Biotransformation Of Pyrene Research Articles

Risk reductions during pyrene biotransformation and mobilization in a model plant-bacteria-biochar system

The productive application of motile microorganisms for degrading hydrophobic contaminants in soil is one of the most promising processes in modern remediation due to its sustainability and low cost. However, the incomplete biodegradation of the contaminants and the formation of the intermediary metabolites in the process may increase the toxicity in soil during bioremediation, and motile inoculants may mobilize the pollutants through biosorption. Therefore, controlling these factors should be a fundamental part of soil remediation approaches. The aim of this study was to evaluate the sources of risk associated with the cometabolism-based transformation of 14C-labeled pyrene by inoculated Pseudomonas putida G7 and identify ways to minimize risk. Our model scenario examined the increase in bioaccessibility to a distant source of contamination facilitated by sunflower (Helianthus annuus L.) roots. A biochar trap for mobilized pollutant metabolites and bacteria has also been employed. The experimental design consisted of pots filled with a layer of sand with 14C-labeled pyrene (88 mg kg−1) as a contamination focus located several centimeters from the inoculation point. Half of the pots included a biochar layer at the bottom. The pots were incubated in a greenhouse with sunflower plants and P. putida G7 bacteria. Pots with sunflower plants showed a higher biodegradation of pyrene, its mobilization as metabolites through the percolate and the roots, and bacterial mobilization toward the source of contamination, also resulting in increased pyrene transformation. In addition, the biochar layer efficiently reduced the concentrations of pyrene metabolites collected in the leachates. Therefore, the combination of plants, motile bacteria and biochar safely reduced the risk caused by the biological transformation of pyrene.

Cometabolic benzo[a]pyrene biotransformation by Sphingobium barthaii KK22 proceeds through the kata-annelated ring and 1-pyrenecarboxylic acid to downstream products.

Benzo[a]pyrene is a hazardous environmental pollutant that is resistant to bacterial biodegradation and biotransformation pathway development has not been advanced. Considering this, cometabolic benzo[a]pyrene biotransformation by a member of the genus Sphingobium was investigated by cell growth on phenanthrene as a carbon and energy source in conjunction with comparative analyses of culture extracts by gas chromatography (GC) and liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Under these culture conditions, approximately 20% of 100 mg/L benzo[a]pyrene (apparent solubility) was biodegraded in 14 days and benzo[a]pyrene biotransformation products were detected and identified to be extradiol and intradiol ring-cleavage products of the benzo[a]pyrene kata-annelated ring. The o‑hydroxy-tetraaromatic acids, 2-hydroxypyrene-1-carboxylic acid, and 1-hydroxypyrene-2-carboxylic acid were identified in addition to 7,8- and 9,10-dihydroxy-benzo[a]pyrenes, carboxyvinylpyrene-carboxylic acids, hydroxypyrene-oxobutenoic acids, pyrene-1,2-dicarboxylic acid, 1,2-dihydroxypyrene and different oxophenalene compounds. 1-Pyrenecarboxylic acid and other ring-opened oxidized compounds were identified as novel benzo[a]pyrene biodegradation products. In separate assays, 2-hydroxypyrene-1-carboxylic acid was found to be produced from 1-pyrenecarboxylic acid and these results reshaped our understanding of bacterial benzo[a]pyrene biotransformation pathways. Overall, the identities of thirteen products were proposed and new benzo[a]pyrene biodegradation pathways were constructed from experimental chemical evidence. This work extends our understanding of benzo[a]pyrene biotransformation by gram-negative bacteria.

Biotransformation of pyrene in soil in the presence of earthworm Eisenia fetida

Abstract Pyrene, a toxic four benzene ring polycyclic aromatic hydrocarbons (PAHs) that persists in the environment, is highly resistant to degradation. Hence, this study aims to investigate the pyrene degradation by the combination of earthworm (Eisenia fetida) and some microbes in the soil. The artificially contaminated soil (200 g) was prepared in a container box by the addition of pyrene and combined with the addition of the earthworms and some microbes. The results showed that no mortality of earthworms in all soil conditions. The largest amount of pyrene (68.7%) was removed by earthworm microbes in the unsterilized soil. Twelve enzymes were produced such as peroxidase, laccase, invertase, glucosidase, phosphatase, phytase, urease, hydrolase, chitinase, nitrogenase, aminopeptidase, and arylsulfatase. The combination of earthworm and microbes transformed pyrene to protocatechuic acid via pyrene-4,5-dione, phenanthrene-4,5-dicarboxylic acid, and phenanthrene-4-carboxylic acid. But, these compounds were not confirmed in our extract.

Performance of Three-Dimensional Rainbow Trout (Oncorhynchus mykiss) Hepatocyte Spheroids for Evaluating Biotransformation of Pyrene.

The aquatic bioconcentration of a chemical is typically determined using conventional fish tests. To foster the approach of alternatives to animal testing, a combination of computational models and in vitro substrate depletion bioassays (e.g., primary hepatocytes) can be used. One recently developed in vitro assay is the three-dimensional (3D) hepatic spheroid model from rainbow trout (Oncorhynchus mykiss). The aim of the present study was to evaluate the metabolic competence of the 3D spheroids from rainbow trout when exposed to pyrene, using 2 different sampling procedures (SP1 and SP2). The results were compared with previously published intrinsic clearance (CL) results from S9 fractions and primary hepatocyte assays. Extraction of pyrene using SP1 suggested that the spheroids had depleted 33% of the pyrene within 4 h of exposure, reducing to 91% after 30 h. However, when applying SP2 a substantial amount (36%) of the pyrene was bound to the exposure vial within 2 h, decreasing after 6 h of exposure. Formation of hydroxypyrene-glucuronide (OH-PYR-Glu) was obtained throughout the study, displaying the metabolic competence of the 3D spheroids. The 2 sampling procedures yielded different CLin vitro , where pyrene depletion using SP2 was very similar to published studies using primary hepatocytes. The 3D spheroids demonstrated reproducibile, log-linear biotransformation of pyrene and displayed formation of OH-PYR-Glu, indicating their metabolic competence for 30 h or more. Environ Toxicol Chem 2019;38:1738-1747. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Alteration of benzo(a)pyrene biotransformation by resveratrol in ApcMin/+ mouse model of colon carcinogenesis.

Epidemiological surveys have revealed that environmental and dietary factors contribute to most of the human cancers. Our earlier studies have shown that resveratrol (RVT), a phytochemical reduced the tumor number, size and incidence of dysplasias induced by benzo(a)pyrene (BaP), an environmental toxicant in the ApcMin/+ mouse model of colon cancer. In this study we investigated to ascertain whether the preventive effects of RVT on BaP-induced colon carcinogenesis is a result of altered BaP biotransformation by RVT. For the first group of mice, 100μg BaP/kg bw was administered in peanut oil via oral gavage over a 60day period. For the second group, 45μg RVT/kg bw was co-administered with BaP. For the third group, RVT was administered for 1week prior to BaP exposure. Blood, colon and liver were collected from control and BaP/RVT-treated mice at 60days post-BaP & RVT exposure. We have assayed activities and expression (protein & mRNA) of drug metabolizing enzymes such as cytochrome P4501A1 (CYP1A1), CYP1B1, and glutathione-S-transferase (GST) in colon and liver samples from the treatment groups mentioned above. An increased expression of CYP1A1 in liver and colon and of CYP1B1 in liver of BaP-treated mice was seen, while RVT inhibited the extent of biotransformation mediated by these enzymes in the respective tissue samples. In the case of GST, an increased expression in colon of BaP alone-treated mice was noted when RVT was administered prior to BaP or simultaneously with BaP. However, there is no change in liver GST expression between BaP and RVT treatment groups. The concentrations of BaP aqueous (phase II) metabolites were found to be greater than the organic (phase I) metabolites, suggesting that RVT slows down the phase I metabolism (metabolic activation) of BaP, while enhancing phase II metabolism (detoxification). Additionally, the BaP-DNA adduct concentrations measured in colon and liver of BaP + RVT-treated mice were low relative to their BaP counterparts. Taken together, our findings strongly suggest that RVT alleviates BaP-induced colon carcinogenesis by impairing biotransformation pathways and DNA adduct formation, and therefore holds promise as a chemopreventive agent.

Effects of saline-alkaline stress on benzo[a]pyrene biotransformation and ligninolytic enzyme expression by Bjerkandera adusta SM46.

Benzo[a]pyrene (BaP) accumulates in marine organisms and contaminated coastal areas. The biotreatment of waste water using saline-alkaline-tolerant white rot fungi (WRF) represents a promising method for removing BaP under saline-alkaline conditions based on WRF's ability to produce ligninolytic enzymes. In a pre-screening for degradation of polycyclic aromatic hydrocarbons of 82 fungal strains using Remazol brilliant blue R, Bjerkandera adusta SM46 exhibited the highest tolerance to saline-alkaline stress. Moreover, a B. adusta culture grown in BaP-containing liquid medium exhibited resistance to salinities up to 20 g l(-1). These conditions did not inhibit fungal growth or the expression of manganese peroxidase (MnP) or lignin peroxidase (LiP). The degradation rate also became higher as salinity increased to 20 g l(-1). Fungal growth and enzyme expression were inhibited at a salinity of 35 g l(-1). These inhibitory effects directly decreased the degradation rate (>24%). The presence of MnSO4 as an inducer improved the degradation rate and enzyme expression. MnP and LiP activity also increased by seven- and fivefold, respectively. SM46 degraded BaP (38-89% over 30 days) in an acidic environment (pH 4.5) and under saline-alkaline stress conditions (pH 8.2). Investigating the metabolites produced revealed BaP-1,6-dione as the main product, indicating the important role of ligninolytic enzymes in initializing BaP cleavage. The other metabolites detected, naphthalene acetic acid, hydroxybenzoic acid, benzoic acid, and catechol, may have been ring fission products. The wide range of activities observed suggests that B. adusta SM46 is a potential agent for biodegrading BaP under saline conditions.

Abstract 1589: Olive oil alters benzo(a)pyrene biotransformation and reduces oxidative DNA damage in colon of ApcMin mouse

Abstract Sporadic colon cancers have been shown to be triggered by diet and environmental factors. Epidemiological studies have shown that colon cancer rates were significantly reduced in Mediterranean countries where olive oil is the main ingredient of diet. Published works have shown that olive oil inhibits cell proliferation and promotes apoptosis in colorectal cancer cell lines. Studies conducted earlier in our laboratory have shown that dietary exposure to the environmental toxicant benzo(a)pyrene (BaP) leads to colon tumor formation. Benzo(a)pyrene biotransformation studies have shown that once BaP is metabolized by cytochrome P450 drug metabolizing enzymes, it forms metabolites such as BaP 7, 8-diol-epoxide (BPDE), BaP 3,6 & 6,12-diones. These metabolites have also been shown to cause double stranded breaks in DNA and cause oxidative damage. Therefore, we hypothesize that olive oil's interaction with BaP will modulate BaP metabolism and reduce colon tumor formation. In this study we investigated the effect of olive oil on BaP-induced colon carcinogenesis in male ApcMin mice. Mice were assigned to a control (n=7) or treatment group (n =7). Treatment consisted of 50 and 100 μg BaP/kg body weight dissolved in tricaprylin (BaP-only group) or olive oil administered daily via oral gavage for sixty days. Post exposure, mice were sacrificed; colon and liver tissues were retrieved from each group of mice and were preserved in formalin and pathological changes were evaluated. The tissues were further analyzed for the induction of drug metabolizing enzymes such as cytochrome 1A, cytochrome 1B1, and glutathione S-Transferase. Additionally, to determine if BaP caused oxidative DNA damage to tissues, genomic DNA was isolated from the respective tissues and we quantified aldehyde lesions. There was a reduced incidence of adenomas in colons of mice that ingested BaP + olive oil compared to mice that received BaP only (p < 0.05). CYP protein expression was reduced while GST protein expression increased in the colon and liver tissues of BaP + olive oil-treated mice compared to BaP only-treated mice. The BaP organic metabolite concentrations decreased, while aqueous metabolite concentrations increased in the BaP + olive oil group compared to BaP only-treatment group. DNA lesions were increased in the colon and liver tissues of BaP-treated mice compared to BaP + olive oil treated mice. In summary, our studies suggest that olive oil exerts a protective effect against BaP-induced colon cancer. Citation Format: Leah D. Banks, Priscilla Amoah, Mohammad S. Niaz, Mary K. Washington, Samuel E. Adunyah, Aramandla Ramesh. Olive oil alters benzo(a)pyrene biotransformation and reduces oxidative DNA damage in colon of ApcMin mouse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2014-1589

Single and combined effects of microplastics and pyrene on juveniles (0+ group) of the common goby Pomatoschistus microps (Teleostei, Gobiidae)

Microplastic particles have increasingly been detected in aquatic biota, from zooplankton to fish, raising concern for potential effects on aquatic organisms. In addition, they may potentially influence the toxicity of other contaminants in the marine environment. The aim of this study was to clarify whether polyethylene microspheres (1–5μm) modulate short-term toxicity of the polycyclic aromatic hydrocarbon pyrene to juveniles (0+ group) of the common goby (Pomatoschistus microps). Fish were exposed for 96h to pyrene (20 and 200μgL−1) in the absence and presence of microplastics (0, 18.4 and 184μgL−1). Mortality, bile pyrene metabolites, and biomarkers involved in neurotransmission, aerobic energy production, biotransformation and oxidative stress were quantified. Microplastics delayed pyrene-induced fish mortality and increased the concentration of bile pyrene metabolites. Microplastics, alone or in combination with pyrene, significantly reduced acetylcholinesterase (AChE) activity, an effect also observed for pyrene alone. The mixture also decreased isocitrate dehydrogenase (IDH) activity. No significant effects were found for glutathione S-transferase activity or lipid peroxidation. Overall, results show that: (i) microplastics modulate either the bioavailability or biotransformation of pyrene; (ii) simultaneous exposure to microplastics and pyrene decrease the energy available through the aerobic pathway of energy production; and (iii) microplastics inhibit AChE activity. The mechanism for AChE inhibition appeared to be different for pyrene and microplastics, since simultaneous exposure to both did not increase significantly the inhibitory effect. The observed neurotoxic effects of microplastics per se and the effects on IDH activity of the two stressors combined are of concern because they may increase mortality in natural fish populations. More studies need to be carried out on possible combined effects of microplastics and polycyclic aromatic hydrocarbons on fish, particularly juveniles.

Abstract 5459: Benzo(a)pyrene biotransformation enzyme expression, activities and metabolite disposition in ApcMin mouse colon and liver is altered by resveratrol exposure.

Abstract Statistics released by the American Cancer society indicates that colon cancer is the third leading cause of cancer cases and related deaths in America. In addition to dietary preferences and lifestyle habits, exposure to toxicants such as benzo(a)pyrene (BaP) is one of the major contributing factors to the development of sporadic colon cancer. Our laboratory has validated the ApcMin mouse model to study BaP-induced colon carcinogenesis. Ongoing studies in our laboratory are focused towards elucidating the anticarcinogenic effects of resveratrol (RVT) against colon tumors. Our studies thus far have shown a decrease in the incidence, size, and number of adenomas formed in the colon of mice exposed to BaP and RVT, compared to BaP exposure alone. Since biotransformation of toxicants is one of the key steps for initiating carcinogenesis, the objective of this study was to investigate whether RVT exposure simultaneously or prior to BaP treatment alters BaP biotransformation and bioavailability in ApcMin mice. The BaP treatment consisted of BaP-only administration (in peanut oil) at a dose of 100 μg/kg body weight (bw) via oral gavage over a 60 day period (group I); BaP (100 μg/kg bw) co-administered with RVT (in 10% ethanol + 90% deionized water) at a dose of 45 μg/kg bw (group II); RVT administered for 1 week prior to BaP dosing (group III). Blood, colon and liver samples were collected at the end of exposure period. The expression of BaP biotransformation enzymes (CYP1A1, CYP1B1 and GST) in liver and colon were assayed at the level of protein and enzyme activities. Plasma, liver and colon tissue samples were analyzed by reverse phase-HPLC for BaP metabolites. Resveratrol exposure both prior to- and concurrent with BaP exposure caused a decrease in the expression and activity of CYP1A1/1B1 enzymes and an increase in GST enzymes both in liver and colon. Additionally, our studies revealed a decrease in concentrations of organic (Phase I) metabolites in plasma, liver and colon in mice that received RVT + BaP compared to mice that received RVT alone. In contrast, an opposite trend was noted with aqueous (Phase II) metabolites registering an increase in mice that received RVT + BaP compared to mice that received RVT alone. Between the two RVT-treatment strategies, concurrent administration of RVT appeared to limit BaP bioactivation compared to RVT treatment prior to BaP exposure. In summary, our results suggest that RVT provides a preventive effect against BaP-induced colon cancer initiation and progression in ApcMin mice (funded by NIH grants 5T32HL007735-12, 1F31ES019432-01A1 and 5RO1CA142845-O2). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5459. doi:1538-7445.AM2012-5459

Inhibition of pyrene biotransformation by piperonyl butoxide and identification of two pyrene derivatives in Lumbriculus variegatus (Oligochaeta)

Using the freshwater annelid Lumbriculus variegatus (Oligochaeta), the presence of cytochrome P450 (CYP) isozymes was investigated by analyzing metabolites of the polycyclic aromatic hydrocarbon (PAH) pyrene in treatments with and without the CYP inhibitor piperonyl butoxide (PBO). The results show a low biotransformation capability of L. variegatus (7% of total pyrene body burden as metabolites at 168 h). Addition of PBO resulted in a significant reduction of metabolites, suggesting the presence of a CYP in L. variegatus. Besides 1-hydroxypyrene, three peaks representing unknown metabolites were detected in LC-FLD (liquid chromatography with fluorescence detection) chromatograms of L. variegatus. Deconjugations showed that sulfonation and glucosidation are involved in the formation of these unknowns. Further studies with the time of flight mass analyzer provided the identification of the glucose-sulfate conjugate of 1-hydroxypyrene. The same metabolites were detected in the solvent-nonextractable fraction by incubation of the tissue residues with proteinase K, suggesting that part of these metabolites are bound to proteins. Overall, the slow biotransformation of pyrene by L. variegatus (involving CYP) supports the use of this species in standard bioaccumulation tests; however, the tissue-bound metabolite fraction described in the current study deserves further investigation for its toxicity and availability to upper trophic levels through diet.

Dissolved organic matter modulating the uptake, biotransformation, and elimination rates of pyrene in Daphnia magna

In the current study, the toxicokinetics of pyrene in Daphnia magna was determined in detail, and the role of dissolved organic matter (DOM) in these processes was investigated. Uptake, biotransformation, parent compound and metabolite elimination rates (k(u), k(m), k(ep), k(em)), depuration rates, bioconcentration factors (BCFs), and biological half-lives were studied in one natural humic water, in two waters made from DOM isolates, and in DOM-free artificial fresh water. The daphnids were exposed to pyrene for 24 h and depurated for another 24 h. To analyze the data, the two-compartment model was used to obtain toxicokinetic parameters, and the depuration data were analyzed by linear regression. Extensive biotransformation of pyrene resulted in high metabolite concentration in the organisms (83% of total radioactivity) after 24 h exposure. Depuration of the parent compound seemed to be biphasic: at first, depuration was rapid, but after 4 to 10 h in uncontaminated water, the disappearance rate came close to zero. After a 24-h depuration time, 7.8% of the pyrene accumulated remained in the organism. Overall, the elimination of parent compound was faster than the elimination of metabolites, which might imply inefficient excretion of water-soluble biotransformation products from daphnids. The effect of DOM on accumulation of pyrene was seen in a decreased uptake rate, indicating lower bioavailability of the compound in waters that contain high-aromaticity DOM. In the same waters, DOM significantly accelerated pyrene biotransformation and overall elimination. Altogether, DOM appears to be able to affect toxicokinetics through mechanisms that are connected to interaction with the contaminant but also directly through interaction with biotransformation systems.

Bioaccumulation and biotransformation of pyrene and 1‐hydroxypyrene by the marine whelk Buccinum undatum

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3% of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of [(14)C]pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue.

Sublethal toxicity and biotransformation of pyrene in Lumbriculus variegatus (Oligochaeta)

The aim of this work was to study the toxicity and biotransformation of polyaromatic hydrocarbon (PAH) pyrene in the oligochaete aquatic worm, Lumbriculus variegatus. PAHs are ubiquitous environmental pollutants that pose a hazard to aquatic organisms, and metabolizing capability is poorly known in the case of many invertebrate species. To study the toxicity and biotransformation of pyrene, the worm was exposed for 15 days to various concentrations of water-borne pyrene. The dorsal blood vessel pulse rate was used as a sublethal endpoint. Pyrene biotransformation by L. variegatus was studied and the critical body residues (CBR) were estimated for pyrene toxicity. The toxicokinetics of pyrene uptake was evaluated. A combination of radiolabeled (14C) and nonlabeled pyrene was used in the exposures, and liquid scintillation counting (LSC) and high-pressure liquid chromatography were employed in both water and tissue residue analyses. The results showed that L. variegatus was moderately able to metabolize pyrene to 1-hydroxypyrene (1-HP), thus demonstrating that the phase-I-like oxidizing enzyme system metabolizes pyrene in L. variegatus. The amount of the 1-HP was 1-2% of the amount of pyrene in the worm tissues. The exposure to pyrene reduced the blood vessel pulse rate significantly (p<0.05), showing that pyrene had a narcotic effect. The estimated CBRs remained constant during the exposure time, varying from 0.120 to 0.174 mmol pyrene/kg worm wet weight. The bioconcentration factors (BCF) decreased as exposure concentration increased. It was suggested that the increased toxicity of pyrene accounted for the decrease in BCFs by lowering the activity of the organism.

Humic substances modify accumulation but not biotransformation of pyrene in salmon yolk-sac fry

Humic substances may influence the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in aquatic environment. Relatively little is known how humic substances affect the biotransformation of PAHs in aquatic animals. Here we report how two different types of humic substances affected the accumulation of pyrene, a four-ringed PAH, to yolk-sac fry of landlocked salmon ( Salmo salar m. sebago). The accumulation of pyrene to yolk-sac fry tissues was inversely related to humic substance concentration in a short term (72 h) exposure. However, the biotransformation of pyrene was not affected by humic substances. Pyrene or humic subtances did not induce CYP1A activity in yolk-sac fry tissues contrasting to β-naphthoflavone, which was used as a positive control. Yolk-sac fry were capable to biotransform pyrene to phase I (1-hydroxypyrene) and phase II (pyrene-1-sulphate) products. Interestingly, glucuronide conjugate (i.e. pyrene-1-glucuronide) was not present in yolk-sac fry tissues. The concentration of parent pyrene and 1-hydroxypyrene remained the same throughout the experiment but the concentration of pyrene-1-sulphate more than doubled from 24 to 72 h. This finding suggests that salmon yolk-sac fry are not capable to excrete phase II biotransformation products or the excretion is very slow. Further, this could indicate that early life stage toxicity of many CYP1A inducing compounds is related to accumulation of phase II conjugates in fry tissues.

Quantitative analysis of benzo[ a]pyrene biotransformation and adduct formation in Ahr knockout mice

Benzo[ a]pyrene (BP) is an ubiquitous environmental pollutant with potent mutagenic and carcinogenic properties. The Ah receptor (Ahr) is involved in the metabolic activation of BP and is therefore important in the induction of chemical carcinogenesis. In this study, the relationship between Ahr genotype and biotransformation of BP in internal organs was investigated in Ahr (+/+), Ahr (+/−) and Ahr (−/−) mice. The mice were treated with BP (100 mg/kg) by gavage. Gene expression was measured after 24 h by real-time RT-PCR and showed induction of Cyp1a1 in liver and lung, and Cyp1b1 in lung in both Ahr (+/+) and Ahr (+/−). No induction of the Cyp genes was observed in the Ahr (−/−). There was a significant basal expression of Cyp1b1 in the liver of all genotypes, and this expression was independent of the BP exposure. Analyzed by HPLC-fluorescence, there were increased levels of protein and DNA adducts, metabolites, conjugates and unmetabolized BP in the internal organs of Ahr (−/−) as compared to Ahr (+/+) and Ahr (+/−) mice. This may be partly explained by a delayed bioactivation of BP in the Ahr deficient mice. The BP metabolism observed in the Ahr (−/−) mice is also evidence of an Ahr independent biotransformation of BP.

Metabolism of pyrene by aquatic crustacean, Daphnia magna

The aquatic crustacean Daphnia magna is an important species for ecotoxicological study, and is often used as a test organism for environmental risk assessment. However, the mechanism of xenobiotic metabolism by this species has not been studied in detail. In the present study, pyrene was used as model substance to investigate the mechanism of xenobiotic metabolism in D. magna. The results of 24-h exposure experiments showed that D. magna could metabolize pyrene and biotransform it into water-soluble metabolites. On the other hand, the metabolism of pyrene was significantly inhibited by SKF-525A as the cytochrome P450 (CYP) inhibitor. These observations indicated that oxidation by CYP participated in the biotransformation of pyrene by D. magna. We also identified the pyrene metabolites formed by D. magna by HPLC with an electrospray ionization triple quadrupole mass spectrometry detector (LC/ESIMS/MS) and de-conjugation by sulfatase, β-glucuronidase, and β-glucosidase. One of the metabolites was ionized in ESI negative mode and formed a dominant mass of m/ z 297 (MS) with the product ion of m/ z 217 (MS 2). Furthermore, this metabolite formed 1-hydroxypyrene on treatment with sulfatase. This metabolite was considered to be a sulfate conjugate of oxidized pyrene (1-hydroxypyrenesulfate). Furthermore, we quantified the deconjugated 1-hydroxypyrene formed by the above enzyme treatment. It showed that 52% of the total metabolized pyrene was biotransformed into 1-hydroxypyrene-sulfate, and more than 73% was biotransformed into oxidized pyrene conjugate. These results indicated that CYP and several conjugation enzymes participate in its biotransformation, and sulfation is important in D. magna for metabolism and elimination of xenobiotics.

Antioxidant responses of microalgal species to pyrene

The antioxidant response of four freshwater microalgal species, Chlorella vulgaris Beij., Scenedesmus platydiscus (G. M. Smith) Chod., Scenedesmus quadricauda(Turp.) Breb., and Selenastrum capricornutum Printz without pyrene addition (control) and at two pyrene concentrations (0.1 and 1.0 mgL−1) were investigated. Under the control condition, the values of the antioxidant parameters differed significantly among species and the difference was seemed not to be related to their susceptibility to pyrene. The antioxidant response to pyrene treatments also varied from species to species. Pyrene led to a significant increase in total glutathione (GSH) content in all species except C. vulgaris, a species did not exhibit any ability to metabolize pyrene. The glutathione-S-transferase (GST) activities also remained unchanged in pyrene treated C. vulgaris, increased greatly in S. platydiscus and Se. capricornutum (the two species with higher pyrene metabolism ability), but inhibited remarkably in S. quadricauda (the only species sensitive to pyrene toxicity). On the other hand, the glutathione reductase (GR) activities increased in C. vulgaris but remained at a similar level as the control in the other three species. The malondialdehyde (MDA) content, an indicator of lipid peroxidation, declined in S. quadricauda but showed no significant change in the other three species. The activities of glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) in pyrene treated cells remained almost the same as the controls for all microalgal species. Results suggested that the alterations of antioxidant systems in microalgae might not be useful indicators of pyrene exposure but pyrene-enhanced GSH metabolism might be important in pyrene biotransformation.

Polycyclic aromatic hydrocarbon biodegradation in extracellular fluids and static batch cultures of selected sub-tropical white rot fungi

Four sub-tropical white rot fungi, Trametes versicolor, Trametes pocas, Trametes cingulata and isolate DSPM95 were studied alongside the well studied white rot fungus, Phanerochaete chrysosporium, for their ability to remove polycyclic aromatic hydrocarbons (PAHs) from culture media. Both static shallow cultures and extracellular fluids were studied using media contaminated with a defined mixture of the PAHs; fluorene, phenanthrene, anthracene, pyrene and benzo(a)anthracene. With all isolates, the total loss of the parent compound in 31 days was high for fluorene, at +60%, phenanthrene at +40% and anthracene at +42%. Biotransformation of pyrene and benzo(a)anthracene by all the isolates was low, with the highest reduction of pyrene of 15.2% and benzo(a)anthracene of 15.8% being achieved with P. chrysosporium. Disappearance of the more condensed PAHs, pyrene and benzo(a)anthracene, increased in shallow static cultures with the addition of glucose and glucose oxidase as a source of additional H 2O 2. The addition of Mn 2+ and ABTS (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) to culture supernatants was associated with higher levels of biotransformation. Comparison of the isolates T. versicolor, T. pocas, T. cingulata and isolate DSPM95 with P. chrysosporium showed that these strains were competitive in the reduction of the PAHs, reducing the PAHs by more or less the same magnitude. Also these sub-tropical isolates did not accumulate a lot of HPLC detectable metabolites as much as P. chrysosporium.

Pyrene biotransformation and kinetics in the hepatopancreas of the isopod Porcellio scaber.

Various techniques exist for polycyclic aromatic hydrocarbon (PAH) determination in environmental samples, but an adequate risk assessment of PAHs should include aspects such as bioavailability of the contaminant and biotransformation capacity of the species under investigation. In this study, we provided an analysis of the kinetics of pyrene in the terrestrial isopod Porcellio scaber. Isopods were exposed to pyrene in their food (10 microg/g d/w) for 7 days followed by an elimination period of 7 days. The animals were dissected, and the hepatopancreases were analyzed for pyrene biotransformation products; nonmetabolised pyrene in the gut was also monitored. Concentrations of 1-hydroxypyrene in the hepatopancreas were very low. Almost all of the pyrene was found as three conjugates: pyrene-1-glucoside, pyrene-1-sulfate, and a third unknown 1-hydroxypyrene conjugate. Concentrations of the metabolites were extremely variable between individuals because of variable feeding activity. An apparent steady state was reached already after 24 hours of exposure, whereas elimination was complete 48 hours after ending the exposure. This rapid response to changes in the exposure concentration shows that terrestrial isopods have a high biotransformation capacity for PAHs. The data show that concentrations of parent PAHs will not provide a good indication of exposure in rapidly metabolizing invertebrates such as isopods; instead, pyrene metabolites may be considered a promising biomarker for bioavailability of PAH contamination in the field.

PAH biotransformation in terrestrial invertebrates—a new phase II metabolite in isopods and springtails

Soil-living invertebrates are exposed to high concentrations of contaminants accumulating in dead organic matter, such as polycyclic aromatic hydrocarbons (PAHs). The capacity for PAH biotransformation is not equally developed in all invertebrates. In this paper, we compare three species of invertebrates, Porcellio scaber (Isopoda), Eisenia andrei (Lumbricidae) and Folsomia candida (Collembola), for the metabolites formed upon exposure to pyrene. Metabolic products of pyrene biotransformation in extracts from whole animals or isopod hepatopancreas were compared to those found in fish bile (flounder and plaice). An optimized HPLC method was used with fluorescence detection; excitation/emission spectra were compared to reference samples of 1-hydroxypyrene and enzymatically synthesized conjugates. Enzymatic hydrolysis after fractionation was used to demonstrate that the conjugates originated from 1-hydroxypyrene. All three invertebrates were able to oxidize pyrene to 1-hydroxypyrene, however, isopods and collembolans stood out as more efficient metabolizers compared to earthworms. In contrast to fish, none of the invertebrates produced pyrene-1-glucuronide as a phase II conjugate. Both Collembola and Isopoda produced significant amounts of pyrene-1-glucoside, whereas isopods also produced pyrene-1-sulfate. A third, previously unknown, conjugate was found in both isopods and springtails, and was analysed further using electrospray and atmospheric pressure chemical ionisation mass spectrometry. Based on the obtained mass spectra, a new conjugate is proposed: pyrene-1- O-(6″- O-malonyl)glucoside. The use of glucose-malonate as a conjugant in animal phase II biotransformation has not been described before, but is understandable in the microenvironment of soil-living invertebrates. In the earthworm, three other pyrene metabolites were observed, none of which was shared with the arthropods, although two were conjugates of 1-hydroxypyrene. Our study illustrates the great variety of the still unexplored metabolic diversity of invertebrate xenobiotic metabolism.