Assessment of the cytotoxic potential of an aqueous-ethanolic extract from Thalassia testudinum angiosperm marine grown in the Caribbean Sea.

OBJECTIVES: Reported antioxidant, anti-inflammatory and neuroprotective properties for one aqueous-ethanolic extract from Thalassia testudinum which grows in the Caribbean Sea compelled us to explore about extract cytotoxic effects. METHODS: Cell viability was assayed on tumour (HepG2, PC12, Caco-2 and 4T1) and non-tumour (VERO, 3T3, CHO, MCDK and BHK2) cell lines. The extract effects upon primary cultures of rat and human hepatocytes and human lymphocytes were assayed. KEY FINDINGS: The extract exhibited cytotoxicity against cancer cells compared to normal cells, and the IC50 values were 102 µg/ml for HepG2, 135 µg/ml for PC12, 165 µg/ml for Caco-2 and 129 µg/ml for 4T1 cells after 48 h, whereas IC50 could not be calculated for normal cells. Additional data from a high-content screening multiparametric assay indicated that after 24-h exposure, the extract (up to 100 µg/ml) induced death in HepG2 cells through oxidative stress-associated mechanism, DNA damage and hypercalcaemia. Comet assay corroborated extract-induced DNA damage. CONCLUSIONS: Thalassia testudinum extract is more cytotoxic and produced more DNA damage on human hepatoma cells than to other non-tumour cells. A possible mechanism is suggested for extract-induced cytotoxicity based on oxidative stress, nuclear damage and hypercalcaemia in HepG2 cells. T. testudinum may be a source for antitumour agents.

New microRNA Biomarkers for Drug-Induced Steatosis and Their Potential to Predict the Contribution of Drugs to Non-alcoholic Fatty Liver Disease.

Background and Aims: Drug-induced steatosis is a major reason for drug failure in clinical trials and post-marketing withdrawal; and therefore, predictive biomarkers are essential. These could be particularly relevant in non-alcoholic fatty liver disease (NAFLD), where most patients show features of the metabolic syndrome and are prescribed with combined chronic therapies, which can contribute to fatty liver. However, specific biomarkers to assess the contribution of drugs to NAFLD are lacking. We aimed to find microRNAs (miRNAs) responsive to steatotic drugs and to investigate if they could become circulating biomarkers for drug-induced steatosis. Methods: Human HepG2 cells were treated with drugs and changes in miRNA levels were measured by microarray and qRT-PCR. Drug-induced fat accumulation in HepG2 was analyzed by high-content screening and enzymatic methods. miRNA biomarkers were also analyzed in the sera of 44 biopsy-proven NAFLD patients and in 10 controls. Results: We found a set of 10 miRNAs [miR-22-5p, -3929, -24-2-5p, -663a, -29a-3p, -21 (5p and 3p), -27a-5p, -1260 and -202-3p] that were induced in human HepG2 cells and secreted to the culture medium upon incubation with model steatotic drugs (valproate, doxycycline, cyclosporin A and tamoxifen). Moreover, cell exposure to 17 common drugs for NAFLD patients showed that some of them (e.g., irbesartan, fenofibrate, and omeprazole) also induced these miRNAs and increased intracellular triglycerides, particularly in combinations. Finally, we found that most of these miRNAs (60%) were detected in human serum, and that NAFLD patients under fibrates showed both induction of these miRNAs and a more severe steatosis grade. Conclusion: Steatotic drugs induce a common set of hepatic miRNAs that could be used in drug screening during preclinical development. Moreover, most of these miRNAs are serum circulating biomarkers that could become useful in the diagnosis of iatrogenic steatosis.

DNA damage induced by an extract of Mangifera indica stem bark using in vitro Comet assay / Daño inducido al ADN por un extracto de la corteza de Mangifera indica mediante un ensayo Cometa in vitro

The aqueous standard extract of Mangifera indica L stem bark (MSBE) is used as a food supplement in Cuba. In this study, the genotoxic effect of MSBE was measured using different variants of the in vitro Comet assay in human lymphocytes and rat hepatocytes incubated with MSBE at 37C for 1 hour. Lymphocytes were incubated with MSBE for the subcellular (at two different pH conditions) and the standard Comet assays, in presence of catalase or S9 microsomal fraction. Hydrogen peroxide, benzo(a)pirene and UV radiation were used as positive controls. Results from standard and subcellular Comet assays clearly showed that MSBE (50 ug/mL) induced primary DNA damage to lymphocytes. This genotoxic effect was slightly reduced when lymphocytes were incubated with MSBE plus catalase, which suggests that hydrogen peroxide is involved in this DNA injury. S9 fraction also decreased MSBE-induced damage to DNA in human lymphocytes. Not genotoxic effect was observed when rat hepatocytes were exposed at MSBE, suggesting that the metabolic activity can be involved in the elimination of the DNA damage generated by the MSBE. In conclusion, MSBE causes primary DNA injury of human lymphocytes in vitro Comet assay, but not in rat hepatocytes in similar conditions.

El extracto acuoso de la corteza de Mangifera indica L. (MSBE) es usado como suplemento alimenticio en Cuba. En este estudio se determinaron los efectos genotóxicos de MSBE mediante diferentes variantes del ensayo Cometa in vitro en linfocitos humanos y hepatocitos de rata incubados con MSBE a 37C por 1 hora. Los linfocitos fueron incubados con MSBE para la realización de los ensayos Cometa subcelular (a dos condiciones de pH diferentes) y estándar, en presencia de catalasa o fracción microsomal S9. Peróxido de hidrógeno, benzo(a)pireno y radiación UV fueron usados como controles positivos. Los resultados de los ensayos Cometa, tanto subcelular como estándar, mostraron que MSBE (50 ug/mL) indujo daño primario al ADN de los linfocitos. Este efecto genotóxico fue ligeramente reducido cuando las células fueron incubadas con MSBE más catalasa, lo que sugiere que el peróxido de hidrógeno está involucrado en este daño al ADN. La fracción S9 también decreció el daño inducido por MSBE al ADN en linfocitos humanos. No fueron observados efectos genotóxicos cuando los hepatocitos de rata fueron expuestos a MSBE, sugiriendo que la actividad metabólica pudiera estar involucrada en la eliminación del daño al ADN generado por MSBE. En conclusión, MSBE causa daño primario al ADN de linfocitos humanos en el ensayo Cometa in vitro, pero no en hepatocitos de rata bajo condiciones similares.

Inhibition of human P450 enzymes by natural extracts used in traditional medicine.

Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts, two natural polyphenol-rich extracts, diminished CYP1A1/2 and 3A4 activities, but not the CYP2D6 activity. These results suggest that these herbs inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions.

Antioxidant and cytotoxic activities of canadine: biological effects and structural aspects.

The cytotoxic effects of four alkaloids, berberine, canadine, anonaine, and antioquine were evaluated using three different cell cultures, a primary culture (rat hepatocytes) and two cell lines (HepG2 and HeLa). Our results indicate that berberine, anonaine, and antioquine possess a significant the cytotoxic effect. In contrast, canadine does not possess cytotoxic effect at concentrations tested here. A molecular modeling study indicates that the quaternary nitrogen, the aromatic polycyclic and planar structure of berberine could be the pharmacophoric patron to produce the cytotoxic effect. In parallel our results demonstrated that canadine possess a significant antioxidant activity. Stereoelectronic aspects of this alkaloid were found to be closely related to those displayed by alpha-tocopherol and its water-soluble analogue trolox. The antioxidant activities of canadine, combined with its low-toxic effect, indicated that the potential of this alkaloid as a novel class of antioxidant agent is very interesting and deserves further research.

Transcription factors involved in the expression of SLC28 genes in human liver parenchymal cells.

Human nucleoside transporters are encoded by SLC28 (hCNTs) and SLC29 (hENTs) genes. These proteins mediate the uptake of anticancer and some antiviral drugs and are also suitable candidates to facilitate nucleoside-derived drug uptake into hepatocytes for detoxification. Despite the putative relevance of these genes in liver physiology, the human SLC28 and SLC29 expression pattern is not known and suitable cell models are not available. These issues have been addressed by examining NT expression in human liver and primary cultures of human hepatocytes. Moreover, the effect of specific liver enriched transcription factors (LETFs) in hCNTs expression has been analyzed. Human hepatocytes express hCNT1, hCNT2, hENT1, and hENT2. Loss of the hepatic phenotype in primary culture is associated with a decrease in hCNT1 and hCNT2 mRNA levels. Selected LETFs are involved in the regulation of SLC28 genes in an isoform-specific manner. HNF4alpha is a major determinant of SLC28A1 expression, whereas C/EBPalpha and HNF3gamma modulate SLC28A2 gene expression.