The VitotoxTM test is a high throughput bacterial genotoxicity assay which
can detect DNA damage caused by genotoxic compounds as light emission changes
depending on SOS DNA repair induction.
The VitotoxTM test is a high throughput bacterial genotoxicity assay, which can
detect DNA damage caused by genotoxic compounds. This test utilizes the
bacterial SOS DNA-repair system induced by genotoxic compounds [2, 3]. Two
genetically engineered Salmonella typhimurium strains are used in this test
system, TA104 recN2-4 (Genox strain) and TA104 pr1 (Cytox strain). The former
strain carries a plasmid containing the bacterial luciferase operon (luxCDABE)
of luminous bacteria Vibrio fischeri under transcriptional control of an recN
promoter (recN2-4). The latter strain constitutively expresses the lux operon.
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 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
MATERIALS AND METHODS
compounds were purchased from GENAUR Molecular Products.
Two tester strains, S. typhimurium TA104 recN2-4 (Genox strain) and TA104 pr1
(Cytox strain) were supplied as components of the Vitotox 10 Kit obtained from
GENTAUR Insrtitute (Kampenhout, Begium). Rat liver S9, purchased from Kikkoman
Co. (Chiba, Japan), was prepared as a 9000x g supernatant fraction of the liver
homogenates prepared from male SD rats treated with phenobarbital and
5,6-benzoflavone. Co-factor I was purchased from Oriental Yeast Co., Ltd (Tokyo,
Japan). The S9 mix was prepared by mixing the S9 fraction and co-factor
solutions at the volume ratio of 1:9.
1. 5 Yellow vials of 0.1ml Genox reagent S. typhimurium TA104 recN2-4 2. 5 Blue
vials of 0.1 ml Cytox reagent S. typhimurium TA104 pr1 (Cytox strain) store at
3. 10 vials 8 ml Rich Growth Solution (with enhancer) -20°C
4. 5 vials of 10 ml Poor Growth Solution -20°C
5. White 96 well plate 5 pcs
6. Clear 96 well strip plates 3pc
7. Reagent basin 20
Equipment Assay procedure
1. Remove 2 vials of 8ml Rich Growth Solution and 1 vials of Genox and Cytox
reagent from the freezer and leave until room temp RT
2. Add 0.1ml of each to their destinated 8 ml Rich Growth Medium
3.Use the Medium Vial as culture vessel and incubate the vials overnight (18h)
on an orbital shaker (160-180 RPM, corresponding to 0.27-0.35G)at 35 to 37°C
Remove 10 ml of Poor Growth Medium from the freezer and leave until room
Measure the optical density at 590nm with 0.5ml that you remove from the 8ml
The optical densitys are
Genox reagent 0.200-0.500
Cytox reagent 0.400-0.600
Store the vials that are in the correct range and store at 4°C until use.
If the densities are below, the growth time can be extended up to 20h.
Dilute each bacterial suspension as described:
Genox reagent: Add 1.1 ml of the 7.5 ml bacterial suspension and add to the 10
ml of the Poor Growth Medium (1/10 dilution). Discard the rest of the Genox
reagent (7.9 ml)
Cytox reagent: Discard 2.5ml of the 7.5 ml bacterial suspension and mix the
remaining 5 ml to 5 ml sterile distilled water. (1/2 dilution)
You will have 11.1ml Genox culture and 10 ml Cytox culture ready for use. You
will only need +- 4.08 ml from each. (2x 1080ul and 2x 960ul)
Dilution of samples
The Vitotox-384 test
The Vitotox-384 test was performed according to the instructions for the Vitotox
10 Kit supplied by the manufacturer, with some minor modifications.
The lyophilized test bacteria (Genox and Cytox strains) in the vials were
hydrated with the growth medium and incubated over night at 37°C with shaking.
Overnight cultures of these bacterial strains were diluted with the medium to
make an optical density 0.03.
Each test substance was dissolved or suspended in DMSO and a series of test
substance solutions was prepared by 2-fold dilutions with the same solvent. Each
preparation was diluted ten-fold with purified water, and the resultant mixtures
were applied to the treatment. For our compounds, 1000 mg/mL was selected as the
highest concentration. When it was impossible or technically difficult to
transfer the test substance preparations to the treatment plate (e.g., inability
of pipetting due to large precipitates), a lower concentration was selected as
the highest concentration. The solvent was used as negative control.
4-Nitroquinoline (4-NQO) and benzp[a]pyrene (B[a]P) were used as the positive
control in the treatment with and without metabolic activation, respectively.
Five mL of the test preparations was added to each well of a 384-well
microplate. Five mL of the S9 mix or purified water and 40mL of the bacterial
cultures were added to each well. The light production from each well was
measured every 15 min for 4 hr at 30°C using a luminometer (Fluoroskan Ascent
The data was analyzed with Ascent Software that automatically calculated the
Genox/Cytox ratio. The genotoxicity of the test substance was evaluated with the
Genox/Cytox ratio. When the ratio increased dose-dependently and became 1.5 or
higher of the solvent control value in non-cytotoxic concentrations, the test
substance was judged positive for genotoxicity (DNA-damaging activity). When the
test substance showed a severe cytotoxicity, an additional test was conducted at
the lower concentrations.
The Ames test
The Ames test was performed with the Salmonella typhimurium TA98, TA100 and
TA2637, and Escherichia coli WP2uvrA, both in the presence and absence of
metabolic activation with rat S9 mix .
RESULTS AND DISCUSSION
Table 1 summarizes the results of the Vitotox-384 test for 61 NTP compounds.
Twenty-six Ames-positive compounds gave positive results in the Vitotox-384
test. Styrene oxide, 1,2-epoxybutane and 8-hydroxyquinoline gave negative
results in the Vitotox-384 assay, as indicated in the Vitotox-96 assay . We
could not examine sufficient higher dose levels for styrene oxide and
1,2-epoxybutane because these chemicals causticized the plastic microplates
used. It was reported that 8-hydroxyquinoline is not genotoxic in the SOS
chromotest and the umu test . Three compounds (2,4-diaminotoluene,
2,6-diaminotoluene and 1,2-dimethylhydrazine) gave inconclusive results because
these compounds enhanced a light emission not only in the Genox strain but also
in the Cytox strain.
Twenty-five of 28 Ames-negative compounds gave negative results in the
Vitotox-384 test. Three compounds, caprolactam, isophorone and thioacetamide,
gave inconclusive results, because they enhanced light emission in both Genox
and Cytox strains.
The sensitivity of Vitotox-384 for NTP selected compounds was about 87% (26/30,
excluding three inconclusive results); negative specificity was 100% (25/25,
excluding three inconclusive results). The concordance between the Vitotox-384
test and the Ames test was about 92%.
We found that Vitotox test sensitivity could be improved by modification of the
treatment time from 180 min, specified by a manufacturer, to 240 min. In many
cases, the induction of light emission in the Genox strain by genotoxins start
at about 60 min after incubation and reaches a maximum within 180 min. However,
we have experienced that some compounds showed “delayed enhancement” of light
emission in the Genox strain. These chemicals can be detected as genotoxic only
with a prolonged treatment (Fig. 1). This fact indicates the advantage of
kinetic analysis in the Vitotox test over end-point assays like the umu test.
We have already screened more than a thousand chemicals synthesized in
Mitsubishi Pharma Corporation for genotoxicity since 2001 with the Vitotox-384
test. A total of 137 compounds were tested with both the Vitotox-384 test and
Ames test (Table 2).
The sensitivity of the Vitotox-384 test for the Ames–positive compounds is
72.4% (21/29). Six of the eight chemicals that gave false negatives in the
Vitotox-384 test belonged to the same medical application category. This might
be caused by some SOS-repair independent mechanism of mutagenicity. All of the
108 Ames-negative compounds gave negative results in the Vitotox-384 test.
Fig. 2 Changes in the rate of positive results in the Vitotox and Ames tests
Fig. 2 shows the rate of positives results in the Vitotox-384 test and Ames
test for our newly synthesized compounds from 1998 to 2005. We screen newly
synthesized chemicals with the Vitotox test first. If the substance gives a
negative result with the Vitotox test, we confirm the result with the Ames test
at a later stage. If the substance gives a positive result in the Vitotox test,
we do not conduct any futher evaluation for the chemical and try to evolve the
structure. The positive rates with the Vitotox-384 test have shown constant
values of about 7 to 11% over the last four years. In contrast, positive rates
with the Ames test have decreased year by year since we introduced the
Vitotox-384 test, indicating that genotoxic chemicals are screened out well
using the Vitotox test in the early stage. In other words, genotoxic
prescreening of genotoxicity with the Vitotox-384 test works well.
The Vitotox-384 test needs less than 10 mg of compound and allows us to test
over 15 chemicals/day, even using a manual procedure. The simple test procedure
involved may make the test easy to automate to improve the throughput.
In conclusion, the Vitotox-384 test is available for genotoxic screening of
newly synthesized chemicals in the early stage of pharmaceutical development.
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