Testing in vitro of an indirect mutagen (cyclophosphamide) with human leukocyte cultures Activation by liver perfusion and by incubation with crude liver homogenate

95

Mutation Research, 54 (1978) 95--99 © Elsevier/North-Holland Biomedical Press

TESTING IN VITRO OF AN INDIRECT MUTAGEN (CYCLOPHOSPHAMIDE) WITH HUMAN LEUKOCYTE CULTURES ACTIVATION BY LIVER PERFUSION AND BY INCUBATION WITH CRUDE LIVER HOMOGENATE

ST..EPHAN MADLE a, DIETER WESTPHAL b, VOLKHARD HILBIG b and GUNTER OBE a a Institut fiir Genetik, Freie Universitd't Berlin, Arnirnallee 5--7, 1000 B E R L I N 33 (B.R.D.), and b Institut fiir Veterin~'rmedizin, Bundesgesundheitsamt Berlin, 1000 Berlin 33 (B.R.D.)

(Received 13 October 1977) (Revision received 27 December 1977) (Accepted 6 January 1978)

Summary The indirect mutagen cyclophosphamide was tested in human whole blood cultures with respect to chromosome-breaking activities and suppression of mitotic indices after activation by liver perfusion and crude liver homogenates with and without cofactors. Both methods produced nearly the same effects, with the exception of liver homogenate without cofactors which had only weak metabolic activities.

Introduction In previous papers we reported that the indirect mutagen cyclophosphamide (CP) induces chromatid translocations in human leukocytes in vitro after metabolic activation by a microsomal preparation of mouse liver [1,3] or by the Udenfriend system [4]. The activation product of CP was added to the cultures by including the activating principle and CP in a dialysis bag and submersing it for 3 h in the blood cultures [3] or by adding the activating principle and CP directly to the cultures for 3 h [4]. In this communication we report on two further possibilities for activating CP and testing the induction of chromatid translocations in human leukocytes in vitro: (1) perfusion of rat liver in situ with CP and direct addition o f the perfusate to the blood cultures, and (2) activation of CP with crude homogenate of mouse liver and addition to the cultures by means of dialysis bags.

96 Materials and Methods

(1) Perfusion o f rat liver The liver-perfusion apparatus was similar to that described elsewhere [2,5-8]. The whole apparatus was included in a thermo-cabinet allowing the perfusion to be carried out at 37°C. Male Wistar rats, 6--8 weeks old and about 300 g, were anesthesized by i.p. injection of 0.3 ml of a 6% solution of nembutal (Abbot}. After injection of 100 units of Liquemin (Hoffman-La Roche), the abdomen and the thorax were opened. The portal vein and the inferior vena cava were cannulated, and the abdominal vena cava and the hepatic artery were ligated. The liver was then perfused by recirculating 100 ml blood-free t y r o d e solution with 3% p-hydroxyethyl starch (Fresenius) which entered the liver by the portal vein and left it by the vena cava. The solution was saturated with oxygen by continuous bubbling of a mixture of 95% 02 and 5% CO2 through the perfusion medium. The perfusion was performed at a hydrostatic pressure of 18--20 cm. CP was added to the perfusion medium to give a final concentration of 20 mM. Before the liver was connected, the circuit was run for some minutes to achieve equilibrium. The first 10 ml of the perfusate were discarded to avoid contamination by blood. Three experiments were performed. The perfusion time was 1 h in the first two experiments, and 5 min in the third, while perfusion-rates of 20, 10 to 1.4 and 10 ml/min, respectively, were maintained. The same type of phytohemagglutinin-stimulated blood cultures was used as in our previous work with CP [3,4] (TC medium 199, 10% fetal bovine serum, 2.5-ml cultures in plastic tubes}. The cultures were incubated for 80 h including 8 h colcemide treatment (0.08 pg/ml). After sterilization by filtration and appropriate dilution with TC medium 199, 1-ml aliquots o f the perfusate were added to the cultures. Since it was n o t known what proportion of CP had been metabolized during the passage through the liver, its concentration in the cultures cannot be given. Disregarding any loss due to metabolism, the final CP concentrations in the cultures were 5.7, 2.9, 1.4, 0.7, 0.4 and 0.2 mM. 3 h after addition of the perfusate with CP the cultures were centrifuged, the pellet was washed once with pre-warmed medium and the cells were re-incubated for a further 29 h with conditioned medium from cultures set up in parallel. Control cultures received unperfused medium without CP. CP alone was not tested in these experiments, because this had been done with the same culture conditions previously [4]. All cultures were set up in parallel. Routine chromosome preparations were stained with Giemsa stain and analyzed with respect to mitotic indices by determining the percentage of metaphases in 2000 cells and frequencies of chromatid translocations by counting 451 up to 1063 mitoses. •

(2) Activation with crude mouse-liver homogenate Female NMRI mice were given phenobarbital (0.1%) for one week. Livers were prepared and frozen at --80°C. Crude liver homogenate was obtained by pressing the frozen liver through a sieve with holes o f l mm 2. This homogenate was mixed with an equal volume of pre-warmed (37°C) 18.6 mM phosphate buffer (pH 7.4) containing 16 mM MgC12 and 20 mM KC1. This activation mix

97 was tested with 5 mM NADP and 10 mM glucose 6-phosphate (both from Boehringer) and w i t h o u t cofactors. Immediately after the addition to the mixture of CP to a final concentrations of 20, 10, 5, 2.5, 1.3, 0.6, 0.3 and 0.2 mM, 1 ml of each mixture was filled into a sterilized dialysis bag and the bag was submersed in the cultures for 3 h -- from 48 to 51 h culture time -- as described [3]. Disregarding any loss due to metabolism, the final CP concentrations in the cultures can be calculated as 5.7, 2.9, 1.4, 0.7, 0.4, 0.2, 0.1 and 0.05 mM. After the incubation for 3 h the bags were removed, the cultures were centrifuged, the pellet was washed once with pre-warmed medium and the cells were re-incubated with conditioned medium from cultures set up in parallel for a further 30 h. In these experiments the culture time was 81 h with 9 h colcemide treatment included. Control cultures received CP-free homogenate with and w i t h o u t cofactors. CP alone was not tested, because this had been done earlier with the same culture conditions [3]. 3 independent experiments were done, and all cultures were set up in parallel. Routine Giemsa-stained preparations were analyzed with respect to mitotic indices by counting 2000 cells and determining the percentages of the mitoses and frequencies of chromatid translocations by counting 173 up to 1500 mitoses. Results and Discussion Table 1 shows the pooled data of the 3 experiments on activation of CP by liver perfusion. The unperfused perfusion medium alone had no influence on the mitotic indices and did not induce chromatid, translocations. Perfused medium containing CP led to a dose
TABLE 1 E F F E C T OF T R E A T M E N T O F 2.5-ml B L O O D C U L T U R E S W I T H 1 ml E A C H OF P E R F U S E D MEDIU M C O N T A I N I N G CP O N M I T O T I C I N D I C E S ( M I ) A N D R A T E S O F C H R O M A T I D T R A N S L O C A TIONS (RB') C u l t u r e t i m e 80 h, t r e a t m e n t t i m e 3 h, r e c o v e r y t i m e 29 h, c o l c e m i d e t r e a t m e n t 8 h. P o o l e d d a t a o f t h r e e experiments. CP c o n c n , in t h e test solution (raM) a

CP c o n c n , in t h e cultures (mM) a

MI in % o f MI in u n t r e a t e d control cultures b

RB' in 1 0 0 m i t o s e s (number of mitoses analyzed)

-- c 20.00 10.00 5.00 2.50 1.25 0.63

-5.71 2.86 1.43 0.71 0.36 0.18

109.3 5.8 17.9 42.6 66.0 88.3 94.6

0.0 (900) _ d 16.2 e (451) 9.8 ( 1 0 6 3 ) 5.5 ( 9 0 0 ) 0.4 (900) 0.0 ( 6 0 0 )

a C a l c u l a t e d f r o m t h e i n i t i a l l c o n c e n t r a t i o n s o f CP in t h e p e r f u s i o n m e d i u m . b T h e M I in u n t r e a t e d c o n t r o l s w a s d e f i n e d a s 1 0 0 % . c T h e s e c u l t u r e s w e r e t r e a t e d w i t h u n p e r f u s e d p e r f u s i o n m e d i u m w i t h o u t CP. d N o t e n o u g h m i t o s e s w e r e f o u n d for a n a l y s i s . e O n l y 2 e x p e r i m e n t s y i e l d e d e n o u g h m i t o s e s for a n a l y s i s .

98 TABLE 2 EFFECT OF TREATMENT OF 2.5-ml BLOOD CULTURES WITH lml EACH OF LIVER HOMOGEN A T E W I T H A N D W I T H O U T C O F A C T O R S A N D L I V E R H O M O G E N A T E A N D CP W I T H A N D W I T H OUT COFACTORS ON MI AND RB' C u l t u r e t i m e 81 h , t r e a t m e n t t i m e 3 h , r e c o v e r y t i m e 3 0 h, c o l c e m i d e t r e a t m e n t 9 h. P o o l e d d a t a o f t h r e e experiments. CP concn, in the test solution (mM)

CP concn, in the cultures (raM) a

Cofactors p r e s e n t (+) or a b s e n t (--)

MI in % of MI in untreated control cultures b

_ c _ c

_ _

-+

92.5 85.8

20.00 10.00 5.00 20.00 10.00 5.00 2.50 1.25

5.71 2.86 1.43 5.71 2.86 1.43 0.71 0.36

---+ + + + +

52.1 54.6 86.2 0.3 1.2 4.1 7.6 12.6

0.18 0.09 0.05

+ + +

8.2 30.0 72.5

0.63 0.31 0.16 f a b c d

Calculated from the The MI in untreated These cultures were Not enough mitoses

R B '1 in 100 mitoses (number of mitoses analyzed) 0.0 (600) 0.0 (600) 2.1 0.0 0.0 _ d _ d 15.7 9.2 10.5

(1500) (600) (600)

e (108) e (173) e (200)

6.4 ( 4 8 8 ) 7.5 (1500) 0.0 (400)

i n i t i a l c o n c e n t r a t i o n s o f CP in t h e liver h o m o g e n a t e . c u l t u r e s w a s d e f i n e d as 1 0 0 % . t r e a t e d w i t h l i v e r h o m o g e n a t e w i t h o u t CP. were found for analysis.

e Only one experiment yielded enough mitoses for analysis. f D a t a are b a s e d o n 2 e x p e r i m e n t s o n l y .

been treated with perfused medium containing 20 mM CP, the mitotic indices were nearly zero; with 10 mM CP, only 2 experiments yielded enough mitoses for analysis. The results of the experiments were the same irrespective of whether the liver was perfused for 1 h or for 5 min, indicating that the metabolic transformation of CP was completed in 5 min or t h a t a steady-state was reached between newly originating chromosome-breaking products and their decompositions. Table 2 shows the pooled data of the 3 experiments on activation of CP with crude liver homogenate. Liver homogenate with and w i t h o u t cofactors had no effect on mitotic indices or on chromosomes. Liver homogenate with CP, but w i t h o u t cofactors, led to a suppression of the mitotic indices at high CP concentrations only (20 and 10 mM in the liver homogenate) and to chromatid translocations at the highest CP concentration tested (20 mM). This activation system was much more effective in the presence of cofactors, suppressing the mitotic indices to nearly zero with 20 and 10 mM CP. With 5, 2.5 and 1.25 mM CP, only 1 of the 3 experiments yielded enough mitoses for analysis. Even with a CP concentration of 0.31 mM, chromatid translocations were induced. The dose--response kinetics in these experiments is unusual. This might be due to different metabolic capacities of the liver homogenates used. These experiments have shown that CP generates chromosome-breaking products in vitro after metabolic activation by rat-liver perfusion and by incu-

99 bation with crude mouse-liver homogenate. As has been shown previously, CP alone induced 1% chromatid translocations with the concentrations of 57 mM [3] and 0.1% with 10 mM [4]. This may be an outcome of a very low "spont a n e o u s " activation of CP (Brock, personal communication). As in our previous experiments with CP [3,4], so in these experiments a 1 : 1 distribution of symmetrical (s) and asymmetrical (a) chromatid translocations was found (perfusion experiments, 124 s and 126 a; liver homogenate experiments, 105 s and 110 a). Exchange aberrations of the chromosome-type (polycentric and ring chromosomes), which can be expected to occur as derived aberration types in second post-treatment mitoses, were not found. With a recovery time of 29-30 h, however, the appearance o f second mitoses in considerable amounts is not very probable. This is because of the cell-cycle