assay of cytotoxicity and mutagenicity of alkylating agents by using neurospora spheroplasts

Mutation Research, 125 (1984) 43-53 43 Elsevier

MTR 03797

Assay of cytotoxicity and mutagenicity of alkylating agents by using Neurospora spheroplasts

Susan Pope, Jennifer M. Baker and J.H. Parish Department of Biochemistry, University of Leeds, Leeds LS2 9JT (Great Britain)

(Received 15 December 1982) (Revision received 3 August 1983)

(Accepted 8 August 1983)

Summary

A system relying on the use of Neurospora crassa spheroplasts has been developed for the assay of cytotoxicity and mutagenicity of chemical compounds. Mutagenicity was assayed by using reversion of alleles in the am gene selected to recognize certain specified transitions and also undefined point mutations. Cytotoxicity was quantified by measuring a 'cytotoxicity parameter', m, which appears in the exponential function that fits the survival/dose curve for each compound (under standard incubation conditions). Of the compounds tested, nitrogen mustard (CI(CH 2)2 NMe(CH2)2 C1) was cytotoxic and non-mutagenic, and ethyl nitrosourea was highly mutagenic but not cytotoxic. Of the remaining compounds tested, methyl nitrosourea, butadiene diepoxide, and cis platin (cis diammonia platinum II chloride) all showed comparable mutagenicity per survivor, although the values of rn covered a wide range. Differences were found between the different compounds in the effects of the uos-2 allele on survival and on the preponderance of G to A transitions.

For studies on the cytotoxicity and mutagenicity of chemicals, microbial systems have the ad- vantages of sensitivity and rapid generation of data. Although the most sensitive systems (for example the 'Ames test', reviewed by Haroun and Ames, 1981) are prokaryotic, in evaluating human risks and the likely mode of action of pharmaceutical preparations, the test organism should contain eukaryotic chromatin. Neurospora crassa is an established test organism for this purpose. An example of this is the use of conidia as targets and forward and reverse mutations of

Abbreviations: BDDE, butadiene diepoxide; CP, cis platin; ENU, ethylnitrosourea; HN2, nitrogen mustard; MNNG, N- methyl-N'-nitro-N-nitrosoguanidine; MNU, methyl nitrosourea.

alleles at the ad-3 locus. This method and the use of alternative genotypes are reviewed by Brock- man et al. (1981).

We have developed an alternative test system based on reversions of alleles at the am locus. The gene product is the ammonia-assimilatory NADP-dependent glutamate dehydrogenase (GDH); the amino acid sequences of the wild-type protein and of several mutants have been determined (Holder et al., 1975; Wootton et al., 1975a, 1975b; Brett et al., 1976). From possible codon utilization, 'true' reversion of four of the alleles requires an unambiguous point mutation: reversion of am-3 to am + requires a G to A transition; reversion of am-19 to am + requires an A to T transversion; reversion of am-7 requires a G to C transversion; and reversion of am-1 re-

002%5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.

44

quires an A to G transition (Brett et al., 1976). However, an additional feature of the am system derives from the multimeric character of the G D H protein: for certain alleles (of which am-3 is an example) reversion can be due to a second mutation in the same gene that complements the con- formational distortion responsible for reducing the G D H activity (under physiological conditions) to an inadequate level for ammonia assimilation. The ' t rue revertants' are referred to as 'first-site', and the second mutations (examples of intragenic sup- pression) are referred to as 'second-site' reversions.

Biochemical and genetic characterization of am-3 revertants by Coddington et al. (1966) established that these could be differentiated on the basis of the kinetic parameters of the G D H enzyme activity and, moreover, that the tests can be performed on crude extracts. The apparent K m for glutamate is much increased in the case of second-site revertants; i.e., the affinity of these mutant enzymes for glutamate is decreased. In the case of most of the revertants, the apparent K m is in fact higher than the g m for the physiologically incompetent form of the enzyme in am-3 itself.

Thus, the am-3 allele can be used to detect specified G to A transitions, non-specified second-site revertants and, additionally, so-called 'escape mutations'. These are recognized as having no apparent K m for glutamate in the G D H assay and (in most cases) poor colonial growth.

Of the four am alleles, second-site revertants are possible for am-3 and am-19, but they are not possible for am-1 and am-7 because these mutations affect coenzyme binding (Brett et al., 1976).

We have developed the Neurospora system for the simultaneous assay of cytotoxicity and mutagenicity of a variety of alkylating agents.

The two monofunctional alkylating agents methyl and ethyl nitrosourea (MNU and ENU) are mutagens and carcinogens and their reaction with DNA involves electrophilic attack at several sites in DNA bases (Lawley and Warren, 1975; Frei et al., 1978). Nitrogen mustard (HN2) is a bifunctional alkylating agent. The major sites of substitution in DNA are likely to be N7 in G residues, and the compound cross-links DNA (Geiduschek, 1961). However, HN2 is capable of cross-linking nucleic acid to protein in ribosomes (Ulmer et al., 1978) and in phage 2~. Moreover, in

the latter case it is likely that the DNA-proteins reaction is responsible for the anti-viral activity of HN2 (Fraser et al., 1982). Butadiene diepoxide (BDDE) is a cross-linking agent established as a mutagen in Neurospora (Auerbach and Ramsey, 1968); from the experiments with phage X, it appears to differ from HN2 in the mechanism of its anti-viral activity, and this is probably not due to DNA-prote in cross-linking (Fraser et al., 1982). Cis platin (cis diammonia platinum II chloride, CP) has a complex reaction with DNA (Roberts and Thomson, 1979; Marcelis et al., 1981). CP is a mutagen (Mattern et al., 1981), and its related compounds are used as antineoplastic drugs (Hill and Speer, 1982).

Materials and methods

Strains The strains of Neurospora crassa used in this

work are listed in Table 1. They were maintained on agar slopes of Vogel's minimal medium (supplemented with 0.2% glutamate in the case of am strains).

Media The minimal medium used was that of Vogel

(1964). Crosses were performed on the medium of Westergaard and Mitchell (1947). Molten agar used for recovery of spheroplasts consisted of Vogel's minimal medium supplemented with sorbose (1.5% w/v), 0.6 M NaC1, agar (1% w/v) and either glutamate (0.2%) or glycine (0.2%). The glutamate supplement was used for counting the total survivors, and the glycine supplement was used for counting am + revertants. For certain purposes (see Results), an enriched recovery medium was em- ployed. This contained, in addition to the NaC1 and agar, the following (g/l): 15 ml glycerol, 2.5 g glucose, 2.5 g malt extract, 0.4 g peptone, 2.5 g yeast extract, 1.0 g KH2PO 4, 0.25 g MgSO 4 • 7H20, 0.1 g CaC12 • 6HzO, 5 #g biotin and 0.5 ml trace element solution (Vogel, 1964); the pH was ad- justed to 7.5.

Preparation, treatment and recovery of spheroplasts Mycelial cultures were grown in Vogel's liquid

medium, and spheroplasts were prepared according to Wiley (1974) with the following modifica-

TABLE 1

SUMMARY OF STRAINS USED

45

Strain Alternative Mating Phenotype Genetic FGSC number or allele type background origin number

St a - a wild-type SL - STA - A wild-type SL 262 a m - 1 32213 a am- SL6 1184 a m - 3 52929 a am- SL6 1 186 a m - 7 K410 a am- SL6 1 189 a m - 19 - a am- SL6 J.C. Woonon uvs - 2 - A uvs M 1694 a m - 1 u v s - 2 - A am-, uvs SL6 this work a m - 3 uvs - 2 - A am-, uvs SL6 this work a m - 7 uvs - 2 - A am-, uvs SL6 this work a m - 19 uos - 2 - A am-, uvs SL6 this work

am-, requires glutamate (amination-defective); uvs, UV-sensitive; SL, St. Lawrence; SL6, back-crossed to SL 6 times; M, mixed.

tions. For certain experiments, fl-glucuronidase was replaced by Novozym 234 (Novo Enzyme Products Ltd, 3 mg/ml ; Hamlyn et al., 1981), and in this case the CaCl 2 was omitted. In all cases 2-mercaptoethanol was used (in place of dithioth- reitol) and incubation was continued until the spheroplasting was complete as judged by phase contrast microscopy.

Spheroplasts were incubated for 1 h (or different periods of time in the case of certain experiments) at 18°C with each mutagen. The mixture consisted of a suspension of spheroplasts (106-]07 spheroplasts per ml; 200/~l), 20 mM K phosphate buffer, pH 6.2 (100 ~tl) and a solution of mutagen in the same buffer (100/~l). In the Results, mutagen concentrations refer to the final concentration in each case. The recovery of spheroplasts was performed by the pour plate method (Wiley, 1974). Revertants, morphological mutants or other survivors required for further testing were trans- ferred to small agar slants.

A s s a y f o r g l u t a m a t e d e h y d r o g e n a s e

The enzyme was assayed in a crude preparation according to Coddington et al. (1966).

M u t a g e n s

The nitrosoureas MNU and ENU were prepared according to Lawley and Warren (1975). HN2 was obtained from Sigma and BDDE from

Aldrich. CP was a generous gift from Johnson and Matthey Ltd.

C u r v e f i t t i n g

Data were fitted to exponential functions by using a program written by Dr. D.G. Herries and implemented on the University of Leeds Amdahl V7 computer.

Results

C o m p a r i s o n o f t a r g e t p r e p a r a t i o n s

Different conidial preparations and spheroplasts were contrasted for sensitivity of HN2. For comparative purposes, we tabulated the inactivation parameter (m, units 1 mmole - t ) for an exponential dose-response (Table 2). The data were fitted directly to the exponential curve because this is statistically safer than performing a linear re- gression (or subjective 'best fit') on a semi-log transformation of the data. (The objection to the semi-log plot is that it gives undue weight to readings at relatively high concentrations of compound.) For a totally innocuous compound, m has a value of 0. The effectiveness of different preparations for evaluating the cytotoxicity of nitrogen mustard was determined (Table 2). Old conidia were essentially insensitive to nitrogen mustard; young conidia were more suitable, but spheroplasts showed the greatest sensitivity in the data

46

TABLE 2

CYTOTOXICITY PARAMETERS (m) FOR NITROGEN MUSTARD AND THREE DIFFERENT PREPARATIONS OF NEUROSPORA STA

Preparation m (1 mmole- ~ ) Concentration range × 102 (mM) over which

inactivation can be measured a

Old conidia b 0.92±0.85 0- 50 Young conidia c 1.75 ± 0.28 0-100

Pre-germinated conidia variable d 0- 10 Spheroplasts 2.79 ±0.16 0- 10

a Under standard incubation conditions, see Materials and Methods.

b More than 1 week.

¢ Less than 1 week. d See text. Dose responses were computer-fitted to the exponential equa-

tion:

S = S O exp ( - m C )

where S is the number of survivors at drug concentration C and S o is the number of survivors at zero C.

shown. The results with conidia pre-germinated to 80% (as checked microscopically) were variable and irreproducible. With certain preparations, these were more sensitive than the spheroplasts; with others they were relatively insensitive. The

problems were apparently due to systematic errors and may reflect an inhomogeneity in the age distribution of any con±dial suspension that is required for pre-germination. From this experience, spheroplast preparations were selected as a relatively sensitive and reproducible system.

Comparison of cytotoxicity of several compounds Some monofunctional and bifunctional alkylat-

ing agents were used to treat spheroplasts of am + and four am strains, and the cytotoxicity parameter m was determined for each. For certain combi- nations of compound and allele, effects of the uvs-2 mutation were determined (Table 3).

Comparison of the data for am + strain shows that for all the compounds tested am alleles have an effect on cytotoxicity and that for MNU, possi- bly ENU, and HN2, am strains are in general more sensitive than am + . This might reflect differences in the properties of spheroplasts obtained from cultures of these different strains.

Of the monofunctional compounds, MNU is more cytotoxic than ENU. Indeed, for ENU in all strains other than am-19 and am-3, the errors are greater than the cytotoxicity parameter; therefore, ENU is not cytotoxic in the concentration range used. This is illustrated in Fig. 1, which is also an example of the computer fitting of data.

The effect of the uvs-2 allele differs from compound to compound. As uvs-2 strains are profi-

TABLE 3

INACTIVATION OF SPHEROPLASTS FROM SEVERAL NEUROSPORA STRAINS BY MONOFUNCTIONAL AND BI- FUNCTIONAL ALKYLATING AGENTS

Compound MNU ENU HN2 BDDE CP

Concentration range (mM) 0-25 Cyto tox ic i t y p a r a m e t e r ( m ) × 10 2 l m m o l e - I

0-25 0-10 0-50

ST,4 13.6 ± 1.81 0.24±0.26 u v s - 2 7.48+ 1.81 1.45 5:1.56 a m - 3 28.1 +12.0 1.355:0.55 a m - 3 u o s - 2 15.2 ± 8.6 -0.585:1.63 a m - 1 9 22.4 ± 1.8 2.655:0.93 a m - 1 9 u v s - 2 14.1 ± 4.6 0.51 ±0.55 a m - 7 10.5 5:1 .5 -0 .65±1 .4 a m - 7 uvs - 2 0.01 ± 1.44 a m - 1 26.9 + 9.1 0.13_+0.2 a m - l u v s - 2 11.1 ± 1.2 -0 .29±1.25

2.79±0.16 10.6 5:3.8 4.94±1.88 3.26±0.67 9.14±1.17 3.35±1.01

7.43±1.67 3.85±0.94

0-1.6

52.0±8.3

42.2±9.3

20.5±6.7 49.0±5.8

The data are values for the cytotoxicity parameter, m 5: S.E.M. Blank entries were not tested.

101 o o

~00. 5

0 5 10 15 Conce~r'~ion(mM)

a O J 25

Fig. 1. An example of direct fitting of data to the exponential function of Table 3. The ratio of am-3 spheroplast survivors (S /S °) is plotted as a function of concentration of ENU (O) and MNU (e). The lines are fitted from the calculated values of m (Table 3).

cient in post-replication repair, it is likely that they are allelic for a gene involed in nucleotide excision repair (Calza and Schroeder, 1982). Of the pairs of strains tested, uvs-2 strains are more sensitive to HN2 than uvs ÷ strains, and the uvs-2 + gene can be implicated in repair of HN2-mediated damage. In contrast, uvs-2 + appears to sensitize strains to MNU. This result might imply that uvs-2 is allelic for a gene that is involved in a repair pathway that for certain DNA lesions caused by MNU can lead to a lethal event. ENU is so slightly cytotoxic that any effect of uvs-2 is difficult to assess, but in the case of am-19, uvs-2 appears to have a similar effect on sensitivity to ENU as it does on sensitivity to MNU. From the data, it is impossible to be certain whether this is a similar phenomenon with BDDE: from the four sets of data (Table 3), the am-7 uvs ÷ strain is seemingly anomalous in being sensitive to BDDE. The same strain is, in contrast, less sensitive than other strains to CP.

The time course of survival with two bifunctional reagents was determined (Fig. 2). The inactivation with CP was over in approximately 45 min; in contrast, the inactivation with HN2 was much more prolonged. This difference could be due to a different rate of chemical reaction or could be due to different repair pathways. The

47

100

~ 10 to

°o 3'0 6*0 9~ 12o Time (minutes)

Fig. 2. Time course of inactivation of am-3 spheroplasts by 0.15 mM CP (O) and 2 mM HN2 (O).

evidence for a shoulder in a semi-log plot of the HN2 data (Fig. 2) might indeed be suggestive of a repair process. However, further data points would be required to justify the significance of such shoulders in survival curves plotted either as a function of time or concentration.

Comparison of mutagenicities of seoeral compounds For several am alleles, revertants were scored,

contrasted with survival, and (for certain selected revertants) assayed biochemically to distinguish first-site and second:site revertants and escape mutants. The biochemical test consisted of assays for G D H activity on crude enzyme preparations. Examples of the enzyme assays are shown in Fig. 3, and the reversion data are summarized in Table 4.

Both monofunctional agents (MNU and ENU) are effective mutagens, and am-3 is the most sensitive am allele for reversion studies. The spontaneous mutation rate is higher in uos-2 than in uvs strains, but the mutagenicity of these alkylating agents is much reduced by uos-2. This establishes

g

TABLE 4

REVERTANTS OBTAINED FROM TREATMENT OF SPHEROPLASTS WITH ALKYLATING REAGENTS

Compound a m - 3 a m - 3 u v s - 2

Concn. % Revertants Number Number lst Number2nd Number Concn. % Revertants Number Number lst Number2nd Number (mM) survivors per 104 tested site site 'escape' (mM) survivors per 104 tested site site 'escape'

survivors survivors (number (number counted) counted)

MNU 0 100 0 (0) 0 0 0 0 1 75 2.47 (12) 7 3 2 2 5 25 27 (51) 22 9 7 6

10 6 44.4 (15) 13 10 1 2 15 1.5 0 (0) 0 0 0 0 25 0.09 0 (0) 0 0 0 0

ENU 0 100 0 (0) 0 0 0 0 1 98 3.06 (10) 10 7 0 3 5 93 4.03 (12) 10 8 2 0

10 87 8.04 (15) 14 12 1 1 25 71 10.54 (24) 15 10 1 4

HN2 0 100 0 1 95 0 2.5 88 0 5 78 0 b

BDDE

CP

0 100 0.29 (2) 0 a 5 85 1.1 (7) 0

10 72 1.1 (7) 0 20 52 2.1 (9) 0 30 38 4.9 (8) 0 50 20 2.3 (1) 0

0 IO0 0 (0) 0 0.2 92 0.86 (16) 12 0.8 71 1.74 (29) 12 1.2 60 2.07 (21) 14 1.6 51 1.50 (16) 12

0 0 0 10 2 0

8 4 0 8 5 1 8 4 0

0 100 3.1 (20) 10 10 0 0 1 86 2.9 (31) 11 7 4 0 5 46 6.8 (40) 15 3 9 3

0 100 3.1 (26) 10 1 101 2.8 (22) 12 5 103 6.1 (26) 10

10 106 3.2 (35) 0 25 115 3.7 (52) 12

0 100 3.1 (26) 0 1 91 0.36 (3) 0 2.5 80 0.27 (2) 0 5 63 0 (0) 0

10 40 0 (0) 0

0 100 0.4 (2) 0 a 2.5 92 0.2 (1) 0 5 85 0 (0) 0

10 72 0 (0) 0 25 43 0 (0) 0

10 0 0 12 0 0

8 2 0 0 0 0 9 3 0

4 9

,..a

< [...,

0 >

E

8 ~

0 >

8 ~

~ g Z ~

"0

Z ~

0 >

8 ~

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

O O 0 0 Q

@@CCE

Z Z Z ~ a.

TABLE 4 (continued)

Compounds a m - 1 9 u v s - 2 a m - 1

conc % revertants conc % (mM) survivors per 104 (mM) survivors

survivors

revertants per 104

survivors

a m - 1 u u s - 2

conc (raM)

%

survivors revertants pert 1 0 4

survivors

MNU

ENU

0 100 0 0 100 0 0 100 0 1 89 0 2.5 44 0 1 90 0 5 51 0 5 23 0 5 58 0

10 25 0 7.5 12 0 10 33 0 15 12 0 10 6 0 15 19 0

25 3 0

0 100 0 0 100 0 0 100 0 1 99 0 1 100 0 1 99 0 5 98 0 5 99 0 5 95 0

10 95 0 10 99 0 10 90 0 25 88 0 25 98 0 15 85 0

25 77 0

a GDH activity was not tested because colonies did not grow in liquid culture. b Tests using germinated conidia and HN2 concentrations of up to 200 mM (1~ survival) failed to detect revertants. 0 revertants per 104 survivors signifies a reversion frequency of < 10-5. Blank entries were not tested.

51

-10 0 10 20 :30 I /G lu

Fig. 3. Example of double reciprocal plots of kinetic data for glutamate dehydrogenase for four different strains; O, STA (results from two independent experiments); e , am-3 (results from two different strains); n, am-3 cp R12 (a second-site CP revertant); and I1, am-3 cp R2 (a first-site CP revertant).

that the mechanism of spontaneous reversion of am-3 is different from that of alkylating agent-induced reversion; this is supported by the fact that in the case of the am + revertants that do arise in uvs-2 strains, MNU produces a shift from first-site to second-site revertants. As the effect of uos-2 is also to enhance survival after MNU treatment, we conclude that the uvs-2 ÷ gene product is involved in an error-prone pathway induced by or respon- sive to nitrosoureas and that this pathway can lead to mutations or death.

As am-1 is not revertible by MNU and ENU, these compounds do not effect A to G transitions at a detectable level. Of the possible transversion test systems, am-19 is not revertible and data from am-7 are difficult to interpret because there is a significant variation in the spontaneous reversion frequency from one batch of spheroplasts to another (compare the MNU and CP data in Table 4). Of the am-7 revertants all were first-site as expected (see Introduction).

Of the bifunctional reagents, HN2 is not mutagenic with am-3; BDDE is weakly mutagenic, in

TABLE 5

SURVIVORS DETECTED USING VOGEL'S M I N I M A L A G A R A N D S U P E R C O N I D I A T I N G A G A R

Concn. of Survivors/ml

M N U (mM) On Vogel's On superconidiating

minimal agar

0 3.52×106 3.54X106 5 2.89 X 106 3.26 × 106

15 1.58 X 106 1.66 X 106 25 3.50 × 105 6.75 X 105

the strains tested, but probably by a different mechanism, because the apparent revertants were weakly growing and could not be characterized (Table 4). CP is a mutagen for reversion of am-3

and generates first- and second-site revertants. From the previous data, both nitrosoureas are

highly mutagenic, but MNU is also apparently highly cytotoxic, whereas ENU is essentially not cytotoxic in the concentration range in which it is mutagenic. A possible explanation might be that MNU induces large numbers of auxotrophic and other conditional lethal mutations that appear as kills when the survival is estimated by using a defined medium in the plates. For this reason, the survivors were measured on both survival medium and a rich supplemented medium (Table 5). Any effect of the supplement was slight and the cytotoxicity is therefore due to either non-repairable damage or the induction of mutations that are unconditionally lethal on both media.

TABLE 6

C O M P A R I S O N OF M U T A G E N I C I T Y WITH CYTO- TOXICITY FOR am-3 TO am + REVERSION

Compound Concentration a Revertants per S/So b

(mM) 104 survivors

M N U 1 2.5 0.75 E N U 1 4 0.98 HN2 100 0 0.007 BDDE 20 1.8 0.55 CP 0.8 1.7 0.71

a Minimum concentration at which mutat ions arise with a frequency of approximately 2 in 104.

b Ratio of survivors at stated concentration(s) to survivors at a concentration of 0 m M (S 0), calculated from Table 3.

52

TABLE 7

INCIDENCE OF MORPHOLOGICAL MUTANTS AMONGST REVERTANTS IN TWO SYSTEMS

Mutagen Mutant Concn. Revertants per Morphological (mM) 104 survivors mutants as per cent

revertants

MNU am-3 uvs-2 0 3.1 0 1 2.9 0 5 6.8 42

10 42.1 53

am - 3 0 0 0 0.2 0.85 33 0.4 1.39 37.5 0.8 1.74 44 1.2 2.07 44 1.6 1.50 62.5

cP

As the cytotoxicity of the compounds varied so greatly, we contrasted the cytotoxicities and concentrations that, with the exception of the non- mutagenic compound HN2, gave comparable yields of revertants/104 survivors (Table 6). HN2 is non-mutagenic, and ENU is exceptional in that mutations arise under conditions in which there is little death. The remaining three compounds are comparable in such a correlation.

Mutants of abnormal morphology were occa- sionally observed. The commonest classes of these were ropy (ro) and colonial (col). MNU-induced revertants of a m - 3 uvs-2 and CP-induced revertants of a m - 3 contained (of those tested) the largest frequencies of morphological mutations. The two conditions both resulted, in general terms, in an increase in morphologicals as a function of dose, but a striking quantitative difference was revealed when the frequency of morphologicals was contrasted with the frequency of a m revertants (Table 7).

Discussion

The use of spheroplasts from a m strains of N e u r o s p o r a crassa as sensitive targets for mutagenicity and cytotoxicity testing in a eukaryotic system has been established in this paper. In the light of the availability of methods for the transformation of spheroplasts (Radford et al., 1981), it will

be possible to examine whether the differences in mechanism of action of HN2 with DNA or intact nucleoprotein, demonstrated so far in the case of phage (Fraser et al., 1982), can be extended to eukaryotic DNA and chromatin.

The effects of the uvs-2 system on the repair of chemically induced D N A lesions are inconsistent from chemical to chemical: although HN2 lesions appear to be partly repairable by the uvs-2 system, the uvs-2 mutants are significantly more resistant than wild-type to MNU. This is contrast with the compound MNNG, for which uvs-2 mutants are more sensitive (De Serres, 1980). The matter requires further study, but the result could be related to the fact that although both compounds are methylating agents, M N U methylates DNA efficiently in oitro (and thus does not require metabolic activation), whereas M N N G does not meth- ylate DNA efficiently in vitro and does rely on metabolic activation (Drake and Abrahamson, 1981). Until this and similar problems have been resolved, it seems that uvs ÷ and uvs-2 strains should be used for cytotoxicity and mutagenesis assays based on spheroplast preparations.

The mutagenicity of CP in Neurospora is con- sistent with the mutagenicity of this compound in Escher i ch ia coli (Brouwer et al., 1981), S a l m o n e l l a

t y p h i m u r i u m and Chinese hamster ovary cells, and also prophage induction (Mattern et al., 1981). It is significant that CP is a mutagen in repair-profi- cient N. crassa strains; in this, the data resemble those in E. coli (in contrast, it is mutagenic only in S. t y p h i m u r i u m strains constitutive for SOS repair; Mattern et al., 1981). By analogy with the Salmonella data one would expect CP to be a frame-shift mutagen. However, the small number of a m - 7 revertants (Table 4) demonstrate for the first time that, although as a point mutagen CP probably generates predominantly transitions, it is capable of generating transversions also.

Acknowledgements

This work was supported by a research grant (to J.H.P.) from the Medical Research Council. We are very grateful to Dr. M.J. Fraser and Mr. A.J. Baron for advice, to Dr. D.G. Herries for computing, and to Johnson Matthey Ltd for a gift of CP and their interest in the project.

References

Auerbach, C., and D. Ramsey (1968) Analysis of a case of mutagen specificity of Neurospora crassa I: Dose-response curves, Mol. Gen. Genet., 103, 72-104.

Brett, M., G.K. Chambers, A.A. Holder, J.R.S. Fincham and J.C. Wootton (1976) Mutational amino acid replacements in Neurospora ¢rassa NADP-specific glutamate dehydrogenase, J. Mol. Biol., 106, 1-21.

Brockman, H.E., C.Y. Hung, F.J. de Serres and T.M. Ong (1981) Mutagenicity of selected chemicals in Neurospora crassa, in: F.J. de Serres and M.D. Shelby (Eds.), Compara- tive Chemical Mutagenesis, Plenum, New York, pp. 109-138.

Brouwer, J., P. van de Putte, A.M.J. Fichtinger-Schepman and J. Reedijk (1981) Base-pair substitution hotspots in GAG and GCG nucleotide sequences in Escherichia coli K-12, induced by cis-diamminedichloroplatinum (II), Proc. Natl. Acad. Sci. (U.S.A.), 78, 7010-7014.

Calza, R.E., and A.L. Schroeder (1982) Postreplication repair in Neurospora crassa, Mol. Gen. Genet., 185, 111-119.

Coddington, A., J.R.S. Fincham and T.K. Sundaram (1966) Multiple active varieties of Neurospora glutamate dehydrogenase formed by hybridization between two inactive proteins in vivo and in vitro, J. Mol. Biol., 17, 503-512.

De Serres, F.J. (1980) Mutation-induction in repair-deficient strains of Neurospora crassa, in: W.M. Generoso, M.D. Shelby and F.J. de Serres (Eds.), DNA Repair and Muta- genesis in Eukaryotes, Basic Life Sciences, Vol. 15, Plenum, New York, pp. 75-84.

Drake, J.W., and S. Abrahamson (1981) Comparative Mutagen- icity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ICR-170, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 883-892.

Fraser, M.J., K. Ainley and J.H. Parish (1982) Inactivation of bacteriophage k and A DNA by nitrogen mustard, Mutation Res., 96, 153-165.

Frei, J.V., D.H. Swenson, W. Warren and P.D. Lawley (1978) Alkylation of deoxyribonucleic acid in vivo in various organs of CS7BL mice by the carcinogens MNUA, ENUA and EMS in relation to induction of thymic lymphoma; Some applications of high-pressure liquid chromatography, Bio- chem. J., 174, 1031-1044.

Geiduschek, E.G. (1961) Reversible DNA, Proc. Natl. Acad. Sci. (U.S.A.), 47, 900-905.

Hamlyn, P.F., R.E. Bradshaw, F.M. Mellon, C.M. Santiago, J.M. Wilson and J.F. Peberdy (1981) Efficient protoplast isolation from fungi using commercial enzymes, Enzyme Microb. Technol., 3, 321-325.

53

Haroun, L., and B.N. Ames (1981) Mutagenicity of selected chemicals in the Salmonella/microsome mutagenicity test, in: F.J. de Serres and M.D. Shelby (Eds.), Comparative Chemical Mutagenesis, Plenum, New York, pp. 27-68.

Hill, J.M., and R.J. Speer (1982) Organoplatinum complexes as anti-tumour agents, Anticancer Res., 2, 173-186.

Holder, A.A., J.C. Wootton, A.J. Baron, G.K. Chambers and J.R.S. Fincham (1975) The amino acid sequence of Neuro- spora NADP-specific GDH; Peptic and chymotryptic peptides and the complete sequence, Biochem. J., 149, 757-773.

Lawley, P.D., and W. Warren (1975) Specific excision of ethyl- ated purines from DNA of Escherichia coli treated with N-methyl-N-nitrosourea, Chem.-Biol. Interact., 11, 55-59.

Marcelis, A.T.M., G.W. Canters and J. Reedijk (1981) Chela- tion of cis-Pt (NH3)2C12 by the deoxynucleotide d(C-C-G- G), J. Roy. Netherl. Chem. Soc., 100, 391-392.

Mattern, I.E., L. Cocchiarella, C.G. van Kralingen and P.H.M. Lohman (1981) Prophage induction and mutagenicity of a series of anti-tumour platinum (II) and platinum (IV) co- ordination complexes, Mutation Res., 95, 79-93.

Radford, A., S. Pope, A. Sazci, M.J. Fraser and J.H. Parish (1981) Liposome-mediated genetic transformation of Neuro- spora crassa, Mol. Gen. Genet., 184, 567-569.

Roberts, J.J., and A.J. Thomson (1979) The mechanism of action of antitumour platinum compounds, Progr. Nucl. Acids Res. Mol. Biol., 22, 71-133.

Ulmer, E., M. Meicke, M. Ross, K. Fink and R. Brimacombe (1978) Chemical cross-linking of protein to RNA within ribosomal subunits from E. coli, Mol. Gen. Genet., 160, 183-193.

Vogel, H.J. (1964) Distribution of lysine pathways in fungi; Evolutionary implications, Am. Naturalist, 98, 435-446.

Westergaard, M., and H.K. Mitchell (1947) Neurospora B, A synthetic medium favouring sexual reproduction, Am. J. Botany, 34, 573-577.

Wiley, W.R. (1974) Isolation of spheroplast and membrane vesicles from filamentous fungi, Methods Enzymol., 31, 609-626.

Wootton, J.C., A.J. Baron and J.R.S. Fincham (1975a) The amino acid sequence of Neurospora NADP-specific GDH; Peptides from digestion with a staphylococcal proteinase, Biochem. J., 149, 749-755.

Wootton, J.C., J.G. Taylor, A.A. Jackson, G.K. Chambers and J.R.S. Fincham (1975b) The amino acid sequence of Neuro- spora NADP-specific GDH; The tryptic peptides, Biochem. J., 149, 739-748.

assay of cytotoxicity and mutagenicity of alkylating agents by using neurospora spheroplasts

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