The mutagenic activity of chlorpromazine

Mutation Research, 208 (1988) 33-38

33

Elsevier MTRL 101

The mutagenic activity of chlorpromazine E.E. Obaseiki-Ebor

and J.O. Akerele

Department ~f Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Benin Cit v (Nigeria)

(Accepted 5 January 1988)

Keywords: Chlorpromazine; Mutagenicity; Salmonella.

Summary The mutagenic activity of chlorpromazine hydrochloride based on the Ames plate incorporation test and the modified fluctuation test in the presence and absence of liver microsomal enzyme ($9 fraction) and N A D P H was determined. The results indicated that chlorpromazine required activation for mutagenic activity for the reversion of some of the tester bacterial strains from tryptophan and histidine dependence to independence respectively. The positive response of Escherichia coli WP2 trp, u v r A , E. coli WP2 trp (pKM 101 and pAQ1) and Salmonella t y p h i m u r i u m his TA102 indicated that chlorpromazine mediates basepair substitution and frame-shift mutagenesis.

Chlorpromazine is popularly used in the m a n a g e m e n t of various mental disorders, allergic conditions and nausea. It was reported to exhibit potent antibacterial activities in vitro (Molnar and Schneider, 1978) and plasmid-curing effects in bacterial cells (Molnar and Schneider, 1978; Molnar et al., 1980). Although its mode of antibacterial activity is not properly understood, it has been reported to intercalate into D N A in vitro (Lerman, 1963; Hirschman and Garfinkel, 1978). It was also reported that chlorpromazine might after all not intercalate into DNA, that its curing effect could have been due to its surface activity on the bacteria (Molnar et al., 1980). However, overCorrespondence: Dr. E.E. Obaseiki-Ebor, Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Benin, Benin City (Nigeria).

whelming evidence indicates that chlorpromazine intercalates into D N A (Ohnishi and McConnell, 1965; Jose and Yielding, 1978; McPhee and Imray, 1974). Chlorpromazine has a structural similarity to the acridines (Lerman, 1963) and cures R-plasmids as the acridines (Molnar and Schneider, 1978; Molnar et al., 1980). The mutagenic potential of chlorpromazine has not been resolved, although it was reported to be non-mutagenic (Molnar et al., 1977; Tosk, 1974), photo-excited chlorpromazine was reported to be mutagenic in Salmonella t y p h i m u r i u m strains (McPhee and Imrey, 1974). Chlorpromazine was found to be less mutagenic in strains with proficient D N A repair compared to DNA-repair-defective strains, suggesting that chlorpromazine activity was at the least repairdependent and some of its induced D N A lesions

0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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could be repaired in the S. typhimurium strains (McPhee and Imrey, 1974). This paper reports studies of the mutagenic potential of chlorpromazine. The mutagenicity test was based on the Ames plate reversion test (Ames et al., 1973, 1975) and the modified fluctuation test procedure (Green et al., 1976). The mutagenic bacterial tester strains used were the latest internationally recognised newer and improved S. typhimurium TA97 and TA102 strains (Maron and Ames, 1983) and the standard E. coli tester strains (Green, 1984). Materials and methods

The S. typhimurium his strains used were TA97 his and TA102 his (Levin et al., 1982; Maron and Ames, 1983). TA97 detects frame-shift mutagens that act at G:C-rich sequences while TA102 detects mutagens that require an intact excision-repair system and oxidative mutagens that preferentially act at A:T base pairs. E. coli WP2 trp; WP2 trp; uvrA. WP6 trp, polyA1; WP67 trp uvrA, polyA1 (Green and Muriel, 1976; Green, 1984) were used. These E. coli strains detect base-pair substitution mutagens at the trp mutant site. The E. coli transconjugant strains EE97 and EEl02 obtained from the conjugal transfer of R-plasmids from the S. typhimurium strains TA97 and TA102 respectively to the E. coli WP2 trp (Obaseiki-Ebor and Obasi, 1986) were also used in the experiments. Single colonies of all the tester strains were subcultured onto nutrient agar slants and stored at 4°C.

Media Nutrient broth No. 2 (Oxoid), nutrient agar (Oxoid), agar No. 1 (Oxoid), Davis and Mingioli (DM) (1950) salts solution were used. The Davis and Mingioli salts solution used for the mutagenicity testing generally contained 62.5 ng/ml tryptophan or histidine in trace amounts except where otherwise stated, and biotin (5/xg/ml). This low concentration of tryptophan or histidine would stimulate the growth of the auxotrophic cell, and once it is used up only trp + or his + revertants could continue to grow.

Drugs and biochemicals Chlorpromazine hydrochloride injection vials (May and Baker Ltd.) were stock solutions of chlorpromazine and were prepared at 10 mg/ml, sterilised using 0.22-tzm membrane filters (Millipore, U.S.A.) and stored at 4°C in an ambercoloured bottle. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) type 1 was obtained from Sigma (London). Animals Albino (Sprague-Dawley) rats weighing 130-200 g were treated with phenobarbital sodium injections (50 mg/kg) for 7 consecutive days before they were killed. The livers of 3 rats were aseptically removed and pooled in a beaker containing Tris-HC1 buffer, pH 7.5 at approximately 3 ml/g wet liver. Preparation of liver homogenate fraction ($9) The procedure of Ames et al. (1975) was modified where necessary. The liver lobes were minced with sterile scissors and homogenised in a ceramic manual mortar and pestle that had been oven-sterilised and cooled to 4°C. The homogenate was centrifuged at 9000 × g for 20 min in an MSE High speed 18 Centrifuge at 4°C. The supernatant was carefully decanted into a sterile universal bottle and kept on ice. The liver extract ($9) was immediately used for the mutagenicity tests after determining its protein concentration. Protein concentration This was estimated using the method of Lowry et al. (1951) with bovine serum albumin as standard. Determination of minimum inhibitory concentration (MIC) o f chlorpromazine The MIC of chlorpromazine against the test bacterial strains was determined by the agardilution protocol as previously described (Obaseiki-Ebor and Obasi, 1986). Wet Davis and Mingioli minimal liquid or agar medium supplemented with tryptophan or histidine (50/~g/ml)

35 containing varying concentrations of chlorpromazine hydrochloride (5 n g / m l to 200 t~g/ml) were inoculated with about 105 colony-forming units of the washed test cultures. The M I C was the lowest concentration of chlorpromazine that completely inhibited growth after 24-h incubation at 37°C.

Mutagenicity testing using the plate incorporation method (without activation) The method of Brusick et al. (1980) was suitably modified. Subinhibitory concentrations of chlorpromazine (5, 10 #g/ml) were added to tubes containing 2.5 ml of molten overlay agar (0.7% agar-agar) held at 45°C. 0.1 ml of undiluted, washed test culture was added to the tubes, the contents of the tubes were gently mixed and immediately poured onto the surface of the Davis and Mingioli agar plates to a total volume of 20 ml and allowed to solidify. Controls, positive (containing tryptophan 50 ~g/ml or histidine 50 ~g/ml and no test agent) and negative (not containing amino acids and test agent), were included. All plates were incubated at 37°C for 48 h and the mean number of colonies growing per plate in triplicate experiments was recorded. Plate incorporation method with activation by $9 mix The, procedure was as above except that N A D P H and the enzyme preparation ($9 mix) were added to the top agar. A subinhibitory concentration of chlorpromazine, 0.1 ml of the undiluted test culture, 0.1 ml of N A D P H 10 m g / m l stock solution, and 0.5 ml of the $9 mix (3.13 m g / m l ) were mixed in sterile universal bottles at 4°C for 15 min. This mixture was mixed with the 2.5 ml molten overlay agar (0.7o70) held at 45°C and immediately poured onto the surface of Davis and Mingioli agar plates to a total volume of 20 ml and allowed to solidify with subsequent incubation at 37°C for 48 h. The positive and negative controls were as described above.

Modified fluctuation test without $9 mix activation The method of Green et al. (1976) was suitably modified. A fresh overnight washed test culture in 10 ml Davis and Mingioli liquid medium appropriately supplemented with either histidine (50 ~g/ml) or tryptophan (50 ~g/ml) and glucose (200 t~g/ml) was grown to log phase, harvested and washed free of the histidine or tryptophan supplements. 0.4 ml of the resuspended culture (1 x 10 9 c f u / m l ) in 10 ml sterile distilled water was inoculated into 400 ml Davis and Mingioli basal salts containing glucose (200 ~g/ml), biotin (5 ~g/ml) and bromocresol purple indicator (12 ~g/ml). The contents was thoroughly mixed and aseptically divided into 4 portions of 100 ml as follows: a 100-ml aliquot containing the test concentration of chlorpromazine (in duplicate); a 100-ml aliquot containing additional histidine or tryptophan (50 t~g/ml); and a 100-ml aliquot containing no histidine, tryptophan or chlorpromazine. The contents of each flask were aseptically dispensed in 2-ml aliquots to 50 small test tubes and incubated at 37°C. F r o m 72 h of incubation, the tubes containing his + or trp + revertants were turbid, which changed their colouration from purple to yellow. Each duplicate test concentration of chlorpromazine was determined on at least 2 occasions. Modified fluctuation test with $9 activation The procedure was basically as described above. The 4 × 100 ml biotin-supplemented minimal salts aliquot (above) contained in addition to the test culture 0.25 ml of the $9 mix (3.13 m g / m l protein), 0.5 ml of 10 t~g/ml N A D P H and the appropriate concentration of chlorpromazine. The controls were the same as for the tests without activation (above). Each 100-ml aliquot was aseptically dispensed in 2-ml aliquots to 50 small test tubes and incubated at 37°C for 72 h. The number of revertant tubes which became turbid and had their colour changed f r o m purple to yellow were noted. Each duplicate test was determined on at least 2 occasions.

36

Stat&tical analysis In the fluctuation test, significance was tested by the chi-square test. Results and discussion

Chlorpromazine hydrochloride was found to exhibit a broad spectrum of antibacterial activity with an MIC between 8 and 15 #g/ml in minimal salts media. The MIC of chlorpromazine in minimum liquid medium was lower (8 #g/ml) than the values obtained from the solid medium (15 #g/ml), probably because of the greater diffusibility in the liquid medium. The mutagenicity tests indicated that the activation of chlorpromazine by the $9 fraction of the liver microsomal enzymes mediated a significant increase in base-pair substitutions and frame-shift mutations in E. coli WP2 trp, uvrA and the S. typhimurium TA102 strains respectively. In the plate incorporation reversion test there was no significant chlorpromazine mutagenesis except in the presence of liver microsomal activation (Tables 1, 3). The necessity for chlorpromazine activation to elicit mutagenesis explained why chlorpromazine per se was found to be non-mutagenic by Molnar et al. (1977) and Tosk (1974). Hence the photoexcited chlorpromazine derivatives mediated

mutagenesis (McPhee and Imrey, 1974). Chlorpromazine reverted S. typhimurium TA102 from H i s- to His ÷ without reverting the TA97 strain. E. coli WP2 trp- was not reverted but the transconjugant EEl02 (obtained from the transfer of R-plasmids pKM101 and pAQ1 from TA102 to WP2 t rp-) was reverted from Trp- to Trp +. The E. coli WPz trp, uvrA mutant was also reverted. The mutagenic reversion of all these susceptible strains occurred in both the plate incorporation and fluctuation tests with a lower concentration of chlorpromazine inducing a higher reversion than a corresponding higher concentration (Tables 2 and 3). The mutagenic activity of chlorpromazine obtained in this study correlates with the induction of mutagenesis in S. typhimurium by photo-activated chlorpromazine (McPhee and Imrey, 1974). The non-discriminatory inhibitory effect of chlorpromazine against DNA-repair-proficient and repair-defective strains indicated that the inhibitory effect was repair-independent. It is plausible to suggest that mutations could have been induced in the susceptible tester strains during the repair processes of the DNA-induced lesions by chlorpromazine. The susceptibility of strain TA102 to chlorpromazine in mediating frame-shift mutations and the non-susceptibility of TA97 in-

TABLE 1 RESULTS OF C H L O R P R O M A Z I N E (CPZ) REVERSION TESTS USING THE AMES PLATE I N C O R P O R A T I O N TEST METHOD, W I T H O U T MICROSOMAL ACTIVATION Strain

Agent

Concentration (t~g/ml)

Number of revertant colonies in test plates

Number of colonies in negative control plates

Number of colonies in positive control plates

EE97 EE97 TA97 TA97 EEl02 EEl02 TAI02 TAI02 WP2 WP2 WP2 her WP2 hcr

CPZ CPZ CPZ CPZ CPZ CPZ CPZ CPZ CPZ CPZ CPZ CPZ

5 10 5 10 5 10 5 10 5 10 5 10

3 2 2 1 3 2 0 0 1 2 3 1

3 1 2 1 3 2 1 0 2 2 3 2

300 280 250 200 330 280 310 230 120 95 210 163

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TABLE 2 RESULTS OF C H L O R P R O M A Z I N E (CPZ) REVERSION TESTS USING T H E AMES P L A T E I N C O R P O R A T I O N TEST METHOD, WITH MICROSOMAL ACTIVATION

Strain

Agent

Concentration (#g/ml)

Number of revertant Number of colonies colonies in in negative test plates control plates

Number of colonies in positive control plates

EE97 EE97

CPZ CPZ

5 10

0 2

2 3

200 193

TA97 TA97 EEl02

CPZ CPZ CPZ

5 10 5

3 1 120

4 2 0

189 170 210

EEl02 TA102 TAI02 WP2 WP2 WPz WP2

CPZ CPZ CPZ CPZ CPZ CPZ CPZ

10 5 10 5 10 5 10

45 80 23 3 2 12 5

0 5 2 2 2 0 0

189 220 200 218 234 203 198

Significance

P<0.01 P<0.01 P<0.0! P<0.01

P<0.01

TABLE 3 F L U C T U A T I O N TEST RESULTS W I T H C H L O R P R O M A Z I N E (CPZ) W I T H LIVER M I C R O S O M A L A C T I V A T I O N

Number of turbid tubes in the test

Agent

Concentration (~g/ml)

EE97 EE97 EEl02 EEl02

CPZ CPZ CPZ CPZ

0.4 0.8 2.0 4.0

3 4 9 6

4 5 0 0

50 50 50 50

P<0.01 P<0.01

WP2 WPz WP2 her WP2 hot

CPZ CPZ CPZ CPZ

0.4 0.8 2.0 4.0

2 6 43 25

1 4 10 10

50 50 50 50

P<0.01 P<0.01

dicated that chlorpromazine has preferential affinity for the A:T-rich sequences of nitrogenous bases of the DNA. Chlorpromazine has a high protein binding activity (92-97%) over the range of the clinical blood concentration of 4-470 ng/ml. This therefore indicates that a probable accumulation of chlorpromazine could induce genotoxic effects since the concentrations eliciting mutagenesis were generally higher than the normal clinical blood concentration. Although there has not been any report of chlorpromazine-induced carcinogenicity, this study provides baseline data that chlorpromazine is mutagenic.

Number of turbid tubes in the negative control

Number of turbid tubes in the positive control

Significance

Strain

Acknowledgement The authors are grateful to the Department of Biochemistry, Nigeria Institute For Oil Palm Research (NIFOR), Benin City for generously allowing the use of their equipment for the preparation of the $9 enzyme fraction. References Ames, B.N., F.D. Lee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786.

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