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Hyperthermia-induced micronucleus formation in a human keratinocyte cell line
Mutation Research 738–739 (2012) 71–74
Contents lists available at SciVerse ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Short communication
Hyperthermia-induced micronucleus formation in a human keratinocyte cell line Henning Hintzsche, Thorsten Riese, Helga Stopper ∗ Universität Würzburg, Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Würzburg, Germany
a r t i c l e
i n f o
Article history: Received 8 February 2012 Received in revised form 6 August 2012 Accepted 15 August 2012 Available online 4 September 2012 Keywords: Hyperthermia Temperature DNA damage Micronucleus test Hsp70
a b s t r a c t Elevated temperature can cause biological effects in vitro and in vivo. Many studies on effects of hypoand hyperthermia have been conducted, but only few studies systematically investigated the formation of genomic damage in the micronucleus test in human cells in vitro as a consequence of different temperatures. In the present study, HaCaT human keratinocytes were exposed to different temperatures from 37 ◦ C to 42 ◦ C for 24 h in a regular cell culture incubator. Micronucleus frequency as a marker of genomic damage was elevated in a temperature-dependent and statistically significant manner. Apoptosis occurred at temperatures of 39 ◦ C or higher. Cell proliferation was unaffected up to 40 ◦ C and decreased at 41 ◦ C and 42 ◦ C. Expression of the heat shock protein Hsp70 was elevated, particularly at temperatures of 40 ◦ C and higher. These findings are in agreement with several in vivo studies and some in vitro studies looking at single, specific temperatures, but a systematically investigated temperature-dependent increase of genomic damage in human keratinocytes in vitro is demonstrated for the first time here. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Changes in temperature can cause functional effects in biological systems. This phenomenon can be quantified on all levels of biological research, ranging from investigations of subcellular structures to studies of whole populations in epidemiology, as demonstrated recently for the effect of high temperature on cause-specific mortality [1]. Basically all biological structures can be affected by hyper- and hypothermia [2]. Besides experimental hyperthermia treatment in oncology, drug-induced temperature changes can be of clinical relevance [3]. The major difference related to temperature between in vivo and in vitro studies is that many organisms have the ability of maintaining a constant core temperature irrespective of the surrounding temperature [4]. This can be achieved by various mechanisms such as blood circulation, tremor, sweating, etc. Under specific circumstances (such as drug-induce hypo- or hyperthermia, fever) it is not possible to correct external influences completely, underlining why investigating toxicological effects of temperature changes is very important. The main primary targets of hyperthermia in cells are proteins. The impact upon proteins caused by temperature changes can subsequently lead to many different consequences, such as apoptosis,
∗ Corresponding author. Tel.: +49 931 20148427; fax: +49 931 20148446. E-mail address: [email protected] (H. Stopper). 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2012.08.004
necrosis or DNA damage [5]. One important part of the heat shock response of the cells is the elevated expression of heat shock proteins, which are also closely associated with genomic damage [6]. The investigation of DNA damage is of particular importance because DNA damage in germ cells can lead to genetic disorders which are passed onto next generations. In somatic cells, DNA damage is closely associated with cancer formation. One of the assays to quantify DNA damage is the micronucleus test [7]. It was developed 40 years ago and later improved for in vitro-testing by the addition of cytochalasin B to account for cell proliferation changes [8,9]. Today it is widely accepted because international validation studies have demonstrated its reliability [10]. Recently, the OECD guideline for the use of the in vitro micronucleus test in mammalian cells has been approved [11]. The micronucleus test quantifies small chromatin bodies which can contain either chromosome fragments or whole chromosomes which have not been distributed to one of the two main daughter nuclei during mitosis. Thus it can reflect structural and numerical chromosomal damage. There are numerous studies investigating the effect of distinct temperature changes in vitro [5]. This also includes studies on cytogenetic effects caused by hyperthermia [12–15]. An interference with the interpretation of toxicological test data has been discovered [16,17], hindering risk assessment of chemicals which influence body temperature. Despite this fact, surprisingly few studies are available which systematically investigate the effect of various different temperatures on micronucleus formation in vitro. Therefore, the aim of this study was to evaluate the DNA damage as
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H. Hintzsche et al. / Mutation Research 738–739 (2012) 71–74
micronucleus formation including its mechanism of emergence as a consequence of different temperatures on human cells and relate it to the occurrence of apoptosis and the cellular heat shock response, quantified as heat shock protein hsp70 expression. A human skin keratinocyte cell line was chosen, because skin cells are directly affected by elevated temperatures in vivo during extended highintensity sunlight exposure.
2.6. Kinetochore analysis For kinetochore staining, cells were incubated with CREST serum (Antibodies Incorporated) for 16 h at 37 ◦ C. The secondary antibody was a FITC-conjugated goat anti-human antibody diluted 1:20 in PBS. For evaluation, the occurrence of kinetochore signals was quantified microscopically in 200 micronuclei for the 37 ◦ Csamples and in 300 micronuclei for the 42 ◦ C-samples. 2.7. Statistics
2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma–Aldrich (Steinheim, Germany), PAA (Pasching, Austria) or Invitrogen Life Technologies (Darmstadt, Germany). HaCaT cells were purchased from Cell Line Service (Eppelheim, Germany). TBS-T consisted of Tris-buffered saline and 0.05% Tween 20. DABCO solution consisted of 250 mg DABCO dissolved in 10 ml PBS and subsequently mixed with 90 ml glycerol. 2.2. Cell culture HaCaT cells, spontaneously immortalized human keratinocytes, were used for the experiments to examine the influence of different incubation temperatures. They were cultivated as adherent cells in DMEM medium (4.5 g/l glucose) supplemented with 10% fetal calf serum, 2 mM l-glutamine, 50 U/ml penicillin and 50 g/ml streptomycin. Exponentially growing HaCaT cells were exposed for 24 h to defined temperature levels in a regular cell culture incubator (CO2 Incubator 1500, Flow Laboratories, Rockville, USA) in a humidified atmosphere of 5.0 % CO2 . Temperature was adjusted by the incubator control unit. The applied temperatures were 37, 38, 39, 40, 41, and 42 ◦ C. These measurements were validated using a TempLog temperature logger (Driesen + Kern, Bad Bramstedt, Germany). During exposure, cells were kept in -dishes (Ibidi, Germany) for the micronucleus test and in culture flasks for heat shock protein quantification. For the micronucleus test, cells were incubated with cytochalasin B (3 g/ml) during exposure. 2.3. Micronucleus test After exposure, the medium of the -dishes was discarded and the cells were washed with PBS buffer twice. Cells were then fixed in methanol (−20 ◦ C) for at least 1 h. For staining, cells were air-dried and stained with 10 l safe-green solution for 3 min and finally covered with DABCO solution. For evaluation, 1000 cells were counted using fluorescence microscopy with a FITC filter to determine the ratio of mono- (MN), bi- (BN) and multinuclear cells (MuN) and the number of apoptotic and mitotic cells. The cytochalasin B proliferation index (CBPI) was calculated as CBPI =
1 × MN + 2 × BN + 3 × MuN . MN + BN + MuN
Afterwards, 2000 binuclear cells were analyzed to evaluate the number of micronucleated cells. Criteria for micronuclei were: location within the cytoplasm, no overlap with the main nucleus, staining pattern similar to the main nucleus, size 1/16 to 1/3 of the main nucleus. Apoptotic cells were determined morphologically as cells with fragmented nuclei. Evaluation was performed with three independently exposed replicate samples. 2.4. Heat shock protein expression Proteins were extracted after exposure to different temperatures and separated by SDS-PAGE. Proteins were transferred to PVDF membranes using a semi-dry blotting technique followed by blocking of the membrane at room temperature for 2 h with 5% milk powder in TBS-T. For immunostaining the membrane was incubated with the primary antibody (anti-Hsp70, 1:4000, Santa Cruz Biotechnology) under gentle shaking for 16 h at 4 ◦ C. The secondary antibody was a goat anti-mouse HRP-conjugated antibody diluted 1:10,000 in 5% milk powder/TBS-T. Signals were visualized by exposure to an x-ray-sensitive film and quantified densitometrically. Results are shown normalized to -actin. 2.5. Analysis of cleaved caspase-3 Proteins were extracted after exposure to 37 ◦ C or 42 ◦ C and separated by SDSPAGE. Proteins were transferred to PVDF membranes using a semi-dry blotting technique followed by blocking of the membrane at room temperature for 2 h with 5% milk powder in TBS-T. For immunostaining the membrane was incubated with the primary antibody (anti-cleaved caspase-3, 1:1000, Santa Cruz Biotechnology) under gentle shaking for 16 h at 4 ◦ C. The secondary antibody was a goat anti-rabbit HRP-conjugated antibody diluted 1:2000 in 5% milk powder/TBS-T. Signals were visualized by exposure to an X-ray-sensitive film and quantified densitometrically. Results are shown normalized to -actin.
The evaluation regarding micronucleus frequency, apoptosis, proliferation, and kinetochore frequency were performed in triplicates. For this reason the data could not be tested for normal distribution and parametric tests could not be applied. To test significance of differences between exposure to different temperatures and the control (37 ◦ C) the non-parametric Mann–Whitney-U-test was applied. Significance was considered for p < 0.05.
3. Results and discussion The influence of temperature on biological systems has been investigated for a long time [2]. This also includes investigations of genomic damage. However, no studies systematically looked at effects of temperature changes in vitro on DNA damage in human keratinocytes measured as micronucleus frequency. Therefore the micronucleus test was applied in the present study to investigate whether ambient temperatures from 37 ◦ C to 42 ◦ C affect chromosomal damage in HaCaT cells. The frequency of micronuclei increased temperaturedependently up to 41 ◦ C to about four-fold of the value of the control samples (Fig. 1a). Only the highest temperature of 42 ◦ C did not further increase the damage, but showed a micronucleus number of just below the one found for 41 ◦ C. All different temperature exposures led to a statistically significant increase of the micronucleus frequency, demonstrating the ability of hyperthermia to induce genomic damage in vitro. Apoptotic cells were quantified by morphologic analysis as fragmented nuclei. There was no increase in cells exposed to 38 ◦ C, but all other samples showed elevated frequencies of apoptotic cells (Fig. 1b). These increases were statistically significant with the exception of the 40 ◦ C sample, which did show elevated levels compared to the control samples, but also a high variability. Cell proliferation was quantified as the cytochalasin B proliferation index, for which a value of 1.0 represents a scenario where no cell has divided and a value of 2.0 represents a scenario where on average every cell has divided once [18]. Temperatures up to 40 ◦ C did not affect proliferation whereas exposure to 41 ◦ C and 42 ◦ C resulted in a clearly and significantly reduced proliferation (Fig. 1c). Interestingly, the proliferation did not show a temperature-dependent decrease, as was observed for the genomic damage, but rather exhibited a clear threshold between 40 ◦ C and 41 ◦ C. Hsp70 expression was quantified and found to be clearly elevated after exposure to temperatures ≥40 ◦ C (Fig. 2), which is known for many other biological systems [19]. To analyze apoptosis more specifically than with the morphologic analysis, cleaved caspase-3 was quantified in cells exposed to the highest and lowest temperature (Fig. 3). The cells incubated at 42 ◦ C showed a small increase of 1.28-fold compared to 37 ◦ C. This increase was lower than what was found in the morphologic analysis, but confirmed the occurrence of apoptosis. Finally, kinetochore analysis was performed to investigate the mechanism of micronucleus formation. The analysis was performed with separate samples which were incubated at the highest and lowest temperature (i.e. 37 ◦ C and 42 ◦ C). The fold-increase of micronuclei was similar to the numbers reported in Fig. 1. The frequencies of kinetochore-positive and kinetochore-negative micronuclei are shown in Fig. 4. The control sample (37 ◦ C) contained 15% kinetochore-positive micronuclei, whereas the sample incubated at 42 ◦ C contained 41% kinetochore-positive micronuclei. The differences between the numbers of 37 ◦ C and 42 ◦ C are also
H. Hintzsche et al. / Mutation Research 738–739 (2012) 71–74
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(a) Hsp70 expression [a. u.]
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Fig. 3. Expression of cleaved caspase-3 after incubation for 24 h at 37 ◦ C and 42 ◦ C.
1.6
Many of these studies investigated the frequency of micronucleated polychromatic erythrocytes as a consequence of drug-induced hypothermia. Increased micronucleus frequencies were mainly demonstrated for treatment with anti-psychotic drugs, e.g. E-5842 [16,17], haloperidol [20] and chlorpromazine [21]. The decrease in rectal temperature was as high as 10 ◦ C resulting in a clear increase in micronucleus frequency. Also, hypothermia induced by decreased ambient temperature resulted in micronucleus formation [22]. In vivo studies investigating hyperthermic effects
1.4 1.2 1.0
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Fig. 1. (a) Number of micronuclei per 1000 binucleated cells after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values of all elevated temperatures (≥38 ◦ C) are significantly different (p < 0.05) from the control (37 ◦ C). (b) Number of apoptotic cells per 1000 cells after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values are significantly different (p < 0.05) from the control (37 ◦ C) for 39 ◦ C, 41 ◦ C and 42 ◦ C. (c) Cytochalasin B proliferation index (CBPI) after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values are significantly different (p < 0.05) from the control (37 ◦ C) for 41 ◦ C and 42 ◦ C.
shown as “Induced” micronuclei. It was found that the number of induced kinetochore-positive micronuclei was similar to the number of induced kinetochore-negative micronuclei, suggesting both clastogenic and aneugenic effects. The results of the present study are in line with many other investigations on cytotoxic effects and DNA damage [5]. They are also in agreement with studies using the micronucleus test in vitro after exposure to one particular temperature. However, no systematic investigations of micronucleus formation in human keratinocyte cells in vitro caused by different temperatures are available. In contrast to this, a number of in vivo studies investigating the effects of different temperatures have been performed.
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Fig. 2. Expression of heat shock protein Hsp70 after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The figure insert shows a blot.
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Fig. 4. Number of kinetochore-positive and kinetochore-negative micronuclei per 1000 cells after incubation for 24 h at 37 ◦ C and 42 ◦ C. The values of 42 ◦ C are significantly different from the control (37 ◦ C), both for kinetochore-positive and kinetochore-negative. Additionally, the numbers of induced micronuclei (calculated by subtracting the values of 37 ◦ C from the values of 42 ◦ C) are shown.
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clearly showed elevated micronucleus frequencies, e.g. induced by opioid treatment [12] and by change in ambient temperature [13,14,23,24]. The findings of the present study are also in line with several in vitro studies. It was demonstrated in rodent cells, that hyperthermic conditions lead to an increase of chromosome aberrations and micronuclei [15]. Micronucleus frequency was increased fourfold and twenty-fold after exposure to 40 ◦ C and 42 ◦ C respectively (24 h), but no other temperatures were investigated. Another study exposed whole blood samples to 43.5 ◦ C (here only 1 h) and micronucleus frequencies in lymphocytes were found to be increased three-fold [25]. In both human and rodent cells it was demonstrated that effects of hyperthermic treatment does not only depend upon temperature and time, but also on the heating method applied. This was demonstrated for ␥H2AX focus formation [26], but probably also applies for other endpoints including the micronucleus test. Interestingly, a recent study demonstrated, that bystander effects can be observed not only with irradiated but also with hyperthermia-treated cells [27]. It was shown that fibroblasts had elevated micronucleus frequencies when they were co-cultured with cells pre-exposed to elevated temperature, suggesting that cells secrete signaling molecules as a consequence of hyperthermia. The results of the present study suggest that this could be cell-cycle-related or mediated by apoptosis pathways or heat shock proteins. All in all, human cells were exposed to hyperthermia for 24 h and the resulting genomic damage was quantified using the micronucleus test. Several temperatures from 37 ◦ C to 42 ◦ C were systematically investigated. Besides increased Hsp70 expression, induction of micronuclei and apoptotic cells was observed. At higher temperatures, proliferation was decreased. These findings are in line with other reports on investigations about DNA damage under particular temperature conditions. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment Excellent technical assistance by Ingrid Richter and Silvana Wunram is greatly appreciated. References [1] A. Gasparrini, B. Armstrong, S. Kovats, P. Wilkinson, The effect of high temperatures on cause-specific mortality in England and Wales, Occup. Environ. Med. 69 (2012) 56–61. [2] C.J. Gordon, Temperature and Toxicology: An Intergrative, Comparative, and Environmental Approach, 1st ed., CRC Press, 2005. [3] F. Eyer, T. Zilker, Bench-to-bedside review: mechanisms and management of hyperthermia due to toxicity, Crit. Care 11 (2007) 236. [4] C.J. Gordon, P.J. Spencer, J. Hotchkiss, D.B. Miller, P.M. Hinderliter, J. Pauluhn, Thermoregulation and its influence on toxicity assessment, Toxicology 244 (2008) 87–97. [5] J.L. Roti Roti, Cellular responses to hyperthermia (40–46 ◦ C): cell killing and molecular events, Int. J. Hyperthermia 24 (2008) 3–15.
[6] G. Chen, W.D. Bradford, C.W. Seidel, R. Li, Hsp90 stress potentiates rapid cellular adaptation through induction of aneuploidy, Nature (2012). [7] M. Kirsch-Volders, G. Plas, A. Elhajouji, M. Lukamowicz, L. Gonzalez, K. Vande Loock, I. Decordier, The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance, Arch. Toxicol. 85 (2011) 873–899. [8] K. Boller, W. Schmid, Chemical mutagenesis in mammals. The Chinese hamster bone marrow as an in vivo test system. Hematological findings after treatment with trenimon, Humangenetik 11 (1970) 35–54. [9] M. Fenech, A.A. Morley, Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation, Mutat. Res. 161 (1986) 193–198. [10] G. Reifferscheid, C. Ziemann, D. Fieblinger, F. Dill, R. Gminski, H.J. Grummt, C. Hafner, H. Hollert, S. Kunz, G. Rodrigo, H. Stopper, D. Selke, Measurement of genotoxicity in wastewater samples with the in vitro micronucleus test: results of a round-robin study in the context of standardisation according to ISO, Mutat. Res. 649 (2008) 15–27. [11] OECD, Guideline For The Testing of Chemicals: 487 In Vitro Mammalian Cell Micronucleus Test, in: OECD (Ed.) 487, 2010. [12] D.L. Shuey, R. Gudi, L. Krsmanovic, R.J. Gerson, Evidence that oxymorphoneinduced increases in micronuclei occur secondary to hyperthermia, Toxicol. Sci. 95 (2007) 369–375. [13] S. Masunaga, K. Nagata, M. Suzuki, G. Kashino, Y. Kinashi, K. Ono, Inhibition of repair of radiation-induced damage by mild temperature hyperthermia, referring to the effect on quiescent cell populations, Radiat. Med. 25 (2007) 417–425. [14] S. Asanami, K. Shimono, The effect of hyperthermia on micronucleus induction by mutagens in mice, Mutat. Res. 446 (1999) 149–154. [15] S. Asanami, K. Shimono, S. Kaneda, Effect of temperature on the frequency of chromosome aberrations and micronuclei in cultured Chinese hamster cells, J. Toxicol. Sci. 26 (2001) 323–326. [16] A. Guzman, C. Garcia, A.P. Marin, M.T. Ruiz, A. Tortajada, A.R. Fernandez de Henestrosa, Induction of micronuclei in mouse bone-marrow erythrocytes in association with hypothermia after administration of the sigma receptor ligand E-5842, Mutat. Res. 565 (2004) 11–22. [17] A. Guzman, A.P. Marin, C. Garcia, A.R. Fernandez de Henestrosa, M.T. Ruiz, A. Tortajada, R. Marcos, Induction of hypothermic conditions associated with increased micronuclei formation in sigma-1 receptor knockout mice after administration of the antipsychotic compound E-5842, Environ. Mol. Mutagen. 49 (2008) 727–733. [18] J. Surralles, N. Xamena, A. Creus, J. Catalan, H. Norppa, R. Marcos, Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures, Mutat. Res. 341 (1995) 169–184. [19] S. Khoei, G.R. Fazeli, A. Amerizadeh, D. Eslimi, B. Goliaei, Elimination of enhanced thermal resistance of spheroid culture model of prostate carcinoma cell line by inhibitors of Hsp70 induction, Yakhteh 12 (2010) 105–112. [20] S. Asanami, K. Shimono, Species-level differences between mice and rats in regards to micronucleus induction with the hypothermia-inducing drug haloperidol, Mutat. Res. 676 (2009) 102–105. [21] A. Guzman, C. Garcia, A.P. Marin, A. Tortajada, M.T. Ruiz, A.R. Fernandez de Henestrosa, R. Marcos, Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis, Mutat. Res. 656 (2008) 8–13. [22] S. Asanami, K. Shimono, Effects of chemically- and environmentally-induced hypothermia on micronucleus induction in rats, Mutat. Res. 471 (2000) 81–86. [23] K.S. Bisht, P.U. Uma Devi, Modification of radiation-induced chromosome damage and micronucleus induction in mouse bone marrow by misonidazole and hyperthermia, Acta Oncol. 34 (1995) 913–918. [24] E.K. Rofstad, K.H. Falkvoll, P. Oftedal, Micronucleus formation in human melanoma xenografts following exposure to hyperthermia, Radiat. Environ. Biophys. 23 (1984) 51–60. [25] T.K. Lee, K. O’Brien, K. Christie, A.L. Wiley, U.L. Karlsson Jr., Effect of ex vivo hyperthermia on radiation-induced micronuclei in lymphocytes of cancer patients before and during radiotherapy, Mutat. Res. 417 (1998) 1–8. [26] W. Wu, C. Zhang, Z. Chen, G. Zhang, J. Yang, Differences in heating methods may account for variation in reported effects on gammaH2AX focus formation, Mutat. Res. 676 (2009) 48–53. [27] M. Purschke, H.J. Laubach, R.R. Anderson, D. Manstein, Thermal injury causes DNA damage and lethality in unheated surrounding cells: active thermal bystander effect, J. Invest. Dermatol. 130 (2010) 86–92.
Contents lists available at SciVerse ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Short communication
Hyperthermia-induced micronucleus formation in a human keratinocyte cell line Henning Hintzsche, Thorsten Riese, Helga Stopper ∗ Universität Würzburg, Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Würzburg, Germany
a r t i c l e
i n f o
Article history: Received 8 February 2012 Received in revised form 6 August 2012 Accepted 15 August 2012 Available online 4 September 2012 Keywords: Hyperthermia Temperature DNA damage Micronucleus test Hsp70
a b s t r a c t Elevated temperature can cause biological effects in vitro and in vivo. Many studies on effects of hypoand hyperthermia have been conducted, but only few studies systematically investigated the formation of genomic damage in the micronucleus test in human cells in vitro as a consequence of different temperatures. In the present study, HaCaT human keratinocytes were exposed to different temperatures from 37 ◦ C to 42 ◦ C for 24 h in a regular cell culture incubator. Micronucleus frequency as a marker of genomic damage was elevated in a temperature-dependent and statistically significant manner. Apoptosis occurred at temperatures of 39 ◦ C or higher. Cell proliferation was unaffected up to 40 ◦ C and decreased at 41 ◦ C and 42 ◦ C. Expression of the heat shock protein Hsp70 was elevated, particularly at temperatures of 40 ◦ C and higher. These findings are in agreement with several in vivo studies and some in vitro studies looking at single, specific temperatures, but a systematically investigated temperature-dependent increase of genomic damage in human keratinocytes in vitro is demonstrated for the first time here. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Changes in temperature can cause functional effects in biological systems. This phenomenon can be quantified on all levels of biological research, ranging from investigations of subcellular structures to studies of whole populations in epidemiology, as demonstrated recently for the effect of high temperature on cause-specific mortality [1]. Basically all biological structures can be affected by hyper- and hypothermia [2]. Besides experimental hyperthermia treatment in oncology, drug-induced temperature changes can be of clinical relevance [3]. The major difference related to temperature between in vivo and in vitro studies is that many organisms have the ability of maintaining a constant core temperature irrespective of the surrounding temperature [4]. This can be achieved by various mechanisms such as blood circulation, tremor, sweating, etc. Under specific circumstances (such as drug-induce hypo- or hyperthermia, fever) it is not possible to correct external influences completely, underlining why investigating toxicological effects of temperature changes is very important. The main primary targets of hyperthermia in cells are proteins. The impact upon proteins caused by temperature changes can subsequently lead to many different consequences, such as apoptosis,
∗ Corresponding author. Tel.: +49 931 20148427; fax: +49 931 20148446. E-mail address: [email protected] (H. Stopper). 0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrfmmm.2012.08.004
necrosis or DNA damage [5]. One important part of the heat shock response of the cells is the elevated expression of heat shock proteins, which are also closely associated with genomic damage [6]. The investigation of DNA damage is of particular importance because DNA damage in germ cells can lead to genetic disorders which are passed onto next generations. In somatic cells, DNA damage is closely associated with cancer formation. One of the assays to quantify DNA damage is the micronucleus test [7]. It was developed 40 years ago and later improved for in vitro-testing by the addition of cytochalasin B to account for cell proliferation changes [8,9]. Today it is widely accepted because international validation studies have demonstrated its reliability [10]. Recently, the OECD guideline for the use of the in vitro micronucleus test in mammalian cells has been approved [11]. The micronucleus test quantifies small chromatin bodies which can contain either chromosome fragments or whole chromosomes which have not been distributed to one of the two main daughter nuclei during mitosis. Thus it can reflect structural and numerical chromosomal damage. There are numerous studies investigating the effect of distinct temperature changes in vitro [5]. This also includes studies on cytogenetic effects caused by hyperthermia [12–15]. An interference with the interpretation of toxicological test data has been discovered [16,17], hindering risk assessment of chemicals which influence body temperature. Despite this fact, surprisingly few studies are available which systematically investigate the effect of various different temperatures on micronucleus formation in vitro. Therefore, the aim of this study was to evaluate the DNA damage as
72
H. Hintzsche et al. / Mutation Research 738–739 (2012) 71–74
micronucleus formation including its mechanism of emergence as a consequence of different temperatures on human cells and relate it to the occurrence of apoptosis and the cellular heat shock response, quantified as heat shock protein hsp70 expression. A human skin keratinocyte cell line was chosen, because skin cells are directly affected by elevated temperatures in vivo during extended highintensity sunlight exposure.
2.6. Kinetochore analysis For kinetochore staining, cells were incubated with CREST serum (Antibodies Incorporated) for 16 h at 37 ◦ C. The secondary antibody was a FITC-conjugated goat anti-human antibody diluted 1:20 in PBS. For evaluation, the occurrence of kinetochore signals was quantified microscopically in 200 micronuclei for the 37 ◦ Csamples and in 300 micronuclei for the 42 ◦ C-samples. 2.7. Statistics
2. Materials and methods 2.1. Materials All chemicals were purchased from Sigma–Aldrich (Steinheim, Germany), PAA (Pasching, Austria) or Invitrogen Life Technologies (Darmstadt, Germany). HaCaT cells were purchased from Cell Line Service (Eppelheim, Germany). TBS-T consisted of Tris-buffered saline and 0.05% Tween 20. DABCO solution consisted of 250 mg DABCO dissolved in 10 ml PBS and subsequently mixed with 90 ml glycerol. 2.2. Cell culture HaCaT cells, spontaneously immortalized human keratinocytes, were used for the experiments to examine the influence of different incubation temperatures. They were cultivated as adherent cells in DMEM medium (4.5 g/l glucose) supplemented with 10% fetal calf serum, 2 mM l-glutamine, 50 U/ml penicillin and 50 g/ml streptomycin. Exponentially growing HaCaT cells were exposed for 24 h to defined temperature levels in a regular cell culture incubator (CO2 Incubator 1500, Flow Laboratories, Rockville, USA) in a humidified atmosphere of 5.0 % CO2 . Temperature was adjusted by the incubator control unit. The applied temperatures were 37, 38, 39, 40, 41, and 42 ◦ C. These measurements were validated using a TempLog temperature logger (Driesen + Kern, Bad Bramstedt, Germany). During exposure, cells were kept in -dishes (Ibidi, Germany) for the micronucleus test and in culture flasks for heat shock protein quantification. For the micronucleus test, cells were incubated with cytochalasin B (3 g/ml) during exposure. 2.3. Micronucleus test After exposure, the medium of the -dishes was discarded and the cells were washed with PBS buffer twice. Cells were then fixed in methanol (−20 ◦ C) for at least 1 h. For staining, cells were air-dried and stained with 10 l safe-green solution for 3 min and finally covered with DABCO solution. For evaluation, 1000 cells were counted using fluorescence microscopy with a FITC filter to determine the ratio of mono- (MN), bi- (BN) and multinuclear cells (MuN) and the number of apoptotic and mitotic cells. The cytochalasin B proliferation index (CBPI) was calculated as CBPI =
1 × MN + 2 × BN + 3 × MuN . MN + BN + MuN
Afterwards, 2000 binuclear cells were analyzed to evaluate the number of micronucleated cells. Criteria for micronuclei were: location within the cytoplasm, no overlap with the main nucleus, staining pattern similar to the main nucleus, size 1/16 to 1/3 of the main nucleus. Apoptotic cells were determined morphologically as cells with fragmented nuclei. Evaluation was performed with three independently exposed replicate samples. 2.4. Heat shock protein expression Proteins were extracted after exposure to different temperatures and separated by SDS-PAGE. Proteins were transferred to PVDF membranes using a semi-dry blotting technique followed by blocking of the membrane at room temperature for 2 h with 5% milk powder in TBS-T. For immunostaining the membrane was incubated with the primary antibody (anti-Hsp70, 1:4000, Santa Cruz Biotechnology) under gentle shaking for 16 h at 4 ◦ C. The secondary antibody was a goat anti-mouse HRP-conjugated antibody diluted 1:10,000 in 5% milk powder/TBS-T. Signals were visualized by exposure to an x-ray-sensitive film and quantified densitometrically. Results are shown normalized to -actin. 2.5. Analysis of cleaved caspase-3 Proteins were extracted after exposure to 37 ◦ C or 42 ◦ C and separated by SDSPAGE. Proteins were transferred to PVDF membranes using a semi-dry blotting technique followed by blocking of the membrane at room temperature for 2 h with 5% milk powder in TBS-T. For immunostaining the membrane was incubated with the primary antibody (anti-cleaved caspase-3, 1:1000, Santa Cruz Biotechnology) under gentle shaking for 16 h at 4 ◦ C. The secondary antibody was a goat anti-rabbit HRP-conjugated antibody diluted 1:2000 in 5% milk powder/TBS-T. Signals were visualized by exposure to an X-ray-sensitive film and quantified densitometrically. Results are shown normalized to -actin.
The evaluation regarding micronucleus frequency, apoptosis, proliferation, and kinetochore frequency were performed in triplicates. For this reason the data could not be tested for normal distribution and parametric tests could not be applied. To test significance of differences between exposure to different temperatures and the control (37 ◦ C) the non-parametric Mann–Whitney-U-test was applied. Significance was considered for p < 0.05.
3. Results and discussion The influence of temperature on biological systems has been investigated for a long time [2]. This also includes investigations of genomic damage. However, no studies systematically looked at effects of temperature changes in vitro on DNA damage in human keratinocytes measured as micronucleus frequency. Therefore the micronucleus test was applied in the present study to investigate whether ambient temperatures from 37 ◦ C to 42 ◦ C affect chromosomal damage in HaCaT cells. The frequency of micronuclei increased temperaturedependently up to 41 ◦ C to about four-fold of the value of the control samples (Fig. 1a). Only the highest temperature of 42 ◦ C did not further increase the damage, but showed a micronucleus number of just below the one found for 41 ◦ C. All different temperature exposures led to a statistically significant increase of the micronucleus frequency, demonstrating the ability of hyperthermia to induce genomic damage in vitro. Apoptotic cells were quantified by morphologic analysis as fragmented nuclei. There was no increase in cells exposed to 38 ◦ C, but all other samples showed elevated frequencies of apoptotic cells (Fig. 1b). These increases were statistically significant with the exception of the 40 ◦ C sample, which did show elevated levels compared to the control samples, but also a high variability. Cell proliferation was quantified as the cytochalasin B proliferation index, for which a value of 1.0 represents a scenario where no cell has divided and a value of 2.0 represents a scenario where on average every cell has divided once [18]. Temperatures up to 40 ◦ C did not affect proliferation whereas exposure to 41 ◦ C and 42 ◦ C resulted in a clearly and significantly reduced proliferation (Fig. 1c). Interestingly, the proliferation did not show a temperature-dependent decrease, as was observed for the genomic damage, but rather exhibited a clear threshold between 40 ◦ C and 41 ◦ C. Hsp70 expression was quantified and found to be clearly elevated after exposure to temperatures ≥40 ◦ C (Fig. 2), which is known for many other biological systems [19]. To analyze apoptosis more specifically than with the morphologic analysis, cleaved caspase-3 was quantified in cells exposed to the highest and lowest temperature (Fig. 3). The cells incubated at 42 ◦ C showed a small increase of 1.28-fold compared to 37 ◦ C. This increase was lower than what was found in the morphologic analysis, but confirmed the occurrence of apoptosis. Finally, kinetochore analysis was performed to investigate the mechanism of micronucleus formation. The analysis was performed with separate samples which were incubated at the highest and lowest temperature (i.e. 37 ◦ C and 42 ◦ C). The fold-increase of micronuclei was similar to the numbers reported in Fig. 1. The frequencies of kinetochore-positive and kinetochore-negative micronuclei are shown in Fig. 4. The control sample (37 ◦ C) contained 15% kinetochore-positive micronuclei, whereas the sample incubated at 42 ◦ C contained 41% kinetochore-positive micronuclei. The differences between the numbers of 37 ◦ C and 42 ◦ C are also
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Many of these studies investigated the frequency of micronucleated polychromatic erythrocytes as a consequence of drug-induced hypothermia. Increased micronucleus frequencies were mainly demonstrated for treatment with anti-psychotic drugs, e.g. E-5842 [16,17], haloperidol [20] and chlorpromazine [21]. The decrease in rectal temperature was as high as 10 ◦ C resulting in a clear increase in micronucleus frequency. Also, hypothermia induced by decreased ambient temperature resulted in micronucleus formation [22]. In vivo studies investigating hyperthermic effects
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Fig. 1. (a) Number of micronuclei per 1000 binucleated cells after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values of all elevated temperatures (≥38 ◦ C) are significantly different (p < 0.05) from the control (37 ◦ C). (b) Number of apoptotic cells per 1000 cells after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values are significantly different (p < 0.05) from the control (37 ◦ C) for 39 ◦ C, 41 ◦ C and 42 ◦ C. (c) Cytochalasin B proliferation index (CBPI) after incubation for 24 h at different temperatures ranging from 37 ◦ C to 42 ◦ C. The values are significantly different (p < 0.05) from the control (37 ◦ C) for 41 ◦ C and 42 ◦ C.
shown as “Induced” micronuclei. It was found that the number of induced kinetochore-positive micronuclei was similar to the number of induced kinetochore-negative micronuclei, suggesting both clastogenic and aneugenic effects. The results of the present study are in line with many other investigations on cytotoxic effects and DNA damage [5]. They are also in agreement with studies using the micronucleus test in vitro after exposure to one particular temperature. However, no systematic investigations of micronucleus formation in human keratinocyte cells in vitro caused by different temperatures are available. In contrast to this, a number of in vivo studies investigating the effects of different temperatures have been performed.
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clearly showed elevated micronucleus frequencies, e.g. induced by opioid treatment [12] and by change in ambient temperature [13,14,23,24]. The findings of the present study are also in line with several in vitro studies. It was demonstrated in rodent cells, that hyperthermic conditions lead to an increase of chromosome aberrations and micronuclei [15]. Micronucleus frequency was increased fourfold and twenty-fold after exposure to 40 ◦ C and 42 ◦ C respectively (24 h), but no other temperatures were investigated. Another study exposed whole blood samples to 43.5 ◦ C (here only 1 h) and micronucleus frequencies in lymphocytes were found to be increased three-fold [25]. In both human and rodent cells it was demonstrated that effects of hyperthermic treatment does not only depend upon temperature and time, but also on the heating method applied. This was demonstrated for ␥H2AX focus formation [26], but probably also applies for other endpoints including the micronucleus test. Interestingly, a recent study demonstrated, that bystander effects can be observed not only with irradiated but also with hyperthermia-treated cells [27]. It was shown that fibroblasts had elevated micronucleus frequencies when they were co-cultured with cells pre-exposed to elevated temperature, suggesting that cells secrete signaling molecules as a consequence of hyperthermia. The results of the present study suggest that this could be cell-cycle-related or mediated by apoptosis pathways or heat shock proteins. All in all, human cells were exposed to hyperthermia for 24 h and the resulting genomic damage was quantified using the micronucleus test. Several temperatures from 37 ◦ C to 42 ◦ C were systematically investigated. Besides increased Hsp70 expression, induction of micronuclei and apoptotic cells was observed. At higher temperatures, proliferation was decreased. These findings are in line with other reports on investigations about DNA damage under particular temperature conditions. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment Excellent technical assistance by Ingrid Richter and Silvana Wunram is greatly appreciated. References [1] A. Gasparrini, B. Armstrong, S. Kovats, P. Wilkinson, The effect of high temperatures on cause-specific mortality in England and Wales, Occup. Environ. Med. 69 (2012) 56–61. [2] C.J. Gordon, Temperature and Toxicology: An Intergrative, Comparative, and Environmental Approach, 1st ed., CRC Press, 2005. [3] F. Eyer, T. Zilker, Bench-to-bedside review: mechanisms and management of hyperthermia due to toxicity, Crit. Care 11 (2007) 236. [4] C.J. Gordon, P.J. Spencer, J. Hotchkiss, D.B. Miller, P.M. Hinderliter, J. Pauluhn, Thermoregulation and its influence on toxicity assessment, Toxicology 244 (2008) 87–97. [5] J.L. Roti Roti, Cellular responses to hyperthermia (40–46 ◦ C): cell killing and molecular events, Int. J. Hyperthermia 24 (2008) 3–15.
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