Evaluation of cytoprotective effects of compounds isolated from Copaifera langsdorffii Desf. against induced cytotoxicity by exposure to methylmercury and lead.

Copaifera langsdorffii L. is one of the most known medicinal species in Brazil. Its leaves are rich in phenolic compounds with potential biological activities as an antioxidant and chelating agent. This paper reports the isolation of four compounds from the hydroalcoholic extract of the leaves of C. langsdorffii and the investigation of their possible cytoprotective effects against heavy metal poisoning. Quercitrin (1), afzelin (2), 3,5-di-O-(3-O-methyl galloyl) quinic acid (3) and 4,5-di-O-(3-O-methyl galloyl) quinic acid (4), were associated with toxic doses of methylmercury and lead and evaluated by Alamar blue cell viability assays in HepG2 and PC12. The compounds displayed significant cytoprotective effect for the HepG2 cell line against both metals. Compounds 1-4 did not protect PC12 cells against methylmercury induced-cytotoxicity, but at lower concentrations, they protected against lead induced-cytotoxicity. The evaluated compounds showed a promising cytoprotection effect against exposure to heavy metals and should be further investigated as protective agents.

Analysis of the cytotoxic, genotoxic, mutagenic, and pro-oxidant effect of synephrine, a component of thermogenic supplements, in human hepatic cells in vitro.

Thermogenic supplements containing synephrine (SN) are widely used to weight loss. SN is a proto-alkaloid naturally found in the bark of immature fruits of Citrus aurantium (bitter orange) that has been added to thermogenic supplements due to its chemical and pharmacological similarity with adrenergic amines, such as ephedrine and amphetamines. Although orally ingested SN is mainly metabolized in the liver, it remains unclear whether it affects the redox status and genetic material of human hepatic cells. The present study aims to examine whether SN affects cell viability, cell cycle, redox balance, genomic stability, and expression of the DNA damage response (DDR)-related genes ATM, ATR, CHEK1, CHECK2, TP53, and SIRT1 in HepG2 cells - used as in vitro hepatocyte model. SN induced overproduction of intracellular reactive oxygen species (ROS) after 6 h of treatment with the three concentrations tested (2, 20 and 200 µM). After 24 h of treatment, SN at 200 µM induced intracellular ROS overproduction and exerted cytostatic effects, while SN at 20 and 200 µM increased the levels of GPx and GSH. SN was not cytotoxic (2-5000 µM), genotoxic, and mutagenic and did not alter the expression of DDR-related genes (2-200 µM), indicating that the fast/specific SN metabolization and upregulation of antioxidant defense components to detoxify intracellular ROS were sufficient to prevent intracellular damage in HepG2 cells. In conclusion, SN showed no cytotoxic, genotoxic, and mutagenic potential at relevant concentrations for thermogenic users in human hepatic cells in vitro, although, it plays pro-oxidative action, and cytostatic effects. Taken together, our results suggest that other investigations about the hazard absence of this thermogenic compound should be performed.

Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-glucoside, Safety and Anti-Inflammatory Effect.

Chamomile is a medicinal plant, which presents several biological effects, especially the anti-inflammatory effect. One of the compounds related to this effect is apigenin, a flavonoid that is mostly found in its glycosylated form, apigenin-7-glucoside (APG), in natural sources. However, the affectivity and safety of this glycoside have not been well explored for topical application. In this context, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC-DAD) method to quantify APG in chamomile preparations. Additionally, the safety and the anti-inflammatory potential of this flavonoid were verified. The RP-HPLC-DAD method was developed and validated with linearity at 24.0-36.0 µg/mL range (r = 0.9994). Intra- and interday precision (RSD) were 0.27-2.66% and accuracy was 98.27-101.21%. The validated method was applied in the analysis of chamomile flower heads, glycolic extract, and Kamillen cream, supporting the method application in the quality control of chamomile preparations. Furthermore, the APG safety was assessed by MTT cytotoxicity assay and mutagenic protocols and the anti-inflammatory activity was confirmed by a diminished TNF-α production showed by mice macrophages treated with APG following LPS treatment.

Bixin and norbixin protect against DNA-damage and alterations of redox status induced by methylmercury exposure in vivo.

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed "annatto" which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 µg/kg/bw/day), BIX (0.1-10 mg/kg/bw/day), NOR (0.01-1.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury.

Dietary carotenoid lutein protects against DNA damage and alterations of the redox status induced by cisplatin in human derived HepG2 cells.

Several epidemiological and experimental studies has been reported that lutein (LT) presents antioxidant properties. Aim of the present study was to investigate the protective effects of LT against oxidative stress and DNA damage induced by cisplatin (cDDP) in a human derived liver cell line (HepG2). Cell viability and DNA-damage was monitored by MTT and comet assays. Moreover, different biochemical parameters related to redox status (glutathione, cytochrome-c and intracellular ROS) were also evaluated. A clear DNA-damage was seen with cDDP (1.0µM) treatment. In combination with the carotenoid, reduction of DNA damage was observed after pre- and simultaneous treatment of the cells, but not when the carotenoid was added to the cells after the exposure to cDDP. Exposure of the cells to cDDP also caused significant changes of all biochemical parameters and in co-treatment of the cells with LT, the carotenoid reverted these alterations. The results indicate that cDDP induces pronounced oxidative stress in HepG2 cells that is related to DNA damage and that the supplementation with the antioxidant LT may protect these adverse effects caused by the exposure of the cells to platinum compound, which can be a good predict for chemoprevention.

Evaluation of the genotoxic and anti-genotoxic activities of silybin in human hepatoma cells (HepG2).

Silybin (SB), a constituent of the medicinal plant Silybum marianum, is reported to be a potent hepatoprotective agent, but little is currently known regarding its genotoxicity, mutagenicity and potential chemopreventive properties. In this study, we evaluated the ability of SB to induce DNA migration and micronuclei (MN) formation in human hepatoma cells (HepG2). Also, possible preventive effects of SB on MN formation induced by three different mutagens, bleomycin (BLEO), benzo[a]pyrene (B[a]P) and aflatoxin B(1) (AFB(1)), were studied. To clarify the possible mechanism of SB antimutagenicity, three treatment protocols were applied: pretreatment, in which SB was added before the application of the mutagens; simultaneous treatment, in which SB was added during treatment and post-treatment, in which SB was added after the application of the mutagens. At concentrations up to 100 microM, SB was non-genotoxic, while at a concentration of 200 microM, SB induced DNA migration, generated oxidized DNA bases, reduced cell viability, decreased the replicative index of the cells and induced oxidative stress. It is noteworthy that SB was able to reduce the genotoxic effect induced by B[a]P, BLEO and AFB(1) in pretreatment and simultaneous treatments but had no significant effect on DNA damage induction in post-treatment. Taken together, our findings indicate that SB presents anti-genotoxic activity in vitro, which suggests potential use as a chemopreventive agent.

Effect of annatto on micronuclei induction by direct and indirect mutagens in HepG2 cells.

Annatto (AN), a natural food colorant rich in carotenoids, has been reported as being an effective antioxidant, but little is known about its potential chemopreventive properties. In this study, we evaluated the ability of AN to protect human hepatoma cells (HepG2) from micronucleus (MN) induction against three different mutagens: benzo(a)pyrene (B(a)P), doxorubicin (DXR), and methyl methanesulfonate (MMS). In an attempt to clarify the possible mechanism of antimutagenicity of AN, three protocols of treatment were applied (pretreatment; simultaneous treatment, and post-treatment with AN following treatment with the mutagens). Also, cells exposed only to AN were assayed for cytotoxicity and mutagenicity. A dosage up to 10 microg/ml of AN was devoid of mutagenic activity. Protective effects were seen on micronuclei induced by B(a)P and DXR using pre and simultaneous treatment, but AN had no significant effect on MN induction by MMS in any of the protocols. Our results also show that exposure of cells to concentrations of AN higher than 10 microg/ml decreased cell viability. Taken together, our findings indicate that AN presents antimutagenic activity in vitro, but its protective effect is dependent on the mutagen and on type of treatment suggesting its potential use as a chemopreventive agent.

Evaluation of mutagenicity and antimutagenicity of cashew stem bark methanolic extract in vitro.

Plants remain to be a great source of medicines for humanity. Among them is the Anacardium occidentale L. species, popularly known as the cashew, which has several therapeutic indications, such as cicatrizing, antihypertensive, hypoglycemic and antitumoral properties. The objective of the present work was to assess the mutagenicity and antimutagenicity of cashew stem bark methanolic extract (CSBME) on cell cultures of Chinese hamster lung fibroblasts (V79). The cultures were treated with different concentrations of the CSBME (500, 1000 and 2000 microg/mL) or with the extract associated with doxorubicin (DXR, 0.75 microg/mL) during the cell cycle phases G1, S, and G2 and throughout continuous treatment. Apart from these treatments, the cell cultures were also treated with PBS (negative-control) and DXR (positive-control). The data obtained in the chromosome aberrations (CA) test showed a significant reduction in CA frequency in the cultures treated with DXR and extract in comparison with those that received only DXR during the cell cycle phases G1 and S and throughout the entire cycle, as well as the absence of mutagenicity in all the treatments realized. The antimutagenic effect observed in this work reinforces the presence of the previously described therapeutic properties of cashew and indicates the safe use of this extract.