Module ID  Description # of Compounds
A2I Salmonella Ames Mutagenicity 2154
A99 Salmonella Mutagenicity of Pesticides 55
A9L Salmonella mutation GeneTox 2003 (SAL+/- S9) 706
A9M Salmonella mutation NTP 2003 (SAL+/- S9) 1549
A2N Salmonella typhimurium (TA97) mutation in absence of S9 1007
A2O Salmonella typhimurium (TA98) mutation in absence of S9 3456
A2P Salmonella typhimurium (TA100) mutation in absence of S9 3781
A9A Salmonella typhimurium (TA102) mutation in absence of S9 522
A9B Salmonella typhimurium (TA104) mutation in absence of S9 276
A2L Salmonella typhimurium (TA1535) mutation in absence of S9 2051
A2M Salmonella typhimurium (TA1537) mutation in absence of S9 1558
A9C Salmonella typhimurium (TA1538) mutation in absence of S9 646
A2T Salmonella typhimurium (TA97) mutation in presence of Hamster S9 639
A2U Salmonella typhimurium (TA98) mutation in presence of Hamster S9 1673
A2P Salmonella typhimurium (TA100) mutation in presence of Hamster S9 1662
A9D Salmonella typhimurium (TA102) mutation in presence of Hamster S9 39
A9E Salmonella typhimurium (TA104) mutation in presence of Hamster S9 33
A2R Salmonella typhimurium (TA1535) mutation in presence of Hamster S9 1476
A2S Salmonella typhimurium (TA1537) mutation in presence of Hamster S9 948
A9F Salmonella typhimurium (TA1538) mutation in presence of Hamster S9 218
A2Y Salmonella typhimurium (TA97) mutation in presence of Rat S9 766
A2Z Salmonella typhimurium (TA98) mutation in presence of Rat S9 2983
A2V Salmonella typhimurium (TA100) mutation in presence of Rat S9 3214
A9G Salmonella typhimurium (TA102) mutation in presence of Rat S9 424
A9H Salmonella typhimurium (TA104) mutation in presence of Rat S9 216
A2W Salmonella typhimurium (TA1535) mutation in presence of Rat S9 1757
A2X Salmonella typhimurium (TA1537) mutation in presence of Rat S9 1469
A9I Salmonella typhimurium (TA1538) mutation in presence of Rat S9 714
A2H Salmonella Ames Mutagenicity updated from NTP, GENETOX, FDA and others 5864*
A6A Aneuploidy in Yeast 93
A6B Bacillus subtilis Mutagenicity of Pesticides 31
A2D Drosophila Mutation 783*
A6E E.Coli mutation (CCRIS) 862*
A6F GENETOX (full database) 808
A62 Micronuclei Induction 904*
A6J Mouse lymphoma L5178Y (TK+/TK-) +/-S9 (NTP, GENETOX, CCRIS) 910*
A61 NTP Chromosomal Aberration  805*
A60 NTP Sister Chromatid Exchange 585*
A6M Saccharomyces cerevisiae Mutagenicity of Pesticides 55
A6N Sister Chromatid Exchanges - In Vivo 176
A6O SOS Chromotest 462
A2E Structural Alerts - Ashby 784*
A64 UDS Induction 299
A7A FDA Mutagenicity Microbial composite (Sal_Ecoli_Bac) 3682*

A7B

FDA Mutagenicity Salmonella t. 5-strains            

3573*

A7C

FDA Mutagenicity E.coli composite                   

526*

A7D

FDA Mutagenicity E.coli WP strains                  

279*

A7E

FDA Mutagenicity Yeast composite                    

603*

A7F

FDA Mutagenicity Yeast Sacharomyces                 

472*

A7G

FDA Mutagenicity Drosophila composite               

591*

A7H

FDA Mutagenicity Drosophila sex linked rec

584*

A7I

FDA Mutagenicity Drosophila heritable translocation 

115*

A7J

FDA Mutagenicity Mammal in vivo composite           

214*

A7K

FDA Mutagenicity Mammal in vivo dominant lethal     

182*

A7O

FDA Mutagenicity Mammal in vitro CHO V79 hgprt loci 

641*

A7S

FDA Clastogenicity Micronucleus in vivo composite     

820*

A7T

FDA Clastogenicity Micronucleus in vivo mouse         

622*

A8J

FDA Clastogenicity Micronucleus in vivo undef. species

441*

A7P

FDA Clastogenicity Chromosome abs in vivo composite   

284*

A7R

FDA Clastogenicity Chromosome abs in vivo Mouse       

152*

A8A

FDA DNA Effects UDS composite                      

376*

A8B

FDA Mutagenicity UDS rat hepatocytes                

142*

A8C

FDA Mutagenicity UDS human fibroblasts              

197*

A8K

FDA Mutagenicity UDS other cell                     

102*

Salmonella TA97,TA98, TA100, TA102, TA104, TA1535, TA1537 and TA1538 strains modules  are developed with four different flavors: experimental call, maximum fold, maximum effective concentration and rev/nmol.

The VITOTOX test is a test which is more rapid and less expensive than the
“classical” Ames test. The test is also at least as sensitive as the Ames test. It is based on
bacteria that contain the lux operon of Vibrio fischeri under transcriptional control of the recN
gene, that is part of the SOS-system (Scheme 6.1). This gene is normally not transcribed (no
light production) but it will be “switched on” when the bacteria are exposed to a genotoxic
compound (mutagen or “SOS-inducing” substance). Genotoxicity is thus expressed as light
production.

Schematic representation of the principle of SOS-induction on
which the VITOTOX test is based (insertion of an operon-less “lux” gene next
to recN results in light production when the bacterial DNA is damaged)

Although any bacterial strain can be used, Salmonella typhimurium strains were chosen as
these are well known for mutagenicity testing and because the same bacteria can also be used
Screening of the Bioactivity of the Isolated Natural Productsfor a classical Ames test.

 However, as all Salmonella constructs gave very comparable results,
only the TA104 construct [called T104 (recN2-4)] is used as it was shown to be sometimes a
little bit more sensitive than the other hybrid strains.
As it was realised that some compounds act directly on the light production (e.g., aldehydes)
or enhance the metabolism of the bacteria creating false-positive results, a constitutive light
producing strain with a lux operon under control of the strong promoter pr1 was also
incorporated. This is used as an internal control system, which also gives important
information on the toxicity of the test compound. Major advantages are less sample used, results in 4h.

Is is used in real time with Agilent or LuminMax luminometer.

Monitoring genotoxicity during the photocatalytic degradation of p-nitrophenol with VitoTOX on Agilent
 
M. Shani Sekler 1, Y. Levi 1, B. Polyak 1, A. Novoa 1, P. S. M. Dunlop 2, J. A. Byrne 2, R. S. Marks 1 3 *
1Institute for Applied Biosciences, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel
2NIBEC, University of Ulster at Jordanstown, Newtownabbey, Northern Ireland BT37 0QB, UK
3Department of Biotechnology Engineering, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel
 
email: R. S. Marks (This email address is being protected from spambots. You need JavaScript enabled to view it.)

*Correspondence to R. S. Marks, Institute for Applied Biosciences, Ben-Gurion University, PO Box 653, Beer-Sheva 84105, Israel.

Funded by:
 EC; Grant Number: EVKI-CT-2000-00069

photocatalysis • genotoxicity • p-nitrophenol • bioassay
p-Nitrophenol is a common structural unit of many pesticides and was chosen as a model compound to monitor genotoxicity during photocatalytic degradation. The genotoxicity of p-nitrophenol (PNP) and its breakdown products was measured using a bioluminescent bacterial bioassay, VitotoxTM. The genotoxic potential decreased with the concomitant photocatalytic degradation of the parent PNP concentration. The rate of genotoxicity reduction was slower than the rate of removal of the parent PNP, due to the formation of genotoxic by-products. After 6 h of photocatalytic treatment the total genotoxicity was removed. These results indicate that bioassays can be used as a simple and highly sensitive method for monitoring the general toxicity of chemical pollutants before, during and after photocatalytic treatment or other destructive processes. Copyright © 2004 John Wiley & Sons, Ltd.

LuminMax-C

Easy to Use Attomole Luminometer

   ►   Introduction

The study and use of bioluminescence and chemiluminescence has increased dramatically in the recent years. The applications have extended from ATP-luciferase assay for cell viability tests, to current DNA, genomic, and proteomic analysis. Unlike a fluorescence system, there is no need for an exogenous light source. Luminescence is generated from chemical reactions. Utilizing photoncounting detector, counting the photons generated from the reaction, luminescence assay has become one of the most sensitive optical detection method. LuminMax-C offers excellent sensitivity, accuracy, ease of use, compactness, and affordability. 

 

   ►   Features

 LuminMax-C is a compact model designed for highly sensitive chemiluminescence and bioluminescence detection. The system accurately quantifies the luminescence intensity in a 96-well microplate (black or white). The microplate has aclear bottom; therefore, the luminescence can be detected from the bottom of the well. Because it uses state-of-the-art photoncounting multiplier tube as detector, the system is extremely sensitive. LuminMax-C has the ability to count the number of photon generated from the reaction, it means that it can detect very small amount of analyte in the samples. A CD, with user-friendly software, is provided for easy installation. LuminMax-C utilizes PC or notebook as its microprocessor. The system is interfaced to a computer by a simple plug-in (USB or serial port) connection. After click on the "Go!" botton, the system automatically and quickly scans all of the selected microwells and displays the results. The resulting data is displayed as a spreadsheet in Microsoft-Excel format. The data, reported as number of photon counts or relative light unit (RLU), is displayed as it is collected.

 

   ►   Specifications

Optical detection: Chemi- or bio-luminescence
Plate format: Microplate (96 wells)
Sensitivity: 1 attomole HRP and ATP
Optical wavelength: 300 ~ 680 nm
Dynamic range: Seven decades
Optical detector: Photoncounting PMT
Cross-talk: < 6 x 10-5
Operation: Automoted scanning any or all wells; Integration time (0.01 ~ 10.0 S) per well; Adjustable number of scans; Adjustable delay for kinetic study
Interface: Serial or USB to PC or Notebook (PC or Notebook not included)
Software: CD with user friendly software; Display all microwell data
Data output: Excel format in MS Windows
Power requirement: 115V, 60Hz
Dimension: 12" W x 11" L x 5.8" H 
(30cm W x 28cm L x 15cm H)
Weight: 19.8 lbs. (9 kgs.)
 

 

   ►   Applications

ATP assay
Luciferase assay
Immunoassay & proteomics
Nucleic acid, DNA assay & Genomics
Clinical diagnostics
Genomic analysis
VitotTox Toxicity test
Cell viability test
Restaurant sanitary test
 

 

   ►   Luminometer Users

Biotechnology research laboratories
University biological and biochemical laboratories
Government biological and biomedical laboratories
Hospital research laboratories
Food industry, environmental or forensic testing
 
 

CELL QUANTIFICATION AND ATP TESTS                                            

LuminMax-Q-Cell:  Luminescent Cell Viability Assay                                                                                                                   155 USD

1 vial LuminMax-ES, Luciferase and Substrate (luciferin) (lyophilized)

10 ml LuminMax-CB, Cell Buffer 

100µl per assay (microwell),

the material is sufficient for 100 assays in 96-well plate  

ATP:  for Sensitivity and Standard Tests                                                                                                                     175 USD

1 vial (400 µl, 100mM) 

20µl for a series of dilution for a standard (calibration) test,

the material is sufficient for standard tests for 20 times

  • The lyophilized LuminMax-ES and Cell Buffer should be stored at -20oC for

            long-term (over one week) storage.  For frequent use, it can be stored at 4oC;

however, it may lose some activity.

ATP Assay Protocol:

Purpose: To generate ATP standard curve

Material: 

1.            ATP 100mM

2.            LuminMax-CB buffer

3.            LumiMax-ES substrate (lyophilized)

4.            Bottom clear 96 well microplate or strip: Must be compatible with the luminometer

5.      Nuclease-free water 

Reagent Preparation: 

1.           Thaw the LuminMax-CB Buffer and equilibrate to room temperature prior to use.

2.           Equilibrate the lyophilized LuminMax-ES Substrate to room temperature prior to

         use.

3.           Transfer the appropriate volume of Buffer into the bottle containing Substrate to

         reconstitute the lyophilized enzyme/substrate mixture.  This is the Quantitative

         Cell (or Signal) Reagent. 

Experiment:

1.            Prepare 1uM ATP in culture medium, buffer or nuclease-free water.

        (100µl of 1µM ATP solution contains 10-10 moles ATP). 

2.            Prepare tenfold serial dilutions of ATP in culture medium, buffer or nuclease-free

         water (e.g. 1µM to 10nM). (Dilute further for sensitivity test of the luminometer.)

3.            Dispense 100µl of ATP solutions in microwell plate.

4.            Add a volume of Q-Cell reagent equal to the volume of ATP.

5.            Mix contents for 2 minutes on a shaker.

6.            Incubate the plate at room temperature for 5 or 10 minutes.

7.            Read luminescence with integration time of 100 ms or 1.0 second.

Example of ATP Standard Curve Assay

Purpose: To Evaluate sensitivity with ATP assay using LuminMax-C

     

Material:

               

1.                   ATP: 100mM, 400ul, store at -20C, thawed, Store 4C.

     
 

Prepare (1)20ul + 1.98ml nuclease-free water = 1mM;

   
 

(2) 20ul of (1) +1.98 ml of water = 10uM

       
 

(3) 0.1ml of (2)+ 0.9ml water =1uM, 100ul 1uM ATP=10 -10moles ATP ;

 
 

(4) 20ul of (3)+1.98ml water =10nM; 

       
 

(5) 20ul of (4)+1.98ml water = 100pM;

     
 

(6) 200ul of (5)+200ul water = 50pM;

       
 

(7) 200ul of (6) +200ul water = 25pM

       

2.       

Cell buffer, 10ml

         
 

Thaw and equilibrate the lyophilized substrate and buffer to RT.

   
 

Transfer all buffer into the substrate bottle. Mix.

     
 

Dispense 1.5ml per vial if needed. Store at 4C.

     

Experiment :

               

1.        Dispense 100ul of ATP in the microwell.    

       

2.        Add 100ul of LuminMax-Q-Cell equal to the volume of ATP standard present in each well.

3.        Mix for 2 minutes on orbital shaker.

         

4.        Allow the plate to incubate at RT for 5 or 10 min.

     

5.        Record luminescent on LuminMax for 100 ms or 1 second.

     

Result:

               

Microwell

1

2

3

4

5

6

7

8

Concentration, 100ul

0

25pM

50pM

100pM

10nM

100nM

1uM

10uM

5 min (100ms)

3

9

20

57

141

590

5662

40497

10 min (1 second)'

40

137

243

433

1861

5925

57954

409921

 

Cell Assay Protocol: 

Cell viability assay is similar to ATP assay protocol, instead of using ATP as analyte, cell sample is input to the microwell.  Depend on the cell types; each cell may contain 10-15

-   10-18   mole of ATP.  Once the cell titer (Cells per microwell) vs. Luminescence Signal is calibrated, the method can be used to correlate cell number with luminescent output.

 

 

3.4. The evaluation of the Vitotox test through 11 blind tested
samples
Jacky Van Gompel and Luc Thilemans
Department of Genetic and in vitro Toxicology
Janssen Research Foundation
Turnhoutseweg 30
B-2340 Beerse
Belgium.
Tel.: 0032(0)14 60 50 18
Fax: 0032(0)14 60 65 15
E-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.
E-mail : mailto:This email address is being protected from spambots. You need JavaScript enabled to view it.
Summary
In order to assess the robustness, sensitivity and specificity of a recently developed screening
assay, 11 coded compounds were tested in the Vitotox assay with strains TA104recn2-4 and
TA104pr1, obtained from the Flemish Institute for Technological Research (VITO). The aim
was to assess the results from the different test systems used by the participants of this
collaborative study – Technotox - and to compare them with known literature data. The most
remarkable fact is the absence of false positive results which we also observed in our own test
panel of 45 Janssen compounds. All of the 11 samples in the current study were correctly
identified with very high sensitivity.
Introduction
New synthesis strategies together with the establishment of high throughput screening
methods in pharmacology departments are increasing the number of compounds entering the
“exploratory development” phase. Due to the necessity to optimally use resources, selections
have to be made in an early phase of development to minimise the risks of failure in later and
more expensive phases of development. Recently emerging screening tests for detecting DNA
damage and gene mutations enable the genetic toxicologist to provide crucial mutagenicity
information for selection of the most promising candidates or in priority setting between
several similar drug candidates. One of the key problems was the compound consumption and
the short time frame in which results should be obtained. Apart from the discussion about the
predictive value of bacterial tests towards human carcinogenicity, the bacterial reverse
mutation test still has a pivotal position in the final regulatory acceptance of new chemical
entities. Bacterial assays, like the SOS chromotest, based on the expression of repair genes
induced by genotoxic agents are very useful screening tools with high predictive power
towards the Ames test results. A promising new screening test, the Vitotox test developed by
VITO, which has the same basic principle as the SOS chromotest test but has the additional
advantage of the kinetic evaluation and higher dynamic range, scores very well both on
compound consumption and testing time schedule. The TA104recN2-4 strain has a lux operon
of Vibrio fisheri under transcriptional control of the recN gene, that is part of the SOS repair
system. If under the influence of a genotoxic compound the recN promotor is depressed, this
will result in expression of the lux operon and thus light production. The other strain,
TA104pr1, has the lux operon under control of a strong constitutive promotor pr1 and is used
as internal control to detect false positive compounds (compounds that act directly on the light
production (aldehydes) or enhance the metabolism). An internal evaluation study at our
laboratories with about 66 new drug candidates or drug intermediates gave very favourable
results as prediction towards Ames results (Sensitivity = 82%, Specificity = 100%,
Concordance = 92%, False negative rate = 18%, False positive rate = 0%).
Material and Methods
Chemicals
The 11 samples under investigation were distributed with a blind code. The S9-fraction from
the rat metabolic activation system was provided by Moltox. Following positive control
substances were used: 4-nitroquinoline-oxide (4-NQO) and benzo(a)pyrene (B(a)P).
Origin of the strains
Salmonella typhimurium strains TA104recN2-4 and TA104pr1 were obtained from VITO,
Mol, Belgium and were kept at –80 °C in a Biofreezer (New Brunswick Scientific ) until use.
Overnight culture
Twenty microliter of each strain (TA104recN2-4 and TA104pr1) is added to a 50 ml falcon
tube, containing 5 ml of a normal bacterial growth medium, supplemented with extra CaCl2 to
allow optimal growth. Bacterial cultures are then incubated overnight in an Innova 4000 (New
Brunswick Scientific) rotative, environmental shaker at 250 rpm and at 37ºC. A falcon tube
with only growth medium was also incubated to check the sterility of the culture medium.
One hour culture
The next morning, OD600 values were taken from the cultures. OD600 should be > 2 otherwise
incubation was continued until sufficient OD values were reached. Cultures were then diluted
10 times. 50 μl of each strain (TA104recN2-4 and TA104pr1) of the overnight culture was
added to a 50 ml falcon tube containing 2.5 ml growth medium. Bacterial cultures were then
incubated on an Innova 4000 environmental shaker at 250 rpm and at 37ºC (1 hour) to obtain
log phase growth.
Preparation of the test and control compounds
During the one hour culture period, the concentration range of the test compounds was made.
Preparation of the 96-well microtitre plate: DMSO was used as standard solvent for unknown
test compounds (1% final concentration). Environmental samples were diluted in an aqueous
buffer provided by VITO. 8 concentrations of the test compound were used together with 2
solvent controls and 2 positive controls. Serial dilutions with a factor of 2 were made. 4-NQO
was used as positive control for the test without metabolic activation in a final concentration of
4 μg/ml and benzo(a)pyrene in the presence of a metabolic activation system in a final
concentration of 800 μg/ml.
S9 mix
The S9 (batch 869) was supplied by Molecular Toxicology Inc. (NC, USA) and was stored in
a biofreezer at –80C until use. Prior to use, the S9 was combined with an Ames mutagenicity
test tablet containing the necessary co-factors such as NADP and G-6-P. The S9 fraction was
10% of the S9-mix volume. In the final measurement plate this was again 10 fold diluted to a
1% final solution. The samples without S9-mix were provided with phosphate buffer to keep
the number of bacteria the same.
Exposure
In each well of the microtitre plate, 90 μl of the one hour culture is mixed with 10 μl of the test
compound.
Luminometry
The 96-well microtitre plate was placed in an Microlumat LB96P luminometer (EG&G
Berthold) or in a Luminoskan Ascent (Labsystems) and measuring was performed with the
following parameters: 1 s/well; cycle time = 5 min; 60 cycles; incubation temperature = 30ºC.
Data handling
When the luminometer measurements were completed, data was copied and pasted into an
Excel macro sheet. The signal-to-noise ratio (S/N), being the light production of exposed cells
divided by the light production of non-exposed cells, was calculated for each measurement.
Evaluation criteria
A test compound was considered genotoxic when
· the max S/N (recN2-4)/ max S/N (pr1) > 1.5;
· A clear dose response curve is generated;
· the signal is not generated in the first 30 s;
Results
Table 1: The un-coded and grouped results for the test compound 4-nitroquinoline-N-oxide (4-
NQO).

Legend to table 1: wo_S9 : without the addition of induced rat liver S9 mix from Moltox.
w_S9: with the addition of induced rat liver S9 mix from Moltox.
Values are the induction factors as compared with the solvent controls. The red colour + bold
code represents the concentrations regarded positive.
4-NQO is a directly acting compound and this was very clearly detected in the three samples.
Only at the very high concentrations of 0.5 and 0.25 mg/ml was there some signal in the
presence of metabolic activation, probably due to overloading of the activation system. If the
sensitivity is compared between the three samples, in the first sample detection is below
0.00037 μg/ml. In sample 2 it is 0.0003 μg/ml and in sample 3 it is 0.00062 μg/ml.
Table 2: The un-coded and grouped results for the test compound 4-N-methyl-n-nitro-Nnitrosoguanidine
(MNNG).

Legend to table 2: wo_S9 : without the addition of induced rat liver S9 mix from Moltox.
w_S9: with the addition of induced rat liver S9 mix from Moltox.
Values are the induction factors as compared with the solvent controls
If the sensitivity is compared between the two samples, in the first sample detection is below
0.3 μg/ml. In sample 2 it is 0.0125 μg/ml in the absence and 0.1 μg/ml in the presence of a
metabolic activation system.
 

Table 3: The un-coded and grouped results for test compound 2- Aminoanthracene (2-AA).

Legend to table 3: wo_S9 : without the addition of induced rat liver S9 mix from Moltox.
w_S9: with the addition of induced rat liver S9 mix from Moltox.
Values are the induction factors as compared with the solvent controls
2-AA is an indirectly acting compound which needs metabolic activation and this was very
clearly detected in the three samples. If the sensitivity is compared between the three samples,
in the first sample detection is below 0.2 μg/ml. In sample 2 it is below 0.312 μg/ml and in
sample 3 it is below 0.05 μg/ml. No activity was observed in the absence of a metabolic
activation system.
Table 4:

Legend to table 4: wo_S9 : without the addition of induced rat liver S9 mix from Moltox.
w_S9: with the addition of induced rat liver S9 mix from Moltox.
Values are the induction factors as compared with the solvent controls
Genotoxic activity was observed only in the industrial effluent,. In the absence of a metabolic
activation system, activity was detected from a 6.25 % dilution onwards. No DNA damaging
activity was detected in the surface water samples.

Table 5: Summary table of the lowest effect levels (LEL)
Compound code solvent Met. Act. LEL (μg/ml)

Legend to table 5: code: blind code; -S9 : without the addition of induced rat liver S9 mix
from Moltox. +S9: with the addition of induced rat liver S9 mix from Moltox. LEL values are
the calculated concentrations from the top concentrations provided. “no” means no effect
observed.
Discussion
With the currently used 96-well luminometer, two compounds can be tested in one 96-well
plate at 8 concentrations with and without a metabolic activation system using the two strains.
Hands-on time for the preparation of the test compounds is about one hour, this is more or less
the time needed for the second incubation. Results are then obtained during the 4 hours of
incubation in the temperature controlled luminometer. Data is then transferred into a specially
developed Excel spreadsheet and the final calculated data, graphs and tables are generated. For
our lab this means that one technician can handle 4 compounds a day with and without a
metabolic activation system and with all necessary controls. If we do not initiate bacterial
cultures during the weekend, we can handle 16 compounds a week. 16 additional compounds a
week can be handled per extra reading device by the same technician. By using 384-well
plates (not used for this Technotox study) and a luminometer that can handle this format, the
number of compounds can be increased to 64 compounds a week or even 80 if the incubator is
programmed for starting on Sunday and to deliver the cultures on Monday morning.
For interpretation of the results from the test compounds, all data were very straightforward.
This means very pronounced dose-response effects and a very clear discrimination between
the effects obtained in the absence and in the presence of a metabolic activation system. The
sensitivity between the three different samples can be considered as equal and no relevant
differences were found between the samples which used DMSO as solvent and the diluted
sample without DMSO. The complete range of effects was detected with our standard and
ordinary 1:2 dilution series using 8 concentrations. This means that our most diluted sample
was 128 times diluted as compared to the first concentration and 12800 times as compared to
the original solution.
The correct prediction of the surface water samples may require a different protocol as they
should perhaps be concentrated instead of diluted to detect any DNA damaging contaminants.
References
1. Gee P., D.M. Maron and B. Ames (1994) Detection and classification of mutagens: A set of base
specific Salmonella tester strains, Proc. Natl. Acad. Sci. USA, 91, 11606-11610.
2. van der Lelie D., Regniers L., Borremans B., Provoost A. and Verschaeve L. (1997) The VITO-Tox
test, a bioluminescent Salmonella typhimurium test to measure genotoxicity kinetics. Mutation Res.
389:279-290.
3. Quillardet P. and Hofnung M. (1993) The SOS chromotest: a review. Mutation Res. 297: 235-279.
4. Verschaeve L., Van Gompel J., Regniers L., Thilemans L., Vanparys P., van der Lelie D.. (1999)
The VITOTOX genotoxicity and toxicity test for the rapid screening of chemicals, Environmental
and Molecular Mutagenesis, 33, 240-248.
List of recent relevant patents
D. van der Lelie, B. Borremans, A. Provoost, L. Regniers, L. Verschaeve (1996). Novel
recombinant nucleic acid sequences, host microorganisms comprising such sequences and
use there of in tests for determining the presence of a toxic compound in a sample, for
determining both genotoxicity and mutagenicity of a sample and determining the kinetics
of genotoxicity of a sample, said method requiring luminescence measurements.
PCT/EP96/01745
D. van der Lelie, L. Regniers, S. Taghavi, P. Corbisier, L. Verschaeve (1999). Diagnostic
system and method for determining the presence of a genotoxic compound in a sample.
PCT/BE99/00049
http://www.the-scientist.com/yr2000/jan/profile_000110.html

NADP Nicotinamide adenine dinucleotide
phosphate, oxidised form
BIB3013  Assay (by titration) >98% 
195 Euro / 100 mg

Glucose-6-Phosphate Dehydrogenase

Source: Leuconostoc mesenteroides

I.U.B.: 1.1.1.49

The Leuconostoc GPDH exhibits dual coenzyme specificity, namely NAD and NADP (Olive and Levy, Biochem., 6, 730 730, 1967). When assayed under conditions that are optimal for the particular coenzyme, the ratio of observed catalytic activity is NAD/NADP = 1.8.

Stability/Storage: The Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase is a relatively stable enzyme in solution. The lyophilized and ammonium sulfate preparations are stable for 12 months when stored at 2-8°C.

Unit Definition: One Unit reduces one micromole of NAD per minute at 37°C, pH 7.8, using glucose-6-phosphate as substrate.

Glucose-6-Phosphate Dehydrogenase, Suspension
Code: ZF
Chromatographically purified. A suspension in 2.4M ammonium sulfate. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities ≤0.02%, 0.003%, 0.002% and 0.002% respectively. Store at 2-8°C. 
Source: Leuconostoc mesenteroides
Minimum Activity: ≥200 NADP units per mg protein
 
Cat# Pack Size Price  
LS003983 500 un 126.00$  
LS003985 5 ku 355.00$  
 
Glucose-6-Phosphate Dehydrogenase, Suspension
Code: ZFD
Chromatographically purified. Same as Code: ZF except assayed using NAD. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities ≤0.011%, 0.002%, 0.0011% and 0.0011% respectively. A suspension in 2.4M ammonium sulfate. Store at 2-8°C. 
Source: Leuconostoc mesenteroides
Minimum Activity: ≥360 NAD units per mg protein
 
Cat# Pack Size Price
LS003992 900 un 126.00$
LS003993 9 ku 355.00$
 
Glucose-6-Phosphate Dehydrogenase, Lyophilized
Code: ZFL
Chromatographically purified. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities ≤0.02%, 0.003%, 0.002% and 0.002% respectively. A lyophilized powder. Store at 2-8°C. 
Source: Leuconostoc mesenteroides
Minimum Activity: ≥200 NADP units per mg protein
 
Cat# Pack Size Price
LS003981 1 ku 152.00$
LS003980 10 ku 590.00$
 
Glucose-6-Phosphate Dehydrogenase, Lyophilized
Code: ZFLD
Chromatographically purified. Same as Code: ZFL except assayed using NAD. Phosphohexose isomerase, phosphogluconate dehydrogenase, adenylate kinase and creatine phosphokinase contaminant activities ≤0.011%, 0.002%, 0.0011% and 0.0011% respectively. A lyophilized powder. Store at 2-8°C. 
Source: Leuconostoc mesenteroides
Minimum Activity: ≥360 NAD units per mg protein
 
Cat# Pack Size Price
LS003997 2 ku 152.00$
LS003998 18 ku 590.00$
 
Catalog #

Description

Price 

  NaR-PkY


Superior Stock YNaR1 
freeze-dried, One vial, 1 unit/vial  
+  NADH for 20-50 assays,
freeze-dried, One vial

75.00$
  NaR-PkAt


Superior Stock AtNaR2
freeze-dried, One vial, 1 unit/vial  
+  NADH for 20-50 assays,
freeze-dried, One vial

75.00$

The Bacterial Ames Test The Ames Salmonella/microsome mutagenicity assay (Salmonella Test; Ames test) is a shortterm bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. The Ames test is the most widely and validated genotoxicity test. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his+) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen (the compound) is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner (166). The sensitivity of the test is enhanced by the use of particular mutant strains preventing, e.g., adequate DNA repair or increasing resistance to toxic compounds. Bacteria are grown on a selective medium in the presence of the tested compound. After 48 h of incubation at 37 °C, mutant colonies are counted and compared to the number of colonies formed in unexposed cultures (spontaneous back mutatons). A compound is considered genotoxic when the mean number of revertants is the double of that found in the solvent control culture (+ dose effect relationship). Negative controls are normally between approximatively 5-50 revertants for TA98 and 100-150 for TA100. Screening of the Bioactivity of the Isolated Natural Products. Positive controls must show a clear increase over the solvent controls.

 

Genotoxiciteit biosensor (VITOtox®)
De VITOtox® is een commercieel beschikbare biosensortest die gebruikt kan worden om de toxiciteit en de genotoxiciteit van stoffen te bepalen. De test maakt gebruik van twee bacteriestammen: MA989 voor het bepalen van de genotoxiciteit(genotoxstam) en MA1007 voor het meten van de toxiciteit (cytotoxstam). Beide stammen zijn afgeleid van de niet pathogene Salmonella typhimuriumstam TA104, die in de farmaceutische industrie wordt gebruikt voor het uitvoeren van de klassieke Amestest. In aanwezigheid van genotoxische stoffen zal de genotoxstam MA989 een lichtsignaal uitzenden, terwijl in de aanwezigheid van toxische stammen de constitutieve lichtproductie van de cytotoxstam zal afnemen.

Voordelen van de VITOtox®-test ten opzichte van andere bacteriële (geno)toxiciteitstests:

  • lage kostprijs: tot tien keer goedkoper dan traditionele tests;
  • snel: traditionele tests gebaseerd op bacteriële groei duren veel langer;
  • eenvoudig: de interpretatie van de resultaten is erg eenvoudig; de test onder de vorm van een commercieel verkrijgbare testkit is geautomatiseerd en vraagt relatief weinig voorbereiding;
  • uitermate geschikt voor de high throughput screenings fase van een drug discovery programma;
  • betrouwbaar: de test werd gevalideerd in verschillende laboratoria (referenties op aanvraag);
  • nauwkeurig: een goede correlatie met de Amestest.

De ®-test is gepatenteerd (PCT/EP96/01745 en PCT/BE99/00049) en is verkrijgbaar bij Labsystems O.Y., Finland.

De karakteristieken en prestatie van 14 toxiciteit en genotoxiciteit testen werden recent geëvalueerd tijdens een BIOSET Technical Workshop on Genotoxicity Sensing met name Technotox. De proceedings van de Technotox workshop georganiseerd in Mol op 8-12 mei 2000.

 

Vitotox is a high-throughput biolumine-scent assay that offers a unique method for rapid and cost effective geno- and cytotoxicity screening.

Unique assay principle
The Vitotox assay is a Salmonella typhimurium test that uses the light emission of bacteria to detect the genotoxicity, cytotoxicity and muta-genic potency of the sample. It is used for the detection of genetic damage caused by the chemical in pharmaceutical, cosmetic, environ-mental, etc., research. The assay is based on a reporter gene system where luciferase activity is used as a function of the genotoxicity. Luciferase expression is activated via a cascade of reactions known as the SOS response.
Benefits of the assay
The entire DNA content of the cell functions as a target for the genotoxin
to display its effect. Therefore, only a few micrograms of the sample is normally required for the assay.
A cytotoxicity assay is also per-formed together with each sample to prevent false positive and nega-tive results.
No cell growth is required for the genotoxicity detection, resulting in very short assay times.

 General principle of SOS induction. Derepression of the RecN gene isfollowed by placing a Lux reporter system under transcriptional control of the RecN promoter. A turnkey solution for the CellularAssay Workstation!

96-well layout for the Vitotox test
 

The test uses a simple “mix and measure” procedure: the com-pounds are pipetted into 96- or 384-well microplates as a dilution series along with blanks, positive control samples, and with or without S9 metabolic extract. The cultured test bacteria are added and the light emission is followed over a three-hour assay period.
Correlation with traditional genotox-icity assays is exceptionally good.

Vitotox Test System
Product#                     Price                         Description
04-6400-000                3300 Euro                  Vitotox 10 Kit, for 10 samples
04-6400-010                6800 Euro                  Vitotox 384 HTS Kit, 1 x package, for 345 samples
04-6400-020                47 518 Euro                 Vitotox 384 HTS Kit, 10 x package, for 3450 samples

ATP Assay Products
The Quantitative ATP Monitoring Kit is used for measur-ing free ATP concentrations. Using the bioluminescence technique, the monitoring kit measures ATP in bacterial, plant or mammalian cells, as well as tissue samples over a concentration range of 10-11 to 10-6 moles per liter. This kit also enables any enzyme or substrate that can be coupled to the production or consumption of ATP to be mea-sured quantitatively. The assay can be freely used with any cells and cell lysis reagents. It is convenient, rapid and reproducible, with results available in just minutes. Individual assay components are also avail-able for those researchers who only have very few samples.

 

Cat# 

Product Name 

Unit Size

30025-2 AlamarBlueTM Cell Viability Assay Kit 100 mL
30025-1 AlamarBlueTM Cell Viability Assay Kit 25 mL
30020-4 ATP-GloTM Bioluminometric Cell Viability Assay Kit 10x100 mL
30020-3 ATP-GloTM Bioluminometric Cell Viability Assay Kit 100 mL
30020-2 ATP-GloTM Bioluminometric Cell Viability Assay Kit 10x10 mL
30020-1 ATP-GloTM Bioluminometric Cell Viability Assay Kit 10 mL
30026 Calcein AM Cell Viability Assay Kit 1000 assays
30006 MTT Cell Viability Assay Kit 1000 assays
30027 Viability/Cytotoxicity Assay kit for Bacteria Live and Dead Cells 100-1000 assays
30002 Viability/Cytotoxicity Staining Kit for Animal Live & Dead Cells 300 assays
30007 XTT Cell Viability Assay Kit 1000 assays
 
30020-1  <actinic:retail_price_text>Price:  $128.00
30020-2  <actinic:retail_price_text>Price:  $284.00
 
30020-3  <actinic:retail_price_text>Price:  $259.00
30020-4  <actinic:retail_price_text>Price:  $1,169.00
ATP Cell Viability Assay Kit
200 assays. Utilizes bioluminescent detection of the ATP level via luciferase catalyzed reaction for a rapid screening of apoptosis and cell viability in mammalian cells. The assay can be done directly in culture plates requiring no harvest/washing/or sample preparations and can be fully automatic for high throughput (10 seconds/sample) and is highly sensitive (detects 10-100 mammalian cells/well). 
Product Name ATP Cell Viability A Stock# K254-200
ATP Cell Viability Assay Kit
1000 assays. See the description above in K254-200.
Product Name ATP Cell Viability A Stock# K254-1000
ADP/ATP Ratio Assay Kit
200 assays. Designed to detect ADP/ATP ratios for a rapid screening of apoptosis/necrosis/growth arrest/cell proliferation simultaneously in mammalian cells. Offers highly consistent results and with excellent correlation to other apoptosis markers (e.g. TUNEL-based assays and caspase assays). Assay can be fully automatic for high throughput (10 seconds/sample) and is highly sensitive (detects 10-100 cells/well).
Product Name ADP/ATP Ratio Assay Stock# K255-200

http://www.bioxys.com/i_Biotium/bioscience_assay_kits.htm

 

Cytotoxicity Bioluminescence Assay Kit, Vitotox™

10 assays
Bioluminescence, Misc. Viability/Proliferation/Cytotoxicity Assays
Vitotox is a new genotoxicity and cytotoxicity test based on genetically modified test organisms for faster and more superior results. The Vitotox assay, an SOS-bioluminescence Salmonella typhimurium test, uses the light emission of bacteria to detect the genotoxicity, toxicity and mutagenic potency of the sample. Increased luciferase activity is measured kinetically. Effects of cytotoxicity can be measured simultaneously with genotoxicity. The test detects any DNA damage and gene mutations.
Vitotox Test System

 

 
The Vitotox kits offer a unique technology for rapid genotoxicity screening with capacity of over 1000 test per day. The assay detects DNA damage and gene mutations in specially designed recombinant bacterial strains. It is a homogeneous assay for 96- or 384-well plates and can be automated for screening purposes. The assay has the shortest assay time and lowest sample consumption compared to all related assay technologies.
 
   Product Detail

 

 
A high-throughput bioluminescent genotoxicity and cytotoxicity test

The Vitotox Test System is a unique method for rapid and cost effective high-throughput genotoxicity and toxicity screening.

Patent-pending assay principle

Two recombinant Salmonella typhimurium TA104 test strains are used in the assay to determine simultaneously both genotoxicity and cytotoxicity of the sample. The test is based on luciferase reporter gene system which activity is measured by light emission and is a function of the genotoxicity of the test compound. Normally, the luciferase reporter gene is strongly repressed, but in the presence of a DNA damaging genotoxic compound the initiation of a cascade of reactions known as the SOS response will take place leading to derepression of the very strong promoter controlling the luciferase gene.

  • Very small amount of sample required                 
  • High reproducibility and sensitivity                 
  • Very good correlation with AMES test                 
  • Rapid answers in only 3 hours                  
  • Low labor costs; automatic reading of kinetic data                 
  • No test organism maintenance required                 
  • 96- or 384-well microplate format 

Benefits of the system

One of the major benefits over the Ames test is that the entire DNA content of the cell functions as a target for the genotoxin to display its effect. In contrast, the Ames test monitors mutations only in genes related to histidine synthesis. This results in a significantly lower sample concentrations (up to 1000 fold) required for the Vitotox test.

Simultaneously cytotoxicity assay

An important control for the system is a cytotoxicity test performed together with each sample to ensure that a positive signal in the genotoxicity test is not due to a nonspecific enhancement in luminescence and that a negative response is not due to toxicity of the test compound resulting in cell death. 

Performing Vitotox assay

The test is very simple and cost efficient to perform. Compounds are pipetted into 96-well or 386-well microplates in a dilution series along with blanks, positive control samples, and with or without S9 metabolic extract. The cultured test bacteria are added and the plate is measured repeatedly for the three-hour assay period. 

Optimal performance with Luminoskan Ascent 

Vitotox can be optimally performed with Luminoskan Ascent. A fully automatic measurement and data analysis system, based on Ascent Software, runs the kinetic Vitotox  assay, then automatically analyses the data and generates the report with all the test results.

Special 384 well format for HTS

With the 384-well microtiter plate setup, 23 samples can be simultaneously tested. Combining this with the Assist robot it is possible to easily test over 800 compounds during one working day. This allows integrating the Vitotox test in a high-throughput screening program.

Small sample amount required

Since the Vitotox test is miniaturized and very sensitive, only small amounts of the test compound are required to perform the test. This makes Vitotox test usefull as a prescreening for the Ames test for early identification of compounds that would ultimately be eliminated by Ames testing in the initial stage of the product discovery phase. 

Application areas of the Vitotox assay

Vitotox is ideal for laboratories that currently use the government standard AMES test. It can be used as: 

  • A prescreening test in pharmaceutical and biotechnology laboratories to speed up the product discovery phase
  • Validation of environmental compliance or in other industrial applications
Please contact us for pricing and availability at This email address is being protected from spambots. You need JavaScript enabled to view it..

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