MOM/026

Letters in Applied Microbiology 1990,10, 161-165

The use of tissue culture for the detection of mycotoxins J E A N ROBB, M A R YN O R V A L&* W . A . N E I L L *Department of Microbiology, East of Scotland College of Agriculture, King’s Buildings, West Mains Road, Edinburgh EH9 3JG and *Department of Bacteriology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, U K Received 4 January 1989 and accepted 16 January 1989

ROBB,J., N O R V A LM. , & N E I L LW.A. , 1990. The use of tissue culture for the detection of mycotoxins. Letters in Applied Microbiology 10, 161-165. After incubation with 20 different mycotoxin standards and extracts from fungi and feed stuffs, fluorescent flow cytometry was used to measure viability of NS.1 cells and compared to microscopic assessment of cytotoxicity on stained HEp-11 monolayers. Both methods gave essentially the same results but the cytometric analysis offered a more quantitative approach and was particularly appropriate in the screening of extracts containing fats. The potential uses of a cytotoxic test in the analysis of feed stuffs and fungal extracts for the presence of mycotoxins are discussed.

The presence of mycotoxins in animal feed stuffs and in food for human consumption has important implications for animal and human health. Fungi can produce at least 800 known toxic metabolites with the Fusarium species alone synthesizing over 80 trichothecenes. There are obvious problems facing the worker in the field who wishes to determine whether or not a feed may contribute to a disease syndrome or to decide whether a feeding stuff is safe for consumption. Generally the significance of mycotoxin contamination is dependent on the toxic properties of the compounds involved. This fact is sometimes overlooked as chemical and immunoassay techniques are developed to detect smaller and smaller amounts of a few wellcharacterized mycotoxins, such as the aflatoxin series, ochratoxin and T-2 toxin. It is known that the type and quantity of mycotoxins present in a feed is dependent on many factors including the species of crop as well as climatic and storage conditions which vary considerably from year to year. It is not a practical proposition to assay chemically for all known mycotoxins and, in addition, substances may be present which have not yet been described and characterized.

In this paper we report on the effects of mycotoxin standards on the viability of NS-1 cells, a line of mouse myeloma cells which grow in suspension, as analysed by fluorescent flow cytometry; comparison is made with the effects of the same standards on HEp-I1 cells assessed microscopically. Extracts from a Chaetomium species implicated in a disease syndrome in horses and cattle, and from feeding stuffs are also studied in both systems.

Materials and Methods MYCOTOXIN S T A N D A R D S A N D EXTRACTS

The pure standards were obtained from Sigma (Poole, Dorset) except for roridin A, T-2 triol, verrucarin A and verrucarol which were obtained from Cambridge Research Biochemicals, and 15-acetoxyscirpenol and moniliformin kindly donated by Dr P.S. Steyn. Each compound was dissolved in ethyl acetate at 1 mg/ml except for sterigmatocystin which was used at 0.25 mg/ml. The Chaetornium extract was provided by Professor G. Bean, University of Maryland; it was fractionated by thin layer chromatography

162

Jean Robb et al.

and seven equal areas scraped off and suspended in ethyl acetate. The feed stuffs consisted of broiler feeds and were extracted by the method of Patterson & Roberts (1979); extract I was assayed. HEP-I1 A S S A Y HEp-I1 cells were cultured in Earles-based Eagles medium (Northumbria Biologicals Ltd, Cramlington) supplemented with 5% newborn calf serum, 200 i.u./ml penicillin and 200 pg/ml streptomycin. Two x lo4 cells in 200 p1 medium were placed in each well of a 96-well microtitre plate (Cel-Cult, Feltham). One pl of each sample was added after the plates had been incubated for 48 h at 37°C in humidified air containing 5% CO, and the incubation continued for a further 24 h. The cells were fixed in alcohol, stained with Giemsa and examined microscopically to assess any cytotoxic effect. F L U O R E S C E N T FLOW C Y T O M E T R Y

NS-I/I.AG4-I mouse myeloma cells (Kohler et al. 1976) were cultured in suspension in RPMI 1640 medium (Northumbria Biologicals Ltd., Cramlington) containing 5% foetal calf serum, 2 mM L-glutamine, 5 x M 2mercaptoethanol, 100 i.u./ml penicillin, 200 pg/ml streptomycin, 100 pg/ml gentamicin and 20 pg/ml fungizone. One ml cell suspension containing 3 x lo5 viable cells/ml, as measured by trypan blue exclusion, was placed in each well of 24-well Costar plates (Cel-Cult, Feltham). One ml fresh medium and 2 p1 of each sample were added. Two p1 of the appropriate solvent was added to control wells at the same time. The plates were incubated at 37°C in humidified air containing 5% CO, for 24 h. The contents of each well were then transferred to tubes, centrifuged for 5 min at 500 g and the pellets resuspended in 100 pl rhodamine 123 (Sigma, Poole, Dorset) at a concentration of 10 pg/ml (Darzynkiewicz et al. 1982). Excess stain was removed by washing the cells in 0.01 M phosphate buffered saline, pH 7.2. Rhodamine 123 is taken up by the mitochondria of live cells resulting in cells with brightly staining cytoplasm while non-viable cells exhibit a weak, diffuse fluorescence. The stained cells were analysed by fluorescent flow cytometry in an EPICS C (Coulter)

equipped with an argon ion laser that used 200 mW power and emitted light at 488 nm. The cells were run at a rate of 400/s until 20000 had been counted. The percentage viable cells was obtained by gateing out weakly staining non-viable cells. The percentage viable cells in the control well was also obtained for each experiment and was between 6@70%. The final percentage of viable cells in each test well was then calculated with reference to this value.

Results and Discussion Table 1 shows the result of assaying the mycotoxin standards by their cytotoxic effect on HEp-I1 cells and by using fluorescent flow cytometry after staining NS-1 cells with rhodamine. The latter test was found to be reproducible and the analysis quick, each sample taking less than one minute. Two typical profiles from the EPICS are shown in Figs l a and b. The two tests were found to give essentially the same results except for tetrahydrodeoxyaflatoxin B, and two of the polyketides (cyclopiazonic acid and secalonic acid) which were toxic in the fluorescent flow cytometric test without showing any effect on HEp-I1 cells. The relative sensitivities of the two methods were not compared. As may be seen in Table 2, all the extracts of feed stuffs were toxic by both tests. With regards to the fractions of Chaetomium prepared by thin layer chromatography, those containing toxic material were detected equally by both methods. As the detection of many mycotoxins by chemical methods is not adequate at present, and the feasibility of testing for all known mycotoxins chemically is not practical, we developed a tissue culture method using HEp-I1 cells which enabled us, firstly, to detect toxic compounds in poultry feeds (Robb et ul. 1982), then to screen a range of mycotoxin standards for their ability to kill this cell type (Robb & Norval 1983). HEp-I1 cells were found to be particularly sensitive to the trichothecenes, as noted earlier by Grove & Mortimer (1969). This group of compounds is difficult to detect by thin layer chromatography and for which there is no general immunoassay available, although specific monoclonal antibodies against a few trichothecenes have been made and characterized recently which may be useful in the future (Hack et al. 1988; Fan et al. 1987). Other groups have

0, no effect;

0

++ 0 ++

0 0 0

0

14-acetoxyscirpenol 3-acetyl deoxynivalenol chaetoglobosin C diacetoxyscirpenol 4,15-diacetylverrucarol deoxynivalenol neosolaniol roridin A T-2 toxin T-2 trio1 T-2 tetraol verrucarin verrucarol

Trichothecenes

+, 25% cell death; + +, 50% cell death; + + +, 75% cell death or more.

100 100 100 100 52 100 52 8

Aflatoxin B , Aflatoxin B, Aflatoxin G I Aflatoxin G , 5-Methoxysterigmatocystin Ochratoxin A Sterigmatocystin Tetrahydrodeoxyaflatoxin B I

HEp-I1

47 100 80 0 13 16 9 0 0 0 64 15 100

live cells

0

++ + + i -+ + + + ++ +++ +++ +++ + +

HEp-I1

patulin penicillic acid citrinin cytochalasin A cyclopiazonic acid ergosterol esculin gossypol luteoskyrin moniliformin mycophenolic acid PR-toxin rubratoxin B scopoletin secalonic acid o-toluidine zearalenol zearalenone

Other compounds

100 100 20 100 80 80

64

33 100 72 100 90 100 31

+ 0 +

40 0

+ 0 + 0 + +++

0 0

0

++ 54

100

+

++

HEp-I1

90

23

live cells

Yo

live cells

Yo

%

Aflatoxins

EPICS

EPICS

EPICS

Table 1. The effect of mycotoxin standards on the viability of cells measured by fluorescent flow cytometry on NS-1 cells (EPICS) and by cytotoxicity on HEp-I1 cells

2

sm

z. m

6' 3

a

(P

Jean Robb et al.

164 (a I H 4 : I P 1024 117

LGFL 255

( b f

H4 : IP 1024 117

LGFL 255

Fig. 1. Result of fluorescent flow cytometry of NS-1 cells stained with rhodamine 123. The area to the left of the CUTSOT represents dead cells, while that to the right represents viable cells. In (a) the cells have been incubated for 24 h with diacetoxyscirpenol,and in (b) with PR-toxin.

also investigated the use of tissue culture to assay mycotoxins, such as mitogen-stimulated lymphocytes for T-2 toxin (Thompson & Wannemaker 1984) and for trichothecenes in corn (Porcher et a!. 1987) and human and mouse fibroblasts for trichothecenes (Abbas et al. 1984). A fast screening method for detection of trichothecenes in maize has also been described using protein synthesis inhibition in cultured fibroblasts (Scossa-Romano et al. 1987). If cells grown as monolayers in tissue culture are used as an assay system for mycotoxins, it is

possible to assess a toxic effect microscopically. A more quantitative test uses incorporation of labelled nucleic acid (Forsell et al. 1985) or protein precursors (Thompson & Wannemaker 1984) into cells as an indication of cell proliferation or death. We wished to develop a method which was more quantitative than microscopy, did not involve the use of radioactive compounds, was simple to perform, quick and reproducible. Fluorescent flow cytometry after rhodamine staining was tried which gives percentages of viable and dead cells in each test.

Detection of mycotoxins in tissue culture

165

Table 2. The effect of extract I of feedstuffs and a fractionated Chaetomium extract on the viability of cells measured by fluorescent flow cytometry on NS-1 cells (EPICS) and by cytotoxicity on HEp-I1 cells EPICS Sample of feedstuff

live cells

HEpII

1 2 3 4 5 6 7 8 9 10

17 33 52 35 41 35 37 19 61 48

+++ +++ +++ ++ +++ +++ +++ ++ +++

0, no effect;

Yo

Fraction of Chaetomium extract from thin layer plate

EPICS live cells

HEp-I1

1 2 3 4 5 6 7

90 100 68 0 22 0 48

0 0 0

Yo

++ ++ ++ 0

++

+ +, 50% cell death; + + +, 75% cell death or more.

HACK,R., MARTLBALJER, E. & TERPLAN, G. 1988 Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A. Applied and Environmental Microbiology 54, 23282330. KOHLER, G., HOWE,S.C. & MILSTEIN, C. 1976 Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines. European Journal of Immunology 6,292-295. PATTERSON, J.P. & ROBERTS, B.A. 1979 Mycotoxins in animal feedstuffs: sensitive thin layer chromatography detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone and T-2 toxin. Journal of the Association of Official Analytical Chemists 62, 1265-1267. PORCHER, J.M., LAFARGE-FRAYSSINET, C., FRAYSSINET, C., NURIE,A., MELCION,D. & RICHARD-MOLARD, References D. 1987 Determination of cytotoxic trichothecenes ABBAS,H.K., SHIER,W.T. & MIROCHA, C.T. 1984 in corn by cell culture toxicity assay. Journal of the Sensitivity of cultured human and mouse fibroblasts Association of Oflcial Analytical Chemists 70, 8 4 4 to trichothecenes. Journal of the Association of Offi849. cial Analytical Chemists 67, 607-610. ROBB,J. & NORVAL, M. 1983 Comparison of cytoDARZYNKIEWICZ, Z., TRAGANOS, F., STAIANO-COICO, toxicity and thin layer chromatography methods for L., KAPUSCINSKI, J. & MELAMED, M.R. 1982 Interthe detection of mycotoxins. Applied and Environactions of Rhodamine 123 with living cells studied mental Microbiology 46,948-950. by flow cytometry. Cancer Research 42,799-806. ROBB,J., KIRKPATRICK, K.S. & NORVAL,M. 1982 FAN,T., ZHANG,G.S. & CHU, F.S. 1987 Production Association of toxin-producing fungi with disease in and characterization of antibodies against HT-2 broilers. Veterinary Record 11 1,389-390. luxin and T-2 tetraol tetraacetate. Applied and SCOSA-ROMANO, D.A., BICKEL,R E , ZWEIFEL,U., Environmental Microbiology 53, 17-21. REINHART, C.A., LUTHY,J.W. & SEHLATTER, C.L. FORSELL,J.H., KATELEY, J.R., YOCHIZAWA, T. & 1987 Fast and sensitive screening method for the PESTKA,J.J. 1985 Inhibition of mitogen-induced detection of trichothecenes in maize by using blastogenesis in human lymphocytes by T-2 toxin protein synthesis inhibition cultured fibroblasts. and its metabolites. Applied and Entironmental Journal of the Association of Official Analytical Microbiology 49, 1523-1526. Chemists 70, 129-132. GROVE,J.F. & MORTIMER, P.H. 1969 The cytotoxicity THOMPSON, W.L. & WANNEMACHER, R.W. 1984 Detecof some transformation products of diation and quantitation of T-2 mycotoxin with a simcetoxyscirpenol. Biochemical Pharmacology 18, plified protein synthesis inhibition assay. Applied 1473-1478. and Environmental Microbiology 48, 11761 180.

The method is simple and quick, offering a n alternative t o radioactive labelling. It was found to be comparable to cytotoxicity, assessed microscopically, in detecting a range of pure mycotoxins and toxic fractions from fungal cultures. In the case of the poultry feeds examined, the EPICS method appeared to be less affected by fats which cause non-specific toxicity of HEp-I1 an d other cell lines. T h u s fluorescent flow cytometry may be a useful test in the screening of extracts from diverse a n d impure sources.

The use of tissue culture for the detection of mycotoxins.

After incubation with 20 different mycotoxin standards and extracts from fungi and feed stuffs, fluorescent flow cytometry was used to measure viabili...
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