P450 Monooxygenase Activity Research Articles

Effect of strychnine hydrochloride on liver cytochrome P450 mRNA expression and monooxygenase activities in rat

Strychnos nux-vomica L. has been frequently used in traditional Chinese medicine but has high acute toxicity. It is commonly taken with Glycyrrhizae radix to decrease its toxicity but the mechanism of this interaction is unknown. In this work, the mRNA expression and the activity of four cytochrome P450 (CYP) enzymes representative of four subfamilies (CYP1A, CYP3A, CYP2C and CYP2E) were determined ex vivo in rat livers from groups of Wistar rats orally administered strychnine hydrochloride (SH) at three doses (0.1, 0.3 and 0.9 mg/kg/day) alone and, at the highest dose, in combination with glycyrrhetinic acid (GA, 25 mg/kg/day) or liquiritin (LQ, 20 mg/kg/day) once a day for 7 consecutive days. Compared to control, the mRNA expressions of CYP3A1, 1A2 and 2E1 were higher in rats receiving the highest dose of SH but lower for CYP3A1 and CYP2E1 in rats receiving the SH+GA and SH+LQ combinations. CYP2E1 activity was higher and CYP2C, CYP3A and CYP1A2 activities were lower in rats receiving the highest dose of SH. In contrast CYP1A2 and CYP2C activities were higher and CYP2E1 and CYP3A activities lower in rats receiving the SH+GA combination. CYP2E1 and CYP3A activities were also lower in rats receiving the SH+LQ combination. The results show that treatment with SH for 7 days affects the expression and the activity of CYP enzymes and that coadministration of GA and LQ modulates these effects. This modulation may explain the role of Glycyrrhizae radix in reducing the acute toxicity of Strychnos nux-vomica L.CYPs enzymes.

Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis

Abamectin resistance was selected in the western flower thrips [ Frankliniella occidentalis (Pergande)] under the laboratory conditions, and cross-resistance patterns and possible resistance mechanisms in the abamectin-resistant strain (ABA-R) were investigated. Compared with the susceptible strain (ABA-S), the ABA-R strain displayed 45.5-fold resistance to abamectin after 15 selection cycles during 18 generations. Rapid reversion of abamectin resistance was observed in the ABA-R strain in the absence of the insecticide selection pressure. Moderate and low levels of cross-resistance to chlorpyrifos (RR 11.4) and lambda-cyhalothrin (3.98) were observed in the ABA-R strain, but no significant cross-resistance was found to spinosad (2.00), acetamiprid (1.47) and chlorfenapyr (0.26). Our studies also showed that the esterase inhibitor S, S, S-tributyl phosphorotrithioate (DEF) and glutathione S-transferase inhibitor diethyl maleate (DEM) were not able to synergize the toxicity of abamectin, whereas the oxidase inhibitor piperonyl butoxide (PBO) conferred a significant synergism on abamectin in the ABA-R strain (SR 3.00). Biochemical analysis showed that cytochrome P450 monooxygenase activity of the ABA-R strain was 6.66-fold higher than that of the ABA-S strain. It appears that enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases was a major mechanism for abamectin resistance in the western flower thrips.

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b5 reductase–cyt b5. In fungi, limited information is available for the cyt b5 reductase–cyt b5 system. Here we characterized the kinetic mechanism of (cyt b5r)–cyt b5 redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b5r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b5 reductases, Pc-cyt b5r lacked the typical ferricyanide reduction activity, a standard for cyt b5 reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b5r–cyt b5 complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b5r–cyt b5 complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b5r–cyt b5) in terms of supporting a P450 monooxygenase activity.

Survey of Susceptibility to Abamectin of Pear Psylla (Hemiptera: Psyllidae) in Northern Italy

The pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), is a relevant pest of pear, Pyrus communis L., trees in Emilia-Romagna region (northern Italy). The susceptibility to the insecticide abamectin was evaluated at different times of the year on C. pyri populations undergoing different control strategies within conventional, integrated, and organic farms. The tests performed were the egg spray and the topic and dip bioassay on adults. The larval mortality was evaluated by dip bioassay on treated leaves. The activity of P450-dependent monooxygenases, a relevant enzyme system involved in insecticide resistance of C. pyri, was also determined in adults by 7-ethoxycoumarin O-deethylation (ECOD assay). Tests on treated eggs and on larvae showed no significant differences in LC50 and LC90, although these values were always lower in individuals collected from organic farms in comparison with all other farms. Tests on overwintering adults revealed differences among populations, probably more related to collection time than to field pest control strategies. Unexpectedly, the ECOD assay on adults showed a slightly higher cytochrome P450 monooxygenase activity in the population undergoing organic control in comparison to others. Our results indicate that egg spray is the most reliable bioassay to verify data of open-field applications. Apparently, no resistance to abamectin has yet been developed by C. pyri in Emilia-Romagna.

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae)

Anopheles funestus, one of the main African malaria vectors, caused a major malaria outbreak in South Africa during 1999/2000, even though South Africa had an effective vector control program in place. The outbreak was due to pyrethroid resistant An. funestus invading KwaZulu/Natal. Increased activity of cytochrome P450 (monooxygenase) was responsible for the pyrethroid resistance in this species. A monooxygenase gene, CYP6P9, was highly overexpressed in the pyrethroid-resistant strain compared with a susceptible strain. Characterization of this gene as well as the redox partners involved in the catalytic cycle of P450s was investigated. The full length of the CYP6P9 sequence was isolated, sequenced and compared between the pyrethroid-resistant and -susceptible strains. Sequence identity between the two strains was 99.3%; the sequence differences were mainly outside of the conserved regions. The functional significance is still unknown, but it is feasible that these variations are associated with differences in insecticide metabolism. A second CYP6 gene (CYP6P13) was also isolated; it shared close similarities with CYP6P9. The putative redox partners, cytochrome b(5) (cyt b(5)) and NADPH-cytochrome P450 reductase (CPR), were isolated from An. funestus (resistant strain) and showed high levels of sequence identity to other insect cyt b(5) and CPRs. Isolation of the coding sequences CYP6P9 and its cognate redox partners enables expression of functional recombinant protein for biochemical and structural analysis.

Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice.

Event Abstract Back to Event Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice. Lyubka Tantcheva1, Eleonora Encheva2*, Silvyia Abarova2, Julia Serkedjieva3, Iliana Dimova1 and Elitza Pavlova4 1 Institute of Neurobiology, Bulgaria 2 Medical University-Sofia, Bulgaria 3 Bulgarian Academy of Sciences, Institute of Microbiology, Bulgaria 4 St Kliment Ohridsky Sofia University, Bulgaria Introduction: Inhibited first phase of drug metabolism is a main reason for numerous side effects and increased drug toxicity, observed in the course of influenza virus infection (IVI). Medical plant Geranium Sanguineum L. (GSL), spread in the Balkans, demonstrated significant antioxidant and antiviral activity. The aim of the study is to evaluate the preventive effect of polyphenol complex (PPC) from GSL on the oxidative drug metabolism in IVI. Methods: The experimental model of IVI (A/Aichi/2/68/(H3N2)(4.5 lg LD50) is developed in male albino ICR mice. PPC is applied nasally (10 mg/kg). In liver 9 000 g supernatant thiobarbituric acid reactive substances (TBARS) and antioxidant total activity (ATA) as well as monooxygenase N-demethylase activity (ethylmorphyne, amidopyrine and analgin) and hydroxylase activity (aniline and p-nitrophenol), NADPH- cytochrtome P-450 reductase and cytochrome P-450 content are determined. Student-Fisher t-test and correlation coefficients are used for statistics. Results: High correlation between increased TBARS and decreased monooxygenase activity (p< 0.001) is found in infected animals. It suggests that activation of free radicals can be one of the main reasons for unspecific monooxygenase inhibition, because all components of cytochrome P-450 system are inhibited (mostly the on the 6th and 9th days). PPC pretreatment demonstrates significant preventive effect against increased levels of TBRS and decreased ATA. Drug metabolism in IVI mice is restored by PPC near to healthy controls. Conclusion: TBARS probably play important role not only in the pathogenesis of IVI, but also in modulation of hepatic monooxygenase activity. All inhibited P-450 monooxygenase activities are significantly restored by PPC pretreatment. The complex mechanism of PPC-protective effect obviously combines several biological activities- antioxidant activity, selective antiviral and protein-bounding effect. In contrast to infected animals, PPC has medium pro-oxidant and weak inhibiting effect on monooxygenases in healthy animals, probably related to its protein–binding effect on membrane proteins and enzymes. Keywords: influenza infection, drug metabolism, cyt P-450 monooxygenases, Polyphenols, Geranium Sanguineum L. Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic metabolism Citation: Tantcheva L, Encheva E, Abarova S, Serkedjieva J, Dimova I and Pavlova E (2010). Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice.. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00203 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Eleonora Encheva, Medical University-Sofia, Sofia, Bulgaria, eleonora.encheva@medfac.acad.bg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Google Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Google Scholar Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova PubMed Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Characterisation of abamectin resistance in a field‐evolved multiresistant population of Plutella xylostella

Plutella xylostella (L.) has evolved resistance to various kinds of insecticide in the field. Reversion and selection, cross-resistance, inheritance and mechanisms of abamectin resistance were characterised in a field-derived multiresistant population of P. xylostella from China. Compared with a susceptible Roth strain, the field-derived TH population showed approximately 5000-fold resistance to abamectin. Rapid reversion of abamectin resistance was observed in the TH population when kept without insecticide selection. The TH-Abm strain, selected from the TH population with abamectin, developed 23 670-fold resistance to abamectin, a high level of cross-resistance to emamectin benzoate and low levels of cross-resistance to spinosad and fipronil. Genetic analyses indicated that abamectin resistance in the TH-Abm strain was autosomal, incompletely dominant and polygenic. P450 monooxygenase activities in the TH-Abm strain were significantly elevated compared with the TH strain. Piperonyl butoxide (PBO) inhibited a small part of abamectin resistance in the TH-Abm strain. Field-evolved high-level resistance to abamectin in the TH population was not stable. Selection of the TH population with abamectin resulted in an extremely high level of cross-resistance to emamectin benzoate and low levels of cross-resistance to spinosad and fipronil. Enhanced oxidative metabolism was involved in, but may not be the major mechanism of, polygenic abamectin resistance in the TH-Abm strain.

Cross‐resistance study and biochemical mechanisms of thiamethoxam resistance in B‐biotype Bemisia tabaci (Hemiptera: Aleyrodidae)

B-biotype Bemisia tabaci (Gennadius) has invaded China over the past two decades. To understand the risks and to determine possible mechanisms of resistance to thiamethoxam in B. tabaci, a resistant strain was selected in the laboratory. Cross-resistance and the biochemical mechanisms of thiamethoxam resistance were investigated in the present study. A 66.3-fold thiamethoxam-resistant B. tabaci strain (TH-R) was established after selection for 36 generations. Compared with the susceptible strain (TH-S), the selected TH-R strain showed obvious cross-resistance to imidacloprid (47.3-fold), acetamiprid (35.8-fold), nitenpyram (9.99-fold), abamectin (5.33-fold) and carbosulfan (4.43-fold). No cross-resistance to fipronil, chlorpyrifos or deltamethrin was seen. Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) exhibited significant synergism on thiamethoxam effects in the TH-R strain (3.14- and 2.37-fold respectively). However, diethyl maleate (DEM) did not act synergistically with thiamethoxam. Biochemical assays showed that cytochrome P450 monooxygenase activities increased 1.21- and 1.68-fold respectively, and carboxylesterase activity increased 2.96-fold in the TH-R strain. However, no difference was observed for glutathione S-transferase between the two strains. B-biotype B. tabaci develops resistance to thiamethoxam. Cytochrome P450 monooxygenase and carboxylesterase appear to be responsible for the resistance. Reasonable resistance management that avoids the use of cross-resistance insecticides may delay the development of resistance to thiamethoxam in this species.

Biochemical mechanisms of insecticide resistance in the diamondback moth (DBM), Plutella xylostella L. (Lepidopterata: Yponomeutidae), in the Sydney region, Australia

AbstractFollowing the detection of resistant diamondback moth (DBM) populations to synthetic pyrethroid, organophosphorus and indoxacarb insecticides in the Sydney Basin, a study of the major biochemical mechanisms was conducted to determine the type of resistance in these populations. The activity of cytochrome P450 monooxygenases increased two‐ to sixfold when compared with the susceptible strain. Up to a 1.9‐fold increase in esterase activity in resistant strains compared with the susceptible strain was observed. In vitro inhibition studies showed that profenofos, methamidophos and chlorpyrifos strongly inhibited the esterases while permethrin and esfenvalerate resulted in less than 30% inhibition. Qualitative analysis of the esterases using native polyacrylamide gel electrophoresis showed four bands in both the susceptible and resistant individuals with more intense staining in the resistant individuals. The development of these bands was inhibited by methamidophos and chlorpyrifos pretreatment of the protein extract while permethrin and esfenvalerate did not exhibit this effect. Glutathione S‐transferase (GST) activity was significantly higher in two field populations compared with the remaining populations. Overall, the study showed that the mechanisms of insecticide resistance in the DBM populations in the area studied were due to cytochrome P450 monooxygenases, esterase and GSTs, and possibly other non‐metabolic mechanisms that were not investigated in the present study.

Toxicogenomics Applied to Cultures of Human Hepatocytes Enabled an Identification of Novel Petasites hybridus Extracts for the Treatment of Migraine with Improved Hepatobiliary Safety

Butterbur extracts (Petasites hybridus) are recommended for the prevention of migraine, but pharmacovigilance reports may be suggestive of rare hepatobiliary toxicity. To evaluate its hepatotoxic potential, a series of in vivo and in vitro studies were carried out. Essentially, there were no signs of hepatocellular toxicity at estimated therapeutic C(max) levels of 60 ng/ml. Nonetheless, in a 28-day toxicity study at approximately 200-fold of therapeutic doses, induced liver transaminases and bilirubin elevations were observed. In a subsequent 6-month chronic toxicity study, the initial hepatobiliary effects were reproduced, but at the end of the study, liver function recovered and returned to normal as evidenced by clinical chemistry measurements. To identify possible mechanisms of hepatotoxicity, we investigated liver function in vitro at > 170-fold of therapeutic C(max) levels, including cytotoxicity (lactate dehydrogenase, MTT, and ATP), transaminase activities (alanine aminotransferase and aspartate aminotransferase), albumin synthesis, urea and testosterone metabolism to assay for cytochrome P450 monooxygenase activity. Only with extracts rich in petasin (37% petasin) and at high and well above therapeutic doses, liver toxicity was observed. A toxicogenomic approach applied to hepatocyte cultures enabled hypothesis generation and was highly suggestive for extracts high in petasin content to impair bile acid transport and lipid and protein metabolism. Importantly, neither chronic rat in vivo nor rat in vitro studies predicted reliably hepatotoxicity, therefore reemphasizing the utility of human-based in vitro investigations for the development of safe medicinal products. Finally, toxicogenomics enabled the characterization of a novel butterbur extract with no signals for hepatotoxicity.

Metabolism of sulphonated anthraquinones in rhubarb, maize and celery: the role of cytochromes P450 and peroxidases

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation, and thus contaminating many industrial effluents and rivers. In the development of a phytotreatment to remove sulphonated aromatic compounds, rhubarb (Rheum rhaponticum), a plant producing natural anthraquinones, as well as maize (Zea mays) and celery (Apium graveolens), plants not producing anthraquinones, were tested for their ability to metabolise these xenobiotics. Plants were cultivated under hydroponic conditions, with or without sulphonated anthraquinones, and were harvested at different times. Either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 towards several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results indicated that the activity of cytochromes P450 and peroxidases significantly increased in rhubarb plants cultivated in the presence of sulphonated anthraquinones. A higher activity of cytochromes P450 was also detected in maize and celery exposed to the pollutants. In these two plants, a peroxidase activity was also detected, but without a clear difference between the control plants and the plants exposed to the organic contaminants. This research demonstrated the existence in rhubarb, maize and celery of biochemical mechanisms involved in the metabolism and detoxification of sulphonated anthraquinones. Taken together, results confirmed that rhubarb might be the most appropriate plant for the phytotreatment of these organic pollutants.

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two‐spotted spider mite Tetranychus urticae (Acari: Tetranychidae)

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono-oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Fipronil resistance in the whitebacked planthopper (Sogatella furcifera): possible resistance mechanisms and cross‐resistance

The whitebacked planthopper (WBPH), Sogatella furcifera (Horváth), is a major rice pest in many parts of Asia. Fipronil has been widely used to control rice pests, and resistance to fipronil has been reported in some important species. A field population (F) of WBPH was collected, with 50.5-fold resistance to fipronil, which increased to 137.5-fold (F-se) after continuous selection for 11 generations. The F-se population did not show significant cross-resistance to the insecticides examined by comparison with the F population. TPP (synergism ratio 1.9), DEM (1.5) and PBO (1.1) showed only slight synergism on fipronil in the F-se population. A large increase was found in esterase and P450 monooxygenase activity in the F-se population. In vitro, PBO inhibited both esterase and P450 monooxygenase activity, and TPP inhibited esterase activity in the F-se population. Synergistic study in vivo and biochemical study in vitro indicated that esterase and P450 monooxygenases might be important factors for fipronil resistance in the selected population F-se. However, these biochemical factors could not lead to such high resistance (137.5-fold) in the F-se population, and target-site insensitivity would be another or more important factor.

Acetylcholinesterase point mutations putatively associated with monocrotophos resistance in the two-spotted spider mite

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene ( Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC 50) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance ( r 2 = 0.712) compared to the F439W mutation ( r 2 = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced ( r 2 = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.

Characterisation of imidacloprid resistance mechanisms in the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae)

Effective control of the brown planthopper, Nilaparvata lugens Stål, across rice-growing regions of Asia has been seriously compromised over the last 2 years by the appearance of widespread resistance to the neonicotinoid insecticide, imidacloprid. Sequence analysis of the ligand-binding domain of the nicotinic acetylcholine receptor α1 subunit from two field-collected resistant strains (CHN-2 and IND-11) did not reveal the Y151S point mutation previously implicated in conferring target-site resistance in this species. This result was supported by ligand-binding studies with [ 3H]-imidacloprid that showed no significant change in insecticide binding to isolated membranes from susceptible and resistant strains. In contrast, there was an approximate 5-fold increase in the mixed function oxidase activity for the two resistant strains suggesting that imidacloprid metabolism by increased cytochrome P450 monooxygenase activity is the major mechanism of resistance in these strains.

The role of cytochromes P450 and peroxidases in the detoxification of sulphonated anthraquinones by rhubarb and common sorrel plants cultivated under hydroponic conditions

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation and thus not eliminated by classical wastewater treatments. In the development of a phytotreatment to remove sulphonated aromatic compounds from dye and textile industrial effluents, it has been shown that rhubarb (Rheum rabarbarum) and common sorrel (Rumex acetosa) are the most efficient plants. Both species, producing natural anthraquinones, not only accumulate, but also transform these xenobiotic chemicals. Even if the precise biochemical mechanisms involved in the detoxification of sulphonated anthraquinones are not yet understood, they probably have cross talks with secondary metabolism, redox processes and plant energy metabolism. The aim of the present study was to investigate the possible roles of cytochrome P450 monooxygenases and peroxidases in the detoxification of several sulphonated anthraquinones. Both plant species were cultivated in a greenhouse under hydroponic conditions, with or without sulphonated anthraquinones. Plants were harvested at different times and either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 toward several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. A significant activity of cytochromes P450 was detected in rhubarb leaves, while no (rhizome) or low (petioles and roots) activity was found in other parts of the plants. An induction of this enzyme was observed at the beginning of the exposition to sulphonated anthraquinones. The results also indicated that cytochromes P450 were able to accept as substrate the five sulphonated anthraquinones, with a higher activity toward AQ-2,6-SS (0.706 nkat/mg protein) and AQ-2-S (0.720 nkat/mg protein). An activity of the cytochromes P450 was also found in the leaves of common sorrel (1.212 nkat/mg protein (AQ-2,6-SS)), but no induction of the activity occurred after the exposition to the pollutant. The activity of peroxidases increased when rhubarb was cultivated in the presence of the five sulphonated anthraquinones (0.857 nkat/mg protein). Peroxidase activity was also detected in the leaves of the common sorrel (0.055 nkat/mg protein), but in this plant, no significant difference was found between plants cultivated with and without sulphonated anthraquinones. Results indicated that the activity of cytochromes P450 and peroxidases increased in rhubarb in the presence of sulphonated anthraquinones and were involved in their detoxification mechanisms. These results suggest the existence in rhubarb and common sorrel of specific mechanisms involved in the metabolism of sulphonated anthraquinones. Further investigation should be performed to find the next steps of this detoxification pathway. Besides these promising results for the phytotreatment of sulphonated anthraquinones, it will be of high interest to develop and test, at small scale, an experimental wastewater treatment system to determine its efficiency. On the other hand, these results reinforce the idea that natural biodiversity should be better studied to use the most appropriate species for the phytotreatment of a specific pollutant.

Endotoxin causes functional endoplasmic reticulum failure, possibly mediated by mitochondria

Inflammatory response has recently been shown to induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which either recovers proper ER function or activates apoptosis. Here we show that endotoxin (lipopolysaccharide = LPS) can lead to functional ER failure tentatively via a mitochondrion-dependent pathway in livers of rats. Histological examination did not reveal significant damage to liver in form of necroses. Electron microscopy displayed transparent rings appearing around morphologically unchanged mitochondria, which were identified as dilated ER. The spliced mRNA variant of X-box protein-1 (XBP1) and also the mRNA of 78 kDa glucose-regulated protein (GRP78) were up-regulated, both typical markers of ER stress. However, GRP78 was down-regulated at the protein level. A pro-apoptotic shift in the bax/bcl-XL mRNA ratio was not accompanied by translocation of apoptosis inducing factor (AIF) to the nucleus, suggesting that the cells entered a pre-apoptotic state, but apoptosis was not executed. Monooxygenase activity of p450, representing the detoxification system in ER, was decreased after administration of endotoxin. Biochemical analysis of proteins important for ER function revealed the impairment of protein folding, transport, and detoxification suggesting functional ER failure. We suggest that functional ER failure may be a reason for organ dysfunction upon excessive inflammatory response mediated by endotoxin.

In vitro metabolism of polychlorinated biphenyls and cytochrome P450 monooxygenase activities in dietary-exposed Greenland sledge dogs

The in vitro metabolism of a polychlorinated biphenyl (PCB) mixture was examined using hepatic microsomes of dietary-exposed Greenland sledge dogs ( Canis familiaris) to an organohalogen-rich diet (Greenland minke whale blubber: EXP cohort) or a control diet (pork fat: CON cohort). The associations between in vitro PCB metabolism, activity of oxidative hepatic microsomal cytochrome P450 (CYP) isoenzymes and concentrations of PCBs and hydroxylated metabolites were investigated. The CON dogs exhibited a 2.3-fold higher depletion percentage for the PCB congeners having at least two pairs of vicinal meta-para Cl-unsubstituted carbons (PCB-18 and -33) relative to the EXP dogs. This depletion discrepancy suggests that there exist substrates in liver of the organohalogen-contaminated EXP dogs that can competitively bind and/or interfere with the active sites of CYP isoenzymes, leading to a lower metabolic efficiency for these PCBs. Testosterone (T) hydroxylase activity, determined via the formation of 6β-OH-T, 16α-OH-T, 16β-OH-T and androstenedione, was strongly correlated with the depletion percentages of PCB-18 and -33 in both cohorts. Based on documented hepatic microsomal CYP isoenzyme substrate specificities in canines, present associations suggest that primarily CYP2B/2C and CYP3A were inducible in sledge dogs and responsible for the in vitro metabolism of PCB-18 and -33.

The Effect of Insecticide Synergists on the Response of Scabies Mites to Pyrethroid Acaricides

BackgroundPermethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.Methodology/Principal FindingsTo determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF (S,S,S-tributyl phosphorotrithioate) and DEM (diethyl maleate) were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.ConclusionsThese findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites.

Effects of Andrographis paniculata extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities after in vivo administration to rats and in vitro in rat and human hepatocyte cultures

The expression of cytochrome P450 (CYP) is regulated by both endogenous factors and foreign compounds including drugs and natural compounds such as herbs. When herbs are co-administrated with a given drug in modern medicine it can lead to drug–herb interaction that can be clinically significant. The ability of Andrographis paniculata extract (APE) and Andrographolide (AND), the most medicinally active phytochemical in the extract, to modulate hepatic CYP expression was examined in vivo in rats and in vitro in rat and human hepatocyte cultures. After in vivo administration, APE at dose levels of 0.5 g/kg/day (i.e. 5 mg/kg/day AND equivalents) and at 2.5 g/kg/day (i.e. 25 mg/kg/day AND equivalents) and AND at dose levels of 5 and 25 mg/kg/day significantly decreased CYP2C11 activity. In primary cultures of rat and human hepatocytes, treatment with AND 50 μM and APE-containing 50 μM AND also resulted in significant decreases in CYP2C expression and activity. In addition, in human hepatocytes, treatment with APE and AND 50 μM resulted in a decrease in CYP3A expression and activity. In conclusion, this study suggests that AND and APE could cause herb–drug interactions in humans through modulation of CYP2C9 and CYP3A4 expression and activities.