Genotoxicity assessment of 1,4-anhydro-4-seleno-D-talitol (SeTal) in human liver HepG2 and HepaRG cells.

1,4-Anhydro-4-seleno-D-talitol (SeTal) is a highly water-soluble selenosugar with interesting antioxidant and skin-tissue-repair properties; it is highly stable in simulated gastric and gastrointestinal fluids and is a potential pharmaceutical ingredient that may be administered orally. Hepatic toxicity is often a major problem with novel drugs and can result in drug withdrawal from the market. Predicting hepatotoxicity is therefore essential to minimize late failure in the drug-discovery process. Herein, we report in vitro studies to evaluate the cytotoxic and genotoxic potential of SeTal in HepG2 and hepatocyte-like differentiated HepaRG cells. Except for extremely high concentrations (10 mM, 68 h-treatment in HepG2), SeTal did not affect the viability of each cell type. While the highest examined concentrations (0.75 and 1 mM in HepG2; 1 mM in HepaRG) were observed to induce primary DNA damage, SeTal did not exhibit clastogenic or aneugenic activity toward either HepG2 or HepaRG cells. Moreover, no significant cytostasis variations were observed in any experiment. The clearly negative results observed in the CBMN test suggest that SeTal might be used as a potential active pharmaceutical ingredient. The present study will be useful for the selection of non-toxic concentrations of SeTal in future investigations.

Modulation of synapse-related gene expression in the cerebellum and prefrontal cortex of rats subjected to the contextual fear conditioning paradigm.

The contextual fear conditioning (CFC) paradigm is the most productive approach for understanding the neurobiology of learning and memory as it allows to follow the evolution of memory traces of a conditioned stimulus and a specific context. The formation of long-term memory involves alterations in synaptic efficacy and neural transmission. It is known that the prefrontal cortex (PFC) exerts top-down control over subcortical structures to regulate behavioural responses. Moreover, cerebellar structures are involved in storing conditioned responses. The purpose of this research was to determine if the response to conditioning and stressful challenge is associated with alterations in synapse-related genes mRNA levels in the PFC, cerebellar vermis (V), and hemispheres (H) of young adult male rats. Four groups of Wistar rats were examined: naïve, CFC, shock only (SO), and exploration (EXPL). The behavioural response was evaluated by measuring the total freezing duration. Real-Time PCR was employed to quantify mRNA levels of some genes involved in synaptic plasticity. The results obtained from this study showed alterations in gene expression in different synapse-related genes after exposure to stressful stimuli and positioning to new environment. In conclusion, conditioning behavioural stimuli change the expression profile of molecules involved in neural transmission.

The Crosstalk between Intestinal Epithelial Cells and Mast Cells Is Modulated by the Probiotic Supplementation in Co-Culture Models.

The intestinal epithelium constitutes a selectively permeable barrier between the internal and external environment that allows the absorption of nutrients, electrolytes, and water, as well as an effective defense against intraluminal bacteria, toxins, and potentially antigenic material. Experimental evidence suggest that intestinal inflammation is critically dependent on an imbalance of homeostasis between the gut microbiota and the mucosal immune system. In this context, mast cells play a crucial role. The intake of specific probiotic strains can prevent the development of gut inflammatory markers and activation of the immune system. Here, the effect of a probiotic formulation containing L. rhamnosus LR 32, B. lactis BL04, and B. longum BB 536 on intestinal epithelial cells and mast cells was investigated. To mimic the natural host compartmentalization, Transwell co-culture models were set up. Co-cultures of intestinal epithelial cells interfaced with the human mast cell line HMC-1.2 in the basolateral chamber were challenged with lipopolysaccharide (LPS), and then treated with probiotics. In the HT29/HMC-1.2 co-culture, the probiotic formulation was able to counteract the LPS-induced release of interleukin 6 from HMC-1.2, and was effective in preserving the epithelial barrier integrity in the HT29/Caco-2/ HMC-1.2 co-culture. The results suggest the potential therapeutic effect of the probiotic formulation.

In vitro toxicological assessment of PhSeZnCl in human liver cells.

Phenylselenenylzinc chloride (PhSeZnCl) is an air-stable selenolate, easily synthesizable through oxidative insertion of elemental zinc into the Se-halogen bond of the commercially available phenylselenyl chloride. PhSeZnCl was shown to possess a marked GPx-like activity both in NMR and in vitro tests, and to effectively react with cellular thiols, and was supposed for a potential use in the chemotherapy of drug-resistant cancers. However, activity of PhSeZnCl in hepatic cells has never been tested before now. In this in vitro approach, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as the effects on cell cycle of PhSeZnCl in two preclinical hepatic models, namely HepG2 and HepaRG cells. Results showed that cell viability of HepG2 and HepaRG cells decreased in a dose-dependent manner, with a more marked effect in HepG2 tumour cells. Moreover, treatment with 50 µg/mL PhSeZnCl caused an increase of primary DNA damage (4 h) and a statistically significant increase of HepG2 cells arrested in G2/M phase. In addition, it altered mitochondrial membrane potential and induced chromosomal DNA fragmentation (24 h). In HepaRG cells, PhSeZnCl was able to determine a cell cycle-independent induction of apoptosis. Particularly, 50 µg/mL induced mitochondrial membrane depolarization after 24 h and apoptosis after 4 h treatment. Futhermore, all PhSeZnCl concentrations tested determined a significant increase of apoptotic cells after 24 h. Apoptosis was also highlighted by the detection of active Caspase-3 by Western Blot analysis after 24 h exposure. In conclusion, this first toxicological assessment provides new insights into the biological activity of PhSeZnCl in preclinical hepatic models that will be useful in future safety assessment investigation of this compound as a potential pharmaceutical. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00148-y.

A Multi-Strain Probiotic Formulation Improves Intestinal Barrier Function by the Modulation of Tight and Adherent Junction Proteins.

In healthy individuals, tight junction proteins (TJPs) maintain the integrity of the intestinal barrier. Dysbiosis and increased intestinal permeability are observed in several diseases, such as inflammatory bowel disease. Many studies highlight the role of probiotics in preventing intestinal barrier dysfunction. The present study aims to investigate the effects of a commercially available probiotic formulation of L. rhamnosus LR 32, B. lactis BL 04, and B. longum BB 536 (Serobioma, Bromatech s.r.l., Milan, Italy) on TJPs and the integrity of the intestinal epithelial barrier, and the ability of this formulation to prevent lipopolysaccharide-induced, inflammation-associated damage. An in vitro model of the intestinal barrier was developed using a Caco-2 cell monolayer. The mRNA expression levels of the TJ genes were analyzed using real-time PCR. Changes in the amounts of proteins were assessed with Western blotting. The effect of Serobioma on the intestinal epithelial barrier function was assessed using transepithelial electrical resistance (TEER) measurements. The probiotic formulation tested in this study modulates the expression of TJPs and prevents inflammatory damage. Our findings provide new insights into the mechanisms by which probiotics are able to prevent damage to the gut epithelial barrier.

Pro-Apoptotic Activity of Artichoke Leaf Extracts in Human HT-29 and RKO Colon Cancer Cells.

(1) Background: Cynara cardunculus L. subsp. scolymus (L.) Hegi, popularly known as artichoke, is an herbaceous plant belonging to the Asteraceae family. Artichoke leaf extracts (ALEs) have been widely used in traditional medicine because of their hepatoprotective, cholagogic, hypoglycaemic, hypolipemic and antibacterial properties. ALEs are also recognized for their antioxidative and anti-inflammatory activities. In this study, we evaluated the cytotoxic, genotoxic, and apoptotic activities, as well as effect on cell growth of ALEs on human colon cancer HT-29 and RKO cells. HT-29 and RKO cells exhibit a different p53 status: RKO cells express the wild-type protein, whereas HT-29 cells express a p53-R273H contact mutant. (2) Methods: Four different ALEs were obtained by sequential extraction of dried artichoke leaves; ALEs were characterized for their content in chlorogenic acid, cynaropicrin, and caffeoylquinic acids. HT-29 and RKO cells were used for in vitro testing (i.e., cytotoxicity and genotoxicity assessment, cell cycle analysis, apoptosis induction). (3) Results: Two out of the four tested ALEs showed marked effects on cell vitality toward HT-29 and RKO tumour cells. The effect was accompanied by a genotoxic activity exerted at a non-cytotoxic concentrations, by a significant perturbation of cell cycle (i.e., with increase of cells in the sub-G1 phase), and by the induction of apoptosis. (4) Conclusions: ALEs rich in cynaropicrin, caffeoylquinic acids, and chlorogenic acid showed to be capable of affecting HT-29 and RKO colon cancer cells by inducing favourable biological effects: cell cycle perturbation, activation of mitochondrial dependent pathway of apoptosis, and the induction of genotoxic effects probably mediated by the induction of apoptosis. Taken together, these results weigh in favour of a potential cancer chemotherapeutic activity of ALEs.