Effects in the comet assay: How can you tell a good comet from a bad comet?




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Effects in the comet assay: How can you tell a good comet from a bad comet?

1999 EEMS-Young Scientist Award Lecture

Dr. Andreas Hartmann, Novartis Pharma AG, Genetic and Experimental Toxicology, WSH-2881-5.14, CH-4002 Basel, Switzerland

Phone +41 61 324 1951; fax +41 61 324 1274;
Email: andreas.hartmann@pharma.novartis.com


Introduction

The single cell gel electrophoresis (comet) assay is an attractive test for measuring genotoxicity, investigating DNA repair, or monitoring populations for exposure against environmental mutagens. The comet assay has widespread applications in genotoxicity testing in vitro and in vivo, human biomonitoring or ecotoxicology research (for reviews see Tice et al., 1995; Anderson et al., 1999; Rojas et al., 1999). Samples can be prepared and evaluated very quickly by visual scoring (Collins et al., 1993) or by using image analysis systems. Moreover, the assay is relatively inexpensive and studies can be performed even with a low budget. Data can be generated fast and efficiently. But sometimes, the interpretation of results might be difficult. Does a strong effect in the comet assay always indicate a strong genotoxic hazard? For example, several investigations show that the comet assay detects oxygen-radical induced genotoxic effects at concentrations far below those, at which cytogenetic effects or mutations are observed. Examples from human studies, furthermore, show that clear DNA effects were detected without an obvious genotoxic exposure of the individuals. Confounding factors such as physical exercise, diabetes or seasonal variation due to sunlight exposure were identified which had so far not been demonstrated to show effects in cytogenetic tests. Mutagen-induced (bad?) comets and comets arising due to physiological processes (good comets?) are of the same microscopical appearance ( they appear not only the same but they are the same: - a consequence of DNA cleavage) and can not be distinguished from each other.


What is a Comet? Underlying mechanisms of comet tail formation

Östling and Johannson (1984) developed a microelectrophoretic technique under neutral conditions to study DNA damage in individual cells after gamma-irradiation. The alkaline version of the comet assay introduced by Singh et al. (1988) was suitable not only to detect DNA breaks but also alkaline-labile sites, crosslinks and transient DNA strand breaks arising due to DNA repair processes. During the last years, studies as well as different applications of the alkaline method have increased exponentially. Interestingly, the test was already heavily used before underlying mechanisms of the comet tail formation were investigated. Östling and Johannson (1984) proposed that strand breaks would enable DNA loops to stretch out upon electrophoresis and form the comet tail. Relaxation of loops was also proposed to be the primary underlying basis for comet formation under alkaline conditions (Collins et al., 1997). Klaude et al (1996) presented an excellent approach to demonstrate that the comet tail under neutral conditions consists of relaxed loops, whereas comet tails under alkaline conditions consist of DNA fragments. However, the rationale of Collins’ proposal was based on the ability of the comet assay to detect break frequencies of a few thousand breaks per cell; using the common alkaline unwinding conditions, which only partially unwind the DNA, free DNA fragments should not be expected at such low break frequencies. Another interesting mechanism was proposed by Singh and Stephens (1997): They speculated that some single strand breaks might reflect DNA-protein complexes. They suggested that residual protein in microgel slides should be removed and, thereby, increases the sensitivity of the assay. Summarizing all the studies it seems that the truth is somewhere inbetween the proposed mechanisms.


The comet assay in genotoxicity testing: Mutagens induce bad comets

The widespread applicability of the comet assay in genotoxicity testing is reflected by the wealth of data published in the last few years. In the beginning of our work with the comet assay we were interested in the sensitivity and the specificity of the test to detect genotoxic agents. We performed comparative investigations with the comet assay and the SCE test, which is known to be very sensitive in detecting chemical mutagens. For most of the chemicals we found a similar sensitivity for the two assays (Hartmann et al., 1994, 1996; Hartmann and Speit 1995). The only exception were chemicals inducing DNA-crosslinks, which were not sensitively detected with the standard version of the comet assay. Meanwhile, modifications were introduced which can not only detect DNA-DNA or DNA-protein crosslinks, but even enable the investigator to distinguish between the different types of crosslinks (Pfuhler et al, 1996; Merk and Speit, 1999). What makes the comet assay even more valuable is the specificity for detecting genotoxicity. Like in other tests, DNA effects induced due to cytotoxicity is a big issue. Data show that cytotoxic effects can be detected (dead cells show specific kinds of comets called 'clouds' or 'hedgehogs') and distinguished from genotoxic effects and, therefore, should have no confounding effects on results (Hartmann and Speit, 1997; Speit et al., 1998; Henderson et al., 1998). Clearly, more work needs to be done on this topic.


From in vitro effects to in vivo situations: human biomonitoring

The in vitro studies we and others conducted demonstrated the sensitivity and the specificity of the test system (for reviews see Anderson et al., 1999; Rojas et al., 1999). Therefore, an application to human monitoring for the sensitive detection of low levels of acute exposure seems to be very attractive. However, before conducting such studies one should consider a few points. For example, it is essential to have data about the variability of results to be expected when samples of the same individual are sampled at different time points. As the comet assay is extremely sensitive and slight variations of the protocol (such as temperature of the unwinding/electrophoresis buffer) can have considerable effects on results, it is of major importance to strictly use the same conditions during a study.

One should be cautious with the interpretation of increased effects in the comet assay. The comet assay detects primary DNA lesions (DNA damage) such as DNA strand breaks. These lesions may not be of much relevance since they may be repaired error-free or represent transient repair sites. In order to study comet-effects in more detail, Collins et al (1997) proposed to use lesion-specific enzymes also in human monitoring studies. Furthermore, comet data from biomonitoring studies should always be discussed in conjunction with results of other test run in parallel. Interestingly, in a study with more than 90 individuals we found increased comet values in leukocytes of waste disposal workers (Hartmann et al., 1998a). We also detected increased chromosome aberrations in the group of workers but found no correlation between these two parameters.

Data obtained in human biomonitoring studies may be biased by confounding factors. One of the best studied and constantly discussed confounding factor in cytogenetic studies is smoking. Some studies found differences, others did not. Similarly, it is yet not clear whether the smoking status of volunteers influences effects in the comet assay performed with blood cells. Contrary results have been reported; while some investigators describe increased comets in leukocytes of smokers (Betti et al., 1995) or show decreasing comets when volunteers quit smoking (Frenzilli et al., 1996), others do not find a difference in smokers versus non-smokers (Collins et al., 1997; Hartmann et al., 1998a). Which may, in part, be attributed to the examination of cells such as leukocytes, which are not directly exposed to cigarette smoke. By using exfoliated buccal cells, Rojas et al. (1996) demonstrated clearly increased DNA damage in smokers. However, more data is needed to shed light on this issue.


DNA effects after physical exercise: good comets?

During our studies on the applicability of the comet assay for human biomonitoring we were constantly seeking individuals providing us with blood samples. In the Medical Genetics group of Ulm everybody knew when it was time to hide: when I was looking around in the hallways with a syringe in my hands. Nevertheless, we always found people willingly giving blood for our various studies (thank you all!). But most of the times I had to give blood myself. Which, in fact, turned out to be very good because we obtained a considerable database on the DNA damage profile of my own leukocytes. The reproducibility of results during several month was excellent - until spring and summer months began. At that time some of my samples showed remarkably high migration values of which we first thought that they arose from technical problems. But the high values showed up repeatedly. The only explanation was that the increased migration values had something to do with my life-style. As I did not consume drugs nor started smoking (which, in fact, is yet not clearly shown to increase DNA migration) the DNA damage must have had some other origin. The origin became clearer when I realised that my comets were always high when my legs were hurting from exercising the other day (at that time I started training for triathlon competitions and was running a lot to get back in shape which I always lost during the winter months). So, we found the causative 'agent'. A connection between running and carrying damaged leukocytes in the blood the following day was established and reproduced with two other individuals. We wrote a first report on that topic which Jim Parry was agreeing to publish in Mutagenesis although one of the reviewers was concerned about the small sample size we were presenting (Hartmann et al., 1994).

We then focussed our further work on physical exercise-induced DNA damage on oxidative stress as a possible explanation for the effects. Vitamin-supplementation of the volunteers reduced DNA damage after exercise (Hartmann et al., 1995; Poch et al., 1996; Niess et al., 1996) but we did not find direct evidence for oxidative DNA damage such as oxidatively modified DNA bases or elevated levels of 8-hydroxy-2'-deoxyguanosine (Hartmann et al., 1998b). So more work needs to be done. On the other hand, there is increasing evidence that regular exercise has a beneficial effect by inducing an adaptive response against oxidative stress. Among others, Radak et al. (1999) showed an increased repair of oxidative DNA damage directly after exercise: the content of 8-hydroxydeoyguanosine in nuclear DNA was decreased in exercised rats compared to sedentary animals. After all, could the elevated level of DNA migration after exercise mean that repair activity of DNA repair enzymes were increased due to exercise-induced oxidative stress? In vitro studies showed that comet effects induced by benzo(a)pyrene or other bulky adduct-forming mutagens are mainly (if not exclusively) due to transient DNA single strand breaks formed during excision repair (Speit and Hartmann, 1995).

Moving on with our exercise-experiments we realized that exercise-treatment of individuals was difficult to use under controlled conditions (also, we were getting short on volunteers). We, therefore, chose a different exposure to oxidative stress: hyperbaric oxygen (HBO) Using HBO conditions (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA) we found clear and reproducible DNA effects in the comet assay with leukocytes of human subjects (Dennog et al., 1996). Interestingly, DNA damage was detected merely after the first treatment and not after further treatments under the same conditions, indicating an increased antioxidant defence. Rothfuss et al. (1998) then demonstrated that blood taken 24 h after HBO treatment is well protected against the in vitro induction of DNA damage by hydrogen peroxide (H2O2). Treating blood cells of volunteers before HBO with H2O2 induced clear DNA effects in the comet assay and micronuclei. In contrast, the same treatment did not induce genotoxic effects 24 h after HBO. This protective effect lasted for at least 1 week. Experiments with isolated lymphocytes gave similar results, indicating that the adaptive response was a cellular effect. The cells were not comparably protected against the genotoxic effects of gamma-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA. It seems, though, that oxidative stress causes DNA effects on the one hand but is, at the same time, also beneficial.


This relieved some of our concerns regarding extremely strong comet effects which we found in individuals after a triathlon competition (Hartmann et al., 1998b). Similar to HBO treatment, evidence for an adaptive response after exercise is also found (Niess et al., 1999). We, furthermore, were also assured by various epidemiological studies investigating the influence of regular physical activity on the incidence of cancer which showed a trend towards a preventive effect of regular exercise in men. A prospective study of 81,516 men and women demonstrated a protective effect of leisure physical activity on lung-cancer risk in male individuals after adjustment for other confounding factors such as smoking habits (Thune and Lund, 1997). A reduced risk of breast cancer was reported more recently in a study of 25,624 women performing a higher level of physical activity at work as well as in their leisure time (Thune et al., 1997). However, little is known about the effect of strenuous exercise. As we detected exercise-induced DNA damage predominantly after intensive exercise considerations should include epidemiological data on the risk of cancer in highly trained athletes, performing extensive training of high volume and intensity over many years. In contrast to the studies mentioned above, only few reliably controlled epidemiological studies are available focussing on the incidence of cancer in highly trained or former athletes. Moreover, most of these studies investigated the all-cause mortality and do not separately evaluate the prevalence of cancer. A study of 8393 athletes participating in competitive sports reported a higher incidence of cancer in active individuals (Poldenak, 1976). Other authors did not confirm this finding, and reported a lower risk of reproductive system cancers in former college athletes. Maybe there exists an dose-effect. What we know today is that athletes do not live longer - but they die more healthy. A detailed review on exercise-induced DNA damage can be found in Hartmann and Niess (1999).


More good comets? DNA effects arising during differentiation processes

There is another source of comets that one would not expect to origin from adversely damaged DNA: strand breaks are believed to arise naturally as part of a developmental program. Using the comet assay Vatolin et al (1997) found a dramatic increase in SSB during the first 2-4 mitoses after the beginning of differentiation of embryonic stem cells in vitro. The increased levels corresponded with increased levels of SCE. It was concluded that about half of the chromatin of the cells had been nicked at those stages, which, however, did not result in a phenotypic manifestation. We used the comet assay to measure DNA effects in embryos from 7.5 day old mice and also found high levels of DNA breaks in the cells (Tebbs et al 1999a; 1999b). It seems, though, that there are comets which do not have a biological significance in terms of mutagenesis.


Conclusion: Comets mean damaged DNA - the relevance needs interpretation

Comet-formation is due to primary DNA lesions. For the interpretation of test results one should elucidate, whether the primary DNA damage is converted into biologically relevant chromosome or gene mutations. From work on the relevance of comet effects for mutation induction, we did not find a relationship between comet effects and gene mutations: comets were induced in concentrations far below those inducing mutations (Speit et al., 1996; Hanelt et al., 1997). Coming back to the relevance of oxidative stress: oxidants readily induce chromosome aberrations. Therefore, I wanted to find out, if reactive oxygen species also induce chromosomal instability (CI). This phenotype is characterised by the persistent accumulation of chromosomal aberrations and is often found in cancer cells. When investigating the effect of oxidative stress on the onset of CI in clonal descendants of treated CHO cells, we soon found that, even at very high toxic doses of oxidants, CI was not induced (Limoli et al., 1998). This was surprising for us because treatment with ionising radiation (which is also associated with oxidative stress) or radiomimetic compounds induced unstable clones to a high extent. The difference might be due to the formation of DNA double strand breaks which are induced by ionizing radiation but not by treating cells with hydrogen peroxide. Evidence accumulates showing that a double strand break plays the major role in the induction of CI (reviewed in Morgan et al., 1998). There may be several examples where there is no clear relationship between DNA migration in the comet assay and other genetic endpoints (which we would consider more biologically relevant). Nevertheless, the comet assay is a valuable tool and is increasingly used in in vitro as well as in vivo studies in genotoxicity testing.


Acknowledgements


This overview is the extended EEMS Young Scientist Award 1999 Lecture presented during the 29th-EEMS Annual Meeting in Kopenhagen. My thanks go out to the Award Committee and the EEMS for inviting me and giving me the opportunity to present this work. Also, I want to thank the following people: Irene Witte (University of Oldenburg) who got me interested into genetic toxicology and supervised my diploma thesis. Günter Speit (University of Ulm) who supervised my PhD thesis and made the extensive excursion from genetic toxicology to sports sciences possible. William F. Morgan (UC San Francisco) who gave me the opportunity to study the effect of oxidative stress on the induction of chromosomal instability.


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