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Pyrethroids and DDT are key insecticides in the control of malaria, yellow fever, and lymphatic filariasis vectors. Knockdown and metabolic resistance mechanisms have been proven to be important in determining the efficacy of insecticides. Here we investigated cytochrome P450 as a resistance mechanism in Anopheles gambiae Giles and Culex quinquefasciatus Say exposed to deltamethrin and DDT. Two- to three-days-old adult female mosquitoes were used for insecticide exposures and PBO synergistic assays using WHO standard guidelines, kits and test papers (DDT 4%, deltamethrin 0.05%, and PBO 4%). Polymerase chain reaction (PCR) assays were used for the identification of the species and for characterization of the kdr allele. Mortality at 24 h post-exposure was 18 and 17% in An. gambiae s.s. exposed to DDT and deltamethrin, respectively; 1 and 5% in Cx. quinquefasciatus exposed to DDT and deltamethrin respectively. Significant (P < 0.01) levels of susceptibility was recorded in mosquitoes pre-exposed to PBO, as KDT50 and 24 h of exposure ranged from 37.6 min to 663.4 min and 27 to 80%, respectively. Presence of a knockdown resistance allele was recorded in An. gambiae s.s., 22.5% for homozygote resistance and 7.5% for heterozygotes, while Cx. quinquefasciatus populations showed no kdr allele despite the high level of resistance to DDT and deltamethrin. Findings from this study indicated that cytochrome P450 mono-oxygenase expression is highly implicated in the resistance phenotype to DDT and pyrethroids in An. gambiae and Cx. quinquefasciatus in the study area.

Neurological diseases (ND) afflict hundreds of millions of people worldwide and constitute 12% of total deaths of the population all around the world. According to a global study conducted by the World Health Organization, 8 out of 10 disorders in the 3 rd highest disability classes are neurologic problems. Commonly they have disabling consequences, compromising severely the quality of the life of affected subjects. Often therapeutic strategies are limited in efficacy. Limitations lie with the enormous fragility of the so complex structure and functions accomplished by the central nervous system, together with the presence of the barrier between the tissue and the blood limiting the penetration of drugs. For these reason new strategies to protect the brain are essential. One way might be the enhancement of local biomolecular mechanisms (metabolizing enzymes, transporters, etc.). However, increasing evidence underline that our knowledge of the brain is still partial, and information cannot simply be derived from other well-known organs such as the liver. This is the case for the brain cytochrome P450 enzymes, showing differential cellular localization, brain regional expression and modulation.

The lung serves as a primary site for xenobiotic metabolism, and many xenobiotics are substrates for cytochrome P450 catalyzed oxidations. Notably, the lung is composed of > 40 different cell types with different levels of metabolic competence (Ding and Kaminsky 2003). Alveolar macrophages and bronchial epithelial cells are part of > 40 different cell types in lung tissue and differ in their biological functions (Hocking and Golde 1979; Hukkanen et al. 2002). Indeed alveolar macrophages clear the airways of tobacco smoke particles and therefore constitute an important defense mechanism (Drath et al. 1979). Furthermore, the lung epithelium takes part in the detoxification of tobacco smoke through metabolic activation by phase I and phase II enzymes (Crawford et al. 1998; Han et al. 2005). Although a number of CYP isoforms have been identified in human lung tissue, a comprehensive survey of most human pulmonary xenobiotic metabolizing enzymes in different human lung tissue compartments has not been performed. Tobacco smoke constitutes a complex mixture of thousands of toxicants, some of which require tissue-specific activation to become genotoxic carcinogens and others become metabolically activated poisons (Smith et al. 2003; Yamazaki et al. 1992). For smokers, the risk of developing lung cancer has been shown to be, at least in part, dependent on pulmonary metabolism of smoke constituents (Rubin 2001). Particular evidence stems from studies with smokers in which genetic polymorphisms of certain xenobiotic metabolizing enzymes were linked to the development of lung cancer (Bartsch et al. 2000; London et al. 1999). Currently available data suggest that genetic variability in coding sequences of P450 enzymes, antioxidants, or DNA repair genes contribute to the risk of developing bronchogenic carcinomas (Caporaso 2002). Cigarette smoke may affect the capacity of lung tissue to dispose of foreign chemicals by changing expression and coded activity of xenobiotic metabolizing enzymes. Additionally, cigarette smoke may affect the pharmacokinetics of inhaled drugs by modulating activity of specific drug metabolizing enzymes (Durak et al. 1996). Therefore, knowledge of expression and activity of xenobiotic and drug metabolizing enzymes in lung tissue of smokers gives us a better understanding of metabolically induced toxicity of tobacco smoke constituents and allows us to assess the capacity for tissue specific detoxification. We investigated the gene expression of 19 cytochrome P450 monooxygenases (CYPs) and 3 flavin-containing monooxygenases (FMOs), as well as uridine diphosphate glucuronosyltransferase (UGT) 2A1 (UGT2A1), epoxide hydrolase (EPHX1), glutathione-S-transferase (GST) A2 (GSTA2), GSTP1, and GSTM1 in lung cells obtained from bronchoalveolar lavage (BAL) fluid and in bronchial biopsies (BBs) of smokers (n = 8) and nonsmokers (n = 10). Additionally, transcriptional regulation of CYPs depends, at least in part, on promoter activation through binding of transcription factors and nuclear receptors to cognate DNA recognition sites (Borlak and Thum 2001; (Pascussi et al. 2003). We therefore studied expression of the aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), liver X receptor (LXR), and glucocorticoid receptor (GR) (Gibson et al. 2002; Tirona et al. 2003) for their established role in CYP gene regulation in BAL cells and BBs derived from smokers and nonsmokers.

4″-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strains with the ability to oxidize avermectin to 4″-oxo-avermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP+ reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in co-expression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly™, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening.

Deltamethrin and other pyrethroids have been extensively used in Argentina since 1980, for the chemical control of Triatoma infestans Klug (Hemiptera: Reduviidae). Recently, resistance to deltamethrin was detected in field populations by the survival of bugs exposed by topical application to the diagnostic dose estimated on the CIPEIN susceptible strain. Results of the current study showed low resistant ratios (RRs) to deltamethrin for the resistant populations (RR ranged from 2.0 for San Luis colony to 7.9 for Salta colony). Biochemical studies were made on the most resistant colony (Salta) and the susceptible strain (CIPEIN), in order to establish the importance of degradative mechanisms as a cause of the detected resistance. Esterase activity was measured on 3 days old first instars through phenylthioacetate and a-naphtyl acetate activities. The results showed a significant difference in no cholinesterase esterase activity from susceptible (7.6 +/- 0,7 micro M S./i.min.) and Salta resistant colony (9.5 +/- 0.8 microM S./i.min.). Cytochrome p450 mono-oxygenase (p450) activity was measured on individual insects through ethoxycoumarine deethylase (ECOD) activity using a fluorescence microplate reader. The dependence of ECOD activity on age and body region of the nymphs, and pH and time of incubation were studied in order to optimize the measurement. As a result, comparative studies were performed on abdomens of 2 days old first instars at pH 7.2 and 4 h incubation time. ECOD activity of first nymphs was significantly lower in the susceptible colony (61.3 +/- 9.08 pg ECOD/ insect) than in the resistant one (108.1+/- 5.7 pg ECOD/ insect). These results suggest that degradative esterases (no-cholinesterase) and mono-oxygenases cytochrome p450, play an important role in the resistance to deltamethrin in Salta colony from Argentina.

Oxidized low-density lipoprotein (oxLDL) is an important risk factor for vascular injury. Its role on coronary vasoconstriction remains speculative. Endothelial monooxygenases (cytochrome P450s [CYPs]) are regulators of vascular tonus through production of epoxy fatty acids. We investigated the effects of oxLDL on CYP monooxygenases in human arterial coronary endothelial cells and explanted healthy and atherosclerotic aortae. We found oxLDL to induce radical oxygen species production via the action of NADPH oxidase NOX4. Intracellular radical oxygen species production prompted reduced protein expression of the transcriptional regulator nuclear factor 1 (NF-1). We identified novel DNA binding sites for NF-1 in promoter regions of CYPs. DNA binding of NF-1 was confirmed by electromobility shift assays. OxLDL repressed DNA binding of NF-1 and diminished transcript level of CYP genes targeted by this factor. The production of endothelial-derived hyperpolarization factor, a key regulator of vascular tonus, was also reduced. Repression of CYP monooxygenases was reversed, and production of endothelial-derived hyperpolarization factor was normalized after treatment of endothelium with the lectin-like oxLDL receptor antagonist kappa-carrageenan or blocking of LOX-1 with a specific antibody. This suggests a mechanistic role of CYP monooxygenases in oxLDL-induced vascular injury. Therapy of endothelial dysfunction through LOX-1 receptor antagonism will be an interesting avenue to explore.

Cytochrome P450 mono-oxygenases (CYP) play an essential role in steroid metabolism, and there is speculation that sex hormones might influence cardiac mass and physiology. As CYP mono-oxygenases activity is frequently altered during disease, we tested our hypothesis that CYP mono-oxygenase expression and testosterone metabolism are altered in cardiac hypertrophy. We investigate major CYP mono-oxygenase isoforms and other steroid-metabolizing enzymes and the androgen receptor in normal, hypertrophic, and assist device-supported human hearts and in spontaneously hypertensive rats (SHR). We show increased and idiosyncratic metabolism of testosterone in hypertrophic heart and link these changes to altered CYP mono-oxygenase expression. We show significant induction of 5-alpha steroid reductase and P450 aromatase gene expression and enhanced production of dihydrotestosterone, which can be inhibited by the 5-alpha reductase inhibitor finasteride. We show increased gene expression of the androgen receptor and increased levels of lipid peroxidation in diseased hearts, the latter being markedly inhibited by CYP mono-oxygenase inactivation. We show alpha-MHC to be significantly repressed in cardiac hypertrophy and restored to normal on testosterone supplementation. We conclude that heart-specific steroid metabolism is of critical importance in cardiac hypertrophy.