1024
Anal. Chem. 1981, 53, 1024-1026
Preliminary Screening of Mutagens with a Microbial Sensor Isao Karube, Tadashi Matsunaga, Takashi Nakahara, and Shuichi Suzuki Research Labaratoty of Resources Utilization, Tokyo Institute of Technology, Nagatsuta-cho, Maori-ku, Yokohama, 227 Japan
Tsuneo Kada Department of Induced Mutation, National Institute of Genetics, Mishima, Shizuoka-ken, 4 1 1 Japan
Preliminary screening of mutagens is achieved with a microbial sensor composed of two microbial electrodes using a recombinant deficient strain, Baclllus subf///s (Rec-), and a wild strain, B. subtills (Rec'). The principle of the sensor is based on the inhibitory action of the mutagens on the respiration of 8. subtllls (Rec-). When chemical mutagens were added to the system, the current of the Rec- electrode gradually increased and that of the Rec' electrode did not increase. Linear relationships were obtained between the rate of the current increase of the Rec- electrode and the mutagen concentration. Such responses were not abtained when the antibiotics, respiration inhibitors, and bactericides were employed to the sensor.
Long-term carcinogenicity tests with laboratory mammals are not only time-consuming but also demanding resources. In practice, it is inconceivable that resources could be made available (either men, monkey, or mice) on the necessary scale to screen all the tens of thousands of substances. Therefore, testing with whole mammals is restricted to certain groups of suspecting substances. If one is to screen for carcinogenic chemicals, one must use a short-term preliminary test with high predictive values. The mutagenic activity of carcinogens has recently been confirmed in a great number of cases (1). The existence of a high correlation between the mutagenicity and carcinogenicity of chemicals is now evident. The use of microbial systems is important for a survey of mutagenic cherpicals. Recently, a number of microbial methods for detecting the various types of mutagens have been developed. Microbial reversion assays using Salmonella typhimurium (2, 3) or Escherichia coli ( 4 ) have been employed for screening tests of chemical carcinogens. A method named "rec-assay" utilizing Bacillus subtilis has also been proposed for screening chemical mutagens and carcinogens (5). These methods are more rapid and simple than the carcinogen test using animals. However, the microbial reversion assays and the "rec-assay" still require a lengthy incubation of bacteria and complicated procedures. Moreover, these methods are not suitable for continuous monitoring of environmental carcinogens and mutagens. A number of microbial electrodes consisting of immobilized microorganisms and electrochemical devices have been developed for amino acid determination (6-8),microbioassays (9, IO), and BOD (biological oxygen demand) estimation (11-13). Recently the inhibitory actio0 of an antibiotic was measured by the microbial electrode using an oxygen electrode (14). This microbial electrode was based on the amperometric determination of microbial respiration change caused by the inhibitory action of the antibiotic. An electrode consisting of the aerobic recombination-deficient bacteria and the oxygen electrode can be applied to the preliminary screening of chemical mutagens and Carcinogens. 0003-2700/81/0Q53-1024$01.25/0
In this paper, the microbial sensor system composed of a B. subtilis Rec- electrode and Rec+ electrode is described and applied to the detection of chemical mutagens.
EXPERIMENTAL SECTION Materials. Beef extract and peptone (from casein) were obtained from Kyokuto Pharmaceutical Co. 2-(2-Furyl)-3-(5nitro-2-fury1)acrylamide(AF-2) (from Kada), mitomycin (Sigma), 4-nitroquinoline N-oxide (4-NQO) (Tokyo Kasei),Captan (Wako Pure Chemicals),N-methyl-N'-nitro-N-nitrosoguanidine(Sigma), and aflatoxin B1 (Sigma) were employed for experimenta. Other reagents were commercially available analytical reagents or laboratory-grade materials. Deionized water was used in all procedures. Microorganisms. The recombination-deficient strain B. subtilis M45 Rec- and the wild strain B. subtilis H17 Rec' were isolated by one of authors. Both strains were aerobically grown at 30 "C for 24 h in 3 mL of the medium containing 30 mg of beef extract, 30 mg of peptone and 15 mg of NaCl (pH 7.0). One milliliter of 50% glycerol (w/v) was added to 3 mL of full-grown bacterial broth culture and stocked at -20 OC. Coqstruction of the Electrode System. Immobilization of B. subtilis Rec- and Rec' was performed as follows: 1 mL of bacteria suspension containing 2.7 X lo8 cells was dropped onto a porous acetylcellulose membrane (Millipore Type HA, 0.45 pm pore size, 25 mm diameter, 150 pm thick) with slight suction, and it was fixed on a Teflon membrane of the oxygen electrode. The microbial electrade system is shown in Figure 1. The electrode system consisted of two microbial electrodes: the electrode of B. subtilis Rec- (Rec- electrode) and the electrode of B. subtilis Rec' (Ret+ electrode). Each electrode was composed of immobilized bacteria and an oxygen electrode (Ishikawa Seisakujo Co., Model A; diameter 1.7 cm, height 7.2 cm; PVC casing). The electrodes were fixed to a 50-mL reaction vessel. Assay Procedure. AF-2,4NQO, and Captan were dissolved in dimethyl sulfoxide (Me2SO)and diluted to the appropriate concentration with 0.1 M phosphate buffer (pH 7.0) containing glucose (1g L-l). The final concentration of MezSO was kept below 0.66%. Both Rec- and Rec+ electrodes containing 2.7 X 108cells, respectively, were inserted into a 30 mL phosphate buffer solution (0.1 M, pH 7.0) containing glucose (1g L-l) which was saturated with dissolved oxygen by stirring. Then, mutagens were added to the solution, and the electrode currents were displayed on a recorder (TOA, Electronics Ltd., Model EPR-2OOA). The rate of the current increase was calculated from the linear portion of the response curve after 20-60 min.
RESULTS The respiration activity of immobilized bacteria can be determined by the oxygen electrode. If sufficient nutrients (e.g., 0.3 g L-l glucose) are present in a sample solution, the constant current is obtained from the electrode. The current depends on the total respiration activity of immobilized cells. Therefore, the total respiration activity of bacteria, the current, depends on the number of viable cells immobilized onto the acetylcellulose membrane. The relationship between the current and the cell numbers on the acetylcellulosemembrane was linear in the range over 0.1-3.0 X lo8 cells. Consequently, 2.7 x lo8 cells of B. subtilis Rec- and Rec+ were immobilized, thereafter, on the membrane of the electrode. 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL.
53, NO. 7,
JUNE
1981 * 1025
Table I. Response to Various Mutagens rate of current increase, 1.1A/h amt of mutagens, RecRec+ fig/mL electrode electrode
6
AF-2 5
mitomycin 2
Captan
4-NQO N-methyl-N-nitro-Nnitrosoguanidine aflatoxin B, Figure 1. Schematic diagram of the electrode system for rapid detection of chemical mutagen: (I) Rec- electrode, (11) Rec+ electrode, (1) Bacillus subti/& Ret', (2) Bacillus subti/is Rec-, (3) membrane filter, (4) Teflon membrane, (5)Pt cathode, (6) Pb anode, (7)recorder.
--4
10 ReC
-
E 3
5
NaN,
cyanide chloramphenicol
0 0
.
1
0 2. I 4.8 0 3.6 12 0 14 0 11 1.0 22 1 12
0 0 4.0 0 0.5 11 0 0 0 0 0.5 1.0 0 1
Table 11. Responses to Antibiotics, Respiration Inhibitors, and Bactericides rate of current increase, &A/h amt of reagent, RecRec+ fig/mL electrode electrode benze thonium chloride streptomycin
Y
0.5 1.6 2.8 0.9 I.2 14.4 0.5 2.0 5.0 16.0 5 20 0.8 12
50 500 20 50 10 50 1 20 1.0 10
8 54 16 50 0 6.0 40 255 0 0
9 53 16 51 0 5.0 41 252 0 0
T i m e (hr)
Figure 2. Response curves of the microbial sensor to AF-2, when 1.6 pg/mL of AF-2 was added to the system. Responses to t h e Chemical Mutagens. When the Recand Rec+ electrodes were inserted into the glucose-buffer solution (0.3 g L-l glucose), steady-state currents were obtained. Then, AF-2, a famous mutagen, was added to the solution. The time course of the electrode currents (response curves) is shown in Figure 2. After 20-40 min, the current of the Rec- electrode began to increase giving a sigmoidal curve. On the other hand, the current of the Rec+ electrode did not increase. The rate of current increase is a measure of the mutagen concentration and is most easily measured as the linear slope at the midpoint of the sigmoidal curve. Table I summarizes the response of the electrode system to various typical chemical mutagens. When chemical mutagens such as AF-2, mitomycin, Captan, 4NQ0,N-methyl-N'-nitro-Nnitrosoguanidine, and aflatoxin B1 were added to the glucose-buffer solution, the rates of the current increase of the Rec- and Rec+ electrodes were measured. The current of the Rec- electrode markedly increased when these reagents were added to the system. Therefore, the mutagenicity of chemicals can be estimated with the electrochemical system. Relationship between t h e Rate of Current Increase a n d t h e Concentration of Mutagen. The relationships between the rate of current increase of the R e t electrode and the concentration of AF-2 and mitomycin were examined. Linear relationships were obtained in the range over 1.6-2.8 kg mL-' for AF-2 and 2.4-7.3 pg mL-' for mitomycin. Responses to Antibiotics, Respiration Inhibitors, and Bactericides. Table I1 shows the responses of the Rec- and the Rec+ electrodes to chloramphenicol, streptomycin, pen-
icillin, azide, cyanide, and benzethonium chloride. Chloramphenicol and streptomycin are known as an inhibitor of protein synthesis in bacteria. When 1.0 and 10 pg mL-l of chloramphenicolwere added to the system, the rates of current increase of both Rec- and Rec+ electrodes were 1 MAh-l, respectively. The currents of both electrodes did not increase when streptomycin (10 pg mL-l) was employed. Therefore, chloramphenicol and streptomycin were not mutagens. Penicillin is known as an inhibitor of the synthesis of bacterial cell wall. As the current increase of the Rec- electrode was not observed, the penicillin is also not a mutagen. Azide and cyanide are inhibitors of cytochrome oxidase. They inhibit the respiration chain in microbial cells. As shown in Table 11, the currents of both the Rec- and Rec+ electrodes increased rapidly. Benzethonium chloride is known as a bactericide. When 20 and 50 mg mL-' of benzethonium chloride were applied to the system, the currents of both the Rec- and Rec+ electrodes increased a t almost the same rates, respectively. Therefore, benzethonium chloride was not a mutagen. DISCUSSION This microbial sensor system is based on the inhibitory action of the mutagens on the respiration of B. subtilis Rec-. Figure 3 shows schematically the principle of the microbial sensor for rapid screening of mutagens. In this study, B. subtilis M45 ( R e d and H17 (Red) were employed. B. subtilis M45 (Rec-) is genetically deficient in the DNA recombination enzyme system, whereas B. subtilis H17 (Ret') is a wild strain which has the ability of repairing damaged DNA through a cellular DNA of Rec- and Rec+ bacteria, leading to the damage of DNA. The subsequent death of Rec- bacteria is preceded
1026
Anal. Chem. 1981, 53, 1026-1030
and 5.0 pg mL-' by the "rec-assay" and 10 pg mL-' by the Ames test for AF-2. Oxygen Oxygen As described above, the recombinant-deficient (Rec-) electrode electrode Bacillus bacteria are killed with mutagens. Therefore, a new bacteria Bacillus FEfIT membrane is needed for each analysis; but the preparation F ~ O $ O ~80 Recl . . ) . "TE . . . O.0"*
[email protected] * NRec- NRec+ 1.$'' .&&'0.8@~@1 *. and exchange of a bacteria membrane are very easy and replacement takes only 1min. Moreover, the bacteria can be + Chemical mutagen (AF-2,Mitomycin) i stored for 6 months a t -20 "C. The electrode current was DNA damage DNA damage reproducible within 5% in the buffer solution containing 1 g L-' glucose when the bacteria membrane used for analysis J. was replaced with a new membrane prepared from the stock Death Recombination bacteria. 4 wpair c The microbial electrode system appears promising and ~ ~ ~~ ~~ ~ ~~ ~- ~ 8 0 8 0 $ 0 5 ) 0 ~ Lo o O o O 8 OJ N attractive for use in the routine preliminary screening of mutagens and carcinogens. Further developmental studies NRect NtRec+ N : Number o f c e l l s NRec->NoRecin this laboratory are being directed toward applying the IRect I : Current o f IRec-
