209
Mutation Research, 261 (1991) 209-216 0 1991 Elsevier Science Publishers B.V. All rights reserved ADONIS 016512189100150X
MUTGEN
01651218/91/$03.50
01718
Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102 F.E. Wiirgler ‘, U. Friederich
‘* and J. Schlatter 2
’ Instituteof Toxicology and ’ Swiss Federal Office of Public Health, Division of Food Science, c/o Institute of Toxicology, Swiss Federal Institute of Technology and CJnil,ersityof Ziirich, CH-8603 Schwerzenbach near Ziirich (Switzerland) (Received 25 January 1991) (Revision received 6 June 1991) (Accepted 11 June 1991)
Keywords: Mutagenicity;
Genotoxicity;
Salmonella typhimurium; Ames test; Ochratoxin;
Citrinin;
Patulin;
Cnestine
Summary
The Aspergillus mycotoxins ochratoxin A and B, citrinin and patulin as well as combinations of ochratoxin A and citrinin did not induce reverse mutations in Salmonella typhimurium strain TA102. Therefore there is no indication for the induction of oxidative damage or crosslinks. The same is true for cnestine, a compound extracted from the plant Cnestis glabra.
There is a great need to test not only man-made chemicals, but also natural compounds for genotoxic activity. Many compounds have already been tested, particularly in reliable and rapid bacterial short-term tests. Data with the standard tester strains in the Salmonella/ mammalian microsome assay have been reported for a number of compounds (Kier et al., 1986). Compounds negative in such tests (with and without metabolic activation) cannot be excluded because they might act
Correspondence: Prof. Dr. F.E. Wiirgler, Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schorenstr. 16, CH-8603 Schwerzenbach near Zurich (Switzerland), Tel. (+41)-l-825 75 11; Fax: (+41)-l-825 04 76. * Present land).
address:
DOW
Europe,
CH-8810
Abbreviations: B(a)P, benzo[a]pyrene; foxide; MMC, mitomycin C.
Horgen
DMSO,
(Switzer-
dimethyl
sul-
as oxidative or crosslinking mutagens. Their mutagenic activity, or that of their potential active metabolites, might depend on an active DNA excision-repair system. This situation was encountered with respect to the potential genotoxic activity of the mycotoxins ochratoxin A and B, citrinin and patulin produced by Aspergillus species. Ochratoxin A is a carcinogen in mice (Kanisawa, 1984; Bendele et al., 1985a) and rats (Boorman, 1989) and human exposure is not completely avoidable; it is therefore of practical importance to test this substance, as well as its dechloro derivative ochratoxin B, for genotoxicity. Citrinin is often found together with ochratoxin A. Since the above-mentioned mycotoxins were negative in the older standard Salmonella typhimurium tester strains (Hayes, 1981; Bendele et al., 1985b), they were checked for potential mutagenic activity in the newer tester strain TA102. The results for these 4 mycotoxins as well as cnestine, a compound extracted from the plant
C’nestis glubrti (Connaraceae) 1985a,b), are reported here. Materials
(Jeannoda
et al.,
(Fluka), danthron (1 17-10-2, l.X-dihydroxyanthraquinone) (Merck) and benzo[a]pyrenc (B(a)P) (50-32-8) (Fluka).
and methods
Chemicals Ochratoxin A (CAS No. 303-47-9) and ochratoxin B (4825-86-9) were isolated by E. Creppy (University of Strasbourg, France) and cnestine (1982-67-8; methionine sulfoximine) by V. Jeannoda (Service de Biologie VegCtale et Biochimie, Etablissement d’Enseignement Superieur des Sciences, C.U.R. de Tananarive, Tananarive, Madagascar). Citrinin (518-75-2) and patulin (149-29-I) were purchased from Sigma. The stock solution of citrinin showed a slight precipitate; therefore, the concentration of the citrinin stock solution in ethanol was verified by spectrophotometric measurements (E = 16 lOO/mM/cm) and was found to be 34.26 mg/ml.
Solr ‘ents used Sodium phosphate buffer (0.1 M, pH 7.4) was used for ochratoxin B, patulin, cnestin and MMC; bicarbonate buffer (pH 7.4) for ochratoxin A; ethanol (25 pi/plate) for citrinin and DMSO (SO pi/plate) for danthron.
Salmonella assay Salmonella typhirnurium TA102
(Levin et al., 1984) obtained from Prof. Bruce Ames (Berkeley, CA, U.S.A.) was grown for lo-12 h (‘overnight culture’). The standard plate procedure with 2 plates per concentration, without preincubation, was used (Maron and Ames, 1983). For tests with metabolic activation 10% (v/v) S9 mix with rat liver S9 from Aroclor 1254-induced male Sprague-Dawley rats (200 g) was added.
Positice controls Positive control (50-07-7) (Sigma),
TABLE
compounds were mitomycin C hydrogen peroxide (7722-84-1)
1
POSITIVE TA102
CONTROLS
Dose
FOR
Salmonella
Total number of revertants per plate h
(pg/plate)
without activation 253
Water control Solvent control (DMSO) H202
Danthron
typhimurium
‘I
with activation
’
A47.6
163.3k
15.1
25 50 100 200
303 i 314.6ir 347.3 + 446.6 +
7 5.5 15.9 27.0
5 10 20 40 80 160
167 + 8.8 193 kl9.9 234.6 + 29.9 286 t27.0 296 +38.1 510.6+23.3
360.6 rtr 30.6
724 894.6 1792 2592 2694 2876
k f ir + k f
36.6 56.9 108.2 60 66 X8
a Danthron was dissolved in 50 ~1 DMSO. ’ Mean values k standard deviation of 3 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
Results In a first set of experiments (Table 1) the sensitivity of strain TA102 was tested with hydrogen peroxide (H,O,) as a direct-acting mutagen and danthron as a promutagen to check metabolic activation. Hydrogen peroxide without addition of S9 mix resulted in a dose-dependent increase of revertants. The frequency of revertants was somewhat lower than the 746 revertants/lOO pg H,O, reported by Levin et al. (1982). With danthron some mutagenic activity was seen without S9 mix, but in the presence of 10% S9 mix the activation was much higher. This confirms the results reported by Liberman et al. (1982). The effect without S9 is the same as reported by Westendorf et al. (19881, but in the presence of S9 under our conditions danthron was clearly more active than in the study of Westendorf et al. (1988). That results obtained with TA102 may vary is substantiated by the low response reported by Hughes et al. (1987): with 1000 pg/plate they found only 382 colonies/plate in the standard plate assay and 442 colonies/plate with the preincubation procedure. In the second experiment (Table 2) ochratoxin A and B were tested for mutagenicity in TA102.
211
ducibility of the mutagenic responses of TA102 under the conditions of our laboratory. In a final experiment (Table 5) the combined action of ochratoxin A plus citrinin was tested at different concentrations because synergistic effects have been reported for hepatorenal carcinogenesis (Kanisawa, 1984). Ochratoxin A and citrinin were used at a ratio of 4: 1. The combination of the 2 mycotoxins did not result in a mutagenic response of TA102, although the positive controls were in the usual range. In Table 6 the statistical analysis based on the non-parametric method of Wahrendorf et al. (1985) is presented. All the positive controls are positive, those from Table 2 at p < 0.05, all the others at p < 0.01. The data for ochratoxin A and B, for citrinin, patulin, cnestine and the combina-
In this and the following experiments the directacting mutagen mitomycin C (MMC) was used at low concentrations to have a sensitive indicator of the mutagenic response of strain TA102. B(a)P (with S9 mix) was used as the positive control for promutagens. MMC showed mutagenic activity in TA102 at 6 and 12 pg/plate. In the case of B(a)P activity was found with 5 and 10 pg/plate. Neither ochratoxin A nor ochratoxin B were mutagenic to TA102. Similarly with citrinin and patulin (Table 3) no mutagenic activity could be detected with TA102. Patulin at concentrations above 100 pg/plate was toxic. Cnestine was also non-mutagenic to TA102 and toxic at higher concentrations (above 80 Fg/plate; Table 4). For the last 2 experiments (Tables 3 and 4) the positive controls MMC and danthron showed repro-
TABLE
2
MUTAGENICITY
TESTS
WITH
A AND B IN Sa/monel/u Total number
Dose (~g/plate)
‘I
Bicarbonate
without 196
buffer
MMC
0.006 0.012
BfalP
5.0 10.0
Ochratoxin
Sodium
OCHRATOXIN
37.0 111.1 333.3 991.2
A
phosphate
buffer
MMC
0.006 0.012
BtalP
10.0 20.0
Ochratoxin
B
22.2 66.6 200.0 600.0
of revertants
activation
of-12.7
typhimurium
TA102
per plate h with activation 361.55
’
9.1
532.5 + 13.4 899 * 18.3 491 558 311 f 329.5+ 316.5 i 241.5 k
4.2 0.7 13.4 10.6
221.5k
9.1
505 587
* 4.24 k25.4
332 + 368 + 375 + 351.5+
7.0 4.2 2.8 10.6
302.5 + 19.0
k57.9 * 1.4 454 *38.1 519.5+ 9.1
254 k 2.8 250.5 + 13.4 200.5* 4.9 221.5 & 27.5
285 294.5 277 282
MMC and B(alP served as positive controls without and with metabolic activation, respectively. J MMC was dissolved in sodium phosphate buffer, B(alP in DMSO, ochratoxin A in bicarbonate in phosphate buffer (pH 7.41. h Mean values + standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
+ f j +
0.0 13.4 7.0 14.1
buffer
(pH 7.41 and ochratoxin
B
TABLE
3
MUTAGENICITY Dose (Kg/plate)
TESTS
WITH
CITRININ
IN Sulmonrllu typhimurium TAIO2
AND PATULIN Total number
L’
without
Solvent controls 250 ~1 ethanol/plate Na phosphate buffer DMSO 100 PI/plate
of revertants
activation
0.025 0.050
Danthron
20 40
Citrinin
4 12 36 108.5 325.5
2x1 316 32s 308 27’)
Patulin
12 35 106 317 YhO
319.3 * 5.0 294 I 13.1 7.3+ x.7
’
402 + I I.3 4X5.3 f 19.4 299.3 k 44.2
527.3 * 13.6 h32.h i 28.0 1 352.6 + 107.‘) 22X5.3& 72.1 i_ 9.0 i-22.6 * 1x.3 * 5.6 f 1.4
38X 381 391 335 358
+ x.4 i 32.5 i 9.9 -t Y.Y i 16.9
371.3 k 27.2 34h i 53 17 + 2.6
“ MMC and patulin were dissolved in sodium phosphate buffer, ” Mean values k standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
danthron
in DMSO,
citrinin
in ethanol
4
MUTAGENICITY Dose (pg/plate)
with activation
208 i 2.x 374.6 * 16.7 170.3 * 26.8
MMC
TABLE
per plate ”
TESTS
WITH
CNESTINE
IN Salmonella fyphimurium TA102 Total number
‘I
without
Sodium phosphate buffer DMSO (100 pi/plate)
355 *
MMC
0.025 0.050
552 k 22.2 788 * X.0
Danthron
20.0 40.0
Cnestine
9.3 27.7 X3.3 250.0 750.0
of revertants
per plate h
activation
with activation
7.0
40’) 244
* i
7.0 0.0
94Y.3 +_x3.3 1642.6 &57.X 297k 8.4 25x*11.3 43+ 1.4 40* 1.4 34-t 2.X
“ MMC and cnestine were dissolved in sodium phosphate ” Mean values k standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
buffer,
373.5 rl_19.0 317.5 + 20.5 170.5+ 0.7 25 * 1.4 31.5+ 4.9 danthron
in DMSO.
’
213
well as in the presence of a metabolic activation system. The same was found for cnestine, a compound extracted from the plant Cnestis glabra. A comparison of the results found in different microbial genotoxicity tests is given in Table 7. Ochratoxin A is negative throughout the battery of microbial assays, whereas citrinin and patulin show some activity in bacterial repair assays. These weak effects can only be demonstrated at concentrations where bacteriostatic effects are observed (Krivobok et al., 1987). It is not clear whether citrinin and patulin have the potential to damage bacterial DNA directly or lead only to secondary DNA damage as a consequence of other toxic effects. Significant frequencies of base-pair substitution mutations have been found in an amber mutant reversion test with phage Ml3 of E. coli with citrinin (Brakhage et al., 1988) and patulin (Burger et al., 1988). Citrinin increased the reversion frequency by a low factor of 1.9-3.1 (Brakhage et al., 19881, but patulin by a factor of 19.5 in a combined treatment of 1 kg/ml patulin to the phage and 5 pg/ml patulin to the host bacteria (Burger et al., 1988). In cultured Chinese hamster ovary (CHO) cells, ochratoxin A induced sister-chromatid exchanges
tion of ochratoxin A plus citrinin are all statistically not significantly different from the corresponding concurrent controls, indicating the absence of mutagenic activity. Discussion The Salmonella typhimurium tester strain TA102 is reverted by changes in an A: T base pair in a DNA repair-proficient genetic background. This strain is therefore able to detect mutations resulting from oxidative damage and DNA crosslinks. In order to test mycotoxins previously found to be negative in the excision-repair-defective Salmonella tester strains (Hayes, 1981; Bendele et al., 1985b) samples of ochratoxin A and B, citrinin and patulin were tested in TA102. Positive controls with low doses of MMC showed a fairly constant response to a directacting DNA crosslinker. Good reproducibility was also observed for the promutagens danthron and B(a)P used with 10% rat liver S9 mix. None of the mycotoxins alone (ochratoxin A and B, citrinin and patulin) nor combinations of ochratoxin A and citrinin induced reverse mutations in TA102. This was true in the absence as
TABLE
5
COMBINED
EXPOSURE
OF Salmonellu fyphimurium TAl02
TO OCHRATOXIN
Total number
Dose
(pg/plate) ”
without
Control Control
261.3f 7.0 213.6 k 29.4
(ethanol) (DMSO)
MMC
Danthron
0.025 0.075
activation
Citrinin
12.3 37.0 Ill.1 333.3 1000.0
3.0 9.0 27.8 83.3 250.0
per plate ’ with activation
’
320 + 20.3 189.6+ 8.0
291.3k22.3’ 550.6+ 79.1 ’
20.0 40.0
Ochratoxin A
” ’ ’ ’
of revertants
A AND CITRININ
868 k47.1 1 922.6 j 84.7
265 k 206.5k 219 + 231 + 199.5 k
MMC was dissolved in sodium phosphate buffer, danthron Mean values+standard deviation of 2 replicate plates. For metabolic activation 10% S9 mix (0.5 ml) was used. Mean values + standard deviation of 3 replicate plates.
7.0 0.7 I.4 7.0 16.2
in DMSO,
277 281 284 321 351 ochratoxin
A in bicarbonate
+ 1.4 + 1.4 + 2.8 k24.0 * 9.9 buffer,
citrinin
in ethanol.
(SCEs) in the presence, but not in the absence, of metabolic activation; it did not significantly increase the number of chromosomal aberrations
TABLE
6
NON-PARAMETRIC OF WAHRENDORF
STATISTICAL ET AL. (19851 S9 ‘3
Compound
Table I l-1,0,
-
Danthron
_
Table 2 Bicarbonate MMC BtalP Ochratoxin
ANALYSIS
h 9AV
LC
OF THE
Td
.3 ‘7-3
DATA
PRESENTED
I’N ’
1.oo 0.97 O.Y9
72.0 454.5 444.0
4.202 4.379
0.001 0.000 0.000
1.oo 1.00 0.40 0.63
13.0 13.0 118.0 100.0
2.138 2.138 0.591 0.739
_
1.oo
+ _
1.oo
13.0 13.0 124.0 130.5
2.138 2.138 1.037
27.0 27.0 27.0 165.5 242.5 162.0 159.5
+
IN TABLES
1-5 USING
Critical
value
’
,1 = 0.0
I
p = 0.0s
THE
METHOD
PM(.’
Mutagenicity ”
o.noo 0.000 0.000
++ ++ ++
85 562 562
93 59X 59X
0.016 0.0 16 0.723 0.230
12 12 7’) 79
14 14 X7 X7
0.01 I 0.01 1 0.724 0.253
+ + _ _
I2 I2 79 79
14 I4 X7 X7
0.01 I 0.0I I 0.847 0.931
+ + _
1.537
0.016 0.016 0.850 0.938
2.683 2.683 2.683 I.385 2.23 I 3.223 3.049
0.004 0.004 0.004 0.0X3 0.9X7 0.999 0.999
2’)
2’) 147 147 x5 X5
33 33 33 I59 IS9 93 03
0.00 I 0.001 0.001 0.089 0.9YO I .ooo 0.999
++ ++ ++ _
.&A_
buffer _ + -
A
+ Sodium phosphate MMC B(alP Ochratoxin B
buffer
+ Tahk 3 MMC B(alP Danthron Citrinin
Table 4 MMC Danthron Cnestine
1.00 0.71 0.16 0.00 0.03
7’)
_
_
1.oo
+
I .oo 0.00 0.03
18.0 265.5 261.0
2.496 2.5 I1 3.2X2 3.094
0.006 0.006 O.Y99 0.999
19 147 147
21 21 15’) 159
0.002 0.002 I .noo 0.999
++ ++ _ _
1.oo 1.00
27.0 27.0
2.683 2.6X3
0.004 0.004
29 29
33 33
0.001 0.000
++ ++
0.14 0.48
305.5 24X.5
2.436 0.141
0.993 0.556
188 188
203
+ Table 5 MMC Danthron Ochratoxin A plus citrinin
0.32 0.24
I .oo I .oo
Patulin
_ + _ +
J ” ’ ’ ’ ’ e ”
with and without metabolic activation (Boorman, 1989). Overall the studies indicated that neither
18.0
19
203
Metabolic activation using S9: - without metabolic activation. + with metabolic activation. The q-estimate is a measure of the consistency of the dose response. Test statistic, low values of L indicate a positive trend. Standardized test statistics. The p-value based on the approximate standard normal distribution for T. Critical values for L corresponding to p-values of 0.01 and 0.05. The p-value obtained by the Monte Carlo evaluation. Overall evaluation of mutagenicity: - non-mutagenic: + mutagenic (p < 0.05); + + mutagenic
0.995 0.551
(p
< 0.01).
_
215
TABLE
I
MUTAGENIC ASSAY, THE
TA91
Mycotoxin
Citrinin Ochratoxin Ochratoxin Patulin
ACTIVITY Exhrrichia
A B
OF SOME
IN .Salmonr/lu typhimurium, THE Bacillus .suhtili.v WC Ml3 TEST WITH f3scherichiu cob
ASPERGILLUS MYCOTOXINS AND THE BACTERIOPHAGE
co/i SOS TEST
TA 100
TAYX
TA1535
TA1537
TAlS3X
TAl02
+
___ -+
B. suhtilis
E. c,o/i SOS
E. coli
WC’ assay
_
+
phage
_
_
NT
~ NT
+ NT NT
(+)
t+)
+
+
-
+
-
+
-
+
-
+
~ -
NT ~
NT _
_ ~
_ _
_ ~
~ _
_ ~
~ _
_ _
~ ~
_ _
_ ~
~ _
_ _
+ ~
NT NT
NT NT
NT _
NT ~
NT _
NT _
NT ~
NT _
NT ~
NT _
NT _
NT _
_
_
NT +
Ml3
References for citrinin and patulin see Hayes (19X1), for ochratoxin A see Hayes (19X1). Bendele et al. (19XSa) and Boorman (19X9); for E. co/i SOS test see Auffray and Boutibonnes (19X8): for E. coli phage Ml3 amber mutation reversion test with citrinin see Brakhage et al. (1988) and with patulin see Burger et al. (1988).
ochratoxin A and B, nor citrinin and patulin are bacterial mutagens in Salmonella typhimurium strain TA102 and that they induce neither oxidative damage nor crosslinks in the DNA of this strain with and without metabolic activation. Acknowledgements We thank Drs. G. Dirheimer and E. Creppy (Institut de Biologie Moltculaire et Cellulaire du C.N.R.S., Strasbourg, France) for their interest in the studies and for providing the samples of the mycotoxins and cnestine. We thank Dr. J. Wahrendorf (Heidelberg) for a copy of the source code of the program MAHON4. We thank Bea Weibel for her competent technical assistance. References Auffray, Y., and P. Boutibonnes (1988) Induction of SOS function in Eschen’chia coli by some mycotoxins, Tox. Assess., 3, 371-378. Bendele, A.M., W.W. Carlton, P. Krogh and E.B. Lillehoj (1985a) Ochratoxin A carcinogenesis in the (C57BL/6J x C3H)Fi mouse, J. Natl. Cancer Inst., 75, 733-739. Bendele, A.M., S.B. Neal, T.J. Oberley, C.Z. Thompson, B.J. Bewsey, L.E. Hill, M.A. Rexroat, W.W. Carlton and G.S. Probst (1985b) Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays, Food Chem. Toxicol., 23, 911-918. Boorman, G.A. (1989) Toxicology and carcinogenesis studies of ochratoxin A (CAS No. 303-47-9) in F344/N rats, National Toxicology Program Technical Report 358, NIH Publication No. 89-2813, 142 pp. Brakhage, A.A., M.G. Burger, E.E. Creppy, G. Dirheimer and R.J. Roschenthaler (1988) Base substitution mutations
induced by the mycotoxin citrinin, Arch. Toxicol., Suppl. 12, 34 I-346. Burger, M.G., A.A. Brakhage, E.E. Creppy, G. Dirheimer and R.J. Rijschenthaler (19X8) Toxicity and mutagenicity of patulin in different test systems. Arch. Toxicol., Suppl. 12, 347-351. Hayes, A.W. (1981) Mycotoxin Teratogenicity and Mutagenicity, CRC Press, Boca Raton, FL, 121 p. Hughes, T.J., D.M. Simmons. L.C. Monteith and L.D. Claxton (19X7) Vaporization technique to measure mutagenic activity of volatile organic chemicals in the Ames/ Salmonella Assay, Environ. Mutagen.. 9, 421-441. Jeannoda. V.L.R., J. Valisolalao, E.E. Creppy and G. Dirheimer (1985a) Identification of the toxic principle of Cnestis &hru as methionine sulphoximine, Phytochemistry, 24, 8544855. Jeannoda, V.L.R., D.A.D. Rakoto-Ranopomalal, J. Valisolalao, E.E. Creppy and G. Dirheimer (IYXSb) Natural occurrence of methionine sulfomixime in the Connaraceae family, J. Ethnopharmacol., 14, I l-17. Kanisawa, M. (1984) Synergistic effect of citrinine on hepatorenai carcinogenesis of ochratoxin A in mice, in: H. Kurata and Y. Ueno (Eds.), Toxic Fungi, Their Toxins and Health Hazard, Elsevier/North Holland, Amsterdam, pp. 2455254. Kier. L.D., D.J. Brusick, A. Auletta, E.S. Von Halle, N.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Prival, T.K. Rao and V. Ray (1986) The Sulmo~iellu typhimurium /mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 168, 69-240. Krivobok. S., Ph. Olivier, D.R. Marzin, F. Seigle-Murandi and R. Steiman (1987) Study of the genotoxic potential of I7 mycotoxins with the SOS Chromotest, Mutagenesis, 2, 4333439. Levi”, D.E., M. Hollstein, M.F. Christman, E.A. Schwiers and B.N. Ames (1982) A new Salmonella tester strain (TA102) with A:T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. (U.S.A.), 79, 74457449.
Levin,
DE,
and
L.J. Marnett
and B.N. Ames
mutagen-induced
Salmonella (U.S.A.). Liberman, A.-A.
tester
strain
TA102.
(19X4) Spontaneous
mechanistic
studies
Proc.
Acad.
Natl.
Maron, in
Sci.
81, 4457-4461. D.F., Stark
hydroxylated crosome
deletions:
R.C.
Fink,
(lY82)
F.L.
Appt.
Environ.
Westendorf.
and B.N. Ames (1483) mutagenicity J., B. Poginsky.
Marquardt
(19x8)
The
and
component
of R&u
anthraquinone
and
component
of ethanolic
in the Ames/Salmonella
mi-
4. 225-23’).
Schaefer,
Mutagenicity
anthraquinones
system,
D.M.,
Salmonella
of
R.J. Mulcahy
Microbial..
43, 1354-IXY.
Revised
test. Mutation H. Marquardt, genotoxicity
tiwtorum Ruhia
methods
for the
Res., 113, 173-215. G. Groth
of lucidin.
and H. a natul-al
L., and lucidinethylethet-. extracts.
Cell Biol. Toxicol..
a
Mutation Research, 261 (1991) 209-216 0 1991 Elsevier Science Publishers B.V. All rights reserved ADONIS 016512189100150X
MUTGEN
01651218/91/$03.50
01718
Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102 F.E. Wiirgler ‘, U. Friederich
‘* and J. Schlatter 2
’ Instituteof Toxicology and ’ Swiss Federal Office of Public Health, Division of Food Science, c/o Institute of Toxicology, Swiss Federal Institute of Technology and CJnil,ersityof Ziirich, CH-8603 Schwerzenbach near Ziirich (Switzerland) (Received 25 January 1991) (Revision received 6 June 1991) (Accepted 11 June 1991)
Keywords: Mutagenicity;
Genotoxicity;
Salmonella typhimurium; Ames test; Ochratoxin;
Citrinin;
Patulin;
Cnestine
Summary
The Aspergillus mycotoxins ochratoxin A and B, citrinin and patulin as well as combinations of ochratoxin A and citrinin did not induce reverse mutations in Salmonella typhimurium strain TA102. Therefore there is no indication for the induction of oxidative damage or crosslinks. The same is true for cnestine, a compound extracted from the plant Cnestis glabra.
There is a great need to test not only man-made chemicals, but also natural compounds for genotoxic activity. Many compounds have already been tested, particularly in reliable and rapid bacterial short-term tests. Data with the standard tester strains in the Salmonella/ mammalian microsome assay have been reported for a number of compounds (Kier et al., 1986). Compounds negative in such tests (with and without metabolic activation) cannot be excluded because they might act
Correspondence: Prof. Dr. F.E. Wiirgler, Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schorenstr. 16, CH-8603 Schwerzenbach near Zurich (Switzerland), Tel. (+41)-l-825 75 11; Fax: (+41)-l-825 04 76. * Present land).
address:
DOW
Europe,
CH-8810
Abbreviations: B(a)P, benzo[a]pyrene; foxide; MMC, mitomycin C.
Horgen
DMSO,
(Switzer-
dimethyl
sul-
as oxidative or crosslinking mutagens. Their mutagenic activity, or that of their potential active metabolites, might depend on an active DNA excision-repair system. This situation was encountered with respect to the potential genotoxic activity of the mycotoxins ochratoxin A and B, citrinin and patulin produced by Aspergillus species. Ochratoxin A is a carcinogen in mice (Kanisawa, 1984; Bendele et al., 1985a) and rats (Boorman, 1989) and human exposure is not completely avoidable; it is therefore of practical importance to test this substance, as well as its dechloro derivative ochratoxin B, for genotoxicity. Citrinin is often found together with ochratoxin A. Since the above-mentioned mycotoxins were negative in the older standard Salmonella typhimurium tester strains (Hayes, 1981; Bendele et al., 1985b), they were checked for potential mutagenic activity in the newer tester strain TA102. The results for these 4 mycotoxins as well as cnestine, a compound extracted from the plant
C’nestis glubrti (Connaraceae) 1985a,b), are reported here. Materials
(Jeannoda
et al.,
(Fluka), danthron (1 17-10-2, l.X-dihydroxyanthraquinone) (Merck) and benzo[a]pyrenc (B(a)P) (50-32-8) (Fluka).
and methods
Chemicals Ochratoxin A (CAS No. 303-47-9) and ochratoxin B (4825-86-9) were isolated by E. Creppy (University of Strasbourg, France) and cnestine (1982-67-8; methionine sulfoximine) by V. Jeannoda (Service de Biologie VegCtale et Biochimie, Etablissement d’Enseignement Superieur des Sciences, C.U.R. de Tananarive, Tananarive, Madagascar). Citrinin (518-75-2) and patulin (149-29-I) were purchased from Sigma. The stock solution of citrinin showed a slight precipitate; therefore, the concentration of the citrinin stock solution in ethanol was verified by spectrophotometric measurements (E = 16 lOO/mM/cm) and was found to be 34.26 mg/ml.
Solr ‘ents used Sodium phosphate buffer (0.1 M, pH 7.4) was used for ochratoxin B, patulin, cnestin and MMC; bicarbonate buffer (pH 7.4) for ochratoxin A; ethanol (25 pi/plate) for citrinin and DMSO (SO pi/plate) for danthron.
Salmonella assay Salmonella typhirnurium TA102
(Levin et al., 1984) obtained from Prof. Bruce Ames (Berkeley, CA, U.S.A.) was grown for lo-12 h (‘overnight culture’). The standard plate procedure with 2 plates per concentration, without preincubation, was used (Maron and Ames, 1983). For tests with metabolic activation 10% (v/v) S9 mix with rat liver S9 from Aroclor 1254-induced male Sprague-Dawley rats (200 g) was added.
Positice controls Positive control (50-07-7) (Sigma),
TABLE
compounds were mitomycin C hydrogen peroxide (7722-84-1)
1
POSITIVE TA102
CONTROLS
Dose
FOR
Salmonella
Total number of revertants per plate h
(pg/plate)
without activation 253
Water control Solvent control (DMSO) H202
Danthron
typhimurium
‘I
with activation
’
A47.6
163.3k
15.1
25 50 100 200
303 i 314.6ir 347.3 + 446.6 +
7 5.5 15.9 27.0
5 10 20 40 80 160
167 + 8.8 193 kl9.9 234.6 + 29.9 286 t27.0 296 +38.1 510.6+23.3
360.6 rtr 30.6
724 894.6 1792 2592 2694 2876
k f ir + k f
36.6 56.9 108.2 60 66 X8
a Danthron was dissolved in 50 ~1 DMSO. ’ Mean values k standard deviation of 3 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
Results In a first set of experiments (Table 1) the sensitivity of strain TA102 was tested with hydrogen peroxide (H,O,) as a direct-acting mutagen and danthron as a promutagen to check metabolic activation. Hydrogen peroxide without addition of S9 mix resulted in a dose-dependent increase of revertants. The frequency of revertants was somewhat lower than the 746 revertants/lOO pg H,O, reported by Levin et al. (1982). With danthron some mutagenic activity was seen without S9 mix, but in the presence of 10% S9 mix the activation was much higher. This confirms the results reported by Liberman et al. (1982). The effect without S9 is the same as reported by Westendorf et al. (19881, but in the presence of S9 under our conditions danthron was clearly more active than in the study of Westendorf et al. (1988). That results obtained with TA102 may vary is substantiated by the low response reported by Hughes et al. (1987): with 1000 pg/plate they found only 382 colonies/plate in the standard plate assay and 442 colonies/plate with the preincubation procedure. In the second experiment (Table 2) ochratoxin A and B were tested for mutagenicity in TA102.
211
ducibility of the mutagenic responses of TA102 under the conditions of our laboratory. In a final experiment (Table 5) the combined action of ochratoxin A plus citrinin was tested at different concentrations because synergistic effects have been reported for hepatorenal carcinogenesis (Kanisawa, 1984). Ochratoxin A and citrinin were used at a ratio of 4: 1. The combination of the 2 mycotoxins did not result in a mutagenic response of TA102, although the positive controls were in the usual range. In Table 6 the statistical analysis based on the non-parametric method of Wahrendorf et al. (1985) is presented. All the positive controls are positive, those from Table 2 at p < 0.05, all the others at p < 0.01. The data for ochratoxin A and B, for citrinin, patulin, cnestine and the combina-
In this and the following experiments the directacting mutagen mitomycin C (MMC) was used at low concentrations to have a sensitive indicator of the mutagenic response of strain TA102. B(a)P (with S9 mix) was used as the positive control for promutagens. MMC showed mutagenic activity in TA102 at 6 and 12 pg/plate. In the case of B(a)P activity was found with 5 and 10 pg/plate. Neither ochratoxin A nor ochratoxin B were mutagenic to TA102. Similarly with citrinin and patulin (Table 3) no mutagenic activity could be detected with TA102. Patulin at concentrations above 100 pg/plate was toxic. Cnestine was also non-mutagenic to TA102 and toxic at higher concentrations (above 80 Fg/plate; Table 4). For the last 2 experiments (Tables 3 and 4) the positive controls MMC and danthron showed repro-
TABLE
2
MUTAGENICITY
TESTS
WITH
A AND B IN Sa/monel/u Total number
Dose (~g/plate)
‘I
Bicarbonate
without 196
buffer
MMC
0.006 0.012
BfalP
5.0 10.0
Ochratoxin
Sodium
OCHRATOXIN
37.0 111.1 333.3 991.2
A
phosphate
buffer
MMC
0.006 0.012
BtalP
10.0 20.0
Ochratoxin
B
22.2 66.6 200.0 600.0
of revertants
activation
of-12.7
typhimurium
TA102
per plate h with activation 361.55
’
9.1
532.5 + 13.4 899 * 18.3 491 558 311 f 329.5+ 316.5 i 241.5 k
4.2 0.7 13.4 10.6
221.5k
9.1
505 587
* 4.24 k25.4
332 + 368 + 375 + 351.5+
7.0 4.2 2.8 10.6
302.5 + 19.0
k57.9 * 1.4 454 *38.1 519.5+ 9.1
254 k 2.8 250.5 + 13.4 200.5* 4.9 221.5 & 27.5
285 294.5 277 282
MMC and B(alP served as positive controls without and with metabolic activation, respectively. J MMC was dissolved in sodium phosphate buffer, B(alP in DMSO, ochratoxin A in bicarbonate in phosphate buffer (pH 7.41. h Mean values + standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
+ f j +
0.0 13.4 7.0 14.1
buffer
(pH 7.41 and ochratoxin
B
TABLE
3
MUTAGENICITY Dose (Kg/plate)
TESTS
WITH
CITRININ
IN Sulmonrllu typhimurium TAIO2
AND PATULIN Total number
L’
without
Solvent controls 250 ~1 ethanol/plate Na phosphate buffer DMSO 100 PI/plate
of revertants
activation
0.025 0.050
Danthron
20 40
Citrinin
4 12 36 108.5 325.5
2x1 316 32s 308 27’)
Patulin
12 35 106 317 YhO
319.3 * 5.0 294 I 13.1 7.3+ x.7
’
402 + I I.3 4X5.3 f 19.4 299.3 k 44.2
527.3 * 13.6 h32.h i 28.0 1 352.6 + 107.‘) 22X5.3& 72.1 i_ 9.0 i-22.6 * 1x.3 * 5.6 f 1.4
38X 381 391 335 358
+ x.4 i 32.5 i 9.9 -t Y.Y i 16.9
371.3 k 27.2 34h i 53 17 + 2.6
“ MMC and patulin were dissolved in sodium phosphate buffer, ” Mean values k standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
danthron
in DMSO,
citrinin
in ethanol
4
MUTAGENICITY Dose (pg/plate)
with activation
208 i 2.x 374.6 * 16.7 170.3 * 26.8
MMC
TABLE
per plate ”
TESTS
WITH
CNESTINE
IN Salmonella fyphimurium TA102 Total number
‘I
without
Sodium phosphate buffer DMSO (100 pi/plate)
355 *
MMC
0.025 0.050
552 k 22.2 788 * X.0
Danthron
20.0 40.0
Cnestine
9.3 27.7 X3.3 250.0 750.0
of revertants
per plate h
activation
with activation
7.0
40’) 244
* i
7.0 0.0
94Y.3 +_x3.3 1642.6 &57.X 297k 8.4 25x*11.3 43+ 1.4 40* 1.4 34-t 2.X
“ MMC and cnestine were dissolved in sodium phosphate ” Mean values k standard deviation of 2 replicate plates. ’ For metabolic activation 10% S9 mix (0.5 ml) was used.
buffer,
373.5 rl_19.0 317.5 + 20.5 170.5+ 0.7 25 * 1.4 31.5+ 4.9 danthron
in DMSO.
’
213
well as in the presence of a metabolic activation system. The same was found for cnestine, a compound extracted from the plant Cnestis glabra. A comparison of the results found in different microbial genotoxicity tests is given in Table 7. Ochratoxin A is negative throughout the battery of microbial assays, whereas citrinin and patulin show some activity in bacterial repair assays. These weak effects can only be demonstrated at concentrations where bacteriostatic effects are observed (Krivobok et al., 1987). It is not clear whether citrinin and patulin have the potential to damage bacterial DNA directly or lead only to secondary DNA damage as a consequence of other toxic effects. Significant frequencies of base-pair substitution mutations have been found in an amber mutant reversion test with phage Ml3 of E. coli with citrinin (Brakhage et al., 1988) and patulin (Burger et al., 1988). Citrinin increased the reversion frequency by a low factor of 1.9-3.1 (Brakhage et al., 19881, but patulin by a factor of 19.5 in a combined treatment of 1 kg/ml patulin to the phage and 5 pg/ml patulin to the host bacteria (Burger et al., 1988). In cultured Chinese hamster ovary (CHO) cells, ochratoxin A induced sister-chromatid exchanges
tion of ochratoxin A plus citrinin are all statistically not significantly different from the corresponding concurrent controls, indicating the absence of mutagenic activity. Discussion The Salmonella typhimurium tester strain TA102 is reverted by changes in an A: T base pair in a DNA repair-proficient genetic background. This strain is therefore able to detect mutations resulting from oxidative damage and DNA crosslinks. In order to test mycotoxins previously found to be negative in the excision-repair-defective Salmonella tester strains (Hayes, 1981; Bendele et al., 1985b) samples of ochratoxin A and B, citrinin and patulin were tested in TA102. Positive controls with low doses of MMC showed a fairly constant response to a directacting DNA crosslinker. Good reproducibility was also observed for the promutagens danthron and B(a)P used with 10% rat liver S9 mix. None of the mycotoxins alone (ochratoxin A and B, citrinin and patulin) nor combinations of ochratoxin A and citrinin induced reverse mutations in TA102. This was true in the absence as
TABLE
5
COMBINED
EXPOSURE
OF Salmonellu fyphimurium TAl02
TO OCHRATOXIN
Total number
Dose
(pg/plate) ”
without
Control Control
261.3f 7.0 213.6 k 29.4
(ethanol) (DMSO)
MMC
Danthron
0.025 0.075
activation
Citrinin
12.3 37.0 Ill.1 333.3 1000.0
3.0 9.0 27.8 83.3 250.0
per plate ’ with activation
’
320 + 20.3 189.6+ 8.0
291.3k22.3’ 550.6+ 79.1 ’
20.0 40.0
Ochratoxin A
” ’ ’ ’
of revertants
A AND CITRININ
868 k47.1 1 922.6 j 84.7
265 k 206.5k 219 + 231 + 199.5 k
MMC was dissolved in sodium phosphate buffer, danthron Mean values+standard deviation of 2 replicate plates. For metabolic activation 10% S9 mix (0.5 ml) was used. Mean values + standard deviation of 3 replicate plates.
7.0 0.7 I.4 7.0 16.2
in DMSO,
277 281 284 321 351 ochratoxin
A in bicarbonate
+ 1.4 + 1.4 + 2.8 k24.0 * 9.9 buffer,
citrinin
in ethanol.
(SCEs) in the presence, but not in the absence, of metabolic activation; it did not significantly increase the number of chromosomal aberrations
TABLE
6
NON-PARAMETRIC OF WAHRENDORF
STATISTICAL ET AL. (19851 S9 ‘3
Compound
Table I l-1,0,
-
Danthron
_
Table 2 Bicarbonate MMC BtalP Ochratoxin
ANALYSIS
h 9AV
LC
OF THE
Td
.3 ‘7-3
DATA
PRESENTED
I’N ’
1.oo 0.97 O.Y9
72.0 454.5 444.0
4.202 4.379
0.001 0.000 0.000
1.oo 1.00 0.40 0.63
13.0 13.0 118.0 100.0
2.138 2.138 0.591 0.739
_
1.oo
+ _
1.oo
13.0 13.0 124.0 130.5
2.138 2.138 1.037
27.0 27.0 27.0 165.5 242.5 162.0 159.5
+
IN TABLES
1-5 USING
Critical
value
’
,1 = 0.0
I
p = 0.0s
THE
METHOD
PM(.’
Mutagenicity ”
o.noo 0.000 0.000
++ ++ ++
85 562 562
93 59X 59X
0.016 0.0 16 0.723 0.230
12 12 7’) 79
14 14 X7 X7
0.01 I 0.01 1 0.724 0.253
+ + _ _
I2 I2 79 79
14 I4 X7 X7
0.01 I 0.0I I 0.847 0.931
+ + _
1.537
0.016 0.016 0.850 0.938
2.683 2.683 2.683 I.385 2.23 I 3.223 3.049
0.004 0.004 0.004 0.0X3 0.9X7 0.999 0.999
2’)
2’) 147 147 x5 X5
33 33 33 I59 IS9 93 03
0.00 I 0.001 0.001 0.089 0.9YO I .ooo 0.999
++ ++ ++ _
.&A_
buffer _ + -
A
+ Sodium phosphate MMC B(alP Ochratoxin B
buffer
+ Tahk 3 MMC B(alP Danthron Citrinin
Table 4 MMC Danthron Cnestine
1.00 0.71 0.16 0.00 0.03
7’)
_
_
1.oo
+
I .oo 0.00 0.03
18.0 265.5 261.0
2.496 2.5 I1 3.2X2 3.094
0.006 0.006 O.Y99 0.999
19 147 147
21 21 15’) 159
0.002 0.002 I .noo 0.999
++ ++ _ _
1.oo 1.00
27.0 27.0
2.683 2.6X3
0.004 0.004
29 29
33 33
0.001 0.000
++ ++
0.14 0.48
305.5 24X.5
2.436 0.141
0.993 0.556
188 188
203
+ Table 5 MMC Danthron Ochratoxin A plus citrinin
0.32 0.24
I .oo I .oo
Patulin
_ + _ +
J ” ’ ’ ’ ’ e ”
with and without metabolic activation (Boorman, 1989). Overall the studies indicated that neither
18.0
19
203
Metabolic activation using S9: - without metabolic activation. + with metabolic activation. The q-estimate is a measure of the consistency of the dose response. Test statistic, low values of L indicate a positive trend. Standardized test statistics. The p-value based on the approximate standard normal distribution for T. Critical values for L corresponding to p-values of 0.01 and 0.05. The p-value obtained by the Monte Carlo evaluation. Overall evaluation of mutagenicity: - non-mutagenic: + mutagenic (p < 0.05); + + mutagenic
0.995 0.551
(p
< 0.01).
_
215
TABLE
I
MUTAGENIC ASSAY, THE
TA91
Mycotoxin
Citrinin Ochratoxin Ochratoxin Patulin
ACTIVITY Exhrrichia
A B
OF SOME
IN .Salmonr/lu typhimurium, THE Bacillus .suhtili.v WC Ml3 TEST WITH f3scherichiu cob
ASPERGILLUS MYCOTOXINS AND THE BACTERIOPHAGE
co/i SOS TEST
TA 100
TAYX
TA1535
TA1537
TAlS3X
TAl02
+
___ -+
B. suhtilis
E. c,o/i SOS
E. coli
WC’ assay
_
+
phage
_
_
NT
~ NT
+ NT NT
(+)
t+)
+
+
-
+
-
+
-
+
-
+
~ -
NT ~
NT _
_ ~
_ _
_ ~
~ _
_ ~
~ _
_ _
~ ~
_ _
_ ~
~ _
_ _
+ ~
NT NT
NT NT
NT _
NT ~
NT _
NT _
NT ~
NT _
NT ~
NT _
NT _
NT _
_
_
NT +
Ml3
References for citrinin and patulin see Hayes (19X1), for ochratoxin A see Hayes (19X1). Bendele et al. (19XSa) and Boorman (19X9); for E. co/i SOS test see Auffray and Boutibonnes (19X8): for E. coli phage Ml3 amber mutation reversion test with citrinin see Brakhage et al. (1988) and with patulin see Burger et al. (1988).
ochratoxin A and B, nor citrinin and patulin are bacterial mutagens in Salmonella typhimurium strain TA102 and that they induce neither oxidative damage nor crosslinks in the DNA of this strain with and without metabolic activation. Acknowledgements We thank Drs. G. Dirheimer and E. Creppy (Institut de Biologie Moltculaire et Cellulaire du C.N.R.S., Strasbourg, France) for their interest in the studies and for providing the samples of the mycotoxins and cnestine. We thank Dr. J. Wahrendorf (Heidelberg) for a copy of the source code of the program MAHON4. We thank Bea Weibel for her competent technical assistance. References Auffray, Y., and P. Boutibonnes (1988) Induction of SOS function in Eschen’chia coli by some mycotoxins, Tox. Assess., 3, 371-378. Bendele, A.M., W.W. Carlton, P. Krogh and E.B. Lillehoj (1985a) Ochratoxin A carcinogenesis in the (C57BL/6J x C3H)Fi mouse, J. Natl. Cancer Inst., 75, 733-739. Bendele, A.M., S.B. Neal, T.J. Oberley, C.Z. Thompson, B.J. Bewsey, L.E. Hill, M.A. Rexroat, W.W. Carlton and G.S. Probst (1985b) Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays, Food Chem. Toxicol., 23, 911-918. Boorman, G.A. (1989) Toxicology and carcinogenesis studies of ochratoxin A (CAS No. 303-47-9) in F344/N rats, National Toxicology Program Technical Report 358, NIH Publication No. 89-2813, 142 pp. Brakhage, A.A., M.G. Burger, E.E. Creppy, G. Dirheimer and R.J. Roschenthaler (1988) Base substitution mutations
induced by the mycotoxin citrinin, Arch. Toxicol., Suppl. 12, 34 I-346. Burger, M.G., A.A. Brakhage, E.E. Creppy, G. Dirheimer and R.J. Rijschenthaler (19X8) Toxicity and mutagenicity of patulin in different test systems. Arch. Toxicol., Suppl. 12, 347-351. Hayes, A.W. (1981) Mycotoxin Teratogenicity and Mutagenicity, CRC Press, Boca Raton, FL, 121 p. Hughes, T.J., D.M. Simmons. L.C. Monteith and L.D. Claxton (19X7) Vaporization technique to measure mutagenic activity of volatile organic chemicals in the Ames/ Salmonella Assay, Environ. Mutagen.. 9, 421-441. Jeannoda. V.L.R., J. Valisolalao, E.E. Creppy and G. Dirheimer (1985a) Identification of the toxic principle of Cnestis &hru as methionine sulphoximine, Phytochemistry, 24, 8544855. Jeannoda, V.L.R., D.A.D. Rakoto-Ranopomalal, J. Valisolalao, E.E. Creppy and G. Dirheimer (IYXSb) Natural occurrence of methionine sulfomixime in the Connaraceae family, J. Ethnopharmacol., 14, I l-17. Kanisawa, M. (1984) Synergistic effect of citrinine on hepatorenai carcinogenesis of ochratoxin A in mice, in: H. Kurata and Y. Ueno (Eds.), Toxic Fungi, Their Toxins and Health Hazard, Elsevier/North Holland, Amsterdam, pp. 2455254. Kier. L.D., D.J. Brusick, A. Auletta, E.S. Von Halle, N.M. Brown, V.F. Simmon, V. Dunkel, J. McCann, K. Mortelmans, M. Prival, T.K. Rao and V. Ray (1986) The Sulmo~iellu typhimurium /mammalian microsomal assay. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 168, 69-240. Krivobok. S., Ph. Olivier, D.R. Marzin, F. Seigle-Murandi and R. Steiman (1987) Study of the genotoxic potential of I7 mycotoxins with the SOS Chromotest, Mutagenesis, 2, 4333439. Levi”, D.E., M. Hollstein, M.F. Christman, E.A. Schwiers and B.N. Ames (1982) A new Salmonella tester strain (TA102) with A:T base pairs at the site of mutation detects oxidative mutagens, Proc. Natl. Acad. Sci. (U.S.A.), 79, 74457449.
Levin,
DE,
and
L.J. Marnett
and B.N. Ames
mutagen-induced
Salmonella (U.S.A.). Liberman, A.-A.
tester
strain
TA102.
(19X4) Spontaneous
mechanistic
studies
Proc.
Acad.
Natl.
Maron, in
Sci.
81, 4457-4461. D.F., Stark
hydroxylated crosome
deletions:
R.C.
Fink,
(lY82)
F.L.
Appt.
Environ.
Westendorf.
and B.N. Ames (1483) mutagenicity J., B. Poginsky.
Marquardt
(19x8)
The
and
component
of R&u
anthraquinone
and
component
of ethanolic
in the Ames/Salmonella
mi-
4. 225-23’).
Schaefer,
Mutagenicity
anthraquinones
system,
D.M.,
Salmonella
of
R.J. Mulcahy
Microbial..
43, 1354-IXY.
Revised
test. Mutation H. Marquardt, genotoxicity
tiwtorum Ruhia
methods
for the
Res., 113, 173-215. G. Groth
of lucidin.
and H. a natul-al
L., and lucidinethylethet-. extracts.
Cell Biol. Toxicol..
a
