Acta pharmacol. et toxicol. 1978,43,28S-290.
From the Department of Animal Nutrition, The Swedish University of Agricultural Sciences, Box 7046, S-750 07 Uppsala, Sweden
Metabolism of Zearalenone in Rat Liver BY K.-H. Kiessiing and H. Pettersson (Received March 3, 1978; Accepted May 23, 1978)
Abstract :The metabolism of zearalenone in rat liver has been investigated. The studies were performed mainly with liver homogenate, though isolated microsomes and hepatocytes have also been used. Zearalenone was metabolized along two principal pathways, conjugation with glucuronic acid, which was the main route and reduction to an isomer of zearalenol. In no case, however, could all zearalenone metabolized be accounted for as conjugated zearalenone and free and conjugated zearalenol. Therefore another, so far unknown metabolite cannot be excluded. Reduction to zearalenol could be increased three times by the addition of NADH (or NADPH) and is probably catalyzed by a hydroxysteroid dehydrogenase. Some 25-50 per cent of the zearalenol could be conjugated, depending on the incubation conditions. The capacity of hepatocytes to eliminate zearalenone was estimated to be about 100 pg per gram of liver in one hour. With a liver homogenate the highest value obtained was 82 pg. Key-words: Mycotoxins - zearalenone - zearalenol - hydroxysteroid dehydrogenase - rat.
Many species of fungi common in various feeds are capable of producing potent toxins, but only a few mycotoxins have been identified chemically. As early as 1928 McNutt et al. (1928) investigated a syndrome called vulvovaginitis involving inflammation of the vulva and the posterior part of the vagina. They associated the disease with the consumption of mouldy maize. Stob et al. (1962) isolated the active component, called F-2, from cultures of Gibberella zeae (Fusarium roseum) and Urry et al. (1966) named it zearalenone. More than a hundred derivatives of zearalenone have been synthesized chemically (for a survey see Shipchandler 1975 and Pathre & Mirocha 1976). A few of these compounds have oestrogenic properties more potent than that of zearalenone itself. As Fusarium is an ubiquitous fungus it easily contaminates crops in the fields or in stores and can therefore constitute a constant health risk to domestic animals. Very little is known about the
metabolism of zearalenone in animals. For this reason the present investigation was carried out on zearalenone metabolism in rat liver and will be followed by a corresponding study on ruminants, swine and poultry. Materials and Methods The animals used were adult male Sprague Dawley rats. Liver homogenate was prepared by removing approximately 6 g liver immediately after the rat was killed, mincing it in ice-cold sucrose (0.25 M); Tris buffer ( 5 mM, pH 7.4); EDTA (1 mM), with scissors and then homogenazing it in a teflon-glass Potter-Elvehjem homogenizer. The homogenate was adjusted to 10% (w/v). When microsomes were prepared, the whole liver, after being weighed, was minced in the same way as described above and microsomes isolated by fractional centrifugation. Isolated hepatocytes were prepared according to Seglen (1976). Liver cell concentration was estimated by centrifugation of a small sample of the cell suspension in a haematocrit centrifuge, and viability by incubation
286
K.-H. KIESSLING AND H. PETTERSSON
of another sample with eosin and then counting the cells in a counting chamber. Incubations were made with additions as stated in the text at 37" in a total volume of 6 ml of liver homogenate and 5 ml of hepatocytes, corresponding to 0.3 g of liver, and with microsomes isolated from one g of liver. The incubations were stopped by addition of 3 ml 0.3 M H,PO, and extracted with 2 x 25 ml chloroform for further analysis of zearalenone and its metabolites (fig. 1) by means of thin-layer chromatography (TLC) or high pressure liquid chromatography (HPLC). The TLC was performed on pre-coated plates with silica gel (Merck) in a benzene-acetic acid (9: 1) system and analysed on an Aminco Bowman Spectrophotofluorometer with a thin-film scanner attachment. A maximum response was obtained with an excitation wavelength of 340 nm and an emission wavelength of 475 nm for both zearalenone and zearalenol. The natural (found in liver homogenate samples) isomer of zearalenol fluoresces twice as intensely as zearalenone. The other zearalenol isomer fluoresces less intensely than zearalenone, while zearalanol has hardly any fluorescence. HPLC was performed using a chromatography apparatus consisting of Waters Model 6000 solvent delivery system, Waters Model U6K septum-less injector and Altex ultraviolet detector (280 nm). By means of a separation system using a mobile phase of isooctanechloroform-methanol (35:25:1.5), a flow rate of 1.5 ml/min. and a Partisil 10 column it was possible to separate zearalenone and the two isomers of zearalenol. The isomers are denoted A and B according to the elution order in this system. A is the natural isomer and is believed to be the isomer with the high melting point. The normal analysis system, however, with methanol-
/ Zearalanol
water (65 :35) as the mobile phase, flow rate 1.O ml/min. on p-Bondapak C,, column (reversed phase) was not suitable for separating zearalenone and the natural metabolite zearalenol. Zearalenone and zearalanol were gifts from Commercial Solvents Corporation, USA and a mixture of the two zearalenol isomers was obtained by reduction of zearalenone with NaBH, as suggested by Hodge (see Shipchandler 1975). Uridine diphosphate glucuronic acid (UDPGA), NAD, NADH and NADPH, ATP, hydroxysteroid dehydropnase, P-glucuronidase (containing sulphatase) and collagenase were purchased from Sigma Chemical Company, and bovine albumin (fraction V) from Armour Pharmaceutical Company.
Results Conjugation of zearalenone. By means of TLC it was shown that about 30 per cent of the zearalenone added to a liver
homogenate was converted into other compounds under the conditions set out in table 1. Addition of UDPGA (uridine diphosphate glucuronic acid) doubled this conversion. With 0-glucuronidase and sulphatase present during the incubation, the bulk of this conversion (60%) could be prevented and is therefore regarded as conjugated zearalenone in the sample without added P-glucuronidasesulphatase (table 1 : Zearalenone conjugated).
Zearalenone
\
NaBHq
Zearalenol
Fig.']. Structure of zearalenone and the two derivatives zearalanol and zearalenol which may occur as a cis and a trans-isomer in the 6-position.
287
ZEARALENONE METABOLISM Table I .
Metabolism of zearalenone by rat liver homogenate. Incubations were carried out at 37" for 30 min. and contained 3 ml homogenate (0.3 g liver), 13.3 pg zearalenone, additions as stated in the table (0.04 mM UDPGA, 0.7 mM NADH, 1 mM Na,SO,, 0.9 mM ATP, 2000 u P-glucuronidase) and phosphate buffer (0.15 M, pH 7.4) in a final volume of 6 ml. The figures for UDPGA, NADH and no addition are mean values of five to six experiments, the remaining figures are from three experiments. Zearalenol formed Zearalenone conjugated** (pg/30 min.)
free
Additions
Zearalenone metabolized* (~g/3Oh n . )
UDPGA SO:- +ATP NADH NADPH UDPGA+NADH
3.9 (1.7-5.2) 9.0 (7.4-10.6) 3.8 9.0 (7.3-10.2) 7.9 9.7 (9.2-12.3)
2.1 (0.8-3.1) 5.5 (3.8-6.4)
0.8 (0.0-1.0) 0.6 (0.3-1.0)
0.2 (0.0-0.4) 0.3 (0.14.5)
2.8 (2.1-3.8)
0.5 2.3 (1.7-3.2)
0.7 (0.5-1.0)
-
*
conjugated** (pg/30 min.)
2.0 4.9 (4.0-6.3)
1.5 (0.9-1.7)
0.5 0.8 (0.5-1.0)
All zearalenone which could not be recovered after the incubation was completed has been regarded as me-
tabolized.
** The figures for conjugated zearalenone and zearalenol are obtained as differences in free zearalenone and zearalenol between incubations in the presence and absence of P-glucuronidase.
Formation of zearalenol. During the incubation of zearalenone with a liver homogenate a compound with a lower Rfvalue than zearalenone could be detected by TLC. The content corresponded to 7 per cent of the added zearalenone and to 25 per cent of the metabolized zearalenone. Addition of NADH (or NADPH) increased the compound about three times. When UDPGA was also present the yield diminished slightly (table l), indicating a minor competition for zearalenone between conjugation and formation of the unknown compound. The TLC revealed the Rf-value of the compound to be nearly the same as that of zearalanol (fig. 1) but, in contrast to zearalanol, it exhibited an intense fluorescence, about twice as marked as zearalenone, when irradiated by UV light. By means of HPLC the compound could be separated from zearalanol as well as from zearalenone but behaved in exactly the same way as isomer A of a synthetically prepared zearalenol. The biotransformation of zearalenone to zearalenol implies a reduction of the keto group in 6'-position (fig. 1). This reaction shows similarities to processes involved in the steroid metabolism catalysed by a steroid dehydrogenase. We there-
fore incubated zearalenone at pH 7.5 with a commercial hydroxysteroid dehydrogenase from Pseudomonas testosteroni in the presence of NADH. Analyses by HPLC showed that the enzyme catalysed the reduction of zearalenone exclusively to the isomer A of zearalenol (fig. 2a), e.g. theisomer which is identicalwith the zearalenol formed from zearalenone in a liver homogenate. Conversely, when the synthetic mixture of the two zearalenol isomers was incubated with the hydroxysteroid dehydrogenase and NAD at pH 9, zearalenone was formed primarily from the isomer A (fig. 2b).
Conjugation of zearalenol. When zearalenone was added to a liver homogenate together with NADH, 25 per cent of the zearalenone which had disappeared could be found again as free zearalenol. When p-glururonidase was also present another 8 per cent was recovered as zearalenol (table 1 : zearalenol conjugated) indicating that some of the zearalenol as well as zearalenone can be conjugated. Zearalenone metabolism with microsomes and isolated hepatocytes. The results obtained with microsomes isolated
288
K.-H. KIESSLING AND H. PETTERSSON
from rat liver were very similar to those with liver homogenate. Eighty per cent of the zearalenone was conjugated within one hour by microsomes
AA.0.1
A
A A.0.1
-
0
5
10
15
20
25
I
:
:
:
;
0
5
10
15
20
: 25 min.
Fig. 2. Incubation of zearalenone and zearalenol with hydroxysteroid dehydrogenase. A. Incubation of zearalenone with hydroxysteroid dehydrogenase and NADH. The incubation contained 0.3 mg zearalenone, 0.3 mg NADH and 50 pg enzyme in 2.5 ml 50 mM phosphate buffer and was performed at 25", pH 7.5. The reaction was registered spectrophotometrically at 340 nm as a decrease in NADH. Samples were taken at A A=0.1 and 0.2 and were analysed for zearalenone and zearalenol with HPLC. The designations of the peaks are: Zn=zearalenone, Z1 A and 21 B=the two isomers A and B of zearalenol. B. Incubation of zearalenol with hydroxysteroid dehydrogenase and NAD. The two isomers of zearalenol, Z1 A and Z1 B, were prepared from zearalenone by reduction with NaBH,. The incubation contained approx. 0.3 mg zearalenol, 6.6 mg NAD and 50 pg enzyme in 2.5 ml 50 mM pyrophosphate buffer and was performed at 25", pH 9.0. The reaction was registered at 340 nm as an increase in NADH. Samples were taken at A A=O (zero time), and on three occasions during the incubation and analysed as described above.
isolated from one gram of liver while 10 per cent was reduced to zearalenol in the presence of UDPGA and NADH. The only difference was that no conjugation of zearalenol could be demonstrated. The activity of the microsomes against zearalenone was lower than in the liver homogenate calculated per gram of liver. The explanation for this is probably that an appreciable loss of microsomes occurs during the isolation. This interpretation is supported by results given in table 2 concerning the NADPH-cytochrome c reductase activity in the microsomal and homogenate preparations. Isolated hepatocytes eliminated zearalenone very rapidly. UDPGA or NADH could not be added because of permeability barriers. However, no zearalenol was detected even after addition of ethanol which considerably increases the extramitochondria1 NADH/NAD ratio. The capacity of the hepatocytes to eliminate zearalenone was estimated to be about 100 pg per gram of liver in one hour (88-110 pg, mean value of three experiments). The highest value obtained with a liver homogenate was 82 pg. NADPH-cytochrome c reductase activity of the various liver preparations. In order to facilitate a comparison of the three liver preparations used - homogenate, microsomes, and isolated hepatocytes - the activity of NADPH-cytochrome c reductase was estimated in each experiment (table 2). The figures show that liver homogenate and isolated hepatocytes are comparable regarding this special function, which indicates that other microsomal activities may also be of the same magnitude in the two preparations. Table 2.
NADPH-cytochrome c reductase activity in the various liver preparations used.
Preparation
NADPH-cytochrome c reductase activity (pmol cyt. c reduced/g liver x min.-')
Liver homogenate Liver microsomes Hepatocytes
2136 935 2707
289
ZEARALENONE METABOLISM
The low activity of the NADPH-cytochrome c reductase in the microsomes is probably attributable to a low recovery during the preparation.
Discussion Our results from in vitro studies with liver homogenate (table 1) and liver microsomes show that zearalenone can be converted along at least two different routes. One is conjugation, probably by glucuronic acid, as added UDPGA (but not SO:-) increases the amount of conjugated zearalenone. The recovery of zearalenone after incubation with p-glucuronidase reveals that zearalenone itself (and not a metabolite) constitutes the bulk of the conjugate. The other mechanism is a reduction of zearalenone to zearalenol, possibly catalysed by a NADH-dependent hydroxysteroid dehydrogenase. Evidence for the product being zearalenol is as follows. When zearalenone is reduced by means of sodium borohydride it is completely converted into two isomers, A and B, of zearalenol as shown in fig. 2b (AA=O). The compound formed when zearalenone is added to a liver homogenate or to liver microsomes in the presence of NADH behaves exactly as the isomer A with TLC and HPLC. When zearalenone is incubated with a commercial u, 0-hydroxysteroid dehydrogenase and NADH at pH 7.5 a compound is formed which coincides in TLC and HPLC with the synthetic isomer A of zearalenol (fig. 2a) and with the metabolite formed from zearalenone by liver. At pH 9.0 and in the presence of NAD primarily the synthetic isomer A is transformed into zearalenone (fig. 2b). The possibility that the biological metabolite may actually be zearalanol is ruled out as the latter exhibits a very weak fluorescencewhen irradiated by UV light and can be separated from zearalenol by means of HPLC. In conclusion, the results show that zearalenone is metabolized by liver homogenate or by isolated microsomes along at least two principal routes. The main one is a conjugation with glucuronic acid. The other, which is a quantitatively less important route, is a reduction of zearalenone to zearalenol, possibly catalysed by a hydroxysteroid
dehydrogenase. The zearalenol formed can also be conjugated, to some extent. The hydroxysteroid reductase which catalyses the reduction of zearalenone to zearalenol is normally involved in the metabolism of steroids. This may lead to a disturbance of the steroid metabolism during prolonged intake of zearalenone, a possibility which is now being investigated. In no case could all the zearalenone metabolized be accounted for as conjugated zearalenone and free and conjugated zearalenol. This is especially obvious in the presence of NADH. Possibly part of this residue is zearalenone which is still conjugated because of the inability of 0-glucuronidase to release zearalenone completely, concurrently with its conjugation. However, the possibility cannot be excluded that the unaccountable residue (21-36%) may constitute another, so far unknown metabolite. During the preparation of this manuscript a review by Hidy et al. (1 977) appeared including reports on the metabolism of ''C-labelled zearalenone administered to rats. They describe how zearalenone is excreted mainly in the faeces and a smaller amount in the urine. Whether zearalenone is excreted in a conjugated or in a free form is not reported. The only metabolite mentioned in this study is zearalenol (Baldwin, unpublished results). t
Acknowledge men t s This work is part of investigations made possible by grants from The Swedish Council for Forestry and Agricultural Research, Dnr A 4683/B 3417. We are indebted to Mrs. Kerstin Tideman for skilful technical assistance and to Mrs. Carina Eriksson for typing of the manuscript.
References Hidy, P. H., R. S. Baldwin, R. L. Greasham, C. L. Keith & J. R. McMullen: Zearalenon and some derivatives : production and biological activities. In : Applied Microbiology 22. Ed.: D. Perlman. Academic Press, New York 1977, pp. 59-82. McNutt, S. H., P. Purwin & C. Murray: Vulvovaginites in swine; preliminary report. J . Am. Vet. Med. Assoc. 1928,73,484492.
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Pathre, S. V. & C. J. Mirocha: Zearalenone and related compounds. Adv. Chem. Ser. 1976,149, 178-227. Seglen, P. 0.:Preparation of isolated rat liver cells. In: Methods in Cell Biology vol. XIII, Academic Press, New York 1976. Shipchandler, M. T. :Chemistry of zearalenone and some of its derivatives. Heterocycles 1975,3,471-520.
Stob, M., R. S. Baldwin, J. Tuite, F. N. Andrews & K. G. Gillette: Isolation of an anabolic uterotropic compound from corn infected with Gibberella zeae. Nature (London) 1962, 1%, 1318. Urry, W. H., H. L. Wehrmeister, E. B. Hodge & P. H. Hidy : The structure of zearalenone. Teirahedron Lett. 1966,27,3109-3114.
From the Department of Animal Nutrition, The Swedish University of Agricultural Sciences, Box 7046, S-750 07 Uppsala, Sweden
Metabolism of Zearalenone in Rat Liver BY K.-H. Kiessiing and H. Pettersson (Received March 3, 1978; Accepted May 23, 1978)
Abstract :The metabolism of zearalenone in rat liver has been investigated. The studies were performed mainly with liver homogenate, though isolated microsomes and hepatocytes have also been used. Zearalenone was metabolized along two principal pathways, conjugation with glucuronic acid, which was the main route and reduction to an isomer of zearalenol. In no case, however, could all zearalenone metabolized be accounted for as conjugated zearalenone and free and conjugated zearalenol. Therefore another, so far unknown metabolite cannot be excluded. Reduction to zearalenol could be increased three times by the addition of NADH (or NADPH) and is probably catalyzed by a hydroxysteroid dehydrogenase. Some 25-50 per cent of the zearalenol could be conjugated, depending on the incubation conditions. The capacity of hepatocytes to eliminate zearalenone was estimated to be about 100 pg per gram of liver in one hour. With a liver homogenate the highest value obtained was 82 pg. Key-words: Mycotoxins - zearalenone - zearalenol - hydroxysteroid dehydrogenase - rat.
Many species of fungi common in various feeds are capable of producing potent toxins, but only a few mycotoxins have been identified chemically. As early as 1928 McNutt et al. (1928) investigated a syndrome called vulvovaginitis involving inflammation of the vulva and the posterior part of the vagina. They associated the disease with the consumption of mouldy maize. Stob et al. (1962) isolated the active component, called F-2, from cultures of Gibberella zeae (Fusarium roseum) and Urry et al. (1966) named it zearalenone. More than a hundred derivatives of zearalenone have been synthesized chemically (for a survey see Shipchandler 1975 and Pathre & Mirocha 1976). A few of these compounds have oestrogenic properties more potent than that of zearalenone itself. As Fusarium is an ubiquitous fungus it easily contaminates crops in the fields or in stores and can therefore constitute a constant health risk to domestic animals. Very little is known about the
metabolism of zearalenone in animals. For this reason the present investigation was carried out on zearalenone metabolism in rat liver and will be followed by a corresponding study on ruminants, swine and poultry. Materials and Methods The animals used were adult male Sprague Dawley rats. Liver homogenate was prepared by removing approximately 6 g liver immediately after the rat was killed, mincing it in ice-cold sucrose (0.25 M); Tris buffer ( 5 mM, pH 7.4); EDTA (1 mM), with scissors and then homogenazing it in a teflon-glass Potter-Elvehjem homogenizer. The homogenate was adjusted to 10% (w/v). When microsomes were prepared, the whole liver, after being weighed, was minced in the same way as described above and microsomes isolated by fractional centrifugation. Isolated hepatocytes were prepared according to Seglen (1976). Liver cell concentration was estimated by centrifugation of a small sample of the cell suspension in a haematocrit centrifuge, and viability by incubation
286
K.-H. KIESSLING AND H. PETTERSSON
of another sample with eosin and then counting the cells in a counting chamber. Incubations were made with additions as stated in the text at 37" in a total volume of 6 ml of liver homogenate and 5 ml of hepatocytes, corresponding to 0.3 g of liver, and with microsomes isolated from one g of liver. The incubations were stopped by addition of 3 ml 0.3 M H,PO, and extracted with 2 x 25 ml chloroform for further analysis of zearalenone and its metabolites (fig. 1) by means of thin-layer chromatography (TLC) or high pressure liquid chromatography (HPLC). The TLC was performed on pre-coated plates with silica gel (Merck) in a benzene-acetic acid (9: 1) system and analysed on an Aminco Bowman Spectrophotofluorometer with a thin-film scanner attachment. A maximum response was obtained with an excitation wavelength of 340 nm and an emission wavelength of 475 nm for both zearalenone and zearalenol. The natural (found in liver homogenate samples) isomer of zearalenol fluoresces twice as intensely as zearalenone. The other zearalenol isomer fluoresces less intensely than zearalenone, while zearalanol has hardly any fluorescence. HPLC was performed using a chromatography apparatus consisting of Waters Model 6000 solvent delivery system, Waters Model U6K septum-less injector and Altex ultraviolet detector (280 nm). By means of a separation system using a mobile phase of isooctanechloroform-methanol (35:25:1.5), a flow rate of 1.5 ml/min. and a Partisil 10 column it was possible to separate zearalenone and the two isomers of zearalenol. The isomers are denoted A and B according to the elution order in this system. A is the natural isomer and is believed to be the isomer with the high melting point. The normal analysis system, however, with methanol-
/ Zearalanol
water (65 :35) as the mobile phase, flow rate 1.O ml/min. on p-Bondapak C,, column (reversed phase) was not suitable for separating zearalenone and the natural metabolite zearalenol. Zearalenone and zearalanol were gifts from Commercial Solvents Corporation, USA and a mixture of the two zearalenol isomers was obtained by reduction of zearalenone with NaBH, as suggested by Hodge (see Shipchandler 1975). Uridine diphosphate glucuronic acid (UDPGA), NAD, NADH and NADPH, ATP, hydroxysteroid dehydropnase, P-glucuronidase (containing sulphatase) and collagenase were purchased from Sigma Chemical Company, and bovine albumin (fraction V) from Armour Pharmaceutical Company.
Results Conjugation of zearalenone. By means of TLC it was shown that about 30 per cent of the zearalenone added to a liver
homogenate was converted into other compounds under the conditions set out in table 1. Addition of UDPGA (uridine diphosphate glucuronic acid) doubled this conversion. With 0-glucuronidase and sulphatase present during the incubation, the bulk of this conversion (60%) could be prevented and is therefore regarded as conjugated zearalenone in the sample without added P-glucuronidasesulphatase (table 1 : Zearalenone conjugated).
Zearalenone
\
NaBHq
Zearalenol
Fig.']. Structure of zearalenone and the two derivatives zearalanol and zearalenol which may occur as a cis and a trans-isomer in the 6-position.
287
ZEARALENONE METABOLISM Table I .
Metabolism of zearalenone by rat liver homogenate. Incubations were carried out at 37" for 30 min. and contained 3 ml homogenate (0.3 g liver), 13.3 pg zearalenone, additions as stated in the table (0.04 mM UDPGA, 0.7 mM NADH, 1 mM Na,SO,, 0.9 mM ATP, 2000 u P-glucuronidase) and phosphate buffer (0.15 M, pH 7.4) in a final volume of 6 ml. The figures for UDPGA, NADH and no addition are mean values of five to six experiments, the remaining figures are from three experiments. Zearalenol formed Zearalenone conjugated** (pg/30 min.)
free
Additions
Zearalenone metabolized* (~g/3Oh n . )
UDPGA SO:- +ATP NADH NADPH UDPGA+NADH
3.9 (1.7-5.2) 9.0 (7.4-10.6) 3.8 9.0 (7.3-10.2) 7.9 9.7 (9.2-12.3)
2.1 (0.8-3.1) 5.5 (3.8-6.4)
0.8 (0.0-1.0) 0.6 (0.3-1.0)
0.2 (0.0-0.4) 0.3 (0.14.5)
2.8 (2.1-3.8)
0.5 2.3 (1.7-3.2)
0.7 (0.5-1.0)
-
*
conjugated** (pg/30 min.)
2.0 4.9 (4.0-6.3)
1.5 (0.9-1.7)
0.5 0.8 (0.5-1.0)
All zearalenone which could not be recovered after the incubation was completed has been regarded as me-
tabolized.
** The figures for conjugated zearalenone and zearalenol are obtained as differences in free zearalenone and zearalenol between incubations in the presence and absence of P-glucuronidase.
Formation of zearalenol. During the incubation of zearalenone with a liver homogenate a compound with a lower Rfvalue than zearalenone could be detected by TLC. The content corresponded to 7 per cent of the added zearalenone and to 25 per cent of the metabolized zearalenone. Addition of NADH (or NADPH) increased the compound about three times. When UDPGA was also present the yield diminished slightly (table l), indicating a minor competition for zearalenone between conjugation and formation of the unknown compound. The TLC revealed the Rf-value of the compound to be nearly the same as that of zearalanol (fig. 1) but, in contrast to zearalanol, it exhibited an intense fluorescence, about twice as marked as zearalenone, when irradiated by UV light. By means of HPLC the compound could be separated from zearalanol as well as from zearalenone but behaved in exactly the same way as isomer A of a synthetically prepared zearalenol. The biotransformation of zearalenone to zearalenol implies a reduction of the keto group in 6'-position (fig. 1). This reaction shows similarities to processes involved in the steroid metabolism catalysed by a steroid dehydrogenase. We there-
fore incubated zearalenone at pH 7.5 with a commercial hydroxysteroid dehydrogenase from Pseudomonas testosteroni in the presence of NADH. Analyses by HPLC showed that the enzyme catalysed the reduction of zearalenone exclusively to the isomer A of zearalenol (fig. 2a), e.g. theisomer which is identicalwith the zearalenol formed from zearalenone in a liver homogenate. Conversely, when the synthetic mixture of the two zearalenol isomers was incubated with the hydroxysteroid dehydrogenase and NAD at pH 9, zearalenone was formed primarily from the isomer A (fig. 2b).
Conjugation of zearalenol. When zearalenone was added to a liver homogenate together with NADH, 25 per cent of the zearalenone which had disappeared could be found again as free zearalenol. When p-glururonidase was also present another 8 per cent was recovered as zearalenol (table 1 : zearalenol conjugated) indicating that some of the zearalenol as well as zearalenone can be conjugated. Zearalenone metabolism with microsomes and isolated hepatocytes. The results obtained with microsomes isolated
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K.-H. KIESSLING AND H. PETTERSSON
from rat liver were very similar to those with liver homogenate. Eighty per cent of the zearalenone was conjugated within one hour by microsomes
AA.0.1
A
A A.0.1
-
0
5
10
15
20
25
I
:
:
:
;
0
5
10
15
20
: 25 min.
Fig. 2. Incubation of zearalenone and zearalenol with hydroxysteroid dehydrogenase. A. Incubation of zearalenone with hydroxysteroid dehydrogenase and NADH. The incubation contained 0.3 mg zearalenone, 0.3 mg NADH and 50 pg enzyme in 2.5 ml 50 mM phosphate buffer and was performed at 25", pH 7.5. The reaction was registered spectrophotometrically at 340 nm as a decrease in NADH. Samples were taken at A A=0.1 and 0.2 and were analysed for zearalenone and zearalenol with HPLC. The designations of the peaks are: Zn=zearalenone, Z1 A and 21 B=the two isomers A and B of zearalenol. B. Incubation of zearalenol with hydroxysteroid dehydrogenase and NAD. The two isomers of zearalenol, Z1 A and Z1 B, were prepared from zearalenone by reduction with NaBH,. The incubation contained approx. 0.3 mg zearalenol, 6.6 mg NAD and 50 pg enzyme in 2.5 ml 50 mM pyrophosphate buffer and was performed at 25", pH 9.0. The reaction was registered at 340 nm as an increase in NADH. Samples were taken at A A=O (zero time), and on three occasions during the incubation and analysed as described above.
isolated from one gram of liver while 10 per cent was reduced to zearalenol in the presence of UDPGA and NADH. The only difference was that no conjugation of zearalenol could be demonstrated. The activity of the microsomes against zearalenone was lower than in the liver homogenate calculated per gram of liver. The explanation for this is probably that an appreciable loss of microsomes occurs during the isolation. This interpretation is supported by results given in table 2 concerning the NADPH-cytochrome c reductase activity in the microsomal and homogenate preparations. Isolated hepatocytes eliminated zearalenone very rapidly. UDPGA or NADH could not be added because of permeability barriers. However, no zearalenol was detected even after addition of ethanol which considerably increases the extramitochondria1 NADH/NAD ratio. The capacity of the hepatocytes to eliminate zearalenone was estimated to be about 100 pg per gram of liver in one hour (88-110 pg, mean value of three experiments). The highest value obtained with a liver homogenate was 82 pg. NADPH-cytochrome c reductase activity of the various liver preparations. In order to facilitate a comparison of the three liver preparations used - homogenate, microsomes, and isolated hepatocytes - the activity of NADPH-cytochrome c reductase was estimated in each experiment (table 2). The figures show that liver homogenate and isolated hepatocytes are comparable regarding this special function, which indicates that other microsomal activities may also be of the same magnitude in the two preparations. Table 2.
NADPH-cytochrome c reductase activity in the various liver preparations used.
Preparation
NADPH-cytochrome c reductase activity (pmol cyt. c reduced/g liver x min.-')
Liver homogenate Liver microsomes Hepatocytes
2136 935 2707
289
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The low activity of the NADPH-cytochrome c reductase in the microsomes is probably attributable to a low recovery during the preparation.
Discussion Our results from in vitro studies with liver homogenate (table 1) and liver microsomes show that zearalenone can be converted along at least two different routes. One is conjugation, probably by glucuronic acid, as added UDPGA (but not SO:-) increases the amount of conjugated zearalenone. The recovery of zearalenone after incubation with p-glucuronidase reveals that zearalenone itself (and not a metabolite) constitutes the bulk of the conjugate. The other mechanism is a reduction of zearalenone to zearalenol, possibly catalysed by a NADH-dependent hydroxysteroid dehydrogenase. Evidence for the product being zearalenol is as follows. When zearalenone is reduced by means of sodium borohydride it is completely converted into two isomers, A and B, of zearalenol as shown in fig. 2b (AA=O). The compound formed when zearalenone is added to a liver homogenate or to liver microsomes in the presence of NADH behaves exactly as the isomer A with TLC and HPLC. When zearalenone is incubated with a commercial u, 0-hydroxysteroid dehydrogenase and NADH at pH 7.5 a compound is formed which coincides in TLC and HPLC with the synthetic isomer A of zearalenol (fig. 2a) and with the metabolite formed from zearalenone by liver. At pH 9.0 and in the presence of NAD primarily the synthetic isomer A is transformed into zearalenone (fig. 2b). The possibility that the biological metabolite may actually be zearalanol is ruled out as the latter exhibits a very weak fluorescencewhen irradiated by UV light and can be separated from zearalenol by means of HPLC. In conclusion, the results show that zearalenone is metabolized by liver homogenate or by isolated microsomes along at least two principal routes. The main one is a conjugation with glucuronic acid. The other, which is a quantitatively less important route, is a reduction of zearalenone to zearalenol, possibly catalysed by a hydroxysteroid
dehydrogenase. The zearalenol formed can also be conjugated, to some extent. The hydroxysteroid reductase which catalyses the reduction of zearalenone to zearalenol is normally involved in the metabolism of steroids. This may lead to a disturbance of the steroid metabolism during prolonged intake of zearalenone, a possibility which is now being investigated. In no case could all the zearalenone metabolized be accounted for as conjugated zearalenone and free and conjugated zearalenol. This is especially obvious in the presence of NADH. Possibly part of this residue is zearalenone which is still conjugated because of the inability of 0-glucuronidase to release zearalenone completely, concurrently with its conjugation. However, the possibility cannot be excluded that the unaccountable residue (21-36%) may constitute another, so far unknown metabolite. During the preparation of this manuscript a review by Hidy et al. (1 977) appeared including reports on the metabolism of ''C-labelled zearalenone administered to rats. They describe how zearalenone is excreted mainly in the faeces and a smaller amount in the urine. Whether zearalenone is excreted in a conjugated or in a free form is not reported. The only metabolite mentioned in this study is zearalenol (Baldwin, unpublished results). t
Acknowledge men t s This work is part of investigations made possible by grants from The Swedish Council for Forestry and Agricultural Research, Dnr A 4683/B 3417. We are indebted to Mrs. Kerstin Tideman for skilful technical assistance and to Mrs. Carina Eriksson for typing of the manuscript.
References Hidy, P. H., R. S. Baldwin, R. L. Greasham, C. L. Keith & J. R. McMullen: Zearalenon and some derivatives : production and biological activities. In : Applied Microbiology 22. Ed.: D. Perlman. Academic Press, New York 1977, pp. 59-82. McNutt, S. H., P. Purwin & C. Murray: Vulvovaginites in swine; preliminary report. J . Am. Vet. Med. Assoc. 1928,73,484492.
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Pathre, S. V. & C. J. Mirocha: Zearalenone and related compounds. Adv. Chem. Ser. 1976,149, 178-227. Seglen, P. 0.:Preparation of isolated rat liver cells. In: Methods in Cell Biology vol. XIII, Academic Press, New York 1976. Shipchandler, M. T. :Chemistry of zearalenone and some of its derivatives. Heterocycles 1975,3,471-520.
Stob, M., R. S. Baldwin, J. Tuite, F. N. Andrews & K. G. Gillette: Isolation of an anabolic uterotropic compound from corn infected with Gibberella zeae. Nature (London) 1962, 1%, 1318. Urry, W. H., H. L. Wehrmeister, E. B. Hodge & P. H. Hidy : The structure of zearalenone. Teirahedron Lett. 1966,27,3109-3114.
