Novel evidence of cytochrome P450-catalyzed oxidation of phenanthrene in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> under ligninolytic conditions

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130 pmol mg⁻¹ in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44 ± 0.02 min⁻¹. One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium.     

Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 ± 14 min⁻¹, and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions.     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P-450

Degradation of 2,4,6-trinitrotoluene (TNT) by the white-rot fungus Bjerkandera adusta DSM 3375 was studied in relation to extracellular ligninolytic activities. The Mn(II)-dependent peroxidase, the only ligninolytic enzyme detectable, reached a maximum activity of 600 ± 159 U/l after incubation in mineral medium with a sufficient nitrogen source. In contrast, the highest extent of [¹⁴C]TNT mineralization was detected in malt extract broth, so that the ability of B. adusta to mineralize TNT did not parallel ligninolytic activity. The microsomal fraction of cells grown in the presence of TNT was found to contain 11 pmol cytochrome P-450/mg protein. In cells grown without TNT, no microsomal cytochrome P-450 could be found. Instead, 14 pmol P-450/mg protein was present in the cytosolic fraction of these cells. Cytochrome P-450 apparently affected the TNT metabolism, as shown by inhibitory studies. Addition of the cytochrome P-450 inhibitor piperonyl butoxide diminished the ¹⁴CO₂ release from 21% to 0.9%, as determined after 23 days of incubation, while 1-aminobenzotriazole and metyrapone decreased the mineralization to 8.6% and 6.3% respectively. Mass-balance analysis of TNT degradation in liquid cultures revealed that, by inhibition of cytochrome P-450, the TNT-derived radioactivity associated with biomass and with polar, water-soluble metabolites decreased from 93.9% to 15.0% and the fraction of radiolabelled metabolites extractable with organic solvents fell to 92.6%. The TNT metabolites of this fraction were identified as aminodinitrotoluenes, indicating that this initial transformation product of TNT may function as a substrate for cytochrome-P-450-dependent reactions in B. adusta. Received: 27 May 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999     

Influence of piperonyl butoxide on the toxicity of organophosphate insecticides to three species of freshwater benthic invertebrates

Toxicity tests were conducted with amphipods (Hyalella azteca), chironomids (Chironomus tentans) and oligochaetes (Lumbriculus variegatus) exposed to a series of organophosphate insecticides in the absence or presence of piperonyl butoxide (PBO), an inhibitor of cytochrome(s) P450. Piperonyl butoxide effectively reduced the toxicity to H. azteca and C. tentans of three organophosphates (diazinon, chlorpyrifos, azinphos-methyl) which undergo metabolic activation by cytochrome(s) P450. Coadministration of PBO with another organophosphate (dichlorvos) which is not activated by cytochrome(s) P450, did not reduce the toxicity to the two species. Lumbriculus variegatus was relatively insensitive to the organophosphates, and PBO did not reduce their toxicity to the oligochaete. These data indicate that both H. azteca and C. tentans possess cytochrome P450-mediated MOs capable of metabolizing organic xenobiotics. However, the degree to which L. variegatus might be capable of the oxidative metabolism of organic xenobiotics is uncertain.     

Fungal cytochrome P450s catalyzing hydroxylation of substituted toluenes to form their hydroxymethyl derivatives

The degradation of a series of nitroaromatic compounds by the lignin-degrading fungus Phanerochaete chrysosporium was examined. From 4-nitrotoluene (4-NT), several metabolic intermediates were identified. Initially, 4-NT was converted to 4-nitrobenzyl alcohol (4-NBA), followed by the oxidation reactions to form 4-nitrobenzaldehyde and 4-nitrobenzoic acid, albeit slowly. Exogenously added 4-nitrobenzaldehyde and 4-nitrobenzoic acid were predominantly reduced to 4-NBA. The fungal formation of 4-NBA was inhibited by piperonyl butoxide, a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450 in the hydroxylation of the methyl group. Similarly, 2-, and 3-nitrotoluenes and 4-chlorotoluene were converted to the corresponding arylalcohols by P. chrysosporium. On the other hand, toluene and 4-methoxytoluene were not converted. Thus, P. chrysosporium possesses an alkyl hydroxylation activity against aromatic compounds substituted with a strong electron-withdrawing group.     

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

The gene cassette (camA⁺ camB⁺ camC) encoding a cytochrome P-450_cam variant was integrated into the nonessential gene pcpM of the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723 by homologous recombination. The recombinant strain could degrade hexachlorobenzene at a rate of 0.67 nmol · mg (dry weight)⁻¹ · h⁻¹, and intermediate pentachlorophenol was also identified.     

A Cytochrome P450 Mediated Naringenin 3'-Hydroxylase from Sweet Orange Cell Cultures

A microsomal flavonoid 3'-hydroxylase (F3'H) catalyzing themetabolism of naringenin to eriodictyol in Citrus sinensis (L.)Osbeck cv. ‘Hamlin’ cell suspension cultures wasshown to be a cytochrome P450 enzyme. This reaction requiredO₂ and NADPH and was inhibited by CO, with partial reversalof CO-inhibition by light at 450 nm. Cytochrome P450 contentranged from 10–20 pmol (mg microsomal protein)^–1.The F3'H reaction was shown to be linear in regard to proteinconcentration between 2.5 and 25 µg of microsomal protein.The optimum pH for the reaction was 7.4–7.6 and the temperatureoptimum was between 30 and 37°C. The apparent K_m and V_maxfor naringenin were 24 µM±3.2 and 81.4±7.9pmol eriodictyol min^–1 (mg protein)^–1, respectively.The microsomal F3'H was also capable of forming dihydroquercetinfrom dihydrokaempferol (40 pmol min^–1 (mg protein)^–1)and of quercetin from kaempferol (3.25 pmol min^–1 (mgprotein^–1). Cytochrome c and ketoconazole were the bestinhibitors of WH activity followed by piperonyl butoxide anda-naphthoflavone. Light was shown to be an inducer of the F3'Halmost doubling the specific activity and increasing the microsomalcytochrome P450 content by 30% over that of dark grown cells.F3'H activity was also confirmed in microsomal preparationsof young (new flush) leaves from ‘Hamlin’ treesand flavedo of ‘Hamlin’ oranges, ‘Marsh’grapefruit, and ‘Lisbon’ lemon. No activity wasobserved in older, hardened leaves and albedo of all the fruittested. Initiation of embryogenesis in the ‘Hamlin’cell suspension cultures by switching from a sucrose mediumto a glycerol-based medium resulted in the down-regulation ofF3'H. ¹Mention of a trademark, warranty, proprietary product, or vendordoes not constitute a guarantee by the U.S. Department of Agricultureand does not imply its approval to the exclusion of other productsor vendors that may also be suitable.     

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC₅₀ values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.     

Studies on the Specificity and Site of Action of alpha-Cyclopropyl-alpha-[p-methoxyphenyl]-5-pyrimidine Methyl Alcohol (Ancymidol), a Plant Growth Regulator

α-Cyclopropyl-α-[p-methoxyphenyl]-5-pyrimidine methyl alcohol (ancymidol) is an inhibitor of ent-kaur-16-ene oxidation in microsomal preparations from the liquid endosperm of immature Marah macrocarpus seeds. The K_i for this inhibitor is about 2 × 10⁻⁹ m. Ancymidol also blocks ent-kaur-16-en-19-ol and ent-kaur-16-en-19-al oxidation by the same preparations with a similar efficiency, but does not significantly inhibit ent-kaur-16-en-19-oic acid oxidation. Ancymidol appears to be specific for this series of oxidations in higher plant tissues. It does not inhibit the oxidation of kaurene nor kaurenoic acid in rat liver microsomes and has no significant effect on the oxidation of cinnamic acid in microsomal preparations from Sorghum bicolor seedlings. Ancymidol also does not inhibit kaurene oxidation in vitro nor in vivo in cultures of the fungus Fusarium moniliforme. The presence of ancymidol did not significantly alter the activities of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, or NADH-cytochrome b₅ reductase. The addition of ancymidol to suspensions of oxidized M. macrocarpus endosperm led to a difference spectrum with an absorption maximum at 427 nm and a minimum at 410 nm.     

Degradation of 4-nitrophenol by the lignin-degrading basidiomycete <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The fungal metabolism of 4-nitrophenol (4-NP) was investigated using the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Despite its phenolic feature, 4-NP was not oxidized by extracellular ligninolytic peroxidases. However, 4-NP was converted to 1,2-dimethoxy-4-nitrobenzene via intermediate formation of 4-nitroanisole by the fungus only under ligninolytic conditions. The metabolism proceeded via hydroxylation of the aromatic ring and methylation of phenolic hydroxyl groups. Although the involvement of nitroreductase in the metabolism of 2,4-dinitrotoluene by many aerobic and anaerobic microorganisms including P. chrysosporium has been reported, no formation of 4-aminophenol was observed during 4-NP metabolism. The formation of 1,2-dimethoxy-4-nitrobenzene was effectively inhibited by exogenously added piperonyl butoxide, a cytochrome P450 inhibitor, suggesting that cytochrome P450 is involved in the hydroxylation reaction. Thus, P. chrysosporium seems to utilize hydroxylation and methylation reactions to produce a more susceptible structure for an oxidative metabolic system.     

Biotransformation of Malachite Green by the Fungus Cunninghamella elegans

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 μmol h⁻¹ (mg of cells)⁻¹. Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.     

Mechanisms of pheromone inactivation in the gut of Periplaneta americana

The sex attractant of the cockroach, Periplaneta americana, has been shown in earlier work to be largely inactivated by dissected midgut from males and from mated females. It is only slightly inactivated by the midgut from virgin females. In this paper, the sex pheromone inactivating system is further studied and shown to be active in late instar larvae of both sexes. In the male this pheromone inactivation is inhibited by piperonyl butoxide, a microsomal oxidase inhibitor. This compound appears to act on the midgut tissue directly. In the mated female, piperonyl butoxide has little effect. When the pheromone inactivating capacity is partitioned into soluble and tissue components, it appears that the soluble component is most active in the male, whereas the tissue component is most active in the female. Evidence from heat inactivation, trichloracetic acid precipitation, and the use of soy bean trypsin inhibitor, as well as the time course of the reaction, suggest that the factor or factors inactivating pheromone are proteins, probably enzymes. Evidence that at least part of the pheromone inactivating capacity is due to microsomal oxidases is considered. It is also observed that both pheromone and piperonyl butoxide absorb to membranes.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Enrichment of an Endosulfan-Degrading Mixed Bacterial Culture

An endosulfan-degrading mixed bacterial culture was enriched from soil with a history of endosulfan exposure. Enrichment was obtained by using the insecticide as the sole source of sulfur. Chemical hydrolysis was minimized by using strongly buffered culture medium (pH 6.6), and the detergent Tween 80 was included to emulsify the insecticide, thereby increasing the amount of endosulfan in contact with the bacteria. No growth occurred in control cultures in the absence of endosulfan. Degradation of the insecticide occurred concomitant with bacterial growth. The compound was both oxidized and hydrolyzed. The oxidation reaction favored the alpha isomer and produced endosulfate, a terminal pathway product. Hydrolysis involved a novel intermediate, tentatively identified as endosulfan monoaldehyde on the basis of gas chromatography-mass spectrometry and chemical derivatization results. The accumulation and decline of metabolites suggest that the parent compound was hydrolyzed to the putative monoaldehyde, thereby releasing the sulfite moiety required for growth. The monoaldehyde was then oxidized to endosulfan hydroxyether and further metabolized to (a) polar product(s). The cytochrome P450 inhibitor, piperonyl butoxide, did not prevent endosulfan oxidation or the formation of other metabolites. These results suggest that this mixed culture is worth investigating as a source of endosulfan-hydrolyzing enzymes for use in enzymatic bioremediation of endosulfan residues.     

Oxidation of dibenzo- p-dioxin,dibenzofuran, biphenyl,and diphenyl ether by the white-rot fungus Phlebia lindtneri

Hydroxylation of dibenzo- p-dioxin (DD), dibenzofuran (DF), biphenyl (BP) and diphenyl ether (DPE) by the white-rot fungus Phlebia lindtneri GB-1027 was studied. DD and DF were rapidly degraded in a culture of P. lindtneri. The initial oxidation products were identified by gas chromatography-mass spectrometry. P. lindtneri oxidized DD to 2-hydroxy-DD, and DF to 2- and 3-hydroxy-DF. BP and DPE were also oxidized to p-hydroxy-BP and p-hydroxy-DPE, respectively. The oxidation catalyzed by P. lindtneri with each substrate was position-specific, because the hydroxyl group was introduced to the molecular edge of every substrate. Significant inhibition of the degradation of DD and DF was observed in incubation with the cytochrome P-450 monooxygenase inhibitors 1-aminobenzotriazole and piperonyl butoxide. These experiments with cytochrome P-450 inhibitors, and formation of the mono-hydroxyl metabolites suggest that P. lindtneri initially oxidizes DD, DF, BP, and DPE by a cytochrome P-450 monooxygenase and that it directly introduces a hydroxyl group to each of these substrates.     

Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624

Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively, after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N ¹-[(6-chloro-3-pyridyl)-methyl]-N ²-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624.     

Causal connection between detoxification enzyme activity and consumption of a toxic plant compound

Insect herbivores can increase their detoxification activities against a particular plant poison in response to prolonged ingestion of the same compound. For example, larval tobacco hornworms (Manduca sexta) experience a dramatic increase in cytochrome P450 activity against nicotine after ingesting nicotine. While it is generally assumed that this induction process permits increased consumption of toxic plant tissues, we are not aware of any direct experimental support for this assumption. Using a two-tiered approach, we examined the functional significance of P450 induction to M. sexta larvae ingesting a toxic but non-deterrent concentration of nicotine. First, we related the time-course of P450 induction in midgut microsomes to changes in nicotine consumption. When offered a nicotine diet, larvae failed to show a significant increase in consumption before 36 h, which was coincident with the time-course of the induction of midgut P450 activities against aldrin and nicotine. Second, we determined whether inhibiting the induced P450 activities affected nicotine consumption. We found that the increase in nicotine consumption following the induction of nicotine metabolism could be strongly inhibited by treatment with piperonyl butoxide, which by itself did not inhibit consumption. These results provide direct evidence for a causal connection between P450-mediated detoxification activity and consumption of a toxic plant compound.Abbreviation PB piperonyl butoxide     

Effects of the synergists piperonyl butoxide and S,S,S-tributyl phosphorotrithioate on propoxur pharmacokinetics in Blattella germanica (Blattodea: Blattellidae).

Effects of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on propoxur pharmacokinetics were examined in the German cockroach, Blattella germanica (L.). Treatment of adult male German cockroaches with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased propoxur toxicity by 2- and 6.8-fold, respectively, implicating hydrolysis as a major detoxification route of propoxur in the German cockroach. However, significant hydrolytic metabolism could not be demonstrated conclusively in vitro resulting in a conflict between in situ bioassay data and in vitro metabolic studies. In vitro propoxur metabolism with NADPH-fortified microsomes produced at least nine metabolites. Formation of metabolites was NADPH-dependent; no quantifiable metabolism was detected with cytosolic fractions. However, microsomal fractions lacking an NADPH source did produce a low, but detectable, quantity of metabolites (1.6 pmol). PBO inhibited NADPH-dependent propoxur metabolism in a dose-dependent fashion, implicating cytochrome P450 monooxygenases as the enzyme system responsible for the metabolism. Interestingly, DEF also inhibited the NADPH-dependent metabolism of propoxur, albeit to a lower extent. Treatment with PBO or DEF also caused a significant reduction in the cuticular penetration rate of propoxur. The data demonstrate that unanticipated effects are possible with synergists and that caution must be exercised when interpreting synergist results.     

Superoxide Production by a Manganese-Oxidizing Bacterium Facilitates Iodide Oxidation

The release of radioactive iodine (i.e., iodine-129 and iodine-131) from nuclear reprocessing facilities is a potential threat to human health. The fate and transport of iodine are determined primarily by its redox status, but processes that affect iodine oxidation states in the environment are poorly characterized. Given the difficulty in removing electrons from iodide (I⁻), naturally occurring iodide oxidation processes require strong oxidants, such as Mn oxides or microbial enzymes. In this study, we examine iodide oxidation by a marine bacterium, Roseobacter sp. AzwK-3b, which promotes Mn(II) oxidation by catalyzing the production of extracellular superoxide (O₂⁻). In the absence of Mn²⁺, Roseobacter sp. AzwK-3b cultures oxidized ∼90% of the provided iodide (10 μM) within 6 days, whereas in the presence of Mn(II), iodide oxidation occurred only after Mn(IV) formation ceased. Iodide oxidation was not observed during incubations in spent medium or with whole cells under anaerobic conditions or following heat treatment (boiling). Furthermore, iodide oxidation was significantly inhibited in the presence of superoxide dismutase and diphenylene iodonium (a general inhibitor of NADH oxidoreductases). In contrast, the addition of exogenous NADH enhanced iodide oxidation. Taken together, the results indicate that iodide oxidation was mediated primarily by extracellular superoxide generated by Roseobacter sp. AzwK-3b and not by the Mn oxides formed by this organism. Considering that extracellular superoxide formation is a widespread phenomenon among marine and terrestrial bacteria, this could represent an important pathway for iodide oxidation in some environments.