Mutation Research, 100 (1982) 133-138 133 Elsevier Biomedical Press

TESTING FOR MITOTIC CROSSINGOVER AND INDUCED ANEUPLOIDY USING Aspergillus nidulans AS PART OF THE UKEMS TEST PROGRAMME

PAUL WATKINS

Imperial Chemical Industries Ltd., Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG (Great Britain)

(Received 29 April 1981) (Accepted 31 July 1981)

SUMMARY

4CMB and BC were shown to induce mitotic crossingover in Aspergillus nidulans, whereas no such activity was shown in the presence of 4HMB. None of the 3 test compounds were able to induce chromosome aneuploidy.

MATERIALS AND METHODS

The strain of Aspergillus nidulans used was strain P as described by Bignami et al. (1974). This diploid strain is heterozygous for the gene determining resistance to p-fluorophenylalanine (PFPA). The conidia can grow on minimal medium supplemented with PFPA only if mitotic crossingover or aneuploidy is induced. Colonies resulting from crossingover are green and may involve nutritional markers. Colonies resulting from non-disjunction are yellow and require p-aminobenzoic acid (paba).

Preparation of liver-homogenate fraction ($9 mix) The $9 mix was prepared according to Ames et al. (1975) from Alderley Park,

Alpk (Wistar derived) rat after induction by Aroclor 1254 500 mg/kg i.p. 5 days before they were killed. During the time of the experiment (7 h) the $9 mix was kept on ice. It has been shown that the $9 mix remains active throughout this time by its ability to activate several indirect-acting mutagens without any noticeable loss of mutagenicity (Watkins, unpublished observations).

0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press

134

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60-

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Fig. 1. Effect o f pre-incubation on the number of PFPA-resistant colonies of Aspergillus nidulans after

treatment with 4CMB.

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Fig. 2. Effect of pre-incubation on the number of PFPA-resistant colonies of Aspergillus nidulans after

treatment with BC.

T A B L E 1

M E A N C O L O N Y C O U N T S FOR D U P L I C A T E P L A T E S P E R T R E A T M E N T

Amount per Chemical plate pg

Untreated 0

5-Fluoro- 250 uracil

Benzyl 100 chloride

1,000

5,000

4-Chloro- 100 methyl-

biphenyl 500

1,000

4-Hydroxy- 100 methyl-

biphenyl 500

1,000

135

S-9 Incubation time prior to treatment (hours) mix

0 i 2 3 4 5 6

+ 3.0 3.5 4.0 8.0 4.0 6.0 7.5 3.5 7.5 5.5 12.0 13.0 11.0 7.5

+ 43.0 70.5 99.5 113.0 78.5 113.5 103.5 85.5 116.0 131.5 180.5 182.0 174.5 174.0

+ 5.0 5.0 4.5 5.5 4.0 4.0 4.5 5.0 7.0 7.0 8.0 8.0 7.5 10.5

+ 1.5 6.0 8.5 7.0 4.5 9.0 5.0 2.0 5.5 11.0 14.5 5.0 8.0 21.0

+ 1.0 1.0 11.5 8.0 3.5 8.0 16.5 2.5 4.0 9.5 13.5 6.0 8.5 13.0

+ 4.0 10.5 20.0 11.0 22.0 19.0 25.5 11.5 33.5 58.0 43.0 58.5 67.0 47.5

+ 8.0 29.5 35.0 24.5 22.0 39.0 25.5

47.5 68.5 73.5 7"2.0 58.5 80.0 59.0

+ 0 0 0 0.5 7.5 6.0 4.5 0 0 1.5 3.0 3.0 10.0 16.5

+ 2.0 8.5 3.0 3.5 6.0 9.5 7.5 5.5 9.0 2.5 12.0 8.5 11.0 9.0

+ 0.5 3.0 1.0 6.0 1.5 3.0 4.5

0.5 7.0 1,5 3.5 4.5 4.0 3.5

+ 0 0 0 0 0 0 0 0 0 1.0 0 0 0.5 0

Test procedures Glasgow minimal media (GMM) plates, supplemented with PFPA, aneurine,

biotin and paba were seeded with 3 x 103 conidia and incubated at 37°C. At hourly time intervals the test chemical as a solution in DMSO was applied in 2.0 ml 0.75% GMM plus supplements with and without $9 mix to the seeded plates and reincubated. The plates were scored 5-7 days later.

R E S U L T S A N D D I S C U S S I O N

Previous work with this strain had shown that a straightforward treat and plate method has a number of disadvantages (Watkins, unpublished results) and one of the parameters being investigated was the effect of pre-incubation in selective and non-selective media when treated with chemicals.

4CMB, BC and 4HMB were tested only on the selective media i.e. containing

136

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Pre incubation time (hour s )

F i g . 3. E f f e c t o f p r e - i n c u b a t i o n o n the n u m b e r o f P F P A - r e s i s t a n t c o l o n i e s o f Aspergillus nidulans a f t e r

t r e a t m e n t w i t h 4 H M B .

PFPA after the Aspergillus conidia already in the media had been incubating for different time periods. It was thought that the conidia would achieve some degree of synchrony after this treatment. PFPA has also been used to synchronise the cell cycle in other organisms (Brostrom and Binkley, 1969). Previous work has shown that the conidia in minimal media plus PFPA did not germinate after 10 h incubation but did show a slight swelling after about 3-5 h. Conidia in minimal media only had started to germinate at about 3.5 h (Watkins, unpublished results).

A qualitative assay similar to that described by Bignami et al. (1974) showed that only 4CMB showed a weak positive result. Further investigation showed this response was the result of mitotic crossingover (results not shown).

The qualitative assay (Table 1) confirmed the previous result with 4CMB and gave a clear indication of the induction of crossingover with increases over the length of

~- 45-

~- 40-

= 35-

8 30- m 25-

20- 15-

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~ 0

._ . . . . . . . . - S-9 mix

I1 I I I I I 2 3 4 5 6

Pre incubation time (hours)

F i g . 4. E f f e c t o f p r e - i n c u b a t i o n o n t h e n u m b e r o f P F P A - r e s i s t a n t c o l o n i e s ofAspergillus nidulans w h e n

u n t r e a t e d .

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~ 1 ~ -

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~ 60- o

~ 40-

~ 20-

0

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, - S -9 mix /

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Fig. 5. Effect of pre-incubation on the number of PFPA-resistant colonies of Aspergillus nidulans when

treated with 5-fluorouracil.

the pre-incubation period (Fig. 1). The inclusion of $9 mix reduced the effect seen. From the bacterial mutagenic studies (Venitt, 1981), it was generally seen that the activity of 4CMB was reduced by the $9 mix. The toxicity of 4CMB to the conidia was not assayed although it was quite evident at 1000/zg per plate.

The variations in the response seen at the different time points may be due to the toxicity of 4CMB combined with its short half-life plus the state of the conidia at

these time points. With BC (Fig. 2) a small induction of crossingover was seen which was only

clearly noticeable when the conidia were treated after 5 and 6 h incubation. The apparent lack of response at previous time points particularly at 4 h pre-incubation may be variations due to the small numbers of plates used and resistant colonies counted rather than a specific time effect. The $9 mix had little or no effect on the

response seen with BC. 4HMB did not show any positive response in the assay (Fig. 3). The untreated

(Fig. 4) and positive control data (Fig. 5) are included for comparison.

CONCLUSION

Of the 3 compounds tested, BC and 4CMB induced crossingover. 4HMB showed no positive result in the assay. No evidence for the induction of aneuploidy was seen with any of the compounds tested.

REFERENCES

Ames , B.N., J. MeCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with

the Salmonel la /mammal ian-microsome mutagenicity test, Mutat ion Res., 31 ,347-364 .

138

Bignami, M., O. Morpugo, R. Pagliani, A. Carere, G. Conti and G. di Giuseppe (1974) Non-disjunction and crossingover induced by pharmaceutical drugs in Aspergillus nidulans, Mutation Res., 26, 159- 170.

Brostrom, M.A., and S.B. Binkley (1969) Synchronous growth of Escherichia coli after treatment with fluorophenylalanine, J. Bacteriol., 98, 3, 1271-1273.

Venitt, S. (1982) U.K.E.M.S. Collaborative Genotoxicity Trial: Bacterial mutation tests of 4-chloro- methylbipheuyl, 4-hydroxymethylbiphenyl and benzyl chloride, analysis of data from seventeen laboratories, Mutation Res., 100, 91-109.