Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Coptotermes gestroi (Isoptera: Rhinotermitidae) in Brazil: possible origins inferred by mitochondrial cytochrome oxidase II gene sequences

The Asian subterranean termite, Coptotermes gestroi, originally from northeast India through Burma, Thailand, Malaysia, and the Indonesian archipelago, is a major termite pest introduced in several countries around the world, including Brazil. We sequenced the mitochondrial COII gene from individuals representing 23 populations. Phylogenetic analysis of COII gene sequences from this and other studies resulted in two main groups: (1) populations of Cleveland (USA) and four populations of Malaysia and (2) populations of Brazil, four populations of Malaysia, and one population from each of Thailand, Puerto Rico, and Key West (USA). Three new localities are reported here, considerably enlarging the distribution of C. gestroi in Brazil: Campo Grande (state of Mato Grosso do Sul), Itajai (state of Santa Catarina), and Porto Alegre (state of Rio Grande do Sul).

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

E2 potentializes benzo(a)pyrene-induced hepatic cytochrome P450 enzyme activities in Nile tilapia at high concentrations

In the aquatic environment, biotransformation enzymes are established biomarkers for assessing PAH exposure in fish, but little is known about the effect of 17β-estradiol (E2) on these enzymes during exposure to benzo(a)pyrene (BaP). In this study, Nile tilapia (Oreochromis niloticus) were exposed for 3, 5, and 10 days to BaP (300 μg L(-1)) and E2 (5 μg L(-1)). These substances were applied isolated or mixed. In the mixture experiment, fish were analyzed pre- and postexposure in order to better understand whether preexposure to the hormone masks the responses activated by PAH or vice versa. Phase I enzymes ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-depenthylase (PROD), and benzyloxyresorufin-O-debenzylase (BROD) activities as well as the phase II enzyme glutathione S-transferase (GST) were analyzed. Isolated E2 treatment decreased EROD activity after 3 days, but this enzyme activity returned to control values after 5 and 10 days of exposure. Isolated BaP treatment significantly induced EROD activity after 3 and 5 days, and the activity returned to control levels after ten exposure days. Combined treatment (E2 + Bap) significantly increased EROD activity, both in the pre- and postexposure. This increase was even higher than in the isolated BaP treatment, suggesting a synergism between these two compounds. When E2 and BaP were used singly, they did not change BROD and PROD activities. However, combined treatment (E2 + Bap) significantly increased PROD activity. Isolated BaP treatment increased GST activity after 10 days. However, this response was not observed in the mixture treatment, suggesting that E2 suppressed the GST induction modulated by BaP. The results put together indicated that E2 altered the biotransformation pathway regarding enzymes activated by BaP in Nile tilapia.

Influence of gasoline inhalation on the enantioselective pharmacokinetics of fluoxetine in rats

Fluoxetine is used clinically as a racemic mixture of (+)-(S) and (-)-(R) enantiomers for the treatment of depression. CYP2D6 catalyzes the metabolism of both fluoxetine enantiomers. We aimed to evaluate whether exposure to gasoline results in CYP2D inhibition. Male Wistar rats exposed to filtered air (n = 36; control group) or to 600 ppm of gasoline (n = 36) in a nose-only inhalation exposure chamber for 6 weeks (6 h/day, 5 days/week) received a single oral 10-mg/kg dose of racemic fluoxetine. Fluoxetine enantiomers in plasma samples were analyzed by a validated analytical method using LC-MS/MS. The separation of fluoxetine enantiomers was performed in a Chirobiotic V column using as the mobile phase a mixture of ethanol:ammonium acetate 15 mM. Higher plasma concentrations of the (+)-(S)-fluoxetine enantiomer were found in the control group (enantiomeric ratio AUC(+)-(S)/(-)-(R) = 1.68). In animals exposed to gasoline, we observed an increase in AUC0-∞ for both enantiomers, with a sharper increase seen for the (-)-(R)-fluoxetine enantiomer (enantiomeric ratio AUC(+)-(S)/(-)-(R) = 1.07), resulting in a loss of enantioselectivity. Exposure to gasoline was found to result in the loss of enantioselectivity of fluoxetine, with the predominant reduction occurring in the clearance of the (-)-(R)-fluoxetine enantiomer (55% vs. 30%). Chirality 25:206-210, 2013. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc.

DNA repair gene polymorphism is associated with the genetic basis of atherosclerotic coronary artery disease

Background: Atherosclerotic coronary artery disease (CAD) is a multifactorial process that appears to be caused by the interaction of environmental risk factors with multiple predisposing genes. It is nowadays accepted that increased levels of DNA damage induced by xenobiotics play an important role in the early phases of atherogenesis. Therefore, in this study, we focus on determining whether genetic variations in xenobiotic-metabolizing [glutathione-S-transferase theta 1 (GSTT1), glutathione-S-transferase mu 1 (GSTM1), cytochrome P450 IIEI (CYP2E1)] and DNA repair [X-ray cross-complementing group 1 (XRCC1)] genes might be associated with increased risk for CAD. Methods: A case-control study was conducted with 400 individuals who underwent subjected to coronary angiography. A total of 299 were patients diagnosed with effective coronary atherosclerosis (case group; >20% obstructive lesion), and 101 (control group) were individuals diagnosed as negative for CAD (<20% obstructive lesions). The polymorphism identifications for GSTM1 and GSTT1, and for CYP2E1 and XRCC1 genes were performed by polymerase chain reaction (PCR) amplification and by PCR-RFLP, respectively. Results and conclusions: The XRCC1 homozygous wild-type genotype Arg/Arg for codon 399 was statistically less pronounced in the case subjects (21.4%) than in controls (38.5%); individuals with the variant XRCC1 genotype had a 2.3-fold increased risk for coronary atherosclerosis than individuals with the wild-type genotype (OR=2.3, 95% CI=1.13-4.69). Conversely, no association between GSTM1, GSTT1, and CYP2E1gene polymorphisms and coronary atherosclerosis was detected. The results provide evidence of the role of DNA damage and repair in cardiovascular disease. © 2011 Elsevier Inc. All rights reserved.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

GSTT1, GSTM1 and CYP2E1 genetic polymorphisms in gastric cancer and chronic gastritis in a Brazilian population

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Advances in prodrug design

The background of prodrug design is presented herein as the basis for introducing new and advanced latent systems, taking into account mainly the versatility of polymers and other macromolecules as carriers. PDEPT (Polymer-Directed Enzyme Prodrug Therapy); PELT (Polymer-Enzyme Liposome Therapy); CDS (Chemical Delivery System); ADEPT(Antibody-Directed Enzyme Prodrug Therapy); GDEPT/VDEPT (Gene-Directed Enzyme Prodrug Therapy/Virus-Directed Enzyme Prodrug Therapy); ODDS (Osteotropic Drug Delivery System) and LEAPT (Lectin-directed enzyme-activated prodrug therapy) are briefly described and some examples are given. © 2005 Bentham Science Publishers Ltd.

Cyp17a1 and cyp19a1 in the zebrafish testis are differentially affected by oestradiol

Oestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17β-oestradiol (E2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology. © 2013 Society for Endocrinology.

Effects of β-glucan extracted from Agaricus blazei on the expression of ERCC5, CASP9, and CYP1A1 genes and metabolic profile in HepG2 cells

The polysaccharide β-glucan has biological properties that stimulate the immune system and can prevent chronic pathologies, including cancer. It has been shown to prevent damage to DNA caused by the chemical and physical agents to which humans are exposed. However, the mechanism of β-glucan remains poorly understood. The objective of the present study was to verify the protective effect of β-glucan on the expression of the genes ERCC5 (involved in excision repair of DNA damage), CASP9 (involved in apoptosis), and CYP1A1 (involved in the metabolism of xenobiotics) using real-time polymerase chain reaction and perform metabolic profile measurements on the HepG2 cells. Cells were exposed to only benzo[a]pyrene (B[a]P), β-glucan, or a combination of B[a]P with β-glucan. The results demonstrated that 50 μg/mL β-glucan significantly repressed the expression of the ERCC5 gene when compared with the untreated control cells in these conditions. No change was found in the CASP9 transcript level. However, the CYP1A1 gene expression was also induced by HepG2 cells exposed to B[a]P only or in association with β-glucan, showing its effective protector against damage caused by B[a]P, while HepG2 cells exposed to only β-glucan did not show CYP1A1 modulation. The metabolic profiles showed moderate bioenergetic metabolism with an increase in the metabolites involved in bioenergetic metabolism (alanine, glutamate, creatine and phosphocholine) in cells treated with β-glucan and to a lesser extent treated with B[a]P. Thus, these results demonstrate that the chemopreventive activity of β-glucan may modulate bioenergetic metabolism and gene expression. © 2013 The Author(s).

Influência da ingestão alimentar nas concentrações plasmáticas de insulina e progesterona, e na expressão de de enzimas hepáticas envolvidas no metabolismo de progesterona em ovelhas pré-púberes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)