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 MU 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 (1.0 mu M) 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. (C) 2011 Elsevier Ltd. All rights reserved.

The protective effect of Canova homeopathic medicine in cyclophosphamide-treated non-human primates

Background: Canova activates macrophages and indirectly induces lymphocyte proliferation. Here we evaluated the effects of Canova in cyclophosphamide-treated non-human primates. Methods: Twelve Cebus apella were evaluated. Four animals were treated with Canova only. Eight animals were treated with two doses of cyclophosphamide (50 mg/kg) and four of these animals received Canova. Body weight, biochemistry and hematologic analyses were performed for 40 days. Micronucleus and comet assays were performed for the evaluation of DNA damage. Results: We observed that cyclophosphamide induced abnormal WBC count in all animals. However, the group treated with cyclophosphamide plus Canova presented a higher leukocyte count than that which received only cyclophosphamide. Cyclophosphamide induced micronucleus and DNA damage in all animals. The frequency of these alterations was significantly lower in the Canova group than in the group without this medicine. Conclusions: Our results demonstrated that Canova treatment minimizes cyclophosphamide myelotoxicity in C. apella. (C) 2012 Elsevier Ltd. All rights reserved.

Genotoxic and mutagenic effects of erythrosine B, a xanthene food dye, on HepG2 cells

Erythrosine (ErB) is a xanthene and an US Food and Drug Administration approved dye used in foods, drugs and cosmetics. Although its utilization is permitted, ErB is described as inhibitor of enzymes and protein-protein interactions and is toxic to pituitary and spermatogenesis processes. However, the genotoxicity and mutagenicity of ErB is inconclusive in the literature. This study aimed to analyze the genotoxicity of this dye using the alkaline comet assay and is the first investigation to evaluate ErB mutagenicity using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay in HepG2 cells. These cells were chosen because they produce phase I and phase II enzymes that can mimic in vivo metabolism. The cells were treated with seven concentrations (0.1-70.0 mu g mL(-1)) of ErB, and the results showed genotoxicity at the two highest concentrations and mutagenicity at six concentrations. Furthermore, as micronuclei result from clastogenic and aneugenic processes, while comet assay is often considered more sensitive and detects DNA single strain breaks, we suggest that an aneugenic is responsible for the observed damage. Although ErB is approved for use in the food, cosmetic and pharmaceutical industries, it must be used carefully because it damages the DNA structure. (C) 2012 Elsevier Ltd. All rights reserved.

Comparative study of beta-carotene and microencapsulated beta-carotene: Evaluation of their genotoxic and antigenotoxic effects

beta-Carotene (BC) is one of the natural pigments that is most commonly added to food; however, the utilization of BC is limited due to its instability. Microencapsulation techniques are commonly used because they can protect the microencapsulated material from oxidization. Nevertheless, the properties of the encapsulated compounds must be studied. We compared the antigenotoxic potential of pure and microencapsulated beta-carotene (mBC) in Wistar rats. Two doses of BC or mBC (2.5 or 5.0 mg/kg) were administered by gavage over a period of 14 days. The final gavage was followed by an injection of doxorubicin (DXR). After 24 h the animals were euthanized. The micronucleus test results showed that when both mBC and DXR were given, only the higher dose was antigenotoxic. The results of the comet assay show that when given in association with DXR, mBC had protective effects in the liver. The differences between the results obtained with BC and mBC suggest that possibly the carotenoid biodisponibility was modified by the process of microencapsulation. In conclusion, mBC does not lose its protective properties, but higher doses must be used to observe antigenotoxic effects. This is the first time that the genotoxicity and antigenotoxicity of a microencapsulated compound was evaluated in vivo. (C) 2012 Elsevier Ltd. All rights reserved.

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3 h old and 1, 3, 5, and 7 days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5 mu M caused the death of all schistosomula within 120 h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent. (c) 2012 Elsevier Inc. All rights reserved.

HEPATOTOXICITY ASSESSMENT OF THE AZO DYES DISPERSE ORANGE 1 (DO1), DISPERSE RED 1 (DR1,) AND DISPERSE RED 13 (DR13) IN HEPG2 CELLS

During the dyeing process in baths approximately 10 to 15% of the dyes used are lost and reach industrial effluents, thus polluting the environment. Studies showed that some classes of dyes, mainly azo dyes and their by-products, exert adverse effects on humans and local biota, since the wastewater treatment systems and water treatment plants were found to be ineffective in removing the color and reducing toxicity of some dyes. In the present study, the toxicity of the azo dyes disperse orange 1 (DO1), disperse red 1 (DR1), and disperse red 13 (DR13) was evaluated in HepG2 cells grown in monolayers or in three dimensional (3D) culture. Hepatotoxicity of the dyes was measured using 3-(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium (MTT) and cell counting kit 8 (CCK-8) assays after 24, 48, and 72 h of incubation of cells with 3 different concentrations of the azo dyes. The dye DO1 only reduced the mitochondrial activity in HepG2 cells grown in a monolayer after 72 h incubation, while the dye DR1 showed this deleterious effect in both monolayer and 3D culture. In contrast, dye DR13 decreased the mitochondrial activity after 24, 48, and 72 h of exposure in both monolayer and 3D culture. With respect to dehydrogenase activity, only the dye DR13 diminished the activity of this enzyme after 72 h of exposure in both monolayer and 3D culture. Our results clearly demonstrated that exposure to the studied dyes induced cytotoxicity in HepG2 cells.

Antigenotoxic Effects of Piquia (Caryocar villosum) in Multiple Rat Organs

This study investigated the in vivo genotoxicity of piquia pulp (Caryocar villosum) and its potential antigenotoxicity on doxorubicin (DXR)-induced DNA damage by comet assay and micronucleus test. In addition, the phytochemicals present in piquia pulp were determined. Piquia fruit pulp (75, 150 or 300 mg/kg b.w.) was administered by gavage to Wistar rats for 14 days, and the animals received an injection of saline or DXR (15 mg/kg b.w., i.p.) 24 h before they were euthanized. The phytochemical analysis revealed the presence of carotenoids; phenolic compounds, including flavonoids; tannins and alpha-tocopherol in piquia pulp. No statistically significant differences were observed in the evaluated parameters, demonstrating the absence of cytotoxic and genotoxic effects of piquia pulp at all tested doses. In liver, kidney, cardiac and bone marrow cells, piquia significantly reduced the DNA damage induced by DXR. Our results showed that the lowest piquia dose caused the largest decrease in DNA damage and the highest dose caused the smallest decrease, demonstrating an inverse dose-response of piquia pulp. Furthermore, we observed a difference in the potential antigenotoxic effects in several tissues. In conclusion, our results demonstrated that piquia pulp was not genotoxic and inhibited the genotoxicity induced by DXR, but some of the protective effects that were observed depended on the doses and experimental conditions. Therefore, further investigations are needed to clarify how piquia pulp positively affects human health.

Evaluation of the mutagenic activity of chrysin, a flavonoid inhibitor of the aromatization process

Chrysin is one of the natural flavonoids present in plants, and large amounts are present in honey and propolis. In addition to anticancer, antioxidation, and anti-inflammatory activities, chrysin has also been reported to be an inhibitor of aromatase, an enzyme converting testosterone into estrogen. The present study evaluated the mutagenicity of this flavonoid using micronucleus (MN) with HepG2 cells and Salmonella. Cell survival after exposure to different concentrations of chrysin was also determined using sulforhodamine B (SRB) colorimetric assay in HepG2 cells and the influence of this flavonoid on growth of cells in relation to the cell cycle and apoptosis. TheMN test showed that from 1 to 15 mu M of this flavonoid mutagenic activity was noted in HepG2 cells. The Salmonella assay demonstrated a positive response to the TA100 Salmonella strain in the presence or absence of S9, suggesting that this compound acted on DNA, inducing base pair substitution before or after metabolism via cytochrome P-450. The SRB assay illustrated that chrysin promoted growth inhibition of HepG2 cells in both periods studied (24 and 48 h). After 24 h of exposure it was noted that the most significant results were obtained with a concentration of 50 mu M, resulting in 83% inhibition and SubG0 percentage of 12%. After 48 h of incubation cell proliferation inhibition rates (97% at 50 mu M) were significantly higher. Our results showed that chrysin is a mutagenic and cytotoxic compound in cultured human HepG2 cells and Salmonella typhimurium. Although it is widely accepted that flavonoids are substances beneficial to health, one must evaluate the risk versus benefit relationship and concentrations of these substances to which an individual may be exposed.

Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levels

As shown in numerous studies, natural compounds may exert adverse effects, mainly when associated with some drugs. The hydroalcoholic extract of Mikania glomerata is the pharmaceutical form present in commercially available syrup used for the treatment of respiratory diseases in popular Brazilian medicine. The objective of the present investigation was (1) to evaluate the preventive effects of standardized hydroalcoholic extract of M. glomerata (MEx) against antitumoral drug doxorubicin (DXR)-induced micronucleated polychromatic erythrocytes (MNPCE) in a subchronic assay in mice, and (2) to determine the liver content of malondialdehyde (MDA) and the antioxidants glutathione (GSH) and vitamin E (VE). Male Swiss mice were treated for 30 d with MEx added to drinking water, combined or not with DXR (90 mg/kg body weight) injected intraperitoneally (ip) 24 h before analysis. The results demonstrated that MEx produced no genotoxic damage, but significantly increased the frequency of MNPCE induced by DXR, indicating a drug-drug interaction. This rise was not accompanied by lipid peroxidation or antioxidants level reduction, as measured by MDA, GSH, and VE. Despite the presence of coumarin (a known antioxidant), MEx may exert adverse effects probably in association with mutagenic compounds, although this effect on DNA damage did not involve oxidative stress.

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Background: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.

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 mu g/kg/bw/day), BIX (0.110 mg/kg/bw/day), NOR (0.011.0 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. Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.

Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.

Citotoxicidade e genotoxicidade de nanotubos de carbono

There are many controversies regarding the cyto- and genotoxicity of carbon nanotubes (CNTs). In this work, we discuss that many of the incongruous arguments are probably associated with the poor physical-chemical characterization of the CNT samples used in many publications. This manuscript presents examples of carbon nanostructures observed under high resolution electron microscopy that can be found in typical CNT samples, and shows which roles in cyto- and genotoxicity need to be better investigated. Issues concerning chemical treatment are addressed and examples of misunderstandings that can occur during the studies of cyto- and genotoxicity of CNT samples are given.

Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Entwicklung neuer Biomarker zur Detektion von Umweltbelastung

Es wurde bis heute meist mit relativ aufwendigen Methoden nach dem Vorkommen von Strangbrüchen in der DNA gesucht. Die hier verwendete Fast-Micromethod steht jetzt neben mehreren bereits etablierten Methoden zur qualitativen und quantitativen Erfassung von DNA-Strangbrüchen zur Verfügung. Die Methode wurde zur schnellen und empfindlichen Messung von DNA-Schäden und deren Reparatur unter Verwendung von PicoGreen, einem spezifisch an DNA-Einzelstränge bindenden Fluoreszenzfarbstoff, durchgeführt. Die Methode wurde in der vorliegenden Arbeit für verschiedene Zellarten wie HeLa-Zellen und Schwamm-Zellen mit Schwermetallionen verwendet. Sie wurde ebenfalls bei verschiedenen Strangbruch-induzierenden Stoffen wie NQO und physikalischen Faktoren wie UV-Bestrahlung und Gammastrahlung erfolgreich eingesetzt. Die Strangbrüche wurden bei HeLa-Zellen mit 10 µM NQO nach 90 Minuten Inkubation und ebenso bei Gammastrahlung mit 16 Gy gemessen. An Schwammzellen wurden Effekte von Schwermetallionen und UV, auch einschließlich nachgeschalteter Reparaturaktivität, gemessen. Dabei war die Reparatur jedoch nicht ganz vollständig. Durch die Fast-Micromethod wurde DNA-Schädigung durch UV-Bestrahlung und Gentoxizität von Schwermetall-Ionen an HeLa-Zellen nachgewiesen. Die Schwammzellen wurden mit verschiedenen Schwermetall-Ionen in verschiedenen Konzentrationen getestet und mit UV bei verschiedenen Dosen bestrahlt. Teilweise wurde nach dem Einfluss die DNA-Reparatur erfasst. Schwammzellen wurden in den drei häufig verwendeten Medien CMFSW, CMFSW+EDTA und Seewasser aufgenommen. Im Seewasser waren die Zellen nach 60 Minuten Inkubationszeit aggregiert (Primmorphe). Im CMFSW+EDTA Medium lagen dagegen nur Einzellen vor, da die Zellen im EDTA-Medium dissoziiert wurden und auch nicht mehr proliferierten.