10T12 cells and induction of EBV-early antigen in Raji cells by altertoxins I and III

Toxic. in Vitro Vol. 2, No. 2, pp. 97-102, 1988 Printed in Great Britain

088%2333/88 $3.00+0.00 Pergamon Press pie

T R A N S F O R M A T I O N OF C3H/10T½ CELLS A N D I N D U C T I O N OF EBV-EARLY ANTIGEN IN RAJI CELLS BY ALTERTOXINS I A N D III L. C. OSBORNE*,V. I. JONES,J. T. PEELER and E. P. LARKIN Division of Microbiology, Food and Drug Administration, Cincinnati, OH 45226, USA (Received 5 May 1987; revisions received 24 September 1987)

Abstract--Moulds of the genus Alternaria are common contaminants of some food crops. Some isolates have been shown to produce mutagenic compounds called altertoxins. Altertoxin I (ATX-I) and altertoxin III (ATX-III) were examined for activity in the Raji cell Epstein-Barr virus early antigen (EBV-EA) induction system and in the C3H/10Tt murine fibroblast cell transformation system. Exposure of Raji cells to ATX-I or ATX-III activated EBV-EA expression by 8- and 9.5-fold, respectively. A single exposure of C3H/10T½ cultures to ATX-I or ATX-III resulted in significant increases in cell transformation, and the response to ATX-I was stronger. Both altertoxins enhanced the transformation of C3H/10T½ cells, and chronic exposure of non-initiated C3H/10T~ cells to ATX-I and ATX-III, starting 6 days after cells were plated, resulted in cell transformation in 8/59 and 12/37 dishes, respectively, compared with transformation in only 2/63 control dishes. Since activation of EBV-EA in Raji cells has been positively correlated with tumour promoters, these data together indicate that ATX-I and ATX-III are not just mutagens but have a potential role in cell transformation.

INTRODUCTION

found AME to be weakly mutagenic without metabolic activation, whereas alternariol was not considered mutagenic. Compounds, tentatively identified as altertoxins I and II, as well as other unidentified compounds, were also found to be mutagenic. Although the mutagenic potential of a compound is generally a good predictor of its carcinogenic potential, mutagenesis and carcinogenesis are not identical processes. The ability of a compound to induce the expression of Epstein-Barr virus early antigen (EBV-EA) in Raji cells has been positively correlated with a compound's tumour-promoting capability (Ito et al. 1981; Takada & zur Hausen, 1984). This system has been used to screen various compounds as tumour promoters (Zeng et al. 1983). In addition, the C3H/10T½ mouse embryo fibroblast cell system is an accepted in vitro system that detects both chemical carcinogens and promoters of cell transformation (Mondal, 1980; Reznikoff et al. 1973a). The study reported here presents data derived from these two in vitro cell systems which suggests that altertoxin I (ATX-I) and the recently discovered compound altertoxin III (ATX-III) (Stack & Prival, 1986) are probably weak transforming agents and can definitely enhance cell transformation.

The Alternaria are a group of moulds that frequently contaminate a variety of plants, some of which are used as food for humans and farm animals. Although some species of the mould are pathogenic for plants, none are pathogenic for man and they are destroyed by most of the processes used in food preparation. However, they produce many metabolites during growth that may not be destroyed during processing, including tenuazonic acid, alternariol, alternariol monomethyl ether (AME), and a group of perylene derivatives called the altertoxins. The toxicity of these componds and their occurrence in the food supply have been reviewed (King & Schade, 1984; Schade & King, 1984; Watson, 1984). Extracts made from Alternaria cultures have been tested in the Salmonella typhimurium mutagenicity assay of Ames. In one report (Harwig et al. 1979), five of five isolates had mutagenic activity, but only with S-9 metabolic activation (from rat liver). Alternariol was detected in one isolate, AME in two, and tenuazonic acid in all five, but the presence of altertoxins was not reported. In another study (Bjeldanes et al. 1978), a strain of A. tenuis was found to be mutagenie, also with S-9 activation. Working with purified metabolites of A. alternata, Scott & Stoltz (1980)

MATERIALS AND METHODS

*Present address: Department of Paternity Evaluation, Roche Biomedical Laboratories, 1447 York Court, Burlington, NC 27215. Abbreviations: AME = alternariol monomethyl ether; ATX-I = altertoxin I; ATX-III = altertoxin III; BME = basal medium Eagle's; EBV-EA = Epstein-Barr virus early antigen; FBS = foetal bovine serum; LTA = lyngyatoxin A; MCA = 3-methylcholanthrene; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine; TI = transformation incidence; TPA= 12-O-tetradecanoylphorbol- 13-acetate. 97

Chemicals. Lyngbyatoxin A (LTA), a known tumour promoter (Fujiki et al. 1981) was kindly supplied by Dr Richard Raybourne, Food and Drug Administration (FDA), Washington, DC. 12-O-tetradecanoylphorbol-13-acetate (TPA), Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG) and 3-methylcholanthrene (MCA) were obtained from Sigma Chemical Co. (St Louis, MO). Methanol, HPLC-grade acetone and Giemsa stain were obtained from Fisher Scientific Co. (Fair Lawn, N J).

98

L. C. OSaORNEet al.

The aitertoxins were isolated by Michael Stack (FDA, Washington, DC) from cultures of A. alternata grown on cooked rice (Stack & Prival, 1986). Acetone was added to a known weight of toxin to give a 104-fold higher concentration in acetone than would be used in the experiments. The acetone solutions were then diluted 10 -4 in medium when used in the studies. (The acetone solutions of altertoxins were not stored because of the instability of the altertoxins in solution.) Negative controls consisted of acetone diluted l0 -4 in medium, or 0.01% acetone. Cultures. Raji cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown at 37°C in RPMI-1640 medium (Whittaker M A Bioproducts, Walkersville, MD) supplemented with 10% heat-inactivated (56°C for 30min) foetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), penicillin (50U/ml), streptomycin (50#g/ml), and gentamicin sulphate (50/tg/ml). The antibiotics were purchased from GIBCO (Grand Island, NY). C3H/10T½ Cl 8 cells were obtained from Dr C. Boreiko, Chemical Industry Institute of Technology (Research Triangle Park, NC), except as noted. Cells were maintained in 25-cm 2 tissue culture flasks (Corning Glass Works, Corning, NY) in Eagle's basal medium (BME: Whittaker M A Bioproducts, Walkersville, MD) supplemented with 10% FBS. Split ratios were in accordance with the methods of Reznikoff et al. (1973b). All experiments were performed in antibiotic-free cultures between passages 7 and 11. Both cell lines used were routinely checked for mycoplasma contamination using the Hoechst staining method (Chen, 1977). Experimental protocol

In the experiments designed to test for the induction of EBV-EA in Raji cells, test substances or controls were added to 25-cm 2 tissue culture flasks containing 5 x 10 6 cells in l0 ml medium. ATX-I and ATX-III were dissolved in acetone and added to cultures at a concentration of 0.1 #g/ml. Acetone, at an equivalent concentration, was added to the control cultures. After cultivation for various time periods, cells were harvested, centrifuged and resuspended in 0.5 ml phosphate-buffered saline containing 2% FBS. (Cells were counted in the presence of trypan blue to test the cytotoxicity of the compounds.) After l0 min, smears of the cell suspension were made, these were air-dried and then fixed in cold (4°C) acetone for l0 min. The smears were incubated for 1 hr at 36°C in a 1 : 100 dilution of a high-titred anti-EBV-EA human serum. (The serum was supplied by Dr Gary Armstrong, FDA, National Center for Drugs and Biologics, Bethesda, MD.) After incubation, the smears were washed and then exposed to fluorescein-labelled rabbit anti-human immunoglobulin serum (Miles Laboratories, Elkhart, IN) for 1 hr at 36°C. They were then washed, air-dried and examined for fluorescence. In each assay 500 cells were counted, and the ratio of EBV-EA-positive cells was recorded. The transformation assays in the C3H/10T l system were performed generally according to the methods of Reznikoff et al. (1973a) with the modifications of Frazelle et al. 0983). Cells were plated onto 60-mm

dishes (Corning Glass Works) at 2000 cells/dish and incubated overnight at 37°C before test or control substances (dissolved in acetone) were added. Cells formed a complete monolayer 8 to 10 days after seeding. In most experiments, a positive control was included which involved treating the cultures with 3.7/~M (l.0#g/ml) MCA. The promotion studies in the C3H/10T½ system were carried out according to the methods of Mondal et al. (1976) as modified by Dorman et al. (1983). Dishes were treated as above, except that after the overnight incubation, the serum-containing plating medium was replaced with serum-free BME and the cultures were treated with 3.4 # M (0.5/~ g/ml) M N N G or control (0.25% acetone). This initiation treatment was terminated after 4 h r by aspiration of the medium and replacement with BME. The test substances were first added to the cultures 5 days after initiation and were then included in all subsequent medium changes which occurred every 4-7 days. LTA was added to groups of dishes as a positive control. The concentration of serum was reduced to 7% (from 10%) when cells became confluent (between 9 and 15 days after initiation) and was further reduced to 5% at the following medium change. After a total incubation time of 6 wk C3H/10T½ cultures were washed with phosphate-buffered saline, fixed with 100% methanol, and stained with Giemsa stain. (Due to the long incubation period, there was sporadic microbial contamination in all groups of dishes. A random distribution of contamination among transformed and non-transformed dishes was assumed.) Dishes were scored for the presence of type II and type III loci as defined by Reznikoff et al. (1973a). Transformation data were expressed as the transformation incidence (TI), which represented the number of dishes containing type II or type III foci divided by the total number of dishes scored for each treatment group. The total number of type II or type III foci observed was also noted. For the C3H/10T ½ assays, the cytotoxicity of the test substances was assessed according to the methods of Reznikoff (1973a). Briefly dishes were seeded with 200 cells and treated as described above. After incubation for 7-10 days, the cultures were fixed and stained and the number of colonies (which represented surviving cells) were counted. Cytotoxicity was expressed as the surviving fraction relative to colony formation in the control solventtreated dishes. The absolute plating efficiency in the dishes receiving only solvent averaged 55-60% for all experiments. For analysis of the C3H/10T½ data, the transformed fraction data were converted to decimal equivalents. Comparisons between test compounds and solvent controls were made using Fisher's exact test (Brownlee, 1965). A test for synergistic increase in transformed cells in promotion experiments was performed according to the procedure described by Frazelle et al. (1983). The level of significance was chosen as P < 0.05. RESULTS

EBV-EA

induction

In initial studies, the effect of time on the induction

Altertoxins and cell transformation of EBV-EA in Raji cells was examined. Cultures of Raji cells were treated with either TPA (25 ng/ml) or solvent control (acetone), and one culture of each was examined daily for EBV-EA for 10 days. The percentage of EBV-EA-positive cells in the solventtreated cultures was consistently around 1% for each of the 10 days. In contrast, the percentage of EBVEA-positive cells in TPA-treated cultures steadily increased with the number of days in culture, peaking at day 8. Other investigators using this system have reported similar findings (Takada & zur Hausen, 1984; zur Hausen et al. 1978), with peaks of EBV-EA observed betweeen 7 and 9 days after addition of tumour promoters. The slight decrease in EBV-EA observed in cells after day 8 in the experiments reported here corresponded to a decrease in cell viability on those days. The steady increase of EBV-EA-positive cells for the first 8 days in response to TPA was also observed with ATX-I and ATX-III (Fig. 1). In some experiments, the peak response was seen on day 7 or 9, but in those cases, the day-8 response was very close to the peak response. Only the day-8 responses are included in the remainder of this report. A summary of the Raji cell day-8 EBV-EA responses to ATX-I and ATX-III is given in Table 1. It is clear that even at 0.3#M, both altertoxins significantly increased the percentage of EBV-EApositive cells. For ATX-I, the effect was greatest at the highest dose tested (6 F M); EBV-EA was detected in 12.0% of the cells treated at this dose compared to only 1.5% in solvent controls; an 8-fold increase. However, doses of 1.2 and 0.3 #M-ATX-I gave responses of about 75 and 50%, respectively, of the response observed with 6 FM-ATX-I. Thus, there was not a linear relationship between the EBV-EA response and the ATX-I dose, at least not within the dose range used in this study. EBV-EA expression increased from an average of 1.3% in solvent controls to 12.3% when cells were treated with 1.5 gM-ATX-III; a 9.5-fold increase. As with ATX-I, no linear relationship was found between the EBV-EA response and ATX-III dose. The 0.3 #M-ATX-III dose gave an EBV-EA response in Raji cells about 80% as high as the response to the 1.5#M-dose, whereas a further reduction in dose to 0.06#M-ATX-III almost totally eliminated the EBV-EA response.

99

11

2

3

4 5 6 Ti me ( days )

10

Transformation of C3H / IOT~ cells ATX-I and ATX-III were first tested for cytotoxicity at concentrations of 1-15 #M and 0.064).3 #M, respectively. C3H/10T½ cells tolerated a 48-hr exposure to 6 #M (2 #g/ml) ATX-I, with a 66% viability relative to the solvent controls; it took 15#M-ATX-I to significantly reduce cell survival. ATX-III was found to be more toxic than ATX-I. Cells became confluent 12 days after treatment with 6 #M-ATX-I, whereas exposure to 0.3 #M (0.1 pg/ml) ATX-III for 18 hr resulted in a 50% loss of cell viability, and cultures did not reach confluency until 16 days after treatment. In the transformation regimen, 48-hr exposure to 6 #M-ATX-I resulted in significantly increased transformation in five separate experiments (Table 2). In ATX-I-treated cells, four type III and 76 type II loci developed in 46 of the 96 dishes; MCA treatment

2

3

Mean

SD

2.4 5.8 ND ND ND

1.0 4.8 8.4 12.6 15.8

1.0 8.4 9.2 l 1.4 14.8

1.5 6.3 8.8 12.0 15.3

0.7 1.4 0.4 0.6 0.5

1.4 1.0 1.4 1.3 0.06 ND 2.4 2.0 2.2 0.3 I 1.0 9.8 9.6 10.1 1.5 11.8 13.4 11.8 12.3 TPA 0.04 ND 15.4 12.6 14.0 ND = not done ATX-I= altertoxin I ATX-I= altertoxin III Cells were exposed to the above treatmentsfor 8 days and then harvested.

0.2 0.2 0.6 0.8 1.4

Experiment...

0.3 1.2 6.0 0.04

Series B

Solventcontrol ATX-III

9

Raji cell growth was not affected by ATX-I at any concentration tested. ATX-III did not appear to be very toxic to Raji cells, but it did affect their growth rate. For example, in Table 1, experiment 3, 83% of cells in the solvent-treated culture were viable compared with 74-78% in ATX-III-treated cultures. However, the cell count showed there were 435 x l05 cells in the solvent-treated culture compared with about 260 x l05 in ATX-III-treated cultures.

l

TPA

7

Fig. l. Effect of altertoxins on EBV-EA induction in Raji cells. Each point is the average of at least three experiments. Control cultures ( ~ ) , ATX-I-treated cultures (R), and ATX-III-treated cultures (+).

Series A Solvent control ATX-I

8

~ o. 3 x 1 rid

Conch

(#M)

T

tu :~ ~ tu 5 .~

Table 1. EBV-EAresponseof Raji cells to 8-day ATX-Iexposure EBV-EA-positiveceils (%) Compound

T

.~ 9

L. C. OSBORNE et al.

100

Table 2. Transformation of C3H/10T~ cells by ATX-I TI Compound

Exposure (hr)

(//M) Acetone ATX-I (6.0) MCA (3.7)

Experiment...

1

48 48 24

0/15 7/19 18/20

2

3

4

5

Total

1/17 7/19 5/13

2/22 11/22 11/15

1/18 11/17 12/15

0/19 10/19 12/16

4/91 (0.04) 46/96(0.48)* 58/79(0.73)*

*Statistically significant increase (by Fisher's exact test) in TI over that observed in acetone-treated controls. Individual experiments were anlaysed by analysis of variance (Ostle & Mensing, 1975), and ATX-I and MCA groups were also found to have significant increases over acetone controls. Cells used in experiments 1, 2 and 4 were obtained from Dr C. Reznikoff, University of Wisconsin Clinical Cancer Center (Madison, WI). The plating efficiency for acetone controls averaged 32% and the surviving fraction after ATX-I treatment (relative to controls) averaged 66%. Table 3. Focus production by ATX-I No. of foci found Experiment... Compound (/~M)

1

Foci type...

Acetone ATX-I (6.0) MCA (3.7)

2

3

4

5

II

III

II

III

II

III

II

III

I1

II1

0 11 26

0 0 3

1 9 9

0 0 2

2 13 20

0 0 6

I 18 29

0 1 1

0 25 28

0 3 1

ATX-I = altertoxin-I

MCA = 3-methylcholanthrene

r e s u l t e d i n 13 t y p e I I I a n d 112 t y p e I I f o c i i n 58 of 79 dishes. No type III and only four type II f o c i d e v e l o p e d i n t h e 91 a c e t o n e - t r e a t e d c o n t r o l s ( T a b l e 3).

Experiments using the transformation regimen w i t h A T X - I I I g a v e u n e x p e c t e d r e s u l t s ( T a b l e 4). A significant increase in transformation was obtained w i t h 0 . 1 2 / a M - A T X - I I I , b u t n o t w i t h 0.3/~M. A l s o ,

Table 4. Transformation of C3H/10T~ cells by ATX-III Compound (~M)

Exposure (hr)

Relative surviving fraction

No. of foci observed Type II

Type III

TI

Solvent control ATX-III (0.12) ATX-III (0.3) MCA (3.7)

24 24 24 24

1.0 0.7 0.5 0.9

1 7 4 61

0 0 0 4

1/54 (0.02) 7/44(0.16)* 2/40(0.05) 30/35(0.86)*

ATX-III = altertoxin III MCA = 3-methylcholanthrene *Statistically significant increase in T1 over that observed for the solvent-treated control. Relative surviving fraction equals the surviving fraction after given treatment divided by the surviving fraction after solvent treatment. Data from two experiments; TI ratios for the individual experiments were similar to this composite.

Table 5. Promotion of C3H/10T~ cell transformation by ATX-I and ATX-III Occurrence of foci in C3H/10T~ cultures pretreated with: Acetone (0.25%) Promoting agent ATX-I LTA ATX-III LTA

Concn (,aM)

IIt

lid

Tit

0.0 1.2 0.0002

2 9 5

0 0 1

Series A 2/63 (0.03) 8/59 (0.14)*:~ 4/38 (0. I 1)

0.0 0.06 0.3 0.0002

1 5 21 4

1 0 0 0

Series B 2/62 (0.03) 4/41 (0.10) 12/37 (0.32)*~ 3/49 (0.06)

MNNG (3.4 ttM) lit

lilt

Tlt

6 43 30

0 0 6

6/63 (0.10) 29/64 (0.45)*§ 32/50 (0.64)*§

7 10 79 43

1 0 1 4

6/66 (0.09) 6/43 (0.14) 32/44 (0.73)*§ 31/47 (0.66)*§

ATX-I = altvrtoxin I ATX-III = altertoxin III LTA = lyngbyatoxin A MNNG = N-methyI-N'-nitro-N-nitrosoguanidine *Statistically significant increase (P < 0.05) in ~focus formation over cells receiving no promotion treatment, and in §focus formation greater than that anticipated for the simple additive effects of separate treatment with initiating and promoting agents. t l l ~ no. of type II foci observed; Ill = no. of type III foci observed; TI = transformation incidence: i.e. total no. of dishes with foci/total no. of dishes scored. Series A is the summary of three experiments, and Series B is from two experiments. TI ratios for the individual experiments were similar to this composite. The relative surviving fraction after treatment with MNNG averaged 0.86.

Altertoxins and cell transformation although the toxicity to cells of 0.12 #M-ATX-III and 6/~M-ATX-I were similar, the TI was much lower (0.16 v. 0.48). Thus, although both compounds functioned as transforming agents in these cells, ATX-I appeared to be the stronger agent. The tumour-promoting effects of ATX-I and ATXIII were also tested, and both were found to increase cell transformation (Table 5). ATX-I at 1.2/ZM enhanced transformed focus formation in M N N G pretreated cultures and to a lesser extent in acetonepretreated cultures. ATX-III at 0.06/z M had no effect on transformation in MNNG-pretreated or acetonepretreated cultures. Although 0.3#M-ATX-III was toxic to cells in experiments described above, it was not toxic when first added to the culture 5 days after pretreatment, or 6 days after plating. With 0.3 #MATX-III, transformation increased with or without M N N G pretreatment, although M N N G pretreatment gave a substantially higher response. Thus, ATX-III functioned as a stronger transforming agent when exposure to it was chronic.

DISCUSSION The study reported here was initiated to determine whether ATX-I and ATX-III, minor metabolites of the Alternaria, might function as carcinogens or tumour promoters in vitro. (A sufficient quantity of altertoxin II for parallel studies was unavailable.) Two cell systems were used: one was the induction of EBV-EA in Raji cells which has been correlated with tumour promoters; and the other was the C3H/10T½ mouse embryo fibroblast system which measures cell transformation in vitro. Both toxins increased EBV-EA in Raji cells (Table 1) and, as predicted from that data, functioned as tumour promoters in the C3H/10T½ system (Table 4). Further, the C3H/10T½ data indicated that they may be complete transforming agents. Consequently, we prefer the use of the term 'transformation enhancement' rather than 'promotion' with these compounds. The effective ATX-I concentrations used in the Raji system were not cytotoxic by any criterion measured. Concentrations of ATX-III which induced EBV-EA did not appear to kill Raji cells, but they did slow cell growth. The lowest observed effective dose of the altertoxins in this system was 0.3 #M while the dose of TPA used as a positive control was 0.04/~M. The induction of EBV-EA was always at a higher level with TPA than with either of the altertoxins tested. Indeed, there was almost no response to 0.06;tM-ATX-III. The altertoxins appear to be, therefore, at least an order of magnitude weaker as tumour promoters than TPA. ATX-I at 6/~M significantly increased transformation of C3H/10T½ cells following a single 48-hr exposure starting 1 day after plating (Tables 2 & 3). The same result was obtained in five experiments but was not obtained with 24-hr exposure or with doses lower than 6 #M (data not shown). In each of the experiments, 3.7/~M-MCA gave a higher TI, even though exposure to MCA was for 24 hr. This indicated that ATX-I was a weaker transforming agent than MCA. ATX-III was weaker still; at 0.12 #M, it significantly increased transformation of cells, but to

101

a lesser extent than MCA (Table 4). The lower response could not be definitely ascribed to differences in toxicity since the relative surviving fraction data for this dose of ATX-III and 6 #M-ATX-I were similar. Both ATX-I and ATX-III enhanced cell transformation at relatively low doses of 1.2 and 0.3/ZM, respectively (Table 5). Given the Raji cell data, and since both altertoxins were transforming agents (Tables 2 4 ) , it is not surprising that they enhanced transformation. For these experiments, LTA was used as a positive control for enhancement. To the best of our knowledge, this is the first report where LTA has been so used in the C3H/10T) system. It was used instead of the traditional promoter TPA as it gave more reproducible results in our laboratory. It was less serum-lot-restrictive, and was not photosensitive. ATX-III was extremely toxic to C3H/10T½ cells when added to cells 1 day after plating. Even when the exposure time was reduced to 24 hr, 0.3 # M-ATXIII killed 50% of the cells (relative to the solventtreated controls). The structure of ATX-III contains two epoxide rings which are not present in ATX-I (Stack & Prival, 1986). This might explain the difference in the toxicity of the compounds. The toxicity of ATX-III in the C3H/10T~ system did not become trivial until the concentration was reduced to 0.06/~M, or 1% of the effective ATX-I concentration. Nesnow et al. (1982) reported that addition of M N N G to low density C3H/10T½ cultures 1 day after plating resulted in a low transformation rate, but that if the cultures were held for 5 days before exposure to M N N G , the transformation rate increased greatly. Huband et al. (1985) showed that the transformation rate could be enhanced with increased cell density. M N N G , an alkylating agent that is unstable in solution, was highly toxic to low density cultures, but as the density was increased, toxicity decreased and the TI increased. MCA, which is a stable procarcinogen, produced foci at all cell densities tested. Gehly et al. (1979) have presented data which suggested that labile diol-epoxides of benzo[a]pyrene may induce weak responses in this cell system due to cytotoxicity, although benzo[a]pyrene itself induced a strong response. It seems likely that ATX-III is a transforming agent that behaves somewhat like M N N G , requiring a higher cell density to compensate for toxicity and increase transformation. The data presented in Table 5 support this hypothesis. A 0.3/tM-concentration of ATX-III, which was toxic to cells when added 1 day after plating, was not toxic when added 5 days later. When cultures received this dose repeatedly starting at the later time, there was increased transformation, with or without M N N G initiation. Like M N N G , ATX-III appeared to be unstable in solution. Solutions of ATX-III were clear immediately after the solvent was added, but become dark brown within 30 min. In the studies reported here, care was taken to ensure that the ATX-III solutions were diluted and added to the cultures as quickly as possible. If ATX-III became denatured soon after its addition to the cultures, as seems likely, then exposure to the compound was intermittent rather than continuous.

L. C. OSBORNEet al.

102

Most altertoxin-mediated foci in these experiments were of type II, whereas a significant number of type III loci was obtained following M C A or M N N G / L T A treatment. Even with the latter treatments, the type II foci greatly outnumbered type III foci. The significance of type III v. type II focus development is unclear. Moulds of the genus Alternaria frequently contaminate a number of foods and food ingredients. The contamination is difficult to detect unless it is quite extensive. Some food processors believe that mould contamination in food poses no cause for concern unless the taste of a food product has been affected. However, Alternaria produce various metabolites during growth, including the altertoxins (King & Schade, 1984; Schade & King, 1984). In the laboratory, altertoxin recovery from mould cultures has been as high as 380/~g/g rice substrate (Stack & Prival, 1986); these levels are much higher than either those used in the study reported here or the levels obtained in naturally contaminated food. A T X - I has been detected in apples naturally infected with Alternaria (Stinson et al. 1981). It has also been found in oranges, lemons, tomatoes and blueberries when they were inoculated with many strains of Alternaria, but the quantity of A T X - I produced was not determined (Stinson et al. 1980; Stinson et al. 1981). (ATX-III had not been described at that time, so it was not studied.) It is therefore unknown whether altertoxin levels in contaminated food approach the effective concentrations used in the study reported here. Such quantitative studies would be necessary in order to determine whether ingestion of altertoxin-contaminated food may have relevance to human cancer. Acknowledgements--We thank Michael Stack for generously supplying us with the altertoxins and Dr Craig Boreiko for his technical advice and review of this manuscript. We thank Susan Grause for assistance in the preparation of the manuscript. REFERENCES

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