Polymorphisms in genes encoding drugs and xenobiotic metabolizing enzymes in a Brazilian population

Polymorphic variations of several genes associated with drugs and xenobiotic metabolism have been linked to the factors that predispose to the carcinogenesis process. As considerable interindividual and interethnic variation in metabolizing enzyme activity has been associated with polymorphic alleles, we evaluated the frequency of the polymorphisms of CYP2D6, EPHX1 and NQO1 genes in 361 Brazilian individuals separated by ethnicity (European and African ancestry), using the polymerase chain reaction-restriction fragment length (PCR-RFLP) method. The allele frequencies of the variants *3 and *4 for the gene CYP2D6 were 0.04 and 0.14 for white subjects and 0.03 and 0.10 for black individuals, respectively. For the both variants of the gene EPHX1, we found higher allele frequencies among white individuals compared with mulatto subjects (0.62 vs 0.54 and 0.18 vs 0.14, respectively); however, these differences were not statistically significant (p = 0.39 and 0.56, respectively). For the NQO1 gene we observed a higher frequency of the homozygous genotype among black individuals (7.9%) compared with white subjects (6.3%) (p = 0.003). The genotype frequencies were within the Hardy-Weinberg equilibrium. We concluded that the allele frequencies of CYP2D6, EPHX1 and NQO1 gene polymorphisms in this Brazilian population showed ethnic variability when compared with those observed in other populations.

Consistent Alterations of Circulating Matrix Metalloproteinases Levels in Untreated Hypertensives and in Spontaneously Hypertensive Rats: A Relevant Pharmacological Target

Inconsistent matrix metalloproteinases (MMPs) levels have been reported in hypertension, with higher, similar and lower MMPs levels reported in hypertensives compared with normotensives. Differences between studies may reflect lack of control of drug effects, accompanying diseases and pre-analytical issues. We compared MMP-2, MMP-8 and MMP-9 levels in 38 untreated hypertensive patients (with no other diseases) with those found in 33 normotensive controls. We also studied endogenous MMPs inhibitors (TIMP-1, TIMP-2 and alpha-2-macroglobulin-A2M). Additionally, we assessed MMPs and A2M levels in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. We hypothesized that similar MMPs/endogenous inhibitors` profiles would be found in this animal model of hypertension and in clinical hypertension. MMPs, TIMPs and A2M were measured in plasma samples with commercially available ELISA and gelatin zymography. We found unaltered MMP-2, MMP-8, TIMP-1, TIMP-2 and A2M levels in hypertension. However, hypertensives had higher MMP-9 levels and MMP-9/A2M ratios than normotensives. Moreover, while we found similar MMP-2 and A2M levels in SHR and WKY rats, we found higher MMP-9 levels and MMP-9/A2M ratios in SHR versus WKY rats. These findings show consistent abnormal net plasma MMP-9 (but not MMP-2) activity in clinical and experimental hypertension. These parallel alterations in clinical hypertension and in SHR suggest an important role for MMPs in hypertension. While MMPs may be a relevant pharmacological target, antihypertensive drugs that down-regulate MMPs may offer advantages in the management of this disease.

Evaluation of IGF-2/ApaI polymorphism in pregnant women infected with human immunodeficiency virus type 1 taking antiretroviral drugs

Objective: Studies carried Out to assess the effects of antiretroviral drugs (ARV) in HIV-1 infected pregnant women have demonstrated carbohydrate intolerance. Some reports also refer to the effect of disturbances in the expression of the insulin-like growth factor (IGF) system on pancreas beta-cell function in humans and IGF-2/ApaI polymorphisms have been associated with obesity and features of the metabolic syndromes. in the present study, we tested the association between IGF-2/ApaI genotype and hyperglycemia in HIV-1 infected pregnant women receiving ARV. Design: We studied IGF-2/ApaI polymorphism in 87 healthy pregnant women, 43 HIV-1 infected pregnant women taking ARV with hyperglycemia during pregnancy, and 43 HIV-1-negative pregnant women with gestational diabetes. Blood samples were obtained for DNA extraction, PCR and genotyping. Data were analyzed statistically by the Kolmogorov-Smirnov normality, ANOVA and chi-square tests. Results: There were no significant differences in genotype frequency among the three groups analyzed. Considering the HIV-1-infected pregnant women, there were no significant differences in genotype frequency between the zidovudine group and the triple antiretroviral treatment group. There were no significant differences in allele frequencies among the groups evaluated. Non-white pregnant women tended to present the GG genotypes compared to white pregnant women. Conclusion: These results contribute to a better understanding of metabolic glycemic disorders in HIV-1 infected pregnant women using ARV, showing that IGF-2/ApaI polymorphisms are not responsible as a single Causative factor of glycemic alterations. These data indicate that other variables should be studied in order to explain these glycemic abnormalities. (C) 2009 Elsevier Ltd. All rights reserved.

Rasmussen encephalitis: long-term outcome after surgery

Rasmussen encephalitis (RE) is characterized by intractable epilepsy, progressive hemiparesis, and unilateral hemispheric atrophy. The progression of the symptoms to significant neurological impairment usually occurs within months to a few years. RE causes are unknown, although evidence of an autoimmune process has been extensively described in the literature. Antiepileptic drugs are usually not effective to control seizures or cerebral atrophy; despite data supporting a beneficial effect of early immunosuppressive and immunomodulatory interventions, for intractable seizures in RE patients with advanced disease, epilepsy surgery in the form of hemispheric disconnection has been considered the treatment of choice. This work describes the clinical and electrographic analyses, as well as the post-operative evolution of patients with RE. This work includes all the patients with RE evaluated from January 1995 to January 2008 by the RibeirA o pound Preto Epilepsy Surgery Program (CIREP), taking variables such as gender; age at epilepsy onset; seizure semiology; seizure frequency; interictal and ictal electroencephalographic (EEG) findings; age at surgery, when done; duration of epilepsy; surgery complications; follow-up duration; anatomo-pathological findings; post-surgery seizure; language and cognitive outcome; and anti-epileptic drug treatment after surgery into account. Twenty-five patients were evaluated; thirteen were female. Mean age of epilepsy onset was 4.4 +/- 2.0 years. There were no differences between patients with slow and fast evolution with respect to age of epilepsy onset (p = 0.79), age at surgery (p = 0.24), duration of epilepsy (0.06), and follow-up (p = 0.40). There were no correlations between the presence of bilateral EEG abnormalities or the absence of spikes and post-operative seizure outcome (p = 0.06). Immunomodulatory therapy was tried in 12 patients (48%). Twenty-three patients underwent surgery. The mean follow-up was 63.3 months. Eleven patients had total seizure control. Twelve individuals persisted with seizures consisting of mild facial jerks (six patients), occasional hemigeneralized tonic-clonic seizures (three patients), and frequent tonic-clonic seizures (three patients). Mental and language impairment was observed in 15 and 12 patients, after surgery, respectively. Eight patients presented post-operative cognitive decline, while only two patients had cognitive improvement. Comparing pre- and post-operative language deficits, 66.7% of the 12 patients with language disturbance did not improve after surgery. This retrospective study reported the clinical and electrographic analysis, as well as the evolution of 23 patients with RE. Patients were divided into two groups: fast evolution and slow evolution to hemiparesis and epilepsia partialis continua. These groups may represent different RE substrates. Fourteen patients achieved satisfactory seizure control, three patients had partial response to surgery, and five patients had maintenance of the pre-operative condition. All patients with left-side involvement presented with some language disturbance, which did not improve after surgery in 66.6% of patients. Cognitive evaluation showed that the majority of the patients did not have any significant improvement, and 38.1% had cognitive deterioration after surgery.

Vascular endothelial growth factor genotypes and haplotypes are associated with pre-eclampsia but not with gestational hypertension

Vascular endothelial growth factor (VEGF) is relevant for normal pregnancy, and abnormalities in VEGF functions are associated with hypertensive disorders of pregnancy. Because there are few studies on how VEGF genetic polymorphisms affect susceptibility to pre-eclampsia (PE), and no studies on how they affect susceptibility to gestational hypertension (GH), we compared VEGF genotype and haplotype distributions in normotensive and hypertensive pregnancies. Genotypes and haplotypes for VEGF polymorphisms (C-2578A, G-1154A and G-634C) were determined in 303 pregnant women (108 healthy pregnant, HP; 101 with GH and 94 with PE). When white and non-white pregnant women were considered together, no significant differences were found in the distributions of VEGF genotypes or haplotypes (P > 0.05) in the three groups. However, with only white subjects, significant differences were found in genotypes distributions for two (C-2578A and G-634C) VEGF polymorphisms (both P < 0.05) between the HP and the PE groups. Importantly, the haplotype including the variants C-2578, G-1154 and C-634, which is associated with higher VEGF gene expression, was less common in the PE group compared with the HP group (4% versus 16%; P = 0.0047). However, we found no significant differences in VEGF haplotypes distributions when the HP and GH groups were compared (P > 0.05). These findings suggest a protective effect for the `C-2578, G-1154 and C-634` haplotype against the development of PE, but no major effects of VEGF gene variants on susceptibility to GH.

Influence of enzyme inducing antiepileptic drugs on the pharmacokinetics of levetiracetam in patients with epilepsy

To assess whether levetiracetam elimination is influenced by enzyme inducing antiepileptic drugs (EIAEDs), serum levetiracetam levels were determined at frequent intervals after a single oral 1000 mg dose in 15 subjects co-medicated with EIAEDs and 15 matched controls. The EIAED group showed a higher levetiracetam oral clearance (p = 0.01) and a shorter half-life (p = 0.02) than controls. Although the magnitude of interaction is relatively modest, it could have clinical significance for some patients. (C) 2011 Elsevier B.V. All rights reserved.

Interchangeability among therapeutics equivalents of lamotrigine in the treatment of refractory epilepsy patients: risks and benefits

Introduction. Epilepsy is a condition characterized by signs and symptoms of neurological disorder. Lamotrigine has been widely used, mainly due to their greater tolerability and lower rate of drug interactions with other antiepileptic drugs however the newest antiepileptic drugs have high cost to patient. In Brazil there are three different sort of pharmaceutical equivalents (reference, generic and similar), and the Brazilian health care authorities offers to users the possibility to receive them free of charge. Moreover these pharmaceutical equivalents can change during the treatment of epilepsy because this authorities buy the cheapest by public tender two or three times a year. Aim. To evaluate the clinical and laboratory findings related to the most frequently used therapeutic equivalents of lamotrigine (reference drugs and similar products). Patients and methods. Two similar formulations (A and B) and one reference (C) were tested in nine epileptic refractory patients. The study was divided into three periods of 42 days, one for each formulation, and medical data about the frequency of seizures, the occurrence of side effects and measurement of plasma concentrations of lamotrigine were collected. Results. The average number of seizures/week and plasma concentration of lamotrigine for formulations A, B and C were not statistically significant differences. Three patients during the use of the formulation C presented mild and transitory side effects. Conclusion. Similar or reference drugs showed satisfactory results, however the interchangeability among the formulations raise the difficulty for the management of seizures in refractory epilepsy.

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro-fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 on In Vitro Produced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 mu l of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

Composite pre-heating: Effects on marginal adaptation, degree of conversion and mechanical properties

Objectives. This study evaluated the effect of composite pre-polymerization temperature and energy density on the marginal adaptation (MA), degree of conversion (DC), flexural strength (FS), and polymer cross-linking (PCL) of a resin composite (Filtek Z350, 3M/ESPE). Methods. For MA, class V cavities (4mmx2mmx2mm) were prepared in 40 bovine incisors. The adhesive system Adper Single Bond 2 (3M/ESPE) was applied. Before being placed in the cavities, the resin composite was either kept at room-temperature (25 degrees C) or previously pre-heated to 68 degrees C in the Calset (TM) device (AdDent Inc., Danbury, CT, USA). The composite was then light polymerized for 20 or 40s at 600mW/cm(2) (12 or 24 J/cm(2), respectively). The percentage of gaps was analyzed by scanning electron microscopy, after sectioning the restorations and preparing epoxy resin replicas. DC (n = 3) was obtained by FT-Raman spectroscopy on irradiated and non-irradiated composite surfaces. FS (n = 10) was measured by the three-point-bending test. KHN (n = 6) was measured after 24h dry storage and again after immersion in 100% ethanol solution for 24 h, to calculate PCL density. Data were analyzed by appropriate statistical analyses. Results. The pre-heated composite showed better MA than the room-temperature groups. A higher number of gaps were observed in the room-temperature groups, irrespective of the energy density, mainly in the axial wall (p < 0.05). Composite pre-heating and energy density did not affect the DC, FS and PCL (p > 0.05). Significance. Pre-heating the composite prior to light polymerization similar in a clinical situation did not alter the mechanical properties and monomer conversion of the composite, but provided enhanced composite adaptation to cavity walls. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.