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Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals. [ABSTRACT FROM AUTHOR]

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases. [ABSTRACT FROM AUTHOR]

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression. [ABSTRACT FROM AUTHOR]

Purpose: Apolipoprotein E2 (ApoE2) gene therapy is a potential disease-modifying therapy for Alzheimer's disease (AD). We investigated the potential of plasmid encoding ApoE2 loaded brain-targeted functionalized-liposomes for treatment of AD. This was achieved via systemic administration of liposomes entrapping therapeutic gene targeting the brain of mice. Methods: Targeting and transfection efficiency of designed liposomes were determined in bEnd.3, primary glial and primary neuronal cells. The ability of liposomal formulations to translocate across in vitro blood-brain barrier (BBB) and, thereafter, transfect primary neuronal cells was investigated using in vitro triple co-culture BBB model. We quantified ApoE expression in the brain of mice after single intravenous injection of brain-targeted liposomes loaded with plasmid ApoE2. Results: Dual surface modification enhanced the in vitro transfection efficiency of designed liposomes. Successful delivery of therapeutic gene overcoming BBB by Transferrin-Penetratin- modified liposomes was demonstrated both in vitro and in vivo. Significant (p < 0.05) increase in ApoE levels in the brain of mice was observed after intravenous administration of Tf-Pen-liposomes encasing plasmid ApoE2. Conclusion: The results indicate that dual-ligand based liposomal gene delivery systems had both enhanced brain targeting and gene delivery efficiencies. Transferrin-Penetratin modified liposomes for delivery of plasmid ApoE2 has great potential for AD treatment. [ABSTRACT FROM AUTHOR]

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Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels. [ABSTRACT FROM AUTHOR]

The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed. [ABSTRACT FROM AUTHOR]