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Genetic activity of metabolites in the ascitic fluid and in the urine of a human patient treated with cyclophosphamide: Induction of mitotic gene conversion in Saccharomyces cerevisiae
Mutation Research, 21 (1973) 257-262
,t) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
257
GENETIC ACTIVITY OF METABOLITES IN T H E ASCITIC F L U I D AND IN T H E U R I N E OF A HUMAN P A T I E N T T R E A T E D W I T H CYCLOPHOSPHAMIDE : INDUCTION OF MITOTIC G E N E CONVERSION IN S A C C H A R O M Y C E S CEREVISIAE
D. SIEBERT AND *URSULA SIMON Forstbotanisches Institut der Universit~t Freiburg i. Br., and * Universitdtsfrauenklinik, Freiburg i. Br. (W. Germany)
(Received April ioth, 1973)
SUMMARY The genetic activity of cyclophosphamide (Cy) metabolites in the ascitic fluid and in the urine of a treated female human patient was compared. The test system used was induction of mitotic gene conversion in two unlinked gene loci of Saccharomyces cerevisiae. With a therapeutic dose of cyclophosphamide (3.1 mg/kg) a highly significant increase of eonvertants in both gene loci was observed in the urine. However, no rise in conversion frequency was observed after treatment of the yeast with ascitic fluid.
INTRODUCTION A possible induction of gene mutations or recombinations, by metabolites that appear in a mammal after treatment with a given substance, is normally tested by an injection of appropriate microorganisms into the ventral cavity of the mammal (hostmediated assay3,4). If such metabolites are excreted in the urine, yeast cells can be used in a "urinary assay". In rats treated with sublethal doses of Cy the induction of suppressor mutations has been observed and, in diploid strains of yeast cells, of mitotic gene conversions (intragenic recombinations)6, 9. In a recent study, the urinary assay was performed with the urine of a human patient treated with therapeutic doses of Cy", but a host-mediated assay in its strict sense cannot be made. In very serious cases of gynaecological cancer, ascitic fluid appears in the ventral cavity of some patients and should be removed. If this is done after a dose of Cy, yeast cells can be exposed to both fluids of the same patient, the ascites and the urine. In this way, not only the genetic activity of excreted metabolites of Cy but also the activity of metabolites existing in the ascites fluid can be determined and compared. In the present paper a comparison of the genetic activity of metabolites in both Abbreviation: Cy, cyclophosphamide.
258
D. S I E B E R T , U. S I M O N
fluids is r e p o r t e d ; the genetic test consisted of the i n d u c t i o n of m i t o t i c gene conversions (intragenic recombinations) in a diploid y e a s t strain, heteroallelic in two gene loci. B e t w e e n t h e convertogenic a n d m u t a g e n i c actions of substances a s t r o n g correl a t i o n exists~2; this has also been d e m o n s t r a t e d for m e t a b o l i t e s in the urine of r a t s t r e a t e d w i t h Cy s. MATERIAL AND METHODS
F o r t h e i n d u c t i o n of m i t o t i c gene conversion the diploid, respiration-deficient s t r a i n D 4 - R D I I (ref. 9) was used which requires adenine or t r y p t o p h a n for growth. Gene conversions p r o d u c e cells no longer requiring either adenine or t r y p t o p h a n or, rarely, cells requiring n e i t h e r adenine nor t r y p t o p h a n . The cells were c u l t u r e d in 5 ml Y E P m e d i u m (1% Difco y e a s t e x t r a c t , 2 % Difco b a c t o peptone, 2 % glucose) in test t u b e s on a r o t a r y s h a k e r at 25 ° u n t i l t h e y just reached the s t a t i o n a r y phase. Samples of o.I ml from each t u b e were s p r e a d on solid m e d i u m selective for conv e r t a n t s to d e t e c t cultures w i t h a low c o n t e n t of s p o n t a n e o u s c o n v e r t a n t s . O n l y these cultures were used in the conversion test. Media selective for c o n v e r t a n t s a n d for scoring s u r v i v a l were s y n t h e t i c m e d i a n s u p p l e m e n t e d with amino acids a n d nucleo-
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Fig. I. Induction of mitotic gene conversion in the unlinked loci ade2 and trp5 in the diploid, respiration-deficient strain D4-RD II of Saccharomyces cerevisiae by cyclophosphamide. Convertogenic activity of the urine of a treated female patient (2 × 2o0 mg i.v. = 2 × 3.1 mg/kg at 2 successive days). O - - O , survivors; ---, trp5; , ade2.
MUTAGENIC ACTION OF CYCLOPHOSPHAMIDE
259
bases s and solidified with 1.5% Difco bacto agar. Convertants were selected on medium lacking either adenine or tryptophan. All samples of urine and aseitic fluid were collected from one female patient (.39 years old, 65 kg) who received, shortly after operation for ovarian carcinoma, 200 mg Cy i.v. (3.1 mg/kg) every day. The renal excretion of the patient was already disturbed: there was a high retention of urea and ereatinine in the blood serum (117. 7 and 2 mg per IOO ml). During the excretion of two successive doses, samples of urine and ascitic fluid were collected. Immediately after collection the samples of urine and aseitic fluid were deepfrozen. Usually i day later they were thawed and filtered to sterilize them (Sartorius membrane filter, type SM 113o7) and then used in a conversion test. For the conversion test, yeast cells were washed twice in distilled water. Ahout lO 8 cells were incubated in test tubes, containing 2 ml of a mixture of 3 parts sterile urine or ascitic fluid, with one part o.I M potassium phosphate buffer (pH 7). This dilution gave the most reliable results with yeasts 1°. In the diluted urine or in the ascitic fluid, the yeasts were treated for 8 h at 25 ° under shaking. The incubation was terminated by dilution, and the cells were washed twice in distilled water, followed by storage in distilled water for 12 h (liquid holding). On medium selective for convertants about lO s cells per plate were spread out; to determine survival o.I ml containing 25o cells was plated on complete medium. The plastic petri dishes were incubated at 25 °, and colonies were counted after 6 days. Statistics were performed with the help of the tables of KASTENBAUMAND BOWMAN5. RESULTS
(z) Conversion frequency in the urine On two successive days, the patient received each day i injection of 200 mg Cy dissolved in glucose solution: this corresponded to a dose of 3.1 mg/kg. The solution was injected every day at io a.m. The samples of urine were collected during two days and one night at intervals of 2 or 4 h. The corresponding conversion frequencies of the samples are shown in Fig. I for the adenine- and tryptophan-gene locus. About 2 h after the first injection the frequencies began to increase, until about 12 h later a peak appeared. The conversion frequency then decreased, and, 24 h after injection, control values were reached again. After the second injection, on the following morning the excretion of genetically active metabolites proceeded more quickly: 4 h later the peak appeared, followed by a decrease which could only be traced until 6 p.m. when the last sample had been collected. The earlier effect of the second injection was probably not caused by a stimulation of the metabolizing enzymes in the liver, since, before the beginning of our investigation, the patient had received several injections of Cy. The frequencies in both gene loci ran strictly parallel, but the tryptophan locus showed a stronger reaction than the adenine locus as soon as higher amounts of active metabolites were present. In the survival curve of our yeast cells unexpected peaks appeared. Between 2 and 3 h after both injections the percentage of survivals increased sharplyto over lOO°/0 and reached a maxinmm of more than 15O°/o. This can be interpreted as a consequence of the excretion of substances which supported growth and division of yeast cells during several hours. The peaks of the survival curves do not
260
D. SIEBERT, U. SIMON
coincide with the peaks of the conversion frequency; after the first injection, the survival reaches its maximum earlier than the curve of conversion frequencies ; after the second injection the conversion comes first.
(2) Comparison of conversion frequencies in urine and ascitic fluid Simultaneously with the collection of urine, 4 and 8 h after the first injection two samples of ascitic fluid of the ventral cavity were taken. 2 h after the second injection a further sample was collected. After the exposure of our yeast cells during 8 h the values in Table I were obtained. These urine values are the same as those in the TABLE I
Saccharomyces cerevisiae, DIPLOID RESPIRATION-DEFICIENT STRAIN D e - R D II C o m p a r i s o n of t h e i n d u c t i o n of m i t o t i c gene c o n v e r s i o n b y ascitic fluid a n d u r i n e d i l u t e d 3 : I of a p a t i e n t t r e a t e d w i t h c y c l o p h o s p h a m i d e (two injections of 2o0 m g in 2 successive days). N u m b e r s in p a r e n t h e s e s indicate a c t u a l n u m b e r s of c o n v e r t a n t colonies c o u n t e d p e r 5 plates. No. Time of after injection inj. (h) I II
A scitic fluid Convertants/Io e survivors loci ade2 trp5
Control 18. 7 (68) 4 6.3 (24) 8 18.7 (44) 2 9.5 (32)
13.8 lO.4 8.1 13.4
(5 o) (4 o) (19) (45)
Survival (%)
Urine Convertants]Io 6 survivors loci ade2 trp5
Survival (%)
ioo.o lO5.4 64.7 92.5
18. 7 77.3 87.2 85. 7
ioo.o 165.1 134.1 99.5
(68) (464) (425) (31o)
13.8 119.1 128.o 118.9
(5 o) (715) (624) (43 o)
curve of Fig. I. They show the range of the single experiments combined in Fig. I. In all urine samples, the active metabolites of Cy exerted a strong effect on the conversion frequencies of both gene loci. In the samples of the ascitic fluid collected synchronously with the urine, the conversion frequencies showed no increase. Therefore in the ascitic fluid the concentration of active metabolites must have been so low that the conversion frequency of both gene loci was not influenced. The percentage of surviving cells in the ascitic fluid seemed to be changed to a lesser degree than in the urine. CONCLUSIONS
(~) The sensitivity of the conversion system Because the results were obtained from one single patient only few conclusions may be drawn by comparison with earlier results in man and mammals. Again, as in another human patient 1°, the conversion system applied to urinary assays showed a high sensitivity. With a dose of only 3.1 mg/kg (total dose 200 mg) a highly significant increase of convertants in both gene loci was observed in the urine. The high activity of the urine seemed not to be caused by a high metabolizing activity of the urinary system. Pharmacological data show that in no organ except the liver was there any activity of enzymes metabolizing Cy 1. Compared with a high effect in the urine, in the ascitic fluid of the same patient no rise in the conversion frequency was seen. This contrast - - h i g h sensitivity in the urine and negative results in the ascitic fluid-- can be explained by several observations and pharmacalogical data. The con-
MUTAGENIC ACTION OF CYCLOPHOSPHAMIDE
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c e n t r a t i o n of active m e t a b o l i t e s in the blood of m a n is a b o u t l O % lower t h a n in e x p e r i m e n t a l animals, e.g. sheep 2. The ratio of c y t o s t a t i c a c t i v i t y of the m e t a b o l i t e s (killing of Y o s h i d a ascites cells) in blood s e r u m a n d urine is i : 128 (ref. I). The conc e n t r a t i o n of active m e t a b o l i t e s in t h e serum a n d in the ascific fluid c o m p a r e d with t h a t in blood is y e t u n k n o w n b u t it can be e x p e c t e d t h a t the ascitic fluid contains fewer m e t a b o l i t e s . F u r t h e r , h o s t - m e d i a t e d assays with b a c t e r i a or yeasts, injected into the v e n t r a l c a v i t y of mice or ratsT, 9, showed t h a t o n l y with v e r y high doses of Cy were (weak) results o b t a i n e d . Hence, w i t h t h e r a p e u t i c (loses, even after t r e a t m e n t of ascitic fluid with sensitive genetic m e t h o d s positive results can h a r d l y be expected. This is stressed b y our n e g a t i v e results even t h o u g h the t r e a t e d p a t i e n t showed increased concent r a t i o n s of m e t a b o l i t e s in the ascitic fluid b y the existing d y s f u n c t i o n of excretion. (2) The excretion of active metabolites in the urine
I n investigations with r a d i o a c t i v e Cy in sheep a n d with the a l k y l a t i o n (NBP)test in m a n a n d several l a b o r a t o r y a n i m a l s ~ a p e a k of active m e t a b o l i t e s a p p e a r e d 4 - 6 h after the a p p l i c a t i o n of the dose. The m a i n excretion of r a d i o a c t i v e m e t a b o l i t e s a n d Cy in sheep lasts 24-48 h 2. Our p r e s e n t results, c o m p a r e d w i t h the urine of a female p a t i e n t t r e a t e d with 4 infusions of Cy TM, are n o t c o n t r a d i c t o r y : peaks of active m e t a b o l i t e s a p p e a r e d in b o t h cases b e t w e e n 4 a n d 12 h after the application. The d u r a t i o n of the e x c r e t i o n seemed to v a r y w i t h the dose. Here, 24 h later, the conversion frequency again reached the control value. I n the other p a t i e n t , who received higher doses 23 h after t h e beginning of infusion, the control value was not y e t reached. The a i m of our pilot e x p e r i m e n t s w i t h urine and ascitic fluid of a h u m a n p a t i e n t was to show t h a t a conversion or m u t a t i o n s y s t e m in m i c r o o r g a n i s m s was sensitive enough to be used to solve some p h a r m a c o k i n e t i c p r o b l e m s otherwise only to be s t u d i e d in vitro. ACKNOWLEDGEMENT
This w o r k was s u p p o r t e d b y the D e u t s c h e Forschungsgemeinschaft. REFERENCES I BROCK, N., AND H. J. HOHORST, ~]ber die Aktivierung yon Cyclophosphamid in vivo und in vitro, Arzneimittel-Forsch., 13 (1963) lO21 xo3i. 2 SCHAUML/3FFEL, E., A. HABERMEHL, N. BROCK UND B. SCHNEIDER, Studies on the pharmacokinetics of cyclophosphamide in sheep, Arzneimittel-Forsch., 23 (1973) 491-5oo. 3 FAHRIG, R., Metabolic activation of aryldialkyltriazenes in the mouse: Induction of mitotic gene conversion in Saccharomyces cerevisiae in the host-nlediated assay, Mutation Res., 13 (1971 ) 436-4394 GABRIDGE, M. G., AND M. S. LEGATOR, A host mediated assay for the detection of nmtagenic compounds, Proc. Soc. Exptl. Biol. Med., 13o (1969) 831 834. 5 I~ASTENBAUM, M. A., AND K. O. BOWMAN, Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9 (197o) 527-549 • 6 MARQUARDT, H., AND D. SIEBERT, Ein neuer host mediated assay (Urinversuch) zum Nachweis mutagener Stoffe mit Saccharomyces cerevisiae, Naturwissenschaften, 58 (1971) 568. 7 PROPPING,P., G. R6HRBORN AND W. BUSELMAIER,Comparative investigations onthe chemical induction of point mutations and dominant lethal mutations in mice, Mol. Gen. Genet., 117 (I972) I97-2o9.
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8 ROMAN, H., A s y s t e m selective for m u t a n t s effecting t h e s y n t h e s i s of a d e n i n e in yeast, Compe. Rend. Trac. Lab. Carlsberg, Ser. Physiol., 26 (1956) 229-314 . 9 SIEBERT, I)., A n e w m e t h o d for t e s t i n g genetically active m e t a b o l i t e s : U r i n a r y a s s a y w i t h c y c l o p h o s p h a m i d e ( E n d o x a n , C y t o x a n ) a n d Saccharomyces cerevisiae, Mutation Res., 17 (1973) 3o7-314 . IO SIEBERT, D., AND W. SIMON, C y c l o p h o s p h a m i d e : pilot s t u d y of genetically active m e t a b o l i t e s in t h e u r i n e of a t r e a t e d h u m a n p a t i e n t . I n d u c t i o n of m i t o t i c gene conversion in yeast, 211utation Res., 19 (1973) 65 72. I I VV'ICKERHAM, L. J., A critical e v a l u a t i o n of t h e n i t r o g e n a s s i m i l a t i o n t e s t c o m m o n l y u s e d in t h e classification of yeast, J. Bacteriol., 52 (1946) 293-3Ol. 12 ZIMMERMANN, V. I~., I n d u c t i o n of m i t o t i c gene c o n v e r s i o n b y m u t a g e n s , Mutation Res., II (1971 ) 327-337.
,t) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
257
GENETIC ACTIVITY OF METABOLITES IN T H E ASCITIC F L U I D AND IN T H E U R I N E OF A HUMAN P A T I E N T T R E A T E D W I T H CYCLOPHOSPHAMIDE : INDUCTION OF MITOTIC G E N E CONVERSION IN S A C C H A R O M Y C E S CEREVISIAE
D. SIEBERT AND *URSULA SIMON Forstbotanisches Institut der Universit~t Freiburg i. Br., and * Universitdtsfrauenklinik, Freiburg i. Br. (W. Germany)
(Received April ioth, 1973)
SUMMARY The genetic activity of cyclophosphamide (Cy) metabolites in the ascitic fluid and in the urine of a treated female human patient was compared. The test system used was induction of mitotic gene conversion in two unlinked gene loci of Saccharomyces cerevisiae. With a therapeutic dose of cyclophosphamide (3.1 mg/kg) a highly significant increase of eonvertants in both gene loci was observed in the urine. However, no rise in conversion frequency was observed after treatment of the yeast with ascitic fluid.
INTRODUCTION A possible induction of gene mutations or recombinations, by metabolites that appear in a mammal after treatment with a given substance, is normally tested by an injection of appropriate microorganisms into the ventral cavity of the mammal (hostmediated assay3,4). If such metabolites are excreted in the urine, yeast cells can be used in a "urinary assay". In rats treated with sublethal doses of Cy the induction of suppressor mutations has been observed and, in diploid strains of yeast cells, of mitotic gene conversions (intragenic recombinations)6, 9. In a recent study, the urinary assay was performed with the urine of a human patient treated with therapeutic doses of Cy", but a host-mediated assay in its strict sense cannot be made. In very serious cases of gynaecological cancer, ascitic fluid appears in the ventral cavity of some patients and should be removed. If this is done after a dose of Cy, yeast cells can be exposed to both fluids of the same patient, the ascites and the urine. In this way, not only the genetic activity of excreted metabolites of Cy but also the activity of metabolites existing in the ascites fluid can be determined and compared. In the present paper a comparison of the genetic activity of metabolites in both Abbreviation: Cy, cyclophosphamide.
258
D. S I E B E R T , U. S I M O N
fluids is r e p o r t e d ; the genetic test consisted of the i n d u c t i o n of m i t o t i c gene conversions (intragenic recombinations) in a diploid y e a s t strain, heteroallelic in two gene loci. B e t w e e n t h e convertogenic a n d m u t a g e n i c actions of substances a s t r o n g correl a t i o n exists~2; this has also been d e m o n s t r a t e d for m e t a b o l i t e s in the urine of r a t s t r e a t e d w i t h Cy s. MATERIAL AND METHODS
F o r t h e i n d u c t i o n of m i t o t i c gene conversion the diploid, respiration-deficient s t r a i n D 4 - R D I I (ref. 9) was used which requires adenine or t r y p t o p h a n for growth. Gene conversions p r o d u c e cells no longer requiring either adenine or t r y p t o p h a n or, rarely, cells requiring n e i t h e r adenine nor t r y p t o p h a n . The cells were c u l t u r e d in 5 ml Y E P m e d i u m (1% Difco y e a s t e x t r a c t , 2 % Difco b a c t o peptone, 2 % glucose) in test t u b e s on a r o t a r y s h a k e r at 25 ° u n t i l t h e y just reached the s t a t i o n a r y phase. Samples of o.I ml from each t u b e were s p r e a d on solid m e d i u m selective for conv e r t a n t s to d e t e c t cultures w i t h a low c o n t e n t of s p o n t a n e o u s c o n v e r t a n t s . O n l y these cultures were used in the conversion test. Media selective for c o n v e r t a n t s a n d for scoring s u r v i v a l were s y n t h e t i c m e d i a n s u p p l e m e n t e d with amino acids a n d nucleo-
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Fig. I. Induction of mitotic gene conversion in the unlinked loci ade2 and trp5 in the diploid, respiration-deficient strain D4-RD II of Saccharomyces cerevisiae by cyclophosphamide. Convertogenic activity of the urine of a treated female patient (2 × 2o0 mg i.v. = 2 × 3.1 mg/kg at 2 successive days). O - - O , survivors; ---, trp5; , ade2.
MUTAGENIC ACTION OF CYCLOPHOSPHAMIDE
259
bases s and solidified with 1.5% Difco bacto agar. Convertants were selected on medium lacking either adenine or tryptophan. All samples of urine and aseitic fluid were collected from one female patient (.39 years old, 65 kg) who received, shortly after operation for ovarian carcinoma, 200 mg Cy i.v. (3.1 mg/kg) every day. The renal excretion of the patient was already disturbed: there was a high retention of urea and ereatinine in the blood serum (117. 7 and 2 mg per IOO ml). During the excretion of two successive doses, samples of urine and ascitic fluid were collected. Immediately after collection the samples of urine and aseitic fluid were deepfrozen. Usually i day later they were thawed and filtered to sterilize them (Sartorius membrane filter, type SM 113o7) and then used in a conversion test. For the conversion test, yeast cells were washed twice in distilled water. Ahout lO 8 cells were incubated in test tubes, containing 2 ml of a mixture of 3 parts sterile urine or ascitic fluid, with one part o.I M potassium phosphate buffer (pH 7). This dilution gave the most reliable results with yeasts 1°. In the diluted urine or in the ascitic fluid, the yeasts were treated for 8 h at 25 ° under shaking. The incubation was terminated by dilution, and the cells were washed twice in distilled water, followed by storage in distilled water for 12 h (liquid holding). On medium selective for convertants about lO s cells per plate were spread out; to determine survival o.I ml containing 25o cells was plated on complete medium. The plastic petri dishes were incubated at 25 °, and colonies were counted after 6 days. Statistics were performed with the help of the tables of KASTENBAUMAND BOWMAN5. RESULTS
(z) Conversion frequency in the urine On two successive days, the patient received each day i injection of 200 mg Cy dissolved in glucose solution: this corresponded to a dose of 3.1 mg/kg. The solution was injected every day at io a.m. The samples of urine were collected during two days and one night at intervals of 2 or 4 h. The corresponding conversion frequencies of the samples are shown in Fig. I for the adenine- and tryptophan-gene locus. About 2 h after the first injection the frequencies began to increase, until about 12 h later a peak appeared. The conversion frequency then decreased, and, 24 h after injection, control values were reached again. After the second injection, on the following morning the excretion of genetically active metabolites proceeded more quickly: 4 h later the peak appeared, followed by a decrease which could only be traced until 6 p.m. when the last sample had been collected. The earlier effect of the second injection was probably not caused by a stimulation of the metabolizing enzymes in the liver, since, before the beginning of our investigation, the patient had received several injections of Cy. The frequencies in both gene loci ran strictly parallel, but the tryptophan locus showed a stronger reaction than the adenine locus as soon as higher amounts of active metabolites were present. In the survival curve of our yeast cells unexpected peaks appeared. Between 2 and 3 h after both injections the percentage of survivals increased sharplyto over lOO°/0 and reached a maxinmm of more than 15O°/o. This can be interpreted as a consequence of the excretion of substances which supported growth and division of yeast cells during several hours. The peaks of the survival curves do not
260
D. SIEBERT, U. SIMON
coincide with the peaks of the conversion frequency; after the first injection, the survival reaches its maximum earlier than the curve of conversion frequencies ; after the second injection the conversion comes first.
(2) Comparison of conversion frequencies in urine and ascitic fluid Simultaneously with the collection of urine, 4 and 8 h after the first injection two samples of ascitic fluid of the ventral cavity were taken. 2 h after the second injection a further sample was collected. After the exposure of our yeast cells during 8 h the values in Table I were obtained. These urine values are the same as those in the TABLE I
Saccharomyces cerevisiae, DIPLOID RESPIRATION-DEFICIENT STRAIN D e - R D II C o m p a r i s o n of t h e i n d u c t i o n of m i t o t i c gene c o n v e r s i o n b y ascitic fluid a n d u r i n e d i l u t e d 3 : I of a p a t i e n t t r e a t e d w i t h c y c l o p h o s p h a m i d e (two injections of 2o0 m g in 2 successive days). N u m b e r s in p a r e n t h e s e s indicate a c t u a l n u m b e r s of c o n v e r t a n t colonies c o u n t e d p e r 5 plates. No. Time of after injection inj. (h) I II
A scitic fluid Convertants/Io e survivors loci ade2 trp5
Control 18. 7 (68) 4 6.3 (24) 8 18.7 (44) 2 9.5 (32)
13.8 lO.4 8.1 13.4
(5 o) (4 o) (19) (45)
Survival (%)
Urine Convertants]Io 6 survivors loci ade2 trp5
Survival (%)
ioo.o lO5.4 64.7 92.5
18. 7 77.3 87.2 85. 7
ioo.o 165.1 134.1 99.5
(68) (464) (425) (31o)
13.8 119.1 128.o 118.9
(5 o) (715) (624) (43 o)
curve of Fig. I. They show the range of the single experiments combined in Fig. I. In all urine samples, the active metabolites of Cy exerted a strong effect on the conversion frequencies of both gene loci. In the samples of the ascitic fluid collected synchronously with the urine, the conversion frequencies showed no increase. Therefore in the ascitic fluid the concentration of active metabolites must have been so low that the conversion frequency of both gene loci was not influenced. The percentage of surviving cells in the ascitic fluid seemed to be changed to a lesser degree than in the urine. CONCLUSIONS
(~) The sensitivity of the conversion system Because the results were obtained from one single patient only few conclusions may be drawn by comparison with earlier results in man and mammals. Again, as in another human patient 1°, the conversion system applied to urinary assays showed a high sensitivity. With a dose of only 3.1 mg/kg (total dose 200 mg) a highly significant increase of convertants in both gene loci was observed in the urine. The high activity of the urine seemed not to be caused by a high metabolizing activity of the urinary system. Pharmacological data show that in no organ except the liver was there any activity of enzymes metabolizing Cy 1. Compared with a high effect in the urine, in the ascitic fluid of the same patient no rise in the conversion frequency was seen. This contrast - - h i g h sensitivity in the urine and negative results in the ascitic fluid-- can be explained by several observations and pharmacalogical data. The con-
MUTAGENIC ACTION OF CYCLOPHOSPHAMIDE
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c e n t r a t i o n of active m e t a b o l i t e s in the blood of m a n is a b o u t l O % lower t h a n in e x p e r i m e n t a l animals, e.g. sheep 2. The ratio of c y t o s t a t i c a c t i v i t y of the m e t a b o l i t e s (killing of Y o s h i d a ascites cells) in blood s e r u m a n d urine is i : 128 (ref. I). The conc e n t r a t i o n of active m e t a b o l i t e s in t h e serum a n d in the ascific fluid c o m p a r e d with t h a t in blood is y e t u n k n o w n b u t it can be e x p e c t e d t h a t the ascitic fluid contains fewer m e t a b o l i t e s . F u r t h e r , h o s t - m e d i a t e d assays with b a c t e r i a or yeasts, injected into the v e n t r a l c a v i t y of mice or ratsT, 9, showed t h a t o n l y with v e r y high doses of Cy were (weak) results o b t a i n e d . Hence, w i t h t h e r a p e u t i c (loses, even after t r e a t m e n t of ascitic fluid with sensitive genetic m e t h o d s positive results can h a r d l y be expected. This is stressed b y our n e g a t i v e results even t h o u g h the t r e a t e d p a t i e n t showed increased concent r a t i o n s of m e t a b o l i t e s in the ascitic fluid b y the existing d y s f u n c t i o n of excretion. (2) The excretion of active metabolites in the urine
I n investigations with r a d i o a c t i v e Cy in sheep a n d with the a l k y l a t i o n (NBP)test in m a n a n d several l a b o r a t o r y a n i m a l s ~ a p e a k of active m e t a b o l i t e s a p p e a r e d 4 - 6 h after the a p p l i c a t i o n of the dose. The m a i n excretion of r a d i o a c t i v e m e t a b o l i t e s a n d Cy in sheep lasts 24-48 h 2. Our p r e s e n t results, c o m p a r e d w i t h the urine of a female p a t i e n t t r e a t e d with 4 infusions of Cy TM, are n o t c o n t r a d i c t o r y : peaks of active m e t a b o l i t e s a p p e a r e d in b o t h cases b e t w e e n 4 a n d 12 h after the application. The d u r a t i o n of the e x c r e t i o n seemed to v a r y w i t h the dose. Here, 24 h later, the conversion frequency again reached the control value. I n the other p a t i e n t , who received higher doses 23 h after t h e beginning of infusion, the control value was not y e t reached. The a i m of our pilot e x p e r i m e n t s w i t h urine and ascitic fluid of a h u m a n p a t i e n t was to show t h a t a conversion or m u t a t i o n s y s t e m in m i c r o o r g a n i s m s was sensitive enough to be used to solve some p h a r m a c o k i n e t i c p r o b l e m s otherwise only to be s t u d i e d in vitro. ACKNOWLEDGEMENT
This w o r k was s u p p o r t e d b y the D e u t s c h e Forschungsgemeinschaft. REFERENCES I BROCK, N., AND H. J. HOHORST, ~]ber die Aktivierung yon Cyclophosphamid in vivo und in vitro, Arzneimittel-Forsch., 13 (1963) lO21 xo3i. 2 SCHAUML/3FFEL, E., A. HABERMEHL, N. BROCK UND B. SCHNEIDER, Studies on the pharmacokinetics of cyclophosphamide in sheep, Arzneimittel-Forsch., 23 (1973) 491-5oo. 3 FAHRIG, R., Metabolic activation of aryldialkyltriazenes in the mouse: Induction of mitotic gene conversion in Saccharomyces cerevisiae in the host-nlediated assay, Mutation Res., 13 (1971 ) 436-4394 GABRIDGE, M. G., AND M. S. LEGATOR, A host mediated assay for the detection of nmtagenic compounds, Proc. Soc. Exptl. Biol. Med., 13o (1969) 831 834. 5 I~ASTENBAUM, M. A., AND K. O. BOWMAN, Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9 (197o) 527-549 • 6 MARQUARDT, H., AND D. SIEBERT, Ein neuer host mediated assay (Urinversuch) zum Nachweis mutagener Stoffe mit Saccharomyces cerevisiae, Naturwissenschaften, 58 (1971) 568. 7 PROPPING,P., G. R6HRBORN AND W. BUSELMAIER,Comparative investigations onthe chemical induction of point mutations and dominant lethal mutations in mice, Mol. Gen. Genet., 117 (I972) I97-2o9.
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