Mutagenesis vol. 19 no. 1 pp. 51-59,
January 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations
Preclinical Safety, 1Modeling and Simulation and 2Genetic Toxicology and Functional Validation, Novartis Pharma AG, CH-4002 Basel, Switzerland
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The alkaline Comet assay was used to investigate the in vivo genotoxicity of 17 compounds. Altogether 21 studies were conducted with these compounds. The investigations were triggered for various reasons. The main reason for performing the studies was to evaluate the in vivo relevance of in vitro genotoxicity findings with 10 compounds. Eight of these compounds showed no effects in the in vivo Comet assay while two compounds induced altered DNA migration patterns in specific organs. The remaining seven compounds were tested to follow up on neoplastic/preneoplastic or chronic toxicity changes as detected in specific target organs identified in rodent studies, to investigate the possibility of site-of- contact genotoxicity and to test the liver as a target organ for a suspected reactive metabolite. For the studies, various organs of rodents were analyzed, depending on the suspected properties of the compounds, including liver, jejunum, leukocytes, stomach mucosa, duodenum, lung and kidney. All tissues were amenable to investigation by gel electrophoresis after simple disaggregation of organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In conclusion, the Comet assay was found to be a reliable and robust test to investigate in vivo genotoxicity in a variety of rodent organs. Therefore, it is concluded that in vivo Comet assay data are useful for elucidating positive in vitro genotoxicity findings and to evaluate genotoxicity in target organs of toxicity.
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Introduction |
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Registration of pharmaceuticals requires a comprehensive assessment of their genotoxic potential in vitro as well as in vivo. In the case of positive genotoxicity data for a drug under investigation, mechanistic in vitro or in vivo studies are usually performed to further elucidate the mechanism of action and to support the risk assessment. The alkaline Comet assay as first introduced by Singh et al. (1988
) is now increasingly used in industrial genotoxicity testing in vitro (Hartmann et al., 2001a,b, 2003a; Kiskinis et al., 2002; Giannotti et al., 2002). Furthermore, the Comet assay is potentially useful as an in vivo genotoxicity test to investigate target organs as well as ‘first-site-of-contact’ genotoxicity. A comprehensive study testing 208 chemicals in eight different organs of mice demonstrated the ability of the Comet assay to detect genotoxic carcinogens in vivo at their important sites of tumorigenic action (Sasaki et al., 2000). These authors and others (Anderson et al., 1998) concluded that the Comet assay is useful to assess the in vivo genotoxic potential of in vitro genotoxic compounds at the site of their deleterious action. It is important to note that such in vivo test approaches are urgently needed since the existing tests with regulatory acceptance have limitations, primarily with regard to the organ from which the cells are derived (ICH S2B, A Standard Battery for Genotoxicity Testing of Pharmaceuticals, available at http://www.ifpma.org/ich1. html.).
The Comet assay has several advantages over other in vivo genotoxicity test methods: cytogenetic evaluations such as the micronucleus test or the chromosome aberration assay are applicable to proliferating cells only and tests such as the liver unscheduled DNA synthesis assay have been extensively used with cells from a single tissue only. Therefore, the Comet assay in vivo could be used as a supplementary assay for mechanistic and/or target cell-specific investigations, similar to mutation analysis in transgenic animals. Provided that a single cell/nuclei suspension can be obtained, the tissue of interest can be investigated for suspected tissue-specific genotoxic activity. Several exploratory studies using the Comet assay have recently been carried out. In these studies, tissue-specific genotoxicity in rodents was evaluated to allow for the assessment of carcinogenic risk. Such examples include the investigation of renal toxicity after inhalation of trichloroethylene (Mensing et al., 2002), liver and bone marrow toxicity after inhalation of benzene (Plappert et al., 1994), effects of ozone on bronchoalveolar lavage cells (Haney et al., 1999; Bornholt et al., 2002), effects of quartz on epithelial lung cells (Knaapen et al., 2002), effects of an Ames-positive nitro compound on cells of the gastrointestinal tract (Suter et al., 2002), effects of equine estrogen on mammary tissues (Zhang et al., 2001) and effects of potassium canrenoate on testes and ovaries (Martelli et al., 2002).
There are relatively few limitations of the Comet assay. Genotoxic insults induced by aneugens are not readily detectable. However, at the stage of development of a compound when the Comet assay would be applied as an in vivo test, the mechanism of action and possible or desired aneugenic activity is usually already known. Very short lived primary DNA lesions such as single-strand breaks, which may undergo rapid DNA repair, could be missed when using inadequate sampling times. However, an appropriate study design should ensure that these lesions are captured at higher dose levels, at which DNA repair may be significantly slowed down or even overwhelmed. Another issue with the Comet assay is that cytotoxicity could potentially lead to false positive results due to DNA fragmentation as a consequence of necrosis or apoptosis. While there is increasing evidence that cytotoxicity can be identified on specific microscopic images and may, therefore, not be a problem in the in vitro Comet assay (Hartmann and Speit, 1997; Henderson et al., 1998; Hartmann et al., 2001a,b, 2003a; Kiskinis et al., 2002; Kiffe et al., 2003), there are insufficient data to exclude a confounding effect in the in vivo situation.
The minimal technical requirements for the conduct of the alkaline Comet assay in vivo have been identified (Tice et al., 2000; Hartmann et al., 2003b). It was acknowledged that the Comet assay procedure bears several sources of variability (e.g. animal-to-animal, slide-to-slide, cell-to-cell, position of the slide in the electrophoresis box) which can have an impact on the results and which have to be accounted for in the design of a study. Simulation studies performed with data sets on liver and white blood cells of mice treated under various conditions resulted in specific recommendations regarding the experimental design and a comparison of the performances of different statistical methods for analysis of Comet assay data (Wiklund and Agurell, 2003).
The aim of the current study was to further evaluate the usefulness of the in vivo Comet assay as a supplementary assay for mechanistic investigations that provide data for critical risk assessment. For this purpose, data from 21 in vivo studies performed with 17 drug candidates are summarized. The main reasons why the Comet assays were carried out included: (i) positive in vitro genotoxicity tests; (ii) neoplastic changes in specific organs in long-term rodent studies; (iii) mechanistic investigations to specify the main genotoxic mechanism of action; (iv) the observation of strong cytotoxic effects on the mucosa of the gastrointestinal tract.
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Materials and methods |
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Test compounds
All test compounds were drug candidates or drugs synthesized by Novartis Pharma. For proprietry reasons, the structures of these compounds are not disclosed. The chemical class of each compound is presented in Table II.
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In vitro genotoxicity tests
All test methods followed current guidelines or have been described in detail previously (Hartmann et al., 2001a, 2003a
; Suter et al., 2002).
Animal husbandry
Animals were housed and treated in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26, 1997, by decision of the OECD Council [C(97)186/Final].
Dose selection and administration schedule
At the time an in vivo Comet assay was performed, sufficient toxicokinetic and/or absorption, distribution, metabolism and excretion data were available to ensure appropriate exposure. In cases where such data were not available, exposure was determined concomitantly with the Comet assay (data not shown). Dose selection for the Comet assay was based on available data from the same species using the same route of exposure or, if not available, dose range-finding tests according to Mackay and Elliot (1992) carried out prior to the study. For the Comet assay, the recommendations of Tice et al. (2000) and Hartmann et al. (2003b) were followed. In cases where a compound did not induce clinical signs of animal toxicity up to the maximum dose of 2000 mg/kg, only this dose was used in the Comet assay (limit test). Otherwise, two or three different doses were administered to the experimental animals, in the majority of cases by oral gavage. The majority of the tests were performed in one of the following two ways.
(i) Animals were treated with the test substance once. In relation to the time of test substance administration, tissue samples were obtained at 3 and 24 h after dosing. The shorter sampling time was deemed sufficient to detect rapidly absorbed as well as unstable or direct acting compounds; the later sampling time was chosen to detect compounds which require a longer time to be absorbed, distributed and metabolized.
(ii) In some cases two treatments within a 24 h interval were used and tissue samples were collected once only. The sampling time was 3 h after last administration of the test substance. Justified deviations from these sampling times were possible.
However, some compounds were tested prior to availability of guidance and in these cases only one sampling time (3 h post-administration) was performed.
Treatment and number of animals
When available data showed no gender difference regarding toxicity of the test compound, four or five male rats were used per dose and sampling time. Otherwise, five males and five females were used per dose group and sampling time. In each test, a vehicle-treated control group consisting of four or five animals (per gender if necessary) and three to five animals treated with a positive control were used.
Preparation of single cell suspensions and microscope slides
A small piece of the respective organ was isolated, rinsed in Hank’s balanced salt solution containing 25 mM EDTA and 10% dimethyl sulfoxide (DMSO) and then minced into fine pieces to obtain a cell suspension. An aliquot of 15 µl of the resulting cell suspension were transferred to a plastic tube and mixed with 135 µl of 0.5% low melting point (LMP) agarose (kept in a 37°C water bath). In the case of leukocytes, 10 µl of whole blood were mixed with 130 µl of 0.5% LMP agarose. Bone marrow samples were diluted 1:30 in fetal calf serum. Aliquots of 45 µl of cell suspension were transferred to each of two slides prepared for each animal and covered with a 24 x 24 mm coverslip; one sample or two different samples were placed on one slide. Regular slides were used which had been coated with 1–1.5% agarose and allowed to dry overnight.
Comet assay
The standard procedure originally described by Singh et al. (1988) with modifications (Hartmann et al., 2001b) was used. Briefly, after gently removing the coverslip, the slide was submersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, 1% Triton X-100, pH 10, 4°C) for at least 1 h. After lysis, the slides were equilibrated for 20 min in a jar containing alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13, 4°C), transferred to an electrophoresis unit with alkaline buffer and subjected to an electric field of 0.86 V/cm for 20 min at 4°C. A balanced design was used to run the electrophoresis, i.e. treated, positive control and vehicle control slides were included in each run. Following electrophoresis, the microgels were neutralized in 0.4 M Tris (pH 7.5), rinsed with water, dehydrated in 100% ethanol for 2 min and allowed to dry at room temperature. The DNA was stained with 2.5 µg/ml propidium iodide. To prevent slides fading and drying out, propidium iodide was first dissolved in distilled water and then further dissolved 1:5 in Vectashield (Vector Laboratories, Burlingame, CA). To detect DNA cross-links the modified procedure as described in detail by Merk and Speit (1999) was used. In brief, cells were treated in vitro with the test compound and processed as described above. After lysis, slides were rinsed with 0.4 M Tris (pH 7.5) and lysed cells were treated with proteinase K for 30 min directly on the slides. Hereafter, the slides were subjected to electrophoresis and further processed as described above.
Evaluation criteria
Examination of coded Comet assay slides was done with image analysis systems. For samples derived from liver, lung, kidney, bone marrow and white blood cells, automated image analysis was used (Frieauff et al., 2001). For tissues derived from the gastrointestinal tract (stomach mucosa, jejunum and duodenum), occasional bacterial contamination of the samples required a semi-manual analysis, which was carried out for samples from stomach mucosa, duodenum or jejunum with the software Comet Assay II (Perceptive Instruments, UK). The parameter used for both image analyses was the tail moment as defined by Olive et al. (1989). The median tail moment of 50 cells/slide was determined and the mean of the two slides per animal was calculated (i.e. 100 cells/animal were analyzed).
Statistical analysis
For determination of whether a test compound was positive in the Comet assay, the following statistical approach was applied, using the animal as the experimental unit (Lovell et al., 1999). The significance level used was 
0.05.
(i) Validation of the experimental procedure by comparing the negative control group with the positive control using a one-sided Mann–Whitney U-test at the 5% significance level. Only in the case of a significant difference being found were further statistical tests performed.
(ii) Comparing individual treatment groups with the negative control group using a Mann–Whitney U-test. A two-sided test was applied because some compounds exhibited a marked decrease in tail moment. In order to obtain the maximum power of these tests, no correction of the significance level to account for multiple comparisons was applied.
(iii) The existence of a dose–response relationship was assessed with the help of linear regression analysis. The slope parameter of the regression model was checked for statistical significance. The detailed results of these analyses will be presented in a forthcoming paper (Schumacher et al., in preparation).
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Positive and vehicle controls
Ethyl methanesulfonate at a dose of 300 mg/kg was used as the positive control throughout all studies. This dose was chosen in order to obtain a positive response in all organs of the treated animals. While in liver, leukocytes, kidney, lung and bone marrow this dose resulted in very strong positive effects, the effects found in cells of the gastrointestinal tract were considerably less pronounced. Depending on the solubility of the test compound, carboxymethyl cellulose (1% aqueous solution) or water was used as the vehicle control. The historical negative and positive control data are summarized in Table I. For the present study, historical data were not used to define acceptability of concurrent positive or negative controls since the database was not considered big enough.
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The chemical class of each compound and the reason why the respective in vivo Comet assay was performed are summarized in Table II.
Compounds tested as a consequence of positive in vitro genotoxicity data
Compounds 1–10 were tested in the in vivo Comet assay to clarify the in vivo relevance of positive in vitro genotoxicity data (Table III). The in vitro tests performed for individual compounds varied. In the case of positive in vitro tests, additional in vitro tests were performed to elucidate potential mechanisms. The summary shows that the majority of these compounds were negative in the in vivo Comet assay carried out with various tissues. These negative results are in agreement with the negative results of the in vivo micronucleus test. One compound (no. 7) was clearly positive in the micronucleus test in vivo, which was expected due to its known aneugenic potential (see below). Compound 7 is a natural tubulin stabilizer with a similar binding site to that of taxol. Its structural features include a lactone ring as well as an epoxide group. The compound was clearly clastogenic and produced a high frequency of polyploidy in vitro and was clearly positive in the bone marrow micronucleus test in rats (Table III). Since the gastrointestinal tract was identified as one major target organ of toxicity for this compound, the Comet assay was conducted to elucidate a potential strand-breaking effect owing to the epoxide function, in addition to its aneugenic potential. Jejunum and liver samples were taken from rats used for the bone marrow micronucleus tests 24 h after the last administration. The doses used (0.5, 2 and 5 mg/kg) produced a high incidence of apoptosis and necrosis of the mucosa along the gastrointestinal tract as well as in liver. However, in the Comet assay with jejunum and liver cells, no effects on DNA migration were seen after 24 h treatment. From these data it seems clear that this natural tubulin stabilizer exerts its effect predominantly in the M phase of the cell cycle and does not produce any relevant DNA strand breakage on top of this action at the sampling time investigated. Moreover, these results also demonstrate that doses of this compound resulting in apoptosis or necrosis in target organs did not lead to increased values for DNA migration.
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Compounds inducing altered DNA migration
Four compounds were found to induce altered DNA migration in treated versus solvent control groups (Table III). Compound 9 induced increased DNA migration while compound 10 retarded DNA migration, which was statistically significant in two of three treated groups. Compound 9 was negative in the bacterial mutagenicity test, positive in the in vitro micronucleus test in V79 cells (with and without S9) and positive in the in vitro Comet assay in V79 cells and human lymphocytes in the presence of S9 (Table III). Furthermore, this compound induced a concentration-related increase in chromosome aberrations in human lymphocytes starting at moderately cytotoxic concentrations. The aberrations included several chromatid exchange type figures. The compound was, however, negative in the in vivo micronucleus test in rats up to doses exhibiting clear bone marrow toxicity. When tested in a Comet assay in vivo in naïve rats, it was negative. However, in vitro tests demonstrated that compound 9 was mainly positive in the presence of S9 from Aroclor 1254-treated rats. These data were supported by the presence of glutathione adducts in rat hepatocytes and a strong metabolism of the compound by cells recombinantly expressing CYP1A1 and CYP1A2 in vitro (data not shown). Consequently, a further in vivo Comet assay was conducted, but this time with Aroclor 1254-pretreated rats. In this in vivo Comet assay a positive effect was seen in liver but not in lung or kidney (Table III).
Compound 10 was weakly positive in a variety of in vitro genotoxicity tests (Table II) but negative in the in vivo micronucleus test in bone marrow cells of rats. In the in vivo Comet assay stomach mucosa was investigated as a site-of-contact tissue. In two of the treated groups, a statistically significant decrease in the mean tail moment was observed, indicating a cross-linking potential of the compound. The mid dose group did not show retarded DNA migration, possibly due to high variability between individual animals. Since retarded DNA migration may indicate cross-linking potential of a compound, a modified in vitro Comet assay was conducted to further clarify the in vivo findings. In this modified assay, cells were treated with the compound and electrophoresis and alkaline unwinding were prolonged in order to achieve a significant extent of DNA migration in control cells. Retarded DNA migration was seen after treatment with certain concentrations of the compound, indicating an induction of cross-links (detailed in vitro data were published in Hartmann et al., 2001a). To investigate whether the cross-links were DNA–DNA or DNA–protein cross-links, a modified protocol as described by Merk and Speit (1999) was used in which lysed cells were post-treated with proteinase K. Using this modification, no difference compared with the control was seen, indicating that DNA–protein cross-links were induced rather than DNA–DNA cross-links (cf. Hartmann et al., 2001a).
Compounds tested to elucidate the contribution of in vivo genotoxicity to induction of target organ toxicity as observed in chronic toxicity studies (Table V)
VFour compounds (nos 11–14) induced hyperplasia in various organs and were tested in the in vivo Comet assay to investigate possible target organ genotoxicity and to support carcinogenicity risk evaluation. All compounds were negative in in vitro genotoxicity tests and in the in vivo bone marrow micronucleus test in rodents (if done). Compounds 11–14 did not alter DNA migration in the organs investigated, confirming the absence of a DNA-damaging potential in the respective target organs under appropriate exposure conditions. Compounds 15 and 16 were tested because they exhibited cytotoxic effects on the bone marrow and induced delayed cytotoxic effects in spermatogonia which were suspected to be related to tissue-specific genotoxicity. Both of these compounds clearly retarded DNA migration in bone marrow and/or spermatogonia and jejunum. However, a follow-up in vitro test demonstrated no direct DNA cross-linking activity of these compounds and the interpretation of the in vivo findings are unclear. Finally, compound 17 was tested because a metabolite generated in vivo was an aromatic amine suspected to pose a genotoxic threat. This compound was negative in the Comet assay with liver and jejunum from treated rats, the liver being the primary organ of formation of the aromatic amine metabolite.
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The Comet assay was used to investigate the in vivo relevance of positive findings of in vitro genotoxicity tests, to study neoplastic changes in specific organs in long-term rodent studies, to further elucidate prior insufficient discrimination of genotoxic mechanism of action and to investigate a possible genotoxic action causing strong cytostatic effects on the mucosa of the gastrointestinal tract. Our studies demonstrate that the alkaline in vivo Comet assay can readily be used to address the above indicated questions.
Ten compounds were tested in the in vivo Comet assay to further investigate the relevance of positive in vitro genotoxicity data. Eight compounds were negative in the in vivo Comet assay, thereby indicating that the positive in vitro data had no relevance for the in vivo situation for these compounds. Two compounds induced altered DNA migration in the Comet assay. One compound retarded DNA migration in stomach mucosa cells of rats, which was indicative of a cross-linking activity. This effect was then further investigated in vitro using modified conditions as described by Merk and Speit (1999). Applying these modifications, an induction of DNA–protein cross-links could be demonstrated. These results show the importance of appropriate experimental conditions, which means that the process of alkaline unwinding and electrophoresis have to be carried out in such a way as to allow for the detection of cross-linking activity. This can be achieved by simply using electrophoretic conditions which result in increased DNA migration in control cells, as described in available guidance (Tice et al., 2000; Hartmann et al., 2003b). Recently, Ranaldi et al. (2002) demonstrated retarded DNA migration in liver and bone marrow of mice treated with melphalan, indicative of a cross-linking activity of the compound, as demonstrated by others (Spanswick et al., 2002). These effects, however, were only detected when the duration of electrophoresis was increased from 25 to 45 min.
The second compound inducing altered DNA migration in the in vivo Comet assay was tested because of various positive in vitro clastogenicity data following incubation in the presence of S9 from Aroclor 1254-pretreated rats. An in vivo micronucleus test in bone marrow cells of rats was negative. Since this compound also induced glutathione adducts in rat hepatocytes and underwent strong metabolism by cells recombinantly expressing CYP1A1 and CYP1A2 in vitro, the in vivo Comet assay was performed under two conditions. First, liver cells of naïve rats treated with this compound were investigated and, second, cells from liver, lung and kidneys of rats pretreated with Aroclor 1254 were studied. Under the latter conditions, a clear induction of increased DNA migration was detected in liver, but not in lung or kidneys. These two examples of Comet assay-positive compounds demonstrate the ability of the Comet assay to elucidate mechanisms of target organ-specific genotoxicity.
Furthermore, with four compounds shown to induce organ-specific neoplastic/preneoplastic changes or hyperplasia after subchronic or chronic treatment of rodents, the contribution of a genotoxic effect to induction of these neoplasms was investigated. For all of these compounds, the toxicological characteristics were not clearly supportive of a non-genotoxic mechanism of tumorigenesis. All compounds were negative in the Comet assay, which indicates that a genotoxic potential in these cases was unlikely to have contributed to the neoplastic changes. In two cases, besides determining DNA migration in target organs of animals treated with a single high dose, samples of animals dosed for 4 weeks were analyzed.
Finally, two compounds with negative in vitro genotoxicity results but delayed toxic effects on spermatogonia in rats were investigated. Interestingly, retarded DNA migration was seen in three different organs of rats, indicating a cross-linking potential. In in vitro Comet assays performed subsequently with the L5178Y mouse lymphoma cell line, no indication of such a potential was seen and, therefore, the reason for the retarded DNA migration remains unclear. One assumption may be that the two compounds investigated confer cross-links with cell proteins under in vivo conditions that cannot be found in the LY5179Y mouse lymphoma cell line.
Our investigations show that the Comet assay can provide important mechanistic insight into target organ genotoxicity and can therefore be used for risk assessment and for identifying genotoxic human carcinogens. For example, ozone damages lung tissue and there are positive clastogenicity data from in vitro and in vivo studies (Viktorin, 1992). Carcinogenicity studies showed increased adenomas and carcinomas in mice but not in rats and it was suggested that ozone is a mouse-specific carcinogen (NTP, 1999). Using the Comet assay, two in vivo studies demonstrated increased DNA damage in bronchoalveolar lavage cells of mice treated with ozone (Haney et al., 1999; Bornholdt et al., 2002), which were presumably not related to secondary effects due to inflammatory reactions. Robbiano et al. (2002) demonstrated that the Comet assay in vivo is amenable to identifying urinary bladder genotoxic carcinogens by investigating cells of urinary bladder mucosa in vivo as well as in vitro. Furthermore, this study showed that the effects can be quantitatively different in rodents compared with humans, corresponding to their tumorigenic activity.
A presumed issue limiting a more widespread use of the Comet assay and its application in the regulatory setting is the potential influence of cytotoxicity on Comet assay results and, hence, the possibility of false positive results. An advantage of the Comet assay compared with the alkaline elution technique is that individual dead or dying cells may be identified by their specific microscopic image, i.e. necrotic or apoptotic cells result in comets with a small or non-existent head and large, diffuse tails (Olive et al., 1995). These cells are commonly called ‘hedgehogs’, ‘ghost cells’, ‘clouds’ or ‘non-detectable cell nuclei (NDCN)’. It was shown for in vitro tests that such cells can be present upon treatment with cytotoxic, non-genotoxic agents (Henderson et al., 1998; Hartmann et al., 1997, 2001a,b; Kiffe et al., 2003). However, since such microscope images can also be seen after treatment with high doses of radiation or high concentrations of strong mutagens, such comets are not uniquely diagnostic for apoptosis/necrosis. In the present study, such cells were seen only occasionally and were excluded from image analysis.
For in vivo tests using the Comet assay, a concurrent assessment of cytotoxicity is recommended, either generally (Tice et al., 2000) or only in the case of a positive response (Hartmann et al., 2003b), since cell death may be associated with increased levels of DNA strand breaks. In the studies described here, concurrent viability measurements were not performed for two reasons. First, histopathology data from studies with animals with the respective compounds were available before the Comet assay was performed. Second, concomitant assessment of cytotoxicity was not practicable since the cell isolation method used resulted in disruption of cell membranes. Dye exclusion methods for assessing cytotoxicity (e.g. Trypan blue exclusion) are only adequate for methods of cell isolation where membrane integrity is not affected, e.g. for hepatocytes derived from liver perfusion (Frei et al., 2001). However, to date it is not clear whether cytotoxicity may confound the results of the Comet assay in vivo, because there have been several studies where clearly established target organ toxicity did not lead to false positive findings: Mensing (2002) described histopathological alterations indicative of an inflammatory reaction in kidneys of rats chronically exposed to trichlorethane but no concomitant effect on DNA migration was seen. Similarly, in testicular cells of rats treated with a high dose of linuron resulting in decreased viability, no increased DNA migration was seen (Scassellati-Sforzolini et al., 1997). In the animals studied, increased DNA migration accompanied by slightly decreased viability (i.e. 
87% as determined by Trypan blue dye exclusion) was observed in liver cells of treated animals. Additionally, the alkaline elution method was used to analyze liver and testis of the animals and an increase in alkali-labile sites and DNA strand breaks was only seen in liver but not in testes. The authors concluded that the DNA-damaging effect on liver cells was due to direct DNA reactivity rather than an effect merely associated with cytotoxicity (Scassellati-Sforzolini et al., 1997). The assumption that cytotoxicity may not be a problem under in vivo conditions is further supported by our data with compound no. 7, which was tested up to doses inducing apoptosis and necrosis in liver and jejunum. In the Comet assay we did not detect increased DNA migration in these tissues.
In conclusion, the Comet assay was very useful to elucidate possible in vivo genotoxicity of compounds. Although we are unable to disclose the chemical structures of the compounds tested, we feel that this study has significant value for the scientific community because it demonstrates that the Comet assay is a very useful tool to investigate target organs or cells at the first site of contact and to obtain insight into possible mechanisms of genotoxicity involved. All tissues of interest were amenable to investigation after simple disaggregation of the organs by means of mincing or, in the case of epithelial cells from the gastrointestinal tract, scraping off cells from the epithelium. In general, the organs chosen were the liver, as the major organ for the metabolism of absorbed compounds, and the gastrointestinal tract, as a site of first contact tissue for orally administered substances (Tice et al., 2000; Hartmann et al., 2003b), or other target organs as identified in rodent chronic toxicity studies. In this context, the Comet assay proved to be extremely useful as an assay that helps to further support a risk evaluation process that is often triggered by neoplastic/preneoplastic findings in rodent chronic toxicity studies. Such further experimentation is deemed necessary and is stipulated by the ICH guidance on genotoxicity and carcinogenicity testing of pharmaceuticals (ICH S1B, Testing for Carcinogenicity of Pharmaceuticals, available at http://www.ifpma.org/ich1.html, and ICH S2B). In these guidelines it is acknowledged that a carcinogenicity test in rodents can yield evidence for a tumorigenic response of a compound that is negative in a genotoxicity test battery in vitro and in vivo. The guidelines ask for further supplemental genotoxicity tests if rodent tumorigenicity is not clearly based on a non-genotoxic mechanism. In the cases described here, the results of the Comet assay mainly contributed to the early termination of a project in the development phase based on the expectation that this compound ultimately has a high risk of being tumorigenic by a genotoxic mechanism of action (compound 9). Con versely, the test provided arguments to support a non-genotoxicity driven risk assessment based on rodent tumorigenesis that is indicative of a lack of human relevance with the inclusion of appropriate safety factors. In this context, the in vivo Comet assay as conducted for compounds 11 and 12 was instrumental in a positive regulatory review.
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3To whom correspondence should be addressed. Tel: +41 61 32 45619; Fax: +41 61 32 49843; Email: andreas.hartmann{at}pharma.novartis.com
Declaration of interest. A.Hartmann holds stock in Novartis Pharma AG and is an employee of the company; M.Schumacher and P.Lowe hold stock in Novartis Pharma AG and are currently conducting research sponsored by this company; U.Plappert-Helbig and L.Mueller are employees of Novartis Pharma AG whose compounds and their effects are described in this article |
References |
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