Selecting cells A specific biological question to be answered is critical to the success of a cell-based bioassay and must be very clear to choose the most adequate cells. The properties and sensitivity of the cells and their growth status are critical factors that affect antioxidant activity evaluation. In addition, cellular response to antioxidant components is quite complex, depending on the component, exposure dose, and time. Several cell types have been used to set up cell-based bioassays for assessing antioxidants activity of foods and dietary supplements at wide concentration ranges, usually selected based on the lack of cytotoxic effect and a realistic concentration range in human diet and serum.
Primary isolated rat hepatocytes have been used to evaluate the protective effects of melanoidins in adriamycin-induced oxidative stress (Valls-Bellés and others 2004). Fusi and others (2010) evaluated the protective effects of alpha-tocopherol in ochratoxin A (OTA) induced oxidative damage in primary porcine fibroblasts. These researchers found a different sensitivity of ear and embryonic fibroblasts both to the oxidative damage and the antioxidant treatment. Primary cells, isolated from human or animal tissue, could be a good cell-based model as they retain the majority of the in vivo functionality, but they survive and maintain their differentiation only few days in cell culture. If the primary cells are chosen, it must be considered that usually they derive from different individuals in each test; consequently, the reproducibility of results may significantly differ from one test to another. Therefore, when phenotypic screens with the use of primary cells are included in designing a cell-based bioassay, it should be taken into consideration that a constant supply of biologically homogenous cells to support a large scale study is necessary. Moreover, isolation procedure for primary cell culture preparation can reduce survival of the cells and cause changes in gene expression, metabolic activity, and the levels of enzymes involved in the oxidant and antioxidant systems. Halliwell (2003), discussing in details how the “culture shock” can affect cells survival and metabolic activity, concludes that the cells which do survive appear to be those that have adapted rapidly, and probably are not representative of the originally cells harvested from a tissue.
The use of immortalized cell lines for the purpose of antioxidant testing may be a desirable approach. A cell line basically comprises a phenotypically and genetically uniform population of individual cells that have been derived from a single tissue. Cell lines are robust, grow, and divide easily in culture, are simple to handle and may provide a good platform for cell-based assays. The availability of a wide number of human and animal cell lines with diverse genotypes and tissue-origins provides broad potential models for the study of various biological processes. In Table 2, examples of different cell lines that have been used for the development of cell-based bioassays for food antioxidant activity analysis are reported.
Baldi and others (2004) evaluated the protective effects of alpha-tocopherol in OTA induced oxidative damage. A panel of 5 well-characterized human and animal cell lines, SK-N-MC (human neuroblastoma), MDCK (Madin Darby canine kidney), AML-12 (mouse liver hepatocytes), LLC-PK1 (pig kidney), and BME-UV1 (bovine mammary epithelium), have been successfully used to investigate the effect of alpha-tocopherol on ROS production on OTA-treated cells. The researchers found that alpha-tocopherol, at concentrations of 10 mM, 10 μM, and 1 nM, significantly inhibited OTA-induced ROS production and that the inhibition was dose dependent. A significant difference in cell sensitivity was also found, with BME-UV1 and MDCK as the most sensitive cell lines. Vitamin E is a well known potent antioxidant and, therefore, is a good positive control to be used to set up a standardized cell-based antioxidant activity assay. Vitamin E function as a peroxyl radical scavenger that terminates chain reactions is well documented (Wang and Quinn 1999; Azzi 2007). The liver is the main target for antioxidant compounds once absorbed from the gastrointestinal tract and also the major place for xenobiotic metabolism. Therefore, studies dealing with the effect of dietary compounds at a physiological level in the liver of live animals and at a cellular level in cultured cells from hepatic origin have been widely used. HepG2 (human liver carcinoma) cells have been used to set up cell-based bioassays for assessing antioxidants and antioxidant activity of foods and dietary supplements (Eberhardt and others 2005; Wolfe and Liu 2007). Wolfe and Liu (2007) tested the antioxidant activity of pure phytochemical compounds and of selected fruit extracts. The antioxidant activity was measured as median effective dose (EC50) for a 50% inhibition of peroxyl radical-induced dichlorofluoorescin oxidation. The researchers found significant differences in the antioxidant activity of both pure phytochemicals (EC50 ranging from 5.92 to more than 250 μM) and fruit extracts, with blueberry showing the higher antioxidant activity (EC50= 3.44 μg/mL). The same researchers compared the activity of antioxidants in the cellular model with the ORAC assay, and they found no consistency in the order of antioxidant activity of fruit extracts in the different assays. Comparable results have been found in a study on the antioxidant activity of broccoli extracts at concentration of 0.5 and 2 mg/mL (Eberhardt and others 2005). Using the same HepG2 cell model, Goya and others (2009) carried out a comparative study, testing different dietary antioxidant groups, like phenolic compounds, coffee melanoidin, and selenomethyl selenocystein at concentrations of 5 to 10 μM, 0.5 μg/mL, and 1 μM, respectively. The defense against oxidative stress has been evaluated using a panel of biomarkers: cell viability, biomarkers of oxidative status, and assessment of the antioxidant non-enzymatic and enzymatic defense. The proposed cell culture model was able not only to evidence differences in the antioxidant activity of the tested compounds but also to assess differences in the mechanisms involved in the antioxidant activity. The choice of the immortalized cell lines for the purpose of antioxidant researches is critical, and it is fundamental that cell lines show no altered functional responses to oxidative stress. In the matter of this topic, the use of HepG2 received the criticism that this cell line has altered functional response (increased catalase mRNA expression) to oxidative stress and performs asymmetrical cell divisions that may cause a proportion of the cells in culture dysfunctional and in various stages of cell death (Honzel and others 2008). A human gastric adenocarcinoma (AGS) cell line has been used in a study to evaluate the antioxidant activity of several lentil cultivars (Xu and Chang 2010). The researchers chose this cell line due to the rapid proliferation properties of AGS and because of criticism received by the use of HepG2. The cell-based assay was able to detect differences in the antioxidant activity of the different cultivars of lentils, with EC50 values ranging from 0.68 to 1.44 mg/mL of lentil extracts. However, the researchers conclude that they do not know if the AGS cell line is the best choice for cellular antioxidant assay and suffers in the same manner as the HepG2 cell lines. Several other cell lines have been tested to set up sensitive cell-based bioassays. A murine fibrosarcoma cell line (L-929) has been used to test the antioxidant activity of fruit and vegetable juices (Girard-Lalancette and others 2009). These reseachers found EC50 values ranging from 14 to 1119 μg/mL for broccoli and peaches, respectively. EC50 values in the same range have been reported for the antioxidant properties of cereal extracts by Hu and others (2007), using a mouse macrophage RAW264.7 cell line. Serra and others (2010) used a human gastric cancer (MKN-45) cell line to test the antioxidant activity of apple varieties. Apple extracts from different varieties were able to inhibit the proliferation of MKN-45 cells (cell viability was measured by MTT assay), with EC50 values ranging from 9 to 26 mg/mL. The intestine, as an important internal environment where a number of processes occurs to nourish the body and protect it against the enteropathogens or harmful substances entering the gut, is another important target for antioxidant compounds. Caco-2 (human epithelial colorectal adenocarcinoma), HT29 (human colon adenocarcinoma grade II), and Int-407 (human embryonic intestinal) cells have been used with good results for food antioxidant activity evaluation (Liu and others 2004; Boyer and others 2004, 2005; Elisia and others 2007; Bellion and others 2009; Serra and others 2010).
Recently, several efforts have been made to set up cell-based bioassays for antioxidant activity evaluation using erythrocytes (RBC) and polymorphonuclear (PMN) cells (Honzel and others 2008; Jensen and others 2008; Blasa and others 2011). According to the researchers, red blood cells may represent a good and biologically relevant model for a cell-based bioassay, as RBC play a critical role in antioxidant protection in the blood, by scavenging reactive oxygen and nitrogen species. As RBC do not have mitochondria, the use of this cell model may reduce the confounding contribution of cellular signaling. The PMN cells type could be also a useful model for assessment of overall antiinflammatory activity against an immune supportive property of a product. Honzell and others (2008) compared chemical (ORAC) and cell-based methods for evaluation of antioxidant activity of 1 animal-based, 1 microbial-based, 1 plant-based, and 1 mixed natural product. Total of 2 cellular models have been used: RBC and PMN cells, to measure antioxidant protection and reactive oxygen species formation, respectively. The researchers found different results comparing the data obtained with the 2 models and concluded that the antioxidants alone could not account for the strong antiinflammatory effect on PMN cells. The same RBC and PMN cell assays have been used by Jensen and others (2008) to evaluate the antioxidant effects of a fruit and belly juice. This juice, at concentration ranging from 0.01 to 10 g/L, was able to protect RBC from oxidative damage and reduce ROS production by PMN cells. Moreover, PMN cells showed altered migratory behavior. The possibility to evaluate the activity of antioxidants in a non-inflammatory red blood cell-based system and in inflammatory PMN cells, open new research field for the development of specific custom bioassays to evaluate specific food properties. Blasa and others (2011), testing the effects of phytochemicals and botanical extracts in a RBC assay, found significant differences in the antioxidant activity of the tested compounds. The model was useful for evaluating synergistic or antagonistic effects of combination of extracts. The phenomenon of the synergy is very important to be evaluated. Food may contain a complex mixture of bioactive compounds which are relevant in health and nutrition. The synergistic effects of phytochemicals in fruits and vegetables have been proposed to be responsible for their potent antioxidant and anticancer activity (Yang and Liu 2009). The possibility to evaluate and quantify the synergy which may occur with different compounds, could be of great interest to formulate fortified food and feed and tailor-made additives. All results taken together, underline how the choice of the cell type is critical to develop standardized cell-based assays for antioxidant research, which allow getting reliable and lab-to-lab reproducible results to be transferred into human and animal nutrition.
Selecting specific biomarkers as an endpoint to measure Antioxidants may operate via multiple mechanisms, briefly described before. Antioxidants can act at the cell membrane and break peroxyl radical chain reactions at the cell surface, or they can be taken up by the cell and react with ROS intracellularly. Therefore, the efficiency of membrane binding and/or cellular uptake, combined with the radical scavenging activity, likely dictates the efficacy of the tested compound. As the cell response to antioxidant components is quite complex, specific biomarkers as endpoints to measure must be identified. Cell-based assay format can be simple viability assays measuring the effect of compounds on cellular growth and/or viability, other can be metabolic assays. Examples of endpoint to measure and of methods and principles of cell-based antioxidant bioassays are reported in Table 3 and Figure 2. For a high throughput screening of food functional properties, biomarkers of cell viability, oxidative status, antioxidant defense, and oxidative damage are the most used.
Table 3–. Examples of endpoints used in cell-based bioassays for food analysis.
|Cell viability||Mitochondrial viability||MTT reduction|
| ||Release of cytoplasmic enzymes||LDH release|
|Cell functionality||Oxidative-reduction status||ROS production|
| || ||Glutathione level|
| || ||Antioxidant enzyme system (SOD, CAD, GPx, GR)|
| ||Intracellular oxidation||Incorporation and oxidation of fluorescent or fluorogenic probes|
| ||Oxidative damage||Malondialdeyde|
Figure 2–. AOx = antioxidants, ROS = reactive oxygen species. Examples of methods and principles of cell-based antioxidant bioassays: A) MTT assay: the yellow MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to a purple formazan by mitochondrial enzymes. Antioxidants scavenge free radicals, singlet oxygen, and electrons in cellular redox reactions; B) LDH (lactate dehydrogenase) release: LDH is released into the culture medium following loss of membrane integrity resulting from oxidative damage. LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of lactate to pyruvate; C) luminescence ATP detection assay system: ATP is a marker for cell viability because it is present in all metabolically active cells and the concentration declines very rapidly when the cells are exposed to cytotoxic stimuli; D) Exogenous probe incorporation: the DCFH-DA (2’,7’-dichlorfluorescein-diacetate) probe diffuses into the cell and it is cleaved by esterases to form DCFH (non-fluorescent 2’,7’-dichlorfluorescein). The peroxyl radicals oxidize the intracellular DCFH to DCF (fluorescent 2’,7’-dichlorofluorescein). Antioxidants bind to the cell membrane and/or enter the cell. Antioxidants prevent oxidation of DCFH and membrane lipids and reduce the formation of DCF.
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The use of fluorescent or fluorogenic probes have been reported in several cell-based bioassays intended for the monitoring and measure of oxidation. Dichlorofluorescein, a compound that once in human cells is easily oxidized to the fluorescent compound dichlorofluorescein, has been used by several researchers in cell-based bioassays (Elisia and others 2007; Wolfe and Liu 2007; Jensen and others 2008; Girard-Lancette and others 2009; Xu and Chang 2010; Blasa and others 2011). Results indicate that the use of fluorescent or fluorogenic probes can be a good approach for a high-throughput measure of antioxidant activity of different compounds. A criticism has been raised by Laguerre and others (2010) in a review on chemical and cell-based methods for evaluating the efficacy of antioxidants. These researchers conclude that these probes may detect a general oxidative stress but cannot be used as markers of a precise oxidative pathway. Regarding this aspect, interesting biomarkers such as intracellular ROS, nonenzymatic antioxidant, and cellular antioxidant enzyme could give more insight into the specific mechanisms involved in the biological activity of antioxidant dietary compounds. Goya and others (2009) presented a HepG2 cell-based bioassay as a model for the study of different antioxidant mechanisms of dietary compounds. The evaluation of several biomarkers have been reported: LDH leakage, low density lipoprotein oxidation assay, ROS evaluation, concentration of reduced glutathione, activity of antioxidant enzymes biomarkers of damage to lipid. This model, with a choice of numerous biomarkers of the cellular antioxidant defense system is a good research model, but it could be too complex for a high-throughput cell-based screening bioassay for dietary compounds’ antioxidant activity evaluation.
In conclusion, while noting that general oxidative stress biomarkers may not be good markers of a precise oxidative pathway, when choosing an endpoint to measure for a cell-based screening antioxidant bioassay, several factors must be considered such as easiness and length of workflow, high-throughput format, specificity and sensitivity of detection, and reproducibility of data.
Selecting cell culture media and environment Cell culture has often been used to study the cellular effects of reactive species and of antioxidants, and many useful data have been obtained. To extrapolate data obtained in cell culture models to the in vivo situation, results must be cautiously discussed and some consideration must be done. The environment in culture conditions can modify the properties of cells and tested compounds. Cells are normally cultured under 95% air/5% CO2, with about 150 mm Hg of O2. In the body, most cells are exposed to O2 concentrations in the range of 1 to 10 mm Hg. Therefore, cells in culture are under an oxidative stress, which can alter their properties in multiple ways. It has been reported that rates of production of ROS by cellular enzymes (for example, xanthine oxidase) or by leakage from electron transport chains (especially in mitochondria) appear to be O2-limited at 10 mm Hg (Halliwell 2003). Another aspect to be considered, when cells are used for studies involving the oxidative stress, is the composition of the cell culture medium and the presence or absence of FCS, which may affects both the status of the cells and the properties of added compounds. Cell culture media are frequently deficient in antioxidants, especially tocopherols, ascorbate, and selenium. Therefore the beneficial effects of added antioxidants can lead to an over-interpretation of the antioxidant efficacy of tested compounds. Other aspects to be considered are the hydrophobicity, stability, and the redox properties of compounds in the culture media. Flavonoids and other polyphenols are instable in commonly-used culture media, especially Dulbecco's Modified Eagle's Medium (DMEM) (Halliwell 2003). It has been demonstrated that different chemical and biological environments can modify the redox properties of carotenoids (Palozza and others 2003; Palozza 2005). Lin and others (2007) demonstrated that the stability and uptake of lycopene in cell culture is improved by the presence of FCS. Several evidences suggest that, in cell culture, the effectiveness of retinoids, which are relatively hydrophobic and unstable, may depend upon the type of medium and the presence or absence of FCS (Klaassen and others 1999; Tsukada and others 2002; Cheli and others 2003).
As discussed previously, the disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. Most of the studies have emphasized their antioxidant effects; however, polyphenols can also exert pro-oxidant activities under certain experimental conditions (Cao and others 1997; Lapidot and others 2002; Rufian-Henares and others 2006; Dai and Mumper 2010). Phenolic compounds in cell culture media undergo rapid oxidation to generate substantial amounts of H2O2 (Long and others 2000; Halliwell 2008). These researchers demonstrated that cell culture media have different “pro-oxidant” activity, with DMEM the most ‘‘pro-oxidant.” Thus, one should always be alert when adding polyphenols to cells in culture and must check for reactions taking place in the culture medium that could lead to artifacts and carefully distinguish effects of oxidation products from ‘‘real” effects of polyphenols. Addition of several components, such as catalase and pyruvate, can be used to scavenge the H2O2 (Babich and others 2009; Long and Halliwell 2009).
To conclude, the effect of cell culture medium and environment seems to be often under-appreciated, but can cause artifacts in the interpretations of the cellular effects of added compounds tested for their antioxidant activity (Halliwell 2003). A numerous examples of artifacts caused by oxidation of compounds added to cell culture media have been reported by Halliwell (2008). Not all the cellular effects of antioxidant are due to artifacts, but it is necessary to consider and taking into account that the cell environment can be a potential source of error for a proper evaluation of the true effects of antioxidant compounds.
Selecting cell culture system Two-dimensional (2-D) cell culture systems are easy and convenient to set up and are very useful for routine screening of food antioxidant properties. However, normal cells experience a 3-dimensional environment, completely surrounded by other cells, extracellular matrix, fibrous layers, and adhesion proteins. Adding the 3rd dimension (3-D) to a cell's environment in “in vitro models” creates significant differences in cellular characteristics and behavior. 3-D cell culture technologies have revolutionized our understanding of cellular behavior. The principal 3-D cell culture systems currently available and employed in basic and applied research are mainly based on the use of extracellular matrix constituents, microporous membranes, bio, and nanomaterials for scaffold construction (Table 4). The current technologies of modeling tissue in 3-D have been recently reviewed by several researchers (Lee and others 2008; Justice and others 2009; Mazzoleni and others 2009). The first description of culturing cells on filters has been reported by Grobstein (1953). Since that time, mammary and intestinal epithelial cells grown on microporous membranes as 3-D cell models of the mammary gland and gut, respectively, has been widely described and used (Cheli and others 2001; Cencič and Langerholc 2010; Diesing and others 2011). Culturing cells in this 3-D model ensures the formation of a functional epithelial barrier in terms of integrity and polarity. The applications for researches in the field of food microbiology and functional food evaluation have been recently reviewed (Cencič and Chingwaru 2010; Cencič and Langerholc 2010; Chopra and others 2010). Results indicate that this cell culture model may be a powerful tool as a bioassay for the functional screening of food, food components, and supplements. Up to now, the 3-D culture technologies developed for research applications do not scale well for screening applications and therefore are still far from a real application as functional bioassays of feed. For screening applications, a significant 3-D culture expansion is still needed.
Table 4–. Examples of most promising currently available 3-D cell culture technologies suitable for cell-based food screening.
|Cell culture models||Advantages||Description|
|Organotypic cultures: extracellular matrix (ECM)-based cultures||Reconstitution of tissue-like organization (polarity, function, viability)||Naturally derived materials (for example, collagen, laminin, fibrin, Matrigel)|
| || ||Synthetic polymers (for example, poly[dimethylsiloxan], poly[DL-lactide-co-glycolide])|
|Organotypic cultures: cells cultured on inserts||Reconstitution of an epithelial barrier-like organization (polarized metabolic processes and function)||Microporous filter membranes (hydrophilic poly[tetrafluoroethylene][PTFE], cellulose esters, polycarbonate membrane, polyethylene teraphthalate [PET])|
| || ||A variety of ECM coating can be considered.|
|Microcarriers-based cultures||Maintenance of cell viability and tissue-like functions||Small spheres with different coatings (gelatin, collagen, laminin)|
In the continuous search for new format for cell-based assays, microfluidic technology and microarray technology represent a further and promising evolution. Microfluidic technologies, that can manipulate samples at nanoliter volumes, are now designed and developed by taking advantage of the microfabrication techniques which were originally used in patterning the electrical circuits on silicon chips. Development of new methods for implementing cell-based assays based on digital microfluidics with no adverse effects on cell viability has been reported (Barbulovic-Nad and others 2008). This method is advantageous for cell-based assays because of automated manipulation of multiple reagents associated with a reduction in reagent use and analysis time. The use of cell-based microarray technology has been investigated and a higher sensitivity than a conventional well plate assay has been demonstrated (Diaz-Mochon and others 2007). These technologies have great potential as an analytical tool for implementing cell-based assays on the microscale. They are still research models and their success, as cell-based bioassays for test screening applications, relies on the development of stable, reproducible and low cost assays.