Cytoprotective activity of amifostine on cultured human lymphocytes exposed to irinotecan

Food and Chemical Toxicology 47 (2009) 2445–2449

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Cytoprotective activity of amifostine on cultured human lymphocytes exposed to irinotecan Th.S. Lialiaris a,*, E. Kotsiou a, S. Pouliliou a, D. Kareli a, H. Makrinou a, A. Kouskoukis a, F. Papachristou a, M. Koukourakis b a b

Department of Genetics, Faculty of Medicine, Demokrition University of Thrace, Alexandroupolis 68100, Greece Department of Radiotherapy and Oncology, Faculty of Medicine, Demokrition University of Thrace, Alexandroupolis 68100, Greece

a r t i c l e

i n f o

Article history: Received 8 May 2009 Accepted 29 June 2009

Keywords: Irinotecan Amifostine Sister Chromatid Exchanges Mitotic index Cultured lymphocytes

a b s t r a c t Irinotecan (camptothecin, CAM) is a topoisomerase-I inhibitor with a well established action in the chemotherapy of colorectal and ovarian cancer. Hematological and intestinal toxicity are commonly noted in patients treated with CAM. In this study, we examined the cytoprotective efficacy of amifostine (ethyol, ETH) against chromosomal damage induced by this drug on cultured peripheral human lymphocytes. Cultured lymphocytes were exposed to CAM (50 and 100 ng/ml of final concentrations) without or with ETH (in concentrations varying between 40 and 800 lg/ml of final culture volume). CAM’s genotoxicity was quantified by counting the Sister Chromatid Exchange (SCEs) rate. The mitotic index (MI) and proliferation rate index (PRI) were also assessed. The SCE rate was increased following incubation with CAM, but the combined treatment of CAM with ETH significantly reduced the SCE formation, especially when ETH added at high concentrations. The MIs and PRIs remained also unaltered in cultures with CAM, but MIs were reduced with the combined treatment at high ETH concentrations. Clinical studies are required to assess the predicted benefits from ETH in patients receiving CAM. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Irinotecan, a semisynthetic analogue of camptothecin-11, (CAM) is a potent inhibitor of topoisomerase-I activity (Mathijssen et al., 2002). Following DNA damage, topoisomerase-I, a monomeric 100-kDa polypeptide, allows the relaxation of the damaged strand in order to facilitate DNA reconstruction. Topoisomerase-I inhibitors block the retirement of the enzyme from its DNA binding site, preventing the rejoining of the DNA strand and therefore stabilize the single strand breaks and increase the probability of cells to undergo apoptosis (Liu et al., 2000). The use of irinotecan is well established in the treatment of patients with metastatic colorectal and ovarian carcinoma (Pizzolato and Saltz, 2003; Hofheinz et al., 2005). Important toxicities, however, are commonly noted, including febrile neutropenia and severe diarrhea (Rothenberg et al., 1993; Shimada et al., 1993). Amifostine (WR-2721) (ethyol, ETH) is a broad spectrum cytoprotective agent approved for the prevention of platinum induced toxicities and radiation induced xerostomia (Koukourakis, 2002). Following dephosphorylation by the alkaline phosphatases, abundantly found in normal tissues (Giatromanolaki et al., 2002), the

* Corresponding author. Address: Department of Genetics, Medical School, Demokrition University of Thrace, Univ. Campus, Dragana, Alexandroupolis 68100, Greece. Tel./fax: +30 25510 30522. E-mail address: [email protected] (Th.S. Lialiaris). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.06.042

active thiolic compound WR-1065 enters rapidly the cells and acts as a potent scavenger of free radicals protecting DNA against strand breaks (Capizzi and Oster, 2000). Oxidation of WR-1065 to the disulfidic compound WR-33278 allows its binding to the DNA. This later compound is believed to accelerate repair of DNA damage. As WR-33278 has the ability to enhance the topoisomerase-I mediated unwinding of supercoiled DNA, it is suggested that ETH may play a protective role against topoisomerase-I inhibitors (Holwitt et al., 1990; Hartmann et al., 2000). On the other hand, the combination of CAM plus ETH in patients with previously treated metastatic colorectal carcinoma did not appear to reduce CAM’s toxicity and ETH did not appear to interfere with the cytotoxic effect of CAM (Delioukina et al., 2002). Also, Souid et al. found that ETH offers some – but not full – myeloprotection for CAM’s toxicity when administered in combination with cisplatin (Souid et al., 2003). In the present study, we investigated the potential effect of amifostine (ETH) on protecting the cellular genome against the activity of topoisomerase-I inhibitors, such as irinotecan (CAM), which may be proved of importance for cancer patients receiving chemotherapy with such drugs. We determined ETH’s antigenotoxic potential using Sister Chromatid Exchanges (SCEs) induced by CAM as genotoxic end points in cultured human lymphocytes (Lialiaris et al., 2007, 2008; Mpountoukas et al., 2008). We tested the effect of ETH, at various concentrations, on the damage exerted by CAM. CAM can initiate genotoxic stress, leading to apoptosis and

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antitumour therapy, but on the other hand can give rise to chromosome instability and mutagenesis. The antioxidant ETH, effectively prevents CAM-induced DNA damage, suggesting that ETH may have preventive or therapeutic effects in CAM-induced chromosome instability and genotoxicity. Presently the protective effect of ETH was evaluated with the simultaneous determination of three cytogenetic parameters: (a) the levels of Sister Chromatid Exchanges (SCEs), (b) the proliferation rate index (PRI) and (c) the mitotic index (MI) in human lymphocyte cultures. The method of Sister Chromatid Exchanges (SCEs) has been proposed as a very sensitive, simple and rapid method for detecting DNA damage and/ or subsequent DNA repair induced by mutagens and/or carcinogens and antimutagenic agents, while its application is very useful for monitoring and improving chemotherapeutic efficacy in vitro and in vivo. SCE methodology is considered a more sensitive method than Chromosome Aberrations, since induced-DNA damage can be demonstrated by the formation of SCEs even at very low concentrations of genotoxic agents (Lialiaris et al., 1989, 1990). Furthermore, the other two indices (PRI and MI) are useful indicators of the cytostatic and cytotoxic properties of various agents, including chemotherapeutic agents (Koukourakis et al., 2003; Fousteris et al., 2006).

3. Results We assessed lymphocytes during the 1st cellular division, where they have both sister chromatids dark stained (Fig. 1), lymphocytes during the 2nd cellular division, where one light-coloured and one dark-coloured chromatid is evident (Fig. 2) and finally, during the 3rd cellular division where a certain percentage of chromosomes have both sister chromatids light-stained (Fig. 3). Cultures were exposed to two different concentrations of CAM (50 and 100 ng/ml) with the presence or absence of ETH in escalated concentrations of 10, 40, 100, 200, 400 and 800 lg/ml (Tables 1 and 2). Tables 1 and 2 illustrate our findings concerning the combined treatment of human lymphocytes with ETH and CAM undertaken to uncover the possible protective effect of ETH on lymphocytes against the genotoxic damage caused by CAM. Data indicate that in vitro administration of CAM in human lymphocytes, at two different concentrations (0.0683 and 0.1366 nV, 50 ng/ml and 100 ng/ml of final concentration respectively) significantly

2. Materials and methods 2.1. Chemicals CAM (Irinotecan, CAS No. 7689-03-4) was provided from Pfizer and ETH (ethyol, amifostine, CAS No. 20537-88-6) from Schering-Plough. 5-Bromo-20 -deoxyuridine (BrdU, CAS No. 59-14-3), bis-benzamide (CAS No. 23491-45-4) and colcemide (CAS No. 64-86-8) were obtained from Sigma. All chemicals were dissolved and diluted further in sterile bidistilled water. 2.2. Cytogenetic studies Heparinized blood samples were collected from six male 18–32 year old healthy donors, none of whom was under any medication treatment, or was a smoker. Cultures of peripheral blood lymphocytes were prepared in universal containers by inoculating 11 drops of whole blood in 5 ml of culture chromosome medium 1A (Gibco). These were incubated at 37 °C for 72 h. Cultures were first treated with ETH (40, 100, 200, 400 and 800 lg/ml) at the 18th hour after PHA stimulation and/or CAM (50 and 100 ng/ml of final concentration). In order to observe SCEs, within each culture, cells were allowed to proliferate for two mitotic cycles in the presence of 5-bromo-20 -deoxyuridine (BrdU) at a final concentration of 5 lg/ml. Cells were harvested after 2 h incubation with colchicine (0.3 lg/ml culture medium). Cultures were incubated and manipulated in a dark environment, in order to prevent, or minimise the photolysis of BrdU. Chromosome preparations were stained using a modified fluorescence plus Giemsa (FPG) technique, as previously described (Goto et al., 1978; Maskaleris et al., 1998). All chemicals were obtained from Sigma, unless otherwise stated. Scoring was performed in a blind fashion. Cells on the 1st, 2nd, 3rd and subsequent mitotic divisions were counted. Three indices were recorded: (1) the mitotic index (MI), which is a qualitative index of cytotoxicity; (2) the proliferation rate index (PRI), which is a qualitative index of cytostaticity; and (3) SCEs, which is a qualitative and quantitative index of genotoxicity. All indices were evaluated in each subject for either treatment. Mean SCE values were evaluated only in suitable 2nd division metaphases. For each experiment, 45–60 metaphases were evaluated. In order to establish the proliferation rate index, 150–300 cells were counted and the following formula was used: PRI = (M1 + 2M2 + 3M3+)/N, where V1 is the percentage of cells in the first division, V2 in the second and V3+ in the third and subsequent divisions, while N is the total number of cells counted i.e. (M1 + M2 + M3+). In addition, mitotic indices (MIs) for 1500–3000 activated lymphocytes were determined for all cultures.

Fig. 1. First division metaphase after FPG staining methodology. All chromosomes have both chromatids dark stained.

2.3. Statistical analysis The evaluation of MI and PRI was based on the v2 test. To compare various treatments, a logarithmic transformation of SCE values was performed using oneway analysis of variance (ANOVA) and the Duncan test, where pair-wise comparisons were concerned. Furthermore, the protective effect of ETH on cells exposed to CAM was evaluated by comparing the expected values (EV) and the observed values (OV). The expected value of CAM was calculated using the formula: OVCAM  (OVETH  OVControl). A probability of P < 0.05 was considered as statistically significant (Lialiaris et al., 1992).

Fig. 2. Second division metaphase after FPG staining methodology (arrows show Sister Chromatid Exchanges). Magnification 1000. All chromosomes have as total one chromatid light and the other dark stained.

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The maximum protection was seen in cultures incubated with the highest ETH concentration used (400 and 800 lg/ml), where the formation of SCEs was significantly reduced by 36%, (P < 0.05). The combined treatment of 400 and 800 lg/ml of ETH and CFV also demonstrated a statistically significant decrease of PRI (P < 0.05), as well as of MI (P < 0.05), compared to equal concentrations of CAM in the control experiment. 4. Discussion

Fig. 3. Third or subsequent division metaphase after FPG staining methodology. There are chromosomes with both chromatids light-stained.

enhances the levels of SCE formation by 3.9 and 6 times, respectively (P < 0 .01, compared to the control experiment). A slight increase of SCEs (1.1–1.5 times) was also noted in cultures exposed to ETH alone at various concentrations (0.1867, 0.467, 0.934, 1.867 and 3.736 mM, or 40 lg/ml, 100, 200, 400 and 800 lg/ml of final concentration respectively), but this was negligible and very close to control levels (P > 0.05 compared to the control experiment). A combined treatment with both CAM and ETH administered at various concentrations, elicited a statistically significant reduction in SCE formation due to reduced cytotoxicity levels, compared to administration of CAM alone (P < 0.01).

Sister Chromatid Exchanges (SCEs) are part of a natural process of reciprocal alterations between the homologous chromatid segments of the same chromosome. They are commonly shown after two rounds of DNA replication in the presence of the base analogue 5-bromo-20 -deoxyuridine (BrdU) (Brogger, 1982; Latt et al., 1984). SCEs are also known to occur at high rates when there is inducedDNA damage, as a response to DNA repair mechanisms (Carrano et al., 1978; Deen et al., 1986; Pantazaki and Lialiaris, 1999). Anti-tumor treatments provide models of controlled mutagen exposure on humans which facilitate the comparison of the sensitivity and specificity of various mutagen–assay systems. Assessment of SCE frequencies is considered a sensitive method of predictive value, able to detect cytogenetic damage induced by low concentrations of genotoxic agents and has been used extensively as a clinical assay for drugs for which a strong correlation between cell killing and induction of SCEs has been established (Solomon and Bobrow, 1975; Perry and Evans, 1975; Dai et al., 2005). A number of previous studies have shown that following administration of CAM to cancer patients, peripheral lymphocytes exhibit increased SCEs, which depend directly upon the dose of the

Table 1 Attenuation of cytogenetic damage produced by CAM in human lymphocytes in vitro exposed to ETH. Agent and concentration

Number of replicate cultures

Mean SCEs ± SEM (range of values)

PRI

MI (%)

1. Control

3

2.68

55.7

2. CAM 50 ng/ml

3

2.62

44.3

3. CAM 100 ng/ml

3

2.54

39.5

4. ETH 40 lg/ml

3

2.66

43.5

5. ETH 40 lg/ml + CAM 50 ng/ml

3

7.71 ± 0.45 (1–14) 38.86 ± 4.25a (12–60) 48.40 ± 11.00a (19–75) 7.52 ± 0.52 (1–12) 39.25 ± 41.00b (11–67) 46.01 ± 5.05b (16–79) 8.64 ± 0.59 (1–19) 33.98 ± 3.85c (9–57) 40.10 ± 4.91c (12–72) 8.92 ± 0.67 (2–20) 27.53 ± 3.50d (5–59) e 31.71 ± 4.09 (2–70)

2.68

40.1

2.65

41.3

2.47

38.4

2.55

37.1

2.50

34.2

6. ETH 40 lg/ml + CAM 100 ng/ml

3

7. ETH 100 lg/ml

3

8. ETH 100 lg/ml + CAM 50 ng/ml

3

9. ETH 100 lg/ml + CAM 100 ng/ml 10. ETH 400 lg/ml 11. ETH 400 lg/ml + CAM 50 ng/ml 12. ETH 400 lg/ml + CAM 100 ng/ml

3 3 3 3

EV = 38.67 EV = 48.21

EV = 39.79 EV = 49.33 2.26f

32.5

f

2.34

28.7g

2.35f

22.8g

EV = 40.07 EV = 49.61

The Sister Chromatid Exchanges (SCEs) frequency was based on 45–60 s-division metaphases for each donor; for proliferation rate index (PRI), 150–300 cells were counted, and for mitotic index (MI) 1500–3000 activated lymphocytes were evaluated. The results were based on three experiments from three different donors. For each experiment 12 cultures were performed. PRI and MI comparisons were made using the v2 test. For SCE comparisons, logarithmic transformation of the data was performed using one-way ANOVA and the Duncan test. TV = expected value if CAM and ETH were acting independently and additively. a P < 0.01 vs. line 1. b P < 0.01 vs. lines 1 and 4. c P < 0.01 vs. lines 1, 4 and 7. d P < 0.01 vs. lines 1, 4, 7 and 10 and P < 0.05 vs. lines 2 and 5. e P < 0.01 vs. lines 1, 4, 7 and 10 and P < 0.05 vs. lines 3 and 6. f P < 0.05 vs. lines 1–9. g P < 0.05 vs. lines 1–8.

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Table 2 Attenuation of cytogenetic damage produced by CAM in human lymphocytes in vitro exposed to ETH. Agent and concentration

Number of replicate cultures

Mean SCEs ± SEM (range of values)

PRI

MI (%)

1. Control

3

2.49

48.9

2. CAM 50 ng/ml

3

2.55

41.5

3. CAM 100 ng/ml

3

2.53

38.5

4. ETH 200 lg/ml

3

2.44

40.2

5. ETH 200 lg/ml + CAM 50 ng/ml

3

6.18 ± 0.33 (1–16) 25.76 ± 2.65a (7–52) 35.40 ± 3.11b (9–65) 6.88 ± 0.47 (1–15) 20.37 ± 2.01c (11–57) 24.11 ± 2.12c (6–48) 7.57 ± 0.52 (1–17) 18.55 ± 1.75d (2–43) 20.13 ± 1.88e (5–52) 6.56 ± 0.45 (1–20) 16.41 ± 1.69f (2–39) 19.83 ± 1.34g (1–45)

2.33

36.4

2.48

33.8

2.17h

34.0

2.25

30.7

6. ETH 200 lg/ml + CAM 100 ng/ml

3

7. ETH 400 lg/ml

3

8. ETH 400 lg/ml + CAM 50 ng/ml

3

9. ETH 400 lg/ml + CAM 100 ng/ml

3

10. ETH 800 lg/ml

3

11. ETH 800 lg/ml + CAM 50 ng/ml

3

12. ETH 800 lg/ml + CAM 100 ng/ml

3

EV = 26.46 EV = 36.10

EV = 24.37 2.29

27.3

EV = 39.79 i

11.5j

1.51i

8.7j

1.20j

6.5j

1.58

EV = 26.14 EV = 35.78

The Sister Chromatid Exchanges (SCEs) frequency was based on 45–60 s-division metaphases for each donor; for proliferation rate index (PRI), 150–300 cells were counted, and for mitotic index (MI) 1500–3000 activated lymphocytes were evaluated. The results were based on three experiments from three different donors. For each experiment 12 cultures were performed. PRI and MI comparisons were made using the v2 test. For SCE comparisons, logarithmic transformation of the data was performed using one-way ANOVA and the Duncan test. TV = expected value if CAM and ETH were acting independently and additively. a P < 0.01 vs. line 1. b P < 0.01 vs. line 1 and P < 0.05 vs. line 2. c P < 0.01 vs. lines 1 and 4. d P < 0.01 vs. lines 1, 4 and 7 and P < 0.05 vs. line 2. e P < 0.01 vs. lines 1, 4 and 7 and P < 0.05 vs. line 3. f P < 0.01 vs. lines 1, 4, 7 and 10 and P < 0.05 vs. line 2. g P < 0.01 vs. lines 1, 4, 7 and 10 and P < 0.05 vs. lines 2, 3 and 6. h P < 0.05 vs. lines 1–6. i P < 0.05 vs. lines 1–9. j P < 0.05 vs. all lines.

injected drug (Degrassi et al., 1989; Tofilon et al., 1985; Kojima et al., 1993). In the present study, we tested the hypothesis that ETH may protect peripheral blood cells against the cytotoxic activity of CAM. Our results are in line with most in vitro studies. Indeed, the SCE frequencies of lymphocyte cultures induced by CAM were significantly reduced in the presence of ETH, an effect that was shown to be dosage dependent. Although the exact mechanism of such a protective effect is unknown, experimental data suggest that WR-33278, an amifostine derivative that appears to follow intracellular oxidation of WR-1065, binds to DNA and accelerates the topoisomerase-I mediated unwinding of supercoiled DNA (Holwitt et al., 1990; Blasiak et al., 2002). Protection of topoisomerase-I activity against its inhibitors and successful DNA repair, prior to S phase removes damage that might otherwise give rise to SCEs, underlying the herein reported findings. Other researchers found that WR-1065 has a protective effect on stem cells causing cytotoxicity reduction, produced by daunorubicin, of 10–20% (Michelutti et al., 2006). The reduction of both the proliferative rate indices and mitotic indices by ETH, but not by CAM, suggests that cell cycle arrest in pre-mitotic phases of the cell cycle may be part of the cytoprotective pathways exploited by ETH. Several previous studies have confirmed the effect of ETH on cell cycle. Lee et al. reported a direct induction of wild-type p53 protein in p53-proficient cells, leading to G1-arrest and cytoprotection against raxanes (Lee et al., 2003). On the contrary, no effect of ETH on the cell cycle of p53-deficient cells was noted and ETH allowed the apoptotic activity of paclitaxel. Similar results have been reported by North et al., where

ETH induced p21 waf-1 and cell cycle arrest in p53-efficient cells (North et al., 2000). As normal cells, unlike tumoral, maintain intact p53 function, protracted pre-mitotic cell arrest induced by ETH may better facilitate DNA repair and recovery of normal cells exposed to CAM. Clinical data regarding the efficacy of ETH in protecting patients against irinotecan activity are scarce. Delioukina et al. found no particular cytoprotective efficacy of ETH over gastrointestinal and haematological toxicities induced by high doses of CAM (250 mg/ ml, every 2 weeks) (Delioukina et al., 2002). It appears that even large amounts of ETH (740 mg/ml) may be not effective in substantiating cytoprotection at the clinical level, when very high doses of CAM are used. As higher doses of ETH are unlikely to be well tolerated by patients, fractionation of the CAM dose and support of each dose with ETH may be necessary to obtain clinical cytoprotection. Indeed, in a phase I study by Souid et al., using weekly administration of CAM and cisplatin in children with refractory solid tumors, the administration of amifostine permitted P30% dose escalation for CAM (Souid et al., 2003). Similarly, in a study of ours in 19 patients with colorectal cancer treated every two weeks with CAM (180 mg/ml, day 1) and 5-fluorouracil (600 mg/ml, days 1 and 2), the subsutaneous administration of 1000 mg of ETH before chemotherapy resulted in minimal haematological and gastrointestinal toxicities (Koukourakis et al., 2003). It is concluded that amifostine protects human lymphocytes against the DNA damaging activity of the topoisomerase-I inhibitor irinotecan. This effect, however, is dose dependent, been detected at high amifostine concentration levels. The mechanisms

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