What are Hepg2 cells

Investigations into the genetic toxicity and chemoprevention of Brassica plant juices in human hepatoma cells (Hep G2)

Table of Contents

List of figures

List of tables

List of abbreviations

1 Introduction

2 Material and Methods
2.1 Materials
2.1.1 Chemicals and reagents
2.1.2 Solutions
2.1.3 Consumables and Devices
2.1.4 Benzo [a] pyrene: mutagen and reference control
2.1.5 Characteristic features of the HepG2 cell line
2.2 Methods
2.2.1 Sample preparation of the plant material
2.2.2 Cultivation and treatment methods of cell cultures
2.2.3 Alkaline single cell gel electrophoresis (cometassay)
2.2.4 Gas chromatography and mass spectrometry

3 results
3.1 Genotoxic effects of Brassica
3.2 Antigen toxic effects of Brassica
3.3 Gene and antigen toxic effects: summary consideration
3.4 Qualitative and quantitative analysis of isothiocyanates
3.4.1 Proportion of dry matter
3.4.2 ITC concentrations in the dry matter
3.4.3 ITC concentrations in the sap

4 discussion
4.1 isothiocyanates in Brassicaceae
4.2 Genotoxicity of Brassicaceae and ingredients
4.2.1 Genotoxic effects of isothiocyanates
4.2.2 Genotoxic mechanisms of action of isothiocyanates
4.2.3 Genotoxicity of Brassicaceae
4.2.4 Test results on the genotoxicity of Brassicaceae versus previous study results
4.3 Antigen toxicity of Brassicaceae and ingredients
4.3.1 Chemoprotective mechanisms of action of isothiocyanates
4.3.2 Antigen toxicity of Brassicaceae
4.3.3 Previous studies on the antigen toxicity of Brassicaceae - vegetable juices
4.3.4 Evidence of the cancer-preventive activity of Brassicaceae and isothiocyanates in animal experiments and in epidemiological studies
4.3.5 Human sap intake 64 Outlook

5 Summary

6 literature

7 Appendix

Original data of the experimental part

List of figures

Fig. 1: Benzo [a] pyrene

Fig. 2: HepG2 cells with 50% growth density

Fig. 3: Comets in HepG2 cells

Fig. 4: Chromatogram of kohlrabi

Fig. 5: AITC calibration function

Fig. 6: OTM after exposure of HepG2 cells to 3.125 - 100 µl / ml cauliflower juice (24 h)

Fig. 7: TL after exposure of HepG2 cells to 3.125 - 100 µl / ml cauliflower juice (24 h)

Fig. 8: OTM after exposure of HepG2 cells to 3.125 - 100 µl / ml kohlrabi juice (24 h)

Fig. 9: TL after exposure of HepG2 cells to 3.125 - 100 µl / ml kohlrabi juice (24 h)

Fig. 10: OTM after exposure of HepG2 cells to 3.125 - 200 µl / ml red cabbage juice (24 h)

Fig. 11: TL after exposure of HepG2 cells to 3.125 - 200 µl / ml red cabbage juice (24 h)

Fig. 12: OTM after exposure of HepG2 cells to 3.125 - 400 µl / ml white cabbage juice (24 h)

Fig. 13: TL after exposure of HepG2 cells to 3.125 - 400 µl / ml white cabbage juice (24 h)

Fig. 14: OTM after exposure of HepG2 cells to 0.156 - 25 µl / ml cauliflower juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 15: TL after exposure of HepG2 cells to 0.156 - 25 µl / ml cauliflower juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 16: OTM after exposure of HepG2 cells to 0.156 - 25 µl / ml kohlrabi juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 17: TL after exposure of HepG2 cells to 0.156 - 25 µl / ml kohlrabi juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 18: OTM after exposure of HepG2 cells to 0.156 - 6.25 µl / ml red cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 19: TL after exposure of HepG2 cells to 0.156 - 6.25 µl / ml red cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 20: OTM after exposure of HepG2 cells to 0.156 - 100 µl / ml white cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Fig. 21: TL after exposure of HepG2 cells to 0.156 - 100 µl / ml white cabbage juice (24 h) and B [a] P, 50 µM (24 h)

List of tables

Tab. 1: Chemicals and reagents

Tab. 2: Solutions and their composition

Tab. 3: Devices used

Tab. 4: B [a] P-metabolizing enzymes in human HepG2 cells

Tab. 5: Parameters of the GC-MS / MS

Tab. 6: Temperature program of the gas chromatograph

Tab. 7: OTM after exposure of HepG2 cells to 3.125 - 100 µl / ml cauliflower juice (24 h)

Tab. 8: TL after exposure of HepG2 cells to 3.125 - 100 µl / ml cauliflower juice (24 h)

Tab. 9: OTM after exposure of HepG2 cells to 3.125 - 100 µl / ml kohlrabi juice (24 h)

Tab. 10: TL after exposure of HepG2 cells to 3.125 - 100 µl / ml kohlrabi juice (24 h)

Tab. 11: OTM after exposure of HepG2 cells to 3.125 - 200 µl / ml red cabbage juice (24 h)

Tab. 12: TL after exposure of HepG2 cells to 3.125 - 200 µl / ml red cabbage juice (24 h)

Tab. 13: OTM after exposure of HepG2 cells to 3.125 - 400 µl / ml white cabbage juice (24 h)

Tab. 14: TL after exposure of HepG2 cells to 3.125 - 400 µl / ml white cabbage juice (24 h)

Tab. 15: OTM after exposure of HepG2 cells to 0.156 - 25 µl / ml cauliflower juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 16: TL after exposure of HepG2 cells to 0.156 - 25 µl / ml cauliflower juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 17: OTM after exposure of HepG2 cells to 0.156 - 25 µl / ml kohlrabi juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 18: TL after exposure of HepG2 cells to 0.156 - 25 µl / ml kohlrabi juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 19: OTM after exposure of HepG2 cells to 0.156 - 6.25 µl / ml red cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 20: TL after exposure of HepG2 cells to 0.156 - 6.25 µl / ml red cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 21: OTM after exposure of HepG2 cells to 0.156 - 100 µl / ml white cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 22: TL after exposure of HepG2 cells to 0.156 - 100 µl / ml white cabbage juice (24 h) and B [a] P, 50 µM (24 h)

Tab. 23: OTM / µl and TL / µl for the examinations of the genotoxicity of Brassicaceae

Tab. 24: OTM / µl and TL / µl for the antigen toxicity studies of Brassicaceae

Tab. 25: Dry matter of the Brassicaceae

Tab. 26: ITC concentrations in the dry matter of the Brassicaceae

Tab. 27: ITC concentrations in Brassicaceae -Plant sap

Tab. 28: Modulation of phase I and phase II enzyme activities by isothiocyanates or Brassica -Vegetables

Tab. 29: Dry matter and sap amount of a fresh serving Brassicaceae -Vegetables

Tab. 30: Plant sap concentrations in human tissue and in the cell cultures of the antigen-toxic investigations

List of abbreviations

Figure not included in this excerpt

1 Introduction

Every year around 395,000 people develop cancer in Germany, 210,000 of them die as a result of their cancer (BERTZ et al. 2004). Malignant neoplasms are the second most common cause of death for both men and women after cardiovascular diseases (DIFE 1999). In addition to genetic determinants, exogenous factors, including above all nutritional factors, play a decisive role in the development of cancer. According to current estimates, medical professionals assume that around 30-35% of all cancers in the western world are caused by nutritional factors (KROKE and BOEING 2000). It is estimated that the number of cancer cases could be reduced by 30 to 40% by changing diet to a healthy diet, combined with exercise and avoiding obesity (WCRF 1997). For Germany this would mean 120,000 to 158,000 fewer cancer cases per year.

According to current knowledge, a high consumption of fruit and vegetables seems to play a protective role in the etiology of various cancers (STEINMETZ and POTTER 1996, BLOCK et al. 1992, KOLONEL et al. 2000). Special bioactive substances such as phytochemicals and dietary fiber seem to be the reason for the chemoprotective effects (STEINMETZ and POTTER 1996). Glucosinolates are among the most important phytochemicals. These are predominantly in plants of the family Brassicaceae (syn. Cruciferaecruciferous vegetables) (VERKERK et al. 1998). These include rapeseed, mustard, cress, individual types of beet and cabbage vegetables (Brassica oleracea). When vegetables are chewed and cut, glucosinolates are metabolized by a plant-specific myrosinase to form isothiocyanates (ITCs). Intestinal degradation of glucosinolates to isothiocyanates is also possible (FAHEY 2001). Epidemiological and experimental data provide evidence that Brassicaceae and isothiocyanates can be held responsible for an inhibition of carcinogenesis (VERHOEVEN et al. 1996; 1997, VAN POPPEL et al. 1999). The chemoprotective effects seem to be based mainly on a modulation of foreign substance metabolizing enzymes (VERHOEVEN et al. 1997, CONAWAY et al. 2002, ZHANG 2004).

On the other hand, could look for high concentrations Brassicaceae - Plant juices and isothiocyanates have been shown to show that they themselves can cause an irreversible transformation of the DNA, i.e. have a genotoxic effect (KASSIE et al. 1996, 1999; KASSIE and KNASMÜLLER 2000). Previous study results have shown that plant saps in particular can induce point mutations and DNA damage in bacterial test systems and chromosomal aberrations in mammalian cells (KASSIE et al. 1996). At present there are only a few data dealing with the genotoxic potential of Brassicaceae Employing juices and making it possible to weigh up the positive and negative effects.

The aim of the present work was on the one hand the sap of the plant Brassica oleracea - Investigate types of cauliflower, kohlrabi, red and white cabbage for their genotoxic effects. For this, the alkaline single cell gel electrophoresis (comet assay) was used. The cometassay enables the detection of DNA migration as an expression of double-strand breaks / single-strand breaks, alkali-labile areas, excision repair sites and DNA-DNA / DNA-protein cross-links under strongly alkaline test conditions at the single cell level (BRENDLER-SCHWAAB et al. 2005) . Metabolically competent human hepatoma cells (HepG2) were used as indicator cells. HepG2 cells express numerous enzymes involved in phase I and phase II foreign substance metabolism. For this reason, they are more suitable for genotoxic experiments than metabolically incompetent cell systems or bacteria (KNASMÜLLER et al. 1998, 2004).

In addition, the chemopreventive potential of the plant juices of cauliflower, kohlrabi, red and white cabbage was examined in a cometassay with HepG2 cells. For this purpose, the cells were first exposed to the plant sap and then to the mutagen benzo [a] pyrene. The reduction in benzo [a] pyrene-induced DNA migration caused by the plant sap was determined as a parameter for a chemopreventive effect. Benzo [a] pyrene was used as a positive control in the genotoxicity tests.

Since isothiocyanates in particular are held responsible for the genotoxic and chemopreventive effects of vegetable juices, the Brassica - Juices examined by gas chromatography with a mass spectrometric detector (GC-MS / MS) for allyl (AITC), benzyl (BITC), methylthiobutyl (MTBITC), phenylethyl (PEITC), phenyl isothiocyanate (PITC), erysoline and sulforaphane. In addition, the amount of sap was determined, which over a Brassica - Vegetable portion can be consumed and their potential distribution in the human organism is calculated. The tissue concentrations were then compared with the antigen-toxic plant sap concentrations.

2 Material and Methods

2.1 Materials

2.1.1 Chemicals and reagents

Table 1 lists the chemicals and reagents as well as their sources of supply that were required for cell cultivation of the HepG2 cells and for performing the cometassay.

Tab. 1: Chemicals and reagents (details of purities in% if available)

Figure not included in this excerpt

2.1.2 Solutions

Table 2 shows the solutions that were made to carry out this study.

Tab. 2: Solutions and their composition

Figure not included in this excerpt

2.1.3 Consumables and Devices

In addition to the usual laboratory equipment and consumables, the equipment listed in Table 3 was used.

Tab. 3: Devices used

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2.1.4 Benzo [a] pyrene: mutagen and reference control

Benzo [a] pyrene is the lead substance of the polycyclic aromatic hydrocarbons (PAH). They arise from incomplete combustion of organic material (coal, wood) as well as from pyrolysis processes and are ubiquitous (LUCH 2005). PAHs could be found in mineral oils

Fig. 1: Benzo [a] pyrene

Figure not included in this excerpt

and their secondary products (bitumen, tar, pitch, soot), in fly ash, as waste in coking plants, in aluminum, iron and steel production, in car exhaust fumes and in tobacco smoke (NAU et al. 2003).

PAHs are bound to particles such as pollen, soot and dust in the air and in this way can lead to contamination of food, especially fresh fruit and vegetables. PAHs can also arise during cooking and preservation processes. Roasted and grilled food as well as smoked goods are the main sources of intake for humans (PHILLIPS 1999). Phase I enzymes of foreign substance metabolism, especially CYP1A1, activate benzo [a] pyrene primarily to benzo [a] pyrene dihydrodiol epoxide (BPDE). BPDE can covalently bind to cellular macromolecules, such as DNA. This can damage DNA replication as well as mutations that can lead to carcinogenic changes (MELENDEZ-COLON et al. 1999, PHILLIPS 1999). Because of its known genotoxic effect, benzo [a] pyrene is used as a positive control in genotoxicological studies (LAKY et al. 2002, UHL et al. 2003). This is also the case in single cell gel electrophoresis with HepG2 cells.

For the experiments carried out, B [a] P was dissolved in sterile DMSO and used in a concentration of 50 μM. This concentration was determined by studies as the optimal effective concentration (original data see appendix) and used in the test series as a reference and positive control.

2.1.5 Characteristic features of the HepG2 cell line

The HepG2 cell line was isolated in 1972 from a primary hepatoblastoma in an 11-year-old Argentine boy (ADEN et al. 1979). The cells have an aneuploid karyotype with an average chromosome number of 52. The generation time is 20 to 28 hours (NATARJAN and DARROUDI 1991). Cytological and histochemical examinations showed that the HepG2 cells show characteristics of normal human hepatocytes. BOUMA et al. (1989) were able to demonstrate a rudimentary intercellular development of bile ducts and Golgi apparatus in HepG2 cells. The cells also synthesize and secrete human plasma proteins such as albumin and transferrin (ADEN et al. 1979, KNOWLES et al. 1980) as well as apolipoproteins, lipoproteins and lipids (DASHTI and WOLFBAUER 1987, ELLSWORTH et al. 1986).

Figure not included in this excerpt

Fig. 2: HepG2 cells with 50% growth density

Numerous in vitro - Studies on the genotoxicity of promutagenic substances are carried out with metabolically incompetent cells (V-79, CHO) or bacteria. In order to compensate for the lack of metabolizing enzyme systems in these experiments, exogenous activation systems (e.g. liver enzyme fractions from rodents (S9 mix)) are usually added. In contrast, HepG2 cells express phase I and phase II enzymes that enable foreign substances to be metabolized in the cells (KNASMÜLLER et al. 1998, 2004). Table 4 shows a selection of metabolizing enzymes that could be detected in HepG2 cells and are relevant for the benzo [a] pyrene metabolism. In addition, the ingredients of the examined Brassicaceae an influence on these enzymes is attributed (see chapter 4.2.1). The activity of the foreign substance-metabolizing enzymes can be significantly influenced by the composition of the culture medium. Cultivation of the cells in “Williams E medium” or “Earle's minimum essential medium” led to an enzyme activity similar to that found in primary human hepatocytes (DOOSTDAR et al. 1988).

Tab. 4: B [a] P-metabolizing enzymes in human HepG2 cells (modified from KNASMÜLLER et al. 1998)

Figure not included in this excerpt

In addition to its ability to metabolize foreign substances, the HepG2 cell line offers further advantages for genetic toxicology. Various biological endpoints such as cytotoxicity and lipid peroxidation as well as the formation of micronuclei, sister chromatid exchange, DNA strand breaks and the formation of DNA adducts can be investigated (MERSCH-SUNDERMANN et al. 2004). In addition, the induction of 6TGr mutations and unscheduled DNA synthesis can also be detected (KNASMÜLLER et al. 1998).

Furthermore, mutagenic and non-mutagenic compounds structurally related to the cells can be distinguished (e.g. benzo [a] pyrene and pyrene) (NATARAJAN and DARROUDI 1991).

In HepG2 cells, genotoxic effects of compounds can be investigated which led to false negative results in test systems with metabolically incompetent indicator cells (e.g. safrole and hexamethylphosphoramide (HMPA)). Compounds that led to false positive results in animal studies could also be recognized as non-carcinogens in test systems with HepG2 cells (e.g. tamoxifen) (UHL et al. 2000).

In addition, the cell line was also used in studies with antigen-toxic and cytoprotective substances, such as ȕ-carotene (SALVADORI et al. 1993), caffeine (SANYAL et al. 1997), polyphenols (JIAO et al. 2003) and diallyl disulfide Allium sativum (KNASMÜLLER et al. 1998). On the one hand, protective mechanisms of action of these substances could be identified in HepG2 cells, such as the inactivation of peroxides, DNA-damaging electrophiles or reactive oxygen species (engl. quenching) (MERSCH-SUNDERMANN et al. 2004). HepG2 cells could also be used to investigate the induction of enzyme systems with an antioxidant effect, such as superoxide dismutase, catalase or glutathione peroxidase. These enzymes are expressed in HepG2 cells to a similar extent as in primary human hepatocytes (LEE et al. 2002). For example, diallyl disulfide induced glutathione-S-transferase (GUDI and SINGH 1991).

2.2 Methods

2.2.1 Sample preparation of the plant material

Production of the vegetable juices

In the present study, the plant saps were as follows Brassica - Species examined:

Figure not included in this excerpt

The Brassica -Arten were bought fresh in supermarkets in Gießen and processed directly (cauliflower, kohlrabi: 03/21/2005; red cabbage, white cabbage: 05/25/2005). The vegetables came from conventional cultivation. To make the raw vegetable juices, each type of cabbage was cut into small pieces with a knife, then further chopped up with a hand mixer and additionally ground with a mortar. This ensured a complete breakdown of the plant matrix. The plant material was then squeezed out using a gauze compress and the juice was collected in a beaker. In order to remove suspended particles from the sap, it was centrifuged for 20 minutes at 14,000 rpm and 4 ° C. The supernatant was first cleared with the aid of a 0.45 µm syringe filter and then sterile filtered through a 0.22 µm syringe filter. The juice was transferred into 0.5 ml microtubes under a workbench, the tubes were sealed and stored at -20 ° C. until exposure.

Sample preparation of plant material for the chromatographic detection of isothiocyanates

10 g of the plant material obtained during the preparation of the plant sap were weighed into a 50 ml centrifuge tube. Of each Brassica -Three samples were taken. 10 ml of solvent (cyclohexane - ethyl acetate, 1: 1) were pipetted onto the plant material, the tube was closed and shaken by hand. The extraction was then carried out on a shaker within 60 minutes. In order to separate the solvent from the plant material, the sample was centrifuged for 5 minutes at 5000 rpm and 20 ° C. 5 ml of the supernatant was removed, cleaned through a 0.45 μm Teflon syringe filter and collected in a 15 ml centrifuge tube. The remaining supernatant was discarded.

In order to extract all ITCs from the sample, another 10 ml of the solvent were pipetted onto the remaining plant material, the sample was shaken and centrifuged. 5 ml of the supernatant were again purified with the aid of a syringe filter and added to the 5 ml of the first extraction. The combined phases were mixed on a vortex. 1 ml of the mixture was transferred to a vial and stored in the freezer at -80 ° C. until GC-MS / MS analysis.

Determination of dry matter

The dry matter of the cabbage vegetables was determined to determine the concentration of ITCs in the various Brassicaceae both to be able to determine and to be able to compare them directly.

First, the exact weight of a plastic weighing pan was determined and noted. 10 g of the plant material were weighed into the dish and the weight was also noted. In order to achieve complete drying, the dish with the contents was placed in the drying cabinet for one week at 38 ° C. The sample was then weighed again and the dry weight in% could be determined as follows:

Figure not included in this excerpt

The water content was determined by subtracting the dry substance from 100%.

2.2.2 Cultivation and treatment methods of cell cultures

Origin of the cell material

The human hepatoma cells were made available by the University of Leiden (Netherlands). The cells were cultivated in a heating cabinet at a temperature of 37 ° C. and a CO2 concentration of 5%. For the in vitro Experiments used cells from passages 10-27.

Culturing the cells

The cell cultures were processed in a sterile environment under a workbench.

Thawing cell cultures

Until they were used, the HepG2 cells were stored in the vapor phase of nitrogen at a temperature of - 180 ° C.

To thaw a cell culture, culture medium was first heated to 37 ° C. in a water bath and 10 ml of it was pipetted into a centrifuge tube. An aliquot of the stored cells was also quickly thawed at 37 ° C., transferred to the medium provided and centrifuged at 1200 rpm and 4 ° C. for 5 minutes. The supernatant was decanted. The cell pellet was resuspended in 10-20 ml of culture medium and placed in a polystyrene cell culture bottle (75 cm2 ) transferred. After a first cultivation phase of 24 hours in the heating cabinet, the culture medium was replaced by fresh medium.

Cultivation and Passenger

The culture medium, trypsin (0.05%) and PBS were warmed to 37 ° C. in a water bath before work began. The culture bottle was first examined macroscopically and microscopically for pathogenic microorganisms or other impurities. The culture medium was then suctioned off and the cell culture was washed twice with 10 ml of PBS each time.

Depending on the bottle size (75 cm2 / 175 cm2 ) 1 or 3 ml of trypsin were evenly distributed on the cell lawn and the cell culture bottle was incubated for 5 minutes in the incubator. After this time, the detachment of the cells from the bottom of the bottle was checked microscopically. In order to stop the trypsin-mediated reaction, culture medium was added and the entire suspension was transferred to a centrifuge tube. The cells were isolated by repeated aspiration using a disposable syringe and injection needle. A sample was then taken from the suspension in order to determine the number of cells. For this purpose, the sample was mixed with erytrosine B in a ratio of 1: 1 and the cells in this mixture were counted in a Neubauer counting chamber. The number of cells per ml could be calculated using the following formula: n x dilution factor x chamber volume x ml of cell suspension.

A portion of the cell suspension was removed and pipetted into a prepared culture bottle filled with medium and placed in the incubator. The amount of seeded cells was based on the number of cells available and the number of cells required for the experiments.

The cells passed through every three to five days, depending on the growth of the cells. Every other day, the cells were washed with PBS and fresh culture medium was added.

Subcultures in cell culture plates

To carry out the experiments, cells were cultivated in cell culture plates with 12 wells each. For this purpose, the cells were treated as in the cell passage. The culture medium was aspirated from the cell culture bottle, the cells were washed twice with PBS, detached from the cell base with trypsin and the reaction was stopped with the medium. The cells were isolated and then the number of cells was determined. In each of the wells of the cell culture plates, 2 ml of culture medium were placed and 3 × 105 Cells sown. The plates were then incubated in the incubator for 24 hours.

Treatment of the cells

A negative control (distilled water) and a positive control (B [a] p; 50 µM) were included in the experiments on genotoxicity. In the experiments on antigen toxicity, distilled water was used on the first day of the experiment. used as negative and reference control, on the second day DMSO as negative control and B [a] p (50 µM) as reference control.

Genotoxicity studies

The cells sown in the culture plates were exposed to the plant sap and incubated for 24 hours in an incubator. The plant sap concentrations used were determined in preliminary tests (data not shown).

Antigen toxicity studies

The sown cells were first incubated with the plant sap for 24 hours. The sap concentrations selected and used were those which did not induce any significant genotoxic effects in the genotoxicity studies. The culture medium was then suctioned off, the cells were washed twice with 1 ml PBS and another 2 ml medium was added. Then B [a] p (50 μM) was added and the cells were incubated for a further 24 hours in the incubator.

2.2.3 Alkaline single cell gel electrophoresis (cometassay)

Basics

Genotoxic substances can cause various lesions on cellular DNA: single / double strand breaks, DNA-DNA or DNA-protein cross-links (engl. crosslinks) and damage to purine and pyrimidine bases. Single cell gel electrophoresis (engl .: single cell gel electrophoresis (SCGE)), also called comet assay, is a test system with which DNA damage at cell level can be detected as DNA migration (TICE and STRAUSS 1995).

The method of the test system is based on suspending cells that have previously been exposed to a genotoxic substance in agarose and placing them on slides. In the case of alkaline lysis, the cells are then lysed in a salt and detergent solution (pH = 10-12). Before electrophoresis, the slides are stored in an alkaline electrophoresis solution (pH> 13). The strongly alkaline environment causes the DNA to unwind due to the breakdown of hydrogen bonds. The cells are then subjected to electrophoresis, with damaged DNA migrating out of the cell nucleus towards the anode through the gel. High molecular weight undamaged DNA cannot leave the cell nucleus. The slides are then washed and the DNA can be stained with a fluorescent dye (e.g. ethidium bromide). When viewed under a fluorescence microscope, cells with damaged DNA show a migration of the DNA that is similar in shape to a comet - the cell nucleus represents the head of this comet (see Fig. 3). The proportion of DNA in the comet's tail and the length of the tail are a measure of the DNA damage caused by a genotoxic substance. Cells with undamaged DNA have no comets (HARTMANN et al. 2003, TICE et al. 2000, FAIRBAIRN et al. 1995, OLIVE et al. 1990).

Fig. 3: Comets in HepG2 cells

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The figures show different levels of DNA migration in HepG2 cells at 400x magnification. The DNA was stained with ethidium bromide.

In the first picture, no DNA migration in the form of a comet can be seen. HepG2 cells were compared to aqua dest. exposed.

Figure two shows the DNA migration after exposure of the cells to B [a] P 25 µM, Figure three after exposure to

ÖSTLING and JOHANSON (1984) carried out a microelectrophoretic test under pH-neutral conditions for the first time. This version enables the detection of DNA double-strand breaks (HU and HILL 1996, FAIRBAIRN et al. 1995). SINGH et al. (1988) used alkaline electrophoresis buffer, which led to an increase in the sensitivity of the test system.

At a pH value greater than 13, DNA single strand breaks, alkaline-labile areas (e.g. abasic areas), excision repair areas and DNA cross-links can also be detected in addition to DNA double-strand breaks (BRENDLER-SCHWAAB et al. 2005). DNA single strand breaks, alkali-labile areas and increased excision repair activities lead to increased DNA migration (BRENDLER-SCHWAAB et al. 2005, TICE et al. 2000, FAIRBAIRN 1995). Since DNA cross-links can stabilize chromosomal DNA, this type of

Damage to reduced DNA migration (TICE et al. 2000, SPEIT and HARTMANN 2005). If the pH value of the electrophoresis buffer is reduced to 12.1, alkali-labile areas can no longer be detected (MIYAMAE et al. 1997). The comets are made differently depending on the electrophoresis conditions. According to KLAUDE et al. (1996) comets that form under alkaline conditions consist of free DNA fragments. Under neutral electrophoresis conditions, the comets are formed from loosened DNA loops that can stretch into the gel. Only with increasing doses of harmful substances do fragments form, which migrate as DNA fragments from the head of the comet into the tail. According to FAIRBAIRN et al. (1995) the length of the tail was not significant. However, the tail intensity is increased as a result of a shift in the DNA concentration from the head to the tail.

The cell cycle phase is another factor that can affect tail formation. S-phase cells show more DNA migration in the alkaline assay and less DNA migration in the neutral assay than G1 and G2 cells (OLIVE and DURAND 2005, OLIVE and BANATH 1993).

The cometassay in its alkaline version (pH> 13) is increasingly used for genotoxicological investigations (BRENDLER-SCHWAAB 2005, HARTMANN et al. 2004, 2001). The reason for this is, on the one hand, the possible high throughput of connections to be checked (KISKINIS et al. 2002). Only a small number of test cells (a few 1000) are required to carry out the cometassay. In addition, the slightest DNA damage can be detected at the individual cell level. The test can be carried out with any eukaryotic cell (BRENDLER-SCHWAAB et al. 2005, TICE et al. 2000, 1995).

Apoptotic cells can partly be detected with the comet assay based on their deviating appearance. They have small heads with a diffuse, fan-like tail (engl. hedgehogs). Large heads with narrow tails characterize necrotic cells - their appearance can hardly be distinguished from genotoxically damaged cells (TICE et al. 2000). Since apoptosis and necrosis can occur both as a result of cytotoxicity and as a result of treatment of cells with strongly mutagenic test substances, the factor cytotoxicity should be excluded in advance (BRENDLAR-SCHWAAB 2005).

In addition, the test is easy to use, quick to perform and inexpensive (TICE et al. 1995).

UHL et al. (1999) developed a standard protocol for carrying out the cometassay, the methods of which were taken into account in the present work.

execution

All work steps from harvesting the cell cultures were carried out under red light in order to prevent cell damage from ultraviolet light.

Preparation of the microscope slide

First, the agarose solutions used (NMP (0.7%), LMP (0.5%), NMP (1%)) were prepared. For this purpose, PBS buffer was heated to 120 ° C. and the agarose in question was dissolved therein with stirring (for volume information see p. 5, tab. 2). The agarose solutions were then heated to 40 ° C. and applied to the slides.

The required amount of roughened slides was cleaned and degreased by rinsing with an ethanol / acetic acid mixture (ratio 1: 1). The first agarose layer (NMP (1%), 85 μl) was then pipetted onto the microscope slide and evenly distributed using cover slips. To allow the agarose to harden, the slides were dried in a drying cabinet at 37 ° C. for 24 h. The cover slip was then removed, the second layer of agarose (NMP (0.7%), 100 μl) applied, again distributed using cover slips and stored in the refrigerator for at least 15 minutes.

Processing of the cell cultures

The medium was aspirated from the cells treated in the cell culture plates and the cells were washed twice with one ml of PBS each time. After removing the buffer, the cells were harvested by means of trypsinization (250 μl trypsin (0.05%) per well, 5 min., 37 ° C.). In order to stop the enzymatic reaction, 750 μl of culture medium were pipetted into the wells of the cell culture plates and the cell suspension was then transferred into centrifuge tubes. The cells were isolated by repeatedly drawing up the suspension with a disposable syringe and injection needle.

One aliquot of each of the treated cells was checked for cytotoxicity with erytrosine B. For this purpose, the cell suspension was mixed with the dye in a ratio of 1: 1 in a microtube and the cells were counted in a Neubauer counting chamber. The percentage of unstained cells was used as an indicator of the cell vitality (% living cells).

Figure not included in this excerpt

The cover slips were removed from the microscope slides, sorted into a Schiefferdecker chamber, mixed with lysis solution and stored in the refrigerator (4 ° C) for at least one hour.

Electrophoresis

The slides were removed from the lysis solution, briefly with chilled aqua dest. washed and sorted into the electrophoresis chamber filled with cold, alkaline buffer solution.

Electrophoresis was started after 20 minutes.The chamber was operated at 300 mA / 25 V for 25 minutes. The electrophoresis conditions were adjusted by adding or removing buffer. By means of a cooling system connected to the chamber, the buffer and the slides stored in it could be cooled during the entire process (approx. 0 ° C).

Neutralization

Following the electrophoresis, the slides were sorted back into the Schiefferdecker chamber, mixed with neutralization solution and stored in the refrigerator for 10 minutes. Rinsing the slide again with distilled water. completed this process.

coloring

After a short drying time (approx. 5 minutes), 80 μl ethidium bromide solution were applied to each slide and a cover slip was placed on it. In order to prevent the gels from drying out, the slides were stored in a moist chamber in the refrigerator and evaluated over a maximum period of three days.

evaluation

The slides were evaluated at 400x magnification on a fluorescence microscope with a camera attached to a computer. With the help of software (Comet 3.1 Europe, Kinetic Imaging), the extent of DNA damage in the cells could be determined. 102 cells per slide were evaluated. The program enables a differentiation between the parameters tail length (µM), proportion of DNA in the tail (in%) and olive tail moment (OTM).

Assessment of cytotoxicity

According to HENDERSON et al. (1998), the maximum concentration of a test substance should generate a cell vitality of> 75% in order to avoid false positive results that could be attributed to cytotoxicity. All attempts to determine the gene and antigen toxicity of the investigated Brassicaceae were carried out up to an average cell vitality of> 72%. The original data of the tests for cytotoxicity are shown in the appendix.

2.2.4 Gas chromatography and mass spectrometry

The qualitative and quantitative detection of isothiocyanates in the Brassicaceae took place by means of gas chromatography with a mass spectrometric detector (GC / MS).

Gas chromatography (GC)

The separation of substances in a moving medium (mobile phase) using the different strengths of adsorption on a fixed medium (stationary phase) is called chromatography (MORTIMER 2001). Gas chromatography is a variant of the chromatographic separation methods in which the mobile phase consists of a gas. Since the gas is only used for transport, it should be chemically inert (SKOOG and LEARY 1996). In this thesis, helium was used as the carrier gas for the analysis of the ITCs.

The stationary phase can be solid (engl. gas solid chromatography, GSC) or liquid (engl. gas liquid chromatography, GLC). A solid stationary phase consisting of phenyl (5%) and dimethylpolysiloxane (95%) was used for the investigations in this work.

A gas chromatograph consists of the components injector, separation column and detector. The task of the injector is to transfer the sample to the column, whereby the sample is first evaporated. With the mobile phase (eluent) the sample is transported through the column (elution) and separated due to the interactions with the stationary phase and the different boiling points. If the interactions between the analyte molecules and the stationary phase are weak, they are only slightly retarded and emerge from the column early. With strong interactions, the molecules elute late. This results in different retention times for the analytes. The documentation of the chromatographic separation takes place on a detector, which converts the eluting substances into an electrical signal. The recorder outputs the signals in the form of peaks (engl. peaks) made visible. The individual peaks are used to identify the substances in the sample; the peak areas can be used for quantification. A series of peaks is called a chromatogram (CAMMANN 2001).

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