The anticarcinogenic effect of eugenol on lung cancer induced by diethylnitrosamine/2-acetylaminofluorene in Wistar rats: insight on the mechanisms of action.

This study was designed to assess the ameliorative effects of eugenol and to propose the possible mechanisms of action of eugenol in diethylnitrosamine (DENA)/acetylaminofluorene (AAF)-caused lung cancer in Wistar rats. To induce lung cancer, DENA at a dose of 150 mg/kg body weight (b.wt) for 2 weeks were intraperitoneally injected once each week and AAF was administered orally at a dose of 20 mg/kg b.wt. four times each week for the next 3 weeks. DENA/AAF-administered rats were orally supplemented with eugenol at a dose of 20 mg/kg b.wt administered once a day until 17 weeks starting from the 1st week of DENA administration. Lung histological lesions, including sheets of tumor cells, micropapillary adenocarcinoma, and apoptotic cells, resulting from the DENA/AAF dosage, were ameliorated by eugenol treatment. However, a significant drop in the levels of LPO in the lungs and a remarkable rise in GSH content and GPx and SOD activities were observed in DENA/AAF-administered rats treated with eugenol compared with those in DENA/AAF-administered controls. Moreover, in DENA/AAF-administered rats, eugenol supplementation significantly reduced TNF-α and IL-1ß levels and mRNA expression levels of NF-κB, NF-κB p65, and MCP-1 but significantly elevated the level of Nrf2. Furthermore, the DENA/AAF-administered rats treated with eugenol exhibited a significant downregulation of Bcl-2 expression levels in addition to a significant upregulation in P53 and Bax expression levels. Otherwise, the administration of DENA/AAF elevated the protein expression level of Ki-67, and this elevation was reversed by eugenol treatment. In conclusion, eugenol has effective antioxidant, anti-inflammatory, proapoptotic, and antiproliferative properties against lung cancer.

Effect of 2-acetylaminofluorene and its genotoxic metabolites on DNA adduct formation and DNA damage in 3D reconstructed human skin tissue models.

In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.

Development of Asialoglycoprotein Receptor-Targeted Nanoparticles for Selective Delivery of Gemcitabine to Hepatocellular Carcinoma.

Selective targeting of anticancer drugs to the tumor site is beneficial in the pharmacotherapy of hepatocellular carcinoma (HCC). This study evaluated the prospective of galactosylated chitosan nanoparticles as a liver-specific carrier to improve the therapeutic efficacy of gemcitabine in HCC by targeting asialoglycoprotein receptors expressed on hepatocytes. Nanoparticles were formulated (G1-G5) by an ionic gelation method and evaluated for various physicochemical characteristics. Targeting efficacy of formulation G4 was evaluated in rats. Physicochemical characteristics exhibited by nanoparticles were optimal for administering and targeting gemcitabine effectively to the liver. The biphasic release behavior observed with G4 can provide higher drug concentration and extend the pharmacotherapy in the liver target site. Rapid plasma clearance of gemcitabine (70% in 30 min) from G4 was noticed in rats with HCC as compared to pure drug (p < 0.05). Higher uptake of gemcitabine predominantly by HCC (64% of administered dose; p < 0.0001) demonstrated excellent liver targeting by G4, while mitigating systemic toxicity. Morphological, biochemical, and histopathological examination as well as blood levels of the tumor marker, alpha-fetoprotein, in rats confirmed the curative effect of G4. In conclusion, this study demonstrated site-specific delivery and enhanced in vivo anti-HCC efficacy of gemcitabine by G4, which could function as promising carrier in hepatoma.

Cells responsible for liver mass regeneration in rats with 2-acetylaminofluorene/partial hepatectomy injury.

BACKGROUND: Whether hepatic progenitor cells (HPCs)/oval cells regenerate liver mass upon chronic liver injury is controversial in mice and has not been conclusively proven in humans and rats. In this study, we examined which cell type-hepatocytes or oval cells-mediates liver regeneration in the classic rat 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) injury where AAF reversibly blocks hepatocyte proliferation, thereby inducing oval cell expansion after the regenerative stimulus of PH. METHODS: We employed lineage tracing of dipeptidyl peptidase IV (DPPIV, a hepatocyte canalicular enzyme)-positive hepatocytes by subjecting rats with DPPIV-chimeric livers to AAF/PH, AAF/PH/AAF (continuous AAF after AAF/PH to nonselectively inhibit regenerating hepatocytes), or AAF/PH/retrorsine injury (2-dose retrorsine after AAF/PH to specifically and irreversibly block existing hepatocytes); through these methods, we determined hepatocyte contribution to liver regeneration. To determine the oval cell contribution to hepatocyte regeneration, we performed DPPIV(+) oval cell transplantation combined with AAF/PH injury or AAF/PH/retrorsine injury in DPPIV-deficient rats to track the fate of DPPIV(+) oval cells. RESULTS: DPPIV-chimeric livers demonstrated typical oval cell activation upon AAF/PH injury. After cessation of AAF, DPPIV(+) hepatocytes underwent extensive proliferation to regenerate the liver mass, whereas oval cells underwent hepatocyte differentiation. Upon AAF/PH/AAF injury where hepatocyte proliferation was inhibited by continuous AAF treatment following AAF/PH, oval cells extensively expanded in an undifferentiated state but did not produce hepatocytes. By substituting retrorsine for AAF administration following AAF/PH (AAF/PH/retrorsine), oval cells regenerated large-scale hepatocytes. CONCLUSIONS: Hepatocyte self-replication provides the majority of hepatocyte regeneration, with supplementary contribution from oval cells in rats under AAF/PH injury. Oval cells expand and maintain in an undifferentiated state upon continuously nonselective liver injury, whereas they can significantly regenerate hepatocytes in a noncompetitive environment.

The Protective Role of Etoricoxib Against Diethylnitrosamine/2-acetylaminofluorene- Induced Hepatocarcinogenesis in Wistar Rats: The Impact of NF-κB/COX-2/PGE2 Signaling.

BACKGROUND: Liver cancer ranks as the 7th and 5th leading cause of cancer morbidity worldwide in men and women, respectively. Hepatocellular Carcinoma (HCC) is the most common type of liver cancer and is associated with an increasing global burden of Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH). OBJECTIVE: The present study aimed to investigate the possible chemopreventive effect of etoricoxib on diethylnitrosamine (DENA) and 2-acetylaminofluorene (2AAF)-induced HCC in male Wistar rats. METHODS: HCC was induced by DENA (150 mg/kg/week; i.p) for 2 weeks, then 2AAF (20 mg/kg; p.o) every other day for three successive weeks. Etoricoxib (0.6 mg/kg, p.o.) was given to DENA/ 2AAF-administered rats for 20 weeks. RESULTS: Etoricoxib significantly suppressed alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19.9) as liver tumor biomarkers. It also decreased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin levels while increased serum albumin levels. Besides, it alleviated DENA/2AAF-induced histopathological abrasions and inflammatory cell infiltration. Furthermore, etoricoxib showed a potent antioxidant effect, supported by a significant lipid peroxide reduction and elevation in superoxide dismutase activity and GSH content. In addition, Etoricoxib significantly down-regulated the protein expression of interleukin 1 beta (IL-1ß), tumor necrosis factor α (TNFα), nuclear Factor-kappa B (NF-κB), phosphorylated nuclear Factor-kappa B (p-NF-κB), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). CONCLUSION: In conclusion, the current results proved that etoricoxib possesses an anticarcinogenic effect via its antioxidant, anti-inflammatory, and modulation of NF-κB/COX-2/PGE2 signaling.

Characteristics of hepatic nuclear-transcription factor-kappa B expression and quantitative analysis in rat hepatocarcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. We analyzed the expression of nuclear-transcription factor-kappa B (NF-kappaB) during hepatocarcinogenesis in order to evaluate its dynamic expression and its clinical value in the development and diagnosis of HCC. METHODS: Hepatoma models were induced by oral administration of 2-acetamidoflurene (2-FAA) to male Sprague-Dawley rats. Morphological changes were observed after hematoxylin and eosin staining. The cellular distribution of NF-kappaB expression during different stages of cancer development was investigated by immunohistochemistry, and the level of NF-kappaB expression in liver tissues was quantitatively analyzed by ELISA. The gene fragments of hepatic NF-kappaB were amplified by nested-polymerase chain reaction assay. RESULTS: Hepatocytes showed vacuole-like degeneration during the early stages, then had a hyperplastic nodal appearance during the middle stages, and finally progressed to tubercles of cancerous nests with high differentiation. The NF-kappaB-positive material was buff-colored, fine particles localized in the nucleus, and the incidence of NF-kappaB-positive cells was 81.8% in degeneration, 83.3% in precancerous lesions, and 100% in cancerous tissues. All of these values were higher than those in controls (P<0.01). Hepatic NF-kappaB expression and hepatic NF-kappaB-mRNA were also higher during the course of HCC development (P<0.01). CONCLUSION: The NF-kappaB signal transduction pathway is activated during the early stages of HCC development, and its abnormal expression may be associated with the occurrence of HCC.

A general dose-response relationship for chronic chemical and other health stressors and mixtures based on an emergent illness severity model.

Current efforts to assess human health response to chemicals based on high-throughput in vitro assay data on intra-cellular changes have been hindered for some illnesses by lack of information on higher-level extracellular, inter-organ, and organism-level interactions. However, a dose-response function (DRF), informed by various levels of information including apical health response, can represent a template for convergent top-down, bottom-up analysis. In this paper, a general DRF for chronic chemical and other health stressors and mixtures is derived based on a general first-order model previously derived and demonstrated for illness progression. The derivation accounts for essential autocorrelation among initiating event magnitudes along a toxicological mode of action, typical of complex processes in general, and reveals the inverse relationship between the minimum illness-inducing dose, and the illness severity per unit dose (both variable across a population). The resulting emergent DRF is theoretically scale-inclusive and amenable to low-dose extrapolation. The two-parameter single-toxicant version can be monotonic or sigmoidal, and is demonstrated preferable to traditional models (multistage, lognormal, generalized linear) for the published cancer and non-cancer datasets analyzed: chloroform (induced liver necrosis in female mice); bromate (induced dysplastic focia in male inbred rats); and 2-acetylaminofluorene (induced liver neoplasms and bladder carcinomas in 20,328 female mice). Common- and dissimilar-mode mixture models are demonstrated versus orthogonal data on toluene/benzene mixtures (mortality in Japanese medaka, Oryzias latipes, following embryonic exposure). Findings support previous empirical demonstration, and also reveal how a chemical with a typical monotonically-increasing DRF can display a J-shaped DRF when a second, antagonistic common-mode chemical is present. Overall, the general DRF derived here based on an autocorrelated first-order model appears to provide both a strong theoretical/biological basis for, as well as an accurate statistical description of, a diverse, albeit small, sample of observed dose-response data. The further generalizability of this conclusion can be tested in future analyses comparing with traditional modeling approaches across a broader range of datasets.

Troxerutin subdues hepatic tumorigenesis via disrupting the MDM2-p53 interaction.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide that lacks proper medical prognosis and treatment. In the present study, the anti-tumoral potential of troxerutin (TX), an ethnomedicine, was examined in relation to its effects on the promoter 2-acetylaminofluorene (2-AAF) in N-nitrosodiethylamine (NDEA) initiated HCC, as compared to its effects on HCC induced by NDEA alone. Liver samples from each experimental group were collected and evaluated for histological, biochemical and cellular characterization. The protein expressions of apoptotic and cell proliferation markers were determined via immunohistochemistry and western blotting. Molecular docking was also performed to delineate the inhibitory mechanism of TX on HCC. The results show that only higher doses of TX showed a significant reduction in the incidence of hepatic nodule formation, and they also counteracted NDEA plus 2-AAF induced alterations in the enzymic status. The frequencies of glutathione-S-transferase and proliferating cell nuclear antigen, markers of S phase progression, were markedly reduced during TX treatment. TX also modulated the imbalance in the MDM2-p53 interaction. The molecular docking results confirmed the interaction of TX with the upstream kinases that regulate apoptosis. This study provides evidence that a copious dose of TX is required to counteract the differential mitoinhibitory effect of 2-AAF in NDEA initiated hepatomas, and TX exhibits an anti-tumoral effect via suppressing oxidative stress, regulating liver function enzymes, inhibiting inflammatory responses and modulating MDM2-p53 interactions, thus inducing apoptosis, and thereby suggesting that TX may provide promising therapeutic effects for the chemoprevention of HCC.

STAT-3 activity in chemically-induced hepatocellular carcinoma.

The signal transducer and activator of transcription (STAT)-3 regulates basic biological processes and it has been reported to be constitutively active in different types of malignant tumours. STAT-3 is active during the regenerative growth of the liver, but there are hardly any data about its presence in liver tumours. We investigated and found a high activity of STAT-3 using an electrophoretic mobility shift assay (EMSA) in chemically-induced rat hepatocellular carcinomas (HCCs). Dexamethasone treatment downregulated both STAT-3 activity and cell proliferation in the tumours. Therefore, the activity of the STAT-3 signal transduction pathway seems to be required for the growth of HCCs and could be a potential new target for therapeutic trials of this tumour type.

The Liverbeads as a tool for the comet assay.

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.

Tryptophan urinary bladder absorption and bladder cancer.

Local vesical absorption of organic compounds may play a role in the pathogenesis of bladder cancer. An associated increased absorption of tryptophan in patients with bladder cancer has been demonstrated.

Hepatocellular ligandin during N-2-fluorenylacetamide carcinogenesis.

Ligandin was decreased by 75% as determined immunologically and by glutathione-S-transferase or steroid isomerase activities in rat hepatocellular carcinomas induced by exposure to N-2-fluorenylacetamide. Minor variable differences in ligandin levels were noted between the putative, premalignant nodules induced by this regimen and normal liver.