APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1991, 0099-2240t91/010180-03$02.00/0 Copyright C) 1991, American Society for Microbiology
p.
Vol. 57, No. 1
180-182
Mutagenicity of Stemphyltoxin
III,
a
Metabolite
of Alternaria alternata VALERIE M.
DAVIS'*
AND MICHAEL E. STACK2
Division of Toxicological Studies1 and Division of Contaminants Chemistry,2 Food and Drug Administration, Washington, D.C. 20204 Received 7 June 1990/Accepted 29 October 1990
Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites, such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.
altertoxin II in thin-layer chromatographic and liquid chromatographic separations. Isolation by silica gel column chromatography and crystallization from chloroform-hexane yielded 154 mg of a brown microcrystalline solid. The purity of the compound was checked by silica gel thin-layer chromatography with benzene-methanol-acetic acid (90:5:5) as the mobile phase. After the plate was viewed under UV light, it was dried and exposed to iodine fumes. Stemphyltoxin III appeared as a single dark spot at an Rf of 0.46 when 10 p,g was applied to the plate. No other spots appeared, indicating that the stemphyltoxin III was free from other mutagenic compounds, including the altertoxins. The other physical properties of the same lot of stemphyltoxin III were previously described by Stack and Mazzola (10) and are consistent with the structure shown in Fig. 1. This lot of stemphyltoxin III was used for all mutagenicity tests. Mutagenicity tests. S. typhimurium TA98, TA100, TA1537, and TA1535 were used to identify reverse mutations from histidine dependence to histidine independence by the plate incorporation method of Ames et al. (1). Bacteria from an overnight culture (0.1 ml), the test substance in dimethyl sulfoxide (0.05 ml), and S-9 mix (0.5 ml when used) were added to 2 ml of molten top agar. The base agar contained 0.5% glucose instead of the 2% glucose used by Ames et al. (1). A total of 50 ,ul of S-9 derived from the livers of Aroclor 1254-induced male Sprague-Dawley rats was used per plate. Stemphyltoxin III was unstable in dimethyl sulfoxide, and dark brown decomposition products were formed. Nitrogen was bubbled into the solutions during the test procedures to reduce the amount of oxygen present (oxygen may have been responsible for the decomposition), and the assay was completed as rapidly as possible in a further attempt to reduce decomposition. The results for all tests, which were run in triplicate with concurrent positive (known mutagen) and solvent controls (Table 1), were within 10% of each other. Plates were incubated for 2 days at 37°C, and all colonies were counted manually. A revertant count equal to twice the background count was considered positive. Bacterial cultures were routinely tested for viable counts and sensitivity to ampicillin and crystal violet.
Molds of the genus Alternaria are widely distributed plant pathogens and decay organisms of fruits and vegetables (7). Alternaria propagules were found in 184 of 230 test samples of grain from the United States (4). Because the mold grows at low temperatures, Alternaria spoilage may also occur in fruits and vegetables kept under refrigeration (12). Thus, small amounts of some toxic metabolites of Alternaria species may be found in food. In less-developed nations, repeated consumption of food heavily contaminated with mold may have chronic toxic effects (7). There is a high incidence of esophageal cancer in areas such as Linxian County, China, where Alternaria contamination of grains is heavier than in areas with a low incidence (2). Dong et al. (5) reported that an extract of an Alternaria culture induced mutations and transformations in mammalian cells and suggested that contamination of grain with metabolites of Alternaria species may contribute to the high incidence of esophageal cancer in China. Alternaria species produce a wide variety of primary and secondary metabolites, e.g., phytotoxins and mycotoxins (6-9). Among the mycotoxins are several perylenequinone metabolites, designated altertoxins I, II, and III, that have been shown to be mutagenic in the Ames Salmonella typhimurium assay (11). Recently, another perylenequinone metabolite was isolated from A. alternata and identified as stemphyltoxin III (10). Previously, stemphyperylenol and stemphyltoxins I, II, III, and IV were isolated from the mold and plant pathogen Stemphylium botryosum var. lactucum (3). Stemphyltoxin II is identical to altertoxin 11 (3, 11). This paper reports on the mutagenicity of stemphyltoxin III with S. typhimurium TA98, TA100, TA1535, and TA1537. MATERIALS AND METHODS Culture conditions and isolation procedure. A. alternata 42 (isolated from cherries) was cultured on autoclaved rice and water and extracted with chloroform as described by Stack and Prival (11). Stemphyltoxin III occurs as a minor metabolite that is midway in polarity between altertoxin I and * Corresponding author. 180
VOL. 57, 1991
STEMPHYLTOXIN III MUTAGENICITY OH
HO
181
OH
HO
OH
OH
0
OH
ALTERTOXIN I
CL
ALTERTOXIN 11
0)
0-
0
00
OH
HO
10 Stemphyltoxin Ill (ug/plate)
1 0
ALTERTOXIN III
STEMPHYLTOXIN III FIG. 1. Structures of stemphyltoxin III and altertoxins I, II, and
FIG. 2. Mutagenicity of stemphyltoxin III with S-9. Symbols: 0, TA98; A, TA100; O, TA1537; El, TA1535.
III.
RESULTS AND DISCUSSION
The results of the mutagenicity assays of stemphyltoxin III are presented in Fig. 2 and 3, and the concurrent positive control data for these tests are given in Table 1. Stemphyltoxin III was tested at twofold dose intervals from 0.6 to 38.4 ,g per plate. Doses of 0.02 to 0.06 ,ug per plate were also tested and caused no mutagenic response in any of the four strains examined. Stemphyltoxin III was highly mutagenic for S. typhimurium TA98 and TA1537 both with and without metabolic activation (Fig. 2 and 3). The mutagenic response to stemphyltoxin III (38.4 ,ug per plate in the presence of S-9) increased 19-fold in strain TA1537 and 7-fold in strain TA98 over that of the dimethyl sulfoxide control. In tests of stemphyltoxin III in the absence of S-9, the number of revertants increased 16-fold with TA1537 and 6-fold with TA98 over that with the solvent control. The compound was negative for TA1535 and marginally mutagenic for TA100 with and without activation. Table 2 lists the minimum doses at which the number of revertants per plate doubled with stemphyltoxin III and presents, for comparison, the results obtained with altertoxins I, II, and III (11). At stemphyltoxin
III doses greater than 38.4 ,ug per plate, toxicity was observed in all S. typhimurium strains, as shown by a decrease in revertant counts. The effect of rat liver S-9 activation is probably due to the ability of the enzymes contained in S-9 to increase the mutagenicity of the epoxidecontaining compounds. The data clearly show that stemphyltoxin III is mutagenic in both the presence and the absence of metabolic activation. The doubling dose of stemphyltoxin III is, in general, higher than those of altertoxins II and III but is about the same as that of altertoxin I (Table 2). Stemphyltoxin III differs from altertoxin II only by a double bond (Fig. 1). The double bond may decrease the mutagenicity of stemphyltoxin III by providing an additional reactive site for metabolism by the bacteria to a less mutagenic derivative. Stemphyltoxins I, II, and IV are likely to be mutagenic as well, although to our knowledge these mycotoxins have not been tested. Altertoxins I, II, and III and stemphyltoxin III are probably the Alternaria metabolites responsible for the mutations and transformations in mammalian cells reported by Dong et al. (5). Alternariol methyl ether has been reported to be weakly mutagenic (2). Because mutagens are often carcino-
TABLE 1. Number of His' revertants in positive and solvent controls in the S. typhimurium mutagenicity assay (1) No. of revertants/ 0
Strain
TA100 TA100 TA98 TA98 TA1537 TA1537 TA1535 TA1535 a
Chemical
Nitrofurantoin 2-Aminoanthracene
4-Nitro-o-phenylenediamine 2-Fluorenylacetamide 4-Nitro-o-phenylenediamine 2-Aminoanthracene Sodium azide 2-Aminoanthracene
-, Not added; +, added.
Dose (,ug/plate)
0.5 0.5 5.0 10 10 2.5 1.25 2.5
plate of: Solvent Positive control
control
766 793 665
142 156 35
-
714 106
38 9
+ +
79 98 84
12 8 9
-
+ +
CL 0)
4-
c c
D) D)
0.2
1
10
Stemphyltoxin Ill (mg/plate) FIG. 3. Mutagenicity of stemphyltoxin III without S-9. *, TA98; A, TA100; *, TA1537; *, TA1535.
Symbols:
182
DAVIS AND STACK
APPL. ENVIRON. MICROBIOL.
TABLE 2. Minimum doses at which a doubling of revertants per plate occurred with stemphyltoxin III and altertoxins I, II, and III (12) Minimum dose (pug/plate) at which a doubling of revertants of the following S. typhimurium strain occurred:
Compound
Stemphyltoxin III Altertoxin I Altertoxin II Altertoxin III
TA98
TA100
TA1535
TA1537
Without S-9
With S-9
Without S-9
With S-9
Without S-9
With S-9
Without S-9
0.6 0.6 0.18 0.018
2.3 18 6 0.18
2.3 6 0.18 0.18
37.5 18 6 1.8
2.3 0.6 0.06 0.06
2.3 18 0.18 0.6
NTb NT NT
With S-9
a
NT NT NT
a _, Negative (no dose caused doubling). NT, Not tested.
b
genic, these perylenequinone mycotoxins may increase cancer risk if they are present in the food supply. Further study of these perylenequinone mycotoxins is planned. REFERENCES 1. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsome mutagenicity test. Mutat. Res. 31:347-367. 2. An, Y.-H., T.-Z. Zhao, J. Miao, G.-T. Liu, Y.-Z. Zheng, Y.-M. Xu, and R. L. Van Etten. 1989. Isolation, identification, and mutagenicity of alternariol monomethyl ether. J. Agric. Food Chem. 37:1341-1343. 3. Arnone, A., G. Nasini, L. Merlini, and G. Assante. 1986. Secondary mould metabolites. Part 16. Stemphyltoxins, new reduced perylenequinone metabolites from Stemphylium botryosum var. lactucum. J. Chem. Soc. Perkin Trans. I 1986:525530. 4. Bruce, V. R., M. E. Stack, and P. B. Mislivec. 1984. Incidence of toxic Alternaria species in small grains from the USA. J. Food Sci. 49:1626-1627. 5. Dong, Z., G. Liu, Z. Dong, Y. Quan, Y. An, J. Miao, and Y. Zhen. 1987. Induction of mutagenesis and transformation by the
extract of Alternaria alternata isolated from grains in Linxian,
China. Carcinogenesis 8:989-991. 6. Ichihara, A., H. Tazaki, and S. Sakamura. 1983. Solanopyrones A, B, and C, phytotoxic metabolites from the fungus Alternaria solani. Tetrahedron Lett. 24:5373-5376. 7. King, A. D., Jr., and J. E. Schade. 1984. Alternaria toxins and their importance in food. J. Food Prot. 47:886-901. 8. Nishimura, A., and K. Kohmoto. 1983. Host-specific toxins and chemical structures from Alternaria species. Annu. Rev. Phytopathol. 21:87-116. 9. Robeson, D., G. Stobel, G. K. Matsumato, E. H. Fisher, M. H. Chen, and J. Clardy. 1984. Alteichin: an unusual phytotoxin from Alternaria eichorniae, a fungal pathogen of water hyacinth. Experientia 40:1248-1250. 10. Stack, M., and E. Mazzola. 1989. Stemphyltoxin III from Alternaria alternata. J. Nat. Prod. 52:426-427. 11. Stack, M. E., and M. J. Prival. 1986. Mutagenicity of the Alternaria metabolites altertoxins I, II, and III. Appl. Environ. Microbiol. 52:718-722. 12. Tiejin, W., and M. Ceponis. 1982. Nature and extent of losses in fresh repacked tomatoes. Phytopathology 72:266-267.
p.
Vol. 57, No. 1
180-182
Mutagenicity of Stemphyltoxin
III,
a
Metabolite
of Alternaria alternata VALERIE M.
DAVIS'*
AND MICHAEL E. STACK2
Division of Toxicological Studies1 and Division of Contaminants Chemistry,2 Food and Drug Administration, Washington, D.C. 20204 Received 7 June 1990/Accepted 29 October 1990
Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites, such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.
altertoxin II in thin-layer chromatographic and liquid chromatographic separations. Isolation by silica gel column chromatography and crystallization from chloroform-hexane yielded 154 mg of a brown microcrystalline solid. The purity of the compound was checked by silica gel thin-layer chromatography with benzene-methanol-acetic acid (90:5:5) as the mobile phase. After the plate was viewed under UV light, it was dried and exposed to iodine fumes. Stemphyltoxin III appeared as a single dark spot at an Rf of 0.46 when 10 p,g was applied to the plate. No other spots appeared, indicating that the stemphyltoxin III was free from other mutagenic compounds, including the altertoxins. The other physical properties of the same lot of stemphyltoxin III were previously described by Stack and Mazzola (10) and are consistent with the structure shown in Fig. 1. This lot of stemphyltoxin III was used for all mutagenicity tests. Mutagenicity tests. S. typhimurium TA98, TA100, TA1537, and TA1535 were used to identify reverse mutations from histidine dependence to histidine independence by the plate incorporation method of Ames et al. (1). Bacteria from an overnight culture (0.1 ml), the test substance in dimethyl sulfoxide (0.05 ml), and S-9 mix (0.5 ml when used) were added to 2 ml of molten top agar. The base agar contained 0.5% glucose instead of the 2% glucose used by Ames et al. (1). A total of 50 ,ul of S-9 derived from the livers of Aroclor 1254-induced male Sprague-Dawley rats was used per plate. Stemphyltoxin III was unstable in dimethyl sulfoxide, and dark brown decomposition products were formed. Nitrogen was bubbled into the solutions during the test procedures to reduce the amount of oxygen present (oxygen may have been responsible for the decomposition), and the assay was completed as rapidly as possible in a further attempt to reduce decomposition. The results for all tests, which were run in triplicate with concurrent positive (known mutagen) and solvent controls (Table 1), were within 10% of each other. Plates were incubated for 2 days at 37°C, and all colonies were counted manually. A revertant count equal to twice the background count was considered positive. Bacterial cultures were routinely tested for viable counts and sensitivity to ampicillin and crystal violet.
Molds of the genus Alternaria are widely distributed plant pathogens and decay organisms of fruits and vegetables (7). Alternaria propagules were found in 184 of 230 test samples of grain from the United States (4). Because the mold grows at low temperatures, Alternaria spoilage may also occur in fruits and vegetables kept under refrigeration (12). Thus, small amounts of some toxic metabolites of Alternaria species may be found in food. In less-developed nations, repeated consumption of food heavily contaminated with mold may have chronic toxic effects (7). There is a high incidence of esophageal cancer in areas such as Linxian County, China, where Alternaria contamination of grains is heavier than in areas with a low incidence (2). Dong et al. (5) reported that an extract of an Alternaria culture induced mutations and transformations in mammalian cells and suggested that contamination of grain with metabolites of Alternaria species may contribute to the high incidence of esophageal cancer in China. Alternaria species produce a wide variety of primary and secondary metabolites, e.g., phytotoxins and mycotoxins (6-9). Among the mycotoxins are several perylenequinone metabolites, designated altertoxins I, II, and III, that have been shown to be mutagenic in the Ames Salmonella typhimurium assay (11). Recently, another perylenequinone metabolite was isolated from A. alternata and identified as stemphyltoxin III (10). Previously, stemphyperylenol and stemphyltoxins I, II, III, and IV were isolated from the mold and plant pathogen Stemphylium botryosum var. lactucum (3). Stemphyltoxin II is identical to altertoxin 11 (3, 11). This paper reports on the mutagenicity of stemphyltoxin III with S. typhimurium TA98, TA100, TA1535, and TA1537. MATERIALS AND METHODS Culture conditions and isolation procedure. A. alternata 42 (isolated from cherries) was cultured on autoclaved rice and water and extracted with chloroform as described by Stack and Prival (11). Stemphyltoxin III occurs as a minor metabolite that is midway in polarity between altertoxin I and * Corresponding author. 180
VOL. 57, 1991
STEMPHYLTOXIN III MUTAGENICITY OH
HO
181
OH
HO
OH
OH
0
OH
ALTERTOXIN I
CL
ALTERTOXIN 11
0)
0-
0
00
OH
HO
10 Stemphyltoxin Ill (ug/plate)
1 0
ALTERTOXIN III
STEMPHYLTOXIN III FIG. 1. Structures of stemphyltoxin III and altertoxins I, II, and
FIG. 2. Mutagenicity of stemphyltoxin III with S-9. Symbols: 0, TA98; A, TA100; O, TA1537; El, TA1535.
III.
RESULTS AND DISCUSSION
The results of the mutagenicity assays of stemphyltoxin III are presented in Fig. 2 and 3, and the concurrent positive control data for these tests are given in Table 1. Stemphyltoxin III was tested at twofold dose intervals from 0.6 to 38.4 ,g per plate. Doses of 0.02 to 0.06 ,ug per plate were also tested and caused no mutagenic response in any of the four strains examined. Stemphyltoxin III was highly mutagenic for S. typhimurium TA98 and TA1537 both with and without metabolic activation (Fig. 2 and 3). The mutagenic response to stemphyltoxin III (38.4 ,ug per plate in the presence of S-9) increased 19-fold in strain TA1537 and 7-fold in strain TA98 over that of the dimethyl sulfoxide control. In tests of stemphyltoxin III in the absence of S-9, the number of revertants increased 16-fold with TA1537 and 6-fold with TA98 over that with the solvent control. The compound was negative for TA1535 and marginally mutagenic for TA100 with and without activation. Table 2 lists the minimum doses at which the number of revertants per plate doubled with stemphyltoxin III and presents, for comparison, the results obtained with altertoxins I, II, and III (11). At stemphyltoxin
III doses greater than 38.4 ,ug per plate, toxicity was observed in all S. typhimurium strains, as shown by a decrease in revertant counts. The effect of rat liver S-9 activation is probably due to the ability of the enzymes contained in S-9 to increase the mutagenicity of the epoxidecontaining compounds. The data clearly show that stemphyltoxin III is mutagenic in both the presence and the absence of metabolic activation. The doubling dose of stemphyltoxin III is, in general, higher than those of altertoxins II and III but is about the same as that of altertoxin I (Table 2). Stemphyltoxin III differs from altertoxin II only by a double bond (Fig. 1). The double bond may decrease the mutagenicity of stemphyltoxin III by providing an additional reactive site for metabolism by the bacteria to a less mutagenic derivative. Stemphyltoxins I, II, and IV are likely to be mutagenic as well, although to our knowledge these mycotoxins have not been tested. Altertoxins I, II, and III and stemphyltoxin III are probably the Alternaria metabolites responsible for the mutations and transformations in mammalian cells reported by Dong et al. (5). Alternariol methyl ether has been reported to be weakly mutagenic (2). Because mutagens are often carcino-
TABLE 1. Number of His' revertants in positive and solvent controls in the S. typhimurium mutagenicity assay (1) No. of revertants/ 0
Strain
TA100 TA100 TA98 TA98 TA1537 TA1537 TA1535 TA1535 a
Chemical
Nitrofurantoin 2-Aminoanthracene
4-Nitro-o-phenylenediamine 2-Fluorenylacetamide 4-Nitro-o-phenylenediamine 2-Aminoanthracene Sodium azide 2-Aminoanthracene
-, Not added; +, added.
Dose (,ug/plate)
0.5 0.5 5.0 10 10 2.5 1.25 2.5
plate of: Solvent Positive control
control
766 793 665
142 156 35
-
714 106
38 9
+ +
79 98 84
12 8 9
-
+ +
CL 0)
4-
c c
D) D)
0.2
1
10
Stemphyltoxin Ill (mg/plate) FIG. 3. Mutagenicity of stemphyltoxin III without S-9. *, TA98; A, TA100; *, TA1537; *, TA1535.
Symbols:
182
DAVIS AND STACK
APPL. ENVIRON. MICROBIOL.
TABLE 2. Minimum doses at which a doubling of revertants per plate occurred with stemphyltoxin III and altertoxins I, II, and III (12) Minimum dose (pug/plate) at which a doubling of revertants of the following S. typhimurium strain occurred:
Compound
Stemphyltoxin III Altertoxin I Altertoxin II Altertoxin III
TA98
TA100
TA1535
TA1537
Without S-9
With S-9
Without S-9
With S-9
Without S-9
With S-9
Without S-9
0.6 0.6 0.18 0.018
2.3 18 6 0.18
2.3 6 0.18 0.18
37.5 18 6 1.8
2.3 0.6 0.06 0.06
2.3 18 0.18 0.6
NTb NT NT
With S-9
a
NT NT NT
a _, Negative (no dose caused doubling). NT, Not tested.
b
genic, these perylenequinone mycotoxins may increase cancer risk if they are present in the food supply. Further study of these perylenequinone mycotoxins is planned. REFERENCES 1. Ames, B. N., J. McCann, and E. Yamasaki. 1975. Methods for detecting carcinogens and mutagens with the Salmonella mammalian-microsome mutagenicity test. Mutat. Res. 31:347-367. 2. An, Y.-H., T.-Z. Zhao, J. Miao, G.-T. Liu, Y.-Z. Zheng, Y.-M. Xu, and R. L. Van Etten. 1989. Isolation, identification, and mutagenicity of alternariol monomethyl ether. J. Agric. Food Chem. 37:1341-1343. 3. Arnone, A., G. Nasini, L. Merlini, and G. Assante. 1986. Secondary mould metabolites. Part 16. Stemphyltoxins, new reduced perylenequinone metabolites from Stemphylium botryosum var. lactucum. J. Chem. Soc. Perkin Trans. I 1986:525530. 4. Bruce, V. R., M. E. Stack, and P. B. Mislivec. 1984. Incidence of toxic Alternaria species in small grains from the USA. J. Food Sci. 49:1626-1627. 5. Dong, Z., G. Liu, Z. Dong, Y. Quan, Y. An, J. Miao, and Y. Zhen. 1987. Induction of mutagenesis and transformation by the
extract of Alternaria alternata isolated from grains in Linxian,
China. Carcinogenesis 8:989-991. 6. Ichihara, A., H. Tazaki, and S. Sakamura. 1983. Solanopyrones A, B, and C, phytotoxic metabolites from the fungus Alternaria solani. Tetrahedron Lett. 24:5373-5376. 7. King, A. D., Jr., and J. E. Schade. 1984. Alternaria toxins and their importance in food. J. Food Prot. 47:886-901. 8. Nishimura, A., and K. Kohmoto. 1983. Host-specific toxins and chemical structures from Alternaria species. Annu. Rev. Phytopathol. 21:87-116. 9. Robeson, D., G. Stobel, G. K. Matsumato, E. H. Fisher, M. H. Chen, and J. Clardy. 1984. Alteichin: an unusual phytotoxin from Alternaria eichorniae, a fungal pathogen of water hyacinth. Experientia 40:1248-1250. 10. Stack, M., and E. Mazzola. 1989. Stemphyltoxin III from Alternaria alternata. J. Nat. Prod. 52:426-427. 11. Stack, M. E., and M. J. Prival. 1986. Mutagenicity of the Alternaria metabolites altertoxins I, II, and III. Appl. Environ. Microbiol. 52:718-722. 12. Tiejin, W., and M. Ceponis. 1982. Nature and extent of losses in fresh repacked tomatoes. Phytopathology 72:266-267.
