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Genotoxicity :describes a deleterious action on a cell's genetic material affecting its integrity. Genotoxic substances are known to be potentially mutagenic or carcinogenic, specifically those capable of causing genetic mutation and of contributing to the development of tumors. This includes both certain chemical compounds and certain types of radiation.

Typical genotoxins like aromatic amines are believed to cause mutations because they are nucleophilic and form strong covalent bonds with DNA resulting with the formation of Aromatic Amine-DNA Adducts, preventing accurate replication.

Genotoxins affecting sperm and eggs can pass genetic changes down to descendants who have never been exposed to the genotoxin.

Cytotoxicity : is the quality of being toxic to cells.

Principle of Genotoxic and Cytotoxic Test

Introduction

The Genox strain carries a plasmid containing the bacterial luciferase operon (luxCDABE) of luminous bacteria Vibrio fischeri under transcriptional control of a mutated recN promoter (recN2-4) that is part of the SOS-system. Genotoxic compounds activate the recN promoter in the Genox strain, which results in transcriptional induction of the lux operon followed by the enhancement of light emission.

The Cytox strain possesses a plasmid containing the Vibrio fischeri lux operon, transcriptionally controlled by the strong pr1 promoter and thus constitutively produces light. This strain is used as an internal control system as some compounds appear to have a direct influence on the light production or enhance the metabolism of the bacteria creating false-positive results.

The cytotoxicity of the compounds is simultaneously assayed with reference to the Cytox strain to identify the non-specific enhancement of light emission. Concomitant use of the Genox and the Cytox strains allows us to identify false positive results caused by non-specific light emission induced by other mechanisms, and not by the genotoxic effect.

Description

The test is based on the original Vitotox test established at the Flemish Institute for Technological Research (van der Lelie et al., 1997, Verschaeve et al. 1999) as a reaction to the conventional bacterial short-term tests to detect genotoxic and toxic agents. The end-result is a test kit that provides a very rapid (with 4- 5 hours) and sensitive answer when screening and prescreening for the genotoxicity and toxicity of new chemicals, pharmaceuticals and intermediate products.

The Salmonella typhimurium bacteria used in the test lack the necessary oxidative enzymes for metabolizing foreign compounds to electrophilic metabolites capable of reacting with DNA, therefore the bacteria are treated with the test agent in the presence and absence of a post-mitochondrial supernatant (S9). This supernatant is prepared from the livers of aroclor-treated rats.

After being incubated overnight in rich growth medium, a dilution of the bacterial suspension is incubated for one more hour on an orbital shaker in poor growth medium the following day. Coincidently, dilutions of different concentrations of the test agent are made and are transferred to a 96 multiwell plate. Next to the dilution series, a blank of the solvent and a positive control are added (e.g. Benzo(α)pyrene with S9 and 4-Nitroquinoline-N-oxide without S9). The genotoxicity and toxicity measurements are performed using a microplate luminometer that enables measurements of emitted light every 5 minutes over a period of 4 hours. After completion, the data are transferred to a spreadsheet application and the signal-to-noise ratio (S/N), i.e. the light production of exposed bacteria divided by the light production of non-exposed bacteria, is calculated for each measurement. The S/N of the Genox and Cytox strain is calculated separately, as well as the ratio between the maximum S/N values of the Genox and Cytox strains. Based on these experimental grounds, an agent is considered genotoxic, cytotoxic or not.

Materials Neededd for the Vitotox test

1. Genox strain S. typhimurium TA104 recN2-4, Five Red vials of 100 µl
Store at -80°C
2. Cytox strain S. typhimurium TA104 pr1, Five Blue vials of 100 µl
Store at -80°C
3. Growth Medium with enhancer, Ten 15 ml tubes of 5 ml
Store at -20°C
4. Test Medium, Ten 15 ml tubes of 2.5 ml
Store at -20°C 5. Test Medium, Ten 15 ml tubes of 4,5 ml Store at -20°C
6. Black 96-well plates, 5 pieces
7. Clear 96-well strip plates, 3 pieces
8. Disposable reagent basins, 15 pieces

General Lab Equipment needed to perform a Vitotox test

1. Microplate Luminometer
2. Orbital Shaker Incubator with Temperature control 35°C
3. 12-channel pipette 5 -50 µl
4. 12-channel pipette 50-300 µl
5. Single channel pipettes 10-100 µl, 20-200 µl, 1-5 ml
6. DMSO and / or other solvents
7. S9 Mixture (see frame →)
8. Positive controls.

Vitotox Procedure

Salamonella Cultures in Vitotox Testing

Preparations the Day before testing

1. Per plate: Take 2 tubes of 5 ml Growth Medium (cf. figure above) and allow defrosting at room Temperature.
2. Remove 1 Red vial of the Genox strain and 1 Blue vial of the Cytox strain from the freezer and bring to room temperature.
3. Transfer 20 µl of the Genox strain to its designated Growth Medium tube.
Discard the vial with remnant in the appropriate waste recipient.
4. Transfer 20 µl of the Cytox strain to its designated Growth Medium tube.
Discard the vial with remnant in the appropriate waste recipient.
5. Use the Growth Medium tubes as culture vessel and incubate the tubes overnight (ON 16 hours) in an orbital shaker incubator in a 45° angle (170 RPM) at 35°C.

Testing Day

1. After the Overnight incubation: Take 2 tubes of 2.5 ml Test Medium and bring to room temperature.
2. Transfer 20 µl of the ON Genox suspension to its designated 2.5 ml Test Medium tube.
Discard the tube with excess bacterial suspension in the appropriate waste recipient.
3. Transfer 20 µl of the ON Cytox suspension to its designated 2.5 ml Test Medium tube.
Discard the tube with excess bacterial suspension in the appropriate waste recipient.
4. Use the Test Medium tubes as culture vessel and incubate the tubes for one hour in an orbital shaker incubator in a 45° angle (170 RPM) at 35°C to obtain the LOG phase.
5. After the LOG Phase Incubation: Take 2 tubes of 4.5 ml Test medium and bring to room temperature.
6. Transfer 500 µl of the LOG Genox suspension to its designated 4.5 ml Test medium tube.
Discard the tube with excess bacterial suspension in the appropriate waste recipient.
7. Transfer 500 µl of the LOG Cytox suspension to its designated 4.5 ml Test medium tube.
Discard the tube with excess bacterial suspension in the appropriate waste recipient.

How to prepare the samples

Dilution of samples

A dilution series of the sample is used for the test. Dilutions of one sample are most easily done into one 96-well clear plate included in the kit. Testing of one sample according to this procedure uses four rows (A to D) of the 96-well plate. It is recommended to test another sample simultaneously using the wells of the four remaining rows E to H. The dilution of the samples is done similarly for both samples; expect that the highest concentration of the sample must be selected individually for both samples. The presentation on the following pages describes the preparation of the one sample dilution series with positive controls. The same procedure should be repeated for the second sample used. The highest concentration used into the test should be close to the maximum solubility of the sample. This concentration is highly dependent on the sample and therefore must be chosen individually for each sample.

Note! The final concentration of the solvent; DMSO, ethanol, methanol, etc. in the measurement plate must be kept lower than 4% (usually 1% final concentration is recommended) and must be identical in all the wells.
Note! All plates and strips used in the VitotoxTM 10 kit are made from polystyrene, which has a limited chemical resistance. Therefore some organic solvents of high toxicity (e.g. hexane or chloroform) cannot be used in the test 10 kit.

1. Add 20 µl of the solvent into wells 1 to 9 of a 12-well microstrip (Strip 1)

2. Add 40 µl of the sample stock solution (in the same solvent) into well 10 of this microstrip.

3. Transfer 20 µl from well 10 to well 9, mix properly and transfer 20 µl to well 8, etc. until coming to well 3, which is the last well for the dilution series. After mixing, discard 20 µl from well 3. This generates a dilution series 1/1, 1/2, …, 1/128. Wells 1 and 2 contain only the solvent.

4. Add 180 µl of purified distilled H2O into each well (1 to 10).

5. Divide this dilution series into two identical 12 well microstrips by transferring 100 µl of the solution with a multichannel pipette into another strip (Strip 2).

Note! For water soluble products make the dilutions as described above by using it as a solvent.

The preparation of the positive controls

4-Nitroquinoline-N-oxide

4-Nitroquinolineoxide (4-NQO) (CAS 56-57-5) is recommended to be used as a positive control for genotoxicity testing without metabolic activation. A suitable control sample is prepared as follows:

• Prepare a stock solution of 4000 ppm in DMSO (4 mg/ml) in a test tube.
This solution can be aliquoted and stored at -20°C for 6 months.
• Dilute this stock solution 1/100 (Add 10 µl of the stock solution to 990 µl DMSO) to get a 40 ppm solution. Further dilute this solution 1/100 to get a 0.4 ppm solution.

1. Transfer 10 µl of the 0.4 ppm solution into well 11 of Strip 1, which contains the previously prepared sample dilution series.

2. Add 90 µl of purified distilled H2O into this well.

Note! The final concentration of 4-NQO in the measurement plate will be 4 ppb.

Prepare the specific blank for the 4-NQO control as follows:

1. Transfer 10 µl of DMSO (solvent of the positive control) into well 12 of Strip 1, which contains the sample dilution series and 4-NQO as a positive control.

2. Add 90 µl of purified distilled H2O into this well.

Benzo(α)Pyrene

Benzo(α)pyrene (B(α)P) (CAS 50-32-8) is recommended to be used as a positive control for genotoxicity testing with metabolic activation. A suitable control sample is prepared as follows:

• Prepare a stock solution of 8000 ppm in DMSO (8 mg/ml) in a test tube.
This solution can be aliquoted and stored at -20°C for 6 months.
• Dilute this stock solution 1/10 (Add 100 µl of the stock solution to 900 µl DMSO) to get a 800 ppm solution.

1. Transfer 10 µl of the 800 ppm solution into well 11 of Strip 2, which contains the previously prepared sample dilution series.

2. Add 90 µl of purified distilled H2O into this well.

Note! The final concentration of B(α)P in the measurement plate will be 8 ppm.

Prepare the specific blank for the B(α)P control as follows:

1. Transfer 10 µl of DMSO (solvent of the positive control) into well 12 of Strip 2, which contains the sample dilution series and B(α)P as a positive control.

2. Add 90 µl of purified distilled H2O into this well.

Note! If some other compounds than 4-NQO and B(α)P are used as controls, the solvent must be chosen according to the solubility of that compound. The final concentration must be kept below 4 % of the final volume (recommended is <1 % of the final volume). The specific blanks must be prepared with the same solvent to get correct blank values.

When this sample dilution and control procedure is repeated for sample 2 of the measuring set, four 12-well strips containing all necessary sample materials will be present according to the figure below.

The dilutions are now ready to be added directly into wells of the black 96-well measuring plate as described in PREPARATION OF THE MEASUREMENT PLATE on page 10

Preparationof the S9 mixture

For samples requiring metabolic activation, a hepatic P450 enzyme containing microsomal homogenate (S9 fraction) from Aroclor-induced rats must be used with the VitotoxTM 10 kit. S9 homogenates are subcellular fractions prepared by a 9000xg centrifugation of liver homogenate, resulting in removal of nuclei and mitochondria. The ribosomal fraction contains both microsomal and cytosolic enzymes (membrane bound and soluble forms). The preparations are capable of both phase I and phase II metabolism. S9 fractions can be prepared in-house or are commercially available.

A fresh S9 mixture should be prepared each time when the test is performed.

To prepare the S9 mixture, mix together the following solutions:

• 50 µl KCl/MgCl2 solution • 50 µl β-NADP solution • 25 µl G6P solution • 1250 µl Phosphate buffer • 1250 µl Distilled water • 66 µl S9 liver homogenate

The composition of the above solutions can be found on the next page. These solutions can be made in advance and stored over a longer period of time. All reagents can be purchased.

PREPARATION OF THE S9 MIXTURE COMPONENTS KCl/MgCl2 solution • 6.15 g KCl • 4.7 g MgCl2 . 6H2O • Sterile distilled H2O to 100 ml Autoclave and store at room temperature. 0.1 M β-NADP solution • 500 mg β-NADP • Sterile distilled H2O to 6.53 ml Divide into aliquots and store in cryotubes at -20°C 1.0 M Glucose-6-phosphate (G6P) solution • 282 mg G6P • Sterile distilled H2O to 1.0 ml Divide into aliquots and store in cryotubes at -20°C 0.2 M Phosphate buffer, pH 7.4 • 60 ml 0.2 M NaH2PO4 . H2O • 440 ml 0.2 M Na2HPO4 Adjust the pH to 7.4 and autoclave in 100 ml portions. Store at room temperature.

The above solutions can be made in advance and stored over a longer period of time.

Note! The concentration of S9 liver homogenate has been selected to give highest assay sensitivity with B(α)P and 2-aminofluorene as a reference chemical. The most effective S9 concentration may be different with other chemicals.
Note! With certain chemicals (for example fluoranthene, phenanthrene and most mixed samples) require a higher amount of S9 liver homogenate for sufficient metabolism. In these cases, the assay should be repeated with higher S9 homogenate concentration, for example 4X concentration (264 µl) of liver homogenate.

Preparation of the measurementplate

The samples from the four 12-well microstrips need to be transferred with a 12 channel pipette to the 96-well plate according to the plate layout in Appendix I, which you can find on page 13.

Mix the contents of the wells with the 12 channel pipette properly before performing the following steps:

1. Transfer 10 µl from the dilution series of Sample I and the 4-NQO positive control
(Sample I / Strip 1) into rows A and B.
2. Transfer 10 µl from the dilution series of Sample I and the B(α)P positive control
(Sample I / Strip 2) into rows C and D.
3. Transfer 10 µl from the dilution series of Sample II and the 4-NQO positive control
(Sample II / Strip 1) into rows E and F.
4. Transfer 10 µl from the dilution series of Sample II and the B(α)P positive control
(Sample II / Strip 2) into rows G and H.

5. Transfer 10 µl of the S9 mixture with a 12 channel pipette from a disposable reagent basin into rows C - D and G – H.

6. Transfer 90 µl of the Genox culture with a 12 channel pipette from a disposable reagent basin into rows A and E.
7. Transfer 80 µl of the Genox culture with a 12 channel pipette from a disposable reagent basin into rows C and G.
8. Transfer 90 µl of the Cytox culture with a 12 channel pipette from a disposable reagent basin into rows B and F.
9. Transfer 80 µl of the Cytox culture with a 12 channel pipette from a disposable reagent basin into rows D and H.

Note! The Final dilutions of the samples in the measurement plate are 1/100, 1/200, 1/400, …, 1/12800 compared to the original stock concentration made at the beginning.

Test procedure

The test is a kinetic assay performed with a microplate luminometer. Light emission from each well is measured every 5 minutes with 1 second integration time, and this for a period of 4 hours.
Before the first measurement, the plate should be mixed for 30 seconds in order to mix all the reagents properly in the wells.

The test should be performed at 30°C (± 1°C)

Measurement Settings: 30°C, 1 sec/well, 48 cycles of 300 sec each.

Calculation of the results

The following calculations have to be made:

• The Signal-to-Noise ratio (S/N). The ratio of a sample value to the corresponding blank value, for each time point, for all assays.
• Use the maximum S/N ratio between 60 and 240 minutes for each sample concentration and for both test agents and treatments to calculate the Genox/Cytox ratio.
• The Genox/Cytox ratio. The ratio of the maximum S/N ratio of the Genox strain to the corresponding maximum S/N ratio of the Cytox strain for each concentration and treatment.

Genotoxicity

A product is considered to be genotoxic when all following criteria are met:

• The maximum S/N ratio of the Genox strain must give a dose-response correlation over a concentration range of at least 3 dilutions.
• The Genox/Cytox ratio is > 1.5 and a dose-response correlation over a concentration range of at least 3 dilutions.
• At least two different concentration are giving a S/N ratio > 1.5
• SOS response only starts after 20 – 45 min. in exponentially growing cultures. Therefore any values obtained from measurements before 60 min. should not be used in any results interpretations.
• If both the Genox strain and the Cytox strain are strongly induced one may not conclude for genotoxicity even as the Genox/Cytox ratio is > 1.5
• If the maximum S/N ratio for the Genox strain is ≤ 1 the result is negative even when the Genox/Cytox ratio is > 1.5

General problems observed when perorming the Vitotox test

Error	Potential Cause(s)
No Signal from the sample wells	Too high original sample concentration Immediate toxic effect Too high concentration of solvent in the wells
No signal from the positive controls	Too high control or solvent concentration No Enhancer Reagent added No test bacteria added
Too low Signal/Noise ratio in positive controls	Incorrect pipetting volumes.

References

• van der Lelie D, Regniers L, Borremans B, Provoost A, Verschaeve L. The VITOTOX test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics. Mutat Res. 1997 Mar 17;389(2-3):279-90.

• Verschaeve L, Van Gompel J, Thilemans L, Regniers L, Vanparys P, van der Lelie D. VITOTOX bacterial genotoxicity and toxicity test for the rapid screening of chemicals. Environ Mol Mutagen. 1999;33(3):240-8.

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