- Email: [email protected]
Analysis of adaptive response to bleomycin and mitomycin C
Mutation Research 513 (2002) 75–81
Analysis of adaptive response to bleomycin and mitomycin C Kamila Schlade-Bartusiak, Agnieszka Stembalska-Kozlowska, Monika Bernady, Marta Kudyba, Maria Sasiadek∗ Department of Genetics, Wroclaw Medical University, Marcinkowskiego 1, 50-368 Wroclaw, Poland Received 18 April 2001; received in revised form 15 August 2001; accepted 16 August 2001
Abstract Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems. The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs). The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Adaptive response; Bleomycin; Mitomycin C; DNA-damage repair
1. Introduction The process of multistep carcinogenesis consists of the accumulation of alterations mainly in protooncogenes, tumour suppressor and mutator genes. A variety of genetic rearrangements in cancer cells ∗ Corresponding author. Tel.: +48-71-784-12-56; fax: +48-71-784-00-63. E-mail address: [email protected] (M. Sasiadek).
are observed both on the molecular and chromosomal level [1]. The primary genetic changes are inherited in 5–10% of cancers, but most of mutations are acquired, e.g. induced by mutagens. As all living organisms are permanently exposed to endogenous and exogenous (environmental) mutagens, they have developed a variety of strategies to protect the integrity of the genome [2]. Therefore, the accumulation of genetic alterations has to result from the imbalance between the induction of DNA-damage and its repair. Genetic
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 2 8 8 - 1
76
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
instability resulting from the disturbances in various mechanisms of DNA-repair is one of the main characteristics of cancer cells. However, there is still an unanswered question how to evaluate the individual repair capacity and its relation to the cancer risk. One group of assays of studying DNA-repair capacity, the so called “mutagen sensitivity assays” is based on the analysis of induced DNA-damage [3]. The most widely used assay is the bleomycin (BLM) test, developed by Hsu et al. [4]. In this test the frequency of BLM-induced chromosome aberrations (CA) provides a measure of chromosome instability. BLM test has been applied to study this phenomenon in cancer patients as well as in various groups of increased cancer risk [5–12]. Chromosome instability has been observed not only in the lymphocytes of patients suffering from both sporadic, e.g. mutagen-induced, and inherited cancers [5–9], but also in those patients’ healthy relatives [10,11], and in Down syndrome children with malignancies [12]. The in vitro susceptibility to BLM has been accepted as a predictor of cancer risk. However, it can be used only for a group not for an individual prognosis. Another possibility to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. The AR has been studied mainly in relation to ionising radiation and radiomimetic chemicals such as BLM [13–15]. Cross-resistance has been demonstrated not only for ionising radiation and radiomimetic agents but also for ionising radiation and an alkylating agent, N-methyl-N-nitro-N -nitrosoguanidine (MNNG) [16,17]. Only few studies concerning AR induced by chemical mutagens have been published so far and the data are not as abundant as those on the radio-AR [18,19]. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems [15]. However, the existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. We present the study on the AR induced by two chemical mutagens, which differ in their action on DNA. BLM
is a clastogenic (inducing CA), radiomimetic agent which complexes with the ferrous ion and molecular oxygen, and then, induces in DNA both single-strand breaks (SSB) and double-strand breaks (DSB) [4]. Mitomycin C (MMC) is a potent bifunctional mutagen acting as an alkylating agent, causing mainly DNA interstrand cross-links, and thus, inducing sister chromatid exchanges (SCEs) [20]. We compared the ARs for the two tested agents in terms of the protective effects. The protective effect, which is expressed as the percentages of the total number of chromosomal alterations [9], provides a measure of the AR, which is independent of the applied cytogenetic endpoint.
2. Materials and methods The experiments were performed on human whole blood lymphocytes (WBL) obtained by venipuncture from 20 volunteers (students of the Wroclaw Medical University). The group consisted of 14 women and 6 men. The mean age of the group was 25.7 ± 3.1 years. All donors were healthy non-smokers and had not been exposed to any known mutagens for 3 months prior to the cytogenetic examination. The lymphocyte cultures were set up in air-tight glass bottles, following the standard protocol. Whole blood (0.5 ml) was added to the growth medium consisting of 4.5 ml of McCoy’s medium with glutamine (Gibco) supplemented with foetal calf serum (10%, Gibco), penicillin (100 UI/ml) and streptomycin (100 g/ml, Gibco), phytohaemagglutynin (PHA; 1%, Wellcome) and 5-bromo-2-deoxyuridine (BrdU; 5 g/ml, Calbiochem). Colcemid (Gibco) in a final concentration 0.02 g/ml was added 2 h prior to the harvesting. The cultures were incubated at 37◦ C for 72 h. The experiment consisted of two simultaneously performed series. In the first one, the lymphocytes were exposed to an adaptive dose (AD) of MMC (0.001 g/ml, Sigma) after 24 h of incubation, and subsequently to a challenging dose (CD) of the tested agent (0.1 g/ml of MMC) after next 24 h. Two other lymphocyte cultures were exposed exclusively to a CD of MMC (0.1 g/ml) after 48 h of incubation. In the second series, an AD of BLM (0.3 M, Nippon Kayaku Co.) was added to two lymphocyte
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
77
78
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
cultures 29 h prior to harvesting, and then, a CD (30 M of BLM) was added after the next 24 h, i.e. 5 h prior to harvesting. Two other cultures were exposed to CD of BLM (30 M) alone, 5 h prior to harvesting. For all concentrations used, tested chemicals (MMC and BLM) were dissolved each in 10 l of ethanol and PBS, respectively. The solvents were also added (10 l/tube) to the control cultures: ethanol after 24 h of incubation for MMC treatment and PBS 29 h prior to harvesting for BLM-treatment. All concentrations of tested chemicals were chosen experimentally (data not shown). Duplicate cultures were used for all treatments. The SCE analysis was performed on slides stained according to the modified fluorescence and Giemsa technique [21]. SCEs were scored in 60 second-division cells per donor and treatment. For the estimation of replication index (RI) the frequencies of the first (M1), second (M2), and third (M3) division metaphases were evaluated from 100 cells per donor/treatment [RI = (M1 + 2M2 + 3M3)/100]. CA were scored in 50 first-division metaphases. Following Hsu et al., chromatid gaps were omitted in scoring and more than 12 chromatid breaks per metaphase were scored as 12. The mean number of chromatid breaks per cell (b/c) and percentage of aberrant metaphases (am%) was used as a measure of mutagen sensitivity [4]. All analyses were performed on coded slides. For statistical analysis Student t–test for pairs was applied.
3. Results In the present study two different cytogenetic endpoints were applied for the assessment of AR induced by the tested chemicals. CA were used for the analysis of BLM-induced AR, and SCE for MMC-induced AR. The results are presented in Tables 1 and 2. There was a statistically significant increase (P < 0.001) in the induction of CA and SCE in cultures treated with BLM or MMC, respectively as compared to the controls. Only the RI value after MMC treatment was not statistically different from the control. The presence of cytogenetic AR was indicated by the statistically significant decrease in the frequency of CAs
79
or SCE after (AD + CD)-treatment in comparison to the CD-treatment. A significant (P < 0.001) cytogenetic AR was observed for both the BLM (am% and b/c) and MMC-treatment (SCE). Therefore, we calculated the protective effect, which provides an objective measure of AR, despite the applied cytogenetic endpoint. For BLM-treatment the protective effect of an AD was almost 50% regarding both of the analysed values. The AR to MMC was not as distinct as to BLM. In the cultures pre-treated with an AD of MMC, the SCE frequency decreased only about 20% in comparison to the cultures treated with a CD alone. The protective effects induced by the tested chemicals were individually variable. In the BLM experiment, the mean value (±S.D.) of the AR was observed for 14 out of 20 donors (70%), while 3 of them showed the AR above and 3 below the mean value. The interindividual differences in the AR have also been observed in MMC experiment, with two donors (10%) showing the AR above and four (20%) below the mean value.
4. Discussion In the present paper we demonstrate the results of cytogenetic analysis of AR induced by two chemical mutagens — BLM and MMC, which differ in their action on DNA. Both tested chemicals are routinely applied for the detection of hidden chromosome instability in human lymphocytes: BLM for the chromosome instability in the lymphocytes of cancer patients and their relatives, and MMC for the increased sensitivity to DNA cross-linking agents in Fanconi anaemia patients [6,7,22]. High individual sensitivity to DNA-damage induced by the tested agents is thought to reflect defective DNA-repair. In the present experiment, the lymphocytes of young healthy donors, without any signs of DNA-repair defects, were exposed to ADs and then to subsequent CDs of BLM and MMC. The protective effect induced by low doses of tested chemicals was analysed by using cytogenetic endpoints as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. In the BLM experiment, the pre-treatment with AD caused almost 50% decrease in CAs frequency
80
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
induced by CD, while in the MMC experiment the protective effect was below 20%. This result may be a consequence of differences in the chemical structure of DNA lesions induced by tested agents, and subsequently, differences in induction of various DNA-repair pathways. In general, the BER pathway relatively easily removes lesions located on one strand such as SSB and the lost material is resynthesised using the undamaged strand as a template [23]. Damages that affect both strands, such as DSBs or interstrand cross-links, are more difficult to repair. It has been demonstrated that the combined action of NER and homologous recombination pathways, as well as non-homologues end-joint recombination are required for the correction of this type of DNA-damage [2,23]. There has also been observed the variability in AR among the young, healthy blood donors, with some of them showing AR higher and lower than average. The observed differences in the ARs induced by BLM and MMC may result from the different repair pathways involved in the processing of BLM and MMC-induced DNA lesions. The individual variability in the AR may reflect the difference in the individual DNA-repair capacity.
Acknowledgements The authors would like to thank Prof. Andrzej Elzanowski for his help in the preparation of the manuscript. References [1] F. Mittelman, Recurrent chromosome aberrations in cancer, Mutat. Res. 462 (2000) 247–253. [2] E. Moustacchi, DNA-damage and repair: consequences on dose–responses, Mutat. Res. 464 (2000) 35–40. [3] M. Berwick, P. Vineis, Markers of DNA-repair and susceptibility to cancer in humans: an epidemiologic review, J. Natl. Cancer Inst. 92 (2000) 874–897. [4] T.C. Hsu, D.A. Johnston, L.M. Cherry, D. Ramkisson, S.P. Schantz, J.M. Jessup, R.J. Winn, L. Shirley, C. Furlong, Sensitivity to genotoxic effects of bleomycin in humans: possible relationship to environmental carcinogenesis, Int. J. Cancer 43 (1989) 403–409. [5] T. Krecicki, K. Schlade, N. Blin, M. Sasiadek, Chromosome instability in head and neck cancer patients, Oncol. Rep. 4 (1997) 1383–1385.
[6] J. Cloos, B.J.M. Braakhuis, I. Steen, M.P. Copper, N. de Vries, J.J.P. Nauta, G.B. Snow, Increased mutagen sensitivity in head and neck squamous-cell carcinoma patients, particularly those with multiple primary tumours, Int. J. Cancer 56 (1994) 816–819. [7] J. Cloos, I. Steen, H. Joenje, J.-Y. Ko, N. de Vries, M.L.T. van der Sterre, J.J.P. Nauta, G.B. Snow, B.J.M. Braakhuis, Association between bleomycin genotoxicity and non-constitutional risk factors for head and neck cancer, Cancer Lett. 74 (1993) 161–165. [8] M. Tzancheva, D. Komitowski, Latent chromosomal instability in cancer patients, Hum. Genet. 99 (1997) 47–51. [9] Z. Trizna, T.C. Hsu, S.P. Schantz, Protective effects of vitamin E against bleomycin-induced genotoxicity in head and neck cancer patients in vitro, Anticancer Res. 12 (1992) 325–328. [10] S.K. Roy, A.H. Trivedi, S.R. Bakshi, S.J. Patel, P.H. Shukla, J.M. Bhatavdekar, D.D. Patel, P.M. Shah, Bleomycin-induced chromosome damage in lymphocytes indicates inefficient DNA-repair capacity in breast cancer families, J. Exp. Clin. Cancer Res. 19 (2000) 169–173. [11] T. Debniak, G. Kurzawski, B. Gorski, J. Kladny, W. Domagala, J. Lubinski, Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer, Eur. J. Cancer 36 (2000) 49–54. [12] R. Ankathil, P. Kusumakumary, M.K. Nair, Increased levels of mutagen-induced chromosome breakage in Down syndrome children with malignancy, Cancer Genet. Cytogenet. 99 (1997) 126–128. [13] J.D. Shadley, G. Dai, Cytogenetic and survival adaptive response in G1 phase human lymphocytes, Mutat. Res. 265 (1992) 273–281. [14] R.E.J. Mitchell, Mechanisms of the adaptive response in irradiated mammalian cells, Radiat. Res. 141 (1995) 117–118. [15] O. Rigaud, E. Moustacchi, Radio adaptation for gene mutation and the possible molecular mechanisms of the adaptive response, Mutat. Res. 358 (1996) 127–134. [16] Vijayalaxmi, W. Burkart, Resistance and cross-resistance to chromosome damage in human lymphocytes adapted to bleomycin, Mutat. Res. 211 (1989) 1–5. [17] S. Wolff, G. Olivieri, V. Afzal, Adaptation of human lymphocytes to radiation or chemical mutagens: differences in cytogenetic repair, in: G. Obe, A.T. Natarajan (Eds.), Chromosomal Aberrations, Basic and Applied Aspects, Springer, Berlin, 1990, pp. 140–150. [18] T. Nikolova, E. Huettner, Adaptive and synergistic effects of a low-dose ENU pre-treatment on the frequency of chromosomal aberrations induced by a challenge dose of ENU in human peripheral blood lymphocytes in vitro, Mutat. Res. 357 (1996) 131–141. [19] M. Sasiadek, M. Paprocka-Borowicz, Correlation between the adaptive response and individual sensitivity to monoepoxybutene in in vitro experiments on human lymphocytes, Mutat. Res. 390 (1997) 239–243.
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81 [20] B.J. Sanderson, A.J. Shield, Mutagenic damage to mammalian cells by therapeutic alkylating agents, Mutat. Res. 355 (1996) 41–57. [21] K.J. Husgafvel-Pursiainen, J. Maki-Paakkanen, H. Norppa, M. Sorsa, Smoking and sister chromatid exchange, Hereditas 92 (1980) 247–250.
81
[22] M. Buchwald, E. Moustacchi, Is Fanconi anaemia caused by a defect in the processing of DNA-damage? Mutat. Res. 408 (1998) 75–90. [23] Braithwaite, X. Wu, Z. Wang, Repair of DNA lesions: mechanisms and relative repair efficiencies, Mutat. Res. 424 (1999) 207–219.
Analysis of adaptive response to bleomycin and mitomycin C Kamila Schlade-Bartusiak, Agnieszka Stembalska-Kozlowska, Monika Bernady, Marta Kudyba, Maria Sasiadek∗ Department of Genetics, Wroclaw Medical University, Marcinkowskiego 1, 50-368 Wroclaw, Poland Received 18 April 2001; received in revised form 15 August 2001; accepted 16 August 2001
Abstract Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems. The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs). The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Adaptive response; Bleomycin; Mitomycin C; DNA-damage repair
1. Introduction The process of multistep carcinogenesis consists of the accumulation of alterations mainly in protooncogenes, tumour suppressor and mutator genes. A variety of genetic rearrangements in cancer cells ∗ Corresponding author. Tel.: +48-71-784-12-56; fax: +48-71-784-00-63. E-mail address: [email protected] (M. Sasiadek).
are observed both on the molecular and chromosomal level [1]. The primary genetic changes are inherited in 5–10% of cancers, but most of mutations are acquired, e.g. induced by mutagens. As all living organisms are permanently exposed to endogenous and exogenous (environmental) mutagens, they have developed a variety of strategies to protect the integrity of the genome [2]. Therefore, the accumulation of genetic alterations has to result from the imbalance between the induction of DNA-damage and its repair. Genetic
1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 ( 0 1 ) 0 0 2 8 8 - 1
76
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
instability resulting from the disturbances in various mechanisms of DNA-repair is one of the main characteristics of cancer cells. However, there is still an unanswered question how to evaluate the individual repair capacity and its relation to the cancer risk. One group of assays of studying DNA-repair capacity, the so called “mutagen sensitivity assays” is based on the analysis of induced DNA-damage [3]. The most widely used assay is the bleomycin (BLM) test, developed by Hsu et al. [4]. In this test the frequency of BLM-induced chromosome aberrations (CA) provides a measure of chromosome instability. BLM test has been applied to study this phenomenon in cancer patients as well as in various groups of increased cancer risk [5–12]. Chromosome instability has been observed not only in the lymphocytes of patients suffering from both sporadic, e.g. mutagen-induced, and inherited cancers [5–9], but also in those patients’ healthy relatives [10,11], and in Down syndrome children with malignancies [12]. The in vitro susceptibility to BLM has been accepted as a predictor of cancer risk. However, it can be used only for a group not for an individual prognosis. Another possibility to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. The AR has been studied mainly in relation to ionising radiation and radiomimetic chemicals such as BLM [13–15]. Cross-resistance has been demonstrated not only for ionising radiation and radiomimetic agents but also for ionising radiation and an alkylating agent, N-methyl-N-nitro-N -nitrosoguanidine (MNNG) [16,17]. Only few studies concerning AR induced by chemical mutagens have been published so far and the data are not as abundant as those on the radio-AR [18,19]. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems [15]. However, the existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. We present the study on the AR induced by two chemical mutagens, which differ in their action on DNA. BLM
is a clastogenic (inducing CA), radiomimetic agent which complexes with the ferrous ion and molecular oxygen, and then, induces in DNA both single-strand breaks (SSB) and double-strand breaks (DSB) [4]. Mitomycin C (MMC) is a potent bifunctional mutagen acting as an alkylating agent, causing mainly DNA interstrand cross-links, and thus, inducing sister chromatid exchanges (SCEs) [20]. We compared the ARs for the two tested agents in terms of the protective effects. The protective effect, which is expressed as the percentages of the total number of chromosomal alterations [9], provides a measure of the AR, which is independent of the applied cytogenetic endpoint.
2. Materials and methods The experiments were performed on human whole blood lymphocytes (WBL) obtained by venipuncture from 20 volunteers (students of the Wroclaw Medical University). The group consisted of 14 women and 6 men. The mean age of the group was 25.7 ± 3.1 years. All donors were healthy non-smokers and had not been exposed to any known mutagens for 3 months prior to the cytogenetic examination. The lymphocyte cultures were set up in air-tight glass bottles, following the standard protocol. Whole blood (0.5 ml) was added to the growth medium consisting of 4.5 ml of McCoy’s medium with glutamine (Gibco) supplemented with foetal calf serum (10%, Gibco), penicillin (100 UI/ml) and streptomycin (100 g/ml, Gibco), phytohaemagglutynin (PHA; 1%, Wellcome) and 5-bromo-2-deoxyuridine (BrdU; 5 g/ml, Calbiochem). Colcemid (Gibco) in a final concentration 0.02 g/ml was added 2 h prior to the harvesting. The cultures were incubated at 37◦ C for 72 h. The experiment consisted of two simultaneously performed series. In the first one, the lymphocytes were exposed to an adaptive dose (AD) of MMC (0.001 g/ml, Sigma) after 24 h of incubation, and subsequently to a challenging dose (CD) of the tested agent (0.1 g/ml of MMC) after next 24 h. Two other lymphocyte cultures were exposed exclusively to a CD of MMC (0.1 g/ml) after 48 h of incubation. In the second series, an AD of BLM (0.3 M, Nippon Kayaku Co.) was added to two lymphocyte
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
77
78
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
cultures 29 h prior to harvesting, and then, a CD (30 M of BLM) was added after the next 24 h, i.e. 5 h prior to harvesting. Two other cultures were exposed to CD of BLM (30 M) alone, 5 h prior to harvesting. For all concentrations used, tested chemicals (MMC and BLM) were dissolved each in 10 l of ethanol and PBS, respectively. The solvents were also added (10 l/tube) to the control cultures: ethanol after 24 h of incubation for MMC treatment and PBS 29 h prior to harvesting for BLM-treatment. All concentrations of tested chemicals were chosen experimentally (data not shown). Duplicate cultures were used for all treatments. The SCE analysis was performed on slides stained according to the modified fluorescence and Giemsa technique [21]. SCEs were scored in 60 second-division cells per donor and treatment. For the estimation of replication index (RI) the frequencies of the first (M1), second (M2), and third (M3) division metaphases were evaluated from 100 cells per donor/treatment [RI = (M1 + 2M2 + 3M3)/100]. CA were scored in 50 first-division metaphases. Following Hsu et al., chromatid gaps were omitted in scoring and more than 12 chromatid breaks per metaphase were scored as 12. The mean number of chromatid breaks per cell (b/c) and percentage of aberrant metaphases (am%) was used as a measure of mutagen sensitivity [4]. All analyses were performed on coded slides. For statistical analysis Student t–test for pairs was applied.
3. Results In the present study two different cytogenetic endpoints were applied for the assessment of AR induced by the tested chemicals. CA were used for the analysis of BLM-induced AR, and SCE for MMC-induced AR. The results are presented in Tables 1 and 2. There was a statistically significant increase (P < 0.001) in the induction of CA and SCE in cultures treated with BLM or MMC, respectively as compared to the controls. Only the RI value after MMC treatment was not statistically different from the control. The presence of cytogenetic AR was indicated by the statistically significant decrease in the frequency of CAs
79
or SCE after (AD + CD)-treatment in comparison to the CD-treatment. A significant (P < 0.001) cytogenetic AR was observed for both the BLM (am% and b/c) and MMC-treatment (SCE). Therefore, we calculated the protective effect, which provides an objective measure of AR, despite the applied cytogenetic endpoint. For BLM-treatment the protective effect of an AD was almost 50% regarding both of the analysed values. The AR to MMC was not as distinct as to BLM. In the cultures pre-treated with an AD of MMC, the SCE frequency decreased only about 20% in comparison to the cultures treated with a CD alone. The protective effects induced by the tested chemicals were individually variable. In the BLM experiment, the mean value (±S.D.) of the AR was observed for 14 out of 20 donors (70%), while 3 of them showed the AR above and 3 below the mean value. The interindividual differences in the AR have also been observed in MMC experiment, with two donors (10%) showing the AR above and four (20%) below the mean value.
4. Discussion In the present paper we demonstrate the results of cytogenetic analysis of AR induced by two chemical mutagens — BLM and MMC, which differ in their action on DNA. Both tested chemicals are routinely applied for the detection of hidden chromosome instability in human lymphocytes: BLM for the chromosome instability in the lymphocytes of cancer patients and their relatives, and MMC for the increased sensitivity to DNA cross-linking agents in Fanconi anaemia patients [6,7,22]. High individual sensitivity to DNA-damage induced by the tested agents is thought to reflect defective DNA-repair. In the present experiment, the lymphocytes of young healthy donors, without any signs of DNA-repair defects, were exposed to ADs and then to subsequent CDs of BLM and MMC. The protective effect induced by low doses of tested chemicals was analysed by using cytogenetic endpoints as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. In the BLM experiment, the pre-treatment with AD caused almost 50% decrease in CAs frequency
80
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81
induced by CD, while in the MMC experiment the protective effect was below 20%. This result may be a consequence of differences in the chemical structure of DNA lesions induced by tested agents, and subsequently, differences in induction of various DNA-repair pathways. In general, the BER pathway relatively easily removes lesions located on one strand such as SSB and the lost material is resynthesised using the undamaged strand as a template [23]. Damages that affect both strands, such as DSBs or interstrand cross-links, are more difficult to repair. It has been demonstrated that the combined action of NER and homologous recombination pathways, as well as non-homologues end-joint recombination are required for the correction of this type of DNA-damage [2,23]. There has also been observed the variability in AR among the young, healthy blood donors, with some of them showing AR higher and lower than average. The observed differences in the ARs induced by BLM and MMC may result from the different repair pathways involved in the processing of BLM and MMC-induced DNA lesions. The individual variability in the AR may reflect the difference in the individual DNA-repair capacity.
Acknowledgements The authors would like to thank Prof. Andrzej Elzanowski for his help in the preparation of the manuscript. References [1] F. Mittelman, Recurrent chromosome aberrations in cancer, Mutat. Res. 462 (2000) 247–253. [2] E. Moustacchi, DNA-damage and repair: consequences on dose–responses, Mutat. Res. 464 (2000) 35–40. [3] M. Berwick, P. Vineis, Markers of DNA-repair and susceptibility to cancer in humans: an epidemiologic review, J. Natl. Cancer Inst. 92 (2000) 874–897. [4] T.C. Hsu, D.A. Johnston, L.M. Cherry, D. Ramkisson, S.P. Schantz, J.M. Jessup, R.J. Winn, L. Shirley, C. Furlong, Sensitivity to genotoxic effects of bleomycin in humans: possible relationship to environmental carcinogenesis, Int. J. Cancer 43 (1989) 403–409. [5] T. Krecicki, K. Schlade, N. Blin, M. Sasiadek, Chromosome instability in head and neck cancer patients, Oncol. Rep. 4 (1997) 1383–1385.
[6] J. Cloos, B.J.M. Braakhuis, I. Steen, M.P. Copper, N. de Vries, J.J.P. Nauta, G.B. Snow, Increased mutagen sensitivity in head and neck squamous-cell carcinoma patients, particularly those with multiple primary tumours, Int. J. Cancer 56 (1994) 816–819. [7] J. Cloos, I. Steen, H. Joenje, J.-Y. Ko, N. de Vries, M.L.T. van der Sterre, J.J.P. Nauta, G.B. Snow, B.J.M. Braakhuis, Association between bleomycin genotoxicity and non-constitutional risk factors for head and neck cancer, Cancer Lett. 74 (1993) 161–165. [8] M. Tzancheva, D. Komitowski, Latent chromosomal instability in cancer patients, Hum. Genet. 99 (1997) 47–51. [9] Z. Trizna, T.C. Hsu, S.P. Schantz, Protective effects of vitamin E against bleomycin-induced genotoxicity in head and neck cancer patients in vitro, Anticancer Res. 12 (1992) 325–328. [10] S.K. Roy, A.H. Trivedi, S.R. Bakshi, S.J. Patel, P.H. Shukla, J.M. Bhatavdekar, D.D. Patel, P.M. Shah, Bleomycin-induced chromosome damage in lymphocytes indicates inefficient DNA-repair capacity in breast cancer families, J. Exp. Clin. Cancer Res. 19 (2000) 169–173. [11] T. Debniak, G. Kurzawski, B. Gorski, J. Kladny, W. Domagala, J. Lubinski, Value of pedigree/clinical data, immunohistochemistry and microsatellite instability analyses in reducing the cost of determining hMLH1 and hMSH2 gene mutations in patients with colorectal cancer, Eur. J. Cancer 36 (2000) 49–54. [12] R. Ankathil, P. Kusumakumary, M.K. Nair, Increased levels of mutagen-induced chromosome breakage in Down syndrome children with malignancy, Cancer Genet. Cytogenet. 99 (1997) 126–128. [13] J.D. Shadley, G. Dai, Cytogenetic and survival adaptive response in G1 phase human lymphocytes, Mutat. Res. 265 (1992) 273–281. [14] R.E.J. Mitchell, Mechanisms of the adaptive response in irradiated mammalian cells, Radiat. Res. 141 (1995) 117–118. [15] O. Rigaud, E. Moustacchi, Radio adaptation for gene mutation and the possible molecular mechanisms of the adaptive response, Mutat. Res. 358 (1996) 127–134. [16] Vijayalaxmi, W. Burkart, Resistance and cross-resistance to chromosome damage in human lymphocytes adapted to bleomycin, Mutat. Res. 211 (1989) 1–5. [17] S. Wolff, G. Olivieri, V. Afzal, Adaptation of human lymphocytes to radiation or chemical mutagens: differences in cytogenetic repair, in: G. Obe, A.T. Natarajan (Eds.), Chromosomal Aberrations, Basic and Applied Aspects, Springer, Berlin, 1990, pp. 140–150. [18] T. Nikolova, E. Huettner, Adaptive and synergistic effects of a low-dose ENU pre-treatment on the frequency of chromosomal aberrations induced by a challenge dose of ENU in human peripheral blood lymphocytes in vitro, Mutat. Res. 357 (1996) 131–141. [19] M. Sasiadek, M. Paprocka-Borowicz, Correlation between the adaptive response and individual sensitivity to monoepoxybutene in in vitro experiments on human lymphocytes, Mutat. Res. 390 (1997) 239–243.
K. Schlade-Bartusiak et al. / Mutation Research 513 (2002) 75–81 [20] B.J. Sanderson, A.J. Shield, Mutagenic damage to mammalian cells by therapeutic alkylating agents, Mutat. Res. 355 (1996) 41–57. [21] K.J. Husgafvel-Pursiainen, J. Maki-Paakkanen, H. Norppa, M. Sorsa, Smoking and sister chromatid exchange, Hereditas 92 (1980) 247–250.
81
[22] M. Buchwald, E. Moustacchi, Is Fanconi anaemia caused by a defect in the processing of DNA-damage? Mutat. Res. 408 (1998) 75–90. [23] Braithwaite, X. Wu, Z. Wang, Repair of DNA lesions: mechanisms and relative repair efficiencies, Mutat. Res. 424 (1999) 207–219.