Mutagenesis by N-acetoxy-2-acetyl aminofluorene of Chinese hamster V79 cells is unaffected by caffeine

Chem.-Biol. Interactions, 21 (1978) 1--18 © Elsevier/North-Holland Scientific Publishers Ltd.

1

MUTAGENESIS BY N-ACETOXY-2-ACETYL AMINOFLUORENE OF CHINESE HAMSTER V79 CELLS IS UNAFFECTED BY CAFFEINE B R I A N C. M Y H R

and J O S E P H A. D I P A O L O

Biology Branch, Somatic Cell Genetics Section, National Cancer Institute,Bethesda, Md. 20014 (U.S.A.)

(Received July 25th, 1977) (Revision received November l l t h, 1977) (Accepted December 13th, 1977)

SUMMARY

8-Azaguanine (AZG)- and 6-thioguanine (TG)-resistant cells (mutants) were induced in Chinese hamster V79-4 cells by 0.1--2.5 pg/ml N-acetoxy-2acetyle aminofluorine (AcAAF) treatments in the presence of 5% fetal bovine serum (FBS). The frequency of resistant colonies increased from 1 to 47 per l 0 s survivors. The effect of caffeine (50--200 ~g/ml) during the mutagenesis expression period was determined by adding caffeine 1--24 h after AcAAF. The medium was replaced after 48 h exposures so that caffeine was absent during subsequent selection with AZG or TG. No significant change in the AcAAF-induced mutant frequency occurred with any treatment combination although caffeine greatly enhanced the lethality associated with AcAAF treatments. Thus, caffeine interferes with postreplication repair in V79-4 cells without affecting the probability of error of the repair process. These results were obtained with a quantitative mutagenesis assay in which the cells were reseeded prior to selection to achieve maximum expression without interference from metabolic crossfeeding. In contrast, the commonly used in situ assay is subject to serious interference from crossfeeding and yields an artifactual enhancement of AcAAF mutagenesis by caffeine. INTRODUCTION

Caffeine has numerous effects on cultured m armmalian cells that differ qualitatively and quantitatively from one cell type to another [1]. One of its actions in most rodent cell cultures is a potentiation of lethality in cells previously damaged by radiation or chemical agents [2,3]. Because caffeine Abbreviations: AcAAF, N-acetoxy-2-acetyl aminofluorene; CE, cloning efficiency; EMS, ethyl methanesulphonate; MNU, N-methyl-N-nitrosourea; UV, ultraviolet; AZG, 8-azaguanine; TG, 6-thioguanine; HGPRT, hypoxanthine: guanine phosphoribosyl transferase; HAT, hypoxanthine, aminopterin, thymidine (see Methods); FBS, fetal bovine serum; DMSO, dimethylsulfoxide; PBS, Dulbecco's phosphate buffered saline; IMF, induced mutation frequency.

does n o t appear to inhibit excision repair in mammalian cells [4], the potentiation o f lethal damage appears best explained by its interference with DNA replication in the presence of lesions in the parental DNA [5,6]. This replication process, termed postreplication repair because of the synthesis of complete daughter strands o f DNA n o t containing lesions, is thought to be the primary mechanism by which mutants arise from cells exposed to DNAdamaging agents, as is known to be the case for bacteria [7]. Postreplication repair in bacteria can be accomplished by a number of distinct pathways which may perform either error-prone or error-free replication, and the same or greater complexities no d o u b t exist in mammalian cells. Inhibition of postreplication repair by caffeine may, in addition to reducing survival, cause changes in the frequency of mutants among the survivors. Knowledge of the effects of caffeine on mutation frequencies may be useful in understanding the components of mammalian cell postreplication repair. Unfortunately, previous studies with Chinese hamster cells have been equivocal and conflicting, yielding either increases or decreases in the frequency of mutants, and the real response to caffeine remains uncertain [8--12]. The previous mutagenesis studies employed an in situ assay introduced by Chu and Malling [13] in which the mutagenic treatment and subsequent selection for m u t a n t colonies are performed on the same set of colonies. The phenotypic marker used was the acquisition of resistance to 8-azaguanine (AZG), which is usually associated with the loss of H G P R T functionality. This laboratory has recently described another assay procedure in which mutagenized cell cultures are reseeded at various times after treatment [ 14]. Selection medium containing AZG is then applied to single cell or multicell colonies, as desired, to determine when the expression of the AZG-resistant p h e n o t y p e is completed. The reseeding of treated cells eliminates the possibility of metabolic cooperation [15,16] within mosaic colonies of wildtype and m u t a n t cells and also allows as much time as necessary for complete expression. With the reseeding procedure, methyl methanesulfonate and N-acetoxy-2-acetyl aminofluorene (AcAAF) treatments produced constant and reproducible induced mutation frequencies {IMFs) approximately 10fold greater than those obtained with the in situ assay [ 14]. It was suggested that the in situ assay for AZG-resistant colonies may frequently lead to serious underestimates o f IMFs due to the unavoidable problems with metabolic cooperation. This conclusion has also been reached by others recently employing the reseeding m e t h o d o l o g y [ 17--21 ]. In the current study, the effect of caffeine on AcAAF mutagenesis o f Chinese hamster V79 cells was determined, using primarily the reseeding assay. Treatments corresponding to a 3-log survival range and caffeine concentrations from 50 to 200 ug/ml (0.25--1 mM) were used. Since it is probable that the discrepancies in the results of the previous investigations are based on the inadequacies of the in situ assay, the in situ procedure was also included for comparison. Whereas the in situ assay indicated a 10-fold enhancement of mutagenesis by caffeine, the reseeding assay showed that caffeine had no significant effect on AcAAF mutagenesis for the corn° binations tested [22].

3 MATERIALS AND METHODS

Cells and culture conditions The Chinese hamster cell line, clone V79-4, was kindly provided b y Dr. E.H.Y. Chu. The cells were maintained in Dulbecco's modified MEM supplemented with 5% fetal bovine serum (FBS) and penicillin G-streptomycin sulfate (100 U and 100 ug/ml, respectively); this medium is referred to as CM. At 3--4~lay intervals, stocks were subcultured to l 0 s cells/100 mm dish in 20 ml CM and incubated at 37°C in a humidified atmosphere containing 10% CO2. The doubling time was consistently 11--12 h. Cultures were routinely discarded after 3--4 months and fresh ones started from stocks of V79-4 held in liquid nitrogen. Tests by Flow Laboratories, RockviUe, Md., using the culture method for detecting Mycoplasma contamination were negative for the cell stock and 4-month-old cultures. Prior to mutagenesis studies, V79 cells were routinely subcultured in HAT medium (CM containing 1 × 10 -s M hypoxanthine, 3.2 × 10 -6 M aminopterin and 5 × 10 -6 M thymidine) for at least 7 days. Although the efficacy of V79-4 held in liquid nitrogen. Tests by Flow Laboratories, Rockville, Md., resistant cells has not been rigorously demonstrated, low backgrounds in the order o f 1--2 × 10 -6 are consistently obtained. The cells were removed from H A T by washing once with HT medium (HAT minus aminopterin) and culturing in HT medium for one day, followed by one day in CM. The population doubling time returned to 12 h after one day in CM. Dialyzed serum for use in selection medium was prepared b y the hollow fiber technique described previously [ 14].

Mutagenesis In the reseeding assay, 4 × 10 s cells were seeded into replicate 150 m m dishes (Falcon) and 16--20 h later, treated with A c A A F . The cell count normally increased to 106 or more cells as logarithmic growth was resumed before treatment. At 1 h (or longer in designated experiments) after A c A A F addition, 5 ml of C M or C M containing caffeine was added; 48 h later, the m e d i u m was replaced with 40 ml fresh CM. The cultures were maintained until confluency, then trypsinized and reseeded at 4 × 104 ceUs/60 m m dish for selection of mutants and at 100 cells/dishfor cloning efficiency (CE) determinations. To minimize possible variations in treatment, three dishes for each mutagenesis condition were combined prior to counting and reseeding. Cell counts were obtained using a hemocytometer. In the in situ assay, a series of 60 mm dishes were seeded with 4 × 104 cells in 4 ml CM and treated 4 h later with AcAAF. Other dishes seeded with 100--200 cells were used to determine the survival to AcAAF, relative to untreated control cells. After 1 h exposure, 2 ml of CM or CM containing 150 #g/ml caffeine was added to the mutagenesis and survival dishes, resulting in a final concentration of 50 ~g/ml caffeine. The medium was replaced after 48 h with CM or selective medium. AcAAF was obtained from Starks Associates and stored desiccated at

4 -- 40°C. Just prior to use, AcAAF was dissolved in spectroscopic grade DMSO (Eastman}. The cells in the 150 mm dishes for the reseeding assay were treated by replacing the medium with 20 ml medium containing 10% FBS, diluting a DMSO solution of AcAAF into serum-free medium at room temperature, and quickly pipeting 20 ml volumes evenly on the dishes. The dishes were immediately swirled to obtain complete, rapid mixing. No more than 4 dishes were treated with a given A c A A F / m e d i u m solution to minimize time-dependent losses of AcAAF activity from hydrolysis. In the in situ assay, cells in the 60 mm dishes were treated directly by micropipet (Eppendorf) with 50 ~1 quantities of AcAAF in DMSO solution. Each plate was vigorously swirled after the addition. Thus, in both procedures, the cells were exposed to AcAAF in the presence of 5% FBS (by volume}. The pH was also maintained at 6.9--7.1 during the treatment operations. At different times after the reseeding of treated cells or the treatment in the in situ assay, the CM in the m u t a t i o n assay dishes was changed to 6 ml of selection medium. This time is referred to as the colony selection time; the expression time refers to the elapsed time after the beginning of the mutagenic t r e a t m e n t and coincides with the colony selection time only in the in situ assay. Three selection media were examined: 10 t~g/ml AZG with 5% dialyzed FBS (10 pg/ml AZG in dCM) and 11 pg/ml TG with 5% dialyzed FBS (11 pg/ml TG in DCM) or with 5% whole FBS (11 pg/ml TG in CM). Most selections were performed with AZG and the TG concentration was chosen to allow equimolar (6.6 X 10 -5 M) comparisons. The AZG medium was replaced twice at 2-day intervals whereas one replacement of the TG medium at 2 days was found sufficient to eliminate wildtype cells. The final volume of selection medium was increased to 8 ml. Thirteen days after seeding, the resistant colonies were washed with PBS, fixed in methanol, stained with Giemsa, and counted. The dishes prepared for CE determinations were refed 24--48 h after plating with 8 ml CM or dCM, depending on the selection medium chosen for the corresponding mutation assay and were stained after 9 days growth. The background m u t a t i o n frequency and CE of untreated V79 cells were determined similarly for each experiment. AZG and TG stock solutions were prepared at 100 X in 0.05 M NaOH and stored at 4°C for a m a x i m u m of two weeks. Sterilization was unnecessary for stocks prepared one day before use. Caffeine stocks (1000 or 1500 ~g/ml) were dissolved in Dulbecco's medium (serum-free), sterilized by filtration through a 0.2 t~m membrane in disposable Nalgene units, and stored at 4°C. The stocks were diluted with CM to 3 to 5 times the desired final concentration and applied to the cell cultures.

Mutant Frequency Calculations The average number of colonies/dish in 6-12 CE dishes containing CM or dCM was used to determine the plating efficiency of cells in the mutagenesis assay. Thus, the m u t a n t frequency per unit number of survivors was obtained by dividing the observed m u t a n t colony frequency by the CE of the cells. The CE was determined each time a treated or control culture was reseeded for

m u t a n t selection or for each treatment condition in the in situ assay. The m u t a n t colony frequency at each selection time was determined from a minimum of 24 dishes (106 total cells plated). Colonies significantly smaller than average size for each experiment were not counted. RESULTS

A cA A F mu tagenesis assays

The induction of TG-resistance in V79-4 cells treated with 1 ~g/ml AcAAF was first studied by using the in situ assay. A seeding density of 4 × 104 cells/dish allows phenotypic expression to continue for about 50 hr before intercolony crossfeeding reduces the efficiency of mutant recovery, as previously determined by reconstruction experiments [14]. With the lethality of the AcAAF treatment reducing the viable cell number to 59% of untreated controls, the time available to study expression was further extended. The frequency of mutants in the treated dishes did n o t exceed the control frequency of 5 X 10 -6 until a b o u t 60 h after the addition of AcAAF (Fig. 1). At 83 h, a maximum frequency of about 1.8 × 10 -s over background was achieved. The decline in observable frequency after 83 h is attributed to metabolic crossfeeding among the colonies that were observed to have grown into contact. In this and subsequent experiments, the resistant colonies surviving the selection conditions are only presumptive HGPRT- mutants since characterization was n o t attempted. Several colonies have previously been isolated and shown to maintain the resistant phenotype after repeated subcultures in nonselective medium [14]. These cells did not survive in HAT medium, which selects against the H G P R T - phenotype. The consistently low control 3

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Fig. 1. In situ e x p r e s s i o n curve for t h e i n d u c t i o n o f T G - r e s i s t a n t c o l o n i e s b y A c A A F . V 7 9 cells were seeded a t 4 × 104 p e r 6 0 m m dish, t r e a t e d w i t h 1 ~ g / m l A c A A F a n d selected a t t h e t i m e s s h o w n w i t h 11 ~ g / m l T G in dCM. Survival relative t o u n t r e a t e d cells was 59%. E a c h p o i n t c o r r e s p o n d s t o t h e n u m b e r of c o l o n i e s in 24 dishes. B a c k g r o u n d (o,~), 1 u g / m l AcAAF (e,,).

6 (background) frequencies of less than 5 × 10 -4 for either TG or AZG selection means t hat cells with wildtype resistance are reliably eliminated. Th e results of a reseeding assay for the f r e q u e n c y of AZG-resistant colonies induced by 2 pg/ml AcAAF are shown in Fig. 2. The cells were reseeded f o r selection at 53 and 103 h after t reat m ent . The insert axes show the c o l o n y selection times f or each reseeding. As with the in situ assay, the f r e q u e n c y o f mutants began to increase over background a b o u t 55--56 h after t r e a t m e n t . However, the induced f r e q u e n c y increased m uch m o r e rapidly and c o n t i n u e d linearly until a definite m a x i m u m of nearly 4 × 10 -4 was attained a b o u t 130 h after t r e a t m e n t . T w o reseedings were necessary t o cover the time course of expression and to d e m o n s t r a t e a plateau. At 83 h expression time, the induced f r e q u e n c y in this e x p e r i m e n t was 8 times the peak f r e q u e n c y shown in Fig. 1 even though the dose of AcAAF was only doubled. The m a x i m u m plateau f r e q u e n c y which occurs with com pl et e expression was 10-fold greater than a doubling of the peak f r e q u e n c y in the in situ assay (Fig. 1). A dose study p e r f o r m e d later with the reseeding p r o c e d u r e showed t hat the m u t a n t f r e q u e n c y is nearly linearly related t o AcAAF dose (Fig. 4). Thus, the in situ assay for AcAAF mutagenesis of V79-4 cells greatly underestimates the f r e q u e n c y of m u t a n t cells among the surviving population. 50

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Fig. 2. Expression curve obtained by the reseeding method for the i n d u c t i o n of drugresistant c o l o n i e s by AcAAF. Approx. 10+ cells in 150 mm dishes were treated with 2 pg/ml A c A A F and the cells were r e s e e d e d to 4 x 104 per 60 mm dish at the expression times shown by the zero points of the inserted time axes. The selection media subsequently used were 10 pg/ml AZG in dCM (e,o), 11 pg/ml TG in dCM (A) and 11 pg/ml TG in CM (o). The open symbols refer to the second reseeding, Each point was determined f r o m the c o l o n i e s in 36 dishes. The cloning efficiencies were 14% and 58% for the first and second reseedings, respectively.The background f r e q u e n c y w a s 5 × 10-+.

7 The in situ and reseeding experiments were performed when different selection media were being routinely employed, so the comparison of the two methods depends on showing the interchangeabflity of these media. The expression curve for the reseeding assay (Fig. 2) therefore includes selections made with 11 pg/ml TG (equimolar to 10 ~g/ml AZG) in medium containing 5 % whole fetal bovine serum (CM) or 5% dialyzed serum (dCM) in addition to the AZG selections in dCM. The maximum induced frequency was the same in all three media. This equivalence of frequencies was also found in subsequent experiments (Table II), suggesting that AZG and TG can be used interchangeably with the medium conditions described. However, the use of TG has several practical advantages in that dialysis of the fetal bovine serum component of the selection medium is not necessary, and replacement of the selection medium is not necessary for complete elimination of wildtype cells. Two or three changes of AZG selection medium are needed to eliminate wildtype growth [14]. The resistant colonies are also much larger after selection in TG, indicating that AZG (or impurities) may be somewhat inhibitory to the growth of the drug-resistant cells. For these reasons, TG was preferentially used for further studies. Effects o f caffeine -- in situ assay The in situ assay was used to determine whether the effect of caffeine on AcAAF mutagenesis would differ markedly from those obtained with the reseeding assay. Table I shows the results of typical experiments in which 50 pg/ml caffeine was added 1 h after 1.0 or 1.5 pg/ml AcAAF. The caffeine was removed after 48 h and selection in 11/~g/ml TG in dCM was begun at TABLE I IN S I T U M U T A T I O N A S S A Y - - E F F E C T O F C A F F E I N E ON A c A A F - I N D U C E D MUTANT FREQUENCY AcAAF (~g/ml)

Caffeine a (~g/ml)

Survival b (%)

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59

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9.6 26.8

a Caffeine a d d e d 1 h a f t e r A c A A F a n d r e m o v e d a f t e r 48-h e x p o s u r e . b C o l o n y survival relative to c o n t r o l CE of 79%. c E x p r e s s i o n t i m e : 8 0 - - 8 3 h ; Selection m e d i u m : 1 1 / ~ g / m l T G in dCM; Averages d e t e r m i n e d f r o m 24 dishes seeded a t 4 × 1 0 ' cells/60 m m dish. d Background frequency subtracted. e R, R a t i o of m u t a n t f r e q u e n c y p e r 105 survivors for A c A A F + caffeine t o t h e f r e q u e n c y for A c A A F only.

8 80--83 h after the beginning of A c A A F treatment. This expression time corresponds to the maximum observable frequency for 1 /~g/ml AcAAF (Fig. 1). Similar results were obtained for two doses of AcAAF. Caffeine at 50 pg/ml caused an absolute increase (2-fold) in the number of TG-resistant colonies per dish despite the increased lethality of the combination treatment. When corrected for survival, the AcAAF-induced mutation frequency was increased 10-fold by caffeine at this particular expression time. No a t t e m p t was made to determine the maximum enhancement caused by caffeine by construction of an expression curve for the AcAAF plus caffeine-treated cells. Effect o f caffeine - - reseeding assay In applying the reseeding assay to mutagenic treatments of varying lethality, it is helpful to know what time may be necessary for complete expression so the workload can be reduced. Previous experience indicated that, for A c A A F and caffeine combinations, the time required for the treated cells to reach confluency in the 150 mm dishes was sufficient for maximum expression. This assumption was tested by reseeding a series of dishes to obtain a colony selection time curve. A plateau region of maximum frequency must be demonstrated. 40

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Fig. 3. S e l e c t i o n o f T G - r e s i s t a n t c o l o n i e s i n d u c e d b y A c A A F a l o n e a n d in c o m b i n a t i o n w i t h caffeine. Mass c u l t u r e s o f 1.5 X 106 cells in 1 5 0 m m dishes were e x p o s e d t o 0.5 p g / m l A c A A F o n l y f o r 4 8 h or t o c a f f e i n e for 4 8 h c o m m e n c i n g 1 h a f t e r A c A A F a d d i t i o n . A t c o n f l u e n c y , 3 c u l t u r e s f o r e a c h t r e a t m e n t c o n d i t i o n were t r y p s i n i z e d , c o m b i n e d a n d r e s e e d e d a t t h e f o l l o w i n g t i m e s a f t e r A c A A F a d d i t i o n : A c A A F , 95 h (e), A c A A F + 50 p g / m l caffeine, 1 0 0 h (A), A c A A F + 2 0 0 p g / m l caffeine, 146 h (o). T h e CEs were 60, 61 a n d 76%, respectively. T h e s e l e c t i o n m e d i u m was 11 u g / m l T G in dCM. E a c h p o i n t is t h e average of 24 dishes ( 6 0 r a m ) s e e d e d a t 4 X 104 cells. C o n t r o l c u l t u r e s (o) were r e s e e d e d 71 h a f t e r t h e t i m e c o r r e s p o n d i n g t o A c A A F a d d i t i o n .

9 Since published studies were performed with caffeine concentrations varying from 50 to 200 ~g/ml (1 mM), this range was used in the present study. Colony selection curves for the effects of 50 and 200 pg/ml caffeine on the mutant frequency induced by 0.5 ~g/ml AcAAF are shown in Fig. 3. The selection medium was 11 ~g/ml TG in dCM. Additional data associated with these experiments is included in Table II. The selection curves in Fig. 3 show that maximum expression was achieved by the various times the treated cultures were reseeded. The mutant frequency normally falls when selection is performed more than 50 h after reseeding, although this didn't happen in the present experiments. In this and subsequent experiments, the results from all selection times in the plateau region were averaged to determine the mutation frequency for each treatment. It is apparent from Fig. 3 and Table II that caffeine did not influence the mutation frequency when applied at 50 or 200 ~g/ml concentrations 1 h after AcAAF. The time of reseeding provides an indication of the lethality of treatment since the cultures were reseeded near confluency. Caffeine at 50 ~g/ml caused almost no delay in culture growth beyond that for AcAAF only, whereas another 2 days were required to reach confluency for 0.5 pg/ml AcAAF plus 200 ~g/ml caffeine. The lethality of the latter treatment could not have exceeded (lh) [6] or about 1.5% of controls and was probably much less because of a delay or slowing of the culture doubling time (12 h in controls). Caffeine by itself was nontoxic at the highest dose of 200 ~g/ml. It should be noted that the CE at the time of reseeding (Table II) was sufficient to avoid large corrections to the observed mutant frequency. The ineffectiveness of caffeine in altering the mutant frequency was not dependent on the choice of selection medium. Selection was performed in some cases at 36--42 h after reseeding with 11 ~g/ml TG in CM and 10 ~g/ml AZG and dCM. The results were the same within experimental error as with 11 ug/ml TG in dCM (Table II). This suggests that serum dialysis did not result in a less efficient recovery of AcAAF plus caffeine-induced mutants and that the specific nature of the resistant colonies was not altered by exposure to caffeine. In one case, however, when 200/~g/ml (1 mM) caffeine was used, the frequency of AZG-resistant colonies was one-half that obtained with TG (Table II).

Dose.response and effect of caffeine Since different concentrations of caffeine did not influence the mutation frequency for a constant dose of A c A A F , the effect of caffeine on increasing doses of A c A A F was investigated. A dose-response Curve for mutagenesis by A c A A F was therefore constructed and the effect of 50 ~g/ml caffeine examined. A c A A F induced TG-resistant colonies in a dose-dependent manner (Fig. 4). The curve as fitted by eye is not quite linear but m a y have a decreasing slope with increasing A c A A F dose. The effect of adding 50 ~g/ml caffeine I h after A c A A F is small. Within individual experiments, caffeine usually caused a small increase in the mutant frequency. The ratio (R) of mutant frequencies,

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a Caffeine added 1 h after A c A A F and removed after 48-h exposure. bTime after A c A A F addition when a combination of 3 confluent cultures in 150 m m dishes was reseeded at 4 × 104 cells/60 mrn dish. c Cloning efficiency at the time of reseeding (100 cells/dish). dSelection media: TG, 11 ~g/ml TG in dCM; TG/CM, 11 ug/ml TG in CM; AZG, 10 ug/ml AZG in dCM. e Range, Total range of frequencies per l 0 s survivors observed over all colony selection times with 24 dishes for each determination. Selection times varied from 8 h to 59 h after reseeding. f R, Ratio of TG-resistant colony frequency per l 0 s survivors for A c A A F + caffeine to the frequency for A c A A F only. The background was subtracted before calculation.

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RESEEDING MUTATION ASSAY -- E F F E C T OF C A F F E I N E ON AcAAF- INDUCED M U T A N T F R E Q U E N C Y

TABLE II

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(pg/ml)

Fig. 4. The induction of TG resistance as a function of AcAAF concentration and the effect of caffeine. (A) Caffeine was added at 50 ug/ml 1 h after AcAAF treatment and removed 48 h later. The open symbols Correspond to AcAAF only and the closed symbols to AcAAF + caffeine. Each symbol type refers to individual experiments; the points at 2.0 ug/ml AcAAF are from earlier studies with AcAAF only. Additional data and experimental details are given in Table III. The line was fitted by eye. (B) The effect of caffeine expressed relative to AcAAF only for each experiment. RffiAcAAF + CAF/AcAAF. ( A c A A F + C A F ) / A c A A F , is given in Fig. 4B a n d T a b l e I I I f o r e a c h experim e n t a l c o m p a r i s o n . T h e d a t a is n o t s u f f i c i e n t l y precise t o d e t e r m i n e w h e t h e r c a f f e i n e h a s n o e f f e c t o r c a u s e d a slight increase in m u t a n t f r e q u e n c y a t l o w A c A A F doses. I n a n y e v e n t , t h e e n h a n c e m e n t d o e s n o t e x c e e d 50% o f t h e A c A A F f r e q u e n c y a n d d o e s n o t a p p r o a c h t h e 10-fold e n h a n c e m e n t n o t e d in t h e in situ assay. A d d i t i o n a l d a t a f o r t h e d o s e - r e s p o n s e s t u d y is p r e s e n t e d in T a b l e I I I . Despite t h e high l e t h a l i t y a s s o c i a t e d w i t h t h e higher d o s e s o f A c A A F used, t h e

12 T A B L E III DOSE-DEPENDENCY OF THE AcAAF-INDUCED MUTANT FREQUENCY PRESENCE AND ABSENCE OF CAFFEINE AcAAF Caffeine a ~g/ml ~g/ml

0.1 0.1 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 1.5 1.5 2.5 2.5

0 50 0 50 0 50 0 50 0 50 0 50 0 50

T i m e of Reseeding a hr

CE a %

78 80 96 97 95 100 101 121 102 148 99 122 169 199

90 71 74 65 60 61 71 63 57 57 50 59 59 66

Total dishes

72 72 72 72 120 120 72 72 84 84 96 72 72 72

IN T H E

Average m u t a n t c o l o n y f r e q u e n c y b Ra p e r 105 ,survivors

Range c

0.78 1.29 11.0 13.3 13.2 17.2 18.1 24.9 25.2 27.1 32.7 28.8 42.7 51.9

(0.36--1.29) (0.75--2.22) (8.9--12.1) (12.7--14.2) (10.8--16.4) (15.5--19.3) (15.8--20.4) (22.5--29.5) (23.7--27.8) (25.6--28.5) (30.0--35.9) (28.1--30.0) (36.2--50.4) (43.2--60.5)

1.6 1.2 1.3 1.4 1.1 0.9 1.2

a See T a b l e II. b Values have t h e b a c k g r o u n d s u b t r a c t e d . S e l e c t i o n m e d i u m was 11 p g / m l T G in dCM. c See T a b l e II. C o l o n y s e l e c t i o n t i m e s varied f r o m 22 h t o 48 h a f t e r reseeding.

T A B L E IV I N F L U E N C E O F C A F F E I N E AS A F U N C T I O N O F T I M E O F A D D I T I O N A F T E R AcAAF ON THE INDUCED MUTATION FREQUENCY AcAAF ug/ml

Time of caffeine addition a (h)

Time of CE b r e s e e d i n g b (%) (h)

T o t a l Average m u t a n t c o l o n y f r e q u e n c y c dishes p e r 105 Range d survivors

1.0

None 1 4 6 12 24

102 148 146 144 124 120

57 57 53 66 55 51

84 84 84 84 84 84

25.2 27.1 28.2 24.5 29.1 32.2

(23.7--27.8) (25.6--28.5) (26.2--30.0) (22.0--27.4) (28.4--29.6) (30.0--34.5)

-1.1 1.1 1.0 1.1 1.3

1.5

None 2

99 123

50 60

96 72

32.7 29.9

(30.0--35.9) (28.6--30.5)

-0.9

a b c d

Rb

50 ;~g/ml caffeine. R e m o v e d a f t e r 4 8 h. See T a b l e II. Values have t h e b a c k g r o u n d s u b t r a c t e d . S e l e c t i o n m e d i u m was 11 u g / m l T G in dCM. R a n g e for t h r e e c o l o n y s e l e c t i o n t i m e s f r o m 17 h to 48 h a f t e r reseeding w i t h 2 4 - - 3 6 dishes p e r d e t e r m i n a t i o n .

13 CE at the time of reseeding was greater than 50%. Thus, for the treatment of 2.5 ~g/ml AcAAF plus 50 ~g/ml caffeine, more than 8 days were required for the cultures to reach confluency. The proportion of cells surviving the treatment was not measured but based on the delay in reaching confluency the survival could not have been lower than (lh) [11 ] or 0.05% of the controls.

Effect of time of addition of caffeine The action of caffeine might depend on interference with DNA repair pathways that may become prominent at different times after the initial mutagenic events. Caffeine enhancement of AcAAF-induced transformation of Syrian hamster embyro cells depends markedly on the time of its addition, the greatest enhancement (10- to 15-fold) being observed when caffeine was added 4 h after AcAAF [23]. Therefore, the influence of caffeine on V79 mutation was studied as a function of elapsed time after AcAAF treatment. Table IV shows the results of experiments in which 50/~g/ml caffeine was present for 48 h commencing 1--24 h after AcAAF. No significant alteration of the AcAAF-induced frequency was observed for any time interval between treatment and incubation in caffeine. The potentiation of AcAAF lethality appeared to decrease, however, when caffeine was added from 12 to 24 h after AcAAF, as indicated by the decreased time needed to obtain confluency. DISCUSSION Reliable measurements of induced mutation frequencies (IMFs) in mamm~dian cells can be obtained only when phenotypic expression is fully completed and the measurement of the maximum IMF is not reduced by assay variables. The reseeding assay used for AZG-resistant colonies (presumed HGPRT deficiency or loss) induced by mutagenic treatments of V79-4 cells meets the requirements for quantitative analysis [14]. Thus, the stringency of the selection procedure is indicated by consistently low background frequencies of less than six colonies per 106 survivors. The reseeding assay operates within the cell density region where intercolony cross-feeding does not reduce the recovery of mutants. The efficiency of recovery of mutant colonies from a reconstructed population was constant (100%) over the range of colony numbers per dish that was encountered under experimental conditions. Finally, the attainment of a constant maximum frequency was demonstrated when cells were reseeded as single cell colonies after a suitable expression time before adding the selection medium. The application of the reseeding method to quantitative studies of mutagenesis also depends on the newly induced mutants being very similar to wildtype cells in regard to doubling time, plating efficiency, and viability of progeny. These conditions will probably be fulfulled to varying extents depending on the cell type, mutagen, or genetic locus being studied. For practical purposes, the attainment of a steady state (constant mutant frequency) within several days following mutagenic treatment and a frequency at least as great as that obtainable with the in situ method can be regarded as

14 evidence for a reliable assay. For V79-4 cells, which have a short doubling time of 11--12 h and an essentially 100% plating efficiency, the reseeding assay operates well for AcAAF induction to AZG- or TG-resistance. A b o u t 130 hr after treatment, the frequency of AcAAF-induced mutants clearly reached a constant level that was approximately 10-fold greater than the peak in situ frequency. The usefulness of the in situ assay for measuring the appearance of AZGor TG-resistant colonies is severely limited by the phenomenon of metabolic cooperation [15,16]. Wildtype cells confer the wildtype p h e n o t y p e on m u t a n t cells when membrane contacts are formed during growth in culture. Thus, in the presence of selection medium, AZG- or TG-nucleotides formed in the wildtype cells, but not in mutant cells, are passed via the membrane junctions to the m u t a n t cells and cause their subsequent death. In the in situ assay, treated cells may give rise to mosaic colonies of cooperating m u t a n t and wildtype cells and individual colonies may grow into contact before sufficient time can be allowed for complete expression. The survival or loss of the colony in selection medium thus depends on its composition and number of cooperating contacts between daughter cells and neighbouring colonies at the time selection is begun. Relatively long expression times, such as seven days [17], are now being found necessary for some mutagenic treatments of V79 cells. The position and height of the peak m u t a n t frequency observed in the in situ assay is therefore dependent on many interacting parameters of u n k n o w n dependence on culture time. These complex interactions are indicated by Fox's [12] recent observations that caffeine apparently lengthens the expression time in V79 cells so that early selection at the standard time of 42 h following UV light treatment appears to show that caffeine decreases the mutant frequency while later selections show an enhancement by caffeine. The conflicting results of earlier studies on the effects of caffeine on UVor MNU-induced mutants were obtained with the in situ assay [8-12]. It therefore was of interest to determine whether the in situ and reseeding methods would yield different results for a given line of Chinese hamster cells. The maximum observable frequency of AZG-resistant colonies occurred 80--83 h after treatment with 1 pg/ml A c A A F in the in situ assay (Fig. 1). This expression time was used to determine the effect of 50 pg/ml caffeine added 1 h after AcAAF and removed after 48 h exposure. Caffeine caused more than a 10-fold increase in the IMF (Table I). This enhancement could have been greater had the o p t i m u m expression time been determined for the combination treatment, b u t the result obtained demonstrates that a large enhancement of A c A A F mutagenesis by caffeine is observed with the in situ assay. When the effect of caffeine was examined with the reseeding assay, no significant change in AcAAF mutagenesis was observable. This conclusion is based u p o n experiments performed with increasing doses of AcAAF treatment, different concentrations of caffeine, and increasing time intervals between AcAAF treatment and caffeine addition. Increasing the dose of

15 A c A A F from 0.1 to 2.5 ~g/ml covers a survival range of about 98%--2%. The addition of 50 ~g/ml caffeine enhances the lethality to a greater degree with higher doses of AcAAF so that survival to 2.5 ~g/ml AcAAF plus 50 ~g/ml caffeine was a b o u t 0.1%. Thus, over a 3-log range of survival to treatment, which represents a wide range of impairment to successful replication, caffeine had no effect on the frequency of mutants among the surviving population. Past studies have also used caffeine concentrations from 50 to 2 0 0 p g / m l and these differences have often been cited as a possible reason for the conflicting reports. With the reseeding procedure, increasing the caffeine from 50 to 200 ~g/ml greatly reduced cell survival b u t did not alter the frequency of mutants induced by 0.5 ~g/ml AcAAF (Table II). A b o u t 90% of the cells survived this AcAAF treatment and an average of 15 mutants per 105 survivors were produced. The repair of the damage was inhibited by caffeine in a dose-dependent manner, so that survival fell to about 2% in the presence of 200 ~ g/ml caffeine, but the frequency of TG-resistant colonies among the survivors remained unchanged. In all cases, caffeine was present for 48 h during the expression period and was removed before selection began, thus avoiding any possible interaction between caffeine and selection agent. Complete expression of the resistant p h e n o t y p e was demonstrated for two doses of AcAAF alone and in combination with 50 ~g/ml and 200 pg/ml caffeine (Fig. 2 and 3). The results were also independent of the choice of selection agent, AZG or TG, under the medium conditions employed (Fig. 2 and Table II). In the one case of 0.5 pg/ml AcAAF plus 200 pg/ml caffeine, the frequency of AZGresistant colonies was about one-half the TG-resistant frequency, b u t anomalously low AZG frequencies are sometimes observed and this result may not be real. Since a number of treatments corresponding to widely different survival levels and selection with either AZG or TG did not reveal any significant effect by caffeine on AcAAF mutagenesis, the results obtained by the reseeding assay appear valid and the in situ results must be considered artifactual. Since no effect by caffeine was found, the stabilities of the induced frequencies at longer times after treatment than those reported were not determined. Very little decay would be expected in view of the finding by Van Zeeland and Simons [17] that EMS-treated V79 cells maintained a constant TGresistant colony frequency for at least 22 days following maximum expression. An observable effect by caffeine on the frequency of mutants at some point in the dose study would be the expected result if the mutant frequency decay rate during the experimental time period was altered by caffeine. The dose response curve for AcAAF-induced mutagenesis is similar to the results of a reseeding assay for UV mutagenesis of CHO cells obtained by Hsie et al. [24]. A more useful form for comparison with other cells or mutagens would be (M--M0) as a function of log (S/S0), where M is the maxim u m IFM, M0 the background frequency, and S/S0 the surviving fraction [ 2 5 ] . Previous comparisons of IMF with survival have been compromised,

16 however, b y n o t performing survival and mutagenesis experiments under identical conditions of mutagen availability per cell. Thus, chemical treatment of 100--200 cells in a dish for survival of colonies is n o t necessarily equivalent to the treatment of 104--106 cells for mutagenesis. To determine the potentiation of lethality by caffeine during mutagenesis, it therefore appears necessary to treat a mixture of normal and X-irradiated cells seeded at the density used for mutagenesis. The X-irradiated cells will react with the mutagen and contribute to the metabolism of caffeine, which is very rapid in V79 cells [26], and thereby mimic the mutagenesis treatment conditions without contributing colonies to obscure the lethality measurement. The lethal potentiation by a given concentration of caffeine is significantly less for 106 cells than for several hundred or thousand cells typically used for survival measurements (unpublished observations). Survivals to treatment are necessary for the calculation of mutants per 106 survivors only in the in situ assay (see Table I). In the reseeding assay, the CE of the treated cells at the time of reseeding is the factor needed to determine mutants. The lethal action of AcAAF and caffeine on V79 cells must be determined under the separate conditions just discussed in order to arrive at the relationship between mutagenesis and survival. AcAAF causes DNA lesions thought to be repairable by the same mechanisms that remove UV light-induced pyrimidine dimers [27]. The sensitivity of cells to UV light has been extensively studied and it is becoming increasingly evident that different rodent cell lines m a y display different degrees of lethal synergism in the presence of caffeine. Thus, Arlett [28] has described two Chinese hamster cell lines that do n o t show increased UV lethality in the presence of 100 ug/ml caffeine and Fox [12] reported that CHO cells showed no potentiation of UV lethality by 150 pg/ml caffeine. Other studies with Chinese hamster and mouse lines usually show high degrees of lethality potentiation by the same concentrations of caffeine [2,3]. Similarly conflicting results have been obtained with heteroploid or malignant human cell lines [29,30]. R o d e n t cell lines are generally lacking in excision repair capability and caffeine does n o t inhibit excision repair in human cells where this repair p a t h w a y operates efficiently [3,6]. Thus, the divergent effects of caffeine on rodent cell survival strongly suggests that different specific mechanisms of postreplication repair operate among different clonal lines of rodent cells. With our V79 Chinese hamster line, caffeine appears to inhibit a postreplication repair process, causing potentiation of AcAAF lethality, without altering the error probability of the remaining postreplication repair activity. The error probability m a y well be altered by caffeine in other Chinese hamster cell lines. Recently, D'Ambrosio and Setlow [31] have obtained evidence for a postreplication DNA repair pathway in V79 cells that is enhanced by AcAAF treatment or UV irradiation. This induced repair was postulated to be responsible for chemical or UV carcinogenesis in mammalian cells by operating in an error-prone manner, thereby producing mutations in the genome. More recently, Rossman et al. [32] presented survival data which indicated CHO

17 cells possess a DNA repair process enhanced by nonlethal exposure to UV light. A 10-fold increase in survival to a lethal dose of UV light was observed when cells were given a nonlethal dose 24 h earlier. The possibility of a caffeine-sensitive, inducible repair in our V79 cells was examined by adding 50 pg/ml caffeine from 1 to 24 h after AcAAF treatment. The caffeine was removed after a 48-h exposure. No changes in the frequency of TG-resistant colonies were observed for any of the time intervals chosen, although less potentiation of AcAAF lethality appeared to occur when caffeine was added at 12 h or later (Table IV). Therefore, if an error-prone inducible repair does operate in these cells, the error probability of this repair is insensitive to caffeine. ACKNOWLEDGEMENT

The technical assistance of Mrs. Joanne Ward is gratefully acknowledged. REFERENCES

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