Mutation Research, 245 (1990) 27-32

27

Elsevier MUTLET 0391

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo J. L / i h d e t i e 1, R . R / i t y 2 a n d M . S o r s a 3 1Department of Medical Genetics, Institute of Biomedicine, University of Turku. SF-20520 Turku (Finland), 2National Public Health Institute, SF-00280 Helsinki (Finland) and 31nstitute of Occupational Health, SF-00250 Helsinki (Finland)

(Accepted27 April 1990)

Keywords: Mesna; Cyclophosphamide;Mutagenicity;Micronuclei

Summary The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.

Cytostatic drugs are powerful tools in treatment of cancer but they often have harmful side effects in normal tissues. Cyclophosphamide (CP), an oxazaphosphorine compound, is metabolically activated producing agents with alkylating ability. The most important metabolite is 4-hydroxy-CP which then generates acrolein and alkylating end-

Correspondence:J. L~ihdetie,Department of Medical Genetics, Institute of Biomedicine,Universityof Turku, Kiinamyllynkatu 10, SF-20520Turku (Finland).

metabolites, e.g., phosphoramide mustard (Brock, 1976). A very common side effect o f treatment with CP is hemorrhagic cystitis which is considered to be caused by acrolein (Brock et al., 1979; Cox, 1979). Urotoxicity o f CP and another oxazaphosphorine, iphosphamide, can be prevented by Mesna (sodium 2-mercaptoethanesulfonate) (Brock et al., 1982a; Scheef et al., 1979). Mesna is rapidly converted to an inactive disulfide form (Dimesna) and is eliminated via the kidneys where Dimesna is reduced back to the active thiol form, Mesna. In the urine Mesna binds to acrolein, and a stable,

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28 harmless thiother is formed. Mesna alos reacts with the primary metabolite 4-hydroxy-CP making a stable and non-toxic condensation product which additionally prevents the breakdown of 4-hydroxyCP and formation of acrolein (Ormstad et al., 1983). With abolition of bladder damage it is possible to administer CP and iphosphamide in higher doses and thus to improve their effectivity in cancer chemotherapy (Bryant et al., 1980). According to IARC (1981) there is sufficient evidence for the carcinogenicity of cyclophosphamide to humans; epidemiological studies have demonstrated excesses of various neoplasms, especially bladder cancer and leukemia. Bladder carcinogenesis may be induced by mutagenic insuits caused by the alkylating metabolites of CP in the urine and by induced cell proliferation due to cytotoxicity. Mesna decreases the incidence of bladder tumors induced in rats and humans by CP (Brock et al., 1982a; Habs and Schm~il, 1983). Recent studies support the view that this is a result of the reduced cytotoxic effects of acrolein (Kunze et al., 1984; Pool et al., 1988; Brock et al., 1982b). We studied the effects of Mesna in combination with CP to elucidate whether Mesna acts by decreasing the mutagenicity of metabolites of CP as suggested by in vitro studies on lymphocytes (Wilmer et al., 1986). Bone marrow mutagenicity was studied because CP therapy has been shown to be carcinogenic in the hematopoietic system in humans and in experimental animals (IARC, 1981; Habs and Schm~il, 1983; Schm~il and Habs, 1979). In addition, a methodological comparison was made between 2 different micronucleus (MN)staining techniques. Malerials and melhods Chemicals CP was obtained from Lfi~ikefarmos (Turku, Finland). Mesna was a gift of Dr. P. Hilgard, Asta Werke (Bielefeld, F.R.G.). Direct effects on Salmonella Mutagenicity of the compounds was tested by reversion to prototrophy with Salmonella

typhimurium strain TA1535 (base-pair substitution strain, kindly provided by Prof. Bruce Ames) with and without metabolic activation and with and without a 30-min pre-incubation of the compounds in solution with bacteria before adding them to agar in the plate-incorporation assay (Ames et al., 1975). Mesna and CP were dissolved in sterile water. Mutagenicities were determined in triplicate. The $9 mix was made from Aroclor 1254induced rat liver homogenate according to Ames et al. (1975). Effects on rats Adult male Wistar rats weighing about 250 g were obtained from the Laboratory Animal Center, University of Kuopio (Finland). They received CP 30 mg/kg i.p. a n d / o r Mesna 15 mg/kg i.p., both dissolved in sterile physiological saline. The injected volume was 5 ml/kg and controls received saline alone. The rats that were treated with both chemicals were first injected with Mesna and 5 min later with CP. The rats were put in metabolic cages (2 rats per cage) and they had access only to 2°70 saccharose water, 100 ml per cage during the experiment. The urine of the metabolic cages was collected during the rest of the life of the animals. The animals were killed with CO2 30 h after the injections. One femur of each rat was cleaned for bone marrow micronucleus analyses (Schmid, 1976). 1.5 ml fetal calf serum was injected into the bone marrow to release the contents into test tubes already containing 3.5 ml fetal calf serum. The cells were smeared on clean slides after suspension by Pasteur pipetting, air-dried, fixed in methanol for 10 min and air-dried. Since it is specially recommended for rat bone marrow MN analyses where Giemsa staining may yield high frequencies of basophilic granules, the double fluorescent staining by Hoechst 33258 and Pyronin Y (MacGregor et al., 1983) was used. MN were scored in both May-Grunwald-Giemsa-stained and fluorescently stained preparations. One smear per animal was stained with 5007o May-Grunwald solution for 3 rain, rinsed in distilled water for 1 min, stained with 10070 Giemsa for 10

29 min and rinsed several times with distilled water until the optimal differentiation between polychromatic (PCE) and normochromatic erythrocytes (NCE) was achieved. Another smear was stained in a foil-wrapped jar containing 60 ml phosphate-buffered saline (PBS), 400 #1 Hoechst 33258 (1.43 /~g/ml), and 600 #1 Pyronin Y (Merck, Darmstadt, F.R.G.) for 1 h at room temperature. Pyronin Y (1000 #g/ml aqueous stock solution) was extracted with chloroform 10 times, and the absorbance of the 1:10 working solution was determined at 545 nm. The slides were rinsed with sterile PBS and sterile water and mounted with sterile water. All solutions were filtered through 0.22/~m filters. Only a few slides were stained at a time. Scoring was performed on coded slides. In May-Grunwald-Giemsa preparations the proportion of PCEs was determined per 1000 NCEs, and the frequency of MN was counted in 1000 PCEs and 1000 NCEs per animal. In Hoechst-Pyronin Y-stained preparations the total number of unnucleated cells (erythrocytes) in each visual field was determined first by phase contrast. Secondly the PCEs were visualized and counted by N2 filter block in a Leitz epi-illumination fluorescence microscope. Thirdly, with filter block A the nuclear fluorescence was visualized. Always when a MN was seen, it was checked whether it was inside a PCE. A total of 1000 PCEs was scored per animal. 12.5 × magnifying oculars and 63 × dry objectives were used. 100 and 200 #I of urine from 2 rats was applied directly after filtration through 0.2-#m membrane filters to the top agar on Salmonella TA1535 with or without $9 mix (Ames et al., 1975). Statistical analysis of mutagenicity assays was done using Student's t-test. MN results were analyzed based on the Poisson distribution (L/ihdetie and Parvinen, 1981). Results of P C E s / N C E s ratio were analyzed by 2-way analysis of variance with repeated measurements by taking'the treatment as a grouping factor and the staining method as the within factor. Pairwise comparisons were performed by Student's t-test with Bonferroni's correction. A BMDP program package was used for these

calculations. The level of significance chosen was 0.05 for all determinations. Results

Mesna alone showed no mutagenicity in the Ames test while CP alone induced a dosedependent increase in the number of revertants but only in the presence of $9 mix (Fig. 1). In the presence of the metabolic activation system, coadministration of Mesna together with CP did not reduce the mutagenicity of CP (Fig. 1). Instead, a small increase in the number of revertants induced was observed which was, however, statistically not significant. There was no significant difference in the results between plates pre-incubated with Mesna for 30 min before adding CP and plates which were treated with Mesna and CP simultaneously (Fig. 1). In rat bone marrow Mesna alone did not induce MN in PCEs (Fig. 2A). CP caused an induction of increased MN frequencies which was not significantly affected by co-administration of Mesna. The same effects were observed irrespective of the staining and scoring method used (Fig. 2A). Slightly lower MN frequencies in controls and slightly higher CP-induced MN frequencies by Hoechst-Pyronin staining were observed (Fig. 2A). The ratio PCEs/NCEs was lowered by CP comNumber of revertants / plate 700 600 500

~]

CP

[]

C P * Mesno (no pf eincubahon j

[]

CP ° Mesno (30 m,n preincubahon ]

z.O0 300 200 100 0

125

250

i

500 ,ug added/plate

Fig. I. Number o f revertants per plate in the Ames test with Salmonella typhimurium strain TA1535 with metabolic activation (mean and SD of triplicate experiments). The a m o u n t of chemicals added is indicated on the horizontal axis. W h e n both cyclophosphamide and Mesna were added, both c o m p o u n d s were added in similar amounts.

30

A

B

Micronuclei / 1000 PCEs

Ratio

PCEs/NCEs

1.6 []

30

1.4 [ ] May Grunwald G,emsastaining [ ] Hoechsl-Pyronin staining -

May-Orunwald- Giemsa staining Hoechst -Pyronin staining

[]

1.2

-

1.0

20

0.8 0.6 10

0.4

[ ~ ~

0.2 CP

C P + Mesna

Mesna

CP

NoCI

CP+ Mesna

Mesna

II NoCI

Fig. 2. (A) Frequency of micronuclei in polychromatic erythrocytes. (B) ratio of polychromatic/normochromatic erythrocytes of rat bone marrow 30 h after injection of cyclophosphamide 30 m g / k g and Mesna 15 m g / k g using either May-Grunwald-Giemsa staining or Hoechst-Pyronin staining. Means and standard deviations are shown.

pared to controls or Mesna-treated rats, indicating bone marrow toxicity (Fig. 2B). This effect was, however, statistically significant only when the CPtreated group was compared with Mesna-treated rats. Co-administration o f Mesna with CP did not significantly improve the P C E / N C E ratio compared to CP alone (Fig. 2B). The P C E / N C E ratio was consistently higher in Hoechst-Pyroninstained slides than in May-Grunwald-Giemsastained slides (Fig. 2B). Two-way analysis o f variance showed no interaction between treatment

and staining method which means that, as to the effect o f treatments, similar results were obtained with both methods. The urine o f rats treated with Mesna showed no mutagenicity to Salmonella either in the presence or in the absence of $9 mix (Table 1). The urine o f rats treated with CP contained both direct and indirect mutagenicity. Rats that received Mesna in addition to CP had a little, but not significantly, higher mutagenic activity in their urine than those w h o received CP alone.

TABLE 1 M U T A G E N 1 C ACTIVITY OF T H E U R I N E OF RATS T R E A T E D W I T H C Y C L O P H O S P H A M 1 D E (CP) A N D / O R MESNA IN

Salmonella typhimurium STRAIN TA1535 Treatment

A m o u n t of urine/plate

Number of revertants/plate (mean of triplicate +_ SEM)

from 2 rats (~1)

With $9

Without $9

Saline control

100 200

11 _-2- 0 10 _+ 1

11 +_ 3 13 _+ 1

Mesna 15 m g / k g

100 200

10 +_ 2 16 +_ 0

12 _+ 1 14 _+ 2

C P 30 m g / k g

100 200

301 4-_ 69 463 _+ 155

216 _+ 69 381 + 89

CP 30 m g / k g and Mesna 15 m g / k g

100 200

329 _+ 156 600 _+ 117

297 _+ 80 509 _+ 140

31

Discussion The results of the Ames test of CP and Mesna are in agreement with earlier studies (Brock et al., 1982b; Pool et al., 1988). Mesna alone was not mutagenic, and when administered together with CP it could not reduce the mutagenicity of metabolites of CP. The number of revertants was, instead, slightly but not significantly higher than with CP alone. This may be due to a reduction of the bactericidal action of acrolein (Pool et al., 1988). In vivo, Mesna did not increase MN frequencies in the bone marrow. The result is consistent with negative findings in vitro when chromosome aberrations and sister-chromatid exchanges (SCEs) were studied in human lymphocytes (Becher et al., 1983; Wilmer et al., 1986). Mesna does not induce neoplasm in carcinogenicity assays (Brock et al., 1982a; Bryant et al., 1980). CP induces an increased frequency of bone marrow MN as has been shown in many studies (e.g., Trzos et al., 1978; Wild, 1978). Contrary to results obtained in vitro with human lymphocytes Mesna could not decrease the incidence of MN caused by CP. In the in vitro study (Wilmer et al., 1986) Mesna was able to decrease SCE frequencies and chromosome aberrations caused by phosphoramide mustard, a reactive intermediate of CP. The discrepancy may be due to the pharmacokinetics of Mesna. In rat blood Mesna is rapidly converted to an inactive disulfide form (Dimesna). Intracellular activation of Dimesna to Mesna has been observed in cytosolic fractions of rat kidney and liver homogenates (Ormstad et al., 1983). However, isolated hepatocytes were not able to take up Dimesna in contrast to isolated intestinal and kidney cells (Ormstad et al., 1983). Our results suggest that erythropoietic cells are not able to take up Dimesna or to reduce it to Mesna, which is supported by observations that Mesna does not interact with the leukotoxic effect of ifosfamide in rats (Brock et al., 1982b). The Hoechst-Pyronin staining recommended for rat bone marrow MN analyses (MacGregor et al., 1983) yielded slightly lower background MN fre-

quencies since the specific DNA staining helps to avoid artifacts. The results of PCE/NCE ratio determinations show the systematic difference between the 2 staining methods used: more PCEs were recognized with Hoechst-Pyronin staining, because NCEs and the background remain dark. However, this method is less practical because each field has to be scored with 3 different illuminations and because with Pyronin Y the obtained fluorescence is weak and variable. The urine of rats treated with CP either with or without Mesna was mutagenic to Salmonella as reported earlier (Pool et al., 1988; Willems and de Raat, 1985). A small but not significant increase in the number of revertants when rats received Mesna together with CP compared to CP alone could be due to reduced toxicity. Our result is different from that of Pool et al. (1988) who observed a slightly lower mutagenicity in the urine compared to treatment with CP alone. However, the authors could not clearly explain this result especially because in similar experiments using ifosfamide, a related compound, co-administration of Mesna to rats did not decrease urine mutagenicity. The present study gives further support to the theory that Mesna acts primarily via the detoxification of acrolein in the urinary tract and the abolition of bladder cancer risk is related to the decrease in hemorrhagic cystitis. Mesna has not been shown to inhibit the cytostatic activity of oxazaphosphorines to a measurable extent (Possinger et al., 1981; Brock et al., 1982b). Therefore, the mechanism by which Mesna exerts its anticarcinogenic action seems to be limited to the urinary tract and does not, in vivo, reduce the mutagenicity of alkylating metabolites of CP.

Acknowledgements We are grateful to Hilkka J/irventaus for technical assistance and Leena Koivusilta for help in the statistical analyses. References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the

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