Anal. Chem. 1982, 54, 1725-1727
1725
Salmonella Electrode for Screening Mutagens Isao Karube, Takastill Nakahara, Tadashl Matsunaga, and Shulchi Suzukl Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsufa-cho, Midori-ku, Yokohama, 227, Japan
Preliminary screening of mutagens was completed within 10 h by using a microbial electrode system composed of an oxygen electrode and a membrane fitter retaining Salmonella typhhurlum revertant. n i e current decrease was correlated with mutagen concentrations. The minimum measurable concentration for AF-2 was 0.001 pg mL-'. The microbial electrode system was applied to the detection of other chemical mutagens such as N-methyl-N'-nltro-N-nltrosoguanidine, nitrofurazone methyl methanesulfonate, and ethyl methanesulfonate.
The mutagenic activity of carcinogens has recently been confirmed in a great number of cases (1). The existence of a high correlation betwleen the mutagenicity and carcinogenicity of chemicals is ntrw evident. The use of the microbial system is important for a survey of mutagenic chemicals. Recently, a number of microbial methods for detecting various types of mutagens have been developed. Microbial reversion assay using Salmonella typhimurium (2, 3) or Escherichia coli (4)have been emplciyed for screening tests of chemical carcinogens. A method named "Rec-assay" using 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 lengthy incubation of bacteria and complicated procedures. The authors have developed a number of microbial sensors consisting of immobilized microorganisms and an oxygen electrode which determined the respiration of microorganisms (6). The microbial sensoir has been used for BOD estimation (6),microbioassay of antibiotics (7), and cell number determination (8). Recently, the microbial electrode using recombinant deficient bacteria Bacillus subtilis Rec- has been applied to the preliminary Eicreening of chemical mutagens and carcinogens (9). In this paper, the microbial electrode using Salmonella typhimurium is describeld and applied to the detection of chemical mutagens. EXPERIMENTAL SECTION Materials. Beef extract, agar, and polypeptone were obtained from Kyokuto Pharmaceutial Co., Tokyo. 2-(2-Furyl)-3-(5nitro-2-fury1)acrylamide (AF-2), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Sigma, Missouri), nitrofurazone (Tokyo Kasei, Tokyo), methyl methanesulfonate (Tokyo Kasei), and ethyl methanesulfonate (Tokyo Kasei) were employed for experimenta. Other reagents were commercially available analytical reagents or laboratory-grade materials. Deionized water was used in all procedures. Microorganisms. Salmonella typhimuriumT A 100 was used for preliminary screening of mutagens. It was maintained on glucose-bouillon agar. It was aerobically grown at 37 O C for 16 h in 80 mL of the medium containing 0.8 g of beef extract, 0.8 g of peptone, and 0.4 g of NaCl (pH 7.0). Then it was centrifuged and resuspended in buffer solution (pH 7.0) for use. Apparatus. The electrode system employed for the screening of mutagens is shown in Figure 1. The system consists of an oxygen electrode (Ishikawa 13eisakujo Co., model A, diameter 1.7 cm, height 7.2 cm, poly(viny1chloride) casing) and a membrane
filter (Millipore type HA, 0.45 pm pore size, 25 mm diameter, 150 pm thick). The oxygen electrode was composed of a Teflon membrane (50 pm thick), a platinum cathode, a lead anode and sodium hydroxide (30%) as the electrolyte. The current of the electrode was converted to the voltage by a 2 kQ resistance and displayed on a recorder (TOA, Electronic Ltd., Model EPR-100A). The Pyrex filter holder (Millipore) was used for retaining microorganismson the membrane fiiter. The apparatus was wrapped and autoclaved in the conventional manner before use. Viable cell number was determined either from the respiratory activity as described in ref 9 or by the conventional colony counting method (IO). Procedure. The histidine free medium (Takara Kosan Co., Tokyo) employed for the rapid test of mutagens had the composition shown in Table I. The bacterial suspension (0.4 mL) was added to the 9.5 mL of medium in a test tube. Then, mutagens dissolved in 0.1 mL of water or dimethyl sulfoxide were added to the mixture and incubated at 37 "C for 10 h. After the incubation was completed, 1mL of broth was dropped onto the membrane filter with slight suction, and the membrane filter retaining S. typhimurium was attached to the surface of the Teflon membrane of an oxygen electrode with a holder. The electrode was inserted into the phosphate buffer solution (pH 7.0, 50 mL) containing 1 g L-' glucose which was saturated with dissolved oxygen by stirring with a magnetic bar. The current was displayed continuously on a recorder and the steady-state current was measured. RESULTS AND DISCUSSION Relationship between the C u r r e n t Decrease a n d the Cell Numbers of S . typhimurium. When the membrane fiiter-electrode containing various amounts of S. typhimurium revertant was inserted into the glucose-buffer solution saturated with oxygen, glucose was assimilated by the bacteria on the membrane. Oxygen was then consumed by the microorganisms so that the oxygen concentration around the membrane decreased and the current of the electrode decreased. The current decreased until it reached a steady state, which indicated that the consumption of oxygen by the microorganisms and the diffusion of oxygen from the solution to the membrane were in equilibrium. It required 10 min to reach the steady state. A linear relationship was obtained between the current decrease and the cell numbers of S. typhimurium revertant in the range lo7-8 X lo8 cells mL-l. Therefore, the revertants of S. typhimurium can be detected by this electrode system. Time Course of Electrode Response. S. typhimurium TA 100 requires histidine for their growth. However, the revertant of this strain can grow on the histidine-free medium. The electrode response depends on the numbers of viable bacteria retained on the membrane filter. Figure 2 shows the time course of electrode response when various amounts of AF-2 were added to the medium. The current was measured at 2-h intervals. After 8 h of incubation, the current decreased with increasing incubation time because the revertants grew above the minimum detectable numbers of cells by the electrode. A 10-h incubation gave the greatest sensitivity. On the other hand, there was no decrease in current from the medium in the absence of AF-2. Relationship between the Current Decrease a n d AF-2 Concentration. Figure 3 shows the relationship between the current decrease and AF-2 concentration. The current de-
0003-2700/82/035~1725$01.25/0 0 1982 American Chemical Society
1726
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
Table I. Composition of Medium for Mutagen Test concn, compound mg L-' L-alanine 200 L-arginine.HC1 200 L-aspartic acid 200 L-cystine 100 L-glutamic acid 5 00 glycine 100 L-isoleucine 100 L-leucine 100 L-lysine .HCI 200 L -methionine 100 L -phenylalanine 100
concn, mg L-'
compound p proline
L-serine threonine L-twptophan L -tyrosine L -valine ducose EH,COONa KH,PO, K,HPO,
100 50 100 50 100 100 2 0 x 103 2 0 x 103 5 00 500
concn , mg L-l
compound NH4C1
MgS04*7H,0 FeSO,. 7H ,O MnS0,.4H20 NaCl adenine guanine uracil xanthine thiamine
3000 200 10 10 10 10 10 10 10 1
concn, mg L'l
compound riboflavine pyridoxine pyridoxal potassium pantothenate nicotinic acid p-aminobenzoic acid biotin folic acid
1 1 1 1 1
0.2 0.01 0.01
m
VI
al m
"
.c m
w al
L
2
/
u
Flgure 1. Schematic diagram of the microblal electrode: (1) Salmo-
nella typhlmurium TA 100, (2) membrane filter, (3) Teflon membrane, (4) pt cathode, (5) Pb anode, (6) alkaline electrolyte.
0.01
0
0.02
AF-2 concentration (pg/ml)
Figure 3. Relationship betwen the current decrease and AF-2 concentratlon. S . typhimurium was Incubated with various concentrations of AF-2 at 37 O C for 10 h.
Table 11. Response of the Electrode to Various Typical Mutagens concn, mutagen no mutagen N-methyl-N' -nitroN-nitrosoguaeidine nitrofurazone 5
10
15
Time ( h )
Flgure 2. Time course of electrode response when ( 0 )0, (0)0.001, and (X) 0.006 pg mL-' of AF-2 was added.
crease became larger with increasing AF-2 concentration. The minimum measurable concentration of AF-2 was 0.001 bm mL-l. The current decrease was reproducible within 5% of the relative error when a sample solution containing 0.006 pg mL-l of AF-2 was employed. Response to Various Mutagens. Table I1 summarizes the responses of the electrode to various typical chemical mutagens. When S. typhimurium was incubated with chemical mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, nitrofurazone, methyl methanesulfonate, and ethyl methanesulfonate, for 10 h the current decrease of the electrode was measured. The response of the electrode increased with increasing concentration of chemical mutagens. Therefore, the mutagenicity of chemicals can be estimated with the microbial electrode. Long-term carcinogenicity testa with laboratory mammals are not only time-consuming but also demanding on resouces. In practice, it is inconceivable that resources could be made available 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
me thy1 methanesulfonate ethyl methanesulfonate
current
Mg mL-l decrease, M A
0 0.005
0
7.0
0.30 0.50 0.007
12.5 11.0 12.5 8.2
0.014 0,010
10.5
0.010
9.0
values. The simple test using S. typhimurium has been developed for testing chemical mutagens by Ames et al. (2, 3, 11). The Ames test showed that about 90% of the organic chemical carcinogens tested thus far are mutagens (12). Now, the Ames test is in current use in over 2000 government, industrial, and academic laboratories throughout the world. Therefore, S. typhimurium was employed for the microbial electrode in this study. S. typhimurium TA 100 is a histidine-requiring bacterial mutant. This strain is reverted back to the wild type by chemical mutagens. Consequently, it can grow on the medium in the absence of histidine. The growth of the revartants can be determined by the electrode system. The conventional Ames test is based on the colony method. Chemical mutagens are dispersed to the agar plate. Therefore, 2 days of incubation is needed for the mutagen test. In this study, a homogeneous bacterial suspension was employed and mutagens were added to the homogeneous suspension. The complete medium containing amino acids, vitamins, and mineral salts was used in place of nutrient broth. Furthermore, employment of the electrode system makes possible a large injection of bacterial suspension. As a result, the time required for mutagen test was shortened to 10 h. The time is longer than that (1h) of
Anal. Chem. 1982, 5 4 , 1727-1729
our previously reported microbial electrode system (9). The sensitivity of the microbial electrode system was higher than the conventional Ames test and our previous system. The minimum measurable mutagen concentration was 0.001 pg mL-l by the microbial electrode, 1.6 pg mL-l by the previous system and 10 fig mL-l by the Ames test for AF-2. The microbial electrode system appears promising and attractive for use in the routine preliminary screening of mutagens and carcinogens. Further developmental studies in this laboratory are beiing directed toward applying immobilized S-9 fraction (microsome from rat liver) to the microbial electrode.
LITERATURE CITED (1) Bridges, B. A. Nature (London) 1976, 261, 195-200. (2) Ames B. N.; Lee, F. D.; Durston, W. E. Roc. Natl. Acad. Sci. U.S.A. 1973, 70, 782-786.
1727
(3) Ames, B. N.; Durston, W. E.; Yamasakl, E.; Lee F. D. R o c . Natl. Acad. Scl. U.S.A. 1973. 70, 2281-2285. (4) Bridges, B. A.; Mothershead, R. P.; Rothweii, M. A.; Green, M. H. L. Chemdiol. Interact. 1972, 5 , 77-84. (5) Kada, T.; Turikawa, K.; Sadaie, Y. Mutat. Res. 1972, 16 165-174. (6) Karube, I.; Matsunaga, T.; Mitsuda, S.; Suzuki, S. Biotechnol. Bioeng. 1977, 19, 1535-1547. (7) Karube, 1,; Matsunaga, T.; Suzuki, S. Anal. Chlm. Acta 1979, 109, 39-44. (8) Matsunaga, T.; Karube, I.; Nakahara, T.; Suzuki, S. Eur. J . Appl. Microbiol. Biotechnoi. 1981, 12, 97-101. (9) Karube, I.; Matsunaga, T.; Nakahara, T.; Suzuki, S. Anal. Chem. 1981, 53, 1024-1026. (10) Postgate, J. R. “Method in Microbiology”; Academic Press: New York, 1969: Vol. 1. (11) Ames, B. N. Science 1979, 204, 587-593. (12) McCann, J.: Choi, E.; Yamazaki. E.; Ames, B. N. Proc. Natl. Acad. Sci. 1975, 72, 5135-5139.
RECEIVED for review January 4,1982. Accepted May 10,1982.
Theory for Double Potential Step Chronoamperometry, Chronocouloimetry, and Chronoabsorptometry with a Quasi-Reversible Electrode Reaction Dennis H. Evans* and Michael J. Kelly Department of Chemistry, University of Wisconsin -Madison,
Madison, Wisconsin 53706
Serles solutions for the current-, charge-, and absorbancetime responses have been obtained for the case where the flrst step Is to a potential where the forward reaction occurs at a dlffuslon-controlled rate and the second step Is to any potential. A slngle equatlon Is adequate for systems ranging from fully reversible to extremely slow electron transfer reactions. Simple data anallysis can be achieved by using Ilmking forms of the solution for times shortly afler the switchlng time.
The analytical signal from most electroanalytical techniques is affected by the rate of the electrode reaction so it is important to be able to mearriire such rates. A simple approach is to apply a potential step rind measure the current ( I ) , charge (2), or (in transmission mode spectroelectrochemistry) the absorbance (3) as a function of time. For example, the current for reduction of a material 0 is given ( I ) by eq 2 k
o -t.ne .&
R
kb
(1)
i = nFACo*kr exp(H2t) erfc kf = k, exp((--anF/RT)(E - E O ) ) kb = k, exp((1 -. a)(nF/RT)(E- E o ) )
(4)
H = kf/i901/2 + kb/DR1f2
(5)
(2)
(3)
where i is the current, A is the electrode area, CO*is the bulk concentration of reactant (product is assumed to be initially absent, CR*= 0), t is time, k , is the standard heterogeneous electron transfer rate contatant, a is the transfer coefficient, E is the electrode potentkill, Eo is the formal potential, DIis the diffusion coefficient of species I, and the other symbols
carry their normal meaning. The discussion will be developed in terms of an initial reduction step but the results can be easily adjusted to cover the converse. When Ht1I2 is small, a linear plot of i vs. t1/2is obtained which may be extrapolated to t = 0 to evaluate the initial current, itlo. When t = 0, eq 2 becomes
it=,, = nFACo*kf
(6) which provides a simple and direct means of determining kf at various potentials. The direct measurement of k b can be achieved by analogous studies of solutions containing R but no 0. Here the potential steps would be from an initial potential where no oxidation occurs to a more positive potential where the oxidation occurs a t a measurable rate. However, it is frequently infeasible to prepare a solution of R either because it is difficult to prepare in a pure form or because R is unstable. In such cases, a double potential step experiment can be implemented. In the first step, the potential is held at a very negative value where 0 is reduced to R at the diffusion-limited rate. This electrolysis produces a well-defined concentration-distance profiie for the product R. At a switching time, T , the potential is changed to a more positive value where oxidation of R occurs and the observed current can be used to evaluate kb. In an earlier application of this technique ( 4 ) , it was necessary to resort to digital simulation (5) to carry out an analysis of the current-time curve during the second step. Digital simulation was also used to find the best fit with experimental absorbance-time curves in a recent spectroelectrochemical study (6). Clearly, an analytical solution would be very useful. In this paper, we report a series solution which provides a useful theoretical basis for data analysis. Several earlier treatments exist. Smit and Wijnen (7)obtained a solution which is valid only for “symmetric” steps, Le., for cases where H on the first step is numerically equal
0003-2700/82/0354-1727$01.25/0 . 0 1982 American Chemlcai Society
