Soluble and microsomal glutatione S-transferase activities in pea seedlings (Pisum sativum L.)

Epicotyl and primary leaves of pea seedlings (Pisum sativum L., var. Alaska) were found to contain soluble and microsomal enzymes catalyzing the addition of glutathione to the olefinic double bond of cinnamic acid. Glutathione S-cinnamoyl transfer was also obtained with enzyme preparations from potato slices and cell suspension cultures of parsley and soybean.The pea transferases had pH-optima between pH 7.4 and 7.8 K_m-values were 0.1–0.4 mM and 1–4 mM for cinnamic acid and glutathione, respectively. V-values were between 2–15 nmol mg^-1 protein x min.Chromatography on Sephacryl S-200 indicated that the soluble pea glutathione S-cinnamoyl transferase activity existed in molecular weight forms of 37,000, 75,000, and 150,000. The glutathione-dependent cleavage of the herbicide fluorodifen was catalyzed by a different soluble enzyme activity which eluted in molecular weight positions of 47,000 and/or 82,000.The microsomal fraction from pea primary leaves also catalyzed the conjugation of the carcinogen benzo[]pyrene with glutathione.Abbreviations GSH glutathione - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDMU l-Chloro-2,2-bis-(4-chlorophenyl)-ethylene     

Metabolism of DDT and Kelthane in cell suspension cultures of parsley (Petroselinum hortense,Hoffm.) and soybean (Glycine max L.)

Cell suspension cultures of parsley and soybean were incubated for 44 to 48 h with¹⁴C-labeled DDT or Kelthane; autoclaved cultures were used as controls. Most of the radioactivity became associated with the cells, and metabolites were isolated by a sequential extraction procedure. The metabolites amounted to 0.6 to 2.2% of the applied pesticide. Relatively non-polar metabolites were identified as DDE in the case of DDT, and remained unidentified in the case of Kelthane. Polar metabolites were also isolated and are as yet unidentified. They were chromatographically different from the known and less polar metabolites of DDT and Kelthane reported from animal and insect studies. [DDT-1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane; Kelthane=(1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol; DDE=1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene.]Abbreviations DDT 1,1,1-Trichloro-2,2-bis-(4-chlorophenyl)-ethane - Kelthane (1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-ethanol - DDE 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethylene - DDA 2,2-bis-(4-chlorophenyl)-acetic acid - DDOH 2,2-bis-(4-chlorophenyl)-ethanol - DDD 1,1-Dichloro-2,2-bis-(4-chlorophenyl)-ethane - DBP 4,4-Dichloro-benzophenone - DDMU 1-Chloro-2,2-bis-(4-chlorophenyl)-ethylene - DDM Bis-(4-chlorophenyl)-methane - FW-152 1,1-Bis-(4-chlorophenyl)-2,2-dichloro-ethanol - SDS sodium dodecylsulphate     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Metabolism and binding of benzo[a]pyrene in randomly-proliferating,confluent and s-phase human skin fibroblasts

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-³H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.Abbreviations BaP benzo[a]pyrene - BaP-4,5-diol trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene - BaP-7,8-diol trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene - Bap-9,10-diol trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene - CM complete medium - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - PAH polycyclic aromatic hydrocarbon - PDL population doubling - RP randomly proliferating     

Mycorrhization alleviates benzo[a]pyrene-induced oxidative stress in an in vitro chicory root model

Among chemicals that are widely spread both in terrestrial and aquatic ecosystems, benzo[a]pyrene is a major source of concern. However, little is known about its adverse effects on plants, as well as about the role of mycorrhization in protection of plant grown in benzo[a]pyrene-polluted conditions. Hence, to contribute to a better understanding of the adverse effects of polycyclic aromatic hydrocarbons on the partners of mycorrhizal symbiotic association, benzo[a]pyrene-induced oxidative stress was studied in transformed Cichorium intybus roots grown in vitro and colonized or not by Glomus intraradices. The arbuscular mycorrhizal fungus development (colonization, extraradical hyphae length, and spore formation) was significantly reduced in response to increasing concentrations of benzo[a]pyrene (35–280 μM). The higher length of arbuscular mycorrhizal roots, compared to non-arbuscular mycorrhizal roots following benzo[a]pyrene exposure, pointed out a lower toxicity of benzo[a]pyrene in arbuscular mycorrhizal roots, thereby suggesting protection of the roots by mycorrhization. Accordingly, in benzo[a]pyrene-exposed arbuscular mycorrhizal roots, statistically significant decreases were observed in malondialdehyde concentration and 8-hydroxy-2′-desoxyguanosine formation. The higher superoxide dismutase activity detected in mycorrhizal chicory roots could explain the benzo[a]pyrene tolerance of the colonized roots. Taken together, these results support an essential role of mycorrhizal fungi in protecting plants submitted to polycyclic aromatic hydrocarbon, notably by reducing polycyclic aromatic hydrocarbon-induced oxidative stress damage.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Evaluation of automated electrospray-TOF mass spectrometryfor metabolic fingerprinting of the plant metabolome

Metabolic fingerprinting is increasingly employed in microbial and plant metabolomics. Identification and evaluation of analytical factors that influence mass spectra produced with automated electrospray time of flight mass spectrometry to support metabolic fingerprinting are described. Instrument resolution of 4000 (FWHM) at mass 200 Da provided detection of ions of the same nominal mass but different monoisotopic masses. Complex mass spectra were obtained from polar extracts of tomato fruit in positive and negative ion mode. These spectra consist of metabolite ions (molecular, adduct and fragment) and those derived from the extraction medium, largely in the form of [M+H]⁺, [M–H]^–, [M+Na]⁺, [M+K]⁺, [2M+H]⁺, [M+Cl]^– and [2M–H]^–. Ionisation suppression reduced sensitivity, although its effect was consistent for a wide range of metabolite concentrations. Variability in ion signal intensity was lower in analytical (2.2–30.1%) compared to biological (within fruit 9.6–27.6%; between-fruit 13.2–34.4%) replicates. The method is applicable to high throughput metabolic fingerprinting and, with accurate mass measurements, is able to provide reductions in data complexity and preliminary identification of metabolites.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Differential protein synthesis in response to sulphate and phosphate deprivation: Identification of possible components of plasma-membrane transport systems in cultured tomato roots

Isolated roots of Lycopersicon esculentum Mill., cultured in axenic conditions were starved of sulphate or phosphate, and uptake capacities for the respective oxyanion-transport systems were observed for several days after sulphate or phosphate withdrawal. Sulphate-uptake capacity of the intact roots, measured in a 20-min period, increased from a control level of 100 nmol · g^–1 · h^–1 to 1100 nmol · g^–1 · h^–1 in 10 d, and phosphate-uptake capacity increased from 500 to 1400 nmol · g^–1 · h^–1 over 4 d. Newly synthesised polypeptides of these root cultures were pulse-labelled in vivo for 2 h, by adding [³H]leucine to the culture medium. The tissue was immediately homogenised and soluble and membrane fractions were prepared. A highly purified plasma-membrane fraction was separated from the crude microsomal membrane fraction using an aqueous two-phase partitioning technique. All fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. A 28-kilodalton (kDa) soluble polypeptide, and 36-, 43-, and 47-kDa plasma-membrane polypeptides were observed to have increased labelling after 4 d of sulphate deprivation. Longer periods resulted in additional polypeptides with increased [³H]leucine incorporation. The synthesis of a 25-kDa membrane polypeptide and a 65-kDa soluble polypeptide was increased after 4 d of phosphate deprivation. Two-dimensional electrophoresis afforded greater resolution of the plasmamembrane polypeptides, confirming increased synthesis of the 36-kDa polypeptide and the presence of the 28-kDa polypeptide in the plasma-membrane preparation from sulphate-starved roots. These polypeptides were also observed in protein-stained two-dimensional gels as low-abundant protein components of the plasmamembrane fraction. It is suggested that the 36-kDa polypeptide may be a component of the plasma-membrane sulphate-transport system and that the 25-kDa polypeptide may be a component of a phosphate-transport system.Abbreviations kDa kilodalton(s) - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - SDS Sodium dodecyl sulphate This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station. We are also grateful for discussions with our colleagues D.T. Clarkson (LARS) and J.-C. Davidian (ENSA/INRA, Montpellier).     

Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18 % in young cultures and 28 % in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27 % in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.     

Fatty-acid composition and biosynthesis in cell suspension cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures — which were normally grown at 25° C — at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [¹⁴C]acetate into [¹⁴C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [¹⁴C] label was distributed mainly between palmitic and oleic acids.     

Neonatal modulation of adult rat hepatic microsomal benzo[a]pyrene hydroxylase activities by Aroclor 1254 or phenobarbital

The constitutive and Aroclor 1254-induced activities of hepatic microsomal benzo[a]pyrene hydroxylases in male and female rats were determined in animals from ages 11 to 120 days. In 11-day-old noninduced male rats, benzo[a]pyrenediones and 9-hydroxybenzo[a]pyrene were the major microsomal metabolites; in 21-day-old males benzo[a]pyrene-diones and benzo[a]pyrene-9,10-dihydrodiol were predominant. In 60- and 120-day-old animals 3-hydroxybenzo[a]pyrene was the major microsomal metabolite. A similar trend was observed for the development of benzo[a]pyrene hydroxylase activities in female rats. With the exception of 4,5-dihydrodiol formation, the highest induction of individual and total benzo[a]pyrene hydroxylase activities by Aroclor 1254 was observed in the 21-day-old immature male rats, in which there was a 330- and 4.5-fold increase in the formation of 3-hydroxybenzo[a]pyrene and quinone metabolites, respectively. The induction of benzo[a]pyrene total metabolite formation by Aroclor 1254 in female rats from 11 to 120 days of age was relatively constant (i.e., 13.3- to 10.1-fold induction); however, the relative induction of the individual benzo[a]pyrene hydroxylases was highly variable. In a second set of experiments, male and female rats were neonatally exposed to phenobarbital (600 mumol/kg) or Aroclor 1254 (100 mumol/kg), and the effects of these xenobiotics on neonatal imprinting of hepatic microsomal benzo[a]pyrene hydroxylase activities were determined in the 120-day-old animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mineralization of benzo[a]pyrene by Marasmiellus troyanus, a mushroom isolated from a toxic waste site.

Mycelia from the mushroom Marasmiellus troyanus were grown in the presence of radiolabeled benzo[a]pyrene in liquid culture. After 15 days, 8.1% of the label from M. troyanus cultures was recovered in CO2 as compared to 1.1% for Phanerochaete chrysosporium and 0.2% for Aspergillus niger. M. troyanus efficiently transformed B[a]P into water soluble metabolites with 64% of the label recovered in the water soluble fraction as compared to 11.7% for P. chrysosporium and 4.1% for A. niger. Glucuronic acid and sulfate conjugates of B[a]P were identified from the aqueous fraction of cultures of M. troyanus, after 17 days.     

Development of an embryogenic suspension culture of soybean (Glycine max Merrill.)

A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1^–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration.     

Pyrene and benzo(a)pyrene Metabolism by an Aspergillus Terreus Strain Isolated from a Polycylic Aromatic Hydrocarbons Polluted Soil

A strain of Aspergillus terreus was isolated from a polycyclic aromatic hydrocarbons (PAHs) polluted soil. The metabolism of pyrene and benzo(a)pyrene by this fungus was investigated in liquid submerged culture added of 50 and 25 ppm respectively of each compound. Depletion of pyrene and Benzo(a)pyrene was evident during the first stages of growth and was 60% and 27.5% respectively of the added amount after nine days of culture. Solvent extracts of the fermentation broth and mycelium were analysed for presence of metabolites by HPLC-MS technique. Under the present cultural conditions pyrene was mainly metabolised to pyrenylsulfate similarly to benzo(a)pyrene that led to benzo(a)pyrenylsulfate. The structure of 1-pyrenilsulfate was determined after purification of extracts and H-NMR analysis. The result show that the isolated A. terreus strain metabolises PAHs by reaction similar to those previously reported for non lignolinolytic fungi with a mechanism that suggests the hydroxylation by a cytochrome P-450 monooxygenase followed by conjugation with sulfate ion.     

Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains

We previously hypothesized that polycyclic aromatic hydrocarbon (PAH)-degrading bacteria that produce laccase may enhance the degree of benzo[a]pyrene mineralization. However, whether the metabolites of benzo[a]pyrene oxidized by laccase can be further transformed by PAH degraders remains unknown. In this study, pyrene-degrading mycobacteria with diverse degradation properties were isolated and employed for investigating the subsequent transformation on the metabolites of benzo[a]pyrene oxidized by fungal laccase of Trametes versicolor. The results confirm the successive transformation of benzo[a]pyrene metabolites, 6-benzo[a]pyrenyl acetate, and quinones by Mycobacterium strains, and report the discovery of the involvement of a O-methylation mediated pathway in the process. In detail, the vast majority of metabolite 6-benzo[a]pyrenyl acetate was transformed into benzo[a]pyrene quinones or methoxybenzo[a]pyrene, via two distinct steps that were controlled by the catechol-O-methyltransferase mediated O-methylation, while quinones were reduced to dihydroxybenzo[a]pyrene and further transformed into dimethoxy derivatives.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Uptake of isolated plant chromosomes by plant protoplasts

For mass isolation of plant metaphase chromosomes, cultured cells of wheat (Triticum monococcum) and parsley (Petroselinum hortense) were synchronized by hydroxyurea and colchicine treatment. This synchronization procedure resulted in high mitotic synchrony, especially in suspension cultures of parsley in which 80% of the cells were found to be at the metaphase stage. Mitotic protoplasts isolated from these synchronized cell cultures served as a source for isolation of chromosomes. The described isolation and purification method yielded relatively pure chromosome suspension. The uptake of the isolated plant chromosomes into recipient wheat, parsley, and maize protoplasts was induced by polyethylene-glycol treatment. Cytological studies provided evidences for uptake of plant chromosomes into plant protoplasts.Abbreviations PEG polyethylene glycol - HU hydroxyruea - C colchicine - HUC hydroxyurea and colchicine - CIM chromosome isolation medium - TCM Tris chromosome medium     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

Formation of sulfate and glucoside conjugates of benzo[e]pyrene by Cunninghamella elegans

 Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-³H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by ¹H and ¹³C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside. Received: 7 September 1995/Received revision: 14 November 1995/Accepted: 11 December 1995