Background: This study focuses on the assessment of the antitumor potential of a combination of doxorubicin hydrochloride and silybin for the treatment of breast cancer. Method: Spectrophotometric analysis, Fourier-transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC) were used to assess the physicochemical characteristics of drugs and their combination. The calibration curve of both drugs obeyed Beer-Lambert’s law. Further spectrophotometric analysis validated the quantification method by confirming the linearity of calibration curves. There were no notable changes in the distinct functional groups observed in the FTIR spectra, suggesting the absence of any chemical interactions. A shifted peak in the drug mixture indicated interaction without degradation, while DSC thermograms showed distinctive thermal transitions. Antiproliferative effects were assessed using cytotoxicity tests on MCF-7 breast cancer cell lines. Results: The silybin-to-doxorubicin mixture in a 2:1 ratio produced the strongest total growth inhibition (TGI = 40 µg/mL) in cytotoxicity tests, which showed modest antiproliferative action. Cellular changes produced by drug concentrations and various ratios were evaluated using morphological analysis, which revealed a concentration-dependent cytotoxic effect. Conclusion: A moderate anticancer activity of a combination of silybin and doxorubicin hydrochloride shows a future direction for continued study.
Introduction: Aerosols generated from heated hair products may pose respiratory risks, particularly for hairdressers due to occupational exposure. While some products show a higher potential for these effects, a clear link toward product and application methods remains unestablished. This study aimed to evaluate a cell-based in vitro inhalation approach to better understand the toxicological impacts, screen existing products, and facilitate the “safe-by-design” approach. Materials and Methods: Four test materials (TM1–TM4), including different products, application techniques, and subtle real-life effects observed after prolonged use, were evaluated, including two methods for TM2 (TM2 and TM2A). Aerosols were generated by product application to real hair strands and sampled by exposure of human lung cells (A549) under optimized conditions (P.R.I.T. ® ExpoCube ® ). Dose–response testing assessed cell viability, mitochondrial function, cellular stress, and interleukin-8 release. Results: Results indicated significant differences in aerosol release based on the test material and application method. Cell viability and mitochondrial function generally remained unaffected; however, there were notable differences in cellular stress and IL-8 release. A score was developed to facilitate a general assessment of the test materials, evaluating both aerosol release and biological effects. It showed promising correspondence between in vitro results and real-life observations. Conclusion: This in vitro –based alternative to animal testing has the potential to provide a strategy to investigate the respiratory effects of cosmetic product aerosols. It might offer a framework for product screening and, as experience with additional test compounds accumulates, could help facilitate the development and acceptance of alternative methods in safe-by-design and safety assessment strategies.
Background: Lanternfish ( Benthosema pterotum ), an abundant deep-sea resource with a unique protein profile, holds significant potential as a source of bioactive peptides. This study was designed to investigate and characterize the immunomodulatory properties of protein hydrolysates derived from lanternfish, prepared using Alcalase (BPHA) and Flavourzyme (BPHF), on the RAW 264.7 murine macrophage cell line. Methods: Lanternfish protein was enzymatically hydrolyzed, and the resulting peptides were fractionated to isolate those with a molecular weight below 3 kDa. RAW 264.7 macrophages were treated with B. pterotum hydrolyzed using Alcalase enzymes (BPHA) and B. pterotum hydrolyzed using Flavourzyme enzymes (BPHF) at concentrations ranging from 100 to 500 µg/mL. The cytotoxicity of the hydrolysates was evaluated using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. To assess immunomodulatory activity, the gene expression levels of pro-inflammatory mediators, including Inducible nitric oxide synthase (iNOS), Tumor necrosis factor alpha (TNF-α), Interleukin-1 beta (IL-1β), and Interleukin-6 (IL-6), were quantified by quantitative real-time PCR in both resting macrophages and in cells stimulated with lipopolysaccharide (LPS) to induce an inflammatory state. Results: The MTT assay confirmed that both BPHA and BPHF were noncytotoxic across all tested concentrations. In resting (nonstimulated) macrophages, both hydrolysates significantly upregulated the expression of iNOS, TNF-α, IL-1β, and IL-6 in a dose-dependent manner, demonstrating a clear immunostimulatory effect. In contrast, when macrophages were exposed to an inflammatory challenge with LPS, the hydrolysates exhibited potent anti-inflammatory activity by significantly downregulating the expression of these same pro-inflammatory genes. Conclusions: Protein hydrolysates derived from lanternfish display a notable dual immunomodulatory capability. They function as immunostimulants in a normal physiological state and act as anti-inflammatory agents during an inflammatory response. These findings underscore the potential of lanternfish hydrolysates as a valuable functional ingredient for applications in nutraceuticals and functional foods aimed at balancing and modulating the immune system.
Introduction: Systematic reviews (SR) collect and integrate data corpuses into consistent, computable, and comparable datasets. The adverse outcome pathway (AOP) framework facilitates the linking of data describing molecular initiating events, through one or more key events (KEs), to adverse biological outcomes. To explore the potential application of data from SRs to the AOP framework, a case study was conducted to explore mapping SR to existing AOP KEs. Methods: SR data consisted of in vitro and in vivo androgen receptor (AR) toxicity information from nonmammalian vertebrate species collected as described by the authors, limiting data comparability. Data were standardized and mapped to terms for Level of Biological Organization, Object, Process, and Action using existing KEs in the AOP-Wiki as a source for endpoint terms. Results: In vitro SR data had 131 of 264 records that mapped to AR transactivation, while in vivo data had 226 of 1891 records directly mappable to 31 different KEs (e.g., increased vitellogenin messenger RNA). When no appropriate terms existed in the AOP-Wiki, standardized terms were proposed for future use. For unstructured data, mapping and standardization required additional interpretation. Conclusions: This study highlights the difficulties in aligning heterogeneously extracted SR data with a structured framework. This work highlights the need for language standardization and the adoption of clear data collection guidance prior to, and during, the SR to enhance data comparability and computability. The adoption of such efforts can advance the ability of resulting data to be reused and applied to frameworks such as AOPs. Lessons learned in this case study are applicable to similar efforts examining the use of automation in data extraction and evaluation.
Volatile organic compounds (VOCs) are ubiquitous in indoor air spaces, including the most sensitive medical environments. In assisted reproductive technology facilities, VOCs are known to greatly decrease embryo implantation rates and may impact embryo growth rate as well as miscarriage rates. There is reason to believe that the observed impacts of VOCs translate into parallel industries as well, such as the emerging cell and gene therapy industry. Previous modeling efforts on VOC transport into cell culture media components have provided estimates on the expected equilibrium concentrations from airborne VOC exposure. However, a fundamental knowledge gap remains regarding the rate of VOC partitioning into cell cultures. In this work, we present an enhanced modeling approach to quantify the partitioning kinetics of selected VOCs in cell cultures consisting of a multiphase system with an oil overlay and a water-based culture media, as well as a water-based media without overlay. Preliminary results from eight prevalent and cytotoxic VOCs indicate rapid equilibration into the system with equilibrium achieved within a timescale of seconds to minutes. These results suggest that practitioners must take steps to maintain VOC-free air for the protection of cell cultures to avoid adverse outcomes, as open-air processes may be compromised even during brief exposure to VOCs. VOCs have been modeled to rapidly equilibrate into cell cultrue systems, potentially disrupting normal cellular development.
Environmental endocrine-disrupting chemicals (EDCs) are increasingly implicated as modifiable risk factors for cardiovascular disease (CVD) and yet their direct impact on human cardiac tissue remains poorly characterized. This study investigated the early transcriptional responses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following exposure to two structurally distinct and widely prevalent EDCs: homosalate (HS) and triphenyl phosphate (TPP). hiPSC-CMs were treated with 10, 30 or 60 µM of HS or TPP for 24 hours. Expression levels of key sarcomeric genes including MYL2, MYH7, TNNT2 and ACTN2 were quantified by RT-qPCR. Both chemicals elicited dose-dependent repression of core contractile transcripts. TPP induced monotonic downregulation beginning at the lowest concentration tested while HS exhibited a biphasic response with mild gene induction at 10 µM and significant suppression at higher doses. Among all targets, MYL2 and MYH7 consistently emerged as the most transcriptionally sensitive markers. These results highlight the sensitivity of sarcomeric gene expression to chemical exposure and suggest MYL2 and MYH7 as potential early indicators of chemically induced cardiac stress. This study underlines the responsiveness of the human cardiac transcriptome to environmental toxicants and supports the application of hiPSC-CMs as a human-relevant platform for mechanistic toxicology and early cardiotoxicity screening.
Genotoxicity testing is critical for evaluating the safety of chemicals, pharmaceuticals, and environmental pollutants. The comet assay, or Single Cell Gel Electrophoresis (SCGE), is a widely employed method for detecting DNA damage at the single-cell level due to its sensitivity and simplicity. However, conventional manual scoring is labor-intensive, prone to observer bias, and limits the assay’s reliability and throughput. This study investigates the application of artificial intelligence (AI) and machine learning (ML) to enhance the comet assay's sensitivity, specificity, and efficiency. HepG2 cells were treated with genotoxic agents, cisplatin and doxorubicin, to induce DNA damage, followed by comet assay analysis with epifluorescence microscopy. Three ML models Support Vector Machine (SVM), Random Forest (RF), and Convolutional Neural Networks (CNN) were employed to classify comet images based on DNA damage severity. Among these, the CNN model demonstrated superior performance, achieving 92.5% accuracy and the highest correlation ( r = 0.94) with expert annotations. The AI model also quantified key parameters, including tail length, tail moment, and DNA content in the tail, offering enhanced sensitivity and specificity over manual scoring. Cross-validation and external testing validated the robustness and generalizability of the CNN model across diverse datasets. Furthermore, the AI-driven approach facilitated high-throughput analysis and minimized inter-observer variability, addressing longstanding challenges in comet assay evaluation. These results underscore the transformative potential of AI in genotoxicity testing, providing a scalable and reliable framework for advancing in vitro toxicology.
Drug-induced liver injury (DILI) is a significant cause of drug attrition and market withdrawal, underscoring the importance of the early assessment of hepatotoxicity during drug development. Disruption of bile acid (BA) homeostasis can precipitate liver injury, making the regulation of BA by the liver essential to prevent DILI. Conventional bile salt export pump (BSEP) inhibition assays have poor predictive value, as they do not consider the BA feedback mechanism that mitigates hepatotoxicity. In this study, we present an innovative approach for the preclinical evaluation of the BA-induced hepatotoxic potential of drug candidates. The C-DILI™ Assay employs two distinct media conditions to differentiate between BA-dependent and BA-independent cytotoxicity by measuring LDH release and ATP depletion in sandwich-cultured human hepatocytes. Seventy-one drugs were evaluated, including 49 unblinded and 22 blinded compounds with varied BSEP inhibition profiles and DILI risk. The assay successfully identified 14 drugs with BA-dependent and 7 with BA-independent hepatotoxicity. For instance, troglitazone (Trog) demonstrated BA-dependent cytotoxicity, whereas cyclosporine A exhibited BA-independent cytotoxicity. Notably, antagonism of farnesoid X receptor (FXR) emerged as a common mechanism underlying BA-dependent toxicity, consistent with FXR’s critical role in BA homeostasis. In the blinded assessment, the assay detected nine drugs with BA-dependent cytotoxicity, affirming its utility in elucidating this mechanism of liver toxicity. Of these, six drugs had documented preclinical or clinical hepatotoxicity findings, thus corroborating the strategy’s value in preclinical safety evaluation. This approach provides a comprehensive and clinically relevant framework for the preclinical prediction of BA-dependent liver toxicity, thereby strengthening preclinical DILI safety assessments. Given the varied clinical presentations and mechanisms of DILI, this strategy should be integrated into a multifaceted preclinical DILI assessment paradigm.
Introduction: Abamectin (ABA) is an insecticide that is commonly used in agricultural spraying. Although it is known to have neurotoxic effects on living organisms in the ecosystem and humans, the details of these effects, including the duration and amount of exposure, are not fully understood. The aim of this study is to evaluate the effect of ABA exposure on human microglia clone 3 (HMC3) cells. These cells were chosen because they are suitable for modeling immunological functions such as cytokine production, cell migration, and inflammation response.
