In vitro and ex vivo angiogenic effects of roxarsone on rat endothelial cells

Roxarsone, a feed additive, is being used worldwide to promote animal growth. However, the potential effect of roxarsone on angiogenesis has not been extensively characterized. We examined the ability of roxarsone to promote angiogenesis of rat endothelial cells in vitro and from rat aorta rings ex vivo. Endothelial cells from rats were exposed to 0.01–10.00 μM roxarsone, 5 ng/mL vascular endothelial growth factor (VEGF) as a positive control or phosphate buffer saline (PBS) as a negative control. Cell proliferation was measured by MTT assay, and the content of VEGF in supernatants was measured by enzyme-linked immunosorbent assay and Western blotting. A Matrigel-induced tube formation assay was used to evaluate the effects of roxarsone on endothelial cells. Additionally, the total number and length of microvessels sprouted from rat aortic rings were measured for ex vivo investigation of angiogenesis. Results showed that the cell viability and total number and length of capillary-like tube formations after roxarsone treatment was significantly higher than that of negative (P < 0.05), with a maximum effect at 1.00 μM exposure. Furthermore, the number of microvessels sprouted from aortic rings treated for 4 h with 0.1–10.0 μM roxarsone was significantly higher than that of PBS treatment, with a peak value of 1.0 μM. These results further demonstrate the potential of roxarsone to promote angiogenesis in vitro and ex vivo.     

Effects of novel brominated flame retardants on steroidogenesis in primary porcine testicular cells

Brominated flame retardants are chemicals with fire quenching properties which are extensively used in manufacturing. Historically, less regulated use of legacy brominated flame retardants (BFRs) for a number of years has resulted in ubiquitous contamination of the environment. As a result, some of the more persistent BFRs have been phased out and are being replaced by a next generation of brominated compounds for which there is little toxicological data. The study investigated effects of 2-ethylhexyl tetrabromobenzoate (TBB), 1,2,5,6-tetrabromocyclooctane (TBCO), and bis-(2-ethylhexyl) tetrabromophthalate (TBPH) on steroidogenesis in a porcine primary testicular cell model. TBB did not affect sex-steroid production in this cell model; rather the data suggest a flux towards synthesis of aldosterone and cortisol via up-regulation of CYP21A2. At the greatest concentrations of TBCO and TBPH tested greater production of sex hormones testosterone (T) and estradiol (E2) was observed. Effects were mediated by regulation of multiple molecular targets in the steroidogenesis pathway; CYP11A in the case of TBPH and CYP17A1 in the case of TBCO. This investigation is the first of its kind to use a testicular mixed population cell model to investigate mechanism(s) of action of three chemically diverse compounds currently used in commercial fire retardants.     

First insights in the metabolism of phosphate flame retardants and plasticizers using human liver fractions

Phosphate flame retardants and plasticizers (PFRs) are additives used in a wide range of polymers. Important representatives, such as tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), have been found in the indoor environment at high levels. Biotransformation of these PFRs needs to be investigated because it can be a major determinant of their bioavailability and toxicity in humans. TBOEP, TPHP, TCEP, TCIPP and TDCIPP were incubated with human liver S9 fraction and microsomes. Supernatants were analyzed using a liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer. Chromatograms were scanned for the presence of Phase-I and Phase-II metabolites and tentatively identified based on mass accuracy of the molecular formula, isotopic pattern, and MS/MS spectra. The two major metabolites of TBOEP were products of O-dealkylation and of hydroxylation, respectively. TPHP was mainly transformed to its diester metabolite by O-dearylation and to a hydroxylated metabolite. TCEP was poorly metabolized into its diester and a product of oxidative dehalogenation. The major metabolite of TCIPP was a product of oxidative dehalogenation. TDCIPP was mainly transformed into its diester and a glutathione S-conjugate. The metabolites identified in the present study are candidate biomarkers for future human biomonitoring studies.     

In vitro assessments of nanomaterial toxicity

Nanotechnology has grown from a scientific interest to a major industry with both commodity and specialty nanomaterial exposure to global populations and ecosystems. Sub-micron materials are currently used in a wide variety of consumer products and in clinical trials as drug delivery carriers and imaging agents. Due to the expected growth in this field and the increasing public exposure to nanomaterials, both from intentional administration and inadvertent contact, improved characterization and reliable toxicity screening tools are required for new and existing nanomaterials. This review discusses current methodologies used to assess nanomaterial physicochemicial properties and their in vitro effects. Current methods lack the desired sensitivity, reliability, correlation and sophistication to provide more than limited, often equivocal, pieces of the overall nanomaterial performance parameter space, particularly in realistic physiological or environmental models containing cells, proteins and solutes. Therefore, improved physicochemical nanomaterial assays are needed to provide accurate exposure risk assessments and genuine predictions of in vivo behavior and therapeutic value. Simpler model nanomaterial systems in buffer do not accurately duplicate this complexity or predict in vivo behavior. A diverse portfolio of complementary material characterization tools and bioassays are required to validate nanomaterial properties in physiology.     

In vitro sensitivity of granulo-monocytic progenitors as a new toxicological cell system and endpoint in the ACuteTox Project

The ACuteTox Project (part of the EU 6th Framework Programme) was started up in January 2005. The aim of this project is to develop a simple and robust in vitro strategy for prediction of human acute systemic toxicity, which could replace animal tests used for regulatory purposes. Our group is responsible for the characterization of the effect of the reference chemicals on the hematopoietic tissue. CFU-GM assay based on the culture of human mononuclear cord blood cells has been used to characterize the effects of the selected compounds on the myeloid progenitors. Previous results have shown the relevance of the CFU-GM assay for the prediction of human acute neutropenia after treatment of antitumoral compounds, and this assay has been recently approved by the ECVAM's Scientific Advisory Committee. Among the compounds included in the study there were pharmaceuticals, environmental pollutants and industrial chemicals. Eleven out of 55 chemicals did not show any cytotoxic effect at the maximum concentration tested. The correlation coefficients of CFU-GM IC50, IC70 and IC90 values with human LC50 values (50% lethal concentration calculated from time-related sublethal and lethal human blood concentrations) were 0.4965, 0.5106 and 0.5142 respectively. Although this correlation is not improve respect to classical in vitro basal cytotoxicity tests such as 3T3 Neutral Red Uptake, chemicals which deviate substantially in the correlation with these assays (colchicine, digoxin, 5-Fluorouracil and thallium sulfate) fitted very well in the linear regression analysis of the CFU-GM progenitors. The results shown in the present study indicate that the sensitivity of CFU-GM progenitors correlates better than the sensitivity of HL-60 cells with human LC50 values and could help to refine the predictability for human acute systemic toxicity when a given chemical may affect to the hematopoietic myeloid system.     

Differential immunotoxic effects of inorganic and organic mercury species in vitro

Despite the fact that humans are exposed to multiple forms of mercury (elemental, inorganic, and organic), most research on mercury toxicity has focused on methylmercury (MeHg) and on neurotoxic outcomes and mechanisms. Recent work has indicated that the immunotoxic effects of mercury compounds may be significant contributors to human disease as well as mechanistically relevant to other target organ toxicities. In this study, we compared the effects of inorganic Hg (iHg) to organic Hg species (MeHg and ethylmercury, EtHg) in human peripheral blood mononuclear cells (PBMCs) in vitro at sub-cytotoxic concentrations, using methods developed to characterize response of human PBMCs to iHg in vitro. PBMCs were isolated from six volunteer blood donors (three males and three females) and cultured in the presence and absence of lipopolysaccharide (LPS) and low levels (up to 200 nM of each Hg species, separately) for 24 h in culture. Cell culture supernatants were analyzed for cytokine concentrations with a bead-based multiplex assay.     

In vitro metabolism of 2-ethylhexyldiphenyl phosphate (EHDPHP) by human liver microsomes

2-ethylhexyl diphenyl phosphate (EHDPHP) is used as flame retardant and plasticizer additive in a variety of consumer products. Since EHDPHP is toxic to aquatic organisms and has been detected in environmental samples, concerns about human exposure and toxicity are emerging. With the aim of identifying human-specific metabolites, the biotransformation of EHDPHP was investigated using human liver microsomes. Using an in silico program (Meteor) for the prediction of metabolites, untargeted screening tools (agilent Mass Hunter) and a suitable analysis platform based on ultra-high performance liquid chromatography (UPLC) and quadrupole time-of-flight high resolution mass spectrometer (QTOF-MS), for the first time a wide variety of phases-I and II metabolites of EHDPHP were identified. Mono- and di-hydroxylated metabolites, keto metabolites, mixed keto and hydroxylated metabolites and diphenyl phosphate were the major phase-I metabolites of EHDPHP. Glucuronidated metabolites of phase-I metabolites of EHDPHP were also formed by human liver microsomes. Using these results, we propose a general metabolism pathway for EHDPHP in humans and a number of candidate biomarkers for assessing the human exposure to this ubiquitous phosphate flame retardant and plasticizer in future biomonitoring studies. Furthermore, we provide a template analytical approach based on the combination of untargeted and targeted screening and UPLC–QTOF-MS analysis suitable for use in future metabolism studies.     

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR.The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.     

In vitro studies on the metabolism of trabectedin (YONDELIS) in monkey and man, including human CYP reaction phenotyping

Trabectedin (YONDELIS^®) is a potent anticancer agent which was recently approved in Europe for the treatment of soft tissue sarcoma. The drug is currently also in clinical development for the treatment of ovarian carcinoma. In vitro experiments were conducted to investigate the hepatic metabolism of [¹⁴C]trabectedin in Cynomolgus monkey and human liver subcellular fractions. The biotransformation of trabectedin was qualitatively similar in 12,000 × g supernatants of both species, and all human metabolites were also produced by the monkey. The trabectedin metabolites were identified by QTOF mass spectrometry, and HPLC co-chromatography with reference compounds. Trabectedin was metabolized via different biotransformation pathways. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation and di-oxidation, carboxylic acid formation with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di- or tri-oxidation. Another metabolite resulted from O-demethylation at the trabectedin C subunit, and in addition, aliphatic ring opening of the methylene dioxybridge at the B domain was detected. Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. Human cDNA expressed CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 in E. coli membranes, but not CYP1B1, 2C19, and 4A11 were able to metabolize [¹⁴C]trabectedin. Experiments with chemical inhibitors and CYP inhibitory antibodies indicated that, at therapeutic levels, CYP3A4 is the main human CYP isoform involved in trabectedin's hepatic metabolism. In monkey and human liver microsomes, trabectedin was not substantially metabolized by glucuronidation.     

In vitro detection of contact allergens: Development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.     

Current in vitro methods in nanoparticle risk assessment: Limitations and challenges

Nanoparticles are an emerging class of functional materials defined by size-dependent properties. Application fields range from medical imaging, new drug delivery technologies to various industrial products. Due to the expanding use of nanoparticles, the risk of human exposure rapidly increases and reliable toxicity test systems are urgently needed. Currently, nanoparticle cytotoxicity testing is based on in vitro methods established for hazard characterization of chemicals. However, evidence is accumulating that nanoparticles differ largely from these materials and may interfere with commonly used test systems. Here, we present an overview of current in vitro toxicity test methods for nanoparticle risk assessment and focus on their limitations resulting from specific nanoparticle properties. Nanoparticle features such as high adsorption capacity, hydrophobicity, surface charge, optical and magnetic properties, or catalytic activity may interfere with assay components or detection systems, which has to be considered in nanoparticle toxicity studies by characterization of specific particle properties and a careful test system validation. Future studies require well-characterized materials, the use of available reference materials and an extensive characterization of the applicability of the test methods employed. The resulting challenge for nanoparticle toxicity testing is the development of new standardized in vitro methods that cannot be affected by nanoparticle properties.     

Systematic comparison of nutraceuticals and antioxidant potential of cultivated, in vitro cultured and commercial Melissa officinalis samples

Melissa officinalis (lemon balm) infusions are used worldwide for digestive, analgesic and other pharmaceutical applications. Herein, the nutraceuticals production and antioxidant potential in garden cultivated, in vitro cultured and two commercial samples (bags and granulated) of lemon balm was compared. The profile of in vitro cultured lemon balm is closer of garden cultivated sample than of both commercial samples (bag or granulate). It presented the highest levels of proteins and ash, and the lowest energetic value. The most favorable n6/n3 ration, as also the highest PUFA (mostly α-linolenic acid), tocopherols (including α-, γ- and δ-isoforms) and ascorbic acid contents were also observed in this sample. Nevertheless, it was the commercial bag lemon balm that gave the highest antioxidant activity and the highest levels of phenolics and flavonoids. As far as we kwon, this is the first comparison of nutraceuticals and antioxidant potential of cultivated, in vitro cultured and commercial lemon balm samples. Moreover, it proved that in vitro culture might be used to stimulate vitamins production.     

Extracts of Halenia elliptica exhibit antioxidant properties in vitro and in vivo

The antioxidant properties of different extracts of Halenia elliptica was investigated by employing various established in vitro systems. The results showed that various extracts possessed strong antioxidant activity in vitro, and the 70% methanol extract (ME) had the strongest antioxidant activity. Based on our in vitro results, ME was used for investigating the antioxidant properties of H. ellipticain vivo. The liver and kidney of CCl₄-intoxicated animals exhibited a significant decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels. Additionally, these organs exhibited a significant increase in the level of malondialdehyde (MDA). These changes were significantly reversed, in a dose-dependent manner, after treatment with ME and the standard treatment Vitamin E. Thus, it may be concluded that the ME possesses potent antioxidant properties, and might be valuable natural source of antioxidants that could be applicable to both the medical and food industries.     

In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells

Epidemiological studies show that exposure to nickel (Ni) compounds is associated with a variety of pulmonary adverse health effects, such as lung inflammation, fibrosis, emphysema and tumours. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. In the current study we used Calu-3, a well differentiated human bronchial cell line, to investigate in vitro the effect of Ni in soluble form (NiCl₂) and in the form of micro-sized Ni particles on the airway epithelium. For this purpose, we evaluated the effect of Ni compounds on the epithelial barrier integrity by monitoring the transepithelial electrical resistance (TEER) and on oxidative stress pathways by measuring reactive oxygen species (ROS) formation and induction of stress-inducible genes. Our results showed that exposure to NiCl₂ and Ni particles resulted in a disruption of the epithelial barrier function observed by alterations in TEER, which occurred prior to the decrease in cell viability. Moreover, Ni compounds induced oxidative stress associated with ROS formation and up-regulation of the stress-inducible genes, Metallothionein 1X (MT1X), Heat shock protein 70 (HSP70), Heme oxygenase-1 (HMOX-1), and gamma-glutamylcysteine synthetase (γGCS). Furthermore, we have demonstrated that the induced effects by Ni compounds can be partially attributed to the increase in Ni ions (Ni²⁺) intracellular levels.     

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data.Isolated human PBMC were exposed for 20 h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10 μg/ml) for 48 h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively.All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.     

In vitro food–drug interaction study: Which milk component has a decreasing effect on the bioavailability of ciprofloxacin?

The purpose of the present work was developing an in vitro dissolution test to highlight the possible molecular background causing ciprofloxacin (CPFX)–milk interaction. The in vitro dissolution of CPFX from film-coated tablets (Ciprinol^® 500 mg) was examined at different pH values, simulating certain parts of the gastrointestinal tract, in the presence of water, low-fat milk, casein- or calcium enriched water. In order to determine the amount of dissolved CPFX, solid phase extraction sample preparation followed by high performance liquid chromatography coupled with mass spectrometry was applied. Comparing the dissolution efficiency values in various media, it can be concluded, that casein has a more pronounced effect on the absorbable amount of the antibiotic at each pH value studied, than calcium. In the case of concomitant intake of CPFX film-coated tablet and milk or other dairy products not only the complexation with calcium, but also the adsorption of CPFX on the surface of proteins decreases the absorbable amount of CPFX.     

Smoke chemistry, in vitro and in vivo toxicology evaluations of the electrically heated cigarette smoking system series K

The Electrically Heated Cigarette Smoking System Series K (EHCSS) produces smoke through the controlled electrical heating of tobacco. Evaluation of the EHCSS was accomplished by comparison with commercial and reference cigarettes, using International Organization for Standardization (ISO) and alternative puffing regimens based on nicotine exposures measured in a short-term clinical study. Using the alternative puffing regimen and compared with conventional cigarettes on a per cigarette basis, the EHCSS had 50–60% reductions in tar and nicotine; at least 90% reductions in carbon monoxide, nitrogen oxides, 1,3-butadiene, isoprene, acrylonitrile, polyaromatic hydrocarbons, hydrogen cyanide, aromatic amines, tobacco specific nitrosamines, and phenol; and least a 40% reduction in 2-nitropropane. Other important smoke constituents in EHCSS smoke were reduced as well. The in vitro studies showed similar large reductions in biological activity. Ames mutagenicity of total particulate matter (TPM) from the EHCSS was reduced by 70–90%; cytotoxicity of the TPM was reduced by approximately 82% and 65% for the gas–vapor phase. In vivo testing under ISO smoking conditions in the mouse skin painting assay demonstrated later dermal tumor onset, lower dermal tumor incidence, reduced dermal tumor multiplicity, and a lower proportion of malignant dermal tumors in EHCSS smoke condensate-exposed mice. Thirty-five day and 90-day nose-only inhalation studies in rats showed reductions in pulmonary inflammation and other biological activity, including histopathological endpoints. We conclude that under the conditions of these in vitro and in vivo studies, the EHCSS demonstrated significantly lower biological activity compared to conventional cigarettes, and may suggest the potential for reductions in human smokers.     

In vivo effects of cnidarian toxins and venoms

Cnidarians (Coelenterates), a very old and diverse animal phylum, possess a wide variety of biologically active substances that can be considered as toxins. Anthozoan toxins can be classified into two chemically very different groups, namely polypeptide toxins isolated from sea anemones and diterpenes isolated from octocorals. Cubozoan and scyphozoan protein toxins have been the most elusive cnidarian toxins to investigate - despite a tremendous effort in the past few decades, very few of these large, relatively unstable protein toxins were isolated, but recently this has been achieved for cubozoan venoms. Hydrozoans mainly contain large proteins with physiological mechanisms of action similar to the sea anemone and jellyfish pore-forming toxins. This article will focus on the in vivo physiological effects of cnidarian toxins and venoms; their actions at the cellular level will only be considered to understand their actions at the organ and whole animal levels. An understanding of mechanisms underlying the in vivo toxic effects will facilitate the development of more effective treatments of cnidarian envenomations.     

Effects of low concentrations of benzene on human lung cells in vitro

Exposure to benzene causes health hazards to humans. The airway epithelium is a physical barrier to inhaled toxicants and particulates. This is an in vitro basic science study to evaluate the effects of benzene on lung cells without the inflammatory responses triggered by inhalation. Dose–response cytotoxicity was assessed using two cell lines: alveolar derived (A549) human epithelial adenocarcinoma and human lung (LL24) fibroblast. A549 cells were more resistant than LL24 fibroblast lung cells to benzene. LL24 cells demonstrated enhanced proliferation with diluted benzene solutions. Moreover, low concentrations of benzene enhanced telomerase activity in LL24 cells while no effects were observed in the adenocarcinoma cells. Proteolysis of lung matrix by matrix metalloproteinases (MMPs) is an early event observed in lung pathologies. Benzene increased MMP-2 and MMP-3 mRNA. Using the ratio (MMP-1 + MMP-2 + MMP-3)/(TIMP-1 + TIMP-2), as an index of prodestructive activity, we observed a dose-dependent increase. The overall higher expression levels of MMPs in benzene treated cells did not appear to be controlled by TIMPs, which are negatively correlated.     

In vitro protective effects of Thymus quinquecostatus Celak extracts on t-BHP-induced cell damage through antioxidant activity

The purpose of this study was to evaluate the antioxidative activities of water and 70% ethanolic extracts from the Thymus quinquecostatus Celak (TQC) for natural antioxidant source. The antioxidant activities were compared with other natural and synthetic antioxidants. The levels of total polyphenols and flavonoids were also determined. The extracts were found to have different levels of antioxidant properties in a few kind of assay. The results showed that higher radical scavenging activity, reducing power and antioxidant capacity in FRAP than those of BHT as a positive control. In addition, the extracts from the TQC leaf and stem showed stronger antioxidant activity than that of vitamin C, α-tocopherol in ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods. Cytoprotective and anti-apoptotic effect of water extracts from TQC was also prevented t-BHP-induced toxicity in Chang liver cells. Therefore, these results indicate that TQC extracts have antioxidant properties through its ability to enhance the cell viability, reduction of production of ROS, inhibition of oxidative damage, mitochondria dysfunction and ultimately inhibition of cell apoptosis. Based on the results described above, it is suggested that TQC has the potential to protect liver on t-BHP-induced cell damage and should be considered as a prospective functional food.