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Objective(s): Insulin resistance (IR) is a critical component of metabolic syndrome, primarily linked to obesity. It contributes to impaired glucose metabolism, beta-cell dysfunction, and the onset of type 2 diabetes. This study aimed to develop a DNAsome nanocarrier designed for the targeted delivery of small interfering RNA (siRNA) to inhibit mRNA of Transforming growth factor beta-like Stimulated Clone 22 D4 (TSC22D4), thereby enhancing insulin sensitivity in hepatocytes. Materials and Methods: The DNAsome was constructed using Y-DNA building blocks derived from three distinct DNA oligonucleotides. Its structural characteristics were analyzed through atomic force microscopy (AFM). The functional efficacy of the DNAsome in delivering siRNA was evaluated by measuring its cellular uptake and ability to down-regulate TSC22D4 expression in HepG2 cells via real-time PCR. Additionally, the cytotoxicity and safety of both the DNAsome and the DNAsomesiRNA complexes were assessed using the MTT assay on HepG2 cells. Results: Findings indicated successful fabrication of the DNAsome nanocarriers, although aggregation was observed at higher concentrations, yielding nanoparticle sizes between 116 and 740 nm. Real-time PCR results confirmed effective siRNA targeting, significant cellular uptake of the nanocomplexes, and successful silencing of TSC22D4 expression. Conclusion: This study suggests that DNAsome-based siRNA delivery systems hold promise for improving insulin sensitivity and addressing IR associated with obesity and metabolic syndrome. [ABSTRACT FROM AUTHOR]

This study assessed the health risks and toxicity of compounds found in the leachate and exhaust air of non-incineration devices used for hospital waste management. Specifically, it measured the levels of potentially toxic elements and pharmaceutical compounds in two disinfection waste treatment devices—hydroclave with shredder (device A) and autoclave without shredder (device B)—at a hospital in Tehran, Iran. Sampling occurred from October 2022 to March 2023. potentially toxic elements were analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), while cytotoxicity was evaluated with an ELISA reader.The results indicated that the exhaust air from device A contained high concentrations of barium (9.80 ± 1.60 µg/m3), zinc (8.60 ± 2.25 µg/m3), and chromium (8.45 ± 2.30 µg/m3). In contrast, barium and zinc were the most abundant potentially toxic elements in device B. Analysis of the leachate from device A showed that nickel and arsenic had the lowest concentrations, while barium, chromium, and zinc had the highest. Additionally, Leachate analysis from Device A also revealed high levels of barium, chromium, and zinc, while nickel and arsenic were found at lower concentrations. Significant concentrations of pharmaceutical compounds, such as azithromycin, ciprofloxacin, diclofenac, and naproxen, were detected in the effluent from both devices, with higher concentrations in Device A, indicating improper segregation and inadequate management of pharmaceutical waste. This underscores the urgent need for continuous training, supervision, and monitoring in pharmaceutical waste management. Cytotoxicity analyses showed that particulate matter and leachate from Device A had a more pronounced negative impact on human cell lines (HepG2 and A549) compared to Device B. Health risk assessments using Monte Carlo simulations indicated that the carcinogenic risks from potentially toxic elements (PTEs) in Device A, particularly arsenic and chromium, exceeded the permissible limits set by the USEPA, while Device B posed significantly lower risks. These findings highlight the importance of educating hospital staff on proper waste segregation, continuous monitoring, and implementing advanced waste management protocols to protect public health and the environment. [ABSTRACT FROM AUTHOR]

Background: Swine hepatitis E (HEV) is a zoonotic infectious disease caused by the swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key virulence factor in swine HEV, playing a crucial role in the release of viral particles, the modulation of the host innate immune response, and regulation of autophagy and apoptosis, etc. However, its main function and pathogenic mechanism remain incompletely understood. Results: In our study, adenoviruses ADV4-ORF3 and ADV4-GFP were successfully constructed and mediated the overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP in HepG2 cells. A total of 217 differentially expressed messenger RNAs (mRNAs) were screened by high-throughput sequencing, and 27 statistically significant differentially expressed genes were screened for further quantitative real-time reverse transcription (qRT-PCR) verification by functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]). They are mainly involved in six pathways: the cellular response to unfolded protein, inflammatory response, cytokine activity, TNF signaling pathway, influenza A, and pathways in cancer. In a comparative analysis of transcriptome and mRNA expression profiles of lncRNA sequencing, the results showed that 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified. Conclusions: Eight differential genes, HSPA1A, HSPA1B, PLD3, RELA, GPI, SAMHD1, RPS6KA4, and PIK3CB, were successfully verified. Comparing and analyzing the results of the two sequencing methods indicated that the 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified in SHEV ORF3-expressing HepG2 cells, which has laid a genetic foundation for the physiological function and mechanism of SHEV ORF3. [ABSTRACT FROM AUTHOR]

Background and aims: Alcoholic hepatitis (AH) and hepatocellular carcinoma (HCC) are common liver diseases. Chronic inflammation caused by AH can progress to alcoholic cirrhosis (AC) and eventually HCC. Methods: This study sought to ascertain potential shared genes between AH and HCC through the utilization of multiple transcriptome databases. Employing an immune infiltration analysis, and calculating the correlation between shared genes and immune infiltration results, in conjunction with independent bulk transcriptome validation sets, led to the identification of core shared genes. Subsequently, single-cell transcriptome data, clinical sample immunohistochemistry experiments, and overexpressed core shared genes in HepG2 cells were employed to validate the core shared genes of AH and HCC. Results: Through the bulk transcriptome discovery sets of AH and HCC, 206 potential shared genes were identified. After screening with two machine learning algorithms, five shared genes remained. Combining the results of the immune infiltration and bulk transcriptome results from an independent validation cohort, the core shared gene was determined to be RASGRF2. Single-cell data further demonstrated that RASGRF2 and its downstream genes were highly expressed in AH, AC, and HCC tissues. Spatial transcriptome data indicated that RASGRF2 was highly expressed in HCC tumor tissues. Compared with the paracancerous tissues, the RASGRF2 gene was significantly overexpressed in HCC tissues. Overexpression of RASGRF2 in HepG2 cells resulted in significantly enhanced migration, invasion, and proliferation abilities. Conclusion: RASGRF2 serve as a pathogenic gene that mediates the progression of AH to AC and potentially to HCC. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (CYP) 3A4 plays a major role in drug metabolism. Its activity could be determined by non-invasive and cost-effective assays, such as breath analysis, for the personalised monitoring of drug response. For the first time, we identify an isotopically unlabelled CYP3A4 substrate, tolterodine that leads to the formation of a non-toxic volatile metabolite, acetone, which could potentially be applied to monitor CYP3A4 activity in humans. In vitro biotransformation of tolterodine by HepG2 cells overexpressing CYP3A4, CYP2D6 or CYP2C9 was investigated by LC-MS analysis of cell culture supernatant for the non-volatile metabolite, N-dealkylated tolterodine, and PTR-ToF-MS analysis of the headspace for acetone. The highest level of the N-dealkylated metabolite was produced by HepG2-CYP3A4. Concentration dependent effects of tolterodine were analysed, resulting in TC50 values of 414 µM and 375 µM for HepG2-CYP3A4 and reference cells, respectively. Acetone and N-dealkylated tolterodine levels increased continuously over 24 h in HepG2-CYP3A4. Treatment with either a pan-CYP inhibitor, 1-aminobenzotriazole, or a CYP3A4 inhibitor, ketoconazole, considerably reduced the production of both metabolites in HepG2-CYP3A4 cells. These findings pave the way for the further development of non-invasive breath tests using unlabelled precursors to determine CYP enzyme activity in individuals. [ABSTRACT FROM AUTHOR]

Introduction: One of the important heterocyclic compounds that exhibit versatile biological activity properties such as anti -inflammatory, antioxidant, and anti-proliferative/anticancer activities are benzimidazole-based compounds. In this study, a new benzimidazolium salt was synthesized. The cytotoxicity of this newly synthesized compound on HepG2 and A549 cell lines was investigated. Methods: In this study, a new benzimidazole salt was synthesized. Then, the cytotoxic effects of this compound 4 on the human liver cancer cell line (HepG2) and human lung cancer cell line (A549) were investigated by the MTT method. Result: According to the results obtained, it was concluded that compound 4, when applied to the human liver cancer cell line and the human lung cancer cell line under in vitro conditions, showed a significant cytotoxic effect on these cells. Cellular proliferation was measured for both lung A549 and liver HepG2 cells. Lung and liver cells exposed to various concentrations of compound 4 and cisplatin were observed at the 72-hour time point. Significant differences were detected in A549 cells treated with different concentrations of compound 4 and cisplatin (p=0.001<0.05). The doses that created the difference were found to be between 200 μM and 25 μM for compound 4 (p = 0.017) and between 200 μM and 50 μM concentrations (p = 0.001). There were also significant differences between doses for cisplatin (p=0.001<0.05). The concentrations that created the difference were found to be between 100 μM and 25 μM (p = 0.042), between 200 μM and 25 μM (p = 0.001), and between 200 μM and 50 μM (p = 0.049). As seen in Figure 1, cell viability in the high-dose compound 4 and cisplatin-treated groups (200 μM, 100 μM) is lower than the viability of cells in the low -dose compound 4 groups (50 μM, 25 μM). There is a difference between compound 4 and cisplatin (p<0.05). In terms of cell viability, the average of cisplatin was lower than compound 4. In the treatment of HepG2 cells with different doses of compound 4 and cisplatin, significant differences were detected between compound 4 and cisplatin (p=0.035<0.05). In terms of cell viability, the average of cisplatin was higher than that of compound 4. Discussion and Conclusion: Benzimidazole derivatives have various biological activities, including antitumor activity. Several investigations have exhibited the bioactivities of benzimidazole derivatives as possible therapies against cancer by focusing on certain molecules or employing non-gene-specific approaches. We tested this compound against two different human cancer cell lines including lung and liver. The results show that compound 4 had cytotoxic effects on both A549 and HepG2. [ABSTRACT FROM AUTHOR]

Liver cancer is a major global health challenge, ranking as the third leading cause of cancer-related deaths worldwide. In this study, we explore the use of paclitaxel-coated ZIF-8 metal-organic framework nanoparticles (ZIF-8 NPs/Pacx) as a novel drug delivery system for enhanced liver cancer treatment. A comprehensive set of analyses, including morphology evaluation, particle size distribution, zeta potential measurement, drug loading capacity, encapsulation efficiency, stability, and In vitro drug release behavior, was conducted to assess the nanoparticles' performance. The ZIF-8 NPs/Pacx demonstrated significant antitumor activity at a concentration of 75 µg/mL, particularly against HepG2 and Hep3B liver cancer cell lines. RT-PCR analysis revealed that ZIF-8 NPs/Pacx stimulated TNF-α expression in HepG2, Hep3B, and normal liver cells (NLCs), with further confirmation through Western blot analysis of TNF-α and β-actin levels. Notably, the nanoparticles exhibited the ability to inhibit cell proliferation and induce apoptosis in liver cancer cells. These findings suggest that ZIF-8 NPs/Pacx could offer an innovative approach for the delivery of paclitaxel to liver cancer cells, maximising treatment effectiveness with minimal side effects, and positioning this system as a promising candidate for future liver cancer treatments. Highlights: Synthesis and characterization of ZIF-8 NPs/Pacx were evaluated through DLS,Zeta potential and TEM analysis. Gene expression of HepG2, Hep3B, and NLC was studied through qRT-PCR. Anti-proliferation, apoptosis, antitumor activity was done for introduces an innovative approach. These results highlights the promise of ZIF-8 NPs/Pacx as a unique strategy for focused delivery mechanisms in liver cancer therapy. [ABSTRACT FROM AUTHOR]

Abstract The leading safety issue and side effect associated with natural herb products is drug-induced liver injury (DILI) caused by bioactive compounds derived from the herb products. Herein, in silico and in vitro analyses were compared to determine the hepatotoxicity of compounds. The results of in silico analyses, which included an integrated database and an interpretable DILI prediction model, identified calycosin, biochanin_A, xanthatin, piperine, and atractyloside as potential hepatotoxic compounds and tenuifolin as a non-hepatotoxic compound. To evaluate the viability of HepG2 cells exposed to the selected compounds, we determined the IC50 and IC20 values of viability using MTT assays. For in-depth screening, we performed hematoxylin and eosin-stained morphological screens, JC-1 mitochondrial assays, and mRNA microarrays. The results indicated that calycosin, biochanin_A, xanthatin, piperine, and atractyloside were potential hepatotoxicants that caused decreased viability and an apoptotic phase in morphology, while these effects were not observed for tenuifolin, a non-hepatotoxicant. In the JC-1 assay, apoptosis was induced by all the predicted hepatotoxicants except atractyloside. According to transcriptomic analysis, all the compounds predicted to induce DILI showed hepatotoxic effects. These results highlighted the importance of using in vitro assays to validate predictive in silico models and determine the potential of bioactive compounds to induce hepatotoxicity in humans.

Rashid Lateef,1,2 Israr Ahmad,2 Abbas Ali Mahdi,3 Neha Lohia,1 Hisham A Alhadlaq,4 Mohd Javed Akhtar,4 Maqusood Ahamed4 1School of Life and Basic Sciences, Jaipur National University, Jaipur, Rajasthan, 302017, India; 2Department of Biotechnology, Era University, Lucknow, Uttar Pradesh, 226003, India; 3Department of Biochemistry, Era University, Lucknow, Uttar Pradesh, 226003, India; 4King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, 11451, Saudi ArabiaCorrespondence: Maqusood Ahamed, King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, 11451, Saudi Arabia, Email mahamed@ksu.edu.saBackground: Researchers have shown substantial interest in bismuth oxide/reduced graphene oxide (Bi2O3/RGO) nanocomposites due to their superior features that are not achievable by each material alone. The growing applications and manufacturing of Bi2O3/RGO nanocomposites have raised concerns regarding their potential human health risks. This work was designed to explore the possible toxicity mechanisms of Bi2O3/RGO nanocomposites in two distinct mammalian cell lines, normal rat kidney cells (NRK52E) and human liver cancer cells (HepG2).Methods: Bi2O3/RGO nanocomposites were prepared by a simple hydrothermal technique. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and dynamic light scattering (DLS) were used to characterize the synthesized nanocomposites. The cytotoxicity of Bi2O3/RGO nanocomposites in NRK52E and HepG2 cells was examined by MTT cell viability assay. Reactive oxygen species (ROS) and glutathione (GSH) were measured as the biomarkers of oxidative stress. The apoptosis study was carried out by measuring several parameters, including cell cycle and caspase-3.Results: High-quality Bi2O3/RGO nanocomposites of &ap;33– 38 nm size without impurities, where crystalline Bi2O3 particles are evenly attached to the RGO sheets. Bi2O3/RGO nanocomposites exhibit cytotoxic effects on NRK52E and HepG2 cells, which were dose- and time-dependent. Interestingly, NRK52E exhibited marginally higher vulnerability to Bi2O3/RGO nanocomposites compared to HepG2. Bi2O3/RGO nanocomposites also cause a dose-dependent increase in ROS production and a decrease in GSH levels. Exposing NRK52E and HepG2 cells to Bi2O3/RGO nanocomposites results in activation of the caspase-3 enzyme and chromosomal condensation. The apoptotic response of Bi2O3/RGO nanocomposites against both types of cells was further confirmed by AO-EB dual staining and altered cell cycle.Conclusion: This study demonstrated that the toxicity of Bi2O3/RGO nanocomposites in both NRK52E and HepG2 cells is attributed to their ability to produce ROS, leading to apoptosis and cell cycle arrest as a consequence of oxidative stress. Keywords: Bi2O3/RGO nanocomposites, health effects, NRK52E, HepG2, Caspase-3, ROS, apoptosis

The leading safety issue and side effect associated with natural herb products is drug-induced liver injury (DILI) caused by bioactive compounds derived from the herb products. Herein, in silico and in vitro analyses were compared to determine the hepatotoxicity of compounds. The results of in silico analyses, which included an integrated database and an interpretable DILI prediction model, identified calycosin, biochanin_A, xanthatin, piperine, and atractyloside as potential hepatotoxic compounds and tenuifolin as a non-hepatotoxic compound. To evaluate the viability of HepG2 cells exposed to the selected compounds, we determined the IC50 and IC20 values of viability using MTT assays. For in-depth screening, we performed hematoxylin and eosin-stained morphological screens, JC-1 mitochondrial assays, and mRNA microarrays. The results indicated that calycosin, biochanin_A, xanthatin, piperine, and atractyloside were potential hepatotoxicants that caused decreased viability and an apoptotic phase in morphology, while these effects were not observed for tenuifolin, a non-hepatotoxicant. In the JC-1 assay, apoptosis was induced by all the predicted hepatotoxicants except atractyloside. According to transcriptomic analysis, all the compounds predicted to induce DILI showed hepatotoxic effects. These results highlighted the importance of using in vitro assays to validate predictive in silico models and determine the potential of bioactive compounds to induce hepatotoxicity in humans. [ABSTRACT FROM AUTHOR]

Fetal Bovine Serum (FBS) is an important ingredient in cell culture media and the current standard for most cells in vitro. However, the use of FBS is controversial for several reasons, including ethical concerns, political, and societal pressure, as well as scientific problems due to the undefined and variable nature of FBS. Nevertheless, scientists hesitate to change the paradigm without solid data de-risking the switch of their assays to alternatives. In this study, HepG2 cells, a human hepatoblastoma cell line commonly used to study drug hepatotoxicity, were adapted to serum-free conditions by using different commercially available media and FBS replacements. After transition to these new culture conditions, the success of adaptation was determined based on cell morphology and growth characteristics. Long-term culturing capacity for each medium was defined as the number of passages HepG2 cells could be cultured without any alterations in morphology or growth behavior. Two media (Advanced DMEM/F12 from ThermoFisher and TCM® Serum Replacement from MP Biomedicals) showed a long-term cultivation capacity comparable to media containing FBS and were selected for further analysis. Both media can be characterized as serum-free, however still contain animal-derived components: bovine serum albumin (both media) and bovine transferrin (only TCM® serum replacement). To assess the functionality of the cells cultivated in either of the two media, HepG2 cells were treated with reference compounds, specifically selected for their known hepatotoxicity characteristics in man. Different toxicological assays focusing on viability, mitochondrial toxicity, oxidative stress, and intracellular drug response were performed. Throughout the different assays, response to reference compounds was comparable, with a slightly higher sensitivity of serum-free cultivated HepG2 cells when assessing viability/cell death and a lower sensitivity towards oxidative stress. Taken together, the two selected media were shown to support growth, morphology, and function of serum-free cultivated HepG2 cells in the early preclinical safety space. Therefore, these results can serve as a starting point to further optimize culture conditions with the goal to remove any remaining animal-derived components. [ABSTRACT FROM AUTHOR]

Ras-related protein Rab-7a (RAB7A) is a member of the RAS oncogene superfamily; it plays an important role in vesicle transport. In this study, we described the mechanism of RAB7A-dependent regulation of glucose metabolism in HepG2 cells under hypoxia. We found that RAB7A increases lactate production, adenosine 5'-triphosphate (ATP) production, and glycogen content in HepG2 cells under hypoxia. RAB7A also stimulates the expression of fructose-bisphosphate aldolase A (ALDOA), an enzyme involved in glucose metabolism. Furthermore, we discovered that RAB7A protein binds to hypoxia-inducible factor 1‑alpha (HIF1A) protein to enhance its binding to ALDOA promoter and elevate expression of ALDOA and promote glycolysis and glycogenesis. Notably, RAB7A and HIF1A did not interfere with each other. These observations preliminarily explain the molecular mechanism by which RAB7A exerts its role in regulating glucose metabolism in HepG2 under hypoxia. Our work may provide a potential direction for studying RAB7A in liver diseases. [ABSTRACT FROM AUTHOR]

Despite significant efforts, cancer remains the second leading cause of mortality worldwide. The medicinal plant Nepeta nuda L. represents a valuable source of biologically active compounds with pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and antiviral. This study aimed to assess the antiproliferative potential and mechanisms of action of aqueous extract from the leaves of wild-grown N. nuda. Cancer cell lines, MDA-MB-231, MCF7 (breast), HT29, Colon 26 (colon), and HepG2 (liver cancer), and a non-cancerous skin cell line, BJ, were assessed for antiproliferative activity by MTT assay and observation of cell morphological alterations. The cancer cell line that was most sensitive to the extract was further studied for apoptotic alterations by Annexin V/propidium iodide staining, colony-forming assay, and qRT-PCR analysis. The results revealed that the plant extract inhibited the proliferation of all investigated cancer cell lines with the strongest cytostatic effect on Colon 26 cells with a half maximal inhibitory concentration (IC50) value of 380.2 μg/mL and a selectivity index (SI) of 3.5. The extract significantly inhibited the ability of cells to form colonies, exhibited considerable proapoptotic potential involving the participation of the CASP8 gene, and increased the expression levels of ATG3 and the BECN1 gene, which suggests a role of autophagic cell death in the antitumor action. [ABSTRACT FROM AUTHOR]

Simple Summary: Artemisia eriantha (Apennine Genepì) is a subendemic species native to the Central Apennines, valued locally for its use in traditional and ancient medicine due to its antibacterial, antifungal, anthelmintic, digestive, and antispasmodic properties. Many of these benefits are shared with other species within the Artemisia genus, which have also recently demonstrated anti-tumor properties. However, the potential cytotoxic and/or antitumor effects of A. eriantha have not yet been explored. The aim of this study was to evaluate the anti-proliferative, antitumor, and anti-metastatic properties of the plant using in vitro models of liver cancer cells. Treatment with an extract of A. eriantha inhibited the proliferation, survival, migration, and metastatic capabilities of liver cancer cell lines. A. eriantha extract appears to influence key factors associated with cancer cell proliferation and invasion, positioning it as a promising candidate for further research as an adjuvant in the prevention or treatment of liver cancer. Artemisia eriantha is a subendemic species of the Central Apennine, valued locally for its applications in ancient and traditional medicine for its antibacterial, antifungal, anthelmintic, digestive, and antispasmodic effects. Several of these properties are also found in other species within the same genus, including recent findings highlighting their anti-tumor actions. However, the presence of cytotoxic or anti-tumor activity has never been studied in A. eriantha. The aim of this study was thus to assess the potential anti-proliferative, pro-apoptotic, and antimetastatic effects of this plant using in vitro models of hepatocarcinoma cells (HepG2 and Huh7 cells). Treatment with A. eriantha extract (130 μg/mL) resulted in the reduced proliferation of HepG2 and Huh7 cells, driven by the dysregulation of proteins involved in the cell cycle (such as cyclin D1, cyclin E, and p27) and the activation of the p53-p21 pathway, leading to an increase in apoptotic activity. In addition, the treatment also lowered the healing capability after scratch wounds in Huh7 cells, which are characterized by a remarkable migratory capacity, suggesting that the A. eriantha extract may also be effective in controlling the migration and spread of metastatic cells. In conclusion, A. eriantha extract 130 μg/mL appears to interfere with key factors of HepG2 and Huh7 cell proliferation and invasion and emerges as a potential new adjuvant for the prevention and/or treatment of hepatocellular carcinoma. Additional research is necessary to establish if the effects observed are cell-specific and to elucidate the mechanisms of action and signaling pathways underlying its effectiveness. [ABSTRACT FROM AUTHOR]

The 21st‐century toxicity testing program recommends the use of cytotoxicity data from human cells in culture for rapid in vitro screening focusing on biological pathways of potential toxicants to predict in vivo toxicity. Liver is the major organ for both endogenous and exogenous chemical metabolism of xenobiotics. Therefore, this review was undertaken to evaluate side by side five different currently used commercial cytotoxicity assay kits for purpose of rapid predictive screening of potential hepatotoxicants. The test compounds for this review were selected from the NIH LiverTox and FDA Liver Toxicity Knowledge Base (LTKB) databases. Human liver HepG2, HepaRG, and rat liver Clone 9 cell cultures were used as the in vitro liver models. Five commercial assay kits representing different biomarkers or pathways were selected for this review. These kits are Vita‐Orange Cell Viability Assay Kit (Sigma‐Aldrich), CellTiter‐Glo Cell Viability Assay Kit (Promega), CytoTox‐ONE Homogeneous Membrane Integrity Assay Kit (Promega), DNA Quantitation Fluorescence Assay Kit (Sigma‐Aldrich), and Neutral Red Based In Vitro Toxicology Assay Kit (Sigma‐Aldrich). This review found that these kits can all be used for rapid predictive cytotoxicity screening of potential hepatotoxicants in human liver HepG2 and rat liver Clone 9 cells in culture as in vitro liver models without compromising quality and accuracy of endpoint measurements as well as the length of toxicity screening time. Unraveling the structure–activity relationship of potential hepatotoxins would help to classify their hepatotoxic effects. Therefore, in addition to the current regulatory hepatotoxicity testing strategies, development and regulatory approval of hepatotoxins need to be discussed in order to identify potential gaps in the safety assessment. The overall results of our study support the hypothesis that a battery of rapid, simple, and reliable assays is an excellent tool for predicting in vivo effects of suspected liver toxins. The human liver HepaRG cells do not appear to be an ideal in vitro liver model for this purpose. Five commercial cytotoxicity assay kits used for screening hepatotoxins were evaluated using HepG2, HepaRG, and Clone 9 cell cultures as in vitro models. This study supports the hypothesis that a battery of these assays is an excellent tool for predicting in vivo effects of liver toxins. [ABSTRACT FROM AUTHOR]

Human African trypanosomiasis (HAT) is an infectious disease which kills humans and animals as a result of hematological distortions, oxidative stress, tissue and neuroinflammations. This study reports on the differences in cytotoxicity, antitrypanosomal, antioxidant, and anti‐inflammatory activities of ethanol extracts from Clausena anisata fruit (CFE), Azadirachta indica (neem) leaf (NLE), and stem bark (NSBE), medicinal plants used to treat HAT in its endemic communities. The extracts remarkably inhibited Trypanosoma brucei brucei (GUTat 3.1) parasite in vitro with CFE recording the highest effect with an IC50 of 0.0055 (0.0955) μg/mL. The IC50 of the standard Coptis japonicum was 0.5957 (0.0693) μg/mL. Also, the antitrypanosomal activity of NLE was 123.34% higher than that of NSBE. The percentage number of wells containing viable T. b. brucei parasites was very significantly (p < 0.001) reduced for all the extracts after 48 h of incubation. Furthermore, the extracts did not show cytotoxicity against the liver (HepG2) cells (CC50s > 100 μg/mL and SI = 13.12–32,025.45). NSBE contained the highest quantity of phenolic compounds and flavonoids and also produced the highest antioxidant and anti‐inflammatory activity in the DPPH free radical scavenging assay (IC50 = 4.99 ± 0.018) and protein denaturation assay (IC50 = 0.1805 ± 0.0002 μg/mL). In addition, phytochemical analysis showed that NLE contained the highest number of classes of phytochemical constituents (seven) among the extracts. These results indicate that CFE, NLE, and NSBE possessed significant antitrypanosomal activity as a result of their antioxidant and anti‐inflammatory actions. However, a different mechanism was also involved in the antitrypanosomal activity of CFE and NLE, since their antitrypanosomal activity is greater than NSBE which demonstrated the highest antioxidant and anti‐inflammatory activities. Due to the remarkable antitrypanosomal action of CFE, its constituents are being isolated for possible development into novel antitrypanosomal agents. [ABSTRACT FROM AUTHOR]

Many researchers are focusing on screening the biological activities of plants owing to their safety and possible pharmacological actions. Consequently, we aimed to explore the antiproliferative and cytotoxic properties of Cycas media methanolic extract on HepG2 cell lines. Moreover, we also explore the antitumor action against the experimentally induced solid Ehrlich carcinoma (SEC) model and investigate the possible involved molecular mechanisms. Also, the antibacterial action of the extract was elucidated. Different concentrations of the extract were incubated with HepG2 to determine cytotoxicity, followed by cell cycle analysis. The in vivo experiment was accomplished by grouping the animals into four different groups (n = 10); normal control, SEC, C. media 100, and C. media 200. The extract was administered at 100 and 200 mg/kg. Tumor volume, tumor inhibition rate, toxicity profile, and antioxidant biomarkers were determined. Moreover, the PI3K/AKT/mTOR signaling pathway was investigated as a possible underlying antitumor mechanism. The tumor control group showed a remarkable upregulation for PI3K, p-AKT, and p-mTOR, along with downregulation for the antioxidant SOD and GPX4, as well as decreased levels of GSH and MDA. C. media extract reversed these parameters to a significant level and the higher dose showed a superior antitumor effect. C. media extract showed antiproliferative effects against HepG2 cells, along with a suppressive action on the PI3K/AKT/mTOR pathway and an antioxidant effect. Additionally, C. media had antibacterial consequences against S. aureus isolates with minimum inhibitory concentrations from 32 to 128 µg/mL. It also caused a noteworthy growth delay as well as a notable reduction in the membrane integrity of S. aureus isolates. These beneficial outcomes suggest C. media to have potential antitumor and antibacterial activities. [ABSTRACT FROM AUTHOR]

Stellaria media L., also called chickweed, is widespread in all parts of the world. In the present study, we investigated the biological properties and chemical profiles of different extracts (ethyl acetate, ethanol, ethanol/water, and water) of S. media. The chemical profiles were examined using UHPLC/MS/MS technique. Regarding the biological properties, antioxidant properties as well as enzyme‐inhibiting and cytotoxic effects of the extracts were demonstrated by in vitro methods. To obtain further information about the structure‐ability relationship, network pharmacology and molecular docking were also performed. Twelve phenolic compounds were identified in the extracts and most of them were flavonoids (apigenin, kaempferol derivatives, etc.). The water extract showed the best free radical scavenging activity, while the ethanol was the most active in reducing power tests. When inhibiting AChE, the ethyl acetate extract showed the best inhibitory effect. The water extract has a good cytotoxic effect on HepG2 (cell viability: 33.9% at a concentration of 100 g/mL). The analysis, performed using the STRING database, included these 45 cancer‐associated targets. The identified hub genes were TP53, CDKN2A, PTEN, KRAS, and HRAS. In molecular docking analysis, acacetin‐O‐hexoside‐O‐deoxyhexoside and napigenin‐7‐O‐hexoside exhibit remarkable binding energies with proteins. Consequently, S. media can be potential raw materials for designing functional formulations in the pharmaceutical, nutraceutical, and cosmeceutical industries. [ABSTRACT FROM AUTHOR]

Background/Objectives: Cell lines do not faithfully replicate the authentic transcriptomic condition of the disease under study. The HepG2 cell line is widely used for studying hepatocellular carcinoma (HCC), but not all biological processes and genes exhibit congruent expression patterns between cell lines and the actual disease. The objective of this study is to perform a comparative transcriptomic analysis of the HepG2 cell line, HCC, and primary hepatocytes (PH) in order to identify genes suitable for research in HepG2 as a model for PH or HCC research. Methods: We conducted a differential expression analysis between publicly available data from HCC patients, PH, and HepG2. We examined specific overlaps of differentially expressed genes (DEGs) in a pairwise manner between groups in order to obtain a valuable gene list for studying HCC or PH using different parameter filtering. We looked into the function and druggability of these genes. Conclusions: In total, we identified 397 genes for HepG2 as a valuable HCC model and 421 genes for HepG2 as a valuable PH model, and with more stringent criteria, we derived a smaller list of 40 and 21 genes, respectively. The majority of genes identified as a valuable set for the HCC model are involved in DNA repair and protein degradation mechanisms. This research aims to provide detailed guidance on gene selection for studying diseases like hepatocellular carcinoma, primary hepatocytes, or others using cell lines. [ABSTRACT FROM AUTHOR]

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (−10.7 KJ/mol−1) than catharanthine (−7.3 KJ/mol−1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR. [ABSTRACT FROM AUTHOR]

Gamma-butyrobetaine hydroxylase (BBOX1) plays a pivotal role in catalyzing the final stage of L-carnitine biosynthesis. Recently, increasing number of studies have reported that BBOX1 is weakly expressed in tumor cells and exhibits antitumor activity. The role of BBOX1 in Hepatoblastoma (HB) has yet to be determined. To substantiate this, we have investigated BBOX1 expression and its clinical relevance in HB, and explored how BBOX1 might inhibit the occurrence and development of HB. The GSE104766 and GSE131329 datasets were used to screen for the core gene BBOX1 in HB and to analyze differences in expression between hepatoblastoma and normal tissues. Based on the clinicopathological features of the GSE131329 dataset, the connections between the expression of BBOX1 and the clinicopathological feature of HB patients were determined. After BBOX1 was overexpressed, CCK-8 and colony formation assays were employed to assess cell proliferation and wound healing experiments were utilized to assess cell migration. The presence of cell apoptosis, cell cycle changes, and reactive oxygen species (ROS) was assayed using flow cytometry. Compared with normal tissues, the expression of BBOX1 in hepatoblastoma tissues was notably decreased. Dysregulated expression of BBOX1 was indicated as a prognostic risk factor closely linked to clinical stag of patients with HB. Furthermore, following BBOX1 overexpression, cell proliferation and migration are decreased, the cell cycle is arrested, and ROS are attenuated. BBOX1 has suppressive effects on HepG2 cells, potentially through its ability to hinder cancer cell proliferation, arrest cell cycle progression, and decrease ROS levels, suggesting its potential as a novel prognostic biomarker and therapeutic candidate for hepatoblastoma. [ABSTRACT FROM AUTHOR]

Rosa Roxburghii (RR), a traditional Chinese medicinal fruit, is rich in bioactive substances that make it a potential natural antioxidant resource. This research aimed to study the antioxidant properties of RR by in vitro experiments and through intracellular assessment in H2O2-induced HepG2 cells. A non-targeted metabolic analysis was conducted to indicate changes in intracellular and extracellular metabolites. Differential metabolites and metabolic pathways were explored using PCA, PLS-DA, and KEGG pathway analysis. The results showed that RR rich in bioactive substances exhibited a significant antioxidative property in vitro and intracellularly. This property may be achieved by scavenging free radicals, increasing the activity of catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and the levels of bicinchoninic acid (BCA) while reducing the reactive oxygen species (ROS) generation. This study identified 13 differential metabolites intracellularly and 7 extracellularly, among which the key differential metabolites included D-glucopyranose, D-mannose, fructose, citric acid, malic acid, cholesterol, and cholestenone. These key metabolites primarily regulated glucose-related metabolism, the citrate cycle, and the primary bile acid biosynthesis pathway in H2O2-induced HepG2 cells. These findings provide potential application evidence of RR in the development of natural resources for functional foods. [ABSTRACT FROM AUTHOR]

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don't tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization. [ABSTRACT FROM AUTHOR]

Background: (E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HMC), a homoisoflavonoid isolated from Portulaca oleracea, has significant anti-adipogenesis potential; it regulates adipogenic transcription factors. However, whether HMC improves hepatic steatosis in hepatocytes remains vague. This study investigated whether HMC ameliorates hepatic steatosis in free fatty acid-treated human hepatocellular carcinoma (HepG2) cells, and if so, its mechanism of action was analyzed. Methods: Hepatic steatosis was induced by a free fatty acid mixture in HepG2 cells. Thereafter, different HMC concentrations (10, 30, and 50 µM) or fenofibrate (10 µM, a PPARα agonist, positive control) was treated in HepG2 cells.Results: HMC markedly decreased lipid accumulation and triglyceride content in free fatty acid-treated HepG2 cell; it (10 and 50 μM) markedly upregulated protein expressions of pAMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. HMC (10 and 50 μM) markedly inhibited the expression of sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-coA desaturase 1, which are the enzymes involved in lipid synthesis. Furthermore, HMC (10 and 50 μM) markedly upregulated the protein expression of peroxisome proliferator-activated receptor alpha (PPARα) and enhanced the protein expressions of carnitine palmitoyl transferase 1 and acyl-CoA oxidase 1. Conclusion: HMC inhibits lipid accumulation and promotes fatty acid oxidation by AMPK and PPARα pathways in free fatty acid-treated HepG2 cells, thereby attenuating hepatic steatosis. [ABSTRACT FROM AUTHOR]

Objective: Exposure to perfluorooctanoic acid (PFOA) is linked to adverse health effects, including cancer and hepatic diseases. PFOA induces reactive oxygen species generation in human hepatic cells, causing oxidative stress and cell death. Resveratrol (RSV) has garnered attention for its protective effects against xenobiotic-induced damage, yet its impact on PFOA-induced oxidative stress and ferroptosis in the liver remains understudied. This study investigates RSV's protective mechanisms against oxidative stress and ferroptosis in HepG2 cells exposed to PFOA. Method: HepG2 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator at 37°C. PFOA was added to the cells at concentrations ranging from 0 to 450 µM and incubated at 37°C for 24 hours. The IC50 was determined to be 457 μM. To examine RSV's protective effects, cells were treated with 60 μM RSV. Following treatment with PFOA, RSV, and the combination of PFOA+RSV, cell lysates were prepared for analysis. Oxidative stress and ferroptosis parameters were measured spectrophotometrically using ELISA. Results: In the PFOA+RSV group, antioxidant capacity increased, and ferroptosis was suppressed compared to the control. Conversely, the PFOA group showed decreased antioxidant capacity, increased oxidant capacity, and induced ferroptosis compared to the control and RSV-treated groups. Conclusion: PFOA exposure heightens oxidative stress and ferroptosis, whereas RSV treatment significantly reduces hepatic oxidative stress and protects against ferroptosis during PFOA exposure. [ABSTRACT FROM AUTHOR]