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Bacterial Biolumininescence as a Bioassay for Micotoxins

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Bioluminiscencia de bacterias para la identificación de micotoxinas
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  APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p. 1072-1075Vol. 44, No. 5 0099-2240/82/111072-04 02.00/0 Bacterial Bioluminescence as a Bioassay for Mycotoxins IDA E. YATES AND JAMES K. PORTER R. B. Russell Agricultural Research Center, Agricultural Research Service, U. S. Department of Agriculture, Athens, Georgia 30613 Received 14 May 1982/Accepted 27 July 1982 The use of bacterial bioluminescence as a toxicological assay for mycotoxins was tested withrubratoxin B,zearalenone, penicillic acid, citrinin, ochratoxin A, PR-toxin, aflatoxin B1, and patulin. The concentrationsof mycotoxins causing 50 light reduction  EC50) in Photobacterium phosphoreum were determined immediatelyand at 5 h after reconstitution of thebacteria from a freeze-dried state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of the above mycotoxins onbioluminescenceweredetermined after 5, 10, 15, and 20 min ofincubation with the bacterial suspensions. The concentrationof rubratoxin B necessary to elicit an EC50 increased with time, whereas the concentrationof citrinin, penicillic acid, patulin, and PR-toxinnecessary decreased with time. There was very little change in the concentration of zearalenone, aflatoxin B1, and ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence assay wasmost sensitive to patulin and least sensitive to rubratoxin B. Mycotoxins aretoxic fungal metabolites found as contaminants in many agricultural products. These compounds cause deleterious effects in biological systems and havebeenimplicated in carcinogenesis, toxicosis, and tera- togenesis in mammalian populations. Mycotox- ins derived from agricultural commodities lead to insidious problems related to the reproduc- tion, health, and growth performances of both animals andhumans. As a result, mycotoxins in the food supply must be evaluated and closely monitored (3, 5). Various in vitro short-term biological assays have been employed to screen forthe presence of several mycotoxins. Lompe and v. Milc zewski (8) demonstrated that 30 ,ug of rubratoxin B per mlwas required to elicit cytocidal effects in Girardiheart cells. All other cell lines re- quired 125 ,ug/ml. Umeda (9) observed cytomor- phological changeson cells cultured from rat liver, kidney, and lung tissues. The hereditable effects for several mycotoxins also havebeen investigated with FM3A cells from mouse mam- mary carcinomas (10). Brine shrimp larvae have been used to evaluate fungal toxins  4 . The concentrationsof mycotoxins that could be de- tected in the chloroform extracts of fungal cul- tures ranged between 10 to 100 ,ug/ml for afla- toxin B1, ochratoxin A, and rubratoxin B and to 500 ,ug/ml for citrinin, penicillicacid, patulin, and zearalenone. Zebra fish larvae were shown to bevery sensitive to several mycotoxins, including aflatoxin B1, ochratoxin A,and patulin  1 . However, penicillic acid was not effective in this system at a concentrationof 5,ug/ml. Addi- tionally, other in vitro systems have utilized onlyone or twomycotoxins to study the bio- chemical andmorphological changesproduced in cell systems. Problems encountered in these various bioassays include the maintenance of animals or cell lines and cultures, technically complex procedures requiring extensiveprepa- ration or assaytimes or both, expensive materi- als, and subjective data analyses. The purpose of this investigation was to determine the effects of mycotoxinson bacterial bioluminescence.Subsequently, such a procedure may serve as a short-term assay for mycotoxins, whichwould circumvent some of the disadvantages of bioas- says currently used. MATERIALS AND METHODS Mycotoxins. Aflatoxin B1 was obtained from Calbio- chem-Behring. Rubratoxin B, zearalenone, penicillicacid, citrinin, ochratoxin A, PR-toxin, and patulin were obtained from Sigma Chemical Co. All mycotox- ins were dissolved in methanol except aflatoxin B, which was dissolved in dimethyl sulfoxide and zeara- lenone whichwas dissolved in each of thesesolvent vehicles. The purity and quantity of each mycotoxin solution was determined by high-performance thin- layer chromatography  HPTLC) (7) and by Xmax  e in methanol with a Varian Cary model 15 UV spectro- photometer. Briefly, the mycotoxins were applied to the HPTLC plates(10 by 10 cm; silica gel 60 F-254, E. Merck) with either a Camag Nanoapplicator (0.2 ,ul or a capillary dispenser system (0.5 ,ul in conjunction with a Camag Nonomat. The HPTLC plates were developed for 5 cm in a linear developing chamber  Camag) with 1.5 ml of developing solvent in eachtrough (toluene-ethylacetate-formic acid, 30:6:0.5 1072  EFFECT OFMYCOTOXINS ON BIOLUMINESCENCE 1073 TABLE 1. EC50 for mycotoxins ECS0 (>g/ml) at: MeanEC50 EC50   Mycotoxin Bacterial (>ig/ml) for change type used 5,10, 15, from 5to 5 min 10 min 15 min 20 min and 20 min 20 min Patulin Fresha 7.533.872.67 2.16c 4.06 -72 Agedb 6.17 3.452.36 1.82c 3.45d -70 o PR-toxin Fresh 7.79 3.262.10 1.72c 3.72 -78 Aged 9.49 3.43 2 26 1.80c 4.25d -81 Penicillic acid Fresh 15.9510.658.72 7.44c 10.69 -53 Aged 14.679.79 7 60 5.91c 9.49d -60 o Citrinin Fresh 27.7420.46 17 07 14.91c 20.05 -46 Aged 30.7019.9916.60 14.46c 20.44 -53 ZearalenoneFresh 14.3713.70 13.21 12.29c13.51 -11 Aged 11.59 11.59 11.3711.30 11.46d -2 Ochratoxin A Fresh 18 49 16.60 16 17 16.27c16.88 -12 Aged 18.53 16.4016.39 16.68c 17.00 -10 o Aflatoxin B1 Fresh 21.97 21.19 19.4420.3520.73 -7 Aged 24.79 24.43 25.66 25.87 25.19d +4 Rubratoxin B Fresh 31 79 33.3635.17 34.73c 33.76 +9 o Aged 26.6831.09 31.82 32.82c 31.23d +23 a Freshly reconstituted bacterial suspension. b Aged bacterial suspension. c Significantly different from the 5-min value at the 0.05 level. d Significantly different from the value forthe freshly reconstituted bacterial suspension at the0.05 level. [vol/vol], 30:14:4.5 [vol/vol], or both). Scanning of the HPTLC plates was performed with a Camag photo- densitometer  monochromator version) attached to a Hewlett-Packard 3390A reporting integrator. The amounts of mycotoxin present in each test solution were determinedimmediately before the bacterial bio- luminescence assay, and quantities reported were the averages of triplicate analyses. Bioluminescence assay. The protocol used forthe bacterial bioluminescence analyses for toxicity was essentially that of Bulich and Isenberg  2 . The freeze- dried bacteria (astrain of Photobacterium phosphore- um), reconstitutionsolution (ultrapure water), and diluent (a solution containing 2 NaCI to provide osmotic protection for the marine bioassay organism) werefrom Beckman Instruments, Inc. The dilution series for each mycotoxin was constructed so that the highest concentration caused a 90 o light decrease and the lowest concentration caused a 10 light decrease. The concentrations of the solvent vehicles, DMSO and methanol  ,ul/ml of diluent), in the assay dilution causing the greatestlight decreases were: PR-toxin, 0.75; penicillic acid, 1.0; citrinin and zearalenone, 2.5; ochratoxin A, 4.5; patulin and rubratoxin B, 5; and aflatoxin Bl, 10. Bioluminescence determinations were made with the Microtox Analyzer  Beckman Instruments) at 5, 10,15, and 20 min after addition of toxin to the bacterial suspensions. Assays were per- formed at 15°Cimmediately afterbacterial reconstitu- tion (freshlyreconstituted suspensions), as recom- mended by Bulich and Isenberg  2 . In addition, bacterial suspensions maintained at 3°C for 5 h after reconstitution (aged suspensions) were tested for sen- sitivity to the mycotoxins. Data analysis. The Microtox datareduction was accomplished by the calculation of gamma (the ratio of light lost to the light remaining) (6) by using a correc- tion factor to accommodate the normal change of light by thebacteria without added toxicant. The correction factor was determined by dividing thediluentcontrol blank reading at the time point analyzed  i.e., 5, 10,15, or 20 min) by the zero time reading. The gammas for each concentration of mycotoxin at a given time point were subjected to linear regression and power curve analyses with the algorithms developed by Hewlett- Packard for the HP41-C calculator. The power curve was used to derive the concentrations  in micrograms per milliter) causing 50 and 20 o light reduction  EC50 and EC20, respectively) for each mycotoxin and repre- sents the mean of at least three separate experiments. Analysis of variance was used to determine significant differences between EC50 values for freshly reconsti- tuted bacteria and aged suspensions and also for significant differences between EC50 values for differ- ent incubation times. RESULTS AND DISCUSSION Bacterial bioluminescence was inhibited by all of the mycotoxins studied (Table 1), with the sensitivity of freshly reconstituted bacteria after5 min ofincubation ranging from 31.79 txg/ml for rubratoxin B to 7.53 ,ug/ml for patulin. Bacterial suspensions aged for 5 hmaintained sensitivity VOL   44, 1982  1074 YATES AND PORTER TABLE 2. Lower limits of detection  EC20) for mycotoxins using bacteria immediately after reconstitution Incubationtime 5 min 20 min Mycotoxin   EC20 Change EC20   Change from from EC50 (>g/m1) EC50 to 4Lg/ml) to EC20 EC20 Patulin 2.56 66 0.89 59 PR-toxin 3.55 57 0.92 47 Penicillic acid 5.34 67 3.14 58 Citrinin11.08 60 7.00 53 Zearalenone 9.35 35 9.69 21 Ochratoxin A 12.61 32 12.56 23 Aflatoxin B1 4.54 71 3.61 82 Rubratoxin B 19.69 38 26.36 24 to the mycotoxins. Patulin, penicillic acid, zea- ralenone, and rubratoxin B were significantly more potent on the aged bacterial suspensions  Table 1) than on freshly reconstituted bacterial suspensions. The increased sensitivity of aged bacterial suspensions was not due to fewer cells since cell counts revealed approximately the same number of cells in both suspensions. The effect of cell number on the EC50 for penicillic acid was examined by using20 ,u and the standard 10 ,ul of aged bacteria per assay tube. With this procedure, the increased number of cells had little effect on the EC50 forpenicillicacid. There was no significant difference be- tween agedand freshly reconstituted bacterial suspensions for citrinin and ochratoxin A  Table 1 . PR-toxin and aflatoxin B1 were less active on the aged bacterial suspension than   thefreshly reconstituted bacteria  Table 1). Reaction rates on the bioluminescence proc- ess were studied for each mycotoxin. The age of the bacterial suspension did not significantly affect the pattern of the response withtime of incubation for penicillic acid, citrinin, ochra- toxin A, aflatoxin Bl, and rubratoxin B  Table 1). For example, the difference between the EC50 values determined with freshly reconsti- tuted bacteria and those determined withaged bacterial suspensions remained constant from 5 to 20 min. There was a significant interaction at the0.05 level with regard to time of incubation of toxin with the age of the bacterial suspension for patulin, PR-toxin, and zearalenone. The in- hibitory action of all mycotoxins on bacterial bioluminescence approached the maximum ef- fect by 15 min  Table 1). Generally, the greatest change in the EC50 occurred between 5 and 10 min, with little change occurring between 15 and20min. Except for aflatoxin B1 and zearalenone, the change in the EC50 withtime was significant for all other mycotoxins studied. Three specific categories could be established on the basis of the change in the EC50 from 5 to 20min. Patulin, PR-toxin, penicillic acid, and citrinin constituted one category. The amounts ofthese toxins re- quired for an EC50 decreased by 46 or greater from 5to 20 min. The concentration of zearale- none, ochratoxin A, and aflatoxin B1 required for an EC50 decreased by 12 or less. Rubra- toxin B was the only mycotoxin analyzed that required a significant increase in concentration with time to elicit a 50 light reduction. The EC20 values for each mycotoxin obtainedwith freshly reconstituted bacteria at 5 and 20 min of incubation are shown in Table 2. These concentrations would be the lower limits of detection of these toxins by the bacterial biolu- minescence assay. The reduction in the concen- tration of toxin required for an EC20 compared to an EC50 was greatest for patulin, PR-toxin, penicillic acid, citrinin, and aflatoxin B1. Like- wise, these were the toxins (with the exception of aflatoxin B1) whichdemonstrated the greatest reduction in the EC50 from 5 to 20min. The EC20 foraflatoxin B1 may be a more definitive expression of thesensitivity of bacterial biolumi- nescence for this toxin because the EC50 foraflatoxin B1 must be extrapolated. The dilution series analyzed foraflatoxin B1 ranged from 0.865 to 13.80 ,ug/ml. Higher concentrations were not analyzed because of thelimited solubil- ity of this toxin. Consequently, concentrations both higher andlower than the EC20 (4.54 ,ug/ml) but none higher than the EC50 (21.97 ,ug/ml) were analyzed.Since aflatoxin B1has limited solubility in methanol, DMSO was used as the solventvehicle for this toxin. Zearalenone is soluble in both methanoland DMSO, and the EC50 valuesobtained for this compound demon- strated that these solvent vehicles had little influence on the toxin's action on the biolumi- nescence process. The results obtained by the bacterial biolumi- nescent procedurehavedemonstrated a reliable short-term method for assessing the toxicity of mycotoxins. The order of toxicity determined by bacterial bioluminescence parallels that reported for mammalian cell cultures. Lompe and v. Milczewski (8) used five mammalian celllines: Flow C II from porcine epithelia, AM II from porcinekidney, Detroit-98 from human bonemarrow, Girardi from human heart, and FHL from human lung. The rangeof concentrations for mycotoxins to elicit cytocidal effects was 0.12 to1.0 jig/ml for patulin, 0.2to 2.5 ,ug/ml for PR-toxin, 5.5to 24 ,ug/ml for penicillic acid, 9 to 50 ,g/ml foraflatoxin B1, 10 to 33 ,ug/ml for ochratoxin A, 30 to 125 ,ug/ml for rubratoxin B, and 50 to 200 ,ug/ml for citrinin. APPL. ENVIRON. MICROBIOL.  EFFECT OFMYCOTOXINS ON BIOLUMINESCENCE 1075 One majoradvantage of the bacterial biolumi- nescence assay over other currently used short- term toxicity assays is that the instrumentation reduces the chance of human error and conjec- ture inherent to many of these cell systems in attempting to determine the extent of cellular alteration. The expressions of toxicity used in the bioluminescence assay varied by only 10 or less. The dataoutput is a mechanical func- tion, and pipetting is the principal laboratory manipulation to introduce error. Furthermore, the system is efficient, inexpensive and simple to perform. For absolute values of toxicity, the time ofincubation of the toxin with thebacteria and the ageof thebacteria themselves are criti- cal for most of the mycotoxins. However, thebacteria do maintain sensitivity to the mycotox- ins even after 5-h reconstitution. Thus, in screening many samples for mycotoxin activity, the srcinal bacterial suspension can be used for an extended time period. In additionthe bioas- say presents many possibilities for a morecom- plete understanding of the mechanism of action of mycotoxins. Future studies with model com-pounds may provide significant correlations be- tween the structures and their toxicities to the bioluminescence process. ACKNOWLEDGEMENTS We thank Joyce L. Lanier, Gary M. Adcock, and Charles J. Herndon for their technical assistance. LITERATURE CITED 1. Abedi,A. H., and P. M. Scott. 1969. Detection of toxicity of aflatoxins, sterigmatocystin, and other fungal toxins by lethal action of zebra fish larvae. J. Assoc. Off. Anal. Chem. 52:963-969. 2. Bulich, A. A., and D. L. Isenberg. 1981. Use of the luminescent bacterial system for the rapid assessment of aquatic toxicity. Instrum. Soc. Am. Trans. 20:29-33. 3. Ciegler, A., H. R. Burmeister, R. F. Vesonder,and C. W. Hesseltine. 1981. Mycotoxins: occurrence in the environ- ment, p. 1-50. In R. C. Shank (ed.) Mycotoxins and N- nitroso compounds: environmental risks, vol. 1. CRC Press, Inc., Boca Raton, Fla. 4. Harwig, J., and P. M. Scott. 1971. Brine shrimp  Artemia salinaL.)larvae as a screening system for fungal toxins. Appl. Microbiol. 21:1011-1016. 5. Hesseltine, C. W. 1979. Introduction, definition, and his- tory of mycotoxins of importance to animal production, p. 3-18. In Interaction of mycotoxins in animal production. National Academy of Sciences, Washington, D.C. 6. Johnson, F. H., H. Eyring, and B. J. Stover. 1974. The theoryof rate processes in biology and medicine. JohnWiley   Sons, New York. 7. Lee, K. Y., C. F. Poole, and A. Zlatkis. 1980. Simulta- neousmulti-mycotoxin determination by high perform- ance thin-layer chromatography. Anal. Chem. 53:837- 842. 8. Lompe, A., and K.-E. v. Milczewski. 1979. Ein zellkultur- test fur den Nachweis von Mykotoxinen. Z. Lebensm. Unters.Forsch. 169:249-254. 9. Umeda, M. 1971. Cytomorphologicalchanges of cultured cells from rat liver, kidney and lung induced by several mycotoxins. Japan. J. Exp.Med. 4:195-207. 10. Umeda, M., T. Tsutsui, and M. Saito. 1977. Mutagenicity and inducibility of DNA single-strand breaks and chromo- some aberrations by various mycotoxins. Gann 69:619- 625. VOL. 44, 1982

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