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Induction of cytogenetic damage in human lymphocytes in vitro and of antineoplastic effects in Ehrlich ascites tumor cells in vivo treated by methotrexate, hyperthermia and/or caffeine

Theodore Maskaleris^a, Theodore Lialiaris^b^, ^* and Constantine Triantaphyllidis^a

^a Department of Pharmacology, Demokritus University of Thrace, Alexandroupolis, Greece
^b Department of Medical Biology and Genetics, Faculty of Medicine, Demokritus University of Thrace, Alexandroupolis, Greece

Received 16 April 1998; revised 16 July 1998; accepted 28 July 1998. Available online 9 December 1998.

1. Introduction

The antimetabolite methotrexate (MTX), a known antineoplastic agent, has been a useful agent for the treatment of human cancer and other diseases, like psoriasis vulgaris or rheumatoid arthritis [1, 2, 3]. MTX limits the intracellular supply of reduced folates (tetrahydrofolate) through inhibition of dihydrofolate reductase (DHFR) and therefore is considered to induce cytotoxic effects with resultant inhibition of new DNA, thymidylate and purine synthesis [4, 5, 6]. In cells treated with MTX, a progressive accumulation of strand breaks in mature DNA (post-replicative DNA) was detected [4, 7]. Hence, DNA strand breaks arise from spontaneous and normally repaired DNA lesions that are not repaired due to a depletion of dTTP, dATP and dGTP. There is also evidence that MTX influences the cellular topoisomerase II content and causes, therefore, an increase in DNA breaks [8, 9]. MTX has been found to be a clastogenic agent in ascites tumor and other mammalian cultured cells [4, 5, 6, 8] and a micronucleus inducer in human and mouse bone marrow cells [7, 10], but the results concerning the induction of chromosomal abnormalities in vivo [2, 10] and SCEs in vitro [11] are conflicting.

Caffeine (CAF) exhibits a variety of modifying effects in mammalian cells which have been treated with DNA-damaging agents: (a) by inhibiting post replication synthesis of DNA, (b) by inhibiting poly(ADP-ribose)polymerase, (c) by increasing number of sites of DNA replication units or (d) by antagonising the DNA-synthesis inhibition that is induced by DNA damage [12, 13]. An alternative mechanism is that CAF acts in G₂ to induce cells to undergo mitosis before the completion of DNA repair [14]. Also, it was reported that CAF enhanced the antitumor activity of different chemotherapeutic agents [15, 16, 17, 18]. Nevertheless, the potential application of CAF to clinical cancer chemotherapy is poorly understood and some studies are contradictory [19, 20].

Hyperthermia (HYP), on the other hand, has an inhibitory effect on RNA and protein synthesis and on DNA repair and synthesis [21, 22, 23, 24]. HYP revealed also a direct cytotoxic effect and potentiation of the action of other chemotherapeutic drugs [25]. Many workers have confirmed the validity of the combination of hyperthermia plus radiation therapy for the treatment of several tumors [26, 27] and cytogenetic trials showed that hyperthermia plus radiation elevated the frequency of SCEs [28].

Sister chromatid exchanges (SCEs) have been proposed as a very sensitive method for detecting mutagens and/or carcinogens, and lately as a valid method for guiding and improving chemotherapy in vitro [29, 30] and in vivo [16, 29]. Furthermore, studies have shown that the determination of proliferation rates (PRIs) and mitotic indices (MIs) in lymphocyte cultures should be a useful and sensitive indicator of the cellular toxicity of chemotherapeutic agents [18, 31].

The present study is a first attempt, to our knowledge, to examine the cytogenetic damage caused by the combined MTX plus CAF and/or HYP treatment in normal human lymphocytes in vitro and by the combination of MTX plus CAF in Ehrlich ascites tumor (EAT) cells in vivo.

2. Materials and methods

2.1. Chemicals

Methotrexate (MTX, CAS No. 59-05-2), caffeine (CAF, CAS No. 58-08-2), 5-bromo-2′-deoxyuridine (BrdU, CAS No. 59-14-3), bis-benzimide (CAS No. 23491-45-4) were obtained from Sigma; colchicine (CAS No. 64-86-8) from Serva. All chemicals were dissolved in bidistilled sterile water. Lower concentrations were prepared by serial dilutions just before treatment. Other chemicals were of the best grade (AR) commercially available.

2.2. In vitro experiments

Heparinized blood samples were obtained from 8 healthy individuals, 18–20 years old, all medical students, none of whom was receiving drugs or was a smoker. Cultures of peripheral lymphocytes were prepared in universal containers by adding 10 drops of whole blood to 5 ml of chromosome medium B (Biochrom KG). These were incubated at 37°C for 96 h. For SCE observations the cultures were first treated with MTX (3.1 to 100 nM final concentration) and CAF (618 μM=120 μg/ml final concentration) at 20 h. Then, some cultures were incubated at 42°C for 2 h. At once, at the same time (at 22 h), 5-Bromo-2′-deoxyuridine (BrdU) was added at a final concentration of 4 μg/ml. After 94 h, colchicine was added for 2 h at a final concentration of 0.5 μg/ml and at the end of the incubation period cultures were harvested. The treatments took place from 20 h till harvesting. Cultures were maintained in the dark to prevent or minimize photolysis of BrdU. The chromosome preparations were stained by a modified fluorescence plus Giemsa (FPG) technique [32].

Scoring was performed blindly. Cells were counted in their first, second or third and subsequent divisions. Mean SCE values were calculated only in suitable 2nd division metaphases, because only in these we could observe and count the exchanges between the sister chromatids. Higher levels of SCEs usually indicate stronger damage on DNA. Thus, the methodology of SCEs provides a tool for evaluating the mutagenic and/or carcinogenic effects of suspected environmental agents. For establishing the PRI, in each case 100 cells were counted according to the formula: PRI=(1M₁+2M₂+3M₃₊)/100, where M₁ is the percentage of cells in the first division, M₂ in the second and M₃₊ in the third and subsequent divisions. In addition, MIs for 1000 activated lymphocytes were determined for all cultures.

2.3. In vivo experiments

Two or 3-month-old male BALB/c mice of an average weight of 25 to 35 g were used. The animals were maintained at a standard pellet diet with water ad libitum. The EAT cells were originally provided from the Theagenion Cancer Institute, Thessaloniki, Greece and maintained by inoculating them to other mice under aseptic conditions every 7 days. EAT cells were diluted in 0.9% NaCl solution, so that the final number of inoculated EAT cells was about 10⁶ cells. Then, 0.2 ml of EAT cells were inoculated to mice by an i.p. injection. We performed two experiments; in each experiment four treatments (four groups of mice). The mice were divided in groups of ten and were marked in their tails with indelible ink, so that they could easily be recognized. The mice were then weighed and the results were marked. The appropriate solutions were prepared and mice were treated on 2nd and 4th day of the experiments with i.p. injection of 0.2 ml of the drugs or 0.9% NaCl, according to the protocol. Mice were weighed every day at the same time and the results were registered and evaluated [16].

In the in vivo experiments we examined the antineoplastic effects of MTX, at the concentration of 0.45 μg/g body weight of mice, alone or in combination with CAF, at the concentration of 120 μg/g body weight. For this purpose we studied: (a) the mean survival of mice for each group and the percentage of increase in mean survival of the groups concerned, compared with that of the control group and (b) the increase of the mean body weight of mice on the 8th day of the experiments for each group and the percentage of the alteration of the increase of body weight of mice on the 8th day of the experiments for each group concerned, compared with that of the control group. The 8th day of the experiments was selected because it was the last day before the first death of mice occurred. We can suppose that the increase of the mean body weight of mice is equal to the weight of the tumor of mice.

2.4. Statistical analysis

Usually according to previous as well as present evaluations of our results, SCE frequencies among individual cells do not follow a normal distribution and therefore is inappropriate to use standard parametric statistical methods. The results need to be transformed in a logarithmic or square root way. So, to compare various treatments, logarithmic transformation of SCEs was performed using the one-way analysis of variance (ANOVA) and subsequently the Duncan test for the calculations concerning pair-wise comparisons. Correlations between SCEs and MIs or SCEs and PRIs were also calculated [18, 31]. Evaluation of MIs and PRIs was based on the χ²-test. Differences in survival time and increase of body weight were evaluated by Wilcoxon test [16].

3. Results

MTX alone, at various concentrations (3.1, 6.25, 12.5, 25 and 50 nM) was tested in vitro (Table 1). It was found that MTX, in human lymphocytes, produced: (a) statistically significant increase in SCE frequencies (P<0.05 at concentration of 6.25 nM and P<0.01 at higher concentrations), (b) reduction of cell proliferation rates (PRIs) but not statistically significant and (c) reduction of MIs at concentrations of 25 and 50 nM statistically significant versus control cultures.

Table 1. Enhancement of cytogenetic damage by various concentrations of MTX in normal human lymphocytes in vitro

We also tested the action of MTX at concentrations higher than 200 nM, but we failed because of the very low number of mitoses and nuclei, which was expected. According to the literature, it is shown that MTX induces low SCE frequencies, but it produces strong cytotoxicity. So, it seems that SCE frequencies and, of course, PRI values are constant except for the cultures which were treated with the triple combination MTX+CAF+HYP (Table 1, Table 2 and Table 3).

Table 2. Enhancement of cytogenetic damage by various concentrations of MTX, alone or in combination with CAF, in normal human lymphocytes in vitro

Table 3. Enhancement of cytogenetic damage by various concentrations of MTX, alone or in combination with CAF and HYP, in normal human lymphocytes in vitro

In Table 2 we tested the combinations of MTX plus CAF on the production of cytogenetic damage. It was found that the presence of CAF in cultures treated with MTX: (a) enhanced the rates of SCEs produced by MTX, at all concentrations used, but statistically significant only at the concentration of 100 nM, (b) delayed the proliferation rates (PRIs) and (c) reduced also all the MI values, but statistically significant only at concentrations of 12.5 nM (P<0.01) and 50 nM (P<0.05).

Table 3 demonstrates the synergistic damage of the triple combination of MTX plus CAF in human lymphocyte cultures treated with HYP. It was found that MTX, in the triple combinations with CAF and HYP: (a) produced enhancement of SCE levels, but statistically significant only at the concentration of 100 nM (P<0.01), (b) delayed the proliferation rates (PRIs) at all concentrations, but statistically significant only at the concentrations of 12.5 nM (P<0.05) and of 50 nM (P<0.01) and (c) reduced the MI values, statistically significant at all concentrations used. From Fig. 1, an indication can be obtained about the synergistic effect of MTX in cultures treated with CAF and HYP.

(3K)

Fig. 1. Synergistic effect of HYP in combination with CAF and MTX in different concentrations on induction of SCEs in human lymphocytes. There is no overlap between SCEs of line IV and each one of the rest of the lines at the highest concentration of MTX. The equation of linear regression was obtained by the method of least squares for each line and for the particular subjects (x=concentration of MTX 10⁻⁸ M, y=SCEs/cell, SE=standard error). I: y=4.79+0.27x (SE=2.37); II: y=6.01+0.21x (SE=1.67); III: y=6.31+0.33x (SE=1.70); IV: y=7.06+0.42x (SE=1.74).

Finally, we examined the effect of MTX, alone and in combination with CAF, on survival time and on body weight of mice inoculated with Ehrlich ascites tumor cells (Table 4). We observed that MTX plus CAF reduced the increase of body weight of treated mice and induced the survival time of Ehrlich ascites tumor bearing mice more than 14.5% compared to controls.

Table 4. Effect of MTX, alone or in combination with CAF, on the survival time and on body weight of mice inoculated with Ehrlich ascites tumor cells

4. Discussion

Antitumor treatments provide models of controlled mutagen exposure on humans which facilitate the comparison of the sensitivity and specificity of various mutagen–assay systems [33]. Of course, the exact mechanism of SCEs has not yet been fully elucidated [34, 35] and some results are contradictory [36, 37]. Attempts to correlate induced SCEs with known repair processes in mammalian cells or with specific types of molecular lesions in DNA have failed to yield consistent results [38]. Nevertheless, the increases on the frequency of SCEs and the reduction of PRIs and MIs caused by genotoxic agents are considered as indicators of cytogenetic damage [39] and therefore SCEs appear to have an application in the clinical prediction of tumor sensitivity to potential chemotherapeutics [16, 18, 29, 30]. Recent studies have shown a possible relation between clastogenicity, DNA repair, cancer and the induction of SCEs since an increase rate of SCEs is accepted as a sensitive indicator of potential mutagenicity [16, 29, 30, 31].

It was found that MTX leads to reduction of new DNA synthesis, causes DNA fragmentations through inhibition of different enzyme mechanisms and that CAF potentiates MTX-induced cell growth inhibition and cell killing [4, 6, 12]. Our results are in agreement with these results. MTX alone at concentrations of 6.25–100 nM produced cytogenetic damage in cultured human lymphocytes and especially together with CAF. Furthermore, we found that MTX plus CAF reduced the ascitic tumor volume in the EAT bearing mice.

HYP seems to produce irreversible membrane structure transitions, induction of cytotoxicity and alteration in DNA synthesis [21, 40, 41]. In our experiments, it was found that hyperthermia, alone or in combination with alkylating drugs, produced cytogenetic damage and higher frequencies of SCEs. It was also found that MTX in combination with CAF and HYP enhanced SCE levels and produced cell division delays and reduction of MIs, which means induction of cytotoxic and cytostatic action in human lymphocyte cultures.

It has been proposed that successful DNA repair, prior to S phase, removes damage that otherwise might give rise to SCEs [15]. In normal human lymphocytes in vitro (Table 1, Table 2 and Table 3) or in EAT cells in vivo (Table 4) interference by CAF with DNA repair of cytostatic-induced (such as MTX) DNA damage would lead to an increase in the number of incompletely repaired lesions at the time the cells reach S phase, lesions which may subsequently give rise to SCEs, cause cell-cycle delays and reduction of MIs (Table 1, Table 2 and Table 3). A strong hypothesis is that CAF inhibits repair processes of DNA by inhibiting poly(ADP-ribose)polymerase [13] which participates in DNA repair of normal or tumor cells after being damaged by alkylation. In our experiments, we found statistically significant reduction of PRIs and MIs by CAF plus MTX or CAF plus MTX and HYP and these results are similar to those referred by other investigators.

The ability to excise and repair various types of DNA damage is probably a general property of living cells. This is of considerable interest in the problem of healing cancer, because it provides a mechanism by which the rate of potential damage induced by chemotherapeutics may be modified [42]. In this context it may be useful to point out that EAT cells like human cells are proficient in excision repair. In our experiments we found that CAF, in combination with MTX, enhanced the survival time of EAT bearing mice and reduced the increase of body weight (e.g., the increase of tumor), significantly compared to controls.

The SCE assay has been proposed as having predictive value as a clinical assay for drugs for which a strong correlation between cell killing and induction of SCEs has been established [30]. In the present study, a sound correlation between SCE enhancement and PRI (r=−0.896, P<0.01) or MI (r=−0.892, P<0.01) suppression was identified. A very good correlation (r=+0.976, P<0.01) between cell division delays and MI suppression was also observed (Table 3). These findings suggest that a common element, possibly a particular type of DNA damage, produced by certain agents is responsible for inducing SCEs and reducing cell survival and cell growth. However, it should be mentioned that the relation of SCEs to toxicity may not hold for all agents, depending on the spectrum of lesions induced [38]

It has been proposed that the effectiveness in SCE induction in vitro [29] and in vivo [16, 29, 30] by antitumor agents, can be positively correlated with in vivo tumor response to these agents. Our present results, concerning the combined treatment of MTX plus CAF and/or HYP in vitro and MTX plus CAF in vivo, show a high correlation between the potency for in vitro and in vivo SCE results. We believe that it is possible to achieve increased effectiveness of MTX, and better therapy results with fewer side effects, if together with MTX, CAF is administered in non-toxic doses and if HYP is applied—either by using the same doses of MTX or even by decreasing them.

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