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Effect of organic solvents on aflatoxin production in cultures of Aspergillus parasiticus
[ 59 1 Trans . Br . mycol. Soc. 84 (4), 591-593 (1985)
]
Printed in Great Britain
EFFECT OF ORGANIC SOLVENTS ON AFLATOXIN PRODUCTION IN CULTURES OF ASPERGILLUS PARASITICUS By C. FANELLI, A. A. FABBRI, S. PIERETTI Dipartimento di Biologia vegetale, Universitii di Roma (L a Sapienza ), Largo Cristina di Svezia 24 00165 Roma, Italy G . PANFILI AND S. PASSI Istituto S. Gallicano, Via S. Gallicano 25a, 00165 Roma, Italy The addition of different organic solvents (benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol and hexane) to cultures of A. parasiticus have shown a high stimulating effect on aflatoxin biosynthesis. The presence of phenobarbital in cultures, greatly increases aflatoxin production induced by organic solvents. No evidence of lipoperoxidation by TBA test on lipid extracts from microsomes of A. parasiticus in the cultures, even in the presence of phenobarbital, was found. We have previously reported that aflatoxin biosynthesis was enhanced in cultures of Aspergillus parasiticus and A .fl auus by substances with epoxide ring (cerulenin, tetrahydrocerulenin, methyl 9, 10 epoxystearate and methyl 9, 10 : 12, 13 diepoxystearate) (Fanelli et al., 1982, 1983a, b), lipoperoxides (F abbri et al., 1983) and carbon tetrachloride (Fanelli et al., 1984). In the case of carbon tetrachloride (CCI 4 ) , we have postulated that aflatoxin production by the fungi could be due to the peroxidation of lipids of endoplasmic reticulum of A. parasiticus by the highly reactive free radicals formed by the interaction of CCl 4 with the NADPH-cytochrome P-450 system of the fungus. This system has been established in the microsomes of A. parasiticus (Bhatnagar et al., 1982). Normally the addition of lipoperoxides to cells or their formation within the cells lead to cell damage and death especially if protective enzymes or antioxidants are lacking. In the case of A. parasiticus and A. fiavus, they do not show an inhibitory effect on fungal growth but highly increase aflatoxin output (F abbri et al., 1983; Passi et al., 1984). In the present work we have investigated the effect of other common organic solvents, i.e. benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol, hexane and methanol. It must be pointed out that several of the solvents studied (alone or in combination) are normally used for extraction and detoxification of aflatoxins in various agricultural commodities (Goldblatt & Dollear, 1977). The metabolism of these solvents has not been extensively studied as in the case of halomethanes, well known hepatotoxins in mammalia (Reynolds & Moslen, 1980). However an involvement of
microsomes and mixed-function oxidase systems was suggested in mammalia for the metabolism of the most part ofthe solvents. In the case of benzene, the first product of its metabolic oxidation by mixed function oxidase system is postulated to be a highly unstable compound, benzene oxide (Jerina & Dayley, 1974). Also ethanol was suggested to be a free radical hepatotoxin: measurement of elevated lipid diene conjugation, the loss of unsaturated fatty acids and the exhalation of ethane (Comporti, Burdino & Raja, 1971) all indicate an ethanol inducer lipid peroxidation. May & McCay (1968) have demonstrated the existence of NADPH stimulated enzymatic processes which trigger lipid peroxidation either in isolated mitochondria or isolated microsomes. Microsomes might be involved also in the formation of toxic metabolites following accidental administration of n-hexane and cyclohexane to men (Perbellini, Brugnone & Pavan, 1980). For the above reasons, in some experiments, we added to cultures supplemented with solvents, phenobarbital, a potent inducer of microsomal enzyme activity, to verify, whether, as in the case of halomethanes, microsomes were really involved in aflatoxin output. MATERIALS AND METHODS
Culture conditions Aspergillus parasiticus Speare (strain NRRL 2999) was used in this study. About 106 conidia from i y-day-old cultures grown on Czapek Dox Agar medium (Difco) were incubated on 100 mlsynthetic medium (SM) (Czapek Dox broth plus 5 mg/l ZnS0 4 ·7H20 and 1 mg/l Na 2Mo0 4 • zH20) sup-
59 2
Organic solvents and aflato xin produ ction
Table 1. Growth and aflatoxin p roduction in 15 days cultu re fi lt rate of A. parasiticus g rown at 30 °C on synt het ic medium (SM) supplement ed with different organic solv ents (0'8 %, v I v ) alone or plu s ph enobarbital (1 mg f ml) (Each result represent s th e mean ± S.E.M. of 5 determinations.)
SM SM+phenobarbital SM+benzene SM +benzene+phenobarbital SM + acetone SM + acetone + phenobarbital SM +dioxan SM+dioxan+phenobarbital SM+cyclohexane SM+ cyclohexane + ph enob arb ital SM + ethyl acetate SM + ethyl acetate + ph enob arbital SM+ethanol SM+ ethanol + phenobarbital SM +hexane SM + hexane + phenobarbital SM + methanol SM + methanol + ph enobarbital
Dry weight of mycelium (rng / eoo ml)
Aflatoxin s (p.g/ 1oo ml
525'6 ±48'7 609'5 ± 50·8 487'9 ±56'8 530'8± 42'2 539'7± 44'4 556'7 ± 50'3 481"7±38' 8 544'4 ±45 '7 571'4±51 '3 598·8± 53.8 572'9±48'1 598·0 ±60'4 647'4 ±68'2 677'2 ± 62'3 477' 2 ±40-6 498'3 ± 48'4 580'3 ± 55'4 620'1 ± 58'9
4'5±3'2 10'9±6'6 167'7 ±19'8 560'8±61 '7 39'7±4'4 82'5 ± 6'9 74'4 ± 8'3 258'8±27'8 196'8±18'8 318'3 ± 30'8 172'9±19'7 439'8±42'8 20'4±3'6 162'5 ± 19'0 47'4±4'6 106'2 ± 10'4 6,8 ± 2'9 52'3 ± 6,8
No significant differences in th e TBA test were found in lipid extracts of microscomes of all mycelia grown with the different organic solvent s either in absence or in pr esence of phenobarbital.
plemented with: 0·8 % (v I v) benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol, hexane and methanol in the presence and absence of 1 mg/rnl phenobarbital (Sigma Ch .), Cultures were incubated at 30 °C for 15 days. Estimation offu ngal growth Fungal growth was est imated as mycelium dry weight as previously described (Fanelli, Fabbri & Passi, 1980). Analysis of aflatoxins The total aflatoxins (B1 + B2 + G 1 + G 2) were extracted from filtered culture medium and individually analysed by high performance liquid chromatography (H P L C) as previously described (F anelli et al ., 1983 a). Lipid peroxidation test
The lipid peroxidation was performed by thiobarbituric test (Buege & Aust, 1978) on microsomes of each mycelium. Microsomes were obtained from an aliquot of the mycelium homogenized in hyposmotic medium (10 mM-EDTA). The absorbtion (A) was measured at 535 nm.
Cellular f ractions separation The cellular fractions were separated by centrifugation: the ' heavy fract ion' (which contains nuclei, plasma membrane and some non -disrupted cells) was separated at 800 g for 15 min ; the mitochondrial fract ion at 7000 g for 20 min and the microsomal fract ions and cytosol at 100000 g for 60 min. Microsomes were then extracted with CHCI 3 CH30H (2 : 1, v I v ) and treated with thiobarbituric acid solution for the lipid peroxidation test. RESUL TS AND DISCUSSION
We previously suggested (Fanelli et al., 1984) that the effect on aflatoxin induction by carbon tetrachloride, chloroform and bromotrichloromethane might be due to th e peroxidation of lipids of endoplasmic ret iculum of A . jiavus and A . parasiticus by highly reactive radicals formed by the interactions of th e halomethanes with the NADPHcytochrome P-450 of the fungus. This hypothesis was supported by our results (Passi et al. , 1984 ) obtained by adding to the cultures, together with CCl 4 a drug, SKF 525A (which at low concentrations inhibits reactions mediated by cytochrome P-450 syst em) or phenobarbital. According to Orrenius & Ernster (1964) in fact, phenobarbital
C. Fanelli and others
593
BUEGE, J. A. & AUST, S. D. (1978). Microsomal Lip id Peroxidation. Methods in Enzymology 51, 301-310. COMPORTI, M ., BURDINO, E. & RAJA, F. (1971). Fany acid composition of mitochondrial and microsomal lipids of rat liver after acute ethanol intoxication. Life Science 10, 855-85 8. FABBRI, A. A., FANELLI, C., PANFlLI, G. , PASSI, S. & FASELLA, P. (1983). Lipoperoxidation and Aflatoxin biosynthesis by A spergillus parasiticus and A . fiavus. J ournal of General Microbiology U9, 3447-3452. FANELLI, c., FABBRI, A. A. & PASSI, S. (1980). Growth requirements and lipid metabolism of A spergillusfiavus. Transactions of the Brit ish My cological Society 75, 371-375 . FANELLI, c., FABBRI, A. A., FINOTTI, E., BELLINCAMPI, D., GUALANDI, G . & PASSI, S. (1982). Induction of the biosynthesis of aflatoxins in Aspergillus parasiticus by cerulenin. Proceedings of Vth International IUPAC Symposium on Mycotoxins and Phycotoxins, Vienna, September 1-3, 1982, 166-169. FANELLI, C., FABBRI, A. A., FINOTTI, E., PANFILI, G. & PASSI, S. (1983 a) . Cerulenin and tetrahydrocerulenin: stimulating factors of aflatoxin biosynthesis. Transactions of the British Mychological Society 81, 201-204 . FANELLI, c., FABBRI, A. A., FINOTTI, E. & PASSI, S. ( 1983 b). Stimulation of aflatoxin biosynthesis by lipophilic epoxides. Journal of General Microbiology U9,1721-1723 · FANELLI, c., FABBRI, A. A., FINOTTI, E., FASELLA, P. & PASSI, S. (1984). Free radical and aflatoxin biosynthesis. Experientia 40, 191-194 · GOLDBLATT, L. A. & DOLLEAR, F . G. (1977). Detoxification of contamined crops. In Mycotoxins in Human and Animal Health (ed. J. V. Rodricks, C. W. Hes seltine & M. A. Mehlan), pp . 139-15°. South Ill.: Park Forest. JERINA, D . H . & DAYLEY, J. R. (1974). Arene oxides: a new aspect of drug metabolism. Science 185, 573-582 . MAY, H . E. & MCCAY, P. B. (1968). Reduced triphosphopyridine nucleot ide oxidase-catalyzed alterations of membrane pho spholipids. Journal of B iological Chemistry 243, 2296-2302. ORRENIUS, S. & ERNSTER, L. (1964). Phenobarbitalinduced synthe sis of the oxidative demethylating enzymes of rat liver microsomes . Biochemical and Biophysical R esearch Communications 16, 60--64. PASSI, S., NAZZARO-PORRO, M., FANELLI, C., FABBRI, A. A. & FASELLA, P. (1984). Role oflipoperoxidation in aflatoxin production. Applied Microbiologyand Biotechnology 19,186-190. PERBELLlNI, L., BRUGNONE, F. & PAVAN, I. (1980). Identification of the metabolites of n-hexane, cycleThis study was supported by fund s from Faculty hexane and their isomers in men's urine. Toxi cology and Applied Pharmacology 53, 22
increases the synthesisofproteins and phospholipids of endoplasmic reticulum with subsequent stimulation of the metabolism of those drugs, the activation of which is mediated by the components of cytochrome P-450 system. In the present work we found that cyclohexane, ethyl acetate, benzene, dioxan, hexane, acetone and ethanol, in decreasing order significantly stimulate aflatoxin biosynthesis when added to cultures of A. parasiticus (T able 1). The explanation of this effect is not easy, because information on the mechanism of action and the fate of these organic solvents is scarce and is mainly concerned with mammals. However the involvement of microsomes of the fungus would appear probable : in fact, the presence of phenobarbital in cultures, further increases aflatoxin production induced by organic solvents (Table 1). On the analogy of halomethanes (F anelli et al., 1984), we believe that the production of aflatoxins in the presence of organic solvents depends on lipoperoxidation of unsaturated lipids of the endoplasmic reticulum of A. parasiticus. The fact that we found no evidence of Iipoperoxidation by TBA test on lipid extracts from microsomes of A . parasiticus in the cultures, even in the presence of phenobarbital, would appear to contradict our suggestion. Nevertheless it must be considered that there is a large difference between mammals and A. parasiticus in the rate of metabolism of organic solvents : very rapid in mammals and very slow , on the contrary in cultures of A . parasiticus. In cultures, in fact, only small amounts of the solvents, depending upon their water solubility, can come into contact with the mycel ium of Aspergillus. Therefore, the possible process of lipoperoxidation would represent the results of this mechanism of inactivation. On the other hand, by -products of lipoperoxidation, such as malondialdehyde and other aldehydes may be inactivated by reacting with -SH and -NH 2 groups of aminoacids, so that the reaction with TBA would not take place. On the other hand according to Plaa & Witschi (1976) in their discussion ofperoxidative mechanism of cell injury' absence of evidence is not necessarily evidence of absence'.
12 5-1 38.
REFERENCE S
BHATNAGAR, R. K. , AHMAD, S., KOHLI, K. K ., MUKERJI, K . G . & VENKITASUBRAMANIAN, T. A. (1982). Induction of polysubstrate monox ygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus. Biochemical and Biophysical Research Communications 104, 1287-1292
REYNOLDS, E. S. & MOSLEN, M . T . (1980). Free radical damage in liver. In Free radicals in Biology, vol. IV (ed. A. A. Pryor), pp. 49-94. New York: Academic Press .
(R eceiv ed for publication 31 August 1984 )
]
Printed in Great Britain
EFFECT OF ORGANIC SOLVENTS ON AFLATOXIN PRODUCTION IN CULTURES OF ASPERGILLUS PARASITICUS By C. FANELLI, A. A. FABBRI, S. PIERETTI Dipartimento di Biologia vegetale, Universitii di Roma (L a Sapienza ), Largo Cristina di Svezia 24 00165 Roma, Italy G . PANFILI AND S. PASSI Istituto S. Gallicano, Via S. Gallicano 25a, 00165 Roma, Italy The addition of different organic solvents (benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol and hexane) to cultures of A. parasiticus have shown a high stimulating effect on aflatoxin biosynthesis. The presence of phenobarbital in cultures, greatly increases aflatoxin production induced by organic solvents. No evidence of lipoperoxidation by TBA test on lipid extracts from microsomes of A. parasiticus in the cultures, even in the presence of phenobarbital, was found. We have previously reported that aflatoxin biosynthesis was enhanced in cultures of Aspergillus parasiticus and A .fl auus by substances with epoxide ring (cerulenin, tetrahydrocerulenin, methyl 9, 10 epoxystearate and methyl 9, 10 : 12, 13 diepoxystearate) (Fanelli et al., 1982, 1983a, b), lipoperoxides (F abbri et al., 1983) and carbon tetrachloride (Fanelli et al., 1984). In the case of carbon tetrachloride (CCI 4 ) , we have postulated that aflatoxin production by the fungi could be due to the peroxidation of lipids of endoplasmic reticulum of A. parasiticus by the highly reactive free radicals formed by the interaction of CCl 4 with the NADPH-cytochrome P-450 system of the fungus. This system has been established in the microsomes of A. parasiticus (Bhatnagar et al., 1982). Normally the addition of lipoperoxides to cells or their formation within the cells lead to cell damage and death especially if protective enzymes or antioxidants are lacking. In the case of A. parasiticus and A. fiavus, they do not show an inhibitory effect on fungal growth but highly increase aflatoxin output (F abbri et al., 1983; Passi et al., 1984). In the present work we have investigated the effect of other common organic solvents, i.e. benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol, hexane and methanol. It must be pointed out that several of the solvents studied (alone or in combination) are normally used for extraction and detoxification of aflatoxins in various agricultural commodities (Goldblatt & Dollear, 1977). The metabolism of these solvents has not been extensively studied as in the case of halomethanes, well known hepatotoxins in mammalia (Reynolds & Moslen, 1980). However an involvement of
microsomes and mixed-function oxidase systems was suggested in mammalia for the metabolism of the most part ofthe solvents. In the case of benzene, the first product of its metabolic oxidation by mixed function oxidase system is postulated to be a highly unstable compound, benzene oxide (Jerina & Dayley, 1974). Also ethanol was suggested to be a free radical hepatotoxin: measurement of elevated lipid diene conjugation, the loss of unsaturated fatty acids and the exhalation of ethane (Comporti, Burdino & Raja, 1971) all indicate an ethanol inducer lipid peroxidation. May & McCay (1968) have demonstrated the existence of NADPH stimulated enzymatic processes which trigger lipid peroxidation either in isolated mitochondria or isolated microsomes. Microsomes might be involved also in the formation of toxic metabolites following accidental administration of n-hexane and cyclohexane to men (Perbellini, Brugnone & Pavan, 1980). For the above reasons, in some experiments, we added to cultures supplemented with solvents, phenobarbital, a potent inducer of microsomal enzyme activity, to verify, whether, as in the case of halomethanes, microsomes were really involved in aflatoxin output. MATERIALS AND METHODS
Culture conditions Aspergillus parasiticus Speare (strain NRRL 2999) was used in this study. About 106 conidia from i y-day-old cultures grown on Czapek Dox Agar medium (Difco) were incubated on 100 mlsynthetic medium (SM) (Czapek Dox broth plus 5 mg/l ZnS0 4 ·7H20 and 1 mg/l Na 2Mo0 4 • zH20) sup-
59 2
Organic solvents and aflato xin produ ction
Table 1. Growth and aflatoxin p roduction in 15 days cultu re fi lt rate of A. parasiticus g rown at 30 °C on synt het ic medium (SM) supplement ed with different organic solv ents (0'8 %, v I v ) alone or plu s ph enobarbital (1 mg f ml) (Each result represent s th e mean ± S.E.M. of 5 determinations.)
SM SM+phenobarbital SM+benzene SM +benzene+phenobarbital SM + acetone SM + acetone + phenobarbital SM +dioxan SM+dioxan+phenobarbital SM+cyclohexane SM+ cyclohexane + ph enob arb ital SM + ethyl acetate SM + ethyl acetate + ph enob arbital SM+ethanol SM+ ethanol + phenobarbital SM +hexane SM + hexane + phenobarbital SM + methanol SM + methanol + ph enobarbital
Dry weight of mycelium (rng / eoo ml)
Aflatoxin s (p.g/ 1oo ml
525'6 ±48'7 609'5 ± 50·8 487'9 ±56'8 530'8± 42'2 539'7± 44'4 556'7 ± 50'3 481"7±38' 8 544'4 ±45 '7 571'4±51 '3 598·8± 53.8 572'9±48'1 598·0 ±60'4 647'4 ±68'2 677'2 ± 62'3 477' 2 ±40-6 498'3 ± 48'4 580'3 ± 55'4 620'1 ± 58'9
4'5±3'2 10'9±6'6 167'7 ±19'8 560'8±61 '7 39'7±4'4 82'5 ± 6'9 74'4 ± 8'3 258'8±27'8 196'8±18'8 318'3 ± 30'8 172'9±19'7 439'8±42'8 20'4±3'6 162'5 ± 19'0 47'4±4'6 106'2 ± 10'4 6,8 ± 2'9 52'3 ± 6,8
No significant differences in th e TBA test were found in lipid extracts of microscomes of all mycelia grown with the different organic solvent s either in absence or in pr esence of phenobarbital.
plemented with: 0·8 % (v I v) benzene, acetone, dioxan, cyclohexane, ethyl acetate, ethanol, hexane and methanol in the presence and absence of 1 mg/rnl phenobarbital (Sigma Ch .), Cultures were incubated at 30 °C for 15 days. Estimation offu ngal growth Fungal growth was est imated as mycelium dry weight as previously described (Fanelli, Fabbri & Passi, 1980). Analysis of aflatoxins The total aflatoxins (B1 + B2 + G 1 + G 2) were extracted from filtered culture medium and individually analysed by high performance liquid chromatography (H P L C) as previously described (F anelli et al ., 1983 a). Lipid peroxidation test
The lipid peroxidation was performed by thiobarbituric test (Buege & Aust, 1978) on microsomes of each mycelium. Microsomes were obtained from an aliquot of the mycelium homogenized in hyposmotic medium (10 mM-EDTA). The absorbtion (A) was measured at 535 nm.
Cellular f ractions separation The cellular fractions were separated by centrifugation: the ' heavy fract ion' (which contains nuclei, plasma membrane and some non -disrupted cells) was separated at 800 g for 15 min ; the mitochondrial fract ion at 7000 g for 20 min and the microsomal fract ions and cytosol at 100000 g for 60 min. Microsomes were then extracted with CHCI 3 CH30H (2 : 1, v I v ) and treated with thiobarbituric acid solution for the lipid peroxidation test. RESUL TS AND DISCUSSION
We previously suggested (Fanelli et al., 1984) that the effect on aflatoxin induction by carbon tetrachloride, chloroform and bromotrichloromethane might be due to th e peroxidation of lipids of endoplasmic ret iculum of A . jiavus and A . parasiticus by highly reactive radicals formed by the interactions of th e halomethanes with the NADPHcytochrome P-450 of the fungus. This hypothesis was supported by our results (Passi et al. , 1984 ) obtained by adding to the cultures, together with CCl 4 a drug, SKF 525A (which at low concentrations inhibits reactions mediated by cytochrome P-450 syst em) or phenobarbital. According to Orrenius & Ernster (1964) in fact, phenobarbital
C. Fanelli and others
593
BUEGE, J. A. & AUST, S. D. (1978). Microsomal Lip id Peroxidation. Methods in Enzymology 51, 301-310. COMPORTI, M ., BURDINO, E. & RAJA, F. (1971). Fany acid composition of mitochondrial and microsomal lipids of rat liver after acute ethanol intoxication. Life Science 10, 855-85 8. FABBRI, A. A., FANELLI, C., PANFlLI, G. , PASSI, S. & FASELLA, P. (1983). Lipoperoxidation and Aflatoxin biosynthesis by A spergillus parasiticus and A . fiavus. J ournal of General Microbiology U9, 3447-3452. FANELLI, c., FABBRI, A. A. & PASSI, S. (1980). Growth requirements and lipid metabolism of A spergillusfiavus. Transactions of the Brit ish My cological Society 75, 371-375 . FANELLI, c., FABBRI, A. A., FINOTTI, E., BELLINCAMPI, D., GUALANDI, G . & PASSI, S. (1982). Induction of the biosynthesis of aflatoxins in Aspergillus parasiticus by cerulenin. Proceedings of Vth International IUPAC Symposium on Mycotoxins and Phycotoxins, Vienna, September 1-3, 1982, 166-169. FANELLI, C., FABBRI, A. A., FINOTTI, E., PANFILI, G. & PASSI, S. (1983 a) . Cerulenin and tetrahydrocerulenin: stimulating factors of aflatoxin biosynthesis. Transactions of the British Mychological Society 81, 201-204 . FANELLI, c., FABBRI, A. A., FINOTTI, E. & PASSI, S. ( 1983 b). Stimulation of aflatoxin biosynthesis by lipophilic epoxides. Journal of General Microbiology U9,1721-1723 · FANELLI, c., FABBRI, A. A., FINOTTI, E., FASELLA, P. & PASSI, S. (1984). Free radical and aflatoxin biosynthesis. Experientia 40, 191-194 · GOLDBLATT, L. A. & DOLLEAR, F . G. (1977). Detoxification of contamined crops. In Mycotoxins in Human and Animal Health (ed. J. V. Rodricks, C. W. Hes seltine & M. A. Mehlan), pp . 139-15°. South Ill.: Park Forest. JERINA, D . H . & DAYLEY, J. R. (1974). Arene oxides: a new aspect of drug metabolism. Science 185, 573-582 . MAY, H . E. & MCCAY, P. B. (1968). Reduced triphosphopyridine nucleot ide oxidase-catalyzed alterations of membrane pho spholipids. Journal of B iological Chemistry 243, 2296-2302. ORRENIUS, S. & ERNSTER, L. (1964). Phenobarbitalinduced synthe sis of the oxidative demethylating enzymes of rat liver microsomes . Biochemical and Biophysical R esearch Communications 16, 60--64. PASSI, S., NAZZARO-PORRO, M., FANELLI, C., FABBRI, A. A. & FASELLA, P. (1984). Role oflipoperoxidation in aflatoxin production. Applied Microbiologyand Biotechnology 19,186-190. PERBELLlNI, L., BRUGNONE, F. & PAVAN, I. (1980). Identification of the metabolites of n-hexane, cycleThis study was supported by fund s from Faculty hexane and their isomers in men's urine. Toxi cology and Applied Pharmacology 53, 22
increases the synthesisofproteins and phospholipids of endoplasmic reticulum with subsequent stimulation of the metabolism of those drugs, the activation of which is mediated by the components of cytochrome P-450 system. In the present work we found that cyclohexane, ethyl acetate, benzene, dioxan, hexane, acetone and ethanol, in decreasing order significantly stimulate aflatoxin biosynthesis when added to cultures of A. parasiticus (T able 1). The explanation of this effect is not easy, because information on the mechanism of action and the fate of these organic solvents is scarce and is mainly concerned with mammals. However the involvement of microsomes of the fungus would appear probable : in fact, the presence of phenobarbital in cultures, further increases aflatoxin production induced by organic solvents (Table 1). On the analogy of halomethanes (F anelli et al., 1984), we believe that the production of aflatoxins in the presence of organic solvents depends on lipoperoxidation of unsaturated lipids of the endoplasmic reticulum of A. parasiticus. The fact that we found no evidence of Iipoperoxidation by TBA test on lipid extracts from microsomes of A . parasiticus in the cultures, even in the presence of phenobarbital, would appear to contradict our suggestion. Nevertheless it must be considered that there is a large difference between mammals and A. parasiticus in the rate of metabolism of organic solvents : very rapid in mammals and very slow , on the contrary in cultures of A . parasiticus. In cultures, in fact, only small amounts of the solvents, depending upon their water solubility, can come into contact with the mycel ium of Aspergillus. Therefore, the possible process of lipoperoxidation would represent the results of this mechanism of inactivation. On the other hand, by -products of lipoperoxidation, such as malondialdehyde and other aldehydes may be inactivated by reacting with -SH and -NH 2 groups of aminoacids, so that the reaction with TBA would not take place. On the other hand according to Plaa & Witschi (1976) in their discussion ofperoxidative mechanism of cell injury' absence of evidence is not necessarily evidence of absence'.
12 5-1 38.
REFERENCE S
BHATNAGAR, R. K. , AHMAD, S., KOHLI, K. K ., MUKERJI, K . G . & VENKITASUBRAMANIAN, T. A. (1982). Induction of polysubstrate monox ygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus. Biochemical and Biophysical Research Communications 104, 1287-1292
REYNOLDS, E. S. & MOSLEN, M . T . (1980). Free radical damage in liver. In Free radicals in Biology, vol. IV (ed. A. A. Pryor), pp. 49-94. New York: Academic Press .
(R eceiv ed for publication 31 August 1984 )