Effects of some mycotoxins on mitogen-induced blastogenesis and SCE frequency in human lymphocytes

Fd Chem. Toxic. Vol. 22, No. 7, pp. 529-534, 1984

0278-6915/84 $3.00+ 0.00 Copyright © 1984 Pergamon Press Ltd

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EFFECTS OF SOME MYCOTOXINS ON MITOGEN-INDUCED BLASTOGENESIS A N D SCE FREQUENCY IN H U M A N LYMPHOCYTES R. COORAY Institute of Zoophysiology, University of Uppsala, P. O. Box 560, S-751 22 Uppsala and Department of Animal Nutrition, The Swedish University of Agricultural Sciences, P.O. Box 7047, S-750 07 Uppsala, Sweden (Received 1 November 1983)

Abstract--The effects of T-2 toxin, diacetoxyscirpenol, ochratoxin A and zearalenone on DNA synthesis in phytohaemagglutinin-stimulated human peripheral blood lymphocytes were studied by assaying the incorporation of [3H]thymidine. Total inhibition was obtained with 8ng "1"-2 toxin/ml, 8ng diacetoxyscirpenol/ml or 30/tg zearalenone/ml, and with 20 #g ochratoxin A/ml inhibition was almost complete; 50% inhibition was produced by 1.5 ng T-2 toxin/ml, 2.7 ng diacetoxyscirpenol/ml, 14pg zearalenone/ml or 14 #g ochratoxin A/ml. The toxicity of the trichothecenes to the lymphocytes was slightly reduced when rat liver cells were present whereas the toxicity of ochratoxin A and zearalenone was unaltered. Low concentrations of trichothecenes did not produce any inhibition of DNA synthesis when the cultures were washed and placed in fresh media containing only phytohaemagglutinin 4 hr after the addition of the test compounds. Sister chromatid exchange frequency was not elevated by T-2 toxin, diacetoxyscirpenol or ochratoxin A. Zearalenone had a weak enhancing effect on the frequency of sister chromatid exchanges. INTRODUCTION

The secondary fungal metabolites referred to as mycotoxins cause pathological changes or physiological abnormalities in man and other warmblooded animals (Ueno & Ueno, 1978). Mycotoxins have a very broad spectrum of proven biological activities and some show evidence of carcinogenicity, mutagenicity and/or teratogenicity in both short- and long-term test systems (Bamberg, 1972; Hayes, Hood & Humphrey, 1973; Kuczuk, Benson, Heath & Hayes, 1978; Ong, 1975; Ueno & Kubota, 1976; Ueno, Kubota, Ito & Nakamura, 1978). Several mycotoxins have been shown to affect various aspects of immunity, including resistance to infectious disease (Richard, Thurston & Pier, 1976). Trichothecenes are metabolites synthesized by Fusarium and related species of fungi (Bamburg & Strong, 1971) which contaminate cereal grains in many parts of the world. Fusarium extracts and its principal components (T-2 toxins, diacetoxyscirpenol (DAS) among others) have profound effects on various aspects of the murine immune system (LafargeFrayssinet, Lespinats, Lafont et al. 1979; Rosenstein, Kretschmer & Lafarge-Frayssinet, 1981). T-2 toxin also causes histopathological changes of the thymus glands in poultry (Richard, Cysewski, Pier & Booth, 1978). Ochratoxin A is a nephrotoxic and hepatotoxic metabolite produced by several species of the genera Aspergillus (Krogh, 1976). Ochratoxin causes lymphoid necrosis in domestic animals (Krogh, 1976) and reduced antibody responses in mice (Prior & Abbreviations: Das = Diacetoxyscirpenol; HPBL = human

peripheral blood lymphocytes; PHA = phytohaemagglutinin; SCE = sister chromatid exchange.

Sisodia, 1979). Zearalenone is a toxic metabolite of Fusarium species. Dietary zearalenone is suspected of inducing spontaneous tumours in laboratory animals. It also exhibits a uterotrophic effect in mice and rats (Ueno & Ueno, 1978). This study was undertaken to elucidate the potential clastogenic and immunotoxic effects of some important mycotoxins on human cells. Since lymphocyte transformation is considered to be a manifestation of cellular immunity, we studied the effects of T-2 toxin, DAS, zearalenone and ochratoxin A on the in vitro transformation of human peripheral blood lymphocytes (HPBL) by phytohaemagglutinin (PHA). We have also studied the influence of these mycotoxins on sister chromatid exchange (SCE) frequency in human lymphocytes, as this is a sensitive indicator of D N A damage. Both assays were performed with and without metabolic activation by isolated hepatocytes. EXPERIMENTAL Cells. Human peripheral blood lymphocytes (HPBL) were prepared from buffy coats obtained from Uppsala Academic Hospital, Uppsala. The blood was diluted 1:1 with sterile saline in plastic tubes containing glass beads for defibrination. The citrate added at the hospital was removed by the addition of CaC12 (1 mmol/buffy coat). The defibrinated blood was centrifuged at 300g for 10rain. The intermediate zone between the supernatant and erythrocytes was carefully withdrawn, washed twice in tissue-culture medium, RPMI 1640 (Flow Lab., Irvine, Ayrshire, UK) supplemented with ~entamycin (Shering Corp. NY, USA) at 50#g/ml 0.01 M-Hepes buffer (Sigma Chemical Co, St Louis, MO, USA). The lymphocytes thus obtained had a

529

530

R. COORAY

viability of 85 to 95~ as measured by trypan blue staining. Rat liver cells were obtained from female Sprague-Dawley rats weighing 250-300 g, and prepared according to Seglen (1973). The intact cells were frozen and stored in liquid nitrogen under the conditions reported by Karlberg & Lindahl-Kiessling (1981). The cells were thawed in a 37°C water bath when required. The supernatant was replaced gradually by RPMI at +4°C supplemented with 0.4~o human albumin (AB Kabi, Stockholm). The cells were washed twice at 10 g and viability was checked with 0.05~o trypan blue. Preparation of cultures. For the assay of D N A synthesis, 4 ml cultures were set up in duplicate in 10ml styrene vials (Cerbo, Trollhfittan at a cell concentration of 0.25 × 106 lymphocytes/ml RPMI 1640, a 2mM-L-glutamine (Flow Lab.) and 0.4~o human albumin. The cells were stimulated at zero hours with PHA (Wellcome, Beckenham, Kent, UK) at 0.5 mg/ml and the test compounds were added at 24 hr. DAS and ochratoxin A were obtained from Makor Chemicals, POB 6570, Jerusalem, Israel. Zearalenone was a gift from Commercial Solvents Corporation, Terre Haute, IN 47808, USA. T-2 toxin was a gift from Northern Regional Research Laboratory, Peoria, IL 6160t, USA. The cells were incubated at 37°C in humidified air containing 5~o CO2 for a further 36 hr. In some cases the cultures were washed 1 or 4 hr after the addition of the test compound and placed in new media containing only PHA. [3H]Thymidine (Amersham International plc, Amersham, Bucks, UK) 0.5 #Ci/ml, 100 Ci/mol was then added 12 hr before cell harvest. The cells were harvested in a semi-automatic cell harvester (Scatron, Lierbyen, Norway) after a total of 72 hr in culture. The filters were counted in a liquid scintillator (Nuclear Chicago Corp, Des Plaines, IL, USA). For the SCE analysis 4ml cultures (1 × 106 lymphocytes/ml) were set up as above. 5-Bromo-2'-deoxyuridine (BudR, Sigma Chemical Co.) at 15#g/ml was added at 24hr. Colcemid (Sigma Chemical Co.) was added to the cultures at 62.5 ng/ml 2hr before cell harvest. The cells were treated with a hypotonic 0.56~o potassium chloride solution and fixed in methanol-acetic acid (3 : 1, v/v). A concentrated suspension of the fixed cells was allowed to flow over wet, alcohol-washed glass slides which were then dried at room temperature. When the cells are exposed to BudR for two cycles of replication secondary metaphase chromosomes possess one chromatid unifilarly substituted with BudR and its sister chromatid bifilarly substituted. Such chromatids stain differentially with the fluorescence plus Giemsa technique of Wolff & Perry (1974). From each culture a total of 20 complete mitoses in the secondary metaphase stage was counted for SCE. Statistically significant differences between SCE frequencies were determined using the Wilcoxon rank test (Colquhoun, 1971). Co-cultures with rat liver cells. To make it possible to add and remove the hepatocytes from the lymphocytes cultures we have constructed a culture vessel consisting of a polypropylene tube (15mm in diameter) with a 5 # m Nuclepor filter (Nuclepor Corp., Pleasonton, CA, USA) glued to one end and a rubber O-ring slipped on the other end. The tube

was then inserted into the lymphocyte culture vessel (outer diameter 20 mm) and the O-ring was adjusted so that the membrane was fixed 1-2 mm above the bottom. The tubes and filters were autoclaved separately and fixed together with silicon rubber (Silastic 732 RTV, Dow Corning SA, Belgium). The pore size was chosen to be as big as possible without letting any cells through. Lymphocytes (4 x 1 0 6 in 2ml culture medium) were added to the outer culture vessel. The membraned tubes were inserted, making sure that the medium passed through the membrane; 300 × 103 hepatocytes in 0.2 ml medium were added and settled rapidly on the membrane. The density of the liver cells has to be kept fairly low (20-30 x 103 cells/cm2) and the distance between the membrane and the medium surface must be short (3--4 mm) to keep the liver cells viable. RESULTS

The addition of trichothecenes (T-2 toxin and DAS), zearalenone and ochratoxin A to PHAstimulated cultures of HPBL resulted in a dosedependent inhibition of [3H]thymidine incorporation compared with control cultures. Complete inhibition was produced by 8 ng T-2 toxin or DAS/ml and by 30 p g zearalenone/ml and almost complete inhibition was caused by 20 pg ochratoxin A/ml (higher doses of ochratoxin A were not tested). Inhibition by 50~o compared with controls results from addition of 1.5 ng T-2 toxin, 2.7 ng DAS or 14#g zearalenone or ochratoxin A/ml (Figs 1, 2 & 3). All of these values were obtained in the absence of metabolic activation. Slides prepared from cultures treated with the trichothecenes and stained with Giemsa, revealed that HPBLs exposed to lower concentrations contained a few blast cells while those treated with the higher concentrations showed no sign of blastogenesis. Cultures were incubated for 4 hr with P H A and the trichothecenes. When the ceils were then washed and ~

lOOe-

•

g

0

0

2

4

6

8

Conch of test compound

10

12

(ng/mL)

Fig. 1. The effect of different doses of T-2 toxin and DAS on DNA synthesis, as measured by [3H]thymidine incorporation, in human peripheral blood lymphocytes in culture in the presence and absence of rat liver cells. T-2 toxin without rat liver ceils (O O); T-2 toxin with rat liver cells ( 0 ' O); DAS without rat liver cells (A A); DAS with rat liver cells (A A).

Mycotoxin effects on DNA synthesis and SCE

Table 1. The effects of T-2 toxin and diacetoxyscirpenol on DNA synthesis as measured by [3H]thymidine incorporation in PHAstimulated human peripheral blood lymphocytes [3H]Thymidine incorporation (cpm as ~ of control) Test compound and concn Washed Unwashed (ng/ml) cultures* cultures Diacetoxyscirpenol 1.5 95.3 76.8 3.0 90.1 21.8 6.0 55.2 9.6 8.0 48.8 0.2 T-2 toxin 1.5 98.10 48.5 3.0 85.21 10.2 6.0 52.89 0.18 12.0 41.89 0.11 *These cultures were washed 4 hr after the addition of the test compounds and placed in new media containing only PHA.

1(30 o

"6 t~

75

g ~~

X

50

~ 25

0

I

]

I

10

15

20

Concn of test compound ( # g / m L )

Fig. 2. The effect of different doses of ochratoxin A on DNA synthesis, as measured by [3H]thymidine incorporation, in human peripheral blood lymphocytes in culture in the presence and absence of rat liver cells. Ochratoxin A with rat liver cells (O 0 ) ; ochratoxin A without rat liver cells (0

0).

placed in new m e d i a c o n t a i n i n g only P H A it was observed, in three such experiments, t h a t washing the cells prevented the inhibition o f D N A synthesis by low c o n c e n t r a t i o n s o f the trichothecenes. Higher c o n c e n t r a t i o n s o f trichothecenes p r o d u c e d a partial inhibition even after washing the toxin-treated cultures (Table 1). W a s h i n g the cells 1 h r after the start o f i n c u b a t i o n h a d a n a l m o s t identical effect (Table 2). As a control o f the metabolic activity o f the rat liver cells a positive control, cyclophosphamide, was used. 80

\ o= 6 0

*

40

2o fr-

o

531

10

15

20

Conch of test compound

25

30

35

(ng/mt)

Fig. 3. The effect of different doses of zearalenone on DNA synthesis, as measured by [3H]thymidine incorporation in human peripheral blood lymphocytes in culture in the presence and absence of rat liver cells. Zearalenone with rat liver cells (O 0 ) ; zearalenone without rat liver cells (0 0).

In the presence o f liver cells c y c l o p h o s p h a m i d e decreased [3H]thymidine i n c o r p o r a t i o n into H P B L . T h e results from co-cultures c o n t a i n i n g H P B L a n d rat liver cells showed t h a t the toxicity o f the trichothecenes was slightly reduced by the rat liver cells, The [3H]thymidine i n c o r p o r a t i o n was 10~o of the control value w i t h o u t rat liver cells at 3 ng/ml o f T-2 toxin b u t increased to 5 3 ~ with rat liver cells. In cultures with the same c o n c e n t r a t i o n of D A S the value increased from 40 to 5 7 ~ (Fig. 1). The inhibitory action o f zearalenone a n d ochratoxin A was n o t markedly altered by the metabolic activity of the rat liver cells (Figs 2 & 3). A t trichothecene or o c h r a t o x i n A c o n c e n t r a t i o n s permitting survival of the H P B L the toxins did not p r o v o k e a n increase in SCE frequency either with or w i t h o u t rat liver cells. All SCE values determined were in the range o f the controls (Tables 3 & 4). Z e a r a l e n o n e e n h a n c e d weakly the SCE f o r m a t i o n in H P B L at a c o n c e n t r a t i o n o f 3 #g/ml. T h e effect was n o t observed at higher c o n c e n t r a t i o n s (6 # g / m l a n d above, Table 5) since very few secondary mitoses were f o u n d at such concentrations. The c h r o m o s o m e m o r p h o l o g y o f H P B L was altered by zearalenone a n d o c h r a t o x i n A in the presence of rat liver cells. T h e c h r o m o s o m e s were m u c h shorter a n d they did n o t separate from one a n o t h e r as c o m p a r e d with control cultures. T h u s the effects o f zearalenone a n d o c h r a t o x i n A o n SCE f o r m a t i o n in H P B L cultures in the presence o f rat liver cells is n o t reported. In mycotoxin-treated cultures the p r o p o r t i o n o f cells in p r i m a r y m e t a p h a s e was higher a n d the proTable 2. The effects of T-2 toxin and diacetoxyseirpenol on DNA synthesis, as measured by [JH]thymidine incorporation, in PHAstimulated human peripheral blood lymphocytes after differing durations of exposure to the test compounds [3H]Thymidine incorporation (cpm as ~ of control of control) Test compound and final concn Washed Washed (ng/ml) after 1 hr* after 4 hr* Diacetoxyscirpenol 3 90 93 8 52 55 T-2 toxin 3 84 86 12 45 46 *The cultures were washed 1 or 4 hr after the addition of the test compounds and placed in new media containing only PHA.

532

R. COORAY

Table 3. The effects of T-2 toxin and diacetoxyscirpenol on the frequency of sister chromatid exchanges in human peripheral blood lymphocytes in culture in the presence and absence of rat liver cells Rat liver cells absent

Rat liver cells present

Percentage of cells, at harvest, in generation Chemical and concn (ng/ml) Controls Negative Cyclophosphamide, 125 x 103 T-2 toxin 1.5 3.0 6.0 12.0 Diacetoxyscirpenol 1.5 3.0 6.0 12.0

Percentage of cells, at harvest, in generation

SCEs/cell*

1

2

3 or later

SCEs/cell*

1

2

3 or later

11.15 ± 3.34 13.80 + 3.50

23 16

61 49

16 35

10.45 ± 3.23 33.0 + 3.25

26 20

47 60

27 20

12.25 ± 3.50 -t -t -t

42 100 100 100

53 0 0 0

5 0 0 0

11.26 + 3.25 10.50 ___3.24 - t --t

29 49 100 100

57 46 0 0

14 5 0 0

11.80___3.8 -t -- t --t

42 100 100 100

44 0 0 0

14 0 0 0

10.45 _ 3.22 12.40 ± 3.52 -t -t

41 50 85 100

46 42 15 0

13 8 0 0

*Values are means + SD for 20 cells. t N o secondary metaphases could be found.

portion in secondary and tertiary metaphase and later division cycles was lower than in control cultures (Tables 3, 4 and 5). DISCUSSION

The results show that trichothecenes (T-2 toxin and DAS) inhibit DNA synthesis, measured as [3H]thymidine incorporation and transformation to lymphoblasts, on PHA stimulated HPBL. These findings agree with those of Ueno & Shimada (1974) who found that trichothecenes are potent inhibitors of protein and DNA synthesis in cells. The administration of T-2 toxin to calves (0.6mg/kg/day) is associated with significant depression of lymphocyte response to the mitogens PHA and Concanavalin A and to poke weed mitogen (Buening, Mann, Hook & Oswiler, 1982). The inhibition of DNA synthesis in HPBL by the trichothecenes was dose dependent. The trichothecenes seemed to act rapidly since the degree of inhibition of DNA synthesis after 1 hr incubation with the trichothecenes was identical to that after 4 hr (Table 2). The C-4 acetyl residue of T-2 toxin can be selectively removed by enzyme(s) localized in liver microsomes. Kinetic studies have shown that HT-2 toxin formed from T-2 toxin and monoacetoxiscirpenol Table 4. The effect of ochratoxin A on the frequency of sister chromatid exchanges in human peripheral blood lymphocytes in cultures Ochratoxin A coucn (/~g/ml)

Percentage of cells, at harvest in generation SCEs/cell*

1

2

3 or later

Experiment 1 Control 5.0 7.5 |0.0

11.15 ± 3.34 11.0+3.31 13.45 _+3.67 10.85 ± 3.29

63 71 69 76

32 21 27 24

5 8 4 0

26 33 39 45

47 47 46 55

27 20 15 5

Experiment 2 Control 5,0 Z5 10.0

10.34 ± 10.50 ± I 1.45 ± 10.35 +

3.25 3.35 3.36 3.28t

*Values are means +_SD for 20 cells. tOnly 18 secondary metaphases were counted.

from DAS are the end products in an in vitro reaction system. As for the biological activity of the C-4 deacetylated metabolites, rabbit reticulocyte bioassay and lethal toxicity tests reveal no marked difference between the parent compounds and the metabolites (Ohta, Matsumoto, Ishii & Ueno, 1978). However, in our experiments the products of trichothecenes formed by the metabolic activity of intact rat liver cells were slightly less toxic than the parent trichothecenes. Some mycotoxins, for example patulin, penicillic acid, and aflatoxin B1 induce DNA breakage in mammalian cells (FM3A) (Umeda, Tsutsui, Itoh & Saito, 1976). Patulin also has a weak enhancing effect on SCE formation in HPBL (Cooray, Kiessling & Lindahl-Kiessling, 1982). The trichothecenes did not induce SCEs in cultured HPBL either with or without metabolic activation by rat liver cells. T-2 toxin is also non-mutagenic in the Salmonella test either with or without metabolic activation (Ueno et al. 1978). Table 5. The effect of zearalenone on the frequency of sister chromatid exchanges in human peripheral blood lymphocytes in culture Zearalenone concn (#g/ml)

Percentage of cells, at harvest in generation SCEs (% of control)t

1

2

3 or later

40 28 1

9 2 0

32 19 29

5 0

30 30 30

11 7 2

35 2 0

16 0 0

Experiment 1 Control 3.0 6.0 Control

1.5 3.0

100 S 3.8 138±4.5" - ~

51 70 99 Experiment 2 100 ± 3.5 63 104 ± 3.4 79 124 +_ 3.8* 71

2

Experiment 3 Control 2.5 5.0

100 ___3.5 112___3.6 139 ± 3.9*

Control 7.5 10.0

100 ± 3.3 - ~: -~

59 63 68

Experiment 4 49 98 100

tValues are calculated from means ( ± SD) for 20 cells except where indicated. Those values marked with asterisks differ significantly (Wilcoxon rank test) from the corresponding control value (*P < 0.01). :~No secondary metaphases could be found.

Mycotoxin effects on DNA synthesis and SCE Both T-2 toxin and DAS were highly efficient in blocking cell division in HPBL. One reason for this could be the reaction of the trichothecene epoxide ring with microfibrillar proteins and in particular their SH-groups. The epoxide groups of many biologically active compounds are able to react enzymatically or non-enzymatically with SH-groups of amino acids and proteins (Ueno, 1977). Such a reaction with the trichothecenes in the dividing HPBL could lead to an increase in the percentage of primary and a decrease in the percentage of secondary, tertiary and later division cycles. T-2 toxin also arrests metaphases in dividing cells of Allium (Linnainmaa, Sorsa & Ilus, 1979). In trichothecenetreated HPBL cultures the chromosomes had a tendency to stick to each other and were much shorter than those in the control cultures. Ochratoxin A inhibited DNA synthesis in cultured HPBL. The effect was dose dependent. The inhibition could be a result of inhibited protein synthesis; ochratoxin A has been shown to inhibit protein synthesis in GBK cells (Prior & Sisodia, 1979). Rat liver microsomes convert ochratoxin A to (4R)- and (4S)-4-hydroxy ochratoxin A (St~rmer, Hansen, Pedersen et al. 1981). According to Hutchison, Steyn & Tompson (1971) (4R)-hydroxy ochratoxin A is nontoxic to rats when given orally (40mg/kg body weight). In contrast we found that the toxicity of ochratoxin A metabolites, formed by intact rat liver cells, was similar to the toxicity of ochratoxin A itself. Ochratoxin A had no effect on the SCE frequency in HPBL. Also according to Kuczuk et al. (1978) ochratoxin is not mutagenic in the Ames test directly or in the presence of the hepatic S-9 enzyme preparation. According to Steyn, Velggaar, DuPreez et al. (1975), who studied monkey kidney epithelial cells, the analogs of ochratoxin A have a toxic effect predominantly in dividing cells during metaphase. It is probable that a similar effect by ochratoxin A on HPBL resulted in the increase in the percentage of primary divisions which we observed. Zearalenone both with and without rat liver cells was equally toxic to HPBL as measured by the inhibition of DNA synthesis. The inhibition was dose dependent and again could be the result of inhibited protein synthesis. There is no direct evidence to support this suggestion but according to Kiessling (1982) zearalenone acts as a growth inhibitor in young rats. The ~t-hydroxy steroid dehydrogenase present in rat liver converts zearalenone to ct- and fl-zearalenol (Olsen, Pettersson & Kiessling, 1981). Mutagenic chemicals are usually considered to cause an elevated frequency of SCE (Keto, 1977; Wolff, 1981). According to Ueno & Kubota (1976) zearalenone modifies bacterial DNA in the "rec assay" using recombination deficient mutant cells of Bacillus subtilis. Our results indicate that zearalenone may also have a mutagenic effect in mammalian cells. In HPBL zearalenone induced a slightly increased SCE formation at low concentrations. Zearalenone arrested cell division at primary metaphase and at high concentrations there were practically no secondary metaphases making it impossible to investigate the effects of high concentrations of zearalenone on SCE formation. From the results of this work it is suggested that

533

zearalenone may have a weak SCE-enhancing effect on mammalian cells, while T-2 toxin, DAS and ochratoxin A do not have any such effect either before or after metabolism. However, T-2 toxin, DAS, ochratoxin A and zearalenone all inhibit DNA synthesis in mammalian cells. Acknowledgements--I am grateful to Professors Kerstin

Lindahl-Kiesslingand Karl-Heinz Kiessling for many valuable suggestions during the course of this work. This study was supported by the University of Uppsala and Carl Trygger's foundation for scientificresearch. The secretarial assistance of Marianne Andersson is gratefully acknowledged.

REFERENCES

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