Involvement of CYP2 and mitochondrial clan P450s of Helicoverpa armigera in xenobiotic metabolism.

Insect CYP2 and mitochondrial clan P450s are relatively conserved genes encoding enzymes generally thought to be involved in biosynthesis or metabolism of endobiotics. However, emerging evidence argues they have potential roles in chemical defense as well, but their actual detoxification functions remain largely unknown. Here, we focused on the full complement of 8 CYP2 and 10 mitochondrial P450s in the generalist herbivore, Helicoverpa armigera. Their varied spatiotemporal expression profiles were analyzed and reflected their specific functions. For functional study of the mitochondrial clan P450s, the redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), were identified from genomes of eight insects and an efficient in vitro electron transfer system of mitochondrial P450 was established by co-expression with Adx and AdR of H. armigera. All CYP2 clan P450s and 8 mitochondrial P450s were successfully expressed in Sf9 cells and compared functionally. In vitro metabolism assays showed that two CYP2 clan P450s (CYP305B1 and CYP18A1) and CYP333B3 (mito clan) could epoxidize aldrin to dieldrin, while CYP305B1 and CYP339A1 (mito clan) have limited but significant hydroxylation capacities to esfenvalerate. CYP303A1 of the CYP2 clan exhibits high metabolic efficiency to 2-tridecanone. Screening the xenobiotic metabolism competence of CYP2 and mitochondrial clan P450s not only provides new insights on insect chemical defense but also can give indications on their physiological functions in H. armigera and other insects.

An evaluation of the levels of organochlorine compounds (OCPs and PCBs) in cultured freshwater and wild sea fish eggs as an exposure biomarker for environmental contamination.

In this study, the eggs of 30 wild Black Sea whiting (Merlangius merlangus euxinus, Nordmann, 1840) and 30 farmed freshwater rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) collected from Samsun Province in Turkey were analyzed to determine the level of contamination by nine organochlorine pesticides (OCPs), namely α-hexachlorocyclohexane (α-HCH), ß-HCH, γ-HCH (lindane), hexachlorobenzene (HCB), aldrin, 2,4'-dichlorodiphenyltrichloroethane (DDT), 4,4'-DDT, 2,4'-dichlorodiphenyldichloroethylene (DDE), 4,4'-DDE, and 15 polychlorinated biphenyls (PCBs) (PCB-28, -70, -74, -81, -99, -101, -118, -138, -153, -156, -170, -180, -183, -187, and -208), and their potential use as biomarkers to monitor levels of environmental contamination. OCPs and PCBs in the fat of fish eggs were extracted cryogenically and their concentrations were determined with a gas chromatography-electron capture detector (GC-ECD). The whiting eggs showed high OCP and PCB levels compared to the rainbow trout eggs. The median ∑ DDT values for whiting and rainbow trout eggs were 1601.62 ng g-1 fat (range 824.87-5049.81) and 406.49 ng g-1 fat (range 199.88-588.82); median ∑Indicator PCBs were 1264.24 ng g-1 fat (range 520.05-6140.32) and 82.11 ng g-1 fat (range 2.85-215.97); and median ∑ HCHs were 155.66 ng g-1 fat (range 35.45-330.40) and 13.48 ng g-1 fat (range 4.44-66.44), respectively. In the whiting eggs, the ∑Indicator PCB level was above the maximum residue limit (MRL) of 200 ng g-1 fat stated in the European Commission Regulation (EC) and Turkish Food Codex (TFC). In addition, there was a significant difference between the contamination levels of the eggs of the two species. In conclusion, it appears that fish eggs can serve as a valuable biomarker for the level of contamination of persistent organochlorine contaminants in different aquatic environments.

Phylogenetic and functional characterization of ten P450 genes from the CYP6AE subfamily of Helicoverpa armigera involved in xenobiotic metabolism.

The cotton bollworm, Helicoverpa armigera, is a generalist herbivore widely distributed over the world and is a major lepidopteran pest on cotton. Studies, especially from Asia, show that it relies on cytochrome P450 monooxygenases with broad substrate specificities to protect itself from pesticides. The number of P450s may have expanded in the processes of coping with the wide diversity of phytochemicals that the insect encounters among its numerous host plants. In order to examine the metabolic capabilities of these P450s, we focused here on all ten P450s of the Helicoverpa armigera CYP6AE subfamily, which can be easily induced by plant toxins and pyrethroids. These P450s, along with cytochrome P450 reductase, were heterologously expressed in insect cells and compared functionally. In vitro metabolism showed that all CYP6AE subfamily members can convert esfenvalerate to 4'-hydroxyesfenvalerate efficiently except CYP6AE20. In contrast, none of the recombinant CYP6AE enzymes could metabolise gossypol under our experimental conditions. Epoxidation capabilities were observed in the CYP6AE subfamily, aldrin can be converted to dieldrin at rates up to 0.45 ±â€¯0.04 pmol/min/pmol P450. Seven P450s in this subfamily can metabolise imidacloprid, but with lower efficiency than Bemisia tabaci CYP6CM1vQ. CYP6AE20 had virtually no metabolic competence to these four compounds but could metabolise several model fluorogenic substrates. These results showed the broad substrate spectrum of H. armigera CYP6AE P450s and suggest a limited role of gossypol upon the evolution of H. armigera CYP6AE genes.

Organochlorine Pesticides Residues in Human Breast Milk from the Middle Governorates in Jordan in 2013/2014.

One hundred samples of mother breast milk were gathered from six middle governorates and districts in Jordan in 2013/2014 to monitor Organochlorine pesticides pollutants. The results showed clearly that banned organochlorine pesticides are still detected in the monitored samples in low concentration despite banning of these persistent pollutants in Jordan since 36 years ago. However, the results indicated that 1% of the contaminated samples contained ß-HCH, 5% γ-HCH, 3% p,p'-DDD, 2% heptachlor, 45% p,p'-DDE and 3% p,p'-DDT. In addition, these monitored samples had no residues of aldrin, dieldrin, α-endosulfan, ß-endosulfan, HCB, o,p'-DD, o,p'-DDT and o,p'-DDE. In conclusion, there was a decline in the residues of Organochlorine pesticides, particularly DDT group members.

Biodegradation of Aldrin and Dieldrin by the White-Rot Fungus Pleurotus ostreatus.

Aldrin and its metabolite dieldrin are persistent organic pollutants that contaminate soil in many parts of the world. Given the potential hazards associated with these pollutants, an efficient degradation method is required. In this study, we investigated the ability of Pleurotus ostreatus to transform aldrin as well as dieldrin in pure liquid cultures. This fungus completely eliminated aldrin in potato dextrose broth (PDB) medium during a 14-day incubation period. Dieldrin was detected as the main metabolite, and 9-hydroxylaldrin and 9-hydroxyldieldrin were less abundant metabolites. The proposed route of aldrin biotransformation is initial metabolism by epoxidation, followed by hydroxylation. The fungus was also capable of degrading dieldrin, a recalcitrant metabolite of aldrin. Approximately 3, 9, and 18% of dieldrin were eliminated by P. ostreatus in low-nitrogen, high-nitrogen, and PDB media, respectively, during a 14-day incubation period. 9-Dihydroxydieldrin was detected as a metabolite in the PDB culture, suggesting that the hydroxylation reaction occurred in the epoxide ring. These results indicate that P. ostreatus has potential applications in the transformation of aldrin as well as dieldrin.

Inflammatory and cardiometabolic risk on obesity: role of environmental xenoestrogens.

CONTEXT: Some chemicals used in consumer products or manufacturing (eg, plastics, pesticides) have estrogenic activities; these xenoestrogens (XEs) may affect immune responses and have recently emerged as a new risk factors for obesity and cardiovascular disease. However, the extent and impact on health of chronic exposure of the general population to XEs are still unknown. OBJECTIVE: The objective of the study was to investigate the levels of XEs in plasma and adipose tissue (AT) depots in a sample of pre- and postmenopausal obese women undergoing bariatric surgery and their cardiometabolic impact in an obese state. DESIGN AND PARTICIPANTS: We evaluated XE levels in plasma and visceral and subcutaneous AT samples of Portuguese obese (body mass index ≥ 35 kg/m(2)) women undergoing bariatric surgery. Association with metabolic parameters and 10-year cardiovascular disease risk was assessed, according to menopausal status (73 pre- and 48 postmenopausal). Levels of XEs were determined by gas chromatography with electron-capture detection. Anthropometric and biochemical data were collected prior to surgery. Adipocyte size was determined on tissue sections obtained during surgery. RESULTS: Our data show that XEs are pervasive in this obese population. Distribution of individual and concentration of total XEs differed between plasma, visceral AT, and subcutaneous AT, and the pattern of accumulation was different between pre- and postmenopausal women. Significant associations between XE levels and metabolic and inflammatory parameters were found. In premenopausal women, XEs in plasma seem to be a predictor of 10-year cardiovascular disease risk. CONCLUSIONS: Our findings point toward a different distribution of XE between plasma and AT in pre- and postmenopausal women, and reveal the association between XEs on the development of metabolic abnormalities in obese premenopausal women.

Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.

Organochlorine pesticides in green mussel, Perna viridis, from the Cienfuegos Bay, Cuba.

The green mussel, Perna viridis, was used to measure bioaccumulated levels of organochlorine pesticides in the marine environment of Cuba. Samples were collected in the Cienfuegos Bay between January and December 2010. The organochlorine pesticides (i.e. DDT, Dieldrin, Chlordane, Endosulfan, HCB, Aldrin, Heptachlor and Lindane) were quantified by gas chromatography. The sum of all organochlorine pesticides in P. viridis was 6.31 ng g(-1). The concentration ranged from 3.53 to 4.42 ng g(-1) dry weight (dw) for DDTs (i.e. sum of pp' DDT, pp' DDD, op' DDE and pp' DDE); 1.7-1.9 ng g(-1) dw for Dieldrin; 0.17-0.20 ng g(-1) dw for Chlordanes; 0.14-0.16 ng g(-1) dw for Endosulfan; 0.11-0.17 ng g(-1) dw for HCB; 0.07-0.11 ng g(-1) dw for Aldrin; 0.046-0.054 ng g(-1) dw for Heptachlor and 0.035-0.039 ng g(-1) dw for Lindane. These levels can be considered as low when compared to reported values from similar studies conducted elsewhere in the world. The concentrations of all organochlorines residues detected in this study fell below the EU Maximum Residue Limits.

Novel metabolic pathways of organochlorine pesticides dieldrin and aldrin by the white rot fungi of the genus Phlebia.

White rot fungi can degrade a wide spectrum of recalcitrant organic pollutants, including polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs). In this experiment, 20 white rot fungi, belonging to genus Phlebia, were investigated for their ability to degrade dieldrin. Based on the screening results, we further investigated Phlebia acanthocystis, Phlebia brevispora, and Phlebia aurea to determine their degradation capacity and metabolic products towards dieldrin and aldrin. The three fungi were able to remove over 50% of dieldrin in a low nitrogen medium, after 42 d of incubation. Three hydroxylated products were detected as metabolites of dieldrin, suggesting that in Phlebia strains, hydroxylation reactions might play an important role in the metabolism of dieldrin. In contrast to dieldrin, aldrin exhibited higher levels of degradation activity. Over 90% of aldrin was removed after 28 d of incubation, and several new metabolites of aldrin in microorganisms, including 9-hydroxyaldrin and two carboxylic acid products, were detected in fungal cultures. These results indicate that the methylene moiety of aldrin and dieldrin molecules might be prone to enzymatic attack by white rot fungi. In this study, we describe for the first time a new metabolic pathway of both compounds by fungi of genus Phlebia.

Polychlorinated biphenyls and organochlorine pesticides in local waterbird eggs from Hong Kong: risk assessment to local waterbirds.

The contamination status of the marine environment in Hong Kong was studied by measuring concentrations of organochlorine (OC) pollutants (i.e., hexachlorobenzene, aldrin, dieldrin, endrin, mirex, total heptachlor, total chlordane, total DDTs, total PCBs, and total toxaphenes) in the eggs of selected waterbird species from different locations around the city: Little Egret (Egretta garzetta) and Chinese Pond Heron (Ardeola bacchus) from Mai Po Village, Great Egret (Ardea alba) and Black-crowned Night Heron (Nycticorax nycticorax) from A Chau, and Chinese Pond Heron (A. bacchus) from Ho Sheung Heung. The mean concentrations of total PCBs and total DDTs ranged from 191-11,100 ng g(-1) lipid and 453-49,000 ng g(-1) lipid, respectively. Recent exposure of waterbirds to technical chlordane was found in Hong Kong. The risk characterization demonstrated potential risks to birds associated with exposure to DDE, which was found to cause a reduction in survival of young in Hong Kong Ardeids based on the endpoint in the risk assessment.

Organochlorine pesticides in Avicennia marina from the Mumbai mangroves, India.

This paper examines the concentration levels of organochlorine pesticides (OCPs) such as hexachlorocyclohexane (HCH) (alpha, beta and gamma), Aldrin, Endosulfan (alpha, beta), DDE, DDD, and DDT in the leaf, root and sediment part of Avicennia marina, a dominantly distributed mangrove species in Mumbai. The overall pattern of accumulation of OCPs was observed as sigmaHCH> sigmaDDT> sigmaEndosulfan>Aldrin. The occurrence of alphaHCH was found to be frequent in all the samples. The leaf and root samples showed better uptake of organochlorine pesticides whereas lower concentration levels of OCPs was observed in case of sediment samples. The mean concentration of OCPs was observed highest in case of root samples. This study indicated towards the capability of A. marina in bioaccumulation of the OCPs. The present data will serve as the baseline against which future study of OCPs uptake in the mangrove plant species may be assessed.

Organochlorines in the Vaccarès Lagoon trophic web (Biosphere Reserve of Camargue, France).

During a decade (1996-2006), ecotoxicological studies were carried out in biota of the Vaccarès Lagoon (Biosphere Reserve in Rhone Delta, France). A multicontamination was shown at all levels of the trophic web due to a direct bioconcentration of chemical from the medium combined with a food transfer. Here, the pollutants investigated were organochlorines, among which many compounds banned or in the course of prohibition (or restriction) (PCB, lindane, pp'-DDE, dieldrin, aldrin, heptachlor, endosulfan...) and some substances likely still used in the Rhone River basin (diuron, fipronil). The results confirmed the ubiquity of contamination. It proves to be chronic, variable and tends to regress; however contamination levels depend on the trophic compartment. A biomagnification process was showed. A comparison of investigation methods used in other Mediterranean wetlands provides basis of discussion, and demonstrates the urgent need of modelling to assess the ecotoxicological risk in order to improve the management of such protected areas.

Aldrin epoxidation and dihydroisodrin hydroxylation as probes of in vivo and in vitro oxidative metabolic capability of some caterpillars.

Comparative biochemical studies are productive means to study factors that limit both beneficial and harmful effects of chemicals. Reactions such as aldrin epoxidation and dihydroisodrin hydroxylation are valuable assays of oxidative metabolism in scientific studies of chemical biology in insects, subhuman primates and other living things. The tissue distribution of activity in caterpillars may have functional significance. Localization of relatively high concentrations of these cytochrome P450 monooxygenases in gut tissue of lepidoptera may represent an important means to minimize absorption of lipophilic foreign chemicals in food. Some polychlorocycloalkanes permit in vivo and in vitro studies owing to their stability, acceptable toxicity and relatively simple pattern of metabolism. In vivo studies to assess the significance of in vitro findings are feasible with substrates such as aldrin, dihydroisodrin (DHI) and oxidative methylenedioxyphenyl inhibitors such as piperonyl butoxide (PBO) or carbon monoxide. Biphasic dose-dependent decreased and increased DHI-OH formation resulted from PBO pretreatment by gut, fat body, head and Malpighian tubule homogenates of cutworms and gut and fat body (the only tissues tested) of cabbage looper Trichplusia ni (Hübner) and black cutworm Agrotis ipsilon (Hüfnagel). The biphasic in vivo responses of caterpillars to PBO are a reminder of the complexity of biochemical and physiological responses of organisms coexposed to chemicals that are classified, often glibly, as toxic substances and metabolic inhibitors and inducers. Knowledge of dose and time relationships demands very careful evaluation in living things in the environment.

Removal of aldrin, dieldrin, heptachlor, and heptachlor epoxide using activated carbon and/or Pseudomonas fluorescens free cell cultures.

Degradation of aldrin (1,2,3,4,10,10-Hexachloro-1,4,4a,5,8,8a-hexahydro-1,4:5-8-dimethanonaphthalene), heptachlor (1H-1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methano indene), dieldrin (1aalpha,2beta,2aalpha,3beta,6beta,6aalpha,7beta,7aalpha)-3,4,5,6,9,9-Hexachloro-1a,2,2a,3,6,6a,7,7a-octahydro-2,7:3,6-d-methanonaphtha[2,3-b]oxirene, and heptachlor epoxide (1aalpha, 1bbeta,2alpha,5alpha,5alphabeta,6beta,6aalpha-2,3,4,5,6,7,7-Heptachloro-1a,1b,5,5a,6,6a-hexahydro-2,5-methano-2H-inden[1,2-b]-oxirene) was tested using free cultures of Pseudomonas fluorescens under controlled conditions. Pesticide concentrations were monitored by gas chromatography during 120 h. Percentages of degradation and biodegradation rates (BDR) were calculated. Data showed a trend suggesting a relation between chemical structure and degradability. Degradation kinetics for each pesticide tested showed that the highest degradation rates were found in the first 24 h. Kinetics data were adjusted to an empirical equation in order to predict their behavior, and the correlation coefficients obtained were satisfactory. Gas chromatography/mass spectrometry (GC/MS) analysis of the final extracts allowed the identification of chlordene and monodechlorodieldrin, which have been reported as final metabolite produced in the biodegradation of this kind of compounds. Regarding adsorption of pesticides on activated vegetal carbon, we concluded that removal efficiencies between 95.45 and 97.18% can be reached, depending on the pesticide and the carbon dose applied. The values for K from the Freundlich equation were quite similar for the four pesticides (between 1.0001 and 1.04), whereas the n values were quite different for each pesticide in the following order of affinity: dieldrin > aldrin > heptachlor epoxide > heptachlor. Equilibrium times, very important for scaling up the process, were between 43 min and 1 h, for the heptachlor epoxide and the heptachlor, respectively.

Determination of oxidative metabolism in Collembolan Proisotoma minuta (Tullberg).

An oxidative metabolism of Collembolan Proisotoma minuta was determined with a model compound of aldrin and dieldrin in this paper. The seven-day LD(50) values for aldrin, dieldrin, and piperonyl butoxide in salt solution were 0.496, 0.367, and 8.346 mg L(-1), respectively. When P. minuta were exposed to aldrin, dieldrin was the sole metabolite. The conversion of aldrin to dieldrin was known to be catalyzed by P450 monooxygenases system. It has been shown that the synergist piperonyl butoxide inhibited the metabolism of aldrin in P. minuta.

A biological loss of endosulfan and related chlorinated organic compounds from aqueous systems in the presence and absence of oxygen.

Endosulfan is a cyclodiene organochlorine currently widely used as an insecticide throughout the world. This study reports that the endosulfan isomers can be readily dissipated from aqueous systems at neutral pH in the absence of biological material or chemical catalysts, in the presence or absence of oxygen. The study showed that aldrin, dieldrin, and endosulfan exhibit bi-phasic loss from water in unsealed and butyl rubber sealed vessels. Half-lives are substantially increased for endosulfan I when oxygen is removed from the incubation vessel. The study conditions, where PTFE was used, were such that loss due to volatilization and alkaline chemical hydrolysis was eliminated. Half-lives determined from these data indicate that the parent isomers are much less persistent than the related cyclodienes, aldrin and dieldrin, confirming the findings of previous studies. The major oxidation product of endosulfans I and II, endosulfan sulfate, is less volatile and can persist longer than either of the parent isomers. Endosulfan sulfate was not formed in any of the treatments suggesting that it would not be formed in aerated waters in the absence of microbial activity or strong chemical oxidants. Since endosulfan sulfate is formed in many environments through biological oxidation, and is only slowly degraded (both chemically in sterile media and biologically), it represents a predominant residue of technical grade endosulfan, which finds its way into aerobic and anaerobic aquatic environments. The data obtained contributes to and confirms the existing body of half-life data on endosulfan I and II and its major oxidation product, endosulfan sulfate. The half-life data generated from the current study can be used in models for predicting the loss of chlorinated cyclodiene compounds from aqueous systems. The findings also highlight the importance of critically reviewing half-life data, to determine what the predominant processes are that are acting on the compounds under study.

In vitro hepatic biotransformation of aldrin and dieldrin in food-producing animals.

The hepatic biotransformation of aldrin (AD) and dieldrin (DD) was studied in liver post-mitochondrial supernatants (S-9s) from laying hens, female cattle and swine. S-9s were incubated with 0.03 nmol of AD or DD for 1 h. After 1 h, AD in the samples was almost epoxidated to DD. This formation was found with all the animal S-9s, and the highest rates occurred in pig S-9 (P < 0.01), followed by cow and hen S-9s. No reduction of DD was found with any of the S-9s.

Conversion of dieldrin to aldrin by intestinal bacteria in rats.

The present study provides the evidence that dieldrin is reductively metabolized to aldrin by intestinal bacteria in rats. When dieldrin was incubated with the cecal contents of rats, aldrin, a reduced metabolite of the epoxide, was isolated from the incubation mixture. The metabolite was identified unequivocally by UV and mass spectral comparison with an authentic sample, and on the basis of its TLC and HPLC behavior. The cecal contents of rats exhibited epoxide reductase activity toward dieldrin under anaerobic conditions. However, only marginal activity was observed under aerobic conditions. Four pure strains of intestinal bacteria exhibited epoxide reductase activities to varying degrees under anaerobic conditions. The highest activity was observed in Clostridium sporogenes. Cell-free extracts of the intestinal bacteria in rat cecal contents showed reductase activity when supplemented with both NAD(P)H and FMN under anaerobic conditions.

Ontogenic expression of detoxication enzymes in an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Marsupials and eutherians display vastly different reproductive strategies. Marsupials are characterised by the production of altricial neonates with little functional capacity. An investigation of the ontogenic expression of phase I (mixed function oxidase) and phase II (glutathione transferase) enzyme systems in the marsupial, the brushtail possum was undertaken. Enzyme expression in the youngest age group studied (60 days old) was between 5% and 10% of the adult level. A gradual increase in expression was then observed until a significant 3-fold increase to adult levels of expression of cytochrome P450, cytochrome b5 and glutathione transferase content and ECOD and AE activity was observed in brushtail possum young between the ages of 150 +/- 15 and 180 +/- 15 days. The expression of EROD activity reached adult levels by the age of 150 +/- days, while the expression of NADPH-cytochrome c reductase activity was delayed and adult levels had not yet been achieved by the oldest group studied (> 200 days). The ontogenic expression of detoxication enzymes was significantly delayed in the marsupial in comparison to eutherians. Adult levels were achieved during the weaning period, suggesting that dietary xenobiotics act as a regulatory mechanism in the developmental expression of these enzymes in the brushtail possum.

Detection of some organochlorine pesticides in cow's milk.

This monitoring study of 192 samples of cow's milk collected from the central region of Veracruz state was conducted to determine the contamination levels of organochlorine pesticides. The results obtained for mean DDT and HCH levels were 0.057 and 0.098 mg/kg respectively expressed on fat basis and are within FAO tolerances and similar to those found in other tropical countries.