Modulatory mechanisms of copperII-albumin complex toward N-nitrosodiethylamine-induced neurotoxicity in mice via regulating oxidative damage, inflammatory, and apoptotic signaling pathways.

N-nitrosodiethylamine (ND) is an extremely toxic unavoidable environmental contaminant. CopperII-albumin (CuAB) complex, a newly developed Cu complex, showed antioxidant and anti-inflammatory potential. Hereby, we explored the plausible neuroprotective role of CuAB complex toward ND-evoked neurotoxicity in mice. Twenty-four male mice were sorted into 4 groups (6 mice each). Control group, mice were administered oral distilled water; and CuAB group, mice received CuAB complex at a dose of 817 µg/kg orally, three times weekly. In ND group, ND was given intraperitoneally (50 mg/kg body weight, once weekly for 6 w). CuAB+ND group, mice were administered a combination of CuAB and ND. The brain was quickly extracted upon completion of the experimental protocol for the evaluation of the oxidative/antioxidative markers, inflammatory cytokines, and histopathological examination. Oxidative stress was induced after ND exposure indicated by a reduction in GSH and SOD1 level, with increased MDA level. In addition, decreased expression of SOD1 proteins, Nrf2, and 5-HT mRNA expression levels were noticed. An apoptotic cascade has also been elicited, evidenced by overexpression of Cyt c, Cl. Casp 3. In addition, increased regulation of proinflammatory genes (TNF-α, IL-6, iNOS, Casp1, and NF-κB (p65/p50); besides, increment of protein expression of P-IKBα and reduced expression of IKBα. Pretreatment with CuAB complex significantly ameliorated ND neuronal damage. Our results recommend CuAB complex supplementation because it exerts neuroprotective effects against ND-induced toxicity.

Sex different effect of antibiotic and probiotic treatment on intestinal microbiota composition in chemically induced liver injury rats.

Differences in the gut microbiota and metabolic processes between males and females may explain differences in the risk of liver injury; however, the sex-specific effects of antibiotics and probiotics on these relationships are not clear. We evaluated differences in the gut microbiota and the risk of liver injury between male and female rats after the oral administration of antibiotics or probiotics followed by a period of diethylnitrosamine treatment to chemically induce liver injuryusing high-throughput sequencing of fecal microbiota combined with histological analyses of liver and colon tissues. Our results suggest that the ratio of gram-positive to gram-negative bacteria in kanamycin-treated rats was significantly higher than that of other groups, and this difference persisted for the duration of the experiment. Antibiotics significantly changed the composition of the gut microbiota of experimental rats. Clindamycin caused more diethylnitrosamine-induced damage to livers of male rats. Probiotics did not influencethe gut microbiota; however, they hadprotective effects against liver injury induced by diethylnitrosamine, especially in female rats. These results strengthen our understanding of sex differences in the indirect effects of antibiotics or probiotics on metabolism and liver injury in hosts via the gut microbiota.

Role of TLR4 signaling pathway in the mitigation of damaged lung by low-dose gamma irradiation.

Organisms frequently suffer negative effects from large doses of ionizing radiation. However, radiation is not as hazardous at lower doses as was once believed. The current study aims to evaluate the possible radio-adaptive effect induced by low-dose radiation (LDR) in modulating high-dose radiation (HDR) and N-nitrosodiethylamine (NDEA)-induced lung injury in male albino rats. Sixty-four male rats were randomly divided into four groups: Group 1 (control): normal rats; Group 2 (D): rats given NDEA in drinking water; Group 3 (DR): rats administered with NDEA then exposed to fractionated HDR; and Group 4 (DRL): rats administered with NDEA then exposed to LDR + HDR. In the next stage, malondialdehyde (MDA), glutathione reduced (GSH), catalase (CAT), and superoxide dismutase (SOD) levels in the lung tissues were measured. Furthermore, the enzyme-linked immunoassay analysis technique was performed to assess the Toll-like receptor 4 (TLR4), interleukin-1 receptor-associated kinase 4 (IRAK4), and mitogen-activated protein kinases (MAPK) expression levels. Histopathological and DNA fragmentation analyses in lung tissue, in addition to hematological and apoptosis analyses of the blood samples, were also conducted. Results demonstrated a significant increase in antioxidant defense and a reduction in MDA levels were observed in LDR-treated animals compared to the D and DR groups. Additionally, exposure to LDR decreased TLR4, IRAK4, and MAPK levels, decreased apoptosis, and restored all the alterations in the histopathological, hematological parameters, and DNA fragmentation, indicating its protective effects on the lung when compared with untreated rats. Taken together, LDR shows protective action against the negative effects of subsequent HDR and NDEA. This impact may be attributable to the adaptive response induced by LDR, which decreases DNA damage in lung tissue and activates the antioxidative, antiapoptotic, and anti-inflammatory systems in the affected animals, enabling them to withstand the following HDR exposure.

Potential of siRNA-Bearing Subtilosomes in the Treatment of Diethylnitrosamine-Induced Hepatocellular Carcinoma.

Therapeutics, based on small interfering RNA (siRNA), have demonstrated tremendous potential for treating cancer. However, issues such as non-specific targeting, premature degradation, and the intrinsic toxicity of the siRNA, have to be solved before they are ready for use in translational medicines. To address these challenges, nanotechnology-based tools might help to shield siRNA and ensure its specific delivery to the target site. Besides playing a crucial role in prostaglandin synthesis, the cyclo-oxygenase-2 (COX-2) enzyme has been reported to mediate carcinogenesis in various types of cancer, including hepatocellular carcinoma (HCC). We encapsulated COX-2-specific siRNA in Bacillus subtilis membrane lipid-based liposomes (subtilosomes) and evaluated their potential in the treatment of diethylnitrosamine (DEN)-induced hepatocellular carcinoma. Our findings suggested that the subtilosome-based formulation was stable, releasing COX-2 siRNA in a sustained manner, and has the potential to abruptly release encapsulated material at acidic pH. The fusogenic property of subtilosomes was revealed by FRET, fluorescence dequenching, content-mixing assay, etc. The subtilosome-based siRNA formulation was successful in inhibiting TNF-α expression in the experimental animals. The apoptosis study indicated that the subtilosomized siRNA inhibits DEN-induced carcinogenesis more effectively than free siRNA. The as-developed formulation also suppressed COX-2 expression, which in turn up-regulated the expression of wild-type p53 and Bax on one hand and down-regulated Bcl-2 expression on the other. The survival data established the increased efficacy of subtilosome-encapsulated COX-2 siRNA against hepatocellular carcinoma.

Examination of the effect of curcumin in experimental liver damage created by diethylnitrosamine in Swiss albino mice to superoxide dismutase and catalase activities and glutathione, malondialdehyde, and advanced oxidation protein products levels.

In this study, the effects of curcumin, glutathione (GSH), malondialdehyde (MDA) levels, advanced protein oxidation products (AOPP), superoxide dismutase (SOD), and catalase (CAT) activities in experimental liver damage with diethylnitrosamine (DEN) in Swiss albino mice were investigated. The subjects (n = 9) used in the study were divided into 5 groups as tumor control 1, tumor control 2, curcumin protective, curcumin treatment and healthy control groups Curcumin oral gavage (in 150 mg/kg of ethylalcohol) was given to the protecting group for 19 days, 5 days before the administration of DEN, and 24 h after the administration of DEN. Hundred microliters of ethylalcohol oral gavage was given to the healthy group for 19 days. While MDA levels decreased significantly in the curcumin preservative group (p < 0.05), (p = 0.002), the decrease was not significant in the treatment groups (p > 0.05), (p = 0.128). AOPP levels decreased significantly in the curcumin protective group (p < 0.05), (p = 0.009) but the decrease in the treatment group was not found significant (p > 0.05), (p = 0.073). SOD activities increased significantly in both groups. It was found as (p < 0.05), (p = 0.001) and (p < 0.05), (p = 0.002), respectively. GSH levels decreased but these reductions were not found statistically significant. CAT activities increased significantly in both groups. It was determined as (p < 0.05), (p = 0.001) for both groups.

Assessment of SENP3-interacting proteins in hepatocytes treated with diethylnitrosamine by BioID assay.

SUMOylation of proteins regulates cell behaviors and is reversibly removed by small ubiquitin-like modifier (SUMO)-specific proteases (SENPs). The SENP family member SENP3 is involved in SUMO2/3 deconjugation and has been reported to sense cell stress and accumulate in several human cancer cells and macrophages. We previously reported that Senp3-knockout heterozygous mice showed smaller liver, but the pertinent mechanisms of SENP3 and SUMOylated substrates remain unclear. Thus, in this study, we investigated the interacting proteins with SENP3 and the alteration in hepatocytes treated with the xenobiotic diethylnitrosamine (DEN), which is specifically transformed in the liver and induces DNA double-strand breaks. Our data revealed that a certain amount of SENP3 was present in normal, untreated hepatocytes; however, DEN treatment promoted rapid SENP3 accumulation. SENP3 was mainly localized in the nuclei, and its level was significantly increased in the cytoplasm after 2 h of DEN treatment. The application of the recent proximity-dependent biotinylation (BioID) method led to the identification of 310 SENP3-interacting proteins that were involved in not only gene transcription but also RNA splicing, protein folding, and metabolism. Furthermore, after DEN exposure for a short duration, ribosomal proteins as well as proteins associated with mitochondrial ATP synthesis, membrane transport, and bile acid synthesis, rather than DNA repair proteins, were identified. This study provides insights into the diverse regulatory roles of SENP3, and the BioID method seems to be efficient for identifying physiologically relevant insoluble proteins.

Dietary Intake of 17α-Ethinylestradiol Promotes HCC Progression in Humanized Male Mice Expressing Sex Hormone-Binding Globulin.

Hepatocellular carcinoma (HCC) is a male-oriented malignancy; its progression is affected by sex hormones. 17α-ethinylestradiol (EE2) is a synthetic estrogen widely used as an oral contraceptive; however, it is unknown whether EE2 regulates sex hormone action in HCC. We investigated whether EE2 influences HCC risk in male androgenic environments, using mice expressing human sex hormone-binding globulin (SHBG). Two-week-old male mice were injected with diethyl-nitrosamine (DEN, 25 mg/kg) and fed an EE2 diet for 10 weeks from 30 weeks of age. Development and characteristics of liver cancer were evaluated in 40-week-old mice via molecular and histological analyses. Although EE2 did not increase HCC progression in wild-type mice, SHBG mice exhibited remarkably higher HCC risk when fed EE2. The livers of EE2-treated SHBG mice exhibited substantially increased pro-inflammatory necrosis with high plasma levels of ALT and HMGB1, and intrahepatic injury and fibers. Additionally, increased androgen response and androgen-mediated proliferation in the livers of EE2-treated SHBG mice and EE2-exposed hepatocytes under SHBG conditions were observed. As a competitor of SHBG-androgen binding, EE2 could bind with SHBG and increase the bioavailability of androgen. Our results revealed that EE2 is a novel risk factor in androgen-dominant men, predisposing them to HCC risk.

Chemopreventive Effect of Cratoxylum formosum (Jack) ssp. pruniflorum on Initial Stage Hepatocarcinogenesis in Rats.

Cratoxylum formosum ssp. pruniflorum (Kurz) Gogelein (CP) is an indigenous plant found mainly in southeast Asia. Several in vitro studies have confirmed its activity against hepatocellular carcinoma; however, in vivo studies of the effect of CP on liver cancer are needed. This study investigated the effect of CP on early-stage hepatocarcinogenesis in rat liver when using diethylnitrosamine (DEN) as a carcinogen. Immunohistochemistry was used to detect (a) upregulation of glutathione S-transferase placental (GST-P) positive foci, (b) the proliferating cell nuclear antigen PCNA, and (c) apoptotic cells in the liver as indicators of early-stage carcinogenesis. Immunohistochemical parameters were observed in rats given CP orally following DEN injection. Rats given DEN presented overexpression of GST-P positive foci, PCNA, and apoptotic cells, indicating the formation of cancerous tissues, and these effects were diminished by CP treatment. CP thus inhibited hepatocarcinogenic effects in an animal model. These results could help plan further in vivo studies and support the use of CP to prevent processes that promote the pathogenesis of hepatocellular carcinoma in humans.

Gut microbiota shape 'inflamm-ageing' cytokines and account for age-dependent decline in DNA damage repair.

OBJECTIVE: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation. DESIGN: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation. RESULTS: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor. CONCLUSIONS: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery.

Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila.

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

Quinazolinone derivative BNUA-3 ameliorated [NDEA+2-AAF]-induced liver carcinogenesis in SD rats by modulating AhR-CYP1B1-Nrf2-Keap1 pathway.

Cytochrome P450 1B1, considered as one of the novel chemotherapeutic targets involved in cancer prevention and therapy is also associated with the conversion of procarcinogens into their active metabolites. The aryl hydrocarbon receptor (AhR) is responsible for mediating different biological responses to a wide variety of environmental pollutants and also causes transcriptional activation of cytochrome P450 enzymes including CYP1B1 and thus plays a pivotal role for initiating cancer and its progression. On the other hand, active carcinogenic metabolites and reactive oxygen species-mediated stress alter different molecular signalling pathways and gene expressions. Quinazoline derivatives are recognized for their diversified biological activities including anticancer properties. The current study was designed for evaluation of chemotherapeutic efficacy of a synthetic quinazolinone derivative BNUA-3 against hepatocellular cancer in Sprague-Dawley (SD) rats. A detailed in vivo analysis was performed by administrating BNUA-3 (15, 30 mg/kg b.w. for 28 days, i.p.) in N-Nitrosodiethylamine + 2-Acetylaminofluorene induced partially hepatectomized liver cancer in SD rats. This was followed by morphological evaluations, biochemical estimations and analysis of different mRNA and protein expressions. The results demonstrated the potency of BNUA-3 in efficient restoration of the altered morphology of liver, its protective effect against lipid peroxidation, enzymic and non-enzymic antioxidants levels in liver tissue which was disrupted after cancer induction. The study also demonstrated downregulation of AhR, CYP1B1 and Keap1 expressions with subsequent augmentation of protective Nrf2, HO-1, NQO1 and GSTA1 expressions thus, revealing the chemotherapeutic potency of BNUA-3 in inhibiting liver carcinogenesis through AhR/CYP1B1/Nrf2/Keap1 pathway.

A Modified Protocol of Diethylnitrosamine Administration in Mice to Model Hepatocellular Carcinoma.

We aimed to create an animal model for hepatocellular carcinoma (HCC) with a short time, a high survival rate, as well as a high incidence of HCC in both males and females than previously reported. The Diethylnitrosamine (DEN) model has an age-related effect. A single dose of DEN treatment is not enough in young mice up to 50 weeks. The same pattern is shown in an adult with multiple-dose trials whether or not there is some promotion agent. In this study, two-week old C57BL6 mice were given a total of eight doses of DEN, initially 20mg/kg body weight, and then 30mg/kg in the third week, followed by 50mg/kg for the last six weeks. The first group is DEN treatment only and the other two groups received thioacetamide (TAA) treatment for four or eight weeks after one week of rest from the last DEN treatment. An autopsy was performed after 24 weeks of the initial dose of DEN in each group. The cellular arrangement of HCC in the entire group was well-differentiated carcinoma and tumor presence with no significant impact on the survival of mice. Increased levels of the biochemical markers in serum, loss of tissue architecture, hepatocyte death, and proliferation were highly activated in all tumor-induced groups. This finding demonstrates an improved strategy to generate an animal model with a high occurrence of tumors combined with cirrhosis in a short time regardless of sex for researchers who want to investigate liver cancer-related.

[High-mobility group protein 1 promotes diethylnitrosamine-induced liver cancer formation in mice by activating mitochondrial biogenesis].

Objective: To study the role of high-mobility group protein 1 (HMGB1) in the promotion of diethylnitrosamine-induced liver cancer formation in C57BL/6 mice and its mechanism. Methods: HMGB1(loxp/loxp)/Alb-Cre(+/-) were used as a liver-specific knockout (KO) of HMGB1 gene in mice. HMGB1(loxp/loxp)/Alb-Cre(-/-), HMGB1(loxp/WT)/Alb-Cre(+/-) and HMGB1(loxp/WT)/Alb-Cre(-/-) born in the same litter were wild-type mice. Six 12-day-old male WT and KO mice were separated and given a single intraperitoneal injection of diethylnitrosamine (25 mg/kg). Six months later, HE staining was used to evaluate the histopathological changes and then the incidence of liver cancer in each mice group was calculated. Serum samples were taken from each mice group to determine alanine aminotransferase levels. Immunohistochemical staining was used to detect the expression and intracellular localizations of HMGB1 protein status in tumor tissue of the two groups of mice. Western blot was used to detect the expressional condition of mitochondrial biogenesis in tumor tissue of the two groups of mice. RT-PCR was used to detect mitochondrial DNA copy number of tumor tissue and normal liver tissue in the two groups of mice. Intra and inter group data comparison was compared using t-tests and one one-way analysis of variance. Results: Compared with WT mice, the liver/body weight ratio of KO mice was decreased significantly (t = 2.634, P = 0.0225). Serum alanine aminotransferase levels in both groups of mice were increased, and the difference was not statistically significant (t = 0.4062, P = 0.6932). There were many visible gray-white nodules of different sizes on the liver surface of WT mice, and the histological type was hepatocellular carcinoma. There was no statistically significant difference in the incidence of liver cancer among different genotypes of WT mice (P > 0.05). The incidence rate of liver cancer in KO mice was significantly reduced (t = 8.521, P < 0.001). Compared with WT mice, the expression levels of HMGB1 and mitochondrial biogenesis (PGC-1α and NRF1) was significantly reduced (t = 6.238, 4.852, P = 0.0335, 0.041) in tumor tissue of KO mice. Mitochondrial DNA copy number was decreased significantly (t = 9.211, P < 0.01). Mitochondrial DNA copy number in tumor tissue of WT mice was significantly higher than that in normal liver tissue (t = 8.305, P = 0.0142). Conclusion: HMGB1 promotes the formation of diethylnitrosamine-induced liver cancer by inducing mitochondrial biogenesis.

An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, mainly because of its poor prognosis. A valid mechanism-based prognostic biomarker is urgently needed. γ-hydroxy-1,N2 -propanodeoxyguanosine (γ-OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ-OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine-injected mice, both prone to HCC development. γ-OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ-OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ-OHPdG and survival or recurrence-free survival. γ-OHPdG levels in liver DNA showed an age-dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ-OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine-injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ-OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence-free survival (P = 0.007). CONCLUSION: These results support γ-OHPdG as a mechanism-based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159-170).

Dual role for inositol-requiring enzyme 1α in promoting the development of hepatocellular carcinoma during diet-induced obesity in mice.

Obesity is associated with both endoplasmic reticulum (ER) stress and chronic metabolic inflammation. ER stress activates the unfolded protein response (UPR) and has been implicated in a variety of cancers, including hepatocellular carcinoma (HCC). It is unclear whether individual UPR pathways are mechanistically linked to HCC development, however. Here we report a dual role for inositol-requiring enzyme 1α (IRE1α), the ER-localized UPR signal transducer, in obesity-promoted HCC development. We found that genetic ablation of IRE1α in hepatocytes not only markedly reduced the occurrence of diethylnitrosamine (DEN)-induced HCC in liver-specific IRE1α knockout (LKO) mice when fed a normal chow (NC) diet, but also protected against the acceleration of HCC progression during high-fat diet (HFD) feeding. Irrespective of their adiposity states, LKO mice showed decreased hepatocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation, even in the face of increased hepatic apoptosis. Furthermore, IRE1α abrogation blunted obesity-associated activation of hepatic inhibitor of nuclear factor kappa B kinase subunit beta (IKKß)-nuclear factor kappa B (NF-κB) pathway, leading to reduced production of the tumor-promoting inflammatory cytokines tumor necrosis factor (TNF) and interleukin 6 (IL-6). Importantly, higher IRE1α expression along with elevated STAT3 phosphorylation was also observed in the tumor tissues from human HCC patients, correlating with their poorer survival rate. CONCLUSION: IRE1α acts in a feed-forward loop during obesity-induced metabolic inflammation to promote HCC development through STAT3-mediated hepatocyte proliferation. (Hepatology 2018).

Hepatoprotective effect of boldine against diethylnitrosamine-induced hepatocarcinogenesis in wistar rats.

Discovering the utmost effective and targeted chemotherapy for hepatocellular carcinoma is still a significant challenge. In the present study, diethylnitrosamine was used as a liver carcinogen and boldine a compound of boldo. We anticipated the hypothesis that boldine endow antiproliferative and promote apoptosis on hepatocarcinoma rats. We analyzed that boldine alters the tumor biomarkers and liver markers enzyme levels. Also, we determined boldine modulate the enzymatic and nonenzymatic antioxidant activities, as well as messenger RNA and protein expressions of Bcl2, Bax, and cleaved caspase 3 by reverse transcription polymerase chain reaction and Western blot analysis, respectively. It was also manifested by histopathology studies in liver tissues of HCC rats. Our finding suggested that boldine has antioxidant activity, and moreover, also contributes apoptotic nature by upregulating the protein expression of Bax, and cleaved caspase 3. Our data accomplishes that boldine a candidate drug has dynamic therapeutic activity and suitable for the treatment of HCC.

Phytofabricated silver nanoparticles of Phyllanthus emblica attenuated diethylnitrosamine-induced hepatic cancer via knock-down oxidative stress and inflammation.

Oxidative stress and inflammation play a pivotal role in the expansion and progression of hepatic cancer. Nanoparticle-based drug delivery can quickly enhance the restorative capability of hepatic cancer. Silver nanoparticles synthesized from plant source are of great importance due to their small size, economic, non-hazardous and different biomedical applications. In the current study, we have evaluated the impacts of oxidative stress and proinflammatory markers of biosynthesized silver nanoparticles of Phyllanthus emblica (PE) leaves against diethylnitrosamine-induced hepatocellular carcinoma (HCC) in wistar rats till 16 weeks with its underlying mechanism. The physico-chemical properties of biosynthesized silver nanoparticles were determined by ultra-visible spectroscopy, Fourier transform infrared spectroscopy, field emission scanning electron microscope, energy dispersive X-ray analysis, transmission electron microscopy and X-ray diffraction studies. Biofabricated silver nanoparticles (PEAgNPs) significantly enhanced the process of recovery from hepatic cancer in animal models, which was ascertained by increased body weight, reduced hepatic knobs on the outer surface of liver, downregulated serum biochemical parameters (ALT: 134.66 ± 2.60; AST: 120.33 ± 3.18; ALP: 153.33 ± 4.25; AFP: 167.33 ± 3.38), decreased hepatic lipid peroxidation (20.22 ± 1.74), increased membrane-bound enzymes (Na+/K+ATPase: 4.18 ± 0.20; Ca2+ATPase: 6.24 ± 0.12), increased antioxidants parameters (CAT: 64.89 ± 4.13; SOD: 6.01 ± 0.11; GPx: 8.55 ± 0.05), alteration in the level of proinflammatory cytokines (TNF-α: 90.15 ± 5.77; NF-κB: 173.29 ± 7.26; IL-6: 178.11 ± 3.16; IL-1ß: 48.26 ± 1.89) and histopathological studies. Our outcomes implicate successfully biofabrication of silver nanoparticles and exhibited a chemoprotective potential in the prevention and intervention of hepatocellular carcinoma.

Genetic inactivation of Nrf2 prevents clonal expansion of initiated cells in a nutritional model of rat hepatocarcinogenesis.

BACKGROUND & AIMS: Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in cancer development. Herein, we examined whether Nrf2 (Nfe2l2) activation occurs at early steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the onset of hepatocellular carcinoma (HCC). METHODS: We used wild-type (WT) and Nrf2 knockout (Nrf2KO) rats treated with a single injection of diethylnitrosamine (DENA) followed by choline-devoid methionine-deficient (CMD) diet. This experimental model causes massive fatty liver and steatohepatitis with fibrosis and enables identification of early stages of hepatocarcinogenesis. RESULTS: We found that Nrf2 activation takes place in early preneoplastic lesions identified by the marker glutathione S-transferase placental form (GSTP). Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to increased liver injury and chronic compensatory hepatocyte regeneration when rats were fed a CMD diet. However, in spite of such a permissive environment, the livers of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. CONCLUSIONS: These results demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of initiated cells, indicating that Nrf2 is critical in the onset of HCC. LAY SUMMARY: Dysregulation of the Keap1-Nrf2 molecular pathway has been observed in human tumors. In a nutritional model of hepatocarcinogenesis, the protein Nrf2 is frequently mutated/activated at early steps of the tumorigenic process. Herein, we show that Nrf2 is mandatory for the development of preneoplastic lesions. These results suggest that Nrf2 has a critical role in the onset of hepatocellular carcinoma.

Higher CYP2E1 Activity Correlates with Hepatocarcinogenesis Induced by Diethylnitrosamine.

Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and the third most frequent cause of cancer death worldwide. Diethylnitrosamine (DEN) is one of the recognized risk factors for hepatocarcinogenesis likely due to CYP2E1-mediated metabolic activation. However, CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients has not been determined; the role of CYP2E1 activity, in particular CYP2E1 constitutive activity and CYP2E1 inhibited activity, with respect to the hepatocarcinogenesis induced by DEN is not yet clear. Herein, we determined CYP2E1-mediated DEN metabolic activity in non-neoplastic liver tissue from HCC patients and found that CYP2E1-mediated DEN metabolic activity was significantly elevated with a 43.3% positive rate, and clinicopathologic parameters did not affect the activity. Then, using a Sprague-Dawley rat liver tumor model induced by DEN, the relationship between CYP2E1 constitutive/inhibited activity and hepatocarcinogenesis was explored. The results showed that the CYP2E1 constitutive activity was strongly correlated with tumor incidence and severity of liver tumorigenesis (nodule numbers and size), whereas inhibition of CYP2E1 activity decreased hepatocyte proliferation, liver injury, and liver carcinogenesis in the presence of DEN. In conclusion, the higher CYP2E1 activity would lead to an increased incidence of HCC as a result of CYP2E1-mediated DEN activation. Therefore, higher CYP2E1 activity might be a risk factor for HCC induced by DEN.

Combined magnetic resonance elastography and collagen molecular magnetic resonance imaging accurately stage liver fibrosis in a rat model.

Hepatic fibrosis is associated with an overproduction of matrix proteins and a pathological increase of liver stiffness. Noninvasive magnetic resonance (MR) quantification of matrix can be assessed with a collagen-binding molecular MR probe and stiffness by MR elastography, complementary techniques. This study used both imaging techniques to more accurately stage hepatic fibrosis in a rat model. Thirty rats with varying levels of diethylnitrosamine-induced liver fibrosis were imaged before and 45 minutes after injection of collagen-specific probe EP-3533. MR elastography was performed in the same imaging session. Changes in liver relaxation rate post-EP-3533 and liver stiffness were compared to the collagen proportional area determined by histology and to Ishak scoring using receiver operating characteristic analysis. Collagen imaging was most sensitive to early fibrosis, while elastography was more sensitive to advanced fibrosis. This complementary feature enabled the formulation of a composite model using multivariate analysis of variance. This model incorporated the discriminating advantages of both MR techniques, resulting in more accurate staging throughout fibrotic progression. CONCLUSION: Collagen molecular MR imaging is complementary to MR elastography, and combining the two techniques in a single exam leads to increased diagnostic accuracy for all stages of fibrosis. (Hepatology 2017;65:1015-1025).