Activities and toxicological significance of hepatic microsomal enzymes of the kestrel (Falco tinnunculus) and sparrowhawk (Accipiter nisus)

Activities of hepatic microsomal monooxygenase and epoxide hydrolase in sparrowhawks and kestrels were determined using organochlorine substrates. Monooxygenase activities were low in the kestrel and very low in the sparrowhawk compared to those in the male rat, down to environmentally realistic substrate concentrations. Epoxide hydrolase activities were very low in both species. These activities are discussed in relation to feeding ecology and susceptibility to organochlorine pollutants.     

Persistent toxic substances in the muscles and liver of the pacific walrus Odobenus rosmarus divergens Illiger, 1815 from the Bering Sea

This study reports data on the contents of organochlorine pesticides and heavy metals in the muscles and livers of eight individuals (five males and three females) of the Pacific walrus Odobenus rosmarus divergens Illiger, 1815, which were captured in the summer of 2011 in Mechigmensky Bay of the Bering Sea. Pesticides, namely α-, β-, and γ-isomers of hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT) and its metabolites dichlorodiphenyldichloroethane (DDD) and dichlorodiphenyldichloroethylene (DDE), as well as the heavy metals Cd, Pb, and Hg were found in all samples studied. The total concentration of organochlorine pesticides in the muscles varied from 200 to 5700 ng/g lipid weight and in the liver from 4900 to 90300 ng/g lipid weight. The concentrations of cadmium, lead, and mercury were 0.04–6.7, 0.13–0.76, and 0.03–0.40 μg/g dry weight, respectively. On the whole, the contents of organochlorine pesticides and heavy metals in the organs of the Pacific walrus were lower compared to those in marine mammals from other regions of the World Ocean.     

Effect of the 1990 die-off in the northern Italian seas on the developmental stability of the striped dolphin Stenella coeruleoalba (Meyen, 1833)

Developmental stability of the community of striped dolphins, Stenella coeruleoalba (Meyen, 1833), that died during the Mediterranean epizootic of 1990 was compared with that of the population prior to and after the epizootic, to assess whether animals that died were the developmentally less stable individuals in the population. Significantly higher levels of fluctuating asymmetry (FA) were found in those individuals that died. Tissue levels of organochlorine pesticide residues and PCBs were determined and the correlation between contaminant concentration and FA was tested. No correlations were found between the contaminant level and FA.     

Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, β endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with β-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.     

Hydropsychid (Trichoptera, Hydropsychidae) gill abnormalities as morphological biomarkers of stream pollution

1. The use of morphological gill abnormalities of hydropsychid larvae was assessed in Hydropsyche siltalai larvae exposed to cadmium in the laboratory and Cheumatopsyche lepida and H. pellucidula larvae collected from a polluted river. Two biomarkers were evaluated: (1) Hydropsychid abnormality incidence (HAI), referring to the proportion of individuals with at least some abnormalities, and (2) Hydropsychid gill abnormality indice (HYI), referring to the average number of abnormal gill tufts for all individuals. 2. Abnormality–contaminant relations for both biomarkers were established by studying gill responses along gradients of increasing cadmium and organochlorine concentrations. A cadmium gradient was verified in laboratory exposures, whereas the concentrations of polychlorinated dibenzo‐p‐dioxin (PCDD), dibenzofuran (PCDF) and diphenyl ether (PCDE) of the aquatic moss Fontinalis antipyretica were used as measures of an organochlorine gradient in the field. 3. Morphological abnormalities were easily distinguished as heavy darkening, malformation and/or reduction of single gill tufts. Darkening of the gills appeared to start either at the basal or distal ends. 4. A marked increase of HYI values with increasing Cd concentration reflected a clear abnormality‐contaminant relation, whereas the mere dicotomic classification of larvae as normal or abnormal (HAI) was less informative. High values of both HAI and HYI were associated with high contamination. A significant positive correlation was found between organochlorine concentration in mosses and biomarker values for H. pellucidula, but not for C. lepida. 5. We conclude that HAI indicates deleterious effects, but fails to quantify the severity of degradation. Use of individual gill tufts, as response units in deriving HYI, revealed a simple solution to the quantification problem. Further research into the ecological meaning, physiological background and patterns of gill abnormality is recommended for assessing the applicability and relevance of hydropsychid gill biomarkers.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.     

Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida

The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components. As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM. Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase. The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.     

Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential control of rat microsomal "aryl hydrocarbon" monooxygenase and epoxide hydratase.

A growing body of evidence implicates epoxide metabolites of mutagenic and carcinogenic polycyclic hydrocarbons as either the only species, or one of the contributing species responsible for these adverse effects. Selective induction of epoxide hydratase(s) catalyzing the transformation of epoxides to electrophilically unreactive dihydrodiols, under conditions not leading to increases in monooxygenase(s) responsible for epoxide formation would, therefore, be of interest. All inducers of rat hepatic epoxide hydratase (determined with [7-3H]styrene oxide as substrate) which have been discovered also induced monooxygenase (determined with benzo(a)pyrene as substrate) suggesting a possible common biosynthetic control of these enzymes. The enzyme levels observed in different sexes and at different stages of the ontogenetic development, possibly dependent on endogenous inducers, strengthened this view. No sex difference is epoxide hydratase activity was observed in young rats (1 to 5 days old) while epoxide hydratase levels were about 3-fold higher in adult males than in females, which was remarkably similar to the behavior of monooxygenase. Moreover, the prenatal development of epoxide hydratase and monooxygenase appeared to be similar--although the low enzyme levels precluded accurate determinations of the latter. Although different types of known monooxygenase inducers all led to epoxide hydratase induction in adult rat liver, their effect of epoxide hydratase and monooxygenase could be dissociated by transplacental treatment. Dissociation was clearest with inducers of the polycyclic hydrocarbon type which led to great induction of monooxygenase while epoxide hydratase remained unchanged. The increases in monooxygenase activity were very different when determined by two methods based on different principles, demonstrating that at least two monooxygenases are involved in oxidative metabolism of benzo(a)pyrene, and that the control of epoxide hydratase is not under common control with either of them.     

Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.     

Biotransformation of p-xylene and 2,6-dimethylnaphthalene by xylene monooxygenase cloned from a Sphingomonas isolate

Sphingomonas strain ASU1 was isolated from an industrial wastewater bioreactor and grew on 2,6-dimethylnaphthalene (2,6-DMN) as the sole carbon/energy source. The genes for a xylene monooxygenase were cloned from strain ASU1. Expression of the ASU1 xylene monooxygenase was compared to expression of the pWWO xylene monooxygenase in Escherichia coli. Both monooxygenases transformed p-xylene and 2,6-DMN by initially hydroxylating one methyl group. In addition, the ASU1 monooxygenase also hydroxylated the second methyl group on p-xylene and 2,6-DMN whereas the pWWO monooxygenase hydroxylated the second methyl group only on p-xylene. Endogenous E. coli enzymes contributed to further oxidation of the resulting aromatic alcohols to form aromatic carboxylates.     

Immunohistochemical demonstration of cytochrome P-450 monooxygenase in regenerating tracheal epithelium: a recapitulation of fetal development

The cytochrome P-450 monooxygenase enzymes, NADPH-reductase and form 2, were demonstrated immunohistochemically in hamster tracheal epithelium that was regenerating after mechanical injury. Bromodeoxyuridine (BrdU), a thymidine analogue, was used to map the location and extent of the wound sites between 8 and 144 h post-injury. In the control and non-wounded areas of the epithelium, the secretory cells were labelled for the monooxygenase enzymes. Label was heaviest in the apical cytoplasm of these columnar cells. At 8 h, secretory cells at the wound margins migrated to cover the wound sites, becoming progressively flattened. Reaction product for monooxygenase enzymes was strong in these flat cells but immunolabelling for BrdU was very low. At 24 h many cells at the wound sites were labelled for BrdU (indicative of a high rate of cell division). Some cells were labelled for monooxygenase but many were not stained at this time. At 48 and 72 h post-injury, none of the cells within the wound sites (regenerating epithelium) were stained. Immunochemical labelling for the monooxygenase enzymes was restored to the nascent secretory cells as they differentiated in the wound sites, beginning at 96 h post-injury. Labelling was stronger at 120 and 144 h post-injury, comparable to that in the control epithelium. The observations suggest that the monooxygenase enzymes were retained by the secretory cells in the wound sites before they divided but were lost from their progeny. Then, the temporal sequence of monooxygenase expression was similar to the pattern of differentiation of nascent secretory cells during fetal development of the tracheal epithelium.     

Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media

The biocatalytic properties of three Baeyer–Villiger monooxygenases (phenylacetone monooxygenase, 4-hydroxyacetophenone monooxygenase and ethionamide monooxygenase) in a variety of aqueous–organic media were studied using organic sulfides as substrates. The influence of the nature and the concentration of the solvents, as well as of the substrates, on the activity and enantioselectivity of the enzymes was investigated in detail. Solvents were found to decrease, to a different extent, enzyme activity. High increases of enantioselectivity and also reversal of enantiopreference were observed depending on the enzyme and on the nature of the solvent and the substrate employed.     

Decreased placental monooxygenase activities associated with birth defects

We have measured monooxygenase activities in placentas from 82 women who smoked throughout their pregnancies and correlated these with the presence or absence of major somatic anomalies. Monooxygenase activities toward benzo(a)pyrene and ethoxyresorufin in placentas from 18 abnormal infants were compared with activities in placentas from 64 concurrently studied normal infants. Placentas from normal infants were found to have high levels of monooxygenase activities and low apparent Kms toward ethoxyresorufin (10(-7) M), reflecting induction of cytochrome P-450 enzymes usually associated with maternal cigarette smoking. Placentas from the abnormal infants, however, had significantly lower monooxygenase activities and higher apparent Kms toward ethoxyresorufin (10(-5) M), indicating that induction of specific cytochrome P-450 systems occurred less frequently among placentas from abnormal infants. The reasons for this association are unclear. Apparent lack of induction of monooxygenase activity occurred most frequently in placentas from anencephalic infants but was neither exclusively nor consistently found with this defect. No specific maternal condition or environmental exposure associated with lack of monooxygenase induction was identified.     

The viable but non-culturable state in the human pathogen Vibrio vulnificus

Abstract Genes encoding paniculate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides ( pmoA and amoA ) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (β-and y-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the γ-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.     

Characterization of two components of the 2-naphthoate monooxygenase system from Burkholderia sp. strain JT1500

2-Naphthoate monooxygenase, a two-protein system, encoded by the nmoA and nmoB genes, was heterologously overexpressed in Escherichia coli. The proteins used for functional characterization were purified to over 90% homogeneity by affinity chromatography. The oxidative component EnmoA (47.9 kDa) lacked substrate catalysis capability on its own, and the reductive component EnmoB (33.4 kDa) and its truncated derivate EnmoB(T) (25 kDa) possessed nearly identical independent flavin reductase activities, c. 130 micromol min(-1) mg(-1) of protein. The inframe fusioned protein EnmoB(T)A (65.2 kDa), containing NmoB(T) and NmoA peptides showed a stable 2-naphthoate monooxygenase activity of 1.2 micromol min(-1) mg(-1) of protein. This is the first report on the purification of a fused form of a two-component flavoprotein monooxygenase. In the specificity experiment, FAD and NADH were shown to be preferred cosubstrates for EnmoB and EnmoB(T). All these data suggest that NmoB(T)A is a two-component flavoprotein monooxygenase, consisting of an oxygenase and a reductase component. NmoA is a member of the class D flavoprotein monooxygenase, and NmoB is an independent NADH:Flavin oxidoreductase.     

Use of skin biopsies for assessing levels of organochlorines in walruses (Odobenus rosmarus)

Skin and blubber samples of ten adult male Pacific walruses (Odobenus rosmarus divergens) from Alaska were used to investigate the relationship between organochlorine (OC) levels in skin and blubber of individuals. For analyses we selected 11 components that were quantified in the blubber of all individuals: hexachlorocyclohexanes (αHCH and βHCH), the DDT (dichlorodiphenyltrichloroethane) metabolite p,p′DDE, oxychlordane, and 7 individual PCB congeners, 28, 99, 105, 118, 138, 153 and 180. The correlation between the levels in the two types of tissues was significant and the relation was isometric for all components. The regression coefficient between levels in blubber (dependent variable) and levels in skin (independent variable) was different from 1 for only four of the components. The mean levels in the two types of tissues were significantly different for 3 of the 11 chemical components (βHCH, oxychlordane, and PCB28). Although this analysis is based on only ten individuals, we propose that skin samples taken by biopsy darts can be used to monitor OC levels in walruses. In August 1993 skin biopsies were collected from 25 adult male Atlantic walruses (O. r. rosmarus) at haul-out sites in southeastern Svalbard in the Norwegian Arctic and from 28 walruses of different sex and age at haul-out sites at Franz Josef Land in the Russian Arctic. The mean levels of OCs were 2–10 times higher at Svalbard than at Franz Josef Land. The dominant OC component was PCB153 in both areas. A principal component analysis detected differences between areas in OC levels but not in patterns. Since the Franz Josef Land samples were mainly taken from females and young individuals and the Svalbard samples were taken largely from adult males, we believe the differences in tissue OC levels observed from these areas can be explained by differences in sex and age of the walrus sampled. Comparable organochlorine levels in skin samples from walruses from other areas are not available. However, compared to the corresponding OC levels found in walrus blubber in other areas, the OC levels from Svalbard and Franz Josef Land are higher. The high levels of OCs in walruses from Svalbard and Franz Josef Land may be a combined effect of high pollution level in the environment and seal-eating habits. In the present study we show that it is possible to use skin biopsies taken by a non-destructive method to assess OC levels in walruses. Accepted: 24 October 1999     

棉铃虫抗药性的生理生化机制研究

本文报道了棉铃虫Helicoverpa armigera田间抗性种群对杀虫剂抗药性的生理生化机制。抗性种群(HJ-R)5龄幼虫羧酸酯酶、谷胱甘肽转移酶、多功能氧化酶活力均明显高于相对敏感种群(HD-S)。两种群乙酰胆碱酯酶对杀虫剂敏感性没有显著差异。HJ-R种群的腹神经索对氰戊菊酯表现了2-3倍的神经不敏感性。HJ-R种群对氨基甲酸酯类杀虫剂的抗性主要是由代谢机制引起,其中多功能氧化酶可能起主导作用;对菊酯的抗性是由多功能氧化酶、酯酶、以及神经不敏感性几个因子综合作用的结果。     

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.