Acute and subchronic toxicity studies of rhein in immature and d-galactose-induced aged mice and its potential hepatotoxicity mechanisms.

Rhein is a key ingredient in many herbal remedies and is widely used. However, herbs containing rhein are frequently associated with poisoning incidents, especially in elderly subjects. Acute and subchronic toxicity of rhein in Kunming mice (KM) was investigated in this experiment. Acute toxicity tests showed a 40% lethality at a given rhein dose of 4000 mg/kg, and the LD50 of rhein was calculated by the bliss method to be greater than 2185.6 mg/kg. In subchronic toxicity, d-gal-induced aged and immature animals were randomized into three groups that were exposed to rhein of 0, 175, and 375 mg/kg/d for 75 days, respectively. No mortality was observed in immature mice group, whereas 55.5% (5/9) subjects in aged mice groups died in the high dosage group. AST, ALT, IL-6, TNF-α levels and typical histopathological changes indicate that rhein causes liver injury. In addition, our investigation explored possible hepatotoxic mechanisms of rhein and experimental results showed increased ROS production, NRF-2 and MDA levels and decreased SOD levels, demonstrating that rhein causes oxidative stress. MMP and mitochondrial swelling levels were able to assess the impact of rhein on mitochondrial function. Furthermore, the effect of rhein on apoptosis can be detected by flow cytometry. Our studies suggested that rhein induces oxidative stress leading to mitochondria dysfunction and apoptotic activation. Multidrug resistance protein (MRP) is an efflux transporter protein and is capable of transporting cellular oxidative stress-related substances. To further clarify the role of MRP in rhein induced oxidative stress, we examined MRP expression in the liver. However, the expression of MRP has no statistical significance.

Aloe-emodin, a hydroxyanthracene derivative, is not genotoxic in an in vivo comet test.

Aloe-emodin, one of the molecules belonging to the group of hydroxyanthracene derivatives, was recently described as genotoxic in vivo. Indeed, the EFSA judged that aloe-emodin, together with other similar molecules (emodin and danthron) and extracts from the leaf of Aloe species containing hydroxyanthracene derivatives, could represent a risk factor for colorectal cancer mediated by a genotoxic effect. Given the marked uncertainty regarding the conclusions in the opinion of the EFSA ANS Panel and conflicts in the epidemiological data on which the opinion is based, a new in vivo study (in vivo alkaline comet assay in mice - OECD 489) was conducted to test the potential genotoxicity of aloe-emodin at doses of 250, 500, 1000 and 2000 mg/kg bw/day on preparations of single cells from the kidney and colon of treated male mice. Following treatment with the test item, no clinical signs were observed in animals in any treatment group. Slight body-weight loss was randomly observed in all groups treated with the test item and was more evident in the groups dosed at 1000 and 2000 mg/kg bw/day. Under these experimental conditions, aloe-emodin showed no genotoxic activity. Possible oxidative damage to colon tissues could not be excluded based on the results obtained after repair enzyme treatment.

Marine Anthraquinones: Pharmacological and Toxicological Issues.

The marine ecosystem, populated by a myriad of animals, plants, and microorganisms, is an inexhaustible reservoir of pharmacologically active molecules. Among the multiple secondary metabolites produced by marine sources, there are anthraquinones and their derivatives. Besides being mainly known to be produced by terrestrial species, even marine organisms and the uncountable kingdom of marine microorganisms biosynthesize anthraquinones. Anthraquinones possess many different biological activities, including a remarkable antitumor activity. However, due to their peculiar chemical structures, anthraquinones are often associated with toxicological issues, even relevant, such as genotoxicity and mutagenicity. The aim of this review is to critically describe the anticancer potential of anthraquinones derived from marine sources and their genotoxic and mutagenic potential. Marine-derived anthraquinones show a promising anticancer potential, although clinical studies are missing. Additionally, an in-depth investigation of their toxicological profile is needed before advocating anthraquinones as a therapeutic armamentarium in the oncological area.

Cytotoxicity and antigenotoxicity evaluation of acetylshikonin and shikonin.

Shikonin (SH) is used as a red pigment for food coloring and cosmetics, and has cytotoxic activity towards cancer cells. However, due to strong toxicity SH has limited potential as an anticancer drug. Acetylshikonin (ASH) is one of the SH derivatives with promising anticancer potential. In present study, we attempted to evaluate and compare the cytotoxicity of SH and ASH towards a normal cell line (V79) and in addition to evaluate their antigenotoxic activity. The evaluation was made with the use of the set of cytotoxicity assays with V79 line and the micronucleus test in vitro performed using clinafloxacin (CLFX), ethyl methanesulfonate (EMS) as direct genotoxins and cyclophosphamide (CPA) as indirect genotoxin. For CPA and EMS the simultaneous protocol was used and for CLFX three different variants were performed: pretreatment, simultaneous, and post-treatment. A higher cytotoxic effect was observed for SH. The EC50 values obtained for SH were approximately twofold lower compared to that of ASH. Moreover, ASH exhibited an antigenotoxic potential against CPA-induced genotoxicity, whereas SH has no activity. However, ASH increased the EMS-induced genotoxicity, when SH exhibited no effect. Both compounds decreased the genotoxicity of CLFX in pretreatment and simultaneous protocol. Based on the results of the present study it can be concluded that ASH is less cytotoxic than SH to normal cells and has comparable antigenotoxic potential.

Mutagenicity of Tectona grandis Wood Extracts and Their Ability to Improve Carbohydrate Yield for Kraft Cooking Eucalyptus Wood.

Considering the toxicity of the impurities of synthesized anthraquinone, this study clarified new catalytic compounds for kraft cooking with improved carbohydrate yield and delignification and less mutagenicity, which are important for ensuring the safety of paper products in contact with food. The 2-methylanthraquinone contents of teak (Tectona grandis) woods were 0.18-0.21%. Acetone extracts containing 2-methylanthraquinone from Myanmar and Indonesia teak woods as additives improved lignin removal during kraft cooking of eucalyptus wood, which resulted in kappa numbers that were 2.2-6.0 points lower than the absence of additive. Myanmar extracts and 2-methylanthraquinone improved carbohydrate yield in pulps with 1.7-2.2% yield gains. Indonesia extracts contained more deoxylapachol and its isomer than 2-methylanthraquinone. The residual content of 2-methylanthraquinone in the kraft pulp was trace. Although Ames tests showed that the Indonesia and Myanmar extracts were mutagenic to Salmonella typhimurium, 2-methylanthraquinone was not. The kraft pulp obtained with the additives should be safe for food-packaging applications, and the addition of 0.03% 2-methylanthraquinone to kraft cooking saves forest resources and fossil energy in industries requiring increased pulp yield.

Chrysophanol Attenuates Manifestations of Immune Bowel Diseases by Regulation of Colorectal Cells and T Cells Activation In Vivo.

Inflammatory bowel disease (IBD) is an immune disorder that develops due to chronic inflammation in several cells. It is known that colorectal and T cells are mainly involved in the pathogenesis of IBD. Chrysophanol is an anthraquinone family member that possesses several bioactivities, including anti-diabetic, anti-tumor, and inhibitory effects on T cell activation. However, it is unknown whether chrysophanol suppresses the activity of colorectal cells. In this study, we found that chrysophanol did not induce cytotoxicity in HT-29 colorectal cells. Pre-treatment with chrysophanol inhibited the mRNA levels of pro-inflammatory cytokines in tumor necrosis factor-α (TNF-α)-stimulated HT-29 cells. Western blot analysis revealed that pre-treatment with chrysophanol mitigates p65 translocation and the mitogen-activated protein kinase (MAPK) pathway in activated HT-29 cells. Results from the in vivo experiment confirmed that oral administration of chrysophanol protects mice from dextran sulfate sodium (DSS)-induced IBD. Chrysophanol administration attenuates the expression of pro-inflammatory cytokines in colon tissues of the DSS-induced IBD model. In addition, we found that oral administration of chrysophanol systemically decreased the expression of effector cytokines from mesenteric lymph nodes. Therefore, these data suggest that chrysophanol has a potent modulatory effect on colorectal cells as well as exhibiting a beneficial potential for curing IBD in vivo.

Electrochemical oxidation of indanthrene blue dye in a filter-press flow reactor and toxicity analyses with Raphidocelis subcapitata and Lactuca sativa.

Alternative routes to degrade dyes are of crucial importance for the environment. Hence, we report the electrochemical removal of indanthrene blue by using a boron-doped diamond anode, focusing on the toxicity of the treated solutions. Different operational conditions were studied, such as current density (5, 10, and 20 mA cm-2) and electrolyte composition (Na2SO4, Na2CO3, and NaNO3). Besides, the pH was monitored throughout the experiment to consider its direct influence on the ecotoxicity effects. The highest electrochemical oxidation efficiency, measured as color removal, was seen in the 180 min condition of electrolysis in 0.033 M Na2SO4, applying 20 mA cm-2, resulting in a color removal of nearly 91% and 40.51 kWh m-3 of energy consumption. The toxicity towards Lactuca sativa depends solely on pH variations being indifferent to color removal. While the inhibition concentration (IC50) for Raphidocelis subcapitata increases 20% after treatment (in optimized conditions), suggesting that the byproducts are more toxic for this specific organism. Our data highlight the importance of analyzing the toxicity towards various organisms to understand the toxic effect of the treatment applied.

Biotransformation and toxicity effect of monoanthraquinone dyes during Bjerkandera adusta CCBAS 930 cultures.

The aim of this study was to evaluate of possibility of biotransformation and toxicity effect of monoanthraquinone dyes in cultures of Bjerkandera adusta CCBAS 930. Phenolic compounds, free radicals, phytotoxicity (Lepidium sativum L.), ecotoxicity (Vibrio fischeri) and cytotoxicity effect were evaluated to determine the toxicity of anthraquinone dyes before and after the treatment with B. adusta CCBAS 930. More than 80% of ABBB and AB129 was removed by biodegradation (decolorization) and biosorption, but biodegradation using oxidoreductases was the main dye removing mechanism. Secondary products toxic to plants and bacteria were formed in B. adusta strain CCBAS 930 cultures, despite efficient decolorization. ABBB and AB129 metabolites increased reactive oxygen species (ROS) production in human fibroblasts, but did not increase LDH release, did not affect the resazurine reduction assay and did not change caspase-9 or caspase-3 activity.

Elucidation of electrocoagulation mechanism in the removal of Blue SI dye from aqueous solution using Al-Al, Cu-Cu electrodes - A comparative study.

This work presents the research on the treatment of an anthraquinone derivatives of disperse dye Blue SI from aqueous solution using aluminium for the optimization of operational parameters like pH, current density, addition of electrolyte, contact time for the color removal efficiency (CRE) and the results are compared with the performance of copper electrodes in electrocoagulation (EC). The parameters for maximum CRE was found with Al at current density 40 Am-2, time 10 min at pH 7, and for Cu at 60 Am-2 15 min, at pH 6 were optimized. The characterization of the treated water using HPLC, MS studies revealed intermediate compounds. From the XPS analysis of the sludge obtained, the mechanism of EC was deduced. Treated aqueous solution was studied for its phytotoxicity with Vigna radiata and ecotoxicity studies were conducted on Artemia salina to study the toxicity effect of the intermediatory products in the treated dye solution. Blue SI dye aqueous solution treated with aluminium electrodes shows no or lesser toxicity in plants as well as in ecotoxic study compared with copper electrodes.

Aloe-emodin: A review of its pharmacology, toxicity, and pharmacokinetics.

Aloe-emodin is a naturally anthraquinone derivative and an active ingredient of Chinese herbs, such as Cassia occidentalis, Rheum palmatum L., Aloe vera, and Polygonum multiflorum Thunb. Emerging evidence suggests that aloe-emodin exhibits many pharmacological effects, including anticancer, antivirus, anti-inflammatory, antibacterial, antiparasitic, neuroprotective, and hepatoprotective activities. These pharmacological properties lay the foundation for the treatment of various diseases, including influenza virus, inflammation, sepsis, Alzheimer's disease, glaucoma, malaria, liver fibrosis, psoriasis, Type 2 diabetes, growth disorders, and several types of cancers. However, an increasing number of published studies have reported adverse effects of aloe-emodin. The primary toxicity among these reports is hepatotoxicity and nephrotoxicity, which are of wide concern worldwide. Pharmacokinetic studies have demonstrated that aloe-emodin has a poor intestinal absorption, short elimination half-life, and low bioavailability. This review aims to provide a comprehensive summary of the pharmacology, toxicity, and pharmacokinetics of aloe-emodin reported to date with an emphasis on its biological properties and mechanisms of action.

Pharmacology, toxicity and pharmacokinetics of acetylshikonin: a review.

CONTEXT: Acetylshikonin, a naphthoquinone derivative, is mainly extracted from some species of the family Boraginaceae, such as Lithospermum erythrorhizon Sieb. et Zucc., Arnebia euchroma (Royle) Johnst., and Arnebia guttata Bunge. As a bioactive compound, acetylshikonin has attracted much attention because of its broad pharmacological properties. OBJECTIVE: This review provides a comprehensive summary of the pharmacology, toxicity, and pharmacokinetics of acetylshikonin focussing on its mechanisms on the basis of currently available literature. METHODS: The information of acetylshikonin from 1977 to 2020 was collected using major databases including Elsevier, Scholar, PubMed, Springer, Web of Science, and CNKI. Acetylshikonin, pharmacology, toxicity, pharmacokinetics, and naphthoquinone derivative were used as key words. RESULTS: According to emerging evidence, acetylshikonin exerts a wide spectrum of pharmacological effects such as anticancer, anti-inflammatory, lipid-regulatory, antidiabetic, antibacterial, antifungal, antioxidative, neuroprotective, and antiviral properties. However, only a few studies have reported the adverse effects of acetylshikonin, with respect to reproductive toxicity and genotoxicity. Pharmacokinetic studies demonstrate that acetylshikonin is associated with a wide distribution and poor absorption. CONCLUSIONS: Although experimental data supports the beneficial effects of this compound, acetylshikonin cannot be considered as a therapy drug without further investigations, especially, on the toxicity and pharmacokinetics.

[Study on potential hepatotoxicity of rhein in Rhei Radix et Rhizoma based on liver metabolism].

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.

Effects of inhibition of hepatic sulfotransferase activity on renal genotoxicity induced by lucidin-3-O-primeveroside.

Sulfotransferase 1A (SULT1A) expression is lower in the liver of humans than that of rodents. Therefore, species differences should be taken into consideration when assessing the risk of rodent hepatocarcinogens metabolically activated by SULT1A in humans. Although some renal carcinogens require SULT1A-mediated activation, it is unclear how SULT1A activity in the liver affects renal carcinogens. To explore the effects of SULT1A activity in the liver on genotoxicity induced by SULT1A-activated renal carcinogens, B6C3F1 mice or gpt delta mice of the same strain background were given lucidin-3-O-primeveroside (LuP), a hepatic and renal carcinogen of rodents, for 4 or 13 weeks, respectively, and pentachlorophenol (PCP) as a liver-specific SULT inhibitor, was given from 1 week before LuP treatment to the end of the experiment. A 4 week exposure of LuP induced lucidin-specific DNA adduct formation. The suppression of Sult1a expression was observed only in the liver but not in the kidneys of PCP-treated mice, but co-administration of PCP suppressed LuP-induced DNA adduct formation in both organs. Thirteen-week exposure of LuP increased mutation frequencies and cotreatment with PCP suppressed these increases in both organs. Given that intact levels of SULT activity in the liver were much higher than in the kidneys of rodents, SULT1A may predominantly activate LuP in the liver, consequently leading to genotoxicity not only in the liver but also in the kidney. Thus, species differences should be considered in human risk assessment of renal carcinogens activated by SULT1A as in the case of the corresponding liver carcinogens.

Apoptotic effects of rhein through the mitochondrial pathways, two death receptor pathways, and reducing autophagy in human liver L02 cells.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is a major component of many medicinal herbs such as Rheum palmatum L. and Polygonum multiflorum. Despite being widely used, intoxication cases associated with rhein-containing herbs are often reported. Currently, there are no available reports addressing the effects of rhein on apoptosis in human liver L02 cells. Thus, the aim of this study is to determine the cytotoxic effects and the underlying mechanism of rhein on human normal liver L02 cells. In the present study, the methyl thiazolyl tetrazolium assay demonstrated that rhein decreased the viability of L02 cells in dose-dependent and time-dependent ways. Rhein was found to trigger apoptosis in L02 cells as shown by Annexin V-fluoresceine isothiocyanate (FITC) apoptosis detection kit and cell mitochondrial membrane potential (MMP) assay, with nuclear morphological changes demonstrated by Hoechst 33258 staining. Detection of intracellular superoxide dismutase activity, lipid oxidation (malondialdehyde) content, and reactive oxygen species (ROS) levels showed that apoptosis was associated with oxidative stress. Moreover, it was observed that the mechanism implicated in rhein-induced apoptosis was presumably via the death receptor pathway and the mitochondrial pathway, as illustrated by upregulation of TNF-α, TNFR1, TRADD, and cleaved caspase-3, and downregulation of procaspase-8, and it is suggested that rhein may increase hepatocyte apoptosis by activating the increase of TNF-α level. Meanwhile, rhein upregulates the expression of Bax and downregulates the expression of procaspase-9 and -3, and it is suggested that the mitochondrial pathway is activated and rhein-induced apoptosis may be involved. In addition, we also want to explore whether rhein-induced apoptosis is related to the autophagic changes induced by rhein. The results showed that rhein treatment increased P62 and decreased LC3-II and beclin-1, which means that autophagy was weakened. The results of our studies indicated that rhein induced caspase-dependent apoptosis via both the Fas death pathway and the mitochondrial pathway by generating ROS, and meanwhile the autophagy tended to weaken.

Metabolomics of Aurantio-Obtusin-Induced Hepatotoxicity in Rats for Discovery of Potential Biomarkers.

Aurantio-obtusin is an anthraquinone derived from Cassia obtusifolia (cassiae semen). It is also used as a tool and a detection index for the identification of cassiae semen, as stipulated by the Chinese Pharmacopoeia. Anthraquinones, the main components in cassiae semen, have been reported to show hepatotoxicity. This study investigates the hepatotoxicity of aurantio-obtusin in male Sprague-Dawley rats. We randomly divided the animals into a blank control group and treated three test groups with different doses of aurantio-obtusin: Low dose (4 mg/kg), medium dose (40 mg/kg), and high dose (200 mg/kg). Each group was treated with aurantio-obtusin for 28 days, whereas the control group was administered an equal volume of 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution. Subsequently, we conducted biochemical, hematological, and pathological investigations and determined the weight of different organs. We used serum metabolomics to identify possible biomarkers related to hepatotoxicity. The low-dose group showed no significant liver injury, whereas the medium- and high-dose groups manifested obvious liver injury. Compared with the control group, the test groups showed an increase in alanine transaminase, aspartate transaminase, and alkaline phosphatase levels. The liver organ coefficient also significantly increased. Additionally, we found significant changes in the hematological indices. Metabolomics analysis showed that aurantio-obtusin induced 28 endogenous markers related to liver injury. Our data indicate that aurantio-obtusin induces hepatotoxicity in rat liver in a dose-dependent manner and is mediated by pathways involving bile acids, fatty acids, amino acids, and energy metabolism. In particular, changes in bile acid content during treatment with therapeutic agents containing aurantio-obtusin deserve increased attention.

Synthesis and Biological Evaluation of Rhein-Based MRI Contrast Agents for in Vivo Visualization of Necrosis.

Early and accurate assessment of therapeutic response to anticancer therapy plays an important role in determining treatment planning and patient management in clinic. Magnetic rseonance imaging (MRI) of necrosis that occurs after cancer therapies provides chances for that. Here, we reported three novel MRI contrast agents, GdL1, GdL2, and GdL3, by conjugating rhein with gadolinium 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic acid (Gd-DOTA) through different linkers. The T1 relaxivities of three probes (7.28, 7.35, and 8.03 mM-1 s-1) were found to be higher than that of Gd-DOTA (4.28 mM-1 s-1). Necrosis avidity of GdL1 was evaluated on the rat models of reperfused liver infarction (RLI) by MRI, which showed an increase of T1-weighted contrast between necrotic and normal liver during 0.5-12 h. Besides, L1 was also labeled with 64Cu to assess its necrosis avidity on rat models of RLI and muscle necrosis (MN) by a γ-counter. The uptakes of 64CuL1 in necrotic liver and muscle were higher than those in normal liver and muscle ( P < 0.05). Then, the ability of GdL1 to assess therapeutic response was tested on rats bearing Walker 256 breast carcinoma injected with a vascular disrupting agent CA4P by MR imaging. The signal intensity of tumoral necrosis was strongly enhanced, and the contrast ratio between necrotic and viable tumor was 1.63 ± 0.11 at 3 h after administration of GdL1. Besides, exposed DNA in necrosis cells may be an important mechanism of three probes targeting to necrosis cells. In summary, GdL1 may serve as a promising MRI contrast agent for accurate assessment of treatment response.

Probing cytochrome P450 bioactivation and fluorescent properties with morpholinyl-tethered anthraquinones.

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring.

Comparative study of eco- and cytotoxicity during biotransformation of anthraquinone dye Alizarin Blue Black B in optimized cultures of microscopic fungi.

The aim of this study was to select optimal conditions (C and N sources, initial pH and temperature) for biodecolorization of 0.03% anthraquinone dye Alizarin Blue Black B (ABBB) by microscopic fungi: Haematonectria haematococca BwIII43, K37 and Trichoderma harzianum BsIII33. The phenolic compounds, phytotoxicity (Lepidium sativum L.), biotoxicity (Microtox), cytotoxicity and yeast viability assay were performed to determine the extent of ABBB detoxification. Biodecolorization and detoxification of 0.03% ABBB in H. haematococca BwIII43 and T. harzianum BsIII33 cultures was correlated with extracellular oxidoreductases activity. In turn, secondary products, toxic to human fibroblasts and respiring sod1 Saccharomyces cerevisiae cells, were formed in H. haematococca K37 strain cultures, despite efficient decolorization.

Subchronic toxicity, immunoregulation and anti-breast tumor effect of Nordamnacantal, an anthraquinone extracted from the stems of Morinda citrifolia L.

BACKGROUND: Morinda citrifolia L. that was reported with immunomodulating and cytotoxic effects has been traditionally used to treat multiple illnesses including cancer. An anthraquinone derived from fruits of Morinda citrifolia L., nordamnacanthal, is a promising agent possessing several in vitro biological activities. However, the in vivo anti-tumor effects and the safety profile of nordamnacanthal are yet to be evaluated. METHODS: In vitro cytotoxicity of nordamnacanthal was tested using MTT, cell cycle and Annexin V/PI assays on human MCF-7 and MDA-MB231 breast cancer cells. Mice were orally fed with nordamnacanthal daily for 28 days for oral subchronic toxicity study. Then, the in vivo anti-tumor effect was evaluated on 4T1 murine cancer cells-challenged mice. Changes of tumor size and immune parameters were evaluated on the untreated and nordamnacanthal treated mice. RESULTS: Nordamnacanthal was found to possess cytotoxic effects on MDA-MB231, MCF-7 and 4T1 cells in vitro. Moreover, based on the cell cycle and Annexin V results, nordamnacanthal managed to induce cell death in both MDA-MB231 and MCF-7 cells. Additionally, no mortality, signs of toxicity and changes of serum liver profile were observed in nordamnacanthal treated mice in the subchronic toxicity study. Furthermore, 50 mg/kg body weight of nordamncanthal successfully delayed the progression of 4T1 tumors in Balb/C mice after 28 days of treatment. Treatment with nordamnacanthal was also able to increase tumor immunity as evidenced by the immunophenotyping of the spleen and YAC-1 cytotoxicity assays. CONCLUSION: Nordamnacanthal managed to inhibit the growth and induce cell death in MDA-MB231 and MCF-7 cell lines in vitro and cease the tumor progression of 4T1 cells in vivo. Overall, nordamnacanthal holds interesting anti-cancer properties that can be further explored.

Chemical marking of European glass eels Anguilla anguilla with alizarin red S and in combination with strontium: in situ evaluation of short-term salinity effects on survival and efficient mass-marking.

This study presents a new chemical double-marking technique for European glass eels Anguilla anguilla by combing alizarin red S (ARS) and strontium chloride hexahydrate (Sr). Marked eels (double marked with ARS and Sr, but also single marked with ARS) were exposed in situ to brackish water (15 g l-1 artificial sea salt) for 14 days and did not exhibit increased mortalities compared with unmarked eels. Indeed, no mortality occurred in a marked group during the experiments. Moreover, an efficient mass-marking approach with low handling effort for both single ARS and double ARS-Sr techniques is described and was proven to be practicable for large-scale stocking programmes.