Hepatocyte DNA Research Articles

Reactivity of the 2-Methylfuran Phase I Metabolite 3-Acetylacrolein Toward DNA.

2-Methylfuran (2-MF) is a well-known industrial chemical and also formed via thermal treatment of food. One main source of 2-MF in the human diet is coffee. 2-MF is known to form 3-acetylacrolein (AcA, 4-oxopent-2-enal) via cytochrome P 450 metabolism and further reacts with amino acids in vivo. Still the reactivity toward other biomolecules is rather scarce. Therefore, AcA was synthesized, and its reaction with 2'-deoxyadenosine (dA), 2'deoxyguanosine (dG), 2'deoxycytosine (dC), and 2'-deoxythymidine (dT) was tested. For this purpose, adduct formation was performed by acid hydrolysis of 2,5-dihydro-2,5-dimethoxy-2-methylfuran (DHDMMF) as well as pure AcA. The structures of these adducts were confirmed by UPLC-ESI+-MS/MS fragmentation patterns and 1H-/13CNMR spectra. Except for dT, which showed no reactivity, all adducts of AcA were characterized, which enabled the development of sensitive quantification methods via (U)HPLC-ESI±-MS/MS. Pure AcA was synthesized by oxidation of 2-MF using dimethyldioxirane (DMDO), and its behavior in aqueous medium was studied. Incubations of AcA and isolated DNA of primary rat hepatocytes (pRH) showed time- and dose-dependent formation of the identified DNA adducts dA-AcA, dG-AcA, or dC-AcA. In contrast, the DNA adducts dA-AcA, dG-AcA, or dC-AcA were not detected on a cellular level when pRH were incubated with 2-MF or AcA. This indicates an efficient detoxification or reaction with biomolecules in the cell, although the induction of other DNA damage, possibly also by other metabolites, cannot be ruled out in principle.

AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model.

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

Juveniles, young adults, and infants with hepatitis B virus infection: A genomic study.

Integration of hepatitis B virus (HBV) DNA into the human genome is recognized as an oncogenic factor and a barrier to hepatitis B cure. In the study, biopsy liver tissues were collected from adolescents and young adults with acute HBV infection younger than or equal to 35 years of age and from HBV-infected infant patients younger than or equal to 6 months of age. A high-throughput sequencing method was used to detect HBV DNA integration. Totally, 12 adolescents, young adults, and 6 infants were included. Among the 12 patients with acute HBV infection, immunohistochemical staining of intrahepatic hepatitis B surface antigen for all displayed negative results, and no HBV DNA integrants in the hepatocyte DNA were confirmed. All infant patients had elevated levels of alanine aminotransferase and high levels of serum HBV DNA. Numerous gene sites of hepatocyte DNA were integrated by HBV DNA for each infant patient, ranging from 120 to 430 integration sites. The fragile histidine triad gene was the high-frequency integrated site in the intragenic region for infant patients. In conclusion, hepatocyte DNA is integrated by HBV DNA in babies with active hepatitis B but seems seldom affected among adolescents and young adults with acute HBV infection. Infantile hepatitis B should be taken seriously considering abundant HBV DNA integration events.

Biological and histological changes and DNA damage in Oreochromis niloticus exposed to oxytetracycline: a potential amelioratory role of ascorbic acid

Antibiotics are crucial in the control of bacterial infections on fish farms. However, the influence of licensed antibiotics on DNA damage, histological changes, and protein differentiation in some farmed fish species is unidentified. The current study aimed to investigate the effect of therapeutic (80 mg/kg body weight/day) and high doses (160 mg/kg body weight/day) of oxytetracycline (OTC) on some biological indices, protein differentiation, histological changes, and DNA damage in Oreochromis niloticus (n = 360; 40 ± 3.5 g) and the potential positive effects of dietary ascorbic acid (AA) and whether it is capable of reducing the deleterious impacts of OTC and working synergistically to increase fish disease resistance and decrease morbidity and mortality rates. In the current study, fish were fed diets with or without OTC (80 or 160 mg/kg body weight/day) and AA (100 mg/kg diet/day) for 28 days, followed by a bacterial stress test with two different virulent strains for 14 days. Treatments include CTR (control; without additives), AA, OTC80, OTC160, OTC80 + AA, and OTC160 + AA. The results revealed that the AA group had the best antioxidative (significant increase in SOD, CAT, and GPx) and immunomodulatory effects (an increase in IgM, nitroblue tetrazolium, and total leucocytic count). Furthermore, the hematological parameters, immune indices (NBT), and antioxidant enzymes were significantly reduced by OTC160 (p ≤ 0.05). The results also showed that OTC160 notably augmented the activity of liver (ALT, AST, and LDH) and kidney (creatinine and urea) damage markers (p ≤ 0.05). OTC160 revealed considerable hepatic and renal tissue damage and significantly higher hepatocyte DNA damage. Pathological changes and DNA damage were directly correlated to the dosage of OTC exposure, where they were more severe in OTC160. OTC80 showed insignificant changes in most parameters. Compared to CTR, the inclusion of AA in the O. niloticus diet significantly restored the biological indices, histological changes, and DNA damage in OTC-treated groups. The morbidity and mortality percentages after bacterial challenge were significantly lower in the OTC80 + AA group, followed by the OTC160 + AA group than in the CTR. These results corroborate the use of AA as a potent antioxidant and immune stimulant and a potential co-adjuvant to OTC in the diet of O. niloticus to reach optimal resistance to disease.

HPB P26 Ex vivo normothermic perfusion of liver, kidney and spleen to predict human pharmacokinetics and assess drug delivery

Abstract Background Many emerging therapeutics fail to provide patient benefit due to unpredictable drug delivery and clearance following clinical translation. Normothermic machine perfusion (NMP) can preserve physiological processes in human-sized organs ex vivo, which may facilitate preclinical characterisation of pharmacokinetics and drug delivery. The performance of ex vivo human and porcine clearance organs (liver, kidney and spleen) to 1) predict human drug pharmacokinetics and 2) assess the delivery of emerging therapeutics was investigated. A small molecule chemotherapy (irinotecan) with well-established pharmacokinetics was delivered to determine the former. Nanotherapeutics and viral vectors, two classes of emerging therapeutics, were delivered to determine the latter. Methods Human liver (n=4), kidney (n=4) and porcine liver (n=11), spleen (n=6) and kidney (n=5) underwent NMP. Organ physiology was measured. Irinotecan (Medac) was delivered to human and porcine liver and kidney. Plasma, tissue, bile and urinary irinotecan and its metabolites were quantified by high performance liquid chromatography. Pharmacokinetic parameters were compared to patient data. Non-PEGylated (ThermoFisher) and PEGylated (in-house), nanoparticles were delivered to liver, spleen, and kidney. Nanoparticles were quantified in biological samples by fluorescence. A lentiviral vector (Oxford Biomedica) was administered to liver and viral kinetics and genomic integration determined using RT-qPCR. Noncompartmental analysis was used for pharmacokinetics. Results Prolonged physiological preservation (up to 38 hours) of human liver, kidney and porcine liver, kidney and spleen was achieved with NMP. Human and porcine organs support the pharmacokinetic processes of drug distribution, metabolism and urinary and biliary excretion of irinotecan. Moreover, porcine organs successfully predicted human hepatic clearance, renal clearance and total body clearance with an absolute fold error of 1.14, 1.71 and 1.04 when compared to human literature values. Finally, predictable kinetics and biodistribution of PEGylated and non-PEGylated nanoparticles were demonstrated in NMP clearance organs, and NMP liver supported the integration of lentiviral genome into the host hepatocyte DNA. Conclusions Ex vivo NMP organs can be used to predict human drug clearance and assess therapeutic delivery; an exciting prospect for preclinical drug development and for the delivery of hepatocyte-targeted therapies, with the potential to reduce the time, cost and risk in developing life-saving treatments for patients.

The Initial Hepatitis B Virus-Hepatocyte Genomic Integrations and Their Role in Hepatocellular Oncogenesis.

Hepatitis B virus (HBV) remains a dominant cause of hepatocellular carcinoma (HCC). Recently, it was shown that HBV and woodchuck hepatitis virus (WHV) integrate into the hepatocyte genome minutes after invasion. Retrotransposons and transposable sequences were frequent sites of the initial insertions, suggesting a mechanism for spontaneous HBV DNA dispersal throughout the hepatocyte genome. Several somatic genes were also identified as early insertional targets in infected hepatocytes and woodchuck livers. Head-to-tail joints (HTJs) dominated amongst fusions, indicating their creation by non-homologous end-joining (NHEJ). Their formation coincided with the robust oxidative damage of hepatocyte DNA. This was associated with the activation of poly(ADP-ribose) polymerase 1 (PARP1)-mediated dsDNA repair, as reflected by the augmented transcription of PARP1 and XRCC1; the PARP1 binding partner OGG1, a responder to oxidative DNA damage; and increased activity of NAD+, a marker of PARP1 activation, and HO1, an indicator of cell oxidative stress. The engagement of the PARP1-mediated NHEJ repair pathway explains the HTJ format of the initial merges. The findings show that HBV and WHV are immediate inducers of oxidative DNA damage and hijack dsDNA repair to integrate into the hepatocyte genome, and through this mechanism, they may initiate pro-oncogenic processes. Tracking initial integrations may uncover early markers of HCC and help to explain HBV-associated oncogenesis.

Genotoxicity assessment of titanium dioxide nanoparticles using a standard battery of in vivo assays

As one representative of nanometal oxides, titanium dioxide nanoparticles (TiO2-NPs) have been widely used, particularly in the food industry. The genotoxicity of TiO2-NPs has attracted great attention over the years. This study was undertaken to investigate the chromosome and DNA damage effects of TiO2-NPs (0, 50, 150, and 500 mg/kg BW) using rodent models. After a comprehensive characterization, we conducted a standard battery of in vivo genotoxicity tests, including the chromosomal aberration test (CA), micronucleus (MN) test, and the comet test. The results of all these tests were negative. There were no structural or numerical chromosomal abnormalities in mice bone marrow cells, no increase in the frequency of micronucleated polychromatic erythrocytes in mice bone marrow cells, and no elevation in % tail DNA in rat hepatocytes. This indicated that TiO2-NPs did not cause chromosomal damage or have a direct impact on DNA. These findings suggested that TiO2-NPs did not exhibit genotoxicity and provided valuable data for risk assessment purposes.

Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment.

Radiation therapy is an effective cancer treatment although damages to healthy tissues are common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damages in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type specific reference DNA methylation atlases. We found that cell-type specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damages were reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.

Celastrol-loaded biomimetic nanodrug ameliorates APAP-induced liver injury through modulating macrophage polarization.

Drug-induced liver injury (DILI) is a major concern in clinical treatment as well as postmarketing surveillance, showing an urgent requirement for the development of protective medications. Celastrol (Cel), a highly active natural product extracted from the roots of Tripterygium wilfordii, has a potential liver protective activity due to its antioxidant and anti-inflammatory effects. However, the further application of Cel to DILI remains a challenge because of its short half-life, low solubility, and toxic side effects. Herein, we developed a Cel-loaded biomimetic nanodrug based on erythrocyte membrane vesicles (EMV) for protecting the liver from acetaminophen (APAP)-induced liver injury. The Cel-loaded EMV (C-EMV) with lower cytotoxicity had a well-sustained release effect and exhibited excellent ability for liver accumulation under physiological and pathological conditions. By suppressing the inflammatory response of pro-inflammatory macrophage M1 polarization while stimulating anti-inflammatory macrophage M2 polarization, C-EMV could significantly alleviate the primary pathological manifestations related to liver injury, including aberrant elevation of biochemical indicators, histopathological alterations, neutrophil infiltration as well as hepatocyte DNA fragmentation. The macrophage depletion experiment further demonstrated that the protective effect of C-EMV on APAP-induced liver injury appeared to be dependent on hepatic macrophages. Therefore, C-EMV as a biomimetic nanodrug exhibits great potential for attenuating the progress of DILI, providing a new approach to protecting the liver from DILI as well as other liver inflammatory diseases through a targeted nanodelivery system. KEY MESSAGES: EMV biomimetic nanocarrier has good monodispersity and sustained-release property. EMV biomimetic nanocarrier displays excellent liver-targeting capability under physiological and pathological conditions. C-EMV biomimetic nanodrug with lower cytotoxicity regulates macrophage polarization in vitro and in vivo. C-EMV biomimetic nanodrug can significantly alleviate APAP-induced liver injury. The protective effect of C-EMV on APAP-induced liver injury is dependent on hepatic macrophages.

The specific core fucose-binding lectin Pholiota squarrosa lectin (PhoSL) inhibits hepatitis B virus infection in vitro

Glycosylation of proteins and lipids in viruses and their host cells is important for viral infection and is a target for antiviral therapy. Hepatitis B virus (HBV) is a major pathogen that causes acute and chronic hepatitis; it cannot be cured because of the persistence of its covalently closed circular DNA (cccDNA) in hepatocytes. Here we found that Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds core fucose, bound to HBV particles and inhibited HBV infection of a modified human HepG2 cell line, HepG2-hNTCP-C4, that expresses an HBV receptor, sodium taurocholate cotransporting polypeptide. Knockout of fucosyltransferase 8, the enzyme responsible for core fucosylation and that aids receptor endocytosis, in HepG2-hNTCP-C4 cells reduced HBV infectivity, and PhoSL facilitated that reduction. PhoSL also blocked the activity of epidermal growth factor receptor, which usually enhances HBV infection. HBV particles bound to fluorescently labeled PhoSL internalized into HepG2-hNTCP-C4 cells, suggesting that PhoSL might inhibit HBV infection after internalization. As PhoSL reduced the formation of HBV cccDNA, a marker of chronic HBV infection, we suggest that PhoSL could impair processes from internalization to cccDNA formation. Our finding could lead to the development of new anti-HBV agents.

Features of gene polymorphism associations linked with sex hormone binding globulin level and breast cancer of various molecular biological subtypes

Aim: to identify specific associations between genes polymorphism associated with sex hormone-binding globulin (SHBG) level and breast cancer (BC) of various molecular biological subtypes.Materials and Methods. The retrospective comparative study was conducted using specimens collected from 261 patients with BC of two molecular biological subtypes – luminal A/B (n = 153) and triple negative (n = 108) as well as 1140 women in control group. All study participants (n = 1401) underwent a molecular genetic study of four single nucleotide polymorphism (SNP) loci, which showed a relationship with circulating SHBG level in previously conducted genome-wide association study (GWAS): rs12150660 SHBG, rs10454142 PPP1R21, rs780093 GCKR, rs17496332 PRMT6.Results. The analysis revealed an association between SHBG SNP candidate genes and a BC risk in patients with luminal A/B subtypes and lacked significant associations between the loci assessed and triple negative BC subtype. CC female genotype of rs10454142 PPP1R21 increased a risk of luminal A/B subtypes BC by more than 2-fold (recessive model [CC vs. TC+TT]; odds ratio = 2.07; 95 % confidence interval = 1.14–3.77; p = 0.017; pperm = 0.018). This SNP is localized in functionally "significant" regions of the genome (enhancers/active enhancers, promoters/active promoters) and affects methylation level in several hepatocyte DNA sites [cg15846641 (chr2:48541264)].Conclusion. The genetic variant rs10454142 PPP1R21 is associated with the risk of developing ВС luminal A/B subtypes, but not with ВС triple negative subtype.

Discovery of a first-in-class orally available HBV cccDNA inhibitor

The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma

Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.

SMUG1 regulates fat homeostasis leading to a fatty liver phenotype in mice

Fatty liver diseases are a major health threat across the western world, leading to cirrhosis and premature morbidity and mortality. Recently, a correlation between the base excision repair enzyme SMUG1 and metabolic homeostasis was identified. As the molecular mechanisms remain unknown, we exploited a SMUG1-knockout mouse model to gain insights into this association by characterizing the liver phenotype in young vs old SMUG1-null mice. We observed increased weight and fat content in one-year old animals, with altered activity of enzymes important for fatty acids influx and uptake. Consistently, lipidomic profiling showed accumulation of free fatty acids and triglycerides in SMUG1-null livers. Old SMUG1-knockout mice also displayed increased hepatocyte senescence and DNA damage at telomeres. Interestingly, RNA sequencing revealed widespread changes in the expression of lipid metabolic genes already in three months old animals. In summary, SMUG1 modulates fat metabolism favouring net lipogenesis and resulting in development of a fatty liver phenotype.

Thymoquinone supplementation mitigates arsenic-induced cytotoxic and genotoxic alterations in rat liver

Arsenic, a widespread environmental toxin, produces multiple organ toxicity, including hepatotoxicity. Thymoquinone (TQ) is known to restore liver functions in several hepatic injury models. This study aims to assess the mitigative potential of TQ against sodium arsenate (NaAs)-induced cytotoxic and genotoxic alterations in the liver. Rats were randomly distributed to control, NaAs, TQ, and NaAs+TQ groups. NaAs+TQ and TQ group of rats were pre-treated with TQ (1.5 mg/kg bwt, orally) for 14 days, and the treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. The deleterious histological alterations in the liver of arsenic intoxicated animals were accompanied by an upsurge in the activities of serum ALT and AST, the diagnostic indicators of liver injury. NaAs caused pronounced alterations in the activities of membrane marker and carbohydrate metabolic enzymes and the enzymatic and non-enzymatic components of hepatic antioxidant defense. Significant hepatocyte DNA damage and hepatic arsenic accumulation were also observed in arsenic-exposed rats. TQ supplementation alleviated these adverse alterations and improved the overall hepatic metabolic and antioxidant status in NaAs-administered rats. Prevention of oxidative injury could be the key mechanism of TQ-elicited protective effects. TQ may have an excellent scope as a dietary supplement in the management of arsenic-induced hepatic pathophysiology.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

Role of hepatitis B virus in development of hepatocellular carcinoma: Focus on covalently closed circular DNA.

Chronic infection with hepatitis B virus (HBV) remains a major global health problem, especially in developing countries. It may lead to prolonged liver damage, fibrosis, cirrhosis, and hepatocellular carcinoma. Persistent chronic HBV infection is related to host immune response and the stability of the covalently closed circular DNA (cccDNA) in human hepatocytes. In addition to being essential for viral transcription and replication, cccDNA is also suspected to play a role in persistent HBV infections or hepatitis relapses since cccDNA is very stable in non-dividing human hepatocytes. Understanding the pathogenicity and oncogenicity of HBV components would be essential in the development of new diagnostic tools and treatment strategies. This review summarizes the role and molecular mechanisms of HBV cccDNA in hepatocyte transformation and hepatocarcinogenesis and current efforts to its detection and targeting.

Pressure Loading Induces DNA Damage in Human Hepatocyte Line L02 Cells via the ERK1/2-Dicer Signaling Pathway.

Alteration of liver tissue mechanical microenvironment is proven to be a key factor for causing hepatocyte injury and even triggering the occurrence of hepatocellular carcinoma; however, the underlying mechanisms involved are not fully understood. In this study, using a customized, pressure-loading device, we assess the effect of pressure loading on DNA damage in human hepatocytes. We show that pressure loading leads to DNA damage and S-phase arresting in the cell cycle, and activates the DNA damage response in hepatocytes. Meanwhile, pressure loading upregulates Dicer expression, and its silencing exacerbates pressure-induced DNA damage. Moreover, pressure loading also activates ERK1/2 signaling molecules. Blockage of ERK1/2 signaling inhibits pressure-upregulated Dicer expression and exacerbates DNA damage by suppressing DNA damage response in hepatocytes. Our findings demonstrate that compressive stress loading induces hepatocyte DNA damage through the ERK1/2–Dicer signaling pathway, which provides evidence for a better understanding of the link between the altered mechanical environment and liver diseases.

Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus.

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract. More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.