The role of rat liver microsomal enzymes in the metabolism of the fungal metabolite fusarin C

Fd Chem. Toxic. Vol. 26, No. 1, pp. 31-36, 1988

0278-6915/88 $3.00+0.00 Copyright © 1988 PergamonJournals Ltd

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THE ROLE OF RAT LIVER MICROSOMAL ENZYMES IN THE METABOLISM OF THE F U N G A L METABOLITE FUSARIN C W. C. A. GELDERSLOMand P. G. THIEL Research Institute for Nutritional Diseases, South African Medical Research Council, Tygerberg 7505 and K. J. VAN DER MERWE Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600, South Africa (Received 31 March 1987; revisions received 7 July 1987)

Abstract--The metabolic activation of fusarin C by a rat liver microsomal monooxygenase resulted in the formation of a water-soluble mutagenic metabolite. However, fusarin C incubated in the presence of a microsomal preparation, but in the absence of an NADPH-generating system, led to the formation of fusarin PM~, a highly water-soluble compound which, like fusarin C, requires metabolic activation to be mutagenic. Enzyme studies using as substrates fusarins A and D, compounds structurally related to fusarin C, together with structural studies of fusarin PM~ indicated that fusarin PM~ was formed by the action of carboxylesterase which hydrolyses the C-20 methyl ester group to a free carboxylic acid.

INTRODUCTION

2-pyrrolidone moiety, more specifically those leading to a loss of the C-13-C-14 epoxide, result in a loss of mutagenicity (Gelderblom et al. 1984c). It has been shown that fusarin C requires metabolic activation by a microsomal monooxygenase in order to express mutagenic activity (Gelderblom et aL 1984c). Several enzymes are known to catalyse the deactivation of mutagenic and carcinogenic compounds. The metabolism of fusarin C by two such enzymes, rat liver microsomal epoxide hydrolase and carboxylesterase, was studied. In addition, some characteristics of the reactive mutagenic metabolite resulting from the monooxygenase-dependent reaction were investigated.

Fusarium moniliforme, a common fungal contaminant

of maize, has been associated with several human and animal diseases (Marasas et al. 1984). Recently a mutagenic compound, fusarin C, was isolated from maize cultures of F. moniliforme and characterized chemically (Gelderblom et al. 1983 & 1984a). This mutagen has also been found to occur naturally on maize in both the Transkei, southern Africa (Gelderblom et al. 1984b) and Pennsylvania, USA (Thiel et al. 1986). Fusarin C appears not to be acutely toxic, since rats survived an oral dose of 200 mg/kg although there was a slight reduction in weight gain (Gelderblom, 1986). The chemical structure of fusarin C (Fig. 1) consists of a polyene chromophore, with all the olefinic bonds in the trans configuration (2E, 4E, 6E, 8E, 10E) and linked in position C-13 to a 2-pyrrolidone moiety (Gelderblom et al. 1984a). Several other compounds structurally related to fusarin C have been isolated and characterized chemically. Fusarins A and D (Fig. 1), which lack the C-1342-14 epoxide group, are produced together with fusarin C by F. moniliforme in culture. Three other compounds, designated the 6Z, 8Z and 10Z isomers are derived from fusarin C after long-wave UV (360 nm) irradiation. These UVinduced compounds exhibit mutagenic activities similar to that of fusarin C, while fusarins A and D are not mutagenic (Gelderblom, 1986). Configurational changes in the polyene side chain do not affect the mutagenic activity, while alterations to the

MATERIALS AND METHODS Chemicals. NADP, glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase were obtained from Boehringer Mannheim (SA) (Pty) Ltd, Randburg. Sodium phenobarbital (PB) was purchased from BDH Chemicals Ltd, Poole, Dorset, UK, and l,l,l-trichloro-2,3-propene oxide (TCPO) was obtained from Sigma Chemical Co., St Louis, MO, USA. Fusarins A, C and D were isolated from maize cultures of F. moniliforme, strain MRC 826, as described previously (Gelderblom et al. 1983). Microsomal preparations. Liver homogenate fractions (S-9) were prepared from PB-induced male BD IX rats weighing approximately 200 g (Ames et al. 1975). The S-9 fraction (15 ml) containing 43.5mg protein/ml was applied to a Sepharose 2B column (40 x 2.5 cm) and equilibrated with 50 mM-Tris HCI buffer (pH 7.4) containing 150 mM-KCI. The purified microsomal fraction eluting in the void volume was collected and stored at -80°C. The microsomal fraction used in mutagenicity determinations was

G-6-P = glucose-6-phosphate; HPLC = high-performance liquid chromatography; NMR = nuclear magnetic resonance; PB = sodium phenobarbital; TCPO = l,l,l-trichloro-2,3-propene oxide.

Abbreviations:

31

32

W. C. A. GELDERBLOMet al.

prepared under sterile conditions from the S-9 fraction by centrifugation at 100,000g for 60min. The microsomal pellet was resuspended in a 50 raM-sodium phosphate buffer (pH 7.4) containing 150 mM-KCI and stored at - 8 0 ° C . Protein concentrations were determined as described by Lowry et al. (1951). Mutagenicity testing. The mutagenicity assay, using Salmonella typhimurium strain TA100, was performed as described by Ames et al. (1975). An activation mixture was prepared in a similar way to the S-9 mix except that the microsomal fraction described above replaced the S-9 fraction. This mixture contained (per ml): MgCI 2 (8/~mol), KC1 (33 #mol), NADP (4/~mol), G-6-P (5 #mol), G-6-P dehydrogenase (1 U) microsomes (540#g protein) and sodium phosphate buffer pH 7.4 (100/~mol). The mutagenic activities of fusarin C (0.2/~g/plate) and fusarin PM~ (5, 10 or 20/~g/plate) were compared using the standard plate incorporation assay. A liquid suspension assay or pre-incubation test was also carried out by pre-incubating the activation mixture (0.5ml), the bac-terial culture (0.1 ml) and fusarin C (0.5/~g) at 37°C for 30 rain before the soft agar (2 ml) was added. This mixture was poured onto plates which were incubated for 48 hr at 37°C.

Stability of the active mutagenic metabolite(s). Fusarin C (0.5 #g/0.5 ml mixture) was pre-incubated with an activation mixture, as described above, without the bacterial culture, and 10 ml of this mixture was then centrifuged under sterile conditions at 100,000g for 1 hr. The supernatant and the resuspended pellet were each made up to the original volume in sterile distilled water and tested (0.6 ml samples) for mutagenicity. In a subsequent experiment the supernatant (10 ml) was fractionated on a C 18 reverse-phase cartridge (Sep-Pak) by consecutive washes (10 ml each) with H20, 20% methanol, 50% methanol and 100% methanol. Each fraction (0.6 ml) was tested for mutagenicity as described. Microsomal conversion of fusarins. Fusarin C (35/,g) was incubated with 5ml of three different incubation mixtures: (1) a 'complete' incubation mixture containing (per ml) MgCl 2 (8#mol), KCI (150/~mol), NADP (1 #mol), G-6-P (5/~mol), G-6-P dehydrogenase (1 U), Tris-HCl (50/~mol) and microsomes (1 mg protein), (2) a microsomal incubation mixture containing (per ml) KC1 (150/lmol), TrisHCI (50 #mol) and microsomes (1 mg protein), and (3) the microsomal incubation mixture but with heat-inactivated microsomes. The mixtures were incubated for 30 min at 37°C in the dark and then extracted with 3 vols chloroform by shaking for 15rain at 4°C. After centrifugation at 2000g for l0 min at 4°C, the top (buffer) layer was removed and the chloroform layer (10ml) was concentrated to 5 ml. These samples were kept at 4°C in the dark for HPLC analysis. Fusarins A and D (501~g) were incubated with a microsomal incubation mixture either without or with microsomes ( ! m g protein/ml) after which the mixtures were treated as described for the fusarin C treatment. Isolation offusarin PMI. Fusarin C (0.25 rag) was incubated with a microsomal incubation mixture (5 ml) containing 2 mg protein/ml for 60 min at 37°C. After extraction with chloroform (3 vols), the mixture

was centrifuged at 2000 g for 10 min. The microsomal protein precipitated at the interface of the chloroform and buffer phases. The buffer phase was passed through a C18 reverse-phase cartridge (Sep-Pak) and washed with distilled water (4 x 5 ml) until free of salt. Fusarin PM~ was eluted in a small volume of methanol and the final purification was achieved by preparative thin-layer chromatography using methanol-ethyl acetate (1:4, v/v) as the developing solvent. This procedure was repeated ten times to yield 0.25 mg of pure fusarin PM~. Proton NMR data (Gelderblom, 1986) were recorded on a Brucker WM-500 spectrometer at the National Chemical Research Laboratory, CSIR, Pretoria. Reaction kinetic studies. Time-course studies indicated that under the conditions used, the rate of formation of fusarin PMI was linear for about 5 min. Reaction kinetic studies were therefore performed by incubating different concentrations of fusarin C (2.5, 3.75, 5.0, 7.5 and 10.0mg/ml) in duplicate with microsomal incubation mixtures (2ml; 2mg protein/ml) at 37°C for 4rain. Zero time samples (1 ml) were taken and the reaction was stopped by the addition of chloroform. Samples were extracted as described and the amount of fusarin PM~ recovered in the buffer phase was determined by reverse-phase HPLC using a standard solution of fusarin PMI (0.02 mg fusarin PMUml methanol) for calibration of the HPLC data. HPLC analyses. A Micromeritics highperformance liquid chromatograph (model 700) equipped with a Micromeritics variable wavelength detector (model 785) was used. The chloroform phases (20/~1) were analysed on an Ultrasphere (Beckman) silica-gel column (4.6 mm × 25 cm; 5/1) using methanol-chloroform (1:19, v/v) as the mobile phase at a flow rate of 1 ml/min. The buffer phases (30 #1) were analysed on a Radial-Pak cartridge (C18, 10#) in a Radial Compression Module (model RCM-100, Waters Associates Inc., Milford, MA, (a)

20 24 Me

"'-,I13

23 Me

Z

°

°

3~O2R~ 4 ~ 2

°

22

OH (b)

20 2t

C02Me

O

24 Me

23 Me

H 13~..-Rg-

8

31 4 ~v'~ 2

6

3e 22

°

is

OH

Fig. 1. Chemical structures of fusarins; (a) fusarin C (R I = Me) or fusarin PM t (R 1= H), (b) fusarin A (R2= H) or fusarin D (R2= OH).

Metabolism of fusarin C by rat liver enzymes USA) using a water/methanol gradient at a flow rate of 2 ml/min. The gradient, using a concave program (degree of curvature = 4), was run from an initial methanol concentration of 10% up to 50% over a period of 6 min, after which the methanol concentration was kept constant for another 9min. The eluates were monitored at 360 nm and the chromatograms were recorded at a chart speed of 0.2 in./min.

1250

33

-

1000 -

] ~1~1

o F, itl

750

-

.---I

',~t'l

i;ill i

o

',!hi 500

RESULTS

r~tt ~,

~

i ,111

Following incubation of fusarin C with microsomes in the presence of the activation mixture, all the mutagenic activity was associated with the supernatant fraction (Fig. 2). Some 50% of this mutagenicity was recovered in the 50% methanol-water eluate after fractionation on a reverse-phase cartridge (Fig. 3). The results of incubating fusarin C in vitro with the complete incubation mixture containing active microsomes and an NADPH-generating system, with a microsomal mixture without NADPH or with a mixture containing heat-inactivated microsomes are shown in Fig. 4. Incubation with active microsomes in the presence or absence of NADPH led to the disappearance of fusarin C from the chloroform phase and the appearance of a new compound called fusarin PM~ in the buffer phase. This microsomal conversion of fusarin C to fusarin PM~ was inhibited by 3 or 6 mM-TCPO (Fig. 5), the degree of inhibition being directly related to the concentration. Fusarins A and D, similarly incubated with a microsomal incubation mixture were also converted to water-soluble products (Fig. 6). Fusarin A gave rise to two water-soluble products. The more polar one (eluting first from the reverse-phase column) co-eluted with the water-soluble compound formed during the fusarin D incubation. Comparison of the mutagenic activities of fusarins C and PM] in S. typhimurium TA100 showed that,

1250

-

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250

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1"1 .~___i . .

.

I =''il .....

.

"; 'l ';I B Q c k Q r ° u n d ~i.~.~..i I. . . . . . . . . .

Fig. 2. Mutagenic activity of fusarin C (0.5/zg/plate) to Salmonella typhimurium TA100: after pre-incubation with bacteria and the activation mixture (1~), and after preincubation without bacteria and with the activation mixture, followed by separation into supernatant ([]) and microsomes ([]) by centrifuging at 100,000g for I hr. Dotted line represents the background level of revertants. like fusarin C, the PMj metabolite was mutagenic only after microsomal activation (Table 1). However, after activation, fusarin PM l was much less mutagenic than fusarin C. Kinetic studies of the enzymic deacylation of fusarin C to fusarin PMI showed (by linear regression) that the K m value was 31.5/zM (limax = 1.2 nmoi fusarin PMl/min/mg protein; Fig. 7). DISCUSSION

A PB-induced microsomal monooxygenase has been shown to convert fusarin C to active mutagenic intermediate(s) (Gelderblom et al. 1984c). The chemical structure of the active metabolite(s), as yet

-

,oool o

/ 750

-

500

-

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250 / /

Background .

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.

.

.

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.

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.

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.

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Fig. 3. Mutagenic activity of fusarin C following pre-incubation with the activation mixture and no bacteria, centrifugation (I00,000 g) and fractionation of the supernatant ([]) on a reverse-phase Sep-Pak cartridge: column eluate (D), water eluate (1~), 20% methanol/water eluate (t~), 50% metanol/water eluate (El) and 100% methanol eluate (i-q). Values are means of triplicate determinations, vertical bars indicate the range and the dotted line represents the background level of revertants.

34

GELDERBLOM et al.

W.C.A.

Table 1. Mutagenic activities of fusarin C and fusarin PM~ using Salmonella typhimurium strain TAI00 with and without microsomal activation Revertants/plate* Concn (#g/plate)

Compound Control:~ Fusarin C Fusarin PM=

Without microsomes

-0.2 5 10 20

100 + 117 ± 101 ± 103 ± 99 ±

With microsomest

7 8 9 6 7

115 ± 680 ± 212 ± 304 ± 557 ±

9 47 10 7 30

*Revertant counts are means ± S D for triplicate determinations. tMicrosomal protein level was 270 #g/plate. :~Dimethylsulphoxide (0.1 ml/plate).

(a) 0.02

Inactivated microsomos

Active microsomes (-- ~NADPH

Active microsomes ( + ~NADPH

Fusarin C

l 0.01

+

T 0 ID

m

I,, 0

0

t, 0.02.

I,, 3

I, 6

, I, 9

I , , I , , I , , I , 0 :3 6 9

I,,I 0

,,I, 3

6

tb

.~_..____._.- Fusorin PMI

I

X'

l

0.01 -

0 -

,I, 9

0

,~1,,I,,I,,I, 3 6

9

12

0

, , I , , I , , [ . . 1 , 3 6 9 12 Retention time (rain)

I, 0

I,, .3

I,, 6

1,, 9

I , 12

Fig. 4. H P L C analyses o f (a) c h l o r o f o r m phase a n d (b) buffer phase from the m i c r o s o m a l c o n v e r s i o n o f fusarin C in the presence or absence of an N A D P H - g e n e r a t i n g system. A control i n c u b a t i o n was carried out using h e a t - i n a c t i v a t e d microsomes.

Metabolism of fusarin C by rat liver enzymes 0.02

[a)

[b)

35 (c)

Fusarin PM 1

/

E c 0 (.o it) 8 c

0.01

I, 0

, I, 3

, I, 6

All 9

i I L '12

ILJ

%

I ,, 0

,I, 12

t, 3

, I,,I, 6 9

I,,t,,I,,I,,1, 0

3

6

9

12

Retention time (rain)

Fig. 5. The effect of (a) 0, (b) 3 and (c) 6 mM concentrations of l,l,l-trichloro-2,3-propene oxide on the HPLC profile of the microsomal formation of fusarin PM~ from fusarin C.

unidentified, would provide more information about this oxidative step and the role of the C-13~C-14 epoxide in the mutagenic behaviour of fusarin C. Our study has indicated that the mutagenic metabolite was water soluble, as all the mutagenic activity was associated with the supernatant fraction following incubation of fusarin C with microsomes and N A D P (Fig. 2). Moreover, the finding that approximately 50% of the mutagenicity present in the supernatant was recovered in the 50% methanol-water eluate after fractionation on a reverse-phase cartridge

(Fig. 3) indicates that the metabolite is relatively stable. This fractionation technique, which removes salts present in the incubation mixture, could be applied to the isolation of the active metabolite(s). However, attempts to isolate this activated metabolite have so far been unsuccessful. In vitro incubation with active microsomes and an N A D P H - g e n e r a t i n g system resulted in the disappearance of fusarin C and the concomitant appearance of a water-soluble c o m p o u n d called fusarin PM~ (Fig. 4). The formation of fusarin PM~ in the presence

0.02

Fusorin A

Fus0rin D

Fusorin A +

c o

Fusorin D

Water soLubLe products

v c 0.01

.a

0

a , l , , l ~ F , t l , , I 3 6 9 12

15

I,,IJ, 0 3

J,, 6

Retention

J~t 9

time

IJ, 12

l 15

ll~ 0

I , , I , , I , , I , , I 3 6 9 12

15

(rain)

Fig. 6. HPLC profile of the water-soluble products of the microsomal conversion of fusarins A and D. For the co-chromatographic separation, samples from the buffer phases of the fusarin A (20/tl) and fusarin D (10/11) incubations were co-injected. FC.T 2 6 [ ~ "

36

W . C . A . GELDERBLOMet al. 7!

vivo activity of fusarin C. The K m value (31.5 #M) for

6I

the enzymatic deacylation of fusarin C to fusarin PMt indicated a high affinity of fusarin C for the carboxylesterase enzyme (Fig. 7). Determination of the kinetic parameters for the monooxygenase-dependent reaction could indicate which of the two reactions is the more likely to dominate under in vivo conditions. This study provides further evidence that the C13-C-14 epoxide is important for determining mutagenicity. All the fusarins containing the epoxide on the 2-pyrrolidone moiety, including fusarin C and fusarin PM~, proved to be mutagenic (Gelderblom, 1986) although they still needed activation by a monooxygenase enzyme. In many cases, the presence of an epoxide group on a molecule is responsible for the carcinogenic and/or mutagenic properties, as binding to D N A often takes place at this electrophilic site (Grover, 1986; Oesch, 1979). Why the fusarins that already contain an epoxide still need further activation by a monooxygenase is not yet known.

,-~7 ~4 it-

=J"

o

j-

v 1

J

I -6

I ~ -4

lif"

-2

0

[

I

I

I

I

I

I

I

I

2

4

6

8

10

12

14

16

18

1/[Fusorin C3 l/~M)xlO -2

Fig. 7. A Lineweaver-Burk plot of the formation of fusarin PMt, determined by the incubation of increasing concentrations of fusarin C with microsomes (2 mg protein/ml). of microsomes without N A D P H indicates that the reaction was not catalysed by the microsomal monooxygenase system. However fusarin PM~ serves as a substrate for the monooxygenase enzymes as it is only mutagenic after metabolic activation (Table 1). This could explain the lower amount of fusarin PM~ recovered after the incubation of fusarin C with both microsomes and N A D P H (Fig. 4b). The conversion of fusarin C to fusarin PM~ was first thought to be catalysed by epoxide hydrolase because of the inhibition of fusarin PMj formation by TCPO, a potent epoxide hydrolase inhibitor (Fig. 5). However, water-soluble products were also formed when fusarins A and D, which lack the C-13-C-14 epoxide (Fig. 1), were incubated with microsomes (Fig. 6) and the original c o m p o u n d disappeared from the corresponding chloroform phase (results not shown). The co-elution of one of the water-soluble derivatives of fusarin A with that formed from fusarin D indicates that in the presence of microsomes fusarin A is partly converted to fusarin D (Fig. 6). The proton N M R data for fusarin PM~ (Gelderblom, 1986) showed no signals for the C-21 protons as observed for fusarin C. Fusarin PM~ is therefore not a product of epoxide hydrolase activity but of a microsomal carboxylesterase (Fig. 1). The microsomal conversion of fusarins A and D, which also contain the C-20 methyl ester, are therefore likely to be catalysed by the same enzyme. The high water solubility of fusarin PM~ compared to fusarin C can be ascribed to the presence of the free carboxylic group at position C-20. It is known that the lipophilicity of compounds is important in their interaction with monooxygenase enzymes and more specifically with cytochrome P-450. As the substrate binding site is concealed inside a hydrophobic pocket (White & Coon, 1980), fusarin C will more readily penetrate the lipoidal barrier to interact with the enzyme than will fusarin PM~. This is in agreement with the observed mutagenic behaviour of fusarin PM ~which was found to be much less mutagenic than fusarin C (Table 1). The relative rate of formation of the active metabolite and the far less mutagenic fusarin PM~ could play an important role in the in

REFERENCES

Ames B. N, McCann J. & Yamasaki E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Res. 31, 347. Gelderblom W. C. A. (1986). The Chemical and Biological Properties of Fusarin C, a Secondary Mutagenic Metabolite Produced by Fusarium moniliforme Sheldon. PhD Thesis. Department of Biochemistry, University of Stellenbosch, South Africa. Gelderblom W. C. A., Marasas W. F. O., Steyn P. S., Thiel P. G., van der Merwe K. J., van Rooyen P. H., Vleggaar R. & Wessels P. L. (1984a). Structural elucidation of fusarin C, a mutagen produced by Fusarium moniliforme. J. Chem. Soc. Commun. p. 122. Gelderblom W. C. A., Thiel P. G., Marasas W. F. O. & van der Merwe K. (1984b). Natural occurrence of fusarin C, a mutagen produced by Fusarium moniliforme, in corn. J. agric. Fd Chem. 32, 1064. Gelderblom W. C. A., Thiel P. G. & Van der Merwe K. J. (1984c), Metabolic activation and deactivation of fusarin C, a mutagen produced by Fusarium moniliforme. Biochem. Pharmac. 33, 1601. Gelderblom W. C. A., Thiel P. G., van der Merwe K. J., Marasas W. F. O. & Spies H. S. C. (1983). A mutagen produced by Fusarium moniliforme. Toxicon 21, 467. Grover P, L. (1986). Pathways involved in the metabolism and activation of polycyclic hydrocarbons. Xenobiotica 16, 915. Lowry O. H., Rosebrough N, J., Farr A. L. & Randall R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. Marasas W. F. O., Nelson P. E. & Toussoun T. A. (1984). Toxigenic Fusarium Species. p. 216. The Pennsylvania State University Press, University Press, University Park. Oesch F. (I 979). Enzymes as regulators of toxic reactions by electrophilic metabolites. Archs ToxicQl. Suppl. 2, 215. Thiel P. G., Gelderblom W. C. A., Marasas W. F. O., Nelson P. E. & Wilson T. M. (1986). Natural occurrence of moniliformin and fusarin C in corn screenings known to be hepatocarcinogenic in rats. J. agric. Fd Chem. 34, 773. White E. & Coon M. J. (1980). Oxygen activation by cytochrome P-450. A. Rev. Biochem. 49, 315.