Identification of xyloside conjugates formed from anthracene by Rhizoctonia solani

509

Mycol. Res. 96 (6): 509-517 (1992) Pn'nfed in Great Britain

Identification of xyloside conjugates formed from anthracene by Rhizoctonia solani

J. B. SUTHERLAND, A. L. SELBY, J. P. FREEMAN, P. P. FU, D. W. MILLER A N D C. E. CERNIGLIA* National Center for Toxicological Research, Food and Drug Administration, Jefferson,Arkansas 72079, U.S.A.

Biotransformation experiments showed that a strain of Rhiwcfonia solani was able to metabolize anthracene, a tricyclic aromatic hydrocarbon. The fungus was grown in a complex liquid medium containing [9-14C]anthracene;after 6 d, 988% of the anthracene had been converted to ethyl acetate-extractable metabolites. These compounds were separated by high-performance liquid chromatography (hplc) and detected by ultraviolet (uv) absorbance and liquid scintillation counting. The major metabolites were identified by their uv, mass, and nuclear magnetic resonance spectra. One of the principal metabolites was identified as trans-1,2dihydroxy-1,2-dihydroanthracene(anthracene trans-1,2-dihydrodiol),which was shown by circular dichroism spectroscopy to be a mixture of two enantiomers. Chiral stationary phase hplc was used to resolve the trans-1,2-dihydrodiol into a (-)-IS,2S enantiomer (60%)and a (+)-IR,ZR enantiomer (40%).The other principal metabolites were novel xyloside conjugates of anthracene: I-0-(2hydroxy-trans-1,2-dihydroanthryl)-~-xylopyranoside, 2-O-(~-hydroxy-frans-~,2-dihydroanthryl)--o-xylopyranoside, and 1-0anthryl-P-D-xylopyranoside. Anthraquinone was found in the culture media but was also present in non-inoculated controls.

Rhizocfonia solani Kiihn inclues a variety of fungi of major ecological and economic importance (Mordue, Currah & Bridge, 1989; Ogoshi, Cook & Bassett, 1990). Several strains of R. solani metabolise naturally occurring aromatic compounds, including phenylacetic acid, trans-cinnamic acid, pcoumaric acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, L-phenylalanine, and phenylpyruvic acid (Kohmoto, Nishimura & Hiroe, 1970; Kalghatgi ef al., 1974). Strains of R. solani also transform the aromatic herbicides chlorbromuron (Weinberger & Bollag, 1972), fluometuron (Weinberger & Bollag, 1972; Rickard & Camper, 1978), alachlor (Smith & Phillips, 1975), and nitralin (Camper & Ellers, 1978). Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that are found in coal and petroleum and are also produced during combustion of fiels (Cemiglia & Heitkamp, 1989). Anthracene, a PAH with three fused aromatic rings, is not generally considered a direct-acting carcinogen but may be methylated by enzymes in the liver to produce a carcinogenic metabolite, 9'10-dimethylanthracene (Myers, Blake & Flesher, 1988). When irradiated with ultraviolet (uv) radiation, anthracene binds to DNA (Sinha & Chignell, 1983) and damages the cell membranes of bacteria (Tuveson ef al., 1990). Anthracene reduces the growth of green algae (Cody, Radike & Warshawsky, 1984) and vascular plants (Mitchell ef al., 1988). It has several known toxic effects in animals, for example, the destabilisation of lysosomes in the digestive cells of mussels (Moore, Lowe & Fieth, 1978). Anthracene is phototoxic to mosquito larvae, crustaceans, fish,

" Corresponding author.

and frog embryos (Kagan, Kagan & Buhse, 1984; Oris ef al., 1984). Bluegill sunfish exposed to anthracene in sunlight are killed by asphyxiation, although fish kept in the shade survive (Bowling ef a/., 1983; Oris ef al., 1984). Whereas several bacteria metabolise anthracene to cis-1,2dihydroxy-1,2-dihydroanthraceneand eventually to CO, (Cemiglia & Heitkamp, 1989), the metabolism of anthracene by fungi is not as well known. Cunninghamella elegans Lendner ATCC 36112 converts anthracene to trans-1,2-dihydroxy-1,2dihydroanthracene (anthracene trans-1,2-dihydrodiol) and 1anthryl sulphate (Cemiglia, 1982; Cemiglia & Yang, 1984). The basidiomes of Auricularia sp. may accumulate 1.0 mg of anthracene kg-' of D.W. (Meier & Aubort, 1988). Phanerochaefe chysosporium Burdsall has been reported to produce CO, from anthracene and other PAHs in a complex mixture (Bumpus, 1989), but the metabolic pathway is unknown. We selected the strain of R. solani used in the present experiments during the screening of a group of fungi for the ability to metabolise PAHs. This paper describes the metabolism of anthracene by R. solani and the identification of four major anthracene metabolites as the trans-1,2-dihydrodiol and three xyloside conjugates of anthracene. The pathways of anthracene metabolism in fungi are also compared with those in mammals.

MATERIALS A N D M E T H O D S Maintenance and growth of cultures Stock cultures of Rhizocfonia solani, strain F-125, were maintained on slopes of malt extract agar (Difco Laboratories,

5 10

Anthracene metabolism by R. sobni Detroit, Mich.). Experimental cultures were grown in a liquid medium containing 6.0 g of malt extract, 6.0 g of D-glucose, 1.8 g of D-maltose, 1.2 g of yeast extract, and 50 mg 1-' of Tween 80 (polyoxyethylene sorbitan monooleate) of deionized water. The pH was adjusted to 5.5. Anthracene was obtained from Aldrich Chemical Co. (Milwaukee, Wisc.) and purified by silica gel column chromatography with hexane as the eluting solvent. For each litre of medium, 840 pg of anthracene was dissolved in 5.0 ml of N,N-dimethylformamide, sterilized separately, and added aseptically. In experiments requiring a labelled substrate, 0.41 pCi of [9-14C]anthracene (54.5 mCi mmol-I, Chemsyn Science Laboratories, Lenexa, Kansas) per litre of medium was added with the unlabelled anthracene. To obtain anthracene metabolites, multiple cultures of R. solani were grown in 2 1 conical flasks, each containing 500 ml of the growth medium with 840 vg 1-' of anthracene. Cultures were inoculated with mycelium from malt extract agar, suspended in sterile saline solution, and chopped in a sterile blender cup. All cultures and non-inoculated controls were incubated in dim light at 25 'C with rotary shaking at 125 rpm. After incubation for 6 d, the mycelium (D.w. ca 2 g I-') was removed by filtration. The spent medium was extracted with ethylacetate, which was dried over anhydrous sodium sulphate. The ethyl acetate was removed in a rotary vacuum evaporator and the solid residue was redissolved in methanol for analysis by liquid chromatography.

Liquid chromatography

Metabolites in the crude ethyl acetate extract were separated by reversed-phase high-performance liquid chromatography (hplc), using a model 420 liquid chromatograph (Beckman Instruments, San Ramon, Calif.) with two Beckrnan model 104 pumps. Analytical hplc was performed with a Beckman Ultrasphere C18 O D s (octadecylsilane) column (25 cm x 4.6 mm i.d.) with a mobile phase consisting of a 40-min linear gradient of methanollwater (from 50:50 to 95:5, v/v) at a flow rate of 1 ml minp'. The absorbance detector was a Beckman model 160 uv detector set at 254 nm. The uv absorption spectra of metabolites eluting from the column were obtained with a model 1040A diode array detector (Hewlett-Packard, Inc., Palo Alto, Calif.) and analysed with a Hewlett-Packard model 300 computer. In experiments using [9-14C]anthracene, fractions eluting from the hplc column were collected at 30 s intervals and counted by liquid scintillation methods (Sutherland ef al., 1990). Selected metabolites were purified by hplc for further analytical studies. compounds were eluted isocratically with a semi-preparative Ultrasphere O D s column (25 cm x 10 mm i.d.), with a mobile phase of methanollwater (80 :20, v/v) at a flow rate of 2 ml min-', and collected in test tubes.

with a model 4023 quadrupole mass spectrometer (Finnigan MAT Corp., San Jose, Calif.) and a direct exposure probe (Sutherland ef al., 1990). Gas chromatography/mass spectrometry (gcms) of alditol acetates was performed with a model 3400 gas chromatograph (Varian Instruments, Walnut Creek, Calif.) fitted with a septum-equipped, programmable injector (SPI) and an SP2330 (15 m x 0.25 mm i.d., film thickness 0.2 pm) fused silica capillary column (Supelco, Inc., Bellefonte, Penn.). The column was connected directly to the ion source of the model 4023 quadrupole mass spectrometer by a temperature-programmable interface (Davco, Inc., San Jose, Calif.). Helium at 69 kPa was the carrier gas. The temperatures of the four heated zones were as follows: SPI, 55' for 0.1 min, heated to 225' at 200' min-' and held for 15 min; column, 65' for 1min, heated to 220' at 20' min-l and held for 12 min; interface, 200' for 5 min, heated to 225' at 5' min-I and held for 10 min; ion source, 270'. Nuclear magnetic resonance spectrometry

Proton nuclear magnetic resonance ('H-NMR) spectrometry was performed with a Bruker model WM 500 NMR spectrometer (Sutherland ef al., 1990). Coupling constants (J,,,) were compared with published values for anthracene metabolites (Jerina ef al., 1976). Proton resonance assignments were made by homonuclear decoupling experiments and nuclear Overhauser effect (NOE) difference experiments. Proton-proton correlation spectra (COSY) were used to confirm some of the proton resonance assignments. Circular dichroism spectroscopy

Circular dichroism (CD) spectra were obtained in methanol with a Jasco model 500a spectropolarimeter (Sutherland ef al., 1990) and compared with CD spectra published previously (Akhtar ef a]., 1975; Cemiglia & Yang, 1984). Catalytic hydrogenation of anthracene trans-1,2dihydrodiol

Anthracene trans-1,2-dihydrodiol (0.6 mg), obtained from the fungal metabolism of anthracene, was dissolved in 1 ml of tetrahydrofuran and 0.1 ml of triethylamine. It then was hydrogenated at 104 kPa in a test tube with 5 mg of a Pd/C catalyst at ambient temperature for 2 h. The reaction mixture was filtered through Celite and washed with acetone. The filtrate was collected and the solvent evaporated under reduced pressure. The residue was purified by injection onto a Zorbax O D s column (25 cm x 9-4 mm i.d., DuPont Co., Wilmington, Del.) and isocratic elution with methanol/water (7:3, v/v) at a flow rate of 2 ml min-'. The product was identified as 1,2,3,4-tetrahydroanthracene trans-1,2-diol by comparison of the hplc retention time and uv/visible absorption spectrum with those of a synthetic standard (Von Tungeln & Fu, 1986).

Mass spectrornetty

Resolution of tefrahydrodiol enantiomers

Mass spectrometry of anthracene metabolites and their acetylated derivatives was performed by electron impact (EI)

Direct resolution of the enantiorners of 1,2,3,4-tetrahydroanthracene trans-1,2-diol was performed with a Pirkle I-A hplc

J. B. Sutherland and others

Elution time (rnin)

Fig. 1. Hplc elution profile of the metabolites produced during the growth of R. solani with [9-14C]anthracene:(a) uv absorbance at 254 nm; (B) radioactivity of the fractions eluting from the column, with the 28 min peak truncated (total height = 1.66 kdpm). Compounds 1-111 are unidentified; IV, 2-O-(1-hydroxy-frans-~,2-dihydroanthryl)--~-xylopyrmoside; V, anthracene trans-1,2-dihydrodiol; VI, 1-0-(2hydroxy-trans-1,2-dihydroanthryl)-~-~-xylopyranoside; VII, 1-0-mthryl-P-D-xylopyranoside; VIII, anthraquinone; IX, anthracene.

column, 25 cm x 4.5 mm i.d. (Von Tungeln & Fu, 1986). The enantiomers were eluted isocratically with a solvent system containing acetonitrile/hexane/ethanol (40: 2 :1, v/v/v). The resolved IR.2R and IS,2S enantiomers were characterized by their uv-visible absorption spectra and the percentage of each was determined by integrating the peak areas (Von Tungeln & Fu, 1986). Deconjuga tion of xylosides

P-Xylosidase (partially purified, 3-6 units mg-' of protein) from Aspergillus niger van Tiegh. (Sigma Chemical Co., St Louis, MO.) was used to deconjugate glycoside metabolites

so that the sugar moieties could be identified by gcms. The method was modified from one used previously to deconjugate naphthalene sulphates and glucuronides (Cemiglia, Freeman & Mitchum, 1982). Purified metabolites that had been identified as glycoside conjugates (0-3-1-7 mg) were suspended in 10 ml of 50 mM sodium acetate buffer (pH 5.0). PXylosidase (0.1 unit) was added and the reaction mixtures were incubated for 2 h at 26' with shaking at 150 rpm. Each of the reaction mixtures was extracted three times with equal volumes of ethyl acetate. The organic solvent phases were combined and dried as described above and the ethyl acetate was evaporated with a rotary vacuum evaporator. The solid residue was dissolved in 0.5 ml of methanol and chromato-

5 12

Anthracene metabolism by R. solani graphed by hplc to confirm the presence of anthracene frans1,2-dihydrodiol or anthrols. Before analysis by gcms, the sugar moieties were converted to alditol acetates by a modification of the method of Albersheim ef al. (1967). To produce the alditols, the aqueous phases from the reaction mixtures were reduced with 10 ml of 500 m ~ - N a B H ,and stirred for 60 min at 26'. Authentic Dxylose in sodium acetate buffer was treated similarly. Glacial acetic acid was added, one drop at a time with constant stirring, until the bubbling stopped. Ten millilitres of a solution containing 0.5 ml of concentrated HCl in 100 ml of methanol was added and evaporated with a rotary vacuum evaporator. This step was repeated until the sample was dry. To acetylate the alditols, 0.2 ml of pyridine and 0.4 ml of acetic anhydride were added to each of the test tubes containing the dried samples. The tubes were sealed and incubated for 16 h at 50'. Excess reagents were allowed to evaporate under a stream of argon. The residues were dissolved in 1 ml of chloroform and centrifuged at 1000 g for 5 min to pellet the chloroform-insoluble material. The supematants, containing the alditol acetates, were decanted and analysed by gcms.

showed that the isomer was anthracene trans-1,2-dihydrodiol. The circular dichroism spectrum, which showed a positive Cotton effect at 282 nm and a negative Cotton effect at 213 nm, indicated that the predominant enantiomer of the dihydrodiol was the (-)-1S,2S form (Cemiglia & Yang, 1984). Direct resolution of anthracene trans-1,2-dihydrodiol into its enantiomers with the chiral hplc column was not possible. Consequently, the dihydrodiol was converted by catalytic hydrogenation into 1,2,3,4-tetrahydroanthracene frans-1,2diol (Fig. 2a), which was resolved successfully into its enantiomers with the chiral column. The hplc results (Fig. 2 b) indicated that the metabolite contained 60% of the IS, 2 s enantiomer and 40% of the IR,2R enantiomer. Thus the optical purity of the anthracene trans-1,2-dihydrodiol formed by R. solani was 20%. The mass spectra of metabolites IV and VI (Table I), both of which show molecular ions (M+)at m/z 344 and fragment ions at m/z 194 (M+-C,H,04-H,O), 181 (M+-C5H,04CH,OH), and 165 (M+-C5H,0,-H,O-CHO), suggested that these compounds were conjugates of anthracene

RESULTS Rhizocfonia solani produced several major metabolites from [914C]anthracene (Fig. I), which were separated by analytical hplc and detected by uv absorbance at 254 nm (fig. la). Fractions collected at 30 s intervals were counted for radioactivity to show which uv-absorbing compounds had been derived from 19-14C]anthracene(Fig. 1b). The four anthracene metabolites eluting from the column at 14.3, 16.1, 17.5 and 22.5 min (compounds IV-VII) accounted for 8.8, 4.5, 24.2, and 4.6%, respectively, of the total radioactivity. The uv absorption spectra of metabolites IV, V, and VI all were similar to that of anthracene trans-1,2dihydrodiol (Cemiglia, 1982). The uv absorption spectrum of metabolite VII, with a peak at 252 nm, was similar to that of I-anthrol (Cemiglia, 1982). The mass spectrum of metabolite V, which showed a molecular ion (M+)at m/z 212 and fragment ions at m/z 194 (Mf-H,O) and 1661165 (M+-H,O-CHO), suggested that compound V was an anthracene 1,2-dihydrodiol. The 'H-NMR coupling constant for the dihydrodiol was I,,, = 10.3 Hz. Since the 'H-NMR coupling constants for the cis and frans isomers of anthracene 1,2-dihydrodiol have been reported as I,, = 4.6 and 10.5 Hz, respectively (Jerina ef al., 1976), this

,

(a)

I

0

20

I

30

I

I

40

I

50

Retention time (min) Fig. 2. (a) Catalytic hydrogenation of anthracene trans-1,2dihydrodiol; (b) chiral stationary phase hplc separation of the dihydrodiol enantiomers produced by R. solani.

Table 1. Mass spectra of the xyloside metabolites produced from anthracene by R. solanin Metabolite

Characteristic mass ions, m/z (with % relative abundance)

2-0-(1-Hydroxy-trans-~,2-dihydroanthryl)-~-o-xylopyranoside (IV)

344 [Mt1 PO), 212 (20),211 (22).196 (21),195 (76).194 (100).183 (15). 182 (11).181 (63),178 (19),177 (12),167 (241,166 (36),165 (45).152 (12), 73 (361,60 (20).57 (19),55 (18)

1-O-(2-Hydroxy-trans-1,2-dihydroanthryl)--D-xylopyranoside (VI)

344 [Mil (3.6),212 (14).211 (15).196 (15),195 (66).194 (100).183 (IZ), 181 (49),178 (21),177 (IZ),167 (24),166 (38),165 (55),152 (13),73 (29), 57 (11) 326 [M'] (4.2),195 (16).194 (loo),165 (29)

l-O-Anthryl-~-~-x~lo~~ranoside (VII) a

All mass spectra were obtained by electron impact with a direct exposure probe.

51 3

J. B. Sutherland and others Table 2. Proton NMR spectral assignments for the xyloside metabolites produced from anthracene by R. solani Metabolite VI Proton assignment

"

8 (p.p.m.)

Metabolite IV ] (Hz)

6 (p.p.m.1

Metabolite VII

I (Hz)

6 (p.p.m.)

I (Hz)

m, multiplet, s, singlet.

m/z

Fig. 3. Mass spectrum of the triacetylated derivative of the I-0-anthryl-P-D-xylopyranoside metabolite of R. solani.

dihydrodiols with five-carbon sugars. To provide further information on the structures of these conjugates, NMR spectra were obtained for compounds IV and VI. The 'H-NMR spectrum of metabolite VI was obtained first (Table 2). The aromatic resonances were consistent with a conjugate of trans-1,2-dihydroxy-1,2-dihydroanthracene (Jerina ef al., 1976). The assignments for HI-FI4 were in excellent agreement with those previously reported for the corresponding protons of anthracene trans-1,2-dihydrodiol (Jerina ef al., 1976). The H5, H6, H7, and H8 resonance assignments were based on the expected A,B, second-order spin-system type of spectra normally observed for aromatic protons in similar environments (Jerina ef al., 1976). The H9 and H I 0 assignments were based on NOE difference spectra (Table 2). The sugar moiety appeared to be in a pyranoside configuration (Capon & Thacker, 1964). A COSY experiment provided the information necessary to make the sugar resonance assignments, which were consistent with the identity of the sugar moiety as xylose. The magnitudes of the three bond-coupling constants associated with the xylose moiety were consistent with a xylopyranoside configuration. Metabolite VI was deduced from these data to be a 1-0-(2hydroxy-frans-l,2-dihydroanthryl)-xylopyranoside.

The 'H-NMR spectrum of metabolite IV (Table 2) was consistent with a 2-0-(I-hydroxy-trans-1,2-dihydroanthry1)xylopyranoside. The assignments for HI-FI4 were in excellent agreement with those previously reported for the corresponding protons of anthracene trans-1,2-dihydrodiol (Jerina ef al., 1976). The chemical shifts of the anthracene trans-1,2dihydrodiol moiety of metabolite IV were similar to those of metabolite VI and were consistent with the decoupling and NOE enhancement results. The homonuclear decoupling results for the xylose moiety were consistent with assignments made by direct comparison to those for metabolite VI. The anomeric proton of the xylose moiety was shifted upfield 0.16 ppm, compared to the anomeric proton of metabolite VI. This shift was attributed to the decrease in the aromatic-ringcurrent effect associated with substitution at the 2 - 0 position versus the 1 - 0 position. The mass spectrum of metabolite VII (Table I), which shows a molecular ion (M+)at m/z 326 and fragment ions at m/z 194 (M+-C5H,0,) and 165 (M+-C5H80,-CHO), suggested that compound VII was a conjugate of an anthrol with a five-carbon sugar. The mass spectrum of the triacetylated derivative (Fig. 3), with a molecular ion (Mf) at m/z 452, was in agreement. The 'H-NMR spectrum of metabolite VII (Table

Anthracene metabolism by R. solani

d

Retention time (min)

s 100

50

--

-

(=)

--1 103 --

---

115

127

158

*I7

187

-

289 m

100

----

145

.

r

-

r

.

l

-

r

-

-

-

p

-

!

l

-

~

~

m

300

200

-

~

-

v

-

~

~

m

-

r

-

~

-

.~ . I- -

l

-

~

-

r

-

400

m/z

Fig. 4. Gcms analysis of alditol acetates. (a) Standard mixture: 1, rhamnitol pentaacetate; 2, fucitol pentaacetate; 3, ribitol pentaacetate; 4, arabitol pentaacetate; 5, xylitol pentaacetate; 6, mannitol hexaacetate; 7, galactitol hexaacetate; 8, glucitol hexaacetate; 9, inositol pentaacetate. (b) Xylitol pentaacetate derivative of the xylose released by p-xylosidase from the 2-0-(1-hydroxy-trans-1,2dihydroanthry1)-P-D-xylopyranosidemetabolite of R. solani. (c) Mass spectrum of the xylitol pentaacetate derivative.

2) was consistent with that of a 1-0-anthrylxylopyranoside. The assignments were accomplished by homonuclear decoupling experiments and comparison to the other two xylosides (compounds IV and VI). The aromatic resonances coupled to each other at 7.13 (doublet), 7 4 0 (doublet of doublets), and 7.72 ppm (doublet) were consistent with substitution at the 1-0position of the anthracene moiety (H2, H3, and H4, respectively) (Table 2). The proton resonances of the xylose moiety were more disperse than those observed with the dihydrodiol xylosides. The anomeric proton was assigned to the most downfield (5.25 ppm) of the sugar resonances. The remainder of the resonance assignments, which were followed by selective homonuclear decoupling, were 3.78 (H2'), 3.59 (H3'), 3.69 (H4'), 4.00 (H5"), and 3.51 (H5') ppm. The observed downfield shift of the anomeric proton was expected because of the aromatic-ring-current effect. The three sugar-proton-proton bond-coupling constants were consistent with those expected for a xylopyranoside. Since compounds IV, VI, and VII had been identified by NMR as conjugates of anthracene with xylose, the nature of the conjugates was investigated further. After incubation with P-xylosidase, compounds IV and VI yielded an ethyl acetatesoluble product with a retention time and a uv absorption spectrum corresponding to those of anthracene trans-1,ddihydrodiol. Compound VII yielded a product with a retention time and a uv absorption spectrum corresponding to those of I-anthrol. The alditol acetate derivatives of the sugar moieties of metabolites IV, VI, and VII were prepared so that gcms could

be used to identify the sugars. The retention time of the major alditol acetate peak (Fig. 4) in each of the three preparations was 12.0 min, which corresponded to that of xylitol pentaacetate prepared from D-xylose by the same procedure. Each of the mass spectra (Fig. 4) lacked a molecular ion. Fragment ions, however, were found at m/z 289, 217, 187, 158, 145, 127, 115, and 103. These mass spectra also corresponded to that of xylitol pentaacetate. From these data, metabolites IV and VI were deduced to be 2-0-(l-hydroxy-trans-1,2-dihydroanthryl)-~-~-xylopyranoside and 1 - 0 - (2 - hydroxy - trans - 1 , 2 - dihydroanthryl) - j3 - D ~ylop~ranoside, respectively. Metabolite VII was deduced to be I-0-anthryl-P-D-xylopyranoside. Five other peaks were detected by hplc. Metabolites I, 11, and 111, eluting at 7.3, 10.3, and 13.1 min and representing 4.4, 4.6 and 3.2% of the 14C-labelled compounds, respectively, were not separated sufficiently from other compounds and were not identified. Compound VIII, eluting at 27.7 min, had 44.5 % of the radioactivity (Fig. 1). It had a retention time and a uv absorption spectrum identical to those of authentic anthraquinone. The same compound was also found in noninoculated controls kept in constant light. A small amount of unchanged anthracene (compound IX), eluting at 37.9 min, accounted for 1.2% of the radioactivity (Fig. I).

DISCUSSION Rhizoctonia solani transformed anthracene initially to anthracene trans-1,2-dihydrodiol, the same product that is formed from anthracene by Cunninghamella elegans (Cemiglia,

~

J. B. Sutherland and others

2-0-(1 -Hydroxy- rrans1,2-dihydroanthryl). PD-xylopyranoside

trans- l,2-dihydrodiol

3

- H,O

0 2

Anthracene Anthracene

I-0-(2-Hydroxy-trans-

1,2-oxide

1,2-dihydroanthry1)ED-xylopyranoside

000 0 Anthraquinone OH I-0-Anthryl-go. xylopyranoside

Fig. 5. Proposed metabolic pathway for the transformation of anthracene by R. solani.

Table 3. Comparison of the enantiomeric composition of the anthracene trans-1,2-dihydrodiols produced by fungi with those produced by mammalian liver microsomes % enantiomers

Optical purity

Enzyme system

S.S

R,R

(%)

Ref.'

C. elegans R. solani Rat microsomes Rat (MC-ind~ced)~ Rat (PB-induced)" Rabbit microsomes

75 60 20

25 40 80 97 88

50 20 60 94 76

this study 2 2 2

34

32

3

3

12 66

1

a References: 1, Cemiglia et al., (1990); 2, Von Tungeln & Fu (1986); 3, Hall & Grover (1987). Induced with 3-methylcholanthrene. Induced with phenobarbital.

1982). However, R. sobni then conjugated the dihydrodiol with xylose instead of sulphate. The proposed metabolic pathway (Fig. 5) is presumed to involve a cytochrome P-450dependent monooxygenation to produce anthracene 1,2oxide, followed by an epoxide hydrolase-dependent reaction to produce the tmns-dihydrodiol (Akhtar et al., 1979). Because epoxide hydrolases are highly active on anthracene 1,2-oxide (Akhtar et al., 1979), the arene oxide did not accumulate in the medium.

It is instructive to compare the stereoselectivity of anthracene trans-1,2-dihydrodiol formation by fungi with that of mammalian enzymes (Table 3). The CD spectrum of the anthracene trans-1,2-dihydrodiol produced by R. sohni was similar, although not identical, to that of the corresponding dihydrodiol produced by C. elegans (Cemiglia & Yang, 1984) and indicated that the major enantiomer had a (+)-IS,2S absolute configuration. The S,S and R,R enantiomers represented 60 and 40%, respectively, of the trans-1,2-dihydrodiol produced by R. solani (Table 3). For C. elegans, they represent 75 and 25 %, respectively (Cemiglia et al., 1990). The anthracene trans-1,2-dihydrodiol produced by rabbits (Boyland & Levi, 1935) also is predominantly (+)-IS,2S (Akhtar et al., 1975; Hall & Grover, 1987) but that produced by rats (Boyland & Levi, 1935) is predominantly (-)-lR,2R (Von Tungeln & Fu, 1986). These results suggest that the predominant cytochrome P-450 isozymes of fungi have a stereoselectivity that is different from that of the predominant isozymes of rats (van Bladeren et al., 1985 ;Hall & Grover, 1987). A more conjectural possibility is that the epoxide hydrolases of fungi preferentially catalyze the hydrolysis of only one of the enantiomers of anthracene 1,2-oxide. Conjugation of PAH metabolites by fungi is generally considered to be a detoxification reaction (Cemiglia et al., 1982). Cunninghamella elegans produces sulphate conjugates from naphthalene, anthracene, benz[a]anthracene, and benzo-

Anthracene metabolism by R. solani

5 16

[alpyrene (Cemiglia & Gibson, 1979; Cerniglia, Dodge & Ankel. H. & Feingold, D. S. (1966). Biosynthesis of widine diphosphate Dxylose. 11. Uridine diphosphate o-glucuronate carboxy-lyase of Cryptococc~ls Gibson, 1980; Cerniglia, 1982; Cerniglia et al., 1982), laurentii. Biochemistry 5 , 182-189. glucuronide conjugates from naphthalene, benz[a]anthracene, Bowling, J. W., Leversee. G. J., Landrum, P. F. & Giesy, J. P. (1983). Acute and benzo[a]pyrene (Cemiglia & Gibson, 1979; Cerniglia et al., mortality of anthracene-contaminated fish exposed to sunlight. Aquatic 1980; 1982), and glucoside conjugates from phenanthrene, Toxicology 3, 79-90. fluoranthene, and pyrene (Cemiglia et al., 1986; 1989; Boyland. E. & Levi, A. A. (1935). Metabolism of polycyclic compounds. I. Product~onof dihydroxydihydroanthracene from anthracene. Biochemical Pothuluri et al., 1990). Rhizocfonia solani detoxified anthracene ]ournal 29, 2679-2683. trans-1,2-dihydrodiol by conjugation with xylose rather than Bumpus, 1. A. (1989). Biodegradation of polycyclic aromatic hydrocarbons by sulphate as in C. elegans (Cerniglia, 1982). Phanerochaete chrysosporrum. Applied and Environmental Microbrology 55, 1- 0 ( 2 - h y d r o x y - trans- 1,2-dihydroanthry1)-P-D-xylo- 154-158. pyranoside represented 45% of the anthracene metabolites Camper, N. D. & Ellers, K. L. (1978). Degradation of nitralin by Rhizodonta solani. Weed Research 18,99-103. and 2-O(1-hydroxy-trans-~,2-dihydroanthryl)-~-~-xylopyranCapon, B. & Thacker, D. (1964). The nuclear magnetic resonance spectra of oside represented 16%. Although no anthrols were detected some aldofuranosides and acyclic aldose acetals. Proceedings of the Chemical reprein the culture medium, I-0-anthryl-P-D-xylopyranoside Society (London) 1964, 369. sented 8 % of the anthracene metabolites. I-Anthrol could Cemiglia, C. E. (1982). Initial reactions in the oxidation of anthracene by Cut~ninghamella elegans. ]ournal of General Microbiology 128. 2055have been produced by rearrangement of the arene oxide or 2061. dehydration of the dihydrodiol (Jerina et al., 1976; Fu, Harvey Cemiglia, C. E., Campbell, W. L., Freeman, J. P. & Evans, F. E. (1989). & Beland, 1978) and then conjugated with xylose. Another Identification of a novel metabolite in phenanthrene metabolism by the possibility would be the dehydration of one of the dihydrodiol fungus Cunninghamella elegans. Applied and Environmental Microbiology 55, 2275-2279. xylosides to form I-0-anthry1-P-D-xylopyranoside. Cemiglia, C. E.. Campbell, W. L., Fu, P. P., Freeman, I. P. & Evans, F. E. (1990). The origin of the xylose in the conjugates is still unknown. Stereoselective fungal metabolism of methylated anthracenes. Applied and Preliminary experiments with R. solani in media containing Environmental Microbiology 56, 661468. lose indicate that the Cemiglia, C. E., Dodge, R. H. & Gibson, D. T. (1980). Studies on the fungal anthracene and ~ - [ ~ - ' ~ C ] x ~(unpublished) 13C-labelledcarbon atom was not incorporated into anthracene oxidation of polycyclic aromatic hydrocarbons. Botanica Marina 23, 121-124. conjugates. It is more likely that UDP-glucuronic acid in the Cerniglia, C. E., Freeman, J. P. & Mitchum, R. K. (1982). Glucuronide and mycelium was decarboxylated to form UDP-xylose, as shown sulfate conjugation in the fungal metabolism of aromatic hydrocarbons. in Cyptococcus spp. (Ankel & Feingold, 1966; Jacobson & Applied and Environmental Microbiology 43. 1070-1075. Payne, 1982), and that the xylosyl moiety of UDP-xylose was Cemiglia, C. E. & Gibson, D. T. (1979). Oxidation of benzolalpyrene by the transferred to the anthracene metabolites. The presence of filamentous fungus Cunninghamella elegans. Journal of Biological Chemistry 254, 12174-12180. xylose in the hyphal walls of R. solani (Kido ef al., 1986) suggests that this fungus produces a xylosyltransferase. Cemiglia, C. E. & Heitkamp, M. A. (1989). Microbial degradation of polycyclic aromatic hydrocarbons (PAH) in the aquatic environment. In Metaboiism of Veratryl P-D-xyloside is produced by Phanerochaete chysoPolycyclic arotnutic Hydrocarbons in the Aquatic Environment (ed. U . Varanasi). sporium and Trametes versicolor (L. : Fr.) Pilit in media containing CRC Press, Boca Raton, Florida. veratryl alcohol and either xylan or holocellulose (Kondo & Cerniglia, C. E.. Kelly, D. W., Freeman, J. P. & Miller, D. W. (1986). Microbial metabolism of pyrene. Chemrco-Biological Interactions 57, 203Imamura, 1989). Xylosides are also produced from xylobiose 216. and either guaiacol or hydroquinone by the P-xylosidase of Aspergillus niger (Shinoyama et al., 1991). No organism has Cerniglia, C. E. & Yang, S. K. (1984). Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans. Applled and previously been shown to detoxify PAHs by conjugation with Environmental Microbiology 47, 119-124. xylose. Cody, T.E., Radike, M. J. & Warshawsky, D. (1984). The phototoxicity of We thank Dr H. S. Fenwick for kindly providing the culture of Rkizoctonia soiani. We also thank Mr T. M. Heinze for

helpful discussions and Miss K. Ramsay for technical assistance.

REFERENCES Akhtar, M. N., Boyd, D. R., Thompson, N. J., Koreeda, M., Gibson, D. T., Mahadevan, V. & Jerina, D. M. (1975). Absolute stereochemistry of the dihydroanthracene cis- and trans-1,2-diols produced from anthracene by mammals and bacterra. lournal of the Chemrcal Soclety Perkin Transactions 1 1975, 2506-2511. Akhtar, M. N., Hamilton. J. G., Boyd, D. R., Braunstein, A., Seifried, H. E. & Jerina, D. M. (1979). Anthracene 1,2-oxide: synthesis and role in the metabolism of anthracene by mammals.]ournal of the Chemical Society Perkrn Transactions 1 1979. 1442-1446. Albersheim, P., Nevins, D. J., English, P. D. & Karr, A. (1967). A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydrate Research 5, 340-345.

benzo[a]pyrene in the green alga Selenastrum capricornutum. Environmental Research 35, 122-132. Fu, P. P., Harvey, R. G. & Beland, F. A. (1978). Molecular orbital theoretical prediction of the isomeric products formed from reactions of arene oxides and related metabolites of polycyclic aromatic hydrocarbons. Tetrahedron 34, 85 7-866. Hall, M. & Grover, P. L. (1987).Differential stereoselectivity in the metabolism of benzo[a]pyrene and anthracene by rabbit epidermal and hepatic microsomes. Cancer Letters 38, 5 7 4 4 . Jacobson, E. S. & Payne, W. R. (1982). UDP glucuronate decarboxylase and synthesis of capsular polysaccharide in Cyptococcus neoformans. Journal of Bacteriology 152, 932-934. Jerina, D. M., Selander, H., Yagi, H., Wells. M. C., Davey, J. F., Mahadevan. V. & Gibson, D. T. (1976). Dihydrodiols from anthracene and phenanthrene. Journal of the American Chemical Socrety 98,5988-5996. Kagan, J., Kagan, P. A. & Buhse, H. E. (1984). Light-dependent toxicity of aterthienyl and anthracene toward late embryonic stages of Rana piplens. Journal of Chemical Ecology 10, 1115-1 122. Kalghatgi, K. K., Nambudiri, A. M. D., Bhat, J. V. & Subba Rao, P. V. (1974). Degradation of L-phenylalanine by Rhizoctonia solani. Indran Iournal of Biochemistry and Biophysics 11, 1 1 6 1 1 8 . Kido. Y., Nagasato, T., Ono, K., Fujimoto, Y., Uyeda, M. & Shibata, M. (1986). Change in a cell-wall component of Rhiwctonia solani inhibited by validamycin. Agrrcultural and Biological Chemistry 50, 1519-1525.

J. B. Sutherland and others Kohmoto, K.. Nishimura, S. & Hiroe, I. (1970). Pathochemical studies on Rhizoctonia disease. 1. Meta-hydroxylation of phenylacetic acid by Rhizoctonia solani. Phytopathology 60, 1025-1026. Kondo, R. & Imamura, H. (1989). Formation of lignin model xyloside in polysaccharides media by wood-rotting fungi. Mokumr Gakkaishi 35, 1001-1007. Meier, P. & Aubort, J.-D. (1988). Polycyclic aromatic hydrocarbons in dried mushrooms. Mitteilungen aus dem Gebiete der Lebensmitteluntersuchung itnd Hygiene 79, 433-439. Mitchell. R. L., Burchett, M. D., Pulkownik, A. & McCluskey, L. (1988). Effects of environmentally hazardous chemicals on the emergence and early growth of selected Australian plants. Plant and Soil 112, 195-199. Moore. M. N., Lowe, D. M. & Fieth, P. E. M. (1978). Lysosomal responses to experimentally injected anthracene in the digestive cells of Mytilus edulis. Marine Biology 48, 297-302. Mordue, J. E. M., Currah, R. S. & Bridge, P. D. (1989). An integrated approach to Rhizoctonia taxonomy: cultural, biochemical and numerical techniques. Mycological Research 92, 78-90. Myers, S. R., Blake, J. W. & Flesher, J. W. (1988). Bialkylation and biooxidation of anthracene, in uitro and in viuo. Biochemical and Biophysical Research Communications 15 1, 1441-1445. Ogoshi, A., Cook, R. J. & Bassett, E. N. (1990). Rhiwctonia species and anastomosis groups causing root rot of wheat and barley in the Pacific Northwest. Phytopathology 80, 784-788. Oris, J. T., Giesy, J. P., Allred, P. M., G h t , D. F. & Landrum, P. F. (1984). Photoinduced toxicity of anthracene in aquatic organisms: an environmental perspective. In The Biosphere: Problems and Soiufions (ed. T. N. Veziroglu). Elsevier Science Publishers, Amsterdam. Pothuluri, J. V., Freeman. J. P., Evans, F. E. & Cemiglia, C. E. (1990). Fungal transformation of fluoranthene. Applied and Enuironmental A4icrobiology 56, 2974-2983.

(Accepted 15 October 1991)

517 Rickard, R. W. & Camper, N. D. (1978). Degradation of fluometuron by Rhizoctonia solani. Pesticide Biochemistry and Physiology 9, 183-189. Shinoyama, H., Ando, A., Fujii, T. & Yasui. T. (1991). The possibility of enzymatic synthesis of a variety of P-xylosides using the transfer reaction of Aspergillus niger P-xylosidase. Agricultural and Biological Chemistry 55, 849-850. Sinha. B. K. & Chignell, C. F. (1983). Binding of anthracene to cellular macromolecules in the presence of light. Photochemistry and Photobiology 37, 33-37. Smith, A. E. & Phillips, D. V. (1975). Degradation of alachlor by Rhizoctonia solani. Agronomy Journal 67, 347-349. Sutherland, J. B., Freeman. I. P., Selby, A. L., Fu, P. P., Miller, D. W. & Cemiglia. C. E. (1990). Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptowiyces fluuovirens. Archives of Microbiology 154, 260-266. Tuveson, R. W., Wang, G.-R., Wang, T. P. & Kagan, J. (1990). Lightdependent cytotoxic reactions of anthracene. Photochemistry and Photobiology 52, 993-1002. van Bladeren, P. J., Sayer, J. M., Ryan, D. E., Thomas, P. E., Levin, W. & Jerina, D. M. (1985). Differential stereoselectivity of cytochromes P-450b and P450c in the formation of naphthalene and anthracene 1,2-oxides. The role of epoxide hydrolase in determining the enantiomer composition of the 1,2-dihydrodiols formed. Journal of Biological Chemistry 260, 1022610235. Von Tungeln, L. S. & Fu, P. P. (1986). Stereoselective metabolism of 9methyl-, 9-hydroxymethyl- and 9.10-dimethylanthracenes. Absolute configurations and optical purities of trans-dihydrodioi metabolites. Carcinogenesis 7, 1135-1141. Weinberger, M. & Bollag, J.-M. (1972). Degradation of chlorbromuron and related compounds by the fungus Rhizoctonia solani. Applied Microbiology 24, 750-754.