Effects of cancer chemotherapeutic agents on testicular DNA synthesis in the rat

275

Mutation Research, 68 (1979) 275--289 © Elsevier/North-Holland Biomedical Press

EFFECTS OF CANCER CHEMOTHERAPEUTIC AGENTS ON TESTICULAR DNA SYNTHESIS IN THE RAT EVALUATION OF A SHORT-TERM TEST FOR STUDIES OF THE GENETIC TOXICITY OF CHEMICALS AND DRUGS IN VIVO

B. LAMBERT and G. ERIKSSON

Toxicology Laboratories, Astra Pharmaceuticals AB, 151 85 S6dertSlje (Sweden) (Received 27 March 1979) (Revision received 29 May 1979) (Accepted 14 June 1979)

Summary Changes in the rate of testicular DNA synthesis in the rat were studied at various times after single doses of 12 cancer chemotherapeutic agents. The animals were given intravenous injections of [~4C]thymidine and [3H]thymidine 24 and 3 h, resp., before they were killed. By combining measurements of the free serum radioactivity and the testicular incorporation of the differentially labelled precursor, different response patterns were obtained for agents with different modes of action. The DNA-damaging agents cyclophosphamide, chlorambucil, thio-TEPA, busulphan, CCNU and procarbacine, after some delay, caused a decrease of testicular thymidine incorporation and a corresponding increase of free serum radioactivity. The non-DNA-damaging agents 5-fluorouracil, methotrexate, hydroxyurea and cytosine arabinoside had a rapid effect on testicular thymidine incorporation and produced diverse response patterns different from that of the DNA-damaging agents. Actinomycin D and also vinblastine caused changes in testicular thymidine incorporation and showed response patterns different from those of the other agents. These results show that simple measurements of testicular DNA synthesis may provide useful information for the evaluation of genotoxic effects of chemical compounds and may help one to distinguish between DNA-damaging agents and metabolic inhibitors of DNA synthesis.

Studies of genetic toxicity and mutagenesis have become increasingly important in the toxicological evaluation of new pharmaceutical compounds [12]. Several short, term tests have been developed to facilitate the detection of genotoxic effects and to improve interpretations of mutagenic mechanisms [7]. The

276 testicular DNA synthesis inhibition (DSI) test [5,11] has promising features as a preliminary assay for genetic toxicity in vivo. The DSI test is simple and provides rapid results. It makes use of administration of the test compound in vivo, which accounts for pharmacokinetics and biotransformation in the intact animal. The testis as target tissue is of obvious relevance for the evaluation of genotoxic and mutagenic effects, and the results can be utilized in the design of subsequent studies on fertility and reproduction. Recent work by Friedman and Staub [5] and Seiler [11] has shown the potential value of the DSI test in the evaluation of mutagenic and carcinogenic effects of chemical agents. The implication of the DSI test response is that damage to DNA usually causes an inhibition of DNA synthesis. However, such an effect may be produced not only by DNA-damaging agents but also by other cytotoxic agents that act by, e.g., reducing the supply of precursors for DNA synthesis. Because it is important to be able to relate the test response to the mechanisms of drug toxicity, we have studied the DNA synthesis in rat testicular cells after treatment of the animals with various cytostatic agents that differ in their modes of action. In this work we show that DNA-damaging agents can be distinguished from non-DNA
Compounds tested and modes of administration The following 12 compounds were studied. Actinomycin-D, Cosmegen, Merck Sharp and Dohme; busulphan, Myleran, Burroughs Wellcome; chlorambucil, Leukeran, Burroughs Wellcome; 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, CCNU, Lundbeck; cyclophosphamide, Sendoxan, Pharmacia; cytosine arabinoside, Cytosar, Upjohn; 5-fluorouracil, Fluoro-uracil, Hoffman-La Roche; hydroxyurea, Sigma; methotrexate, Lederle; procarbazine, Natulanar, Hoffman-La Roche; thio-TEPA, Tifosyl, Astra; vinblastine, Velbe, Lilly. The compounds were administered according to clinical practice. Busulphan, chlorambucil and procarbazine were suspended in water and CCNU in 0.7% Metocel ® and given orally. The other compounds were dissolved or diluted in physiological saline and injected into a tail vein. Experimental design Male Sprague-Dawley rats were used in all studies. The experimental design is shown in Fig. 1. [3H]Thymidine and [14C]thymidine ([Me-3H]thymidine, 5 Ci/mM, 1 mCi/ml and [Me-14C]thymidine, 52 mCi/ mM, 50 #Ci/ml, the Radiochemical Centre, Amersham) were given by i.v. injection into a tail vein at the times indicated. The test compounds were always given before or concurrently with the injection of [14C]thymidine, and no administration of test compound took place during the 24 h before the rats were killed (Fig. 1). This timing of injections of [14C]thymidine and [3H]thymidine in relation to the drug administrations were chosen to obtain a maximal differentiation between immediate and delayed effects of the test compounds on the testicular DNA synthesis.

277

14C-thymidine (lo luCi) A d m i n i s t r a t i o n of test compounds

~ I

I

Days

I

~

21h

I

I

I

I

I

9

7

5

3

1

3H-thymidine (loo

iPCi) 3h i~,

0 Sacrifice

Fig. 1. T i m e s c h e d u l e o f d r u g a d m i n i s t r a t i o n a n d i n j e c t i o n s o f r a d i o a c t i v e t h y m i d i n e . I n d o s e - - e f f e c t s t u d i e s t h e t e s t s u b s t a n c e s w e r e given o n d a y 1 c o n c u r r e n t l y w i t h [ 1 4 C ] t h y m i d i n e " In t i m e - - e f f e c t s t u d i e s a single dose of t h e t e s t s u b s t a n c e w a s given 2 o r m o r e d a y s b e f o r e t h e rats w e r e killed.

Sampling and extraction procedures Immediately before the animals were killed they were anaesthetized in ether and a 0.5-ml sample of blood was taken b y a microcapillary in the orbital veins to determine serum radioactivity (see below). The animals were then killed b y bleeding; the testes were removed, freed of adhering material and weighed. The two testes from an animal were similarly treated b u t kept separate throughout the entire procedure. After decapsulation, the testis was homogenized b y 5 strokes of a glass pestle in a Dounce glass homogenizer containing 1 ml of 0.9% NaC1. The homogenate was transferred to a centrifuge tube, and extracted with 20 ml of ice-cold 5% TCA for 30 min under continuous shaking at 4°C. After centrifugation (1500 rev./min at 4°C) an additional extraction with 20 ml of 5% TCA was carried out. The pellet was then washed in 20 ml of ice-cold 70% ethanol and finally resuspended in 5 ml of water. To this suspension was added 0.5 ml of 2 M NaOH. The solution was shaken vigorously then left for 10 rain at room temperature to clarify. Samples containing 1 ml of this solution were transferred to scintillation vials, and 10 ml of Instagel (Packard) were added. The blood samples for serum activity measurements were left to coagulate for a b o u t 1--2 h at room temperature and then centrifuged. Duplicate serum samples of 100 #1 each were transferred to scintillation vials containing 1 ml of Soluene 350 (Packard) and kept at 60°C for 2 h. 10 ml of toluene scintillator (5.5 g of Permablend III, Packard, in 1000 ml of toluene, Merck) were added, and the activity was measured as described below.

Radioactivity measurement The radioactivity was measured in a Packard Tri-carb scintillation spectrometer. Quenching curves for measurements of 3H and 14C were established and provided for a channel setting that was optimal for the simultaneous measurement of 3H and 14C activity. Quenching was monitored with the aid of the external standard. Spill-over of 3H activity into the 14C channel was negligible. A b o u t 20% of the 14C activity was recovered in the 3H channel. This spill-over was corrected for and did n o t exceed 10% of the measured activity in the 3H channel in the control animals. The efficiency of the 3H activity measurement was 24%, and of the ~4C activity 61%.

Recovery of macromolecular radioactivity Enzymic digestions and h o t acid hydrolysis of the testicular tissue homo-

278

TABLE 1 R E C O V E R Y O F C O L D - T C A - I N S O L U B L E A C T I V I T Y I N T E S T I C U L A R H O M O G E N A T E S A F T E R ENZYMIC D I G E S T I O N AND H O T ACID H Y D R O L Y S I S Treatment

None Ribonuclease Pronase Deoxy Iibonuclease Acid h y d r o l y s i s

R e s i d u a l a c t i v i t y (%) 3H

14 C

IO0 95 93 9 11

96 94 73 19 16

genates were carried o u t to determine the proportion of the measured radioactivity that was incorporated into various macromolecular fractions. Animals were given [I4C]thymidine (10 #Ci) and [3H]thymidine (100 pCi) 24 and 3 h, respectively, before they were killed. The testicular homogenate was extracted with cold TCA and ethanol as described above, and then resuspended in 1 ml of 0.1 M acetate buffer (pH 5.0). Enzymic digestion was for 2 h at 37°C with ribonuclease (RNAase-IA, 1 mg/ml, Sigma), protease (ribonuclease-free pronase, 1 mg/ml, Calbiochem) or deoxyribonuclease (DNAase I, 1 mg, Sigma, in 1 ml of acetate buffer containing 0.005 M MgSO4). Samples incubated in acetate buffer without enzymes served as controls. After incubation, the tissue samples were extracted twice with cold TCA. The total activities in the pellet and TCA extracts were measured. Acid hydrolysis was carried out in 5% TCA at 90°C for 2 h and repeated 3 times. Table 1 shows the remaining 3H and I4C activities in the pellet as a percentage of the total activity of the sample (pellet + TCA-soluble activity). The results show that more than 90% of the 3H activity after 3 h incorporation in vivo, and 80--85% of the '4C activity after 24 h of incorporation, is DNAasesensitive. Thus only a very small proportion of the 3H activity is likely to be incorporated into other macromolecules than DNA, while a b o u t 10--15% of the '4C activity is probably confined to protease-sensitive material (Table 1). Measurements o f total radioactivity in serum and testicular tissue The conditions of incorporation were studied by measuring the total radioactivity in serum and testicular tissue after tail-vein injections of either 100 #Ci of [3H]thymidine or 10 #Ci of ['4C]thymidine. At times indicated in Fig. 2 the animals were anaesthetized with ether, and samples of blood were taken from the orbital plexus. Serum activities were measured as described above. The testes were removed and decapsulated. Small tissue samples of a b o u t 0.1 g were c u t out, weighed and immediately p u t into solubilizer (1.0 ml of Soluene 350, Packard) for 2.5 h at 60°C. After the addition of 10 ml of toluene scintillator the radioactivity was measured as described above. As shown in Fig. 2, there was a rapid decrease of the initial free serum radioactivity after injection of [3H]- as well as [I4C]-thymidine, indicating a fast tissue uptake. 3 and 24 h after injection the total radioactivity in the testicular tissue was close to that in serum.

279

25

cpm x I()3

~,5min

? 20

I

\ \

°°\°-. o 15

I0

. 5 rain

\ \

dk

0

3 5

8

24

Time after thymidine injection (h)

Fig. 2. Total radioactivity in serum and testis various times after intravenous injection of 100 #Ci of [3H]t h y m l d i n e and 10 #Ci of [ 1 4 C ] t h y m i d i n e . The symbols indicate me a ns of double samples from 2 animals: o, 3H activity in 100 #I of serum; @, 3H activity in 100 mg of testicular tissue; ~, 14C activity in serum; A, 14C activity in testis.

Kinetics of incorporation The kinetics of incorporation of [14C]thymidine into testicular DNA were measured to provide a rough basis for comparison with the [3H]thymidine incorporation. 10/~Ci of [14C]thymidine were given by tail-vein injection and the animals were killed at times indicated in Table 2. The 14C radioactivity of TCA-extracted testicular tissue was measured as described above. The data shown in Table 2 indicate that only 3 h after injection of [~4C]thymidine the TABLE 2 COLD-TCA-INSOLUBLE 14C ACTIVITY IN RAT TESTIS AT VARIOUS TIMES A F T E R INTRAVENOUS INJECTION OF [14C] THYMIDINE Time after injection (h)

3 5 24

Radioactivity cpm/g of testis ± S.D.

%

4079 ± 104 4807 ± 367 4863 ± 113

84 99 100

280

TABLE 3 CONTROL ANIMAL DATA a Number

Weight (g)

350 _+ 30 (311-424)

Serum activity c p m / 1 0 0 ~l

3H

3H

14 C

16 9 4 9 _+ 1 5 5 2 (13 9 2 9 - 21 1 8 5 )

1217 +_ 139 (952-1533)

Testis

Animal

61

Testicular incorporation c p m / g o f tissue w e i g h t

1.71 + 0.07 (1.60-1.89)

11 0 8 5 2 1750 (7943-15 1 5 0 )

14 C 5045 +- 6 3 8 (3883-6373)

a M e a n v a l u e s + S.D. I n p a r e n t h e s e s , r a n g e o f v a l u e s o b s e r v e d .

testicular incorporation a m o u n t e d to more than 80% of the level at 24 h. Accordingly, changes in the 14C incorporation observed 24 h after injection of [ ~4C]thymidine mainly reflect influences on DNA synthesis during the initial 3 h after injection.

Results from control animals One or more control animals were included in every dose--effect and time-effect study of each compound. Pooled data from all control animals are shown in Table 3, to provide information about the variability of the m e t h o d . The standard deviation of the serum activities is in the range of +10% of the mean value for 3H activity as well as 14C activity in spite of the wide range of animal weights. As will be shown later, the serum activity is a useful parameter for the interpretation of mechanisms of drug-induced inhibition of testicular DNA synthesis (see results). The testicular DNA incorporation of every experimental and control animal is expressed as the average cpm per g of testicular weight, and is based on double determinations of each testicle. As a rule, the measurements from the two testicles of a single animal were in close agreement (less than 10% difference), but differences between control animals were greater. The standard deviation is in the range of +15% of the mean values (Table 3). There was no correlation between incorporation value expressed as described and animal weight. Because several control animals were used in the evaluation of a single drug, data shown in the result section will be related to these average control data and n o t to current controls. To give some indication of the significance of changes observed, the standard deviation of the control values is shown in each figure, and the range of values recorded at the highest dose levels of each drug is indicated in the legend. Results

DNA-damaging agents The effects of 6 DNA-damaging agents 24 h after their administration are shown in Fig. 3. Cyclophosphamide caused a dose-dependent decrease of [3H]t h y m i d i n e incorporation into testicular tissue and a corresponding increase of free [3H]thymidine activity in the serum. No significant change was observed in the [ 14C]thymidine incorporation, or in the free 14C activity in the serum. Similar response patterns were observed after treatment with chlorambucil, thio-

281 -3

25 cpm x 10

/ ~0--- ~ ._.0 j > . _ - - --O

20

15

/

/

/

/

25'

f

/

/

20

/

Io /

c~// Chlorambucil

Cyclophosphamide

15

Thio-TEPA

z Q.._ I

2'5

7'5

300

~

615

1'2

~

_

_.o-- -- ---a

~

1'9

~6

116

m~kg

25"

cpm x 103

Io

/o.. /

20-

10'

/o / /

/ /

~'0

/

20

/o /

/

15"

2s,

/

15

Busulphan

CCNU

\

/

/

Procarbazine

---.... -

~ zD--

-

,.-EP-

-.-c,--

1~

2~

--~

3~

ZDl

~

~

--o--

;o

~',

3~

li5

3is

ai5

mg/kg

Fig. 3. S e r u m a c t i v i t y and testicular i n c o r p o r a t i o n o f radio-labelled t h y m i d i n e in t h e rat 2 4 h a f t e r a d m i n i s t r a t i o n o f D N A - d a m a g i n g a g e n t s in vivo. T h e t e s t s u b s t a n c e and [ 1 4 C ] t h y m i d i n e w e r e given 2 4 h b e f o r e rats w e r e killed and [ 3 H ] t h y m i d i n c 3 h b e f o r e . D a t a are t h e m e a n s o f d o u b l e s a m p l e s f ~ o m 2 t o 4 animais, e x p r e s s e d as c p m per g o f testicular tissue or 1 0 0 ~I o f s e r u m . S y m b o l s : 3H a c t i v i t y in testis ( o ) and s e r u m ( o ) ; 14C a c t i v i t y in testis ( s ) and s e r u m (D). T h e bars r e p r e s e n t m e a n values + S . D . o f p o o l e d c o n trol data. A t t h e h i g h e s t d o s e o f e a c h c o m p o u n d , all data f o r 3H activities in t h e s e r u m and testis w e r e higher and l o w e r , r e s p e c t i v e l y , t h a n any c o n t r o l d a t a r e c o r d e d (cf. Table 3).

TEPA, busulphan and CCNU. Procarbazine produced the same changes of testicular [SH]thymidine incorporation and serum activity as the other DNA-damaging agents, but in addition this drag caused a decrease of the [ 14C]tbymidine incorporation into testicular tissue (Fig. 3). These results suggest that the DNA-damaging agents studied inhibit testicular DNA synthesis in vivo. The increased level of [3H]thymidine in serum probably reflects a reduced utilization of the supplied precursor and indicates that the DNA synthesis is inhibited in other tissues as well. The absence of changes in 14C activity in testes and serum after administration of cyclophosphamide, chlorambucil, thio-TEPA, CCNU or busulphan suggests that with the limits of

282

25-

cprn J

25"

20-

Cp

BUS

cpm x 1()3

/

20"

\ \

/

o.. "" -.. O._ _.i ~- ---O

TT CCNU

15-

15" Procarbazine

10S

5'

T

1 '~ ~I .4 Days after dosage

[3--[3- --

~

(a)

--0-

-

-(3-

.~ ,~ .~ Days after dosage

-i ~

II

--[3

1'8

(b)

Fig. 4. T i m e course o f the effects o f D N A - d a m a g i n g agents o n s e r u m activity and testicular i n c o r p o r a t i o n o f radio-labelled t h y m i d i n e . (a) [ 3 H ] T h y m i d i n e activities for: c y c l o p h o s p h a r n i d e (CP), 75 m g / k g ; chlora m b u c i l (CA), 19 m g / k g ; b u s u l p h a n (BUS), 3 0 m g / k g ; CCNU, 3 0 m g / k g ; t h i o - T E P A (TT), 1.6 mgfkg. O p e n s y m b o l s d e n o t e c p m / 1 0 0 /~1 o f s e r u m and closed s y m b o l s c p m / g o f testicular tissue. (b) Procarbazine, 3 7 5 m g / k g . S y m b o l s as in Fig. 3.

resolution o f this method, there is a delay of several hours in the action of these drugs (cf. Fig. 2, Table 2). Similarly, procarbazine, which caused a significant reduction o f testicular incorporation o f [14C]thymidine, probably has a more rapid action than the 5 other drugs (Fig. 3). To study the time required to recover normal levels of testicular DNA synthesis, single doses of the test substances were given at increasing numbers of days before the injection of 3H- and 14C-labelled thymidine. The dose of each drug was o f the size that, according to the results of Fig. 3, would produce about 50% inhibition of testicular DNA synthesis 24 h after administration. As shown in Fig. 4, normal testicular DNA synthesis and serum activities were approached within 2--4 days for 5 o f the DNA-damaging agents. In contrast, procarbazine caused a significant reduction in testicular incorporation of [3H] thymidine and increase in 3H activity in serum for 7 days or more (Fig. 4b). Metabolic inhibitors o f DNA syn thesis

The predominant effect produced by hydroxyurea and cytosine arabinoside 24 h after administration was a decrease in the incorporation of [14C]thymi-

283 cpm x 103

25 cpm x 103

20

Cytosine arabinoside

Hydroxyurea

~'-"(N..

0.//

10,

100

200

400

mg/k,

()

3'0

1()0

1"/0

mg/kg

Fig. 5. S e r u m a c t i v i t y a n d t e s t i c u l a z i n c o r p o r a t i o n o f r a d i o - l a b e n e d t h y m i d i n e in t h e r a t 2 4 h a f t e r a d m i n i s t r a t i o n o f h y d r o x y u r e a a n d c y t o s i n e a r a b i n o s i d e . E x p r e s s i o n o f d a t a a n d s y m b o l s as in Fig. 3. All d a t a f o r 1 4 C a c t i v i t y in t h e testis a t t h e h i g h e s t d o s e levels o f b o t h c o m p o u n d s w e r e l o w e r t h a n a n y c o n t r o l d a t a r e c o r d e d (cf. T a b l e 3).

20- cpm x I03

15-

10

Days after dosage Fig. 6 . T i m e c o u r s e o f t h e e f f e c t s o f h y d r o x y u r e a ( 1 0 0 m g / k g , [] a ) a n d c y t o s i n e a~rabinoaide ( 3 0 m g / k g , o e) o n t e s t i e u l a z i n c o r p o r a t i o n o f [ 3 H ] t h y m i d i n e (m e ) a n d [ 1 4 C ] t h y m i d i n e (o o). D a t a axe e x p r e s s e d as e p m / g o f t e s t i c u l a r tissue.

284

25.

20-

c p m x 1o3

5- f l u o r o /

25" c p m x 103

/

/

.

uracil

Methotrexate

20.

~::~ //C~'-.- ~ 15.

15"

10,

10

~,01 /

i=

/ ~

=J'~

5

~_

__Q.-

D--

s'0

"/s

--D

z 13----- ~c~.- ~ ~(~__ -- ~

~

1"~o

io

mg/kg

3'o mg/kg

(a) -3 25" c p m x 10

/ 20

• /

~'

25.~ ,'pro x IG~

\ \

20

s-FU

15

Methotrexate

"D

= ~.._ O--- C-- - - --O-- - - __ __ ~:]

Days a f t e r d o s a g e

(b) Fig. 7. S e r u m 5-fluorouracil the exception

activity and testicular incorporation of radio4abelled thymidine after administration of and methotrexate. E x p r e s s i o n o f d a t a a n d s y m b o l s a s i n Fig. 3. (a) D o s e d e p e n d e n c e . With of 14C activity in the serum, all data at the highest dose levels of 5-fluorouracfl were higher

than any control data recorded. The 3H and 14C activities in the testis at the highest dose level of methotrexate were higher than any control data recorded'(cf. T a b l e 3). (b) T i m e c o u r s e o f e f f e c t s o f 5 - f l u o r o uracil (75 mg/kg) and methotrexate (10 mg/kg).

dine into testicular tissue. N o consistent changes were observed in the other parameters studied (Fig. 5). As soon as 2 days after administration, testicular incorporation was again normal (Fig. 6). These results suggest that hydroxyurea and cytosine arabinoside have a rapid onset and short duration of action, which may also explain the absence of change in free serum activity (cf. Fig. 5). Methotrexate and 5-fluorouracil showed patterns of response different from those of the other drugs studied (Fig. 7). They caused an increase in incorporation of both [ 3H] thymidine and [ 14C]thymidine into testicular tissue 24 h after administration. The free [3H]thymidine activity in the serum was raised after 5-fluorouracfl administration, whereas no such effect was observed with metho-

285 trexate (Fig. 7a). Both drugs had a rapid onset of action as evidenced by the change in [14C]thymidine incorporation (Fig. 7a), but the time for recovery of normal incorporation levels appeared to be longer for 5-fluorouracil than for methotrexate (Fig. 7b). Actinomycin D and vinblastine Both compounds caused a dose-dependent increase in the serum levels of free [3H]thymidine and [14C]thymidine 24 h after administration (Fig. 8a). In addition, vinblastine gave rise to an increase of [ 14C]thymidine incorporation into testicular tissue, but no other change of incorporation was observed with either drug (Fig. 8a). At later times after drug administration, when the serum

30' cpm x 103

25¸icpm x 103

/20

/

/

//O

.o

J

J 15,

Vinblestine

Act. D

10

:D'-"

~

~

--D

mg/kg

,~kg

(a)

cpm x 103

"J J' 19:

{T

IS,

~\\

Act. D

1o. ~

@

\

IS

~

I0

z O, .13--t;I---"13--~43-.--- _.O

Days after dosage

Days after dosage

(b)

Fig. 8. S e r u m activity and testiettlar i n c o r p o r a t i o n o f radio-labelled t h y m i d i n e after administration of a c t i n o m y c i n D and vinblastin'e. E x p r e s s i o n o f data and s y m b o l s as in Fig. 3. (a) Dose dependence. All data for s e r u m activities at the highest d o s e levels o f b o t h c o m p o u n d s w e r e higher t h a n a n y c o n t r o l d a t a r e c o r d e d (cf. Table 3), (b) T i m e course o f the effects o f a c t i n o m y c i n D (0.46 mg/ kg) and vinblastine (1.0 mg/kg).

286 activities had declined towards normal levels, the incorporation of [3H]- and [ 14C]-thymidine showed a transient increase of similar magnitude and duration for both actinomycin D and vinblastine (Fig. 8b). Discussion The toxicological evaluation of new pharmaceutical c o m p o u n d s usually involves extensive observations of drug toxicity in laboratory animals after administration in vivo. Lately, much effort has been p u t into studies of genetic toxicity, carcinogenesis and mutagenesis. The intention of this work was to evaluate the DSI test [5,11] as a convenient assay for genetic toxicity in combination with other animal studies used in the general toxicity evaluation of pharmaceutical compounds. Thus, the primary objective was n o t to improve the sensitivity or specificity of the test system, b u t to work o u t a simple procedure that allows some conclusions to be drawn a b o u t the possible interactions of the test substance or its metabolites with the genetic material in vivo. It is implicit b y our choice of test system that inhibition of DNA synthesis is considered a c o m m o n response to many types of potential genotoxic and mutagenic influence. Painter [10] recently showed that the rate of inhibition and recovery of DNA synthesis in mammalian cell cultures could be used as a rapid test to detect genotoxic damage and to distinguish between chemicals with different mechanisms of action. To examine this idea under conditions in vivo, we studied 4 simple parameters after dosing with exogenously supplied [3H]-and [14C]-thymidine. The incorporation of [3H]thymidine was used to measure DNA synthesis during a 3-h period one day or longer after administration of a drug. The incorporation of [ 14C] thymidine, which was measured during a 24-h period, mainly reflects DNA synthesis during the initial 3--5 h of this period (cf. Fig. 2 and Table 2). The serum activities of [3H]£hymidine and [14C]thymidine were measured at the time the rats were killed (i.e. 3 and 24 h, resp., after injection of the labelled precursors) and were used to obtain a rough indication of drug-induced changes in the uptake of precursors into b o d y tissues. Exogenously supplied thymidine is utilized for DNA synthesis via the thymidylate-kinase pathway. Metabolic conversion in vivo may, however, yield products that are available for synthesis of macromolecules other than DNA. Thus, radioactivity from labelled thymidine m a y be incorporated into protein, R N A and lipid material [14]. The control experiments (Table 1) show that this is a minor source of error in the present work, although the longer time available for incorporation of ['4C]thymidine results in some radioactivity in protease-sensitive material. Other sources of error in the DSI test could be drug-induced changes in precursor pools or precursor pathways leading to spurious alterations of thymidine incorporation into DNA. Evidence from work of Tew and Taylor [13] suggests that the pathway for thymidylate-kinase salvage operates with different efficiencies in various tissues. The results of the 5-fluorouracil and methotrexate experiments in this work (cf. Fig. 7) show that the testicular tissue of the rat is capable of utilizing the salvage p a t h w a y efficiently when de novo synthesis of thymidine is inhibited. Furthermore, the DSI test has a disadvantage in that effects due to changes in size of precursor pool can n o t be distinguished

287 TABLE TEST

4 RESPONSE

PATTERNS

Type of substance

a

Testicular incorporation 3H

DNA-damaging

14 C

Serum activity 3H

14 C

agents

C y clo p h o s P h a m i d e

~

--

t

Chloramb ucil

~

--

I"

Thio-TEPA

~

--

1"

Busulphan

~

--

I"

CCNU

~

--

1"

I

Proeazbazine

~

~

i'

i

I I

Metabolic

inhibitors

Hydzoxyurea arabinosid e

--

~

--

Cytosine

--

~

--

5-Fluorouracil

~

J"

?

Methotrexate

t

1'

--

--

--

t

--

1'

t'

Others Actinomycin Vinblastine a Symbols:

D

I" m e a n s i n c r e a s e , ~ d e c r e a s e and - - n o

change.

from changes due to a direct inhibition of DNA synthesis. Fractional incorporation, as described by Taylor et al. [6,13,14], would be a better estimate of true changes in DNA synthesis, although at the expense of a more demanding experimental procedure. The approach described in the present work is rapid, and as shown by the results compiled in Table 4, it is possible to distinguish between inhibitors of DNA synthesis with different mechanisms of action, which adds a further dimension to the original DSI test as described by Friedman and Staub [5] and applied by Seiler [11]. 5 of the DNA
288

TdR

5FU UDP

I

HU

UT P

~' d U D P

,- d U M P ~

dTMP

dCMP

dTDP l dT T P-

~' dCDP

~" dCTP-

~' dADP

~ dATP-

• d GDP

~' dGTP-

I

CT P CDP

J

/

l

HU

ADP

}

ar~C DNA /"~ ~

HU

GDP

[ HU

DNA-damage

Fig. 9. S i m p l i f i e d s c h e m e of p r e c u r s o r p a t h w a y s a n d the a p p r o x i m a t e s t e p s at w h i c h v a r i o u s i n h i b i t o r s m a y act. 5FU, 5 - f l u o r o u r a c i l ; MTX, m e t h o t r e x a t e ; araC, c y t o s i n e a r a b i n o s i d e ; HU, h y d r o x y u r e a .

natural precursors of DNA synthesis (Fig. 9). H y d r o x y u r e a inhibits the ribonucleoside diphosphate reductases and deprives the cells of deoxyribonucleotides [1,16]. Cytosine arabinoside m a y have a similar a c t i o n b u t its major effect is ascribed to competitive inhibition of DNA polymerase [ 1,3]. 5-Fluorouracil is converted intracellularly into the corresponding deoxynucleotide monophosphate which is a p o t e n t inhibitor of thymidylate synthetase and prevents the formation of thymidine de novo [2,9]. Methotrexate is a folic acid analogue which binds to the enzyme dihydrofolate reductase and thereby decreases in the intracellular pool of reduced folates required for biosynthesis o f thymidylate and purine [1,2]. The response pattern shown by h y d r o x y u r e a and cytosine arabinoside is in accord with the expectations o f these agents, which interact with the general supply of precursors for DNA synthesis or with the immediate incorporation of precursors into DNA. There was an immediate decrease of testicular incorporation of [14C]thymidine. No effect was observed on the incorporation o f [3H]thymidine, or on the serum activity levels, which indicates that the actiOn of these drugs is rapid and o f short duration (Table 4). Inhibition by 5-fluorouracil and b y methotrexate of thymidylate synthesis de novo caused an increase of testicular thymidine incorporation. This effect would be expected t ° be of variable extent and duration depending on the efficiency and duration b y which ithe salvage p a t h w a y is able to rescue some residual DNA synthesis. As indicated in Table 4, both 5-fluorouracil and methotrexate caused an increased incorporation of [3H]thymidine as well as of [14C]thymidine, suggesting a rapid onset of action and considerable duration of the effects. In the case of 5-fluorouracil the incorporation was n o t normalized even 7 days after a single dose of the drug (Fig. 7b) which supports the concept of a tight and almost irreversible binding between the active metabolite and the

289 enzyme [2,9]. In addition, 5-fluorouracil gave rise to increased 3H and 14C activities in serum up to several days after its administration (Fig. 7b), indicating that this drug reduces the utilization of exogenously supplied thymidine in other tissues. In testes, DNA synthesis seems to be rescued to a considerable extent, presumably owing to an efficient salvage via the thymidine-kinase pathway. The effect o f methotrexate on testicular incorporation is apparently less extensive and shorter, and no effect on serum activity levels was recorded (Fig. 7). The response patterns obtained by agents that may not affect DNA synthesis directly, but only via other cytotoxic mechanisms, is of course highly unpredictable. Nevertheless, it is interesting that both actinomycin D and vinblastine caused response patterns different from those of the other drugs discussed (Table 4). Taken together, these results show that simple measurements of thymidine incorporation into testicular DNA and of serum activity levels provide useful information for the evaluation of genotoxic agents and may help one to distinguish drugs acting by metabolic mechanisms from those causing structural damage to DNA. The major drawback of this approach would seem to be the relative insensitivity of the method, i.e. high doses of drugs are required to obtain measurable effects. However, this may not be a serious disadvantage when these studies are made in combination with the conventional toxicological LDs0 or dose-range-finding studies. Further investigations are necessary to explore the usefulness of this approach in the toxicological evaluation of the subchronic and chronic effects of lower doses. References 1 Calabresi, P., and R.E. Parks, in: L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 5th edn., MacMillan, New York, 1975, pp. 1254--1307. 2 Chabner, B.A., C.E. Myers, C.N. Coleman and D.G. Johns, The clinics/ pha rma c ol ogy of antineoplastic a g e n t s (First of two parts), N. Engl. J. Med., May 22 (1975) 1107--1158. 3 ibid., (Second of two parts), N. EngL J. Med., May 22 (1975) 1159--1168. 4 Ehling, U.H., Differential spermatogenic response of mice to the i n d u c t i o n of m u t a t i o n s by antineoplastic drugs, Mutation Res., 26 (1974) 285--295. 5 Friedman, M.A., and J. Staub, Inhibition of mouse testicular DNA synthesis by m u t a g e n s and carcinogens as a p o t e n t i a l simple mammAliAn assay for mutagenesis, Mut a t i on Res., 37 (1976) 67--76. 6 Houghton, P.J., and D.M. Taylor, Fractional incorPoration of [ 3 H ] t h y m i d i n e and DNA specific activity as assays of i n h i b i t i o n of turnout growth, Br. J. Cancer, 35 (1977) 68--77. 7 Klibey, B.J., M. Legator, W. Nichols and C. Ramel (Eds.), H a n d b o o k of Mutagenicity Test Procedures, Elsevier, Amsterdam, 1977. 8 Lee, I.P., and R.L. Dixon, Mutagenicity, earcinogenicity and teratogenicity of procarbazine, Mut a t i on R e s . , 55 (1978) 1--14. 9 Myers, C.E., R.C. Young and B.A. Chabner, Biochemical d e t e r m i n a n t s of 5-fluorouracfl response in vivo, J. Clin. Invest., 56 (1975) 1231--1238. 10 Painter, R.B., Rapid test to d e t e c t a g e n t s t h a t damage h u m a n DNA, Nature (London), 265 (1977) 650---651. 11 Seiler, J.P., Inhibition of testicular DNA synthesis by chemical mutagens and carcinogens, Preliminary results in the validation of a n o v e l s h o r t t e r m test, Mutation Res., 46 (1977) 305--310. 12 de Serres, F.J., Prospects for a revolution in t h e m e t h o d s of toxicological evaluation, Mut a t i on Res., 38 (1976) 165--176. 13 Tew, K.D., and D.M. Taylor, T h e e f f e c t o f m e t h o t r e x a t e o n t h e u p t a k e of de novo and salvage preeur* sors in to the DNA of rat t u m o u r s and normal tissues, Eur. J. Cancer, 13 (1977) 279--289. 14 Tew, K.D., and D.M. Taylor, The relationship of t h y m i d i n e me t a bol i s m t o t h e u s e of fractional incorp o r a t i o n as a measure of DNA synthesis and tissue proliferation, Eur. J. Cancer, 14 (1978) 153--168. 15 Weinkan, R.J., and D.A. Shlba, Metabolic activation of procarbazine, Life Sci., 22 (1978) 937--946. 16 Young, C.W., G. Sehochetman and D.A. Karnosky, H y d r o x y u r e a - i n d u c e d i n h i b i t i o n of de oxyri bonucleotide synthesis: Studies in i n t a c t cells, Cancer Res., 27 (1967) 526---534.