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Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are formed in the environment by combustion processes (24) and by atmospheric reactions of PAHs with hydroxyl and nitrate radicals (1). Because nitro-PAHs are not only widespread environmental contaminants but are also genotoxic, they pose a health risk to humans (17, 25).

6-Nitrochrysene (19) is weakly mutagenic in bacteria and initiates tumors in mice; three possible trans-dihydrodiol metabolites may be responsible for the mutagenic and tumorigenic activities (13). Although it is not as mutagenic as 1-nitropyrene (35), 6-nitrochrysene has extraordinary potency as a lung and liver carcinogen in mice (36). The covalent binding of the primary metabolites of 6-nitrochrysene to DNA in vitro suggests that the metabolites may also produce carcinogen-DNA adducts in mice in vivo (10-12, 21, 36). In rats, both aromatic ring oxidation and nitroreduction have been implicated in the activation of 6-nitrochrysene as a colon carcinogen (8).

The microbial metabolism of nitro-PAHs was recently reviewed (7, 26, 33). Many anaerobic and aerobic bacteria reduce nitro-PAHs to mutagenic amino-PAHs (7, 23, 32). The intestinal microflora is thought to play an important role in the toxicity of 6-nitrochrysene because nitroreduction is the critical step that leads to DNA binding (10, 22).

The filamentous fungus Cunninghamella elegans, which has been studied extensively for its ability to biotransform PAHs (2, 26, 34), metabolizes nitro-PAHs via cytochrome P-450 monooxygenases to form nitroarene oxides (7). The arene oxides can either undergo nonenzymatic rearrangement to form nitrophenols, which can be conjugated with sulfate, glucose, or glucuronic acid, or else be enzymatically hydrated by epoxide hydrolase to form nitroarene trans-dihydrodiols (5, 23, 26). Fungi have been shown to metabolize 1-nitropyrene (5), 6-nitrobenzo[a]pyrene (23), 2- and 3-nitrofluoranthenes (27, 28), and 2-nitrofluorene (29). C. elegans metabolizes nitro-PAHs to products that are generally less mutagenic than the parent compounds (5), whereas mammalian systems metabolize them to products that are often more mutagenic (7, 13, 24). Several fungi have already been shown to metabolize unsubstituted chrysene (20, 31); in the present study, we report the metabolism by C. elegans of 6-nitrochrysene.

Cultures of C. elegans ATCC 36112 were grown as described previously (31) except that 2.0 mg of 6-nitrochrysene, dissolved in 0.5 ml of dimethyl sulfoxide, was added to each culture and to noninoculated controls. All flasks were incubated for an additional 96 h in the dark. Ethyl acetate was used for extraction as previously described (31).

6-Nitro[U-³H]chrysene (specific activity, 2.98 mCi/mmol; radiochemical purity, >98%) and unlabeled 6-nitrochrysene (purity, 99%) were purchased from Chemsyn Science Laboratories (Lenexa, Kans.). All other chemicals were of reagent grade and the highest purity available.

Biotransformation experiments were conducted by adding 1.58 µCi of 6-nitro[³H]chrysene and 2 mg of unlabeled 6-nitrochrysene to culture flasks. Fungal metabolites of 6-nitrochrysene were separated by high-performance liquid chromatography (HPLC) (28). The percent metabolism to various products was quantified by fraction collection and liquid scintillation methods (31). Metabolites were collected from repeated injections; fractions with similar retention times were pooled and concentrated in vacuo. The metabolites that eluted near each other were further purified by HPLC (28).

UV-visible absorption spectra of the purified metabolites in methanol were determined with a Shimadzu (Kyoto, Japan) model UV-2101PC spectrophotometer. Mass spectral analyses were performed with a Finnigan Corp. (San Jose, Calif.) MAT TSQ 700 mass spectrometer. Samples were dissolved in methanol and analyzed by direct exposure probe-electron ionization-mass spectrometry (DEP-EI-MS). The first quadrupole of the mass spectrometer was scanned from 50 to 650 Da in 1 s. The ion source temperature was 150°C, and the electron energy was 70 V. The DEP current was ramped from 0 to 800 mA at 5 mA/s. Nuclear magnetic resonance (NMR) spectra were recorded in the ¹H configuration at 500.13 MHz on a Bruker Instruments (Billerica, Mass.) AM500 spectrometer at 28°C. Samples were dissolved in deuterated acetone; chemical shifts are reported on the  scale by assigning the residual proton resonance of the deuterated solvent to 2.05 ppm (15).

Metabolites that were thought to be sulfates were deconjugated by adding equal quantities to two test tubes, each containing 2 ml of 0.2 M Tris-HCl buffer (pH 7.2). To one tube, 3.0 U (210 µl) of arylsulfatase (type V; Sigma Chemical Co., St. Louis, Mo.) was added; the second tube served as a control. The products were extracted with ethyl acetate (28).

When the cultures of C. elegans incubated with 6-nitro[³H]chrysene were extracted, about 40% of the total radioactivity added was recovered in the organic-soluble phase; the remainder was bound to the mycelia. The ³H-labeled fractions, which were separated by HPLC and detected by liquid scintillation methods, eluted at 7 to 10 min and at 43 min (Fig. 1). The elution profile of the ethyl acetate-soluble metabolites formed during incubation of 6-nitrochrysene with C. elegans, with the UV detector at 254 nm (Fig. 1), shows two major peaks, at 8.0 and 9.0 min. The residual 6-nitrochrysene peak eluted at 43.0 min (Fig. 1). The kinetics of the disappearance of 6-nitro[³H]chrysene and the appearance of the two labeled metabolites during incubation with C. elegans for 8 days are shown in the Fig. 1 inset. Since 62% of the radioactivity recovered in the organic phase at time zero was found in the 6-nitrochrysene peak, the absolute recovered radioactivity of 6-nitrochrysene was adjusted to 100% to correct for extraction efficiency. At 48 h, the 6-nitrochrysene peak had decreased to about 54% of the radioactivity at time zero, while the two metabolites together accounted for about 36%. At 144 h, metabolites I and II together accounted for 74% of the recovered radioactivity and 26% remained as 6-nitrochrysene (Fig. 1, inset). Sterile control flasks dosed with 6-nitrochrysene showed no changes.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   HPLC elution profile (A254) and radioactivity (disintegrations per minute) of the ethyl acetate-soluble metabolites formed by C. elegans in 4 days from 6-nitro[3H]chrysene. Fractions eluting from the column were collected at 0.5-min intervals, and the radioactivity was measured by liquid scintillation counting. The inset shows the disappearance of 6-nitro[3H]chrysene and formation of 3H-labeled metabolites in cultures of C. elegans.

The structures of metabolites I and II were analyzed by NMR spectroscopy and MS. In the ¹H-NMR analysis (Table 1), a comparison of the chemical shifts of the protons in metabolites I and II with those in 6-nitrochrysene shows small upfield shifts of the protons ortho to the C-1 and C-2 substitutions, respectively. These chemical shifts are inconsistent with simple phenol substitution but consistent with sulfate conjugation. The absence of aliphatic resonances eliminates the possibility of conjugation with glucose or glucuronic acid. The DEP-EI mass spectra of both metabolites (Table 1) reveal apparent molecular ions at m/z 289 and characteristic fragment ions at m/z 259 (M⁺  30). At a slightly higher probe current, additional ions for metabolites I and II, which may have been produced by heating of sulfates under vacuum during the DEP-MS analysis, were observed.

Because the NMR data for metabolites I and II suggested the presence of sulfate groups, metabolites I and II were treated with arylsulfatase and the HPLC, UV-visible, and mass spectral analyses were repeated. The HPLC elution profiles of metabolite I before and after treatment with arylsulfatase show an increase in retention time from 8.0 to 20.5 min. Similarly, the elution profiles of metabolite II before and after treatment with arylsulfatase show an increase in retention time from 9.0 to 21.0 min. The UV spectra of the two metabolites, which were similar to each other before arylsulfatase treatment, had changed to two other similar spectra after treatment. The mass spectra of the arylsulfatase-treated metabolites (data not shown) had molecular and fragment ions characteristic of hydroxy-6-nitrochrysenes. Based on the increases in HPLC retention times after deconjugation with arylsulfatase, on analyses of the deconjugated metabolites by UV-visible spectroscopy and MS, and on analyses of the untreated conjugates by NMR, metabolites I and II were identified as 6-nitrochrysene 1-sulfate and 6-nitrochrysene 2-sulfate, respectively.

The metabolism of PAHs and nitro-PAHs by C. elegans often includes both phase I (oxidation) and phase II (conjugation) steps (4, 26, 37). C. elegans metabolizes unsubstituted chrysene to the sulfate conjugates of 2-hydroxychrysene and 2,8-dihydroxychrysene (31). In the present study, C. elegans oxidized 6-nitrochrysene to two hydroxylated intermediates, which it then conjugated with sulfate (Fig. 2). The mechanism presumably involved a cytochrome P-450 monooxygenase reaction (16) to form the 1,2-epoxide, followed by a nonenzymatic rearrangement via a National Institutes of Health shift mechanism (3) to form the two isomeric phenols. Subsequent transfer of sulfate groups formed the conjugates of 6-nitrochrysene, as previously demonstrated with C. elegans for other xenobiotics (4, 6, 23, 37). The same fungus also transforms 2- and 3-nitrofluoranthenes to the 8- and 9-sulfates (27, 28); the nitro group shifts the oxidation to the C-8 and C-9 positions of the 3-nitrofluoranthene molecule (28). It also forms a sulfate conjugate from 1-hydroxy-6-nitrobenzo[a]pyrene (23). However, C. elegans forms glucoside conjugates from 6- and 8-hydroxy-1-nitropyrene (5) and from 1- and 3-hydroxy-6-nitrobenzo[a]pyrene. The sulfates and glucosides of PAHs and nitro-PAHs are usually considered detoxification products (4, 30). For several other xenobiotic compounds, however, sulfate conjugation results in bioactivation (18).

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Proposed pathways for the fungal metabolism of 6-nitrochrysene by C. elegans. The epoxide structure in brackets is a proposed intermediate that has not been detected.

In mice, the major activation pathway of 6-nitrochrysene leads through the proximate tumorigen trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene and then to 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-aminochrysene (14). The formation of the tumorigenic trans-dihydrodiol can lead to formation of carcinogen-DNA adducts (8, 11, 12, 21). Human liver and lung tissues also metabolize 6-nitrochrysene to carcinogenic metabolites via ring oxidation and nitroreduction mediated by cytochrome P-450 enzymes (9). In contrast to the mammalian activation pathways, the formation of sulfate metabolites from 6-nitrochrysene by C. elegans is likely to lead toward detoxification. Previously, C. elegans was shown to reduce the mutagenicity of 1-nitropyrene (5) and several other xenobiotics (2, 7, 26, 30, 37). Considering that the mammalian metabolism of 6-nitrochrysene forms mutagens and carcinogens, the formation of less toxic phenols and conjugates by fungi is desirable and C. elegans may prove to be useful in the bioremediation of wastes containing nitro-PAHs.