies was employed for immunophenotyping of the developed neoplastic lesions in the carcinogen control group, including HepPar1, CD34, cytokeratin 7, cytokeratin 19 and pan-cytokeratin MoAbs. All stained cases of HCA (100%) were immunostained by HePar-1 MoAb. Of the 24 described cases of HCC, 21(87.5%) were positive for HePar-1 MoAb. The 3 negative cases for HePar-1 MoAb included one poorly-differentiated macrotrabecular HCC and 2 poorly-differentiated clear cell HCCs.
CD34 MoAb was employed as endothelial marker of neovascularization to expose the sinusoidal capillarization which occurred concurrently with the neoplastic hepatic lesions. All stained cases of HCC (100%) with CD34 MoAb expressed diffuse positive membranous immunoreaction, while HCA showed negative immunostaining.
CK7 MoAb was used to confirm the histopathologically described FL-HCC which characteristically stained with this antibody. All stained cases of FL-HCC (100%) revealed strong and diffuse cytoplasmic positivity with marked heterogeneity in the staining pattern between neoplastic cells. Cytokeratin 19 as a marker of biliary differentiation stained all cases of cholangiocellular carcinoma (100%) with strong positive cytoplasmic staining pattern. In addition, small positive ductular structures infiltrated within hepatic parenchyma were seen. Pan-cytokeratin (AE1/AE3) MoAb stained (86%) of HCA cases with week cytoplasmic and membranous immunoreaction. Strongly stained cells were observed at the periphery of the adenomatous lesion. On the other hand, (62.5%) of the stained cases of HCC were decorated with pankeratin MoAb as brown-colored cytoplasmic and membranous immunoreaction. The negatively immunostained cases included one poorly-differentiated trabecular HCC as well as all stained cases of pseudoglandular, steatotic, clear cell and fibrolamellar HCCs.
The second group (AgNPs-treated group) consisted of 25 rats which received AgNPs targeted therapy. The therapeutic protocol started as soon as hepatic preneoplastic lesions were confirmed (31 week post NDEA dosing) and continued for 10 successive weeks in a daily dose 0.5 mg/kg b.wt. Five rats were sacrificed after 32, 34, 36, 38 and 40 weeks post NDEA dosing in 5 sacrifices representing different subgroups of 2, 4, 6, 8 and 10 weeks post AgNPs-treatment. The therapeutic impact of AgNPs targeted therapy was evaluated via gross and histopathological examinations as well as immunophenotyping of the developed neoplastic lesions in this group.
The gross findings revealed that only one rat present in subgroup 2 showed discrete grayish-white nodule with irregular outline and projected from the hepatic surface, while other rats of this group showed no grossly visible hepatic lesions.
Histopatholigically, the foci of cellular alteration were observed in (48%) of rats of this experimental group. They were detected as early as 2 weeks post treatment in all 5 examined rats and were expressed as tigroid basophilic, eosinophilic, vacuolated, clear cell and mixed cells foci. The number of rats showed such foci decreased starting from week 4 post-treatment till the end of the experiment where it appeared in 2 rats for each sacrifice corresponding to 4, 6 and 8 weeks post treatment and in only one rat at week 10.
Trabecular hepatocellular carcinoma was detected only in subgroup 2 (4 weeks post treatment) in one rat (4%). Such carcinomatous lesion appeared well-circumscribed and was not accompanied by intrahepatic satellite lesions (intrahepatic metastasis) which elucidated the role of AgNPs treatment in the impairment of intrahepatic metastasis of HCCs. This cancerous growth revealed strong diffuse immunoreactivity with HapPar-1 and CD34 MoAbs, while displayed only foci of cellular grouping with Pan-CK MoAb. Also, our results revealed absence of the evidence of lung metastasis in this case of trabecular hepatocellular carcinoma. Such noteworthy finding indicated that biosynthesized AgNPs impaired extrahepatic metastasis of liver cancer.
Hepatocellular adenoma was observed only in one rat (4%) in subgroup 1 and expressed marked sporadic areas of strong granular cytoplasmic immunoreactivity with anti-HepPar-1 MoAb. The periphery of the adenomatous lesion also displayed strong reaction.
The cholangiocellular proliferative changes detected in this experimental group were expressed as oval cell hyperplasia (28%), bile duct hyperplasia (32%) and cholangiofibrosis (12%).
In the current research work, the therapeutic potential of AgNPs was further evaluated by biochemical assessing of serum liver marker enzymes as well as hepatic tissue supernatant levels of total proteins and oxidative stress indices in comparison with those of the carcinogen control group. The serum levels of ALT showed highly significant decrease started from subgroup 3 till subgroup 5 when the difference was very high significant in comparison to the carcinogen control group. The levels of AST in serum of AgNPs-treated rats revealed very high significant down-regulation starting from subgroup 3 till the end of the experiment. The total protein content in the hepatic tissue supernatant of rats of this group displayed significant increment starting from subgroup 3 till the end of study. The hepatic tissue supernatant levels of lipid peroxidation showed highly significant decrease starting from subgroup 2 and slowly decreased till the end of study when the difference was very high significant. The total antioxidant capacity displayed gradual increase through the time course of the treatment, while significant up-regulation in its level was detected in subgroup 5.