Introduction 

The purpose for detecting antimetabolic activity in compounds, is for searching prospective anticancer agents (Goodman and Gilman,2006). There are several bacterial prescreens available to detect either antimetabolic property associated with anticarcinogenicity (Hanka et al,1978) or mutagenicity which involves carcinogenicity (Ames et al 1973,1982); prescreens which represent in vitro tests are preferred not only because they are simple, cheap and less time consuming, but also because these substantially correlate with the in vivo systems.

The anti-microbial property of many drugs, including several members of the phenothiazines have been reported from our laboratory (Saha et al,1976; Chattopadhyay et al 1988, Ray et al 1990). The present study was carried out with several chemotherapeutic agents for detecting their antimetabolic activity and then proceeding with determination of mutagenicity.

Materials  and  Methods :

Drugs—All the drugs screened are described in Table 1 below. These were all obtained from their respective manufacturers in the pure dry state.

 Table 1: List of drugs screened for in-vitro Antimetabolic Property.

Bacteria 

For detection of antimetabolic property, B.subtilis UC 564 and E. coli UC51, were obtained from Dr. L.J. Hanka, Upjohn company, Michigan, USA. For determination of mutagenicity, Salmonella typhimurium TA 102 was received from Prof. B. Ames, University of California, Berkeley, USA (Hossain et al 1987)

Media 

The composition of various media are given in Tables 2 and 3.

Table 2: Assay  Media  For  Antimetabolic  Activity Nutrient Agar.

Synthetic  Agar

Table 3 : Media  and  buffer used  for  detection  of  mutagenicity.

Vogel Bonner medium : K2HPO4  500 mg, Na(NH4)2SO4  175 mg, citric acid monohydrate 100 mg, MgSO4 1O mg, warm distilled water 100ml.

Soft agar : Difco agar 0.6%  and Nacl 0.5%  with a trace of histidine.

Minimal glucose agar : Difco  agar  5%  and  glucose  2%  in Vogel Bonner medium

2 (M) Na-Phosphate buffer : 0.2 (M) NaH2PO4 ( 13.8 gm/500 ml ) + 0.2 (M) Na2HPO4 ( 14.2 gm/500ml ), Ph 7.4

Test  for detection of Antimetabolite Activity 

Sterile synthetic and nutrient agar plates were overlaid with 2 ml of the same media containing 1 ml of 18 hr old peptone water ( 1% Difco peptone + 0.5% Nacl, ph 7.4) cultures so as to produce a semiconfluent lawn of growth. The plates were dried and discs containing the drugs (100, 200, 400 ug/disc) were placed at suitable distances apart . readings were taken after overnight incubation at 37c. The drug which failed to produce any inhibition on nutrient agar but showed a large zone of inhibition on synthetic agar was considered to contain a potential antimetabolite (Hanka,1978).

Mutagenicity Test 

Drug solutions were taken in sterile tubes kept on ice to which was added 0.5ml of 0.2 M sodium phosphate buffer, followed by 0.1ml of overnight peptone water culture of the test strain; incubation was at 37c for 20 mins with shaking . Soft agar(2ml) was added to the tubes and the contents vortexed and poured over minimal glucose agar (Vogel Bonner) plates. After setting, the plates were inverted and kept at 37c for 48hr, following which the revertant colonies were counted. Control plates containing bacteria, histidine and buffer was studied to give the number of colonies arising due to spontaneous mutation (Ames et al,1973).

Results 

Table 4 describes the antimetaboliic activity of the chemotherapeutic agents. Of 32 compounds tested, nalidixic acid (20ug) and cotrimoxazole (20ug) discs produced inhibition zones 17.0 and 25.0 mm respectively against  E.coli UC51, and 14.0 and 30.0 mm wide zones in case of B.subtilis UC564 on synthetic agar only. Similarly  chlorcyclizine discs  (100, 200 and 400ug) also produced inhibition reaction  on synthetic agar and not nutrient agar. The known antimetabolite, 5-fluorouracil served as the control antimetabolic agent in this study.

Table  4 : Antimetabolic  activity  of  known and  unknown agents.

Following Ame’s test, it was noted that in case of Salmonella typhimurium TA 102, due to spontaneous mutation i.e when no drug is used, 268  colonies were formed mutation (Table 5). Nalidixic acid, and cotrimoxazole were  found to be mutagenic since they produced significantly high number of revertant colonies. However , the antihistamine  chlorcyclizine  failed to score a detectable mutagenic effect as it scored a very low count of revertant colonies

Table 5: Effect  of  different drugs on S.typhimurium TA 102 to produce histidine revertant colonies.

Discussion 

Although, chlorcyclizine and chlorpromazine, differ with each other chemically, however, possess functional similarities, both of them being antihistaminic and antimicrobic. In addition, chlorcyclizine can act as an antimetabolite, whereas chlorpromazine is a powerful psychotropic drug. This functional difference between the two compounds may be explained in terms of their structural dissimilarities.

Thus, it was found that the phenothiazine exhibit a variety of activities which ranged from modulation of different components of the nervous system to antimicrobial and antiplasmid activity. All these functions appeared to depend on the type of attachment to the phenothiazine tricyclic ring structure. The structurally related yet distinctive chemical compound chlorcyclizine shows further variation of the range of functions in exhibiting a potentially antimetabolic and non-mutagenic effect. This shows the possibilities of detecting compounds with greater anticarcinogenic property.

References  

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