Rapid transfection assay for screening mutagens and carcinogens

Mutation Research, 91 (1981) 9-14 © Elsevier/North-Holland Biomedical Press

RAPID TRANSFECTION CARCINOGENS

ASSAY FOR SCREENING

9

MUTAGENS

AND

H.A. VASAVADA and J.D. PADAYATTY Department of Biochemistry, Indian Institute of Science, Bangalore-560 012 (India)

(Accepted 8 August 1980)

Testing for mutagenicity and carcinogenicity has become an integral part of the toxicological evaluation o f drugs and chemicals. Standard carcinogenicity tests in vivo require both large numbers of animals and prolonged experiments. To circumvent these problems, several rapid tests have been developed for preliminary screening of mutagens and carcinogens in vitro. Ames and his associates, the first to develop a mutation test, used mutant strains of Salmonella typhimurium [1]. Mutation tests with Escherichia coli, Bacillus subtilis, Neurospora crassa and Saccharomyces cerevisiae, and DNA-repair tests with E. coli and B. subtilis, have been developed. Cytogenetic assays, in vivo as well as in vitro, in both plant and animal systems, are also used to detect potential mutagens and carcinogens. Transfection is inhibited by base mutation, cleavage of DNA, loss of cohesive ends, interaction with histones, spermidine, nalidixic acid, etc. [3]. The efficiency of transfection is affected by temperature, DNA structure and the condition of the competence of the recipient cells [3]. Transfection assays with phages MS: RNA and ~i, x 174-DNA have been reported [15]. A fast and easy transfection assay using colitis bacteriophage DNA is reported in this communication. The colitis bacteriophage was detected in the stools of colitis patients [7]. The electronmicrographs of a purified preparation of the phage showed the presence of a hexagonal head (90 ,~ × 180 ~,) and a wavy tail (400 ~, x 25 ~,) [131. The head contained 35°7o DNA and 6507o protein. This phage is a small DNA phage of molecular weight 22 x 10 6 and contains double-stranded DNA of molecular weight 10 x 10 6 with a G + C content of 53.3°70 [14]. Materials and methods Phage and bacterial strains. E. coli B lysate of colitis bacteriophage was a gift from Dr. B.M. Gupta, Central Drug Research Institute, Lucknow [7]. It was purified according to the method o f Ramakrishna and Padayatty [13]. The phage DNA was not transfective on wild-type E. coli B cells. E. coil CR63, a suppressor strain of E. coli K12, was used for the transfection. Chemicals. Hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethidium bromide, acrylamide, benzo[a]pyrene, 3-methylcholanthrene, NADP,

10 glucose 6-phosphate, glucose-6-phosphate dehydrogenase, DNAase, RNAase and protease were purchased from Sigma Chemical Co., St. Louis (U.S.A.). Aflatoxin B1 was a gift from Jagdeep Buch, Department o f Microbiology, M.S. University of Baroda. Casamino acids were purchased from Difco Laboratories, Detroit (U.S.A.). All other chemicals were o f analytical grade.

Media. and 0.5 g and 1 ml casamino

M9 medium contained 2.8 g o f Na2HPO4, 3 g o f KH2PO4, 1 g of NH4CI of NaC1 per I. To this we added 20 ml o f 20%0 glucose, 1 ml o f 1 M MgSO4 of 10 mM FeCl3. M9-CCA medium was made by adding 10 ml o f 20% acids to 1 l of M9 medium.

Preparation o f DNA. Colitis phage DNA was isolated by the phenol extraction method as described by B¢vre and Szybalaski [4] with some modifications. Purified phage (1 ml, 8 × 1012p.f.u.) was transferred to a 15-ml corex centrifuge tube, 100/A of 10% SDS were added, and the mixture was heated at 65°C for 5 min. The opalescence due to phage disappeared almost completely. The tube was chilled in ice, 1 ml of phenol saturated with 0.15 M sodium chloride and 15 mM trisodium citrate, pH 7.2 (SSC), was added, and the tube was shaken gently for 10 rain at 4°C. The upper aqueous layer was collected, treated with an equal volume of phenol saturated with SSC, and extracted twice more. The final aqueous phase was dialysed against 3 I of SSC at 4°C. The final solution was highly viscous. The yield of DNA was 60 O.D. units at 260 nm. Protein contamination in the DNA was evidently negligible because the ratio of A260 nm t o A 2 8 0 nm was more than 1.8. Transfection assay. E. coli CR63 was grown at 37°C over the mid-log phase (5 × 108 cells/ml) in M9-CAA medium. Competent cells were prepared, and transfection was carried out according to the method of Mandel and Higa [8]. The phage DNA (0.5/zg in 0.1 ml of SSC) was mixed with 0.1 ml of competent cells (5 × 109). A final volume of 0.5 ml was made up with 10 mM T r i s - H C l buffer (pH 7.5) containing 10 mM CaC12 and 10 mM MgCI2, which was kept in ice for 30 min, incubated at 37°C for 5 min and-then for 10 min at 25°C. For the indication of phage growth, cells from a stationary-phase culture of E. coli CR63 (2 drops) were added, mixed with 3 ml of soft agar at 45°C, plated and incubated at 37°C. Plaques were observed within 3-3.5 h. A direct mutagen, such as nitrous acid, hydroxylamine, ethidium bromide or acrylamide (10/zg in 0.1 ml H20) or M N N G (10/~g in 0.1 ml acetone) was incubated with DNA (0.5 /~g in 0.1 ml SSC) at 37°C for 30 min, and then transfection was carried out. Pro-mutagens that require metabolic activation, such as aflatoxin Bl, benzo[a]pyrene or 3-methylcholanthrene (10 #g in 0.1 ml acetone) and ~-naphthylamine (10/zg in 0.1 ml ethanol) were activated by incubating with 105 000 × g rat-liver microsomal fraction (100/zg of protein in 0.25% sucrose), 0.32 mM NADP, 3 mM glucose 6-phosphate, 1.3 units of glucose-6-phosphate dehydrogenase and 5 mM MgC12, in a total volume of 0.2 ml of 0.1 M potassium phosphate buffer (pH 7.4) for 10 min at 37°C. The DNA (0.5 #g in 0.1 ml SSC) was added and the mixture was incubated for 30 min before the transfection assay was carried out. For the preparation o f liver microsomes, male albino rats (Indian

11

Institute of Science strain) 45 days old and weighing 6 0 - 6 5 g were given daily intraperitoneal injections of phenobarbitone (80 mg/kg). After 4 days, the rats were starved overnight and killed by cervical dislocation; 105 000 × g microsomal fraction from livers was prepared according to the method of Rajamanickam et al. [121. Results and discussion The phage D N A (0.5/zg) gave 1100 plaques within 3 - 3 . 5 h of incubation at 37°C. When the numbers of plaques were plotted against the amount of DNA, linearity was observed up to 1 t~g of D N A (data not shown). The transfection was abolished on treatment of the D N A with DNAase, but not with RNAase, protease or nonmutagens such as acetone, ethanol or NaC1 (Table 1). Mutagens such as nitrous acid, hydroxylamine and M N N G cause deamination, TABLE 1 E F F E C T OF N U C L E A S E S , P R O T E A S E , N O N - M U T A G E N S , M U T A G E N S , P R O - M U T A G E N S A N D A C T I V A T E D M U T A G E N S ON T R A N S F E C T I O N Compound

N u m b e r of plaques (°70 inhibition) Pre-treated cells

Without activation

None DNAase RNAase

-

1100 (0) 18 (98) 1047 (5)

Protease Sodium chloride Acetone

-

1034 (6) 1064 (3) 1018 (8)

Ethanol Nitrous acid Hydroxylamine

-

MNNG Ethidium bromide Acrylamide

1008 (8) 1059 (4) 1029 (7)

390 (65) 445 (60) 547 (50)

Aflatoxin Bt Benzo[a]pyrene 3-Methylcholanthrene

963 (12) 1054 (4) 1029 (8)

1018 (8) 1008 (8) 996 (10)

96 (91) 171 (80) 90 (92)

f3-Naphthylamine Microsomal fraction

1086 (11) -

1061 (4) 869 (21)

696 (36)

• 979 (11) 968 (12)

With activation

1058 (4) 445 (60) 371 (66)

The pre-treatment of the cells was done by incubating sensitized E. coli CR63 cells (5 × 109 cells/0.5 ml) with 10 #g of mutagen, pro-mutagen or activated m u t a g e n at 37°C for 30 rain. D N A (0.5 ~tg) was added and transfection was carried out as described under Materials and Methods. The phage D N A w a s incubated with protease IV (5 ~g), pancreatic RNAase (10 ug), DNAase (1 teg), NaCI (10 #g), ethanol or acetone (0.1 ml), m u t a g e n or activated mutagen (10 #g), or microsomal fraction (100 ~g in 0.2 ml) at 37°C for 30 min and the transfection assay was carried out. The pro-mutagens were activated as described earlier. The values given are the averages from 3 Expts.

12 transition and alkylation, resp., whereas ethidium bromide and acrylamide cause mutation by intercalation with D N A [6]. Each mutagen (10 #g) was pre-incubated with D N A at 37°C for 30 rain, and transfection was carried out on sensitized competent E. coli CR63 cells. Transfection was inhibited in each case to the extent of 50-67o70 as determined by the number of plaques formed (Table 1). These results are in agreement with those of Ames and associates [9], except for those with hydroxylamine which behaved as a non-mutagen in Salmonella typhimurium although it acted as a strong inhibitor in this transfection assay. There was no reduction in the number of plaques formed when the transfection was carried out immediately after the addition of phage D N A to the sensitized cells pre-incubated with mutagen at 37°C for 30 min (Table 1). This showed that transfection was solely due to mutation in transfecting D N A and not due to interference with infection processes. Pre-incubation of D N A with mutagen was required for the reduction in the number of plaques formed in the transfection assay. Mutagens that require enzymatic activation to manifest their mutagenic activity were treated with a rat-liver microsomal fraction isolated from phenobarbitonetreated rats for induction of mixed-function oxidases [2]. In these experiments, a 75

50

25

I

1

I

I

I

I

(b) 75 O

0

5C

25

L

I

I

I

4 8 CONCENTRATION (ug/0.5 ml) l I

I

I

12

Fig. 1. Percentage inhibition of transfection vs. concentration of mutagen/activated mutagen. Various amounts of mutagen or activated mutagen were pre-incubated with DNA (0.5/~g) at 37°C for 30 min, and transfection was carried out as described under Materials and Methods. The percentage inhibition of transfection was calculated and plotted against the concentration of the mutagen/activated mutagen. (a) o, hydroxylamine;z~, nitrous acid; A, ethidium bromide; [J, MNNG; o, acrylamide. (b) o aflatoxin Bi; •, fl-naphthylamine; D, benzo[a]pyrene.

13 105 000

×

g m i c r o s o m a l fraction was used instead o f the crude 9000

×

g

s u p e r n a t a n t f r a c t i o n of rat-liver h o m o g e n a t e . This was d o n e to p r o d u c e a higher activity o f m i x e d - f u n c t i o n oxidases a n d to m i n i m i z e the c o n t a m i n a t i o n o f D N A a s e [12]. W h e n D N A was p r e - i n c u b a t e d with m i c r o s o m a l fraction a n d N A D P H g e n e r a t i n g system, 870 p l a q u e s were observed in the t r a n s f e c t i o n assay, s h o w i n g an i n h i b i t i o n o f 21% in c o m p a r i s o n with the n o r m a l value o f 1100 plaques w i t h o u t activation. This m a y be due to the d e g r a d a t i o n o f D N A by the action of a small a m o u n t o f D N A a s e present in the m i c r o s o m a l fraction. U n a c t i v a t e d carcinogens did not i n h i b i t t r a n s f e c t i o n . T h e use o f a f l a t o x i n Bl, b e n z o [ a ] p y r e n e a n d 3m e t h y l c h o l a n t h r e n e resulted in the i n h i b i t i o n o f t r a n s f e c t i o n to the extent of 8 0 - 9 0 % , b u t ¢3-naphthylamine resulted in the i n h i b i t i o n of 36% (Table 1). These results are in a g r e e m e n t with those f o u n d in the S a l m o n e l l a / m i c r o s o m e test [9]. These chemical carcinogens are k n o w n to f o r m epoxide derivatives, after a c t i v a t i o n with m i x e d - f u n c t i o n oxidases, which b i n d to g u a n i n e a n d a d e n i n e [5,10]. In the cases o f m u t a g e n s a n d activated carcinogens, i n h i b i t i o n o f t r a n s f e c t i o n was linear up to 10 #g in 0.5 ml o f t r a n s f e c t i o n assay mixtures except with ¢~n a p h t h y l a m i n e (Fig. 1). T h e c o r r e l a t i o n between the percentage of i n h i b i t i o n a n d c o n c e n t r a t i o n c a n be used to d e t e r m i n e the p o t e n c y o f a particular m u t a g e n . T h e t r a n s f e c t i o n assay o n colitis b a c t e r i o p h a g e D N A m a y be used in the screening of potential m u t a g e n s a n d c a r c i n o g e n s for genetic activity. It is rapid, inexpensive, sensitive a n d easy to p e r f o r m , in c o m p a r i s o n with other assays in vitro.

REFERENCES 1 Ames, B.N., F.D. Lee and W.E. Durston, An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 782-786. 2 Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens: A simple test system combining liver homogenate for activation and bacteria for detection. Proc. Natl. Acad. Sci. (U.S.A.), 70 (1973) 2281-2285. 3 Benzinger, R., Transfection of entero-bacteriaceae and its application, Microbiol. Rev., 42 (1978) 194-236. 4 BCvre, K., and W. Szybalaski, Multistep DNA-RNA techniques, in: L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. 21, Academic Press, New York, 1974, pp. 337-383. 5 D'Andrea, A.D., and W.A. Haseltine, Modification of DNA; aflatoxin Bj creates alkali-labile lesions in DNA at position of guanine and adenine, Proc. Natl. Acad. Sci. (U.S.A.), 75 (1978) 4120-4124. 6 Freese, E., Molecular mechanisms of mutations, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for Their Detection, Vol. 1, Plenum, New York, 1971, pp. 1-56. 7 Gupta, B.M., and D.P. Johari, Human intestinal bacteriophage: Morphology and host range of crude virus from colitis patient, Indian J. Med. Res., 43 (1955) 171-177. 8 Mandel, M., and A. Higa, Calcium dependent bacteriophage DNA infection, J. Mol. Biol., 53 (1970) 159-162. 9 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmonella/microsome test, Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 (1975) 5135-5139. 10 Meehan, T., and K. Straub, Double stranded DNA stereoselectively binds benzo[alpyrene diol epoxide, Nature (London), 277 (1979) 410-412.

14 11 Nagao, M., T. Sugimura and T. Matsushima, Environmental mutagens and carcinogens, Annu. Rev. Genet., 18 (1978) 117-139. 12 Rajamanickam, C., M.R.S. Rao and G. Padmanaban, On the sequence of reactions leading to cytochrome P-450 synthesis - effect of drugs, J. Biol. Chem., 250 (1975) 2305-2310. 13 Ramakrishna, N., and J.D. Padayatty, Characterization of colitis bacteriophage, Indian J. Biochem. Biophys., 14 (1977) 158-162. 14 Ramakrishna, N., and J.D. Padayatty, Characterization of proteins and nucleic acids after colitis phage infection, Indian J. Biochem. Biophys., in press. 15 Wen-Tab, H., R.G. Harvey, E.J.S. Lin and S.B. Weiss, A bacteriophage system for screening and study of biologically active polycyclic aromatic hydrocarbon and related compounds, Proc. Natl. Acad. Sci. (U.S.A.), 74 (1977) 1378-1382.