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Review |
National Institute of Occupational Health, DK-2100 Copenhagen Ø, Denmark [P. M., L. E. K., H. W.], and Institute of Public Health, University of Copenhagen, DK-2200 Copenhagen N, Denmark [P. M., L. E. K., S. L.]
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Abstract |
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 Top Abstract IntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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Introduction |
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TopAbstractIntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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The comet assay has been developed in an empirical way, with two basically different protocols described in the literature by Singh et al. (1) in 1988 and Olive (2) in 1989. The former version was developed to measure low levels of strand breaks with high sensitivity, whereas the latter was optimized to detect a subpopulation of cells with varying sensitivity to drugs or radiation (3) . The version of Singh et al. (1) has been the protocol of choice in biomonitoring studies (3) . In its most simple form, the comet assay requires few steps. First, cells are embedded in agarose on a microscope slide. In the agarose, the cells and nuclear membranes are lysed, and the DNA is subjected to alkaline electrophoresis. Cellular DNA is visualized using a fluorescence microscope after staining cells with an appropriate dye. Except for these basic steps, there is currently no standard protocol for the comet assay. Different laboratories have modified the single steps to meet their particular needs. Three large reviews have been published that describe the different methodologies of the comet assay in detail (3, 4, 5) . A recent publication has presented recommendations on the test procedures of the comet assay in relation to in vitro and in vivo genetic toxicology testing (6) . In the present review, we have regarded these modifications as parts of the traditional comet assay procedure. In a modified protocol, an additional step of enzyme treatment is included to incise the DNA strand at the location of the damage. For instance, detection of oxidative DNA damage is possible with the FPG enzyme, which recognizes 8-oxodG and formamidopyrimidine lesions, or endonuclease III, which recognizes mostly cytosine hydrates (7) . We will refer to DNA damage detected by the enzymes as FPG-sensitive sites and endonuclease III-sensitive sites.
The end point measured by the traditional comet assay is a mixture of direct strand breaks and DNA damage that is converted to strand breaks by alkaline treatment. We use the term "DNA damage" to encompass all types of damage detected by the traditional comet assay. The level of DNA damage is determined largely by the length of the comet tail or by the "tail moment" (the length of the comet tail multiplied by the intensity of fluorescence in the tail). Alternatively, the level of DNA damage can be scored in arbitrary units, according to the type or grade of DNA damage. To ease the comparison between different kinds of measurements, we have calculated a mean arbitrary level of DNA damage in studies that have provided the results as type or grade of DNA damage. There is a good relationship between the level of DNA damage obtained by a visual score and that obtained by a continuous measurement (8) .
The survey consists of results from 61 studies in which the comet assay has been used in biomonitoring studies. It includes a broad range of exposure circumstances, some of which address the behavior of the comet assay in healthy populations, and others that study specific confounding factors such as smoking. Many studies report the use of the comet assay to detect a DNA-damaging effect in various occupational exposure situations or after treatment with well-known agents used in chemotherapy. In most of the biomonitoring studies, the DNA damage has been detected in isolated lymphocytes or unspecified nucleated blood cells, which will not be distinguished in the present review because of occasionally missing information. We have structured the review under sections defined by the variable reported, for instance: (a) age; (b) air pollution; (c) diet; (d) gender; and (e) infections. One section discusses the surprising finding that exercise dramatically increases the level of DNA damage by the comet assay. Our initial efforts to use the comet assay in biomonitoring led, by coincidence, to the discovery that there was seasonal variation by the assay. We suggested that exposure to sunlight was a determinant for the seasonal variation (9) , and we discuss this in one section.
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Origin of DNA Damage Measured in the Comet Assay |
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TopAbstractIntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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In most situations, intermediate repair sites could be the most important contributors to the DNA damage detected by the comet assay. A detection of cell death by apoptosis is also possible because the hallmark of apoptosis is degradation of nuclear DNA by endonuclease enzymes that cut the DNA helix in the linker region between histone proteins (13) . It has been argued that an image having a small head and a long tail (usually with >90% of the DNA in the tail) is apoptotic because the fragmentation of the DNA in apoptotic cells is so extensive that most DNA is located in the tail of the image (3) . However, it is questionable whether apoptotic cells can be distinguished as a special kind of image by the comet assay.
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Factors That Influence the Level of DNA Damage Detected in the Comet Assay |
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TopAbstractIntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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. The variation between the studies seems to be considerable if only
the range of the lowest and the highest measurement is compared
(1.2–26-fold). By calculation of the dispersion coefficient
(dispersion coefficient = SD divided by the mean), the studies
display a variation with the SD being 36% of the mean (95% CI,
27–46%). Holz et al. (14)
has reported
an intraindividual variation of 42% (95% CI, 18–72%), an
interindividual variation of 26%, and an assay variation of 20%. It
is not possible at the present time to give an estimate of the relative
contributions of physiological, environmental, or assay variation to
the overall variation of the comet assay. This is due in part to the
fact that there have been no satisfactory descriptions of the effect of
single factors or of the manner in which single factors interact with
each other. Some possible factors that cause variation in the
level of DNA damage in healthy untreated individuals are described
below.
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However, a few studies have detected a small effect of age by the comet assay. An American study comprising 41 individuals (age range, 24–93 years) detected a 12% increase in the basal level of DNA damage among individuals >60 years compared with individuals <60 years, which could be ascribed to a 5-fold higher content of highly damaged cells among older individuals (30) . A similar effect of age was observed in hepatocytes from rats, where the subset of highly damaged cells was greater in older individuals, although the mean basal value did not increase with age (31) . Also, a study of 80 individuals from Greece showed that men at the age of 55–60 years had an average of 14.5% more DNA damage than men at the age of 20–25 years (32) .
Overall, the age of the individual appears to have little effect on the mean basal level of DNA damage. An interesting observation by Singh et al. (15) suggested that cells from older individuals have less resistance to DNA damage by ex vivo X-ray exposure. This suggests that the collection and processing of blood samples may produce oxidative DNA damage and that this is a more important determinant for the level of DNA damage in older individuals than in younger individuals.
Air Pollution
Studies on the DNA-damaging effect of air pollution by the comet
assay have been reported from the Czech Republic and Mexico
City, two areas with some of the most severe air pollution
problems in the world. One of the studies from the Czech republic
investigated the effect of air pollution in postal workers and
gardeners from a highly industrialized district in Bohemia (Teplice).
There was an association between ambient polyaromatic hydrocarbon
concentration and the DNA damage detected by the comet assay
(18)
. The effect appeared to depend on the
GSTM1 phase 2 metabolizing enzyme genotype
(GSTM1 null genotype was the genotype associated with the
highest level of DNA damage). The effect of air pollution was later
questioned in a larger study of 542 people, showing that people from a
region with a low level of pollution (Prachatice) and people from
Teplice had a similar level of DNA damage, even when the statistical
analysis was adjusted for GSTM1 polymorphism, smoking, and
ethnic background (19)
.
The southwest metropolitan Mexico City area has one of the most severe air pollution problems in the world, with a daily ozone concentration of 0.12 ppm or more. Studies of children living in Mexico City showed that they had more DNA damage in nasal epithelium than children living in a port by the Pacific Ocean with a low level of pollution (33) . The effect of air pollution in Mexico City was also investigated in young nonsmoking adults who moved to the southwest metropolitan Mexico City area from a Pacific port with a low level of pollution. It was found that within 2 weeks of arrival, the level of DNA damage increased in nasal epithelial cells and remained high during the following 10-week period (34) .
The discrepancy between the Czech study and the Mexican study may be explained by the different types of air pollution in the two regions. Respirable particles and hydrocarbon compounds are the most important constituents of the air pollution in the Teplice region in the Czech Republic, whereas the air pollution in Mexico City has a high content of ozone and other directly oxidizing components. A study from Mexico City suggested that ozone and other directly oxidizing components of urbane air pollution are more likely to be detected by the comet assay in nasal epithelial cells and leukocytes than hydrocarbons and respirable particles (25) . The southern area of Mexico City has a high ambient concentration of ozone, whereas hydrocarbon compounds and respirable particles are the main components of the air pollution in the northern part of Mexico City. It was found that young adults who lived in the southern part of Mexico City had more DNA damage in nasal epithelial cells and leukocytes than people living in the northern part of the city (25) . Although this singles out ozone as a significant contributor to the effect of urban air pollution in directly exposed tissues such as nasal epithelial cells and leukocytes, hydrocarbons and respirable particles could exert an effect in the alveoli and bronchial tree rather than in nasal epithelial cells and leukocytes.
Diet
A number of studies have investigated the effect of nutrients,
antioxidants, or a combination of antioxidants on the level of DNA
damage as assessed by the comet assay in humans (20
, 35, 36, 37, 38, 39)
. No effect of daily supplements of up to 350 mg of
vitamin C was shown on DNA damage and resistance to
H2O2 or ionizing radiation
ex vivo (20
, 36)
, whereas increased ex
vivo resistance to
H2O2 and ionizing radiation
was reported 2–4 h after a single dose of 500-2000 mg of vitamin C
(35
, 40
, 41)
. An 8-week dietary supplementation of vitamin
E had no effect on the basal level of DNA damage and ex vivo
H2O2 resistance
(41)
. However, after 20 weeks with a daily mixture of
vitamins C and E and ß-carotene, the number of endonuclease
III-sensitive sites decreased, and ex vivo resistance to
H2O2 increased
(36)
. Administration of individual carotenoids had no
effect on DNA damage or FPG or endonuclease III-sensitive sites,
although there initially was correlation between the levels of oxidized
pyrimidines and some plasma carotenoids, and that a single dose of
vitamin C immediately increased
H2O2 resistance ex
vivo (38
, 40)
. As compared with a period of
carotenoid depletion, carotenoid-rich vegetable products decreased DNA
damage, and carrot juice also decreased the number of endonuclease
III-sensitive sites. Most of the positive effects of these dietary
antioxidants have been associated with ex vivo resistance of
the lymphocytes. Accordingly, it should be considered whether
differences measured by the comet assay induced by antioxidant
supplementation actually reflect the resistance to handling of samples
ex vivo, e.g., oxidation during sample work-up or
with respect to frozen storage. Moreover, it cannot be determined
whether any change in the actual level of DNA damage is the result of
an altered damage rate, e.g., due to improved antioxidant
defense or to an altered DNA repair capacity.
Exercise
In general, exercise is regarded as promoting good health and
well-being, but excessive exercise is associated with oxidative stress,
reflected by higher levels of oxidative DNA damage (8-oxodG) and lipid
peroxidation (42, 43, 44)
. It is controversial whether or not
the indication of oxidative stress is related to an increased risk of
disease. However, the cautionary approach is to register exercise
habits and analyze these in biomonitoring studies.
Tice et al. (45) were the first to report an effect of exercise by the comet assay. After jogging 5 km, one of three persons had an increased level of DNA damage in the blood sample obtained 5 min after the run (45) . Although the result indicates that the level of DNA damage is increased in blood cells after exercise, it must be considered anecdotal, and no record concerning the intensity of the run was reported. A series of studies from Günter Speit’s laboratory have addressed various aspects of the effect of exercise on the comet assay. They showed that jogging below the aerobic-anaerobic threshold did not result in an increased level of DNA damage (46) . Persons subjected to strenuous exercise above the aerobic threshold did not have an increased level of DNA damage immediately after the exercise (6 min), but the level was increased after 6 h and reached a maximum 24 h after the run (an approximately 3-fold increase). Baseline values were reached after 72 h (46) . The higher level of DNA damage in the hours after exercise was interpreted as an effect caused by the DNA repair system, i.e., excisions of damaged nucleotides, although there was no evidence of cytogenetic damage, as assessed by SCE (46) . A greater level of DNA damage (up to a 100-fold increase) was observed in well-trained athletes after a triathlon race, and the high level of DNA damage remained for a longer period after the race (47) . Curiously, there was no alternation in FPG-sensitive sites, urinary 8-oxodG levels, or MN in the time period of 5 days after the triathlon competition, suggesting that the DNA damage seen by the comet assay is not the result of a direct effect of reactive oxygen species related to exercise-induced oxidative stress (47) . This is in good agreement with the observation that multivitamin supplementation had no effect on the exercise-induced DNA damage, although the individuals receiving vitamin E supplementation 14 days before the exercise had less DNA damage after exercise (48) . As evidenced by the negative result in the SCE and micronucleus assays, exercise apparently does not result in chromosome damage. The increase in DNA damage has been attributed to tissue damage, activation of neutrophils, and inflammation.
It is worth noting that an effect of exercise by the comet assay has not been reported in studies other than those published by Speit’s laboratory, although a higher level of strand breaks in leukocytes after exercise has been reported by fluorometric analysis of DNA unwinding (49) . In recent years, reports have often stated that individuals were asked about their level of exercise in the days before the blood samplings. However, these reports do not state whether exercise was associated with a higher level of DNA damage, possibly because people who exercise heavily are not selected for the studies or because moderate exercise is not an important determinant for the basal level of DNA damage. Before the effect of exercise is known in sufficient detail, biomonitoring studies should obtain information about the type and intensity of the exercise and the time since the last bout of exercise. The training status of the test person may also be important. Again, a study from Speit’s laboratory (50) has shown that trained men had less net induction of DNA damage 24 h after a bout of exhaustive exercise on a treadmill.
Gender
The large majority of published biomonitoring studies consist of
individuals of both sexes. A statistical analysis is routinely carried
out to evaluate any difference between the sexes in the study
population. In general, at present, an effect of gender must be
regarded as a matter of controversy. The results from a large
cross-sectional study of healthy individuals reported that men had more
DNA damage than women, displayed as a wider span of DNA damage in the
group of men (23)
. In contrast to the results of the
Italian study (23)
, we recently found that women
had more basal DNA damage than
men.4
Apart from these cross-sectional studies, other somewhat coincidental
findings from a Polish study (26)
of shoe factory workers
reported that men had a higher level of DNA damage than women, whereas
a study of aflatoxin-exposed individuals from Gambia reported that
women had more DNA damage than men (51)
. It is possible
that study design may offer an explanation for the contradictory
results reported. For instance, the Italians used single
measurements for each individual, whereas we used repeated
measurements. However, a German study using repeated measurements did
not detect an effect by gender (14)
. It is worth noting
that analysis of 8-oxodG in leukocytes also has provided inconclusive
results of differences in oxidative DNA damage according to
gender (52)
. In contrast, there is strong evidence
that women have a higher number of MN than men do (53)
.
Therefore, it seems more likely for women to have the highest level of
DNA damage, if there is an effect of gender.
Infection
It might be expected that the comet assay would detect more DNA
damage in individuals suffering from infectious diseases. It is
necessary to discriminate between random infections such as the common
cold or influenza and chronic infections. Curiously, we have only found
one study in which severe infections in infants were associated with
more DNA damage (54)
. As we saw for exercise, we have
noted that in several biomonitoring studies, data on health status were
recorded, but the contribution of health status to the variation in
statistical analyses is never provided.
Residential Radon Exposure
Exposure to residential radon was associated with a higher level
of DNA damage among family members living in houses with a radioactive
radon concentration above 200 Bq/m3
(55)
.
Smoking
As seen with the development of other tests for DNA damage,
smoking is always one of the first exposure circumstances to which
researchers turn their attention as a source of an agent that should
produce a positive effect. In addition, biomonitoring studies often
include both smokers and nonsmokers. A study of 200 individuals from
Italy showed that smoking increased the extent of migration in
lymphocytes (10% increase in migration among smokers), but there was
no effect detected by SCE or no relationship to the amount of
cigarettes smoked per day (16
, 17
, 23)
. Individuals who
quit smoking had reduced basal levels of DNA damage 1 year after
cessation (17)
. A Greek study consisting of men
ages 20–25 years or 55–60 years showed that smoking was associated
with an increased level of DNA damage (40%; based on an average of the
whole study) for both the young age group and the old age group
(32)
. A number of occupational studies from Turkey
(27
, 56) , France (57)
, and Poland
(58)
have also reported an effect of smoking. Recently, a
study from China found that both smokers and employees in a cigarette
factory had more DNA damage than nonsmoking controls who were not
occupationally exposed to tobacco dust (59)
. A study of
Scottish men ages 50–59 years showed that smokers had 1.6-fold more
endonuclease III-sensitive sites than nonsmokers, although there were
no convincing effects of smoking on the basal level of DNA damage
(36)
. In recent years, an overwhelming amount of
publications (usually reports from occupational studies) have failed to
show any effect of smoking (9
, 19
, 22
, 24
, 28
, 29
, 55
, 60, 61, 62, 63, 64)
. One study has even reported that smokers had less DNA
damage than nonsmokers (65)
. The discrepancy in the
reports is not surprising because smokers had only a slightly higher
level of DNA damage in the positive studies. Several of the negative
studies have been small and have detected the effects of occupational
exposure as well. Thus, the failure to show an effect of smoking could
be due to low statistical power. However, it is striking that
determination of 8-oxodG in leukocytes has produced a similar
discrepancy (52)
. We have noted that the positive studies
seem to cluster in the southern part of Europe (Italy, France, Greece,
and Turkey; Refs. 16
, 17
, 23
, 27
, 32
, 56,
and
57
). It is tempting to speculate that geographical
variation in smoking habits may be an underlying factor that can
explain the positive results of smoking is some parts of Europe. It is
possible that brands of cigarettes containing dark tobacco and high tar
content are smoked more frequently in the southern part of Europe. The
effect of smoking may also be explained by differences in the
composition of the diet in the southern part of Europe, including foods
rich in antioxidants or vitamin supplementation.
Sunlight
Seasonal variation has been reported by the comet assay in some
laboratories. Longitudinal studies by Betti et al.
(16)
and Frenzilli et al. (17)
found that more DNA damage was detected in samples obtained during the
summer months than in samples obtained during other times of the year.
Likewise, unpublished observations from studies carried out by the
group of Ray Tice in the United States indicate that more
DNA damage was present in samples obtained during the summer than in
those collected during the
winter.5
We have also reported a seasonal effect by both the comet assay and
UVC-induced unscheduled DNA synthesis, and we were able to correlate
the seasonal variation to the exposure of the individuals to sunlight
(9)
. To further characterize the seasonal variation, we
set up a large study among healthy individuals, and we have found that
exposure to sunlight is the factor that best accounts for the
variation.6
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Use of the Comet in Assessment of DNA-damaging Exposures in Occupational Settings |
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TopAbstractIntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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summarizes the occupational studies in which the comet assay has been
used as a measurement of effect. For each study, we have calculated an
effect ratio and the 95% CI between the exposed and control groups,
based on the t distribution. There is a statistically
significant effect ratio if 1.0 is not included in the 95% CI.
Information on the mean and SD has been collected directly from
studies that have stated these values; otherwise, we have approximated
the values from graphs. In four studies (56
, 58
, 61
, 66)
,
the level of DNA damage was reported as grade or type, and we have
calculated a mean arbitrary score on basis of the whole cell population
(a detailed description of the calculation of the mean arbitrary score
for these four studies is outlined in the footnotes of Table 2
).
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Farmers
In a study of French farmers, blood samples were obtained the day
before and 1 day after spraying fields with pesticides. There was no
statistically significant difference between samples taken before and
after spraying (difference in tail moment, P = 0.26,
Student’s t test; Ref. 68
). It was also
reported that samples obtained during the summer had more DNA damage
than samples obtained during the winter, and this was suggested to be
due to ambient exposure to pesticides during the summer
(57)
. We have suggested that there is an effect of
exposure to solar radiation by the comet assay (see "Sunlight"),
and we speculate that the seasonal variation might be due to exposure
to sunlight.
Hair Colorists
The effect of occupational exposure to hair dyes has been studied
among professional hair colorists who had 2–25 years of working
experience with hair dye products and did not use gloves during the
application of the dyes. There was no difference in the level of DNA
damage among the hair colorists and referents, and the results of SCE
and urine mutagenicity were negative as well (27)
.
Hospital Workers
Two studies have assessed health effects on hospital workers who
were exposed to either antineoplastic agents (61)
or
anesthetic gases (56)
. In the first study, nurses who
worked in the oncology department had more DNA damage than unexposed
controls (61)
. Use of safety protection equipment such as
gloves, masks, gowns, and eye glasses was associated with a lower level
of DNA damage among exposed nurses. In the second hospital worker
study, a higher level of DNA damage was observed among operating room
personnel, who were exposed to anesthetic gases (mostly halothane,
nitrous oxide, and isoflurane; Ref. 56
).
Ionizing Radiation
The effect of chronic occupational exposure to low-dose ionizing
radiation has been investigated among hospital technicians in Turkey
(66)
and among industry workers in Poland (26
, 69)
. The exposed hospital technicians had a trend toward a
higher level of DNA damage (P < 0.10, Student’s
t test, based on the whole population of scored cells; Ref.
66
). There was a 2-fold higher level of DNA damage among
the Polish industry workers, but no difference in endonuclease III- or
FPG-sensitive sites was seen between exposed and unexposed workers
(69)
. This was unexpected because both endonuclease III
and the FPG protein detect oxidative DNA damage. However, the effect
may be due to an adaptive response toward oxidative stress conferred by
repeated exposures to ionizing radiation. For instance, it has been
reported that leukocytes obtained from children living in the Chernobyl
region are protected from (or have a lower responsiveness to)
ex vivo damage of bleomycin (70)
. Up-regulation
of antioxidant and/or DNA repair enzymes is a possible mechanism behind
the protective effect.
Organic Solvents and Hydrocarbons
Three studies have investigated occupational exposure to mixtures
of organic solvents and hydrocarbons. The exposed groups included
airport personnel (63)
, gasoline station attendants
(28)
, and shoe factory workers (29)
. Both the
airport personnel and the gasoline attendants were exposed to variety
of DNA-damaging agents such as motor vehicle exhausts and gasoline
vapors, including benzene. The gasoline station attendants and the
airport workers had more DNA damage than the control groups (28
, 63)
. There was not a clear pattern between the DNA damage and
the level of exposure in the gasoline station attendant study
(28)
. The airport workers had similar SCE but less MN than
the control group (63)
. This unexpected result was
suggested to be due to the type of exposure or to the fact that the
control group members were, on average, 13 years younger than the
members of the exposed group (63)
.
The study of shoe factory workers included employees from two shoe factories. In both factories, acetone, gasoline, and toluene were detected in the air of the workplaces, and in one of the factories, ethylacetate and diisocyanate were also detected. There was no difference in the level of DNA damage between exposed workers and controls from the factory. The study did not find any relationship between GSTM1 polymorphism and the level of DNA damage as detected by the comet assay (29) .
Plastic Industry.
Vinyl chloride is a potent carcinogen that is activated by cytochrome
P-450 enzymes in the liver to chloroethylene oxide, which spontaneously
rearranges to chloroacetaldehyde (71)
. This active
metabolite reacts with DNA to form
N7-(2-oxoethyl)guanine and exocyclic
ethenoadducts. Vinyl chloride-exposed workers in a plastic production
plant had more DNA damage than members of a referent group
(72)
. Air samples taken by personal air samplers of
exposed workers detected vinyl chloride levels that were below the
permissible value (<5 ppm). The level of increased DNA damage between
exposed workers and controls was in the range of 1.9-fold (0.5–2 years
of employment) and 3.2-fold (5–10 years of employment; Ref.
72
). It was speculated that the association of DNA
damage level and duration of employment was due to the accumulation of
highly persistent vinyl chloride-generated ethenoadducts. However, if
ethenoadducts can be detected by the comet assay, they should be
alkaline labile, and to our knowledge, this is not known.
Rubber Industry.
A large number of chemicals are used in the production of rubber,
including vulcanization agents, colorants, solvent, accelerating
agents, and activating agents. Two studies have shown that workers in
rubber production plants tended to have more DNA damage than controls.
In one of the studies, the level of DNA damage in 30 workers in a
rubber tire factory in the Slovak Republic was more than 2-fold higher
than that of controls (73)
. A 1-year follow-up
study showed that the rubber tire workers still had more DNA damage
than the controls, as well as increased frequency of MN and CA
(74)
. In the second study, lymphocytes obtained from 19
male rubber workers at a plant in northern Italy had slightly more DNA
damage than 20 age-matched controls, although the difference was not
statistically different at the 5% level (24)
. The
cytogenetic assays revealed that exposed workers had more MN, whereas
there was no difference in SCE between the exposed and unexposed groups
(24)
.
Sewage and Waste Disposal Occupations.
Studies of workers exposed to waste materials in a waste disposal site
(22)
and sewage workers (75)
have been
carried out. Twofold more DNA damage was observed in the waste disposal
workers than in the control group (22)
. There was also an
increased frequency of CA, yet there was no correlation between the CA
and the comet assay. In contrast, SCE was not increased in waste
workers as compared with the control group (22)
.
The study of sewage workers did not show any effect related to exposure to waste materials (75) . The sewage workers were classified into three different groups of exposure, based on self-reported levels of exposure, and the group with the highest exposure was defined as those workers who had worked in sewage-contaminated environments for at least 8 h in the preceding 2 weeks. This may indicate that the sewage workers are not exposed to waste matter to an extent that causes DNA damage in lymphocytes. However, the assessment of exposure may not accurately discriminate between different exposure circumstances. It would have been of great value if some sort of definition of the DNA-damaging agents and measurements of exposure had been included in the study.
Styrene Exposure
Styrene has been classified as a possible carcinogen to humans
(group 2B) by the IARC (76)
. It is used in the production
of reinforced plastic fabrications used in boats, corrosion-resistant
tanks, pipes, and car parts, and 10% of styrene may evaporate during
lamination (76)
. There have been reports from two
studies examining styrene-exposed workers in plastic lamination plants
(64
, 77
, 78)
. In one study, a higher level of DNA damage
and CA was observed among exposed workers (64)
. More DNA
damage was also detected in the lamination workers than in the referent
group in the second study (78)
. A study performed 3 years
earlier had indicated an effect, although the difference was not
statistically significant at the 5% level (77)
. The
lamination workers also had more styrene-specific
(O6
-guanine) adducts detected by
32P postlabeling, and a higher HPRT mutation
frequency was seen in the lamination workers compared with unexposed
factory workers and a second control group outside the factory
[laboratory group (77
, 78)
]. The general impression from
the studies is that styrene-exposed workers had more DNA damage, as
determined by the comet assay.
Wood Dust
The IARC has assessed wood dust as being carcinogenic to humans
(group 1), based primarily on the observation that occupational
exposure to wood dust is associated with cancer in the nasal cavities
and paranasal sinuses (79)
. The results of a Polish study
showed that workers employed in a wooden furniture factory had more DNA
damage in lymphocytes than a control group (58)
. The data
support a previous study performed by the authors (80)
that showed that woodworkers had more DNA damage than controls.
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Discussion |
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TopAbstractIntroductionOrigin of DNA Damage...Factors That Influence the...Use of the Comet...DiscussionReferences |
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A point of consideration in biomonitoring is the discriminative power
of the comet assay i.e., to be truly negative in situations
of no exposure or exposures that are not DNA damaging and to be truly
positive where an effect of exposure is detected in the comet
assay. In four of the negative occupational studies (29
, 66 , 68
, 75)
, there have been no parallel determinations of
genotoxic effect with other assays. In the study of the hair colorists,
SCE and urine mutagenicity were negative (27)
, indicating
that it could be a true negative result. There have been mixed results
in the remaining negative studies by the comet assay (24
, 65
, 77) . It is striking to observe that on average, there were 47
individuals (median, 46 individuals) enrolled in the negative studies,
whereas there were 72 individuals (median, 60 individuals) in the
positive studies. This is also reflected by comparing the variation of
the control groups; the negative studies had a dispersion coefficient
of 0.49 (95% CI, 0.09–0.89), and the positive studies had a
dispersion coefficient of 0.33 (95% CI, 0.17–0.48). This clearly
suggests that the negative studies often suffer from low statistical
power rather than reflecting a situation of no biological effect of
exposure. In fact, we have estimated the statistical power in all of
the negative studies to be below 30%. The question that obviously
emerges is how many individuals should be enrolled in future studies to
provide a good chance of detecting a positive result. In an attempt to
answer this question, we have calculated the variation in control
populations outlined in Table 1
, and we have estimated a mean
dispersion coefficient of 0.36 (95% CI, 0.27–0.46). This means that
if we expect to detect 1.5-fold more DNA damage in an exposed group, it
will require that each group consist of 13 individuals (and 20
individuals for the upper 95% CI) to detect a statistically
significant difference at a level of 5%. (This is calculated under the
assumption that the type II error risk ß = 10% and that the SD
is the same in the exposed and the referent group).
We have found 11 studies that are positive by the comet assay in
various occupational studies. Two of the studies reported positive CA
results (22
, 64)
and two studies reported negative SCE
results (22
, 63)
. Eight of the 11 studies had no
parallel determination of effect by other assays (26
, 28
, 56
, 58
, 59 , 61
, 72
, 73)
. Consequently, it is difficult to provide a firm
conclusion of the positive results by the comet assay in investigations
of occupational settings. Furthermore, it disturbs the general picture
of the comet assay as a sensitive test of DNA-damaging effects when a
large number of studies have not found an effect of smoking, an
exposure circumstance that is otherwise regarded as a positive control
for a DNA-damaging exposure. Also, the comet assay did not detect an
effect of aflatoxin exposure in a population from Gambia
(51)
. There have been reports from some studies
investigating the effect of chemotherapy and radioactive iodine therapy
by the comet assay (Table 3)
. It is reassuring to note that an effect has been observed by the
comet assay in these few studies where known DNA-damaging agents are
given to patients in the course of treatment. However, the effect
rarely exceeded a 2-fold increase above the pretreatment level of DNA
damage, and there was no dose-response relationship between the
administered chemical and DNA damage. This may indicate that the comet
assay should not be used to compare DNA-damaging hazards between
exposure levels. A positive result by the comet assay should be
regarded as an indication of a biological effect related to some
environmental or occupational agent. It is worth stressing that the
comet assay seems to be well suited as a fast and inexpensive test of
biological effect, but at present, it should not be used in the
assessment of individual health risk of humans. This observation is
further underlined by our current knowledge that the comet assay is
susceptible to confounding factors such as sunlight, exercise,
infections, air pollution, and dietary factors, which are not easily
assessed.
|
The comet assay is suitable for monitoring levels of DNA damage in cell types other than lymphocytes. In biomonitoring studies, nasal epithelial cells and buccal cells have drawn the most attention because they are cells from tissues that come into direct contact with ingested or inhaled compounds. It has been reported that smokers have higher levels of DNA damage in both nasal epithelial cells and buccals cells (82) and nasal epithelial cells (83) . Also, the effects of air pollution have been detected in nasal epithelial cells and buccal cells (25 , 33 , 34) .
In conclusion, the experiences of the comet assay in biomonitoring studies are still relatively new, although the use of the assay has been exploited in many laboratories. An impressive, wide variety of exposures has been tested with success by the comet assay, and this supports our basic notion that the comet assay is an excellent screening test for exposures that may be DNA damaging. Many reports of confounding factors have emerged in the studies. It appears that usage of the comet assay is in a phase where we have identified numerous confounding factors that may or may not be relevant for determination of the basal level of DNA damage. The problem we are facing now is that the effect of a confounding factor usually has been tested in a study designed for the purpose, whereas the factor may have no effect in cross-sectional studies. This makes it difficult to pinpoint one or a few factors that we would recommend should always be incorporated into study designs, e.g., by matched control populations. However, samples from exposed and unexposed populations should be collected at the same time to avoid seasonal variation. Likewise, we recommend that age, gender, and smoking status be used as criteria in the selection of populations. Information on the level of exercise, infection, and diet should be recorded on the day of the sampling.
|
Footnotes |
|---|
1 Supported by grants from the Danish Cancer
Society and the Working Environment Fund, Denmark. 
2 To whom requests for reprints should be
addressed, at the Institute of Public Health, University of Copenhagen,
Panum Institute 18-5, DK-2200 Copenhagen N, Denmark.
3 The abbreviations used are: CA, chromosome
aberration; FPG, formamidepyrimidine DNA glycosylase; MN,
micronuclei; 8-oxodG, 8-oxo-2'-deoxyguanosine; SCE, sister
chromatid exchange; CI, confidence interval; HPRT, hypoxanthine
phosphoribosyltransferase.
4 P. Møller, H. Wallin, E. Holst, and L.
E. Knudsen, unpublished observations.
5 Ray Tice, personal communication.
6 P. Møller, H. Wallin, E. Holst, and L.
E. Knudsen. Sunlight-induced DNA damage in lymphocytes from humans,
manuscript in preparation.
Received 12/ 2/99; revised 6/ 6/00; accepted 7/13/00.
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