No cyclosporin-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro

Mutation Research, 320 (1994) 217-221 © 1994 Elsevier Science B.V. All rights reserved 0165-1218/94/$07.00

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MUTGEN 1967

No cyclosporin-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro T.S.B. Zwanenburg * and A. C o r d i e r Sandoz Pharma Ltd., Drug Safety Assessment, Toxicology, 4002 Basel, Switzerland (Received 15 June 1993) (Revision received 5 August 1993) (Accepted 6 August 1993)

Keywords: Cyclosporin A; Mitotic index; Human peripheral blood lymphocytes

Summary Continuous in vitro treatment of human peripheral blood lymphocytes with cyclosporin A for 20 h and 44 h in the absence of metabolic activation decreased the mitotic index in a concentration-dependent way (MI about 35% at 50/xg/ml). In the presence of 10% $9 lymphocytes were exposed to cyclosporin A at concentrations which formed cloudy suspension, i.e., above 100 /zg/ml. A 2 h treatment in the presence of metabolic activation showed a depression of the mitotic index at the 44 h fixation time only. Based upon results concerning the depression of the mitotic index, slides of the 5, 10, 25 and 5 0 / z g / m l ( - $ 9 , 20 h and 44 h continuous treatment) and 5, 150, 250 and 300/xg/ml (+$9, 2 h pulse treatment) treatment groups were selected to be analyzed in the chromosomal aberration assay. At no concentration and at no fixation time did cyclosporin A increase the frequency of cells with chromosomal aberrations.

Cyclosporin A is an important immunosuppressive agent isolated from the fungus Tolypocladium inflaturn, which is used in the prevention of rejection of organ transplants. In vitro as well as in vivo experiments were performed during the past years to assess the genotoxic potential of cyclosporin A (Matter et al., 1982; Zwanenburg et al., 1988). Briefly, the data show that cyclosporin A, up to maximum tolerated dose levels, did not induce gene mutations in the standard microsome-Salmonella test using five different strains, nor in the V79 HGPRT mutation assay. No treatment related increases in

* Corresponding author.

SSDI 0165-1218(93)E0092-3

chromosome mutations were observed in the micronucleus test in mice and Chinese hamsters, the chromosomal aberration test in Chinese hamster bone marrow cells, or the dominant lethal mutation test in male mice (Matter et al., 1982; Zwanenburg et al., 1988). Pharmacokinetic investigations showed that cyclosporin A was well absorbed and penetrated well into the bone marrow of mice and hamsters (Matter et al., 1982). Peak plasma levels ranged from 4.8 +__0.5 to 13.6 + 4.5 /xg/ml. The absence of genotoxic properties of cyclosporin A was confirmed when DNA repair was measured in sperm heads of mice after treatment with cyclosporin A or azathioprine. While the latter clearly increased the incorporation of [3H]thymidine, cyclosporin A had no such effect (Matter et al., 1982).

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It has been reported that in comparison to other immunosuppressive agents (6-mercaptopurine, methotrexate, and mizoribine), SCE frequencies are highest after methotrexate and the lowest after cyclosporin A treatment at concentrations that decrease the mitotic index drastically (Yuzawa et al., 1986, 1987). Chromosomal aberration frequency has been reported to be increased in peripheral blood lymphocytes of patients treated with prednisolone, either together with azathioprine or with cyclosporin A (Fukuda et al., 1987, 1988). The present paper reports the results of a study carried out to investigate whether cyclosporin A has an effect on the chromosomes of human peripheral lymphocytes in vitro at cytotoxic concentrations both in the absence of $9 and at high, precipitating concentrations in the presence of 10% $9. Materials and methods

Test compound Cyclosporin A, batch 86001. Positive control chemicals EMS (ethyl methanesulfonate), 5 mM CP (cyclophosphamide), 55 /zM in the presence of 10% $9. Blood collection Peripheral blood from healthy donors was collected in tubes containing the anticoagulant heparin, and immediately incubated with culture medium at 37°C. Blood culture Blood cultures were initiated by adding 0.5 ml blood to 5 ml culture medium (Ham's F10) which contained 20% fetal calf serum, 27.3 ml/1000 ml NaHCO 3 (4.4%). Phytohemagglutinin-M, 10 m l / l of a 200 mM glutamine stock solution and 20 ml/1 of a penicillin and streptomycin solution (final concentration 200 I U / m l and 200/xg/ml, respectively). Blood treatment About 48 h after initiation of the cultures, the ceils were treated with cyclosporin A, either con-

tinuously (without $9) or for 2 h (in the presence of 10% $9). Treatment with EMS and CP was for 3 h and 2 h, respectively. The chemicals, dissolved and diluted in DMSO (cyclosporin A) or in PBS (EMS, CP), were applied by adding 50 /zl solution to the culture flasks. Fixation occurred 20 h or 44 h after start of the treatment (after 2 h in colcemid). Treatment in the presence of $9 occurred in 15 ml tubes after transferring the cells from the culture flasks and replacing the culture medium by 5 ml of the $9 mixture after centrifugation. After 2 h incubation at 37°C and 5% CO 2 with the test substance, the cells were washed once with 5 ml F10 and reincubated in fresh culture medium at 37°C and 5% CO 2 for various time periods.

Blood fixation The cell cultures were transferred to centrifuge tubes and the flasks were washed once with 5 ml Hanks' BSS. After 5 min 1000 rpm centrifugation, the cells were treated with hypotonic solution (10 min at 37°C in 0.075 M KCI). The cells were fixed in methanol/acetic acid (3/1), with three changes. Air-dried slide preparations were made from the fixed cell suspensions. Blood analysis Slides were stained with Giemsa 2% in a 40 mM sodium phosphate buffer, and mounted in Depex, coded and analyzed for the presence of chromosomal aberrations according to well-known methods (Savage, 1976; Scott et al., 1983; Dean and Danford, 1985). When possible, 100 metaphases per culture (46 chromosomes per cell) were analyzed and the following structural aberrations were recorded: chromatid breaks (including deletions), isolocus breaks, chromosome breaks, all the various forms of chromatid exchanges, dicentrics, tricentrics etc., ring chromosomes, and interstitial deletions. Cells with more than five aberrations were recorded as multiply aberrant cells. Chromatid gaps (G') and isolocus gaps (G") were also recorded. 1000 cells were counted per slide to determine the mitotic index.

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Activation system The $9 liver homogenate was prepared according to the procedure of Maron and Ames (1983). 7-9 week old male OFA rats (Biological Research Laboratories, BRL, Ffillinsdorf) were injected with 500 m g / k g Aroclor 1254, 5 days before being sacrificed. The livers were homogenized and centrifuged for i0 min at 9000 rpm (approximately 9000 × g). The supernatant was

frozen in small aliquots and stored at -70°C to -80°C until use. The $9 mix was made in the following way: NADP (2.25 g/l) and glucose-6-phosphate (G-6P, 5.07 g/l) were dissolved in distilled water on the day of treatment and sterilized by filtration. One volume of $9 was diluted with two volumes of 0.15 M KC1. Then, per liter, 100 ml Hanks' BSS (10 × concentrated), 300 ml NADP/G-6-P solution, 292 ml H20, 8.00 ml NaHCO 3 (4.4%) and 300 ml $9 fraction were mixed and stored on ice. The final concentration of the $9 fraction was therefore 10% of the treatment mixture (v/v).

TABLE 1 MITOTIC INDEX AFTER T R E A T M E N T WITH CYCLOSPORIN A First fixation

Second fixation

8.0% 5.8% 8.1%

6.1% 5.4% 9.4%

0 8 1 2 6 36 105 118 99 -

19 19 38 69 67 76 74 85 79

104 100 107 111 127 119 147 117 108

57 79 104 89 90 92 100 68 109

Control a

Medium Medium + 1% DMSO 10% $9+ 1% DMSO Without $ 9 b

300/zg/ml 250/zg/ml 200/~g/ml 150/x g / m l 100/~ g / m l 75/,g/ml 50/zg/ml 25/zg/ml 10/zg/ml 7.5/zg/ml 5.0/.t g / m l 2.5 btg/ml 1.0 p,g/ml With 1 0 % $ 9 b

300/.t g / m l 250/zg/ml 200/~g/ml 150/~g/ml 100/zg/ml 50/.Lg / m l 25/zg/ml 10/zg/ml 5/~g/ml

Without $9, continuous treatment; With $9, 2 h treatment. Cells were fixed 20 h and 44 h after start of the treatment at the first and second fixation, respectively. a Mitotic indices as % of mitotic cells within the total population of mitotic and interphase cells. b Mitotic indices as percentage of the control values. - , not determined.

Results Solubility and stability Cyclosporin A dissolved well in DMSO. However, cloudy suspensions were obtained at concentrations > 100 /zg/ml in culture medium at 1% DMSO. Mitotic index Continuous treatment of lymphocytes with cyclosporin A (20 h and 44 h) in the absence of metabolic activation decreased the mitotic index in a concentration-dependent way (Table 1, MI about 35% at 50 /zg/ml). Cyclosporin A was relatively nontoxic in the presence of 10% $9 (Table 1). The compound was therefore tested in a concentration range above the visible solubility limit. Based upon results concerning the depression of the mitotic index, slides of the 5, 10, 25 and 50 /zg/ml ( - $ 9 , 20 h and 44 h continuous treatment) and 5, 150, 250 and 300/zg/ml (+$9, 2 h pulse treatment) treatment groups were selected to be analyzed in the chromosomal aberration assay. Chromosomal aberrations Table 2 shows that cyclosporin A, tested in two separate experiments, did not increase the frequency of cells carrying structural chromosomal aberrations at concentrations which decreased the mitotic index in a concentration-dependent way ( - $9, 20 h and 44 h continuous treatment) nor at concentrations in the presence of 10% $9 which were well above the solubility limit.

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Discussion

The lack of chromosomal aberration induction obtained in this study supports the previously established absence of genotoxic potential for cyclosporin A in experimental systems (Matter et al., 1982; Zwanenburg et al., 1988). In this context it is difficult to understand the results of Fukuda et al. (1988, 1987). In their study 62% and 68% of the kidney transplant patients undergoing long-term treatment with prednisolone in combination with azathioprine or cyclosporin A, respectively, had an increased frequency of abnormal cells. Since these percentages seem unusually high they need some consideration. (i) In these studies gaps and breaks were pooled. Unfortunately, the presentation of the data does not allow an evaluation without gaps. Since gaps are not considered to represent true chromosomal aberrations, the number of patients with an increased frequency of cells with true

chromosomal aberrations is, therefore, likely to be overestimated. (ii) The data suggest that when a patient was encountered who showed one or more cells with gaps only, this patient was scored as abnormal. No statistical analysis was performed in order to decide objectively whether patients had significantly enhanced numbers of cells with chromosomal aberrations in comparison to historical control data. (iii) A frequency of 1.42 + 0.96% lymphocytes containing chromosomal aberrations (without gaps) has been reported in healthy volunteers (calculated from Ashby and Richardson, 1985), while the frequency of gaps might vary from 0 to 13% and from 0 to 6% in males and females, respectively (Richardson at al., 1984). A frequency of 0% abnormal cells in 50 control individuals, as observed by Fukuda et al., is in sharp contrast to the cited control frequency. (iv) The presence of some cells with chromosome-type aberrations (dicentric, ring and trans-

TABLE 2 CHROMOSOMAL ABERRATION ANALYSIS AFTER CYCLOSPORIN A TREATMENT First fixation Cells analyz,

Second fixation

% Abn. cells + gaps

% Abn. cells - gaps

% Cells with exch.

Cells analyz,

% Abn. cells + gaps

% Abn. cells - gaps

% Cells with exch.

Control Medium Medium + 1% DMSO 10% $9 + 1% DMSO

200 200 200

0.50 0.50 0.00

0.00 0.50 0.00

0.00 0.00 0.00

200 200 200

1.00 1.50 2.00

1,00 1.00 0.50

0.00 0.00 0.00

Without $9 5 t~g/ml 10/~ g / m l 25/zg/ml 50/z g / m l EMS, 5 mM

200 200 200 200 200

0.50 0.50 2.00 1.00 13.50

0.00 0.50 1.00 0.00 4.00

0.00 0.00 0.00 0.00 0.50

200 200 200 200 126

0.50 1.50 0.50 1.50 30.16

0.00 0.00 0.00 1.00 19.84

0.00 0.00 0.00 0.00 5.56

With 10% $9 5 ~g/ml 150/z g / m l 250 ~ g / m l 300 ,u,g/ml CP, 55/zM

200 200 . 200 200

0.50 0.00 . 1.50 16.50

0.00 0.00

0.00 0.00

0.50 13.50

0.00 3.00

200 200 200 200 200

0.50 3.00 1.50 0.50 10.50

0.00 0.50 0.50 0.50 5.50

0.00 0.00 0.00 0.00 2.00

.

.

Without $9, continuous treatment; with $9, 2 h treatment. Ceils were fixed 20 h and 44 h after start of the treatment at the first fixation and second fixation, respectively. -, not determined.

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location) suggests that some patients may have received radiotherapy. In the experimental setup blood was used from healthy individuals or hemodialysis patients. Since it is known that chromosomal aberrations may be detected in lymphocytes of individuals a long time after exposure to clastogens, blood from the patients before start of treatment should have been used as a patientrelated control. (v) None of the patients received azathioprine or cyclosporin A alone. The role of the accompanying chemical (prednisolone) has not been evaluated. A knowledge-based structure-activity relational expert system (CASE) has recently predicted that cyclosporin A is devoid of mutagenicity, clastogenicity and DNA-modifying activity (Rosenkranz and Klopman, 1992), which is in concordance with the experimental results obtained over the past years. References Ashby, J., and C.R. Richardson (1985) Tabulation and assessment of 113 human surveillance cytogenetic studies conducted between 1965 and 1984, Mutation Res., 154, 111133. Dean, B.J., and N. Danford (1985) Assays for the detection of chemically-induced chromosome damage in cultured mammalian cells, in: S. Venitt and J.M. Parry (Eds.), Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, pp. 187-232.

Fukuda, M., I. Aikawa, Y. Ohmori, N. Yoshimura, I. Nakai, S. Matui and T. Oka (1987) Chromosome aberrations in kidney transplant recipients, Transplant. Proc., 19, 22452247. Fukuda, M., Y. Ohmori, I., Aikawa, N. Yoshimura and T. Oka (1988) Mutagenicity of cyclosporine in vivo, Transplant. Proc., 20, 929-930. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Matter, B.E., P. Donatsch, R.R. Racine, B. Schmid and W. Suter (1982) Genotoxicity evaluation of cyclosporin A, a new immunosuppressive agent, Mutation Res., 105, 257264. Richardson, C.R., C.A. Howard, T. Sheldon, J. Wildgoose and M.G. Thomas (1984) The human lymphocyte in vitro cytogentic assay: positive and negative control observations on 30,000 cells, Mutation Res., 141, 59-64. Rosenkranz, H.S., and G. Klopman (1992) A structural analysis of the genotoxic and carcinogenic potentials of cyclosporin A, Mutagenesis, 7, 115-118. Savage, J.R.K. (1976) Annotation: Classification and relationships of induced chromosomal structural changes, J. Med. Genet., 13, 103-122. Scott, D., N.D. Danford, B.J. Dean, D. Kirkland and C.R. Richardson (1983) In vitro chromosome aberration assays, in: B.J. Dean (Ed.), Report of the Sub-Committee on Guidelines for Mutagenicity Testing, United Kingdom Environmental Mutagen Society, Swansea, pp. 41-64. Yuzawa, K., I. Kondo, K. Fukao, Y. Iwasaki and H. Hamaguchi (1986) Mutagenicity of cyclosporine, Induction of sister chromatid exchange in human cells, Transplantation, 42, 61-63. Yuzawa, K., K. Fukao, Y. Iwasaki and H. Hamaguchi (1987) Mutagenicity of cyclosporine against human cells, Transplant. Proc., 19, 1218-1220. Zwanenburg, T.S.B., W. Suter and B.E. Matter (1988) Absence of genotoxic potential for cyclosporine in experimental systems, Transplant. Proc., 20, 435-437.