Mutagenesis pp. 1–11, 2014
Mutagenesis Advance Access published May 6, 2014
doi:10.1093/mutage/geu015
The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay
Günter Speit*, Alexandra Vesely, Petra Schütz, Regina Linsenmeyer and Julia Bausinger Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany *To whom correspondence should be addressed. Universität Ulm, Institut für Humangenetik, Oberer Eselsberg, D-89069 Ulm, Germany. Tel: +49-731-500 65440; Fax: +49-731-500 65402; Email: [email protected]
The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of ‘hedgehogs’ (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results. Introduction The comet assay is a well-established in vitro and in vivo genotoxicity test. Under alkaline (pH > 13) conditions, the assay can detect DNA single and double strand breaks, alkali labile sites, incomplete DNA excision repair sites and— possibly with protocol modifications—DNA–DNA cross links and DNA–protein cross links (DPX). However, there has always been concern that the comet assay (as other DNA strand break assays) may possibly detect exposure-related cytotoxic rather than genotoxic effects because DNA degradation is frequently involved in processes leading to cell death (1). Concurrent measures for cytotoxicity are therefore required for standard genotoxicity testing to assess genotoxicity under appropriate (non-toxic) conditions and to address possible effects of cytotoxicity in comet assay data interpretation. Various methods for measuring cytotoxicity have been used in combination with the comet assay, e.g.: so-called viability assays [trypan blue (TB) staining; fluorescein diacetate/ethidium bromide (FDA/EB) staining] that test membrane integrity, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Materials and methods Cell culture TK6 cells, a human p53 wild-type human B-lymphoblastoid cell line, were cultured in RPMI 1640 media supplemented with 10% heat-inactivated horse serum, 1% sodium pyruvate, 1% glutamine and 0.5% gentamycin. The A549 cell line is an epithelial-like human lung cell line derived through explant culture of carcinomatous lung tissue. Adherent cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 1% glutamine and 0.5% gentamycin. Both cell lines were maintained in a humidified incubator at 37°C with 5% CO2. If not specifically indicated, the chemicals used in these experiments were purchased from Sigma (Munich, Germany). The test substances used were: methyl methanesulfonate (MMS, CAS number 66-27-3, purity 99%); sodium dodecyl sulphate (SDS, CAS number 151-21-3, purity 99%); menthol
© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: [email protected].
Page 1 of 11
Downloaded from http://mutage.oxfordjournals.org/ at University of California, Los Angeles on May 8, 2014
Received on March 5, 2014; revised on March 25, 2014; accepted on March 27, 2014
bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo xymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays that test specific metabolic functions, proliferation assays and relative cloning efficiency. For the in vivo comet assay, histopathology is regarded as the ‘Gold Standard’ for assessing levels of necrosis and apoptosis but other methods are also in use (1). The low molecular weight DNA diffusion assay (LMW assay) has been proposed as an alternative or a supplementation for histopathology (2). It is assumed that the LMW assay detects the pre-lethal DNA strand breaks that are generated by endonuclease activity and/or inflammation during the earliest stages of cell injury before the cell dies and necrosis/apoptosis is histologically evident (3). However, experience with the LMW assay is limited and systematic investigations on the biological significance of effects measured by the LMW assay are missing. Therefore, we now performed in vitro studies with the comet assay and the LMW assay and compared the effects measured with results from other cytotoxicity tests that were performed under the same experimental conditions. We comparatively investigated known genotoxins and nongenotoxins and measured their effects on DNA migration in the comet assay. We also determined the frequency of ‘hedgehogs’ on comet assay slides. Hedgehogs (also called ‘clouds’) are here defined as comets with almost all DNA in the tail that are not precisely measured by image analysis (4). It has recently been discussed that hedgehogs frequently just represent heavily damaged cells and are not diagnostic of apoptosis. Therefore, they should not be regarded as a measure for cytotoxicity (5). We wanted to find out whether there is any relationship between the occurrence of hedgehogs in the comet assay and DNA diffusion in the LMW assay. As an indicator for cytotoxicity, we applied viability tests (TB and FDA/EB staining) although they only measure membrane integrity as one aspect of cellular dysfunction (6). However, we also determined cell proliferation that is the preferred measure of cytotoxicity in genotoxicity testing, particularly in combination with cytogenetic assays (7,8). Our in vitro experiments should provide further evidence for the usefulness of the LMW assay, when concurrently conducted with the comet assay, as an indicator for cytotoxicity and for possible effects of cytotoxicity on comet assay results.
G. Speit et al. (CAS number 89-78-1, purity 99%); sodium arsenite (0.05 M, CAS number 7784-46-5) and (±)-benzo[a]pyrene diol epoxide (BPDE, supplied by BIU, Grosshansdorf, Germany, CAS number 58917-67-2, purity 99%). Cell culture media and ingredients were obtained from Invitrogen (Karlsruhe, Germany). Agarose (NEEO) was supplied by Roth (Karlsruhe, Germany) and low melting agarose (SeaPlaque, ‘GTG’) was from Biozym (Hameln, Germany).
LMW assay The LMW assay was performed according to Vasquez et al. (2). Briefly, an extra comet assay slide was prepared for each sample and all slides were placed in lysis solution. After 1 h, the LMW slide was removed from lysis, neutralised and fixed without electrophoresis. Fixed slides were stained with SYBR GOLD (Invitrogen, Eugene, OR, USA) and 500 cells per slide were scored visually for the percentage of diffused versus condensed cells. Cytotoxicity Cell viability was analysed at the end of treatment using the TB assay and the FDA/EB assay. For the TB assay, cell suspensions were stained for 2 min with 0.5% TB and the percentage of dead (stained) cells was determined among 200 cells per slide. The FDA/EB assay was performed as previously described (4). The FDA/EB solution was mixed with the same volume of cell suspension. Cells were stained for 1 min and the percentage of dead (red) cells was determined among 500 cells. As another measure of cytotoxicity, relative cell counts (RCCs) at 24 and 48 h after the treatment of TK6 cells with the test substances were determined. Three parallel cultures with 150 000 TK6 cells each were set up, treated 24 h later with the test compound in RPMI 1640 media. Cells were counted three times using a cell counter (Cellcounter Model 2000; AL-Systeme, Karlsruhe, Germany). Three independent experiments were performed and the RCCs (mean % of control) were determined (8). Statistical analysis Each of the experiments was independently performed three times under the same conditions. All slides were coded and scored without knowledge of their identity. Results were evaluated with regard to the biological significance of an observed effect (i.e. whether the effect was clear and reproducible). In addition, differences between the mean values of the independently repeated experiments were tested for significance using Student’s t-test. A statistically significant difference was set at P 13. Slides were coded and stained with EB. Images of 100 randomly selected cells were analysed from one slide per culture. Measurements were made by image analysis (Comet Assay IV, Perceptive Instruments, Haverhill, UK) and DNA migration was determined by measuring the ‘tail intensity’ (% tail DNA). Hedgehogs (comets with almost all DNA in the tail that could not be precisely measured by image analysis) were separately scored on each comet assay slide. Hundred comets were analysed per slide and the percentage of hedgehogs was determined.
could not be determined any more by image analysis at the two highest concentrations (640 and 800 µM) because the majority of cells appeared as hedgehogs. Despite the high damage levels seen in the comet assay, diffused cells were not seen in the LMW assay after 2 and 4 h. Interestingly, diffused cells appeared in a concentration-related manner after treatment of TK6 cells with MMS for 24 h (Figure 1B). Similar to the effects in the LMW assay, the incidence of dead cells (as indicated by FDA/EB staining and TB staining) also clearly increased after treatment for 24 h but only marginal differences were measured after treatment for 2 or 4 h (Figure 1C). In further experiments with MMS and the LMW assay, we included two more preparation times (8 and 16 h). It can be seen in Figure 2 that the frequency of diffused cells increased after 16 h but not after 8 h. We also treated TK6 cells with MMS for 4 h, washed the cultures and performed the LMW assay after another 4-h recovery time. This protocol did not lead to an induction of diffused cells (data not shown). The frequency of diffused cells was not influenced by a longer lysis time. We performed parallel experiments with MMS treatment followed by 1- and 24-h lysis. Four-hour MMS exposure did not induce diffused cells under both conditions and 24-h exposure lead to a similar amount of diffused cells under both conditions (data not shown). Lower MMS concentrations (40 and 80 µM) enhanced the tail intensity in a concentration-related manner but had no effect on the frequency of diffused cells in the LMW assay and no clear effect on cell viability measured by FDA/EB staining (data not shown). To see whether the effects measured in experiments with TK6 cells can also be observed in another human cell line, we performed similar experiments with A549 cells. The results are summarised in Figure 3. Treatment of A549 cells with MMS for 2 or 4 h caused a concentration-related increase in tail intensity. Hedgehogs were seen at high concentrations (320 and 480 µM). At 480 µM MMS and 4-h exposure, the majority of cells appeared as hedgehogs. Effects in the LMW assay and the FDA/EB assay were apparent after 24 h but not after 2 or 4 h. These results basically agree with the results obtained with TK6 cells but the A549 cell line seems to be a little bit more sensitive towards the genotoxic and cytotoxic action of MMS. As a second genotoxic substance, BPDE was tested under the same conditions. The results show a similar pattern: tail intensity and the incidence of hedgehogs increase after short-term treatment and effects in the LMW assay and a viability assay occur later (after 24 h) at the same concentrations (Figure 4). In contrast to MMS, the incidence of diffused cells did not increase with increasing BPDE concentrations (2–10 µM). We also tested two non-genotoxic substances, SDS and menthol. Interestingly, the pattern of effects was quite different from that of the two genotoxic substances. SDS (500 µM) induced DNA migration but all cells with increased migration were hedgehogs. Exposure to 1000 µM SDS for 2 h caused intense but measurable DNA migration in some cells but the majority of cells with enhanced migration were hedgehogs. After 4-h exposure to 1000 µM SDS, tail migration could not be determined and all cells with enhanced DNA migration seen on comet assay slides appeared as hedgehogs (Figure 5A). However, diffused cells in the LMW assay and dead cells in the viability assays already occurred after 2 and 4 h and not only after 24 h (Figure 5B and C). A very similar pattern of effects was measured for menthol. A steep increase in DNA migration was seen at 2 mM and nearly all cells were hedgehogs (Figure 6A). The frequency of diffused cells (Figure 6B)
LMW assay and cytotoxicity
A
Tail intensity
Hedgehogs
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4h
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90
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Downloaded from http://mutage.oxfordjournals.org/ at University of California, Los Angeles on May 8, 2014
MMS [µM]
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Diffused cells [%]
80 70 60 50 40 30 20 10 0 Co
160 320 480 640 800
Co
160 320 480 640 800
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FDA/EB
Trypan blue
100 90
2h
24h
4h
80
Dead cells [%]
70
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*
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30
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10 0 Co 160 320 480 640 800
Co 160 320 480 640 800
Co 160 320 480 640 800
MMS [µM]
Fig. 1. The effect of MMS on DNA effects (tail intensity and hedgehogs) in the comet assay (A), DNA diffusion in the LMW assay (B) and cell viability in the TB assay and the FDA/EB assay (C) with TK6 cells. Mean values (with standard deviations) of three independent experiments; *P
Mutagenesis Advance Access published May 6, 2014
doi:10.1093/mutage/geu015
The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay
Günter Speit*, Alexandra Vesely, Petra Schütz, Regina Linsenmeyer and Julia Bausinger Universität Ulm, Institut für Humangenetik, D-89069 Ulm, Germany *To whom correspondence should be addressed. Universität Ulm, Institut für Humangenetik, Oberer Eselsberg, D-89069 Ulm, Germany. Tel: +49-731-500 65440; Fax: +49-731-500 65402; Email: [email protected]
The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of ‘hedgehogs’ (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results. Introduction The comet assay is a well-established in vitro and in vivo genotoxicity test. Under alkaline (pH > 13) conditions, the assay can detect DNA single and double strand breaks, alkali labile sites, incomplete DNA excision repair sites and— possibly with protocol modifications—DNA–DNA cross links and DNA–protein cross links (DPX). However, there has always been concern that the comet assay (as other DNA strand break assays) may possibly detect exposure-related cytotoxic rather than genotoxic effects because DNA degradation is frequently involved in processes leading to cell death (1). Concurrent measures for cytotoxicity are therefore required for standard genotoxicity testing to assess genotoxicity under appropriate (non-toxic) conditions and to address possible effects of cytotoxicity in comet assay data interpretation. Various methods for measuring cytotoxicity have been used in combination with the comet assay, e.g.: so-called viability assays [trypan blue (TB) staining; fluorescein diacetate/ethidium bromide (FDA/EB) staining] that test membrane integrity, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Materials and methods Cell culture TK6 cells, a human p53 wild-type human B-lymphoblastoid cell line, were cultured in RPMI 1640 media supplemented with 10% heat-inactivated horse serum, 1% sodium pyruvate, 1% glutamine and 0.5% gentamycin. The A549 cell line is an epithelial-like human lung cell line derived through explant culture of carcinomatous lung tissue. Adherent cells were cultured in minimal essential medium supplemented with 10% fetal calf serum, 1% glutamine and 0.5% gentamycin. Both cell lines were maintained in a humidified incubator at 37°C with 5% CO2. If not specifically indicated, the chemicals used in these experiments were purchased from Sigma (Munich, Germany). The test substances used were: methyl methanesulfonate (MMS, CAS number 66-27-3, purity 99%); sodium dodecyl sulphate (SDS, CAS number 151-21-3, purity 99%); menthol
© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: [email protected].
Page 1 of 11
Downloaded from http://mutage.oxfordjournals.org/ at University of California, Los Angeles on May 8, 2014
Received on March 5, 2014; revised on March 25, 2014; accepted on March 27, 2014
bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo xymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays that test specific metabolic functions, proliferation assays and relative cloning efficiency. For the in vivo comet assay, histopathology is regarded as the ‘Gold Standard’ for assessing levels of necrosis and apoptosis but other methods are also in use (1). The low molecular weight DNA diffusion assay (LMW assay) has been proposed as an alternative or a supplementation for histopathology (2). It is assumed that the LMW assay detects the pre-lethal DNA strand breaks that are generated by endonuclease activity and/or inflammation during the earliest stages of cell injury before the cell dies and necrosis/apoptosis is histologically evident (3). However, experience with the LMW assay is limited and systematic investigations on the biological significance of effects measured by the LMW assay are missing. Therefore, we now performed in vitro studies with the comet assay and the LMW assay and compared the effects measured with results from other cytotoxicity tests that were performed under the same experimental conditions. We comparatively investigated known genotoxins and nongenotoxins and measured their effects on DNA migration in the comet assay. We also determined the frequency of ‘hedgehogs’ on comet assay slides. Hedgehogs (also called ‘clouds’) are here defined as comets with almost all DNA in the tail that are not precisely measured by image analysis (4). It has recently been discussed that hedgehogs frequently just represent heavily damaged cells and are not diagnostic of apoptosis. Therefore, they should not be regarded as a measure for cytotoxicity (5). We wanted to find out whether there is any relationship between the occurrence of hedgehogs in the comet assay and DNA diffusion in the LMW assay. As an indicator for cytotoxicity, we applied viability tests (TB and FDA/EB staining) although they only measure membrane integrity as one aspect of cellular dysfunction (6). However, we also determined cell proliferation that is the preferred measure of cytotoxicity in genotoxicity testing, particularly in combination with cytogenetic assays (7,8). Our in vitro experiments should provide further evidence for the usefulness of the LMW assay, when concurrently conducted with the comet assay, as an indicator for cytotoxicity and for possible effects of cytotoxicity on comet assay results.
G. Speit et al. (CAS number 89-78-1, purity 99%); sodium arsenite (0.05 M, CAS number 7784-46-5) and (±)-benzo[a]pyrene diol epoxide (BPDE, supplied by BIU, Grosshansdorf, Germany, CAS number 58917-67-2, purity 99%). Cell culture media and ingredients were obtained from Invitrogen (Karlsruhe, Germany). Agarose (NEEO) was supplied by Roth (Karlsruhe, Germany) and low melting agarose (SeaPlaque, ‘GTG’) was from Biozym (Hameln, Germany).
LMW assay The LMW assay was performed according to Vasquez et al. (2). Briefly, an extra comet assay slide was prepared for each sample and all slides were placed in lysis solution. After 1 h, the LMW slide was removed from lysis, neutralised and fixed without electrophoresis. Fixed slides were stained with SYBR GOLD (Invitrogen, Eugene, OR, USA) and 500 cells per slide were scored visually for the percentage of diffused versus condensed cells. Cytotoxicity Cell viability was analysed at the end of treatment using the TB assay and the FDA/EB assay. For the TB assay, cell suspensions were stained for 2 min with 0.5% TB and the percentage of dead (stained) cells was determined among 200 cells per slide. The FDA/EB assay was performed as previously described (4). The FDA/EB solution was mixed with the same volume of cell suspension. Cells were stained for 1 min and the percentage of dead (red) cells was determined among 500 cells. As another measure of cytotoxicity, relative cell counts (RCCs) at 24 and 48 h after the treatment of TK6 cells with the test substances were determined. Three parallel cultures with 150 000 TK6 cells each were set up, treated 24 h later with the test compound in RPMI 1640 media. Cells were counted three times using a cell counter (Cellcounter Model 2000; AL-Systeme, Karlsruhe, Germany). Three independent experiments were performed and the RCCs (mean % of control) were determined (8). Statistical analysis Each of the experiments was independently performed three times under the same conditions. All slides were coded and scored without knowledge of their identity. Results were evaluated with regard to the biological significance of an observed effect (i.e. whether the effect was clear and reproducible). In addition, differences between the mean values of the independently repeated experiments were tested for significance using Student’s t-test. A statistically significant difference was set at P 13. Slides were coded and stained with EB. Images of 100 randomly selected cells were analysed from one slide per culture. Measurements were made by image analysis (Comet Assay IV, Perceptive Instruments, Haverhill, UK) and DNA migration was determined by measuring the ‘tail intensity’ (% tail DNA). Hedgehogs (comets with almost all DNA in the tail that could not be precisely measured by image analysis) were separately scored on each comet assay slide. Hundred comets were analysed per slide and the percentage of hedgehogs was determined.
could not be determined any more by image analysis at the two highest concentrations (640 and 800 µM) because the majority of cells appeared as hedgehogs. Despite the high damage levels seen in the comet assay, diffused cells were not seen in the LMW assay after 2 and 4 h. Interestingly, diffused cells appeared in a concentration-related manner after treatment of TK6 cells with MMS for 24 h (Figure 1B). Similar to the effects in the LMW assay, the incidence of dead cells (as indicated by FDA/EB staining and TB staining) also clearly increased after treatment for 24 h but only marginal differences were measured after treatment for 2 or 4 h (Figure 1C). In further experiments with MMS and the LMW assay, we included two more preparation times (8 and 16 h). It can be seen in Figure 2 that the frequency of diffused cells increased after 16 h but not after 8 h. We also treated TK6 cells with MMS for 4 h, washed the cultures and performed the LMW assay after another 4-h recovery time. This protocol did not lead to an induction of diffused cells (data not shown). The frequency of diffused cells was not influenced by a longer lysis time. We performed parallel experiments with MMS treatment followed by 1- and 24-h lysis. Four-hour MMS exposure did not induce diffused cells under both conditions and 24-h exposure lead to a similar amount of diffused cells under both conditions (data not shown). Lower MMS concentrations (40 and 80 µM) enhanced the tail intensity in a concentration-related manner but had no effect on the frequency of diffused cells in the LMW assay and no clear effect on cell viability measured by FDA/EB staining (data not shown). To see whether the effects measured in experiments with TK6 cells can also be observed in another human cell line, we performed similar experiments with A549 cells. The results are summarised in Figure 3. Treatment of A549 cells with MMS for 2 or 4 h caused a concentration-related increase in tail intensity. Hedgehogs were seen at high concentrations (320 and 480 µM). At 480 µM MMS and 4-h exposure, the majority of cells appeared as hedgehogs. Effects in the LMW assay and the FDA/EB assay were apparent after 24 h but not after 2 or 4 h. These results basically agree with the results obtained with TK6 cells but the A549 cell line seems to be a little bit more sensitive towards the genotoxic and cytotoxic action of MMS. As a second genotoxic substance, BPDE was tested under the same conditions. The results show a similar pattern: tail intensity and the incidence of hedgehogs increase after short-term treatment and effects in the LMW assay and a viability assay occur later (after 24 h) at the same concentrations (Figure 4). In contrast to MMS, the incidence of diffused cells did not increase with increasing BPDE concentrations (2–10 µM). We also tested two non-genotoxic substances, SDS and menthol. Interestingly, the pattern of effects was quite different from that of the two genotoxic substances. SDS (500 µM) induced DNA migration but all cells with increased migration were hedgehogs. Exposure to 1000 µM SDS for 2 h caused intense but measurable DNA migration in some cells but the majority of cells with enhanced migration were hedgehogs. After 4-h exposure to 1000 µM SDS, tail migration could not be determined and all cells with enhanced DNA migration seen on comet assay slides appeared as hedgehogs (Figure 5A). However, diffused cells in the LMW assay and dead cells in the viability assays already occurred after 2 and 4 h and not only after 24 h (Figure 5B and C). A very similar pattern of effects was measured for menthol. A steep increase in DNA migration was seen at 2 mM and nearly all cells were hedgehogs (Figure 6A). The frequency of diffused cells (Figure 6B)
LMW assay and cytotoxicity
A
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Hedgehogs
2h 100
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Downloaded from http://mutage.oxfordjournals.org/ at University of California, Los Angeles on May 8, 2014
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160 320 480 640 800
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Fig. 1. The effect of MMS on DNA effects (tail intensity and hedgehogs) in the comet assay (A), DNA diffusion in the LMW assay (B) and cell viability in the TB assay and the FDA/EB assay (C) with TK6 cells. Mean values (with standard deviations) of three independent experiments; *P
