Download Microbiological Assays for Mutagens

Journal of Applied Microbiology 2003, 95, 1175–1181
doi:10.1046/j.1365-2672.2003.02121.x
A REVIEW
Detection of mutagenic pollution of natural environment using
microbiological assays
G. We˛grzyn1,2 and A. Czyz_ 3
1
Department of Molecular Biology, University of Gdańsk, Kładki, Gdańsk, Poland, 2Institute of Oceanology,
Polish Academy of Sciences, Św. Wojciecha, Gdynia, Poland, and 3Laboratory of Molecular Biology (affiliated
with the University of Gdańsk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kładki,
Gdańsk, Poland
2002/0009: received 7 November 2002, revised 16 April 2003 and accepted 9 September 2003
1. Summary, 1175
2. The problem of mutagenic pollution of natural environment, 1175
3. Biological mutagenicity assays, 1176
4. Using bacteria to detect mutagenes – a general view, 1176
5. Testing environmental samples for the presence of
mutagens, 1177
1. SUMMARY
One of the most important and serious ecological problems
is mutagenic pollution of the natural environment. Therefore, detection of mutagenic compounds in samples taken
from natural habitats is of special interest. Microbiological
mutagenicity tests seem to be very useful tools for such
detection. In this review article, a general view on the tests
employing genetically modified bacterial strains designed for
detection of low concentrations of mutagenic compounds is
presented. Moreover, a comparison of advantages and
disadvantages of selected assays, developed early on and
more recently, and features of these assays are discussed. It
appears that none of the currently available mutagenicity
tests is perfect or optimal for all purposes. Thus, a choice for
the particular assay must depend on the nature of studies
and specific tasks of the experiments to be performed.
2. THE PROBLEM OF MUTAGENIC
POLLUTION OF NATURAL ENVIRONMENT
Mutagenic pollution of the natural environment is
undoubtedly a serious and general problem. Reports of
various agencies indicate that the presence of mutagenic
compounds in different habitats is a common phenomenon
Correspondence to: Grzegorz We˛grzyn, Department of Molecular Biology,
University of Gdan´sk, Kładki 24, 80-822 Gdan´sk, Poland (e-mail: wegrzyn@
biotech.univ.gda.pl).
ª 2003 The Society for Applied Microbiology
5.1.
5.2.
5.3.
5.4.
The Ames test, 1177
Mutatox, 1178
Vitotox, 1178
A newly developed Vibrio harveyi mutagenicity
assay, 1179
6. Concluding remarks, 1179
7. Acknowledgements, 1180
rather than an exception (see, for e.g. Davey 1999). The list
of known mutagenic chemicals is very long. The Environmental Mutagen Information Center database (http://
www.nlm.nih.gov/pubs/factsheets/emicfs.html) contains
over 20 000 citations to literature on agents that have been
tested for mutagenic activity. The problem of the presence
of mutagenic chemicals in natural habitats is very important
because such compounds are capable of inducing serious
diseases, including cancer. Moreover, they can potentially
damage the germ line of higher organisms, which may lead
to fertility problems and to negative genetic changes in
future generations (reviewed by Mortelmans and Zeiger
2000). Therefore, detection of mutagens in the natural
environment is very important.
As chemical mutagens elicit deleterious effects on living
organisms at extremely low concentrations, their detection
in natural habitats, at levels that can be dangerous for
animals or humans, may be difficult and complicated.
Chemical methods for detection of mutagenic compounds
are expensive and time consuming. For e.g. to analyse the
presence of mutagenic compounds in a water sample,
material from a few hundred litres must be concentrated
before an actual analysis. Moreover, such methods are useful
mainly in assays for particular chemicals. As there are
hundreds of different mutagenic chemicals, it is a problem
when performing a quick and preliminary assay to determine mutagenic contamination in the tested environmental
sample.
1176 G . W E˛ G R Z Y N A N D A . C Z Y Z_
Biological assays may be an alternative to chemical
analysis when detecting the presence of mutagenic compounds in the environment. Although no currently available
biological test can provide detailed and precise information
about the chemical nature of detected mutagens, such tests
provide a possibility to answer the question whether
examined samples contain mutagens at levels potentially
dangerous for organisms. Therefore, it seems that the most
reasonable strategy for testing environmental samples is to
use a biological assay as a preliminary test to detect the
presence of mutagenic compounds. Then, if necessary,
subsequent detailed chemical analysis can be performed to
determine the specific type of mutagen(s).
3. BIOLOGICAL MUTAGENICITY ASSAYS
Biological mutagenicity assays are based on the detection of
specific phenotypes of tester organisms that appear after
their contact with mutagens. Obviously, changes in phenotypes (for e.g. the ability to survive under specific conditions) result from changes in biochemical properties of cells
that are caused by genetic changes. Genetic material is a
target for mutagens. Thus employing an organism whose
alternative phenotypes may be easily distinguished, it is
potentially possible to determine the presence or absence of
mutagens in a tested sample on the basis of the frequency of
changes in the specific feature of this organism. As usually
only one such feature is assessed, the problem of sensitivity
of biological assays appears. An assay should be sensitive
enough to allow an investigator to detect levels of mutagens
capable of causing relatively low numbers of genetic changes
in the whole genome. However, one phenotypic feature
often means one locus that represents a very small piece of
the genome. Therefore, tester organisms should be significantly more sensitive to mutagenic compounds than wildtype ones.
Because basic mechanisms of mutagenesis are common for
all organisms, it is generally accepted that compounds
causing mutations in one type of cell should also be
considered mutagenic for other cells. This is one of the
reasons why bacteria are the most commonly used tester
organisms in mutagenicity assays. Results obtained in such
tests can be usually extrapolated to eukaryotic cells,
including human cells. Furthermore, prokaryotic cells grow
significantly faster than eukaryotic cells, their cultivation is
simpler and cheaper, and genetic manipulations in bacterial
cells are considerably easier than in eukaryotic cells.
4. USING BACTERIA TO DETECT
M U T A G EN S : A G E N E R A L V I E W
Many different mutagenicity tests employing genetically
modified bacterial strains have been described. However, the
most widely used assay to assess mutagenicity of tested
compounds is still the Ames test (for reviews see Maron and
Ames 1983; Mortelmans and Zeiger 2000). In this test,
Salmonella typhimurium strains unable to produce one of the
amino acids, histidine, are used. Such defects, causing
inability of bacteria to grow in minimal media lacking
histidine, arise from the presence of various point mutations.
Reverse mutations that restore ability of these cells to form
colonies in the absence of histidine may occur spontaneously, however, they are significantly more frequent upon
contact with mutagenic agents. A conclusion relating to the
mutagenicity of tested samples is based on the number of
revertants (or pseudorevertants), appearing after incubation
of the tester bacteria with the tested material, relative to the
number of spontaneous mutants able to grow on minimal
agar plates without histidine. Pseudorevertants can be
discounted after imprinting a plate without histidine with
a sterile velvet disc. Only true revertants will grow.
The phenomenon of bioluminescence (emission of light
by organisms) is employed in a number of different
mutagenicity and toxicity tests. The use of bioluminescent
bacteria in the detection of toxic and genotoxic chemicals
was proposed a long time ago (Ulitzur et al. 1980; Bulich
and Isenberg 1981). Then, several bioluminescent assays
were described.
Toxicity of different chemicals could be determined by
measuring a decrease in the efficiency of light emission by a
marine luminescent bacterium Vibrio harveyi after addition
of toxic compounds (Thomulka and Lange 1995, 1996;
Lange and Thomulka 1997). Nevertheless, it is important to
distinguish toxicity assays from mutagenicity tests as toxic
chemicals are not always mutagens, and mutagenic agents
are often toxic only at relatively high concentrations. As
many mutagens occur in natural environments at concentrations too low to provoke serious toxic effects in bacterial
cells, the assays described above can be used for detection of
toxic substances rather than mutagenic agents.
In an other group of tests, fusions consisting of lux
operons (encompassing genes coding for proteins involved
in the process of bioluminescence) under the control of one
of LexA-repressed promoters are used (Bar and Ulitzur
1994; Ptitsyn et al. 1997; van der Leile et al. 1997; BenIsrael et al. 1998; Min et al. 1999; Verschaeve et al. 1999).
LexA is a repressor of the SOS regulon. Expression of this
regulon is stimulated by agents causing DNA damage, due
to activation of the RecA protein (upon its binding to singlestranded DNA regions) which is a co-protease in the
reaction of LexA autocleavage (Little and Mount 1982).
Strains bearing the above-mentioned fusions emit light upon
contact with agents that induce the SOS response, i.e. with
agents causing DNA damage.
Genotoxicity may be assessed using tests which can
monitor restoration of luminescence of dark bacterial
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x
DETECTION OF MUTAGENIC POLLUTION
mutants (Ulitzur et al. 1980; Ben-Itzhak et al. 1985; Levi
et al. 1986; Ulitzur and Barak 1988; Sun and Stahr 1993).
Such a restoration occurs upon contact with a mutagen
which causes a reversion of the mutation in the region
controlling an expression of lux genes. These assays have
been demonstrated to be useful in environmental studies
(Brenner et al. 1993a,b, 1994; Belkin et al. 1994).
Treatment of luminescent bacteria with mutagenic agents
causes induction of light emission even in wild-type strains
(Czy_z et al. 2002a). Therefore, the use of wild-type
luminescent bacteria, especially Photobacterium leiognathi
strains, for modification and improvement of mutagenicity
assays was suggested, although not yet demonstrated (Czy_z
et al. 2002a).
A new simple mutagenicity assay, in which appearance of
V. harveyi neomycin-resistant mutants upon contact with
mutagens is monitored, has recently been developed (Czy_z
et al. 2000a). This assay is sensitive and potentially useful in
environmental studies, especially for testing samples of
marine water (Czy_z et al. 2002b).
Only a fraction of the battery of mutagenicity bio-assays
described in the literature has been mentioned above.
However, although most (if not all) of these assays are very
useful in testing the mutagenic potential of particular
chemicals in a laboratory, only a relatively low number of
tests seems to be useful in environmental studies. This
problem is discussed in the next section.
5. TESTING ENVIRONMENTAL SAMPLES
F O R T H E P R E S EN C E O F M U T A G E N S
Some specific features of biological assays for detection of
mutagens are important in testing samples derived from
natural environments. First, the assay should be sufficiently sensitive to detect usually very low, but still
harmful, concentrations of mutagenic chemicals present in
various habitats. This is in contrast to tests designed to
assess mutagenicity of defined chemicals in the laboratory,
when the amounts of investigated chemicals are not a
limiting factor. Secondly, as environmental samples must
be tested, the tester organism should be able to survive
well after contact with such a material. For e.g. in testing
marine water samples it would be ideal to use a marine
bacterium, as survival of other organisms, like enteric
bacteria, in marine water may be poor. For e.g. strains of
S. typhimurium that are used in the Ames test, die
relatively quickly in marine water (Czy_z et al. 2002b).
Moreover, various growth conditions that are nonmutagenic per se for e.g. salinity of the growth medium, should
not be stress factors for tester bacterial strains, as otherwise
the results of tests could be affected by such unspecific,
stress-inducing factors. It would be a great advantage if the
tester organisms were very tolerant to various environ-
1177
mental conditions, which is however difficult to achieve if
they should be also very sensitive to mutagenic agents.
Thirdly, the test should be simple because it is expected
that many samples might be tested simultaneously. Finally,
for the same reason, the test should not be expensive. This
may be especially important in developing countries, where
environmental monitoring and protection should be significantly improved, and where funds for such projects are
very limited.
In the following section, four selected assays that may be
used for the detection of mutagenic pollution of natural
habitats are described briefly and discussed in the light of
their advantages and disadvantages as tests oriented towards
environmental studies. The selected tests are either well
developed and precisely characterized or recently described
and opening new possibilities.
5.1. The Ames test
The Ames test, originally described over 30 years ago (Ames
1971), is undoubtedly the most commonly used mutagenicity assay. It was subsequently modified and improved several
times (Ames et al. 1973, 1975; McCann et al. 1975; Maron
and Ames 1983; Mortelmans and Zeiger 2000), but its
principles have remained the same. In this test, genetically
modified S. typhimurium strains are used. The presence of
mutations in the his genes, causing defects in a metabolic
pathway leading to the production of histidine, allows
positive selection of his+ revertants on minimal agar plates
lacking histidine. Only mutants in which restoration of his
function occurred are able to form colonies on such plates.
Usually the plates containing the tested compound and
tester bacteria are incubated for 48 h and bacterial colonies
are counted.
A deletion of the uvrB gene in most of the tester strains
ensures a higher efficiency of mutagenesis. This is due to an
inactivation of a major bacterial DNA repair system (Ames
et al. 1973). In addition, these bacteria bear the rfa mutation,
which causes partial loss of the lipopolysaccharide (LPS)
barrier that coats the surface of the bacteria. Permeability of
the cell envelope of these mutants to relatively large
molecules (including many mutagens) is significantly
increased relative to wild-type S. typhimurium (Ames et al.
1973). Some of the tester strains also harbour a plasmid
pKM101. This plasmid bears mucA and mucB genes, which
are analogues of bacterial umuC and umuD genes and their
expression causes an enhancement of an error-prone DNA
repair system (McCann et al. 1975; Walker and Dobson
1979; Shanabruch and Walker 1980). Some chemicals, for
e.g. 2-aminofluorene and benzo[a]pyrene, are not mutagenic
agents themselves but require metabolic activation to act as
mutagens. Contrary to reactions catalysed in animal liver,
bacterial cells are unable to perform such activation.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x
1178 G . W E˛ G R Z Y N A N D A . C Z Y Z_
Therefore, a fraction of rat liver homogenate, called S-9 mix
and containing microsomal enzymes and cofactors, is often
added to the bacteriological medium (Maron and Ames
1983).
Thousands of chemicals have already been tested for their
mutagenicity using the Ames test (Mortelmans and Zeiger
2000). This is an unquestionable advantage as no other
currently available mutagenicity assay may provide a similar
amount of data for comparative purposes. The Ames test is
excellent for testing mutagenic activity of defined chemicals
under laboratory conditions. However, there are some
problems with using it in studies on samples taken from
the natural environment. First, survival of S. typhimurium
tester strains under some growth conditions is dramatically
reduced relative to that in standard media used in laboratory. For e.g. these strains are particularly sensitive to water
samples containing high salt concentrations (Czy_z et al.
2002b). Secondly, sensitivity of the test is absolutely
acceptable for assessing mutagenicity of known chemicals
under laboratory conditions, however it may be too low for
the detection of low amounts of mutagens in environmental
samples (Czy_z et al. 2002b). Finally, S. typhimurium is a
potentially pathogenic bacterium, which may be a disadvantage of tests that use this bacterium. Wild-type strains of
this microbe can cause diarrhoea and food poisoning.
Although mutant strains used in the Ames test are less
pathogenic, caution should be exercised at all times the assay
is performed (Mortelmans and Zeiger 2000). However, it
has been used for over 30 years without repeated untoward
effects.
5.2. Mutatox
The Mutatox test is a commercially available (from AZUR
Environmental) microbiological mutagenicity assay, in
which a special dark strain (named M169) of luminescent
bacteria is used (Ulitzur et al. 1980; Bulich and Isenberg
1981; Ulitzur and Weiser 1981). The M169 strain is a dark
mutant, in which the primary genetic lesion appears to be
in the regulatory system rather than in one of the coding
sequences of the lux structural genes. In the presence of
relatively low concentrations of genotoxic agents in a
growth medium, light production is restored in this strain.
Various mutagens can be detected using Mutatox, including chemicals causing base substitution, DNA synthesis
inhibitors, DNA-damaging agents and DNA intercalating
agents.
There are some specific materials and equipment required
to perform the Mutatox test. They include the Microtox
Model 500 Analyzer (Strategic Diagnostics Inc., Newark,
DE, USA), Mutatox reagent (freeze-dried M169 cells),
dried assay media with and without rat-liver microsomal
enzymes (S-9 mix) and appropriate co-factors. The tester
bacterial cells are exposed to dilutions of the assessed sample
in hydrated Mutatox media. The samples are incubated for
24 h and light readings are taken using the Microtox Model
500 Analyzer.
The sensitivity of Mutatox was reported to be comparable
with that of the Ames test for both pure compounds and
environmental samples. The simplicity of the procedure is
an advantage of Mutatox, however special equipment (see
preceding paragraph) is necessary (this may be a practical
problem if performing rare analyses rather than routine
genotoxic studies). Although 24 h are needed to perform the
whole assay, which is an advantage relative to the Ames test
(in which incubation of plates for 48 h is recommended),
this is still not the quickest assay.
5.3. Vitotox
Thermo Electron Corporation (Marietta, OH, USA) is a
manufacturer of the commercially available Vitotox Test
System (van der Leile et al. 1997; Verschaeve et al. 1999).
In this test, two recombinant S. typhimurium TA104 strains
are used to determine both genotoxicity and cytotoxicity of
the tested sample. Vitotox test is based on the lux reporter
gene system, whose activity is measured by light emission
and is a function of the genotoxicity of the tested
compound. Under normal growth conditions, the luciferase
reporter gene is repressed, however, in the presence of a
DNA-damaging agent, the SOS response is stimulated and
leads to derepression of the strong promoter controlling the
expression of the luciferase gene. Luminescence is then
measured, and the Luminoskan Ascent equipment is
recommended at this step. A fully automatic measurement
and data analysis system, based on Ascent Software (MTX
Labs Inc., Vienna, VA, USA), is also available.
In contrast to the Ames test, where mutations only in genes
related to the histidine synthesis pathway are monitored, in
Vitotox the entire DNA content of the cell functions as a
target for genotoxic agents. An induction of the SOS system
is the basis of this assay. This system responds to any DNA
damage. Therefore, up to 1000-fold lower sample concentration is required for the Vitotox test than for the Ames test.
Apart from this high sensitivity, a rapid procedure (ca 3 h) is
another important advantage of Vitotox.
There are two potential problems with using Vitotox for
testing mutagenic pollution of the natural environment.
First, S. typhimurium strains are used, whose survival may
be decreased upon contact with some environmental samples. This problem might perhaps be minimized due to the
fact that a small amount of the sample is required for this
assay. The second problem seems to be more significant.
Namely, as the system is able to detect only agents inducing
the SOS response, some mutagens that do not cause DNA
damage cannot be detected.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x
DETECTION OF MUTAGENIC POLLUTION
5.4. A newly developed V. harveyi mutagenicity
assay
A new mutagenicity assay, in which a series of genetically
modified strains of marine bacterium V. harveyi is used, has
recently been developed (Czy_z et al. 2000a). Vibrio harveyi
possesses three features making this bacterium a useful
bioindicator of mutagenic pollution (Czy_z et al. 2000a,b).
First, it is not pathogenic to humans, and thus it is
completely safe to work with. Secondly, this bacterium is
significantly more sensitive to mutagenic agents than E. coli.
Thirdly, its LPS is significantly more permeable for large
molecules than LPS of S. typhimurium.
Vibrio harveyi is naturally sensitive to neomycin, but
mutants of V. harveyi strains resistant to this antibiotic can
be easily isolated. The frequency of the appearance of such
mutants increases in the presence of mutagens (Czy_z et al.
2000a). To enhance sensitivity of the assay, wild-type
V. harveyi strains have been genetically modified. A
transposon mutant, very sensitive to mutagenic factors,
was isolated (Czy_z et al. 2000a,b). This mutant bears an
insertion in the cgtA gene, coding for the common GTPbinding protein (Czy_z et al. 2001), which is involved in the
regulation of many chromosomal functions (Czy_z et al.
2001; Sikora-Borgula et al. 2002; Słomińska et al. 2002).
The second modification was the introduction of a plasmid
bearing mucA and mucB genes, analogous to the Ames test.
The V. harveyi mutagenicity assay consists of detection of
neomycin-resistant mutants on plates either containing a
mutagen in the solid medium or after incubation of bacterial
cultures in a liquid medium, in the presence of tested
compounds or environmental samples. Neomycin is an
aminoglycoside antibiotic that interferes with decoding at
1179
the ribosomal A site during translation (Dahlberg 1989).
Resistance to this antibiotic occurs as a result of various
rRNA modifications in the decoding site. Therefore, a large
spectrum of mutagenic agents, causing different types of
mutations, may lead to the appearance of neomycin-resistant
mutants, which can be detected by the V. harveyi mutagenicity assay. This makes the V. harveyi strains perhaps more
suitable as general tester organisms than each particular
S. typhimurium strain used in the Ames test (although the
Ames test, as a complete procedure with using several
strains, detects a very wide range of mutagenic chemicals).
Using the V. harveyi assay it is possible to detect
significantly lower concentrations of typical chemical mutagens than when employing the Ames test (Czy_z et al.
2002b). The high sensitivity of the V. harveyi mutagenicity
assay might be to its advantage, besides its low cost and the
fact that no special equipment is necessary. The test is also
useful in environmental studies as detection of mutagens in
marine water samples from different geographical regions is
possible (Czy_z et al. 2003). Disadvantages of the V. harveyi
mutagenicity assay are as follows: (i) a relatively long time to
perform the analysis (similar to the Ames test, an incubation
of plates for 48 h is recommended), and (ii) the relatively
low number of chemicals tested using this assay.
6. CONCLUDING REMARKS
Table 1 summarizes the advantages and disadvantages of the
four microbiological mutagenicity assays, selected and compared in this article, in the light of their usefulness in testing
mutagenic pollution of natural environments. It can be
concluded that there is no ideal test that would posses all the
most desirable features. For e.g. the Ames test is the best assay
Table 1 Comparison of advantages and disadvantages of different microbiological mutagenicity tests
Description
Feature
Ames test
Mutatox
Vitotox
Vibrio harveyi assay
Tester organism
Salmonella typhimurium
(pathogenic)
S. typhimurium (pathogenic)
V. harveyi (nonpathogenic)
Sensitivity to nonmutagenic
environmental factors
Time necessary to perform
the assay
Sensitivity
Relatively high
The M169 luminescent
bacterial strain
(nonpathogenic)
Relatively low
Relatively high
Relatively low
48 h
24 h
3h
48 h
Acceptable for laboratory
tests
Comparable with the
Ames test
Up to 1000-fold higher than
the Ames test
Generally all kinds
Generally all kinds
No
Yes
Agents inducing
the SOS response
Yes
2–25 times higher than the
Ames test (depending on the
nature of the mutagen)
Generally all kinds
Kinds of detectable
mutagenic agents
Requirement for special
equipment
No
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x
1180 G . W E˛ G R Z Y N A N D A . C Z Y Z_
for comparative studies as references to a large number of
previous analyses are available. However, a relatively long
time is necessary to perform the assay, its sensitivity is not the
highest (but it is 90% to genotoxic chemicals) and bacterial
strains used in this test are potentially pathogenic (but have
not been found to be so) and relatively sensitive to nonmutagenic environmental factors. Mutatox has many advantages,
but it is not the quickest test. However, Vitotox is a very
sensitive and quick assay, but only a fraction of mutagenic
agents (i.e. those inducing the SOS response) may be detected
and potentially pathogenic bacterial strains are used. The
newly developed V. harveyi mutagenicity assay is sensitive,
simple and employs nonpathogenic bacterial strains that
survive well in water samples taken from natural habitats, but
it requires 48 h to be completed and to date a relatively low
number of chemicals has been tested using this assay.
In conclusion, as none of the mutagenicity tests is perfect,
the choice of a particular assay must depend on specific
experimental tasks. Thus, a proper choice may have a
significant influence on the results. It is expected that
further development of microbiological tools for the detection of mutagenic compounds will lead to considerable
improvement on already existing tests and perhaps appearance of new mutagenicity assays.
7. ACKNOWLEDGEMENTS
We apologize to all authors whose many articles on the
development of microbiological mutagenicity assays were not
cited in this article due to space limitation. We are very
grateful to an anonymous reviewer of this manuscript for his/
her extremely useful comments and suggestions. This work
was supported by grants from the Polish State Committee for
Scientific Research (project grant no. 6 P04G 033 19 to
Dr Hanna Szpilewska from the Institute of Oceanology of the
Polish Academy of Sciences) and the Provincial Nature
Protection Fund in Gdańsk (project grant no. WFOŚ/D/
210/3/2001 to G.W.). The authors acknowledge financial
support from the Foundation for Polish Science (subsidy no.
14/2000 to G. W. and a stipend to A. C.).
8. REFERENCES
Ames, B.N. (1971) The detection of chemical mutagens with enteric
bacteria. In Chemical Mutagens, Principles and Methods for Their
Detection ed. Hollaender, A. pp. 267–282. New York, NY: Plenum.
Ames, B.N., Lee, F.D. and Durston, W.E. (1973) An improved
bacterial test system for the detection and classification of mutagens
and carcinogens. Proceedings of the National Academy of Sciences of
USA 70, 782–786.
Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for
detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31, 347–364.
Bar, R. and Ulitzur, S. (1994) Bacterial toxicity of cyclodextrins:
luminous Escherichia coli as a model. Applied Microbiology and
Biotechnology 41, 574–577.
Belkin, S., Steiber, M., Tiehm, A., Frimmel, F.H., Abeliovich, A.,
Werner, P. and Ulitzur, S. (1994) Toxicity and genotoxicity
enhancement during polycyclic aromatic hydrocarbons biodegradation. Environmental Toxicology and Water Quality 9, 303–309.
Ben-Israel, O., Ben-Israel, H. and Ulitzur S. (1998) Identification and
quantification of toxic chemicals by use of Escherichia coli carrying
lux genes fused to stress promoters. Applied and Environmental
Microbiology 64, 4346–4352.
Ben-Itzhak, J., Levi, B.Z., Shor, R., Lanir, A., Bassan, H.M. and
Ulitzur, S. (1985) The formation of genotoxic metabolites of
benzo[a]pyrene by the isolated perfused rat liver, as detected by the
bioluminescent assay. Mutation Research 147, 107–112.
Brenner, A., Belkin, S., Ulitzur, S. and Abeliovich, A. (1993a) Fast
assessment of toxicants adsorption on activated carbon using a
luminous bacteria bioassay. Water Science and Technology 27, 113–120.
Brenner, A., Belkin, S., Ulitzur, S. and Abeliovich, A. (1993b)
Evaluation of activated carbon adsorption capacity by a toxicity
bioassay. Water Science and Technology 27, 1577–1583.
Brenner, A., Belkin, S., Ulitzur, S. and Abeliovich A. (1994)
Utilization of a bioluminescence toxicity assay for optimal design
of biological and physico-chemical wastewater treatment processes.
Environ. Toxicology and Water Quality 9, 311–316.
Bulich, A.A. and Isenberg, D.L. (1981) Use of luminescent bacterial
system for the rapid assessment of aquatic toxicity. ISA Transactions
20, 29–33.
Czy_z, A., Jasiecki, J., Bogdan, A., Szpilewska, H. and We˛grzyn, G.
(2000a) Genetically modified Vibrio harveyi strains as potential
bioindicators of mutagenic pollution of marine environments.
Applied and Environmental Microbiology 66, 599–605.
Czy_z, A., Wróbel, B. and We˛grzyn, G. (2000b) Vibrio harveyi
bioluminescence plays a role in stimulation of DNA repair.
Microbiology 146, 283–288.
Czy_z, A., Zielke, R., Konopa, G. and We˛grzyn, G. (2001) A Vibrio
harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose
homologues are present in diverse organisms ranging from bacteria
to humans and are essential genes in many bacterial species.
Microbiology, 147, 183–191.
Czy_z, A., Plata, K. and We˛grzyn, G. (2002a) Induction of light
emission by luminescent bacteria treated with UV light and chemical
mutagens. Journal of Applied Genetics 43, 377–389.
Czy_z, A., Szpilewska, H., Dutkiewicz, R., Kowalska, W., BiniewskaGodlewska, A. and We˛grzyn, G. (2002b) Comparison of the Ames
test and a newly developed assay for detection of mutagenic pollution
of marine environments. Mutation Reserach 519, 67–74.
Czy_z, A., Kowalska, W. and We˛grzyn, G. (2003) Vibrio harveyi
mutagenicity assay as a preliminary test for detection of mutagenic
pollution of marine water. Bulletin of Environmental Contamination
and Toxicology 70, 1065–1070.
Dahlberg, A.E. (1989) The functional role of ribosomal RNA in protein
synthsis. Cell 57, 525–529.
Davey, K. (1999) EPA’s strategy for priority persistent, bioacumulative, and toxic (PBT) pollutants. In Chemicals in Our Community, vol.
2, ed. McDonald, G., Sheridan, D. and Wigginton, M. p. 9.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x
DETECTION OF MUTAGENIC POLLUTION
Washington, DC: United States Environmental Protection Agency –
Office of Pollution Prevention and Toxics.
Lange, J.H. and Thomulka, K.W. (1997) Use of the Vibrio harveyi
toxicity test for evaluating mixture interactions of nitrobenzene
and dinitrobenzene. Ecotoxicology and Environmental Safety 38,
2–12.
van der Leile, D., Regniers, L., Borremans, B., Provoost, A. and
Verschaeve, L. (1997) The VITOTOX test, and SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics.
Mutation Research 389, 279–290.
Levi, B.Z., Kuhn, J.C. and Ulitzur, S. (1986) Determination of the
activity of 16 hydrazine derivatives in the bioluminescence test for
genotoxic agents. Mutation Research 173, 233–237.
Little, J.W. and Mount, D.W. (1982) The SOS regulatory system of
Escherichia coli. Cell 29, 11–22.
McCann, J., Springarn, N.E., Kobori, J. and Ames, B.N. (1975)
Detection of carcinogens and mutagens: bacterial tester strains with
R factor plasmids. Proceedings of the National Academy of Sciences of
USA 72, 979–983.
Maron, D.M. and Ames, B.N. (1983) Revised methods for the
Salmonella mutagenicity test. Mutation Research 113, 173–215.
Min, J., Kim, E.J., LaRossa, R.A. and Gu, M.B. (1999) Distinct
responses of a recA::luxCDABE Escherichia coli strain to direct and
indirect DNA damaging agents. Mutation Research 442, 61–68.
Mortelmans, K. and Zeiger, E. (2000). The Ames Salmonella/
microsome mutagenicity assay. Mutation Research 455, 29–60.
Ptitsyn, L.R., Horneck, G., Komova, O., Kozubek, S., Krasavin, E.A.,
Bonev, M. and Rettberg, P. (1997). A biosensor for environmental
genotoxin screening based on an SOS lux assay in recombinant
Escherichia coli cells. Applied and Environmental Microbiology 63,
4377–4384.
Słomińska, M., Konopa, G., We˛grzyn, G. and Czy_z, A. (2002)
Impaired chromosome partitioning and synchronisation of DNA
replication initiation in a Vibrio harveyi insertional mutant in the
cgtA gene coding for a common GTP-binding protein. Biochemical
Journal 362, 579–584.
1181
Shanabruch, W.G. and Walker, G.C. (1980) Localization of the
plasmid (pKM101) gene(s) involved in recA+ lexA+ -dependent
mutagenesis. Molecular and General Genetics 179, 289–297.
Sikora-Borgula, A., Słomińska, M., Trzonkowski, P., Zielke, R.,
Myśliwski, A., We˛grzyn, G. and Czy_z, A. (2002) A role for the
common GTP-binding protein in coupling of chromosome replication to cell growth and cell division. Biochemical and Biophysical
Research Communications 292, 333–338.
Sun, T.S. and Stahr, H.M. (1993) Evaluation and application of a
bioluminescent bacterial genotoxicity test. J. AOAC International 76,
893–898.
Thomulka, K.W. and Lange, J.H. (1995) Use of the bioluminescent
bacterium Vibrio harveyi to detect biohazardous chemicals in soil and
water extractions with and without acid. Ecotoxicology and Environmental Safety 32, 201–204.
Thomulka, K.W. and Lange, J.H. (1996) A mixture toxicity study
employing combinations of tributylin chloride, dibutylin dichloride,
and tin chloride using the marine bacterium Vibrio harveyi as the test
organism. Ecotoxicology and Environmental Safety 34, 76–84.
Ulitzur, S. and Barak, M. (1988) Detection of genotoxicity of metallic
compounds by the bacterial bioluminescence test. Journal of
Bioluminescence and Chemiluminescence 2, 95–99.
Ulitzur, S. and Weiser, I. (1981) Acridine dyes and other DNAintercalating agents induce the luminescence system of luminous
bacteria and their dark variants. Proceedings of the National Academy
of Sciences of USA 78, 3338–3342.
Ulitzur, S., Weiser, I. and Yannai, S. (1980) A new, sensitive and
simple bioluminescent test for mutagenic compounds. Mutation
Research 74, 113–124.
Verschaeve, L., Van Gompel, J., Thilemans, L., Regniers, L.,
Vanparys, P. and van der Lelie, D. (1999) VITOTOX bacterial
genotoxicity and toxicity test for the rapid screening of chemicals.
Environmental and Molecular Mutagenesis 33, 240–248.
Walker, G.C. and Dobson, P.P. (1979) Mutagenesis and repair
deficiencies of Escherichia coli umuC mutants are suppressed by the
plasmid pKM101. Molecular and General Genetics 172, 17–24.
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1175–1181, doi:10.1046/j.1365-2672.2003.02121.x