SNPs from BCHE and DRD3 genes associated to cocaine abuse amongst violent individuals from Sao Paulo, Brazil.

Violence and drug abuse are highly destructive phenomena found world-wide, especially in Brazil. They seem to rise proportionally to one another and possibly related. Additionally, genetics may also play a role in drug abuse. This study has focused on identifying the use of cocaine within postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo as well as the presence of certain single nucleotide polymorphisms (SNPs) to better understand one's susceptibility to abuse the drug. Both hair and blood samples have been extracted through a simple methanol overnight incubation or a rapid dilute-and-shoot method, respectively. The samples were then analyzed using an UPLC-ESI-MS/MS and genotyped through RT-PCR. Statistical analyses were performed via SPSS software. From 105 postmortem cases, 53% and 51% of the cases shown to be positive for cocaine in hair and blood, respectively. Genetic wise, a significant difference has been observed for SNP rs4263329 from the BCHE gene with higher frequencies of the genotypes A/G and G/G seen in cocaine users (OR=8.91; 95%CI=1.58-50.21; p=0.01). Likewise, also SNP rs6280 from the DRD3 gene presented a significant association, with both genotypes T/C and C/C being more frequent in users (OR=4.96; 95% CI=1.07-23.02; p=0.04). To conclude, a rather high proportion of cocaine has been found, which may suggest a connotation between the use of the drug and risky/violent behaviors. Additionally, significant associations were also found within two SNPs related to cocaine use, however, due to several inherent limitations, these must be confirmed.

Alcohol in combination with illicit drugs among fatal injuries in Sao Paulo, Brazil: An epidemiological study on the association between acute substance use and injury.

Injury deaths have a major impact on public health systems, particularly in the Latin American region; however, little is known about how different drugs, in combination or not with alcohol, interact with each injury type. We tested an epidemiological protocol for investigating alcohol and other drug acute use among fatally injured victims taking into account the injury context for all injury causes in Sao Paulo, Brazil. Blood alcohol and drug content were fully screened and confirmed following a probability sample selection of decedents (n = 365) during 19 consecutive months (2014-2015). Drug concentrations, including benzodiazepines, cannabis, cocaine, and opioids were determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS/MS). Toxicology data were interpreted in combination with injury context retrieved from police records regarding cause, place of injury, and victims' criminal history. More than half of all fatally injured victims studied were under the influence of at least one substance (55.3%). Alcohol was the leading substance consumed before a fatal injury event (30.1%), followed by cocaine (21.9%) and cannabis (14%). Illicit drug use (cocaine and cannabis) comprised more than two thirds of all drug-related deaths. Alcohol-positive deaths are over-represented among road traffic injuries, while drug-positive deaths are more prevalent among intentional injuries. Victims who had previous criminal convictions were significantly more likely to have used illicit drugs compared to those who did not have a criminal background. We estimated that one in every two fatal injuries in the city of Sao Paulo is associated with acute substance use by the victim. The health burden attributed to alcohol- and drug-related fatal injury events has reached significant higher levels in Latin American cities such as Sao Paulo compared globally.

Determination of cocaine and its derivatives in hair samples by liquid phase microextraction (LPME) and gas chromatography-mass spectrometry (GC-MS).

Hair testing is a recognized approach when it comes to accessing historical drug use. According to the World Drug Report of United Nations Office on Drugs and Crime (UNODC) 2015, Brazil is the largest cocaine (COC) market in South America. New analytical methodologies to detect crack/cocaine analytes in hair samples are highly desirable. Here, a method consisting of a liquid-phase microextraction (LPME) as a clean-up step, followed by gas chromatography-mass spectrometry (GC-MS) analysis has been proposed. The new validated method consisted of a washing step; an overnight incubation with methanol and a quick derivatization with butylchloroformate. Once derivatized, the samples were then submitted to the LPME procedure. Limits of detection (LoD) and quantitation (LoQ) obtained were of 0.1 and 0.5ng/mg for COC 0.4 and 0.5ng/mg for anhydroecgonine methyl ester (AEME); 0.03 and 0.05 for cocaethylene (CE), respectively and 0.05ng/mg for both LoD and LoQ for benzoylecgonine (BZE). All calibration curves were linear over the scope applied, from LoQ up to 20ng/mg, with a r2>0.99. Precision and accuracy assays showed acceptable %RSD values, according to international guidelines. Twelve postmortem head hair samples stemming from the Institute of Legal Medicine of Sao Paulo (IML-SP) have been analyzed, from which seven have shown to be positive for COC (0.75->20ng/mg) and BZE (0.1->20ng/mg). Apart from COC's main metabolite, four samples were also positive for CE (0.1-3.9ng/mg) and three samples for AEME (0.5-4.9ng/mg). To conclude, the LPME technique together with GC-MS analysis have shown promising results and were able to meet the demand of the laboratory of analyzing postmortem hair samples to look for all four analytes.

Octopamine and Dopamine differentially modulate the nicotine-induced calcium response in Drosophila Mushroom Body Kenyon Cells.

In Drosophila associative olfactory learning, an odor, the conditioned stimulus (CS), is paired to an unconditioned stimulus (US). The CS and US information arrive at the Mushroom Bodies (MB), a Drosophila brain region that processes the information to generate new memories. It has been shown that olfactory information is conveyed through cholinergic inputs that activate nicotinic acetylcholine receptors (nAChRs) in the MB, while the US is coded by biogenic amine (BA) systems that innervate the MB. In this regard, the MB acts as a coincidence detector. A better understanding of the properties of the responses gated by nicotinic and BA receptors is required to get insights on the cellular and molecular mechanisms responsible for memory formation. In recent years, information has become available on the properties of the responses induced by nAChR activation in Kenyon Cells (KCs), the main neuronal MB population. However, very little information exists on the responses induced by aminergic systems in fly MB. Here we have evaluated some of the properties of the calcium responses gated by Dopamine (DA) and Octopamine (Oct) in identified KCs in culture. We report that exposure to BAs induces a fast but rather modest increase in intracellular calcium levels in cultured KCs. The responses to Oct and DA are fully blocked by a VGCC blocker, while they are differentially modulated by cAMP. Moreover, co-application of BAs and nicotine has different effects on intracellular calcium levels: while DA and nicotine effects are additive, Oct and nicotine induce a synergistic increase in calcium levels. These results suggest that a differential modulation of nicotine-induced calcium increase by DA and Oct could contribute to the events leading to learning and memory in flies.

Amphetamine, cocaine and cannabinoids use among truck drivers on the roads in the State of Sao Paulo, Brazil.

Drugs are important risk factors for traffic accidents. In Brazil, truck drivers report using amphetamines to maintain their extensive work schedule and stay awake. These drugs can be obtained without prescription easily on Brazilian roads. The use of these stimulants can result in health problems and can be associated with traffic accidents. There are Brazilian studies that show that drivers use drugs. However, these studies are questionnaire-based and do not always reflect real-life situations. The purpose of this study was to demonstrate the prevalence of drug use by truck drivers on the roads of Sao Paulo State, Brazil, during 2009. Drivers of large trucks were randomly stopped by police officers on the interstate roads during morning hours. After being informed of the goals of the study, the drivers gave written informed consent before providing a urine sample. In addition, a questionnaire concerning sociodemographic characteristics and health information was administered. Urine samples were screened for amphetamines, cocaine, and cannabinoids by immunoassay and the confirmation was performed using gas chromatography-mass spectrometry (GC-MS). Of the 488 drivers stopped, 456 (93.4%) provided urine samples, and 9.3% of them (n=42) tested positive for drugs. Amphetamines were the most commonly found (n=26) drug, representing 61.9% of the positive samples. Ten cases tested positive for cocaine (23.8%), and five for cannabinoids (11.9%). All drivers were male with a mean age of 40 ± 10.8 years, and 29.3% of them reported some health problem (diabetes, high blood pressure and/or stress). A high incidence of truck drivers who tested positive for drug use was found, among other reported health problems. Thus, there is an evident need to promote a healthier lifestyle among professional drivers and a need for preventive measures aimed at controlling the use of drugs by truck drivers in Brazil.

Descripción histológica de las glándulas anexas del aparato digestivo de avestruz (Struthio camelus var. domesticus) / Histologic description of the annexed glands from the ostrich digestive system (Struthio camelus var. domesticus)

En la última década, la crianza del avestruz en Chile ha ido aumentando sustancialmente, por lo cual se ha hecho necesario incrementar los estudios para mejorar la producción de esta especie. La literatura señala sólo estudios sobre la fisiología y anatomía de esta especie. Nuestro objetivo es aportar al conocimiento de la histología normal de las glándulas anexas al aparato digestivo. Esta investigación se realizó con 6 avestruces clínicamente sanas, de las que se obtuvieron muestras representativas del hígado, pro ventrículo y páncreas. Se realizaron cortes histológicos, los que fueron teñidos y montados para su análisis comparativo bajo microscopio de luz, entre avestruz y gallina. La histología de las glándulas anexas del aparato digestivo es semejante a la descrita en la gallina. Sin embargo, en el hígado los cordones de hepatocitos se disponen en forma radial, tanto alrededor de la vena central como de los espacios porta, característica no observada en otras especies. Con respecto al pro ventrículo, en la mucosa se observan glándulas tubulares simples o ramificadas, semejantes a las glándulas fúndicas de los mamíferos. En la submucosa se observan glándulas túbuloalveolares compuestas con células parietales. El páncreas no presenta diferencias destacables.

In the last decade the upbringing of the ostrich in Chile has increased substantially, for this reason it is necessary to increase the studies to improve the production of this species. The literature only points out studies on the physiology and anatomy of this species. The purpose of this work is to contribute to the knowledge of the normal histology from the annexed glands to the digestive system. This study was carried out clinically using 6 healthy ostriches, of which representative samples of the liver, proventricle and pancreas. Histological sections were realized, mounted and stained for their comparative analysis under low light microscope to describe with those cited in the hen. The histology of the annexed glands from the digestive system is similar to the described in the hen. However, in the liver the arrangement hepatic cords so is in the central vein such as the portal spaces is radial, characteristic not observed in other species. In relation to the histology of the proventricle, the tubular glands are quite, similar to the fundic glands of the mammals. In the submucosa compound tubulosacular glands are observed, with parietal cells. The pancreas doesn't present prominent differences.

Descripción histológica de los diferentes segmentos del aparato digestivo de avestruz (Struthio camelus var. domesticus) / Histologic description of the different segments from the ostrich digestive system (Struthio camelus var. domesticus)

Struthio camelus domesticus (Swart, 1987) es descrita como reflejo del híbrido natural de avestruces de granja en Sudáfrica, la que actualmente se conoce como avestruz de cuello negro o African Black. Esta ave fue desarrollada mediante programas de mejoramiento genético, con el objetivo de aumentar el valor comercial de la especie. Se caracteriza por ser de menor talla y más fértil que las otras subespecies, estructura del plumaje bien desarrollada, carácter dócil, y de fácil crianza en granjas, ya que es tremendamente curiosa y amigable con los humanos (Deeming, 2001; Camiruaga, 2004). En relación a las características anatómicas generales del tracto digestivo, Camiruaga & Simonetti, (2003) señalan que el avestruz presenta semejanzas y diferencias, tanto con otras aves, como con los rumiantes y otros herbívoros (equinos). Del análisis comparativo con la gallina, presenta ciertas diferencias anatómicas, una de ellas es no presentar buche, órgano almacenador de alimento que existe en otras aves. El proventrículo y el estómago muscular (molleja), en el avestruz, pueden cumplir dicha función (Angel, 1996). No presentan vesícula biliar, por lo que el vaciamiento de la bilis se realiza directamente al intestino delgado. Además, el intestino grueso del avestruz, a diferencia de otras especies, representa el 50 % del largo total del tubo digestivo y el intestino delgado corresponde sólo al 35,5%. (Camiruaga, 2004). En el presente trabajo se analizó la histología normal de los diferentes segmentos del tubo digestivo del avestruz: esófago, proventrículo, estómago muscular (molleja o ventrículo), intestino delgado (duodeno, yeyuno e íleon) e intestino grueso (ciego, colon y recto), y se analizó comparativamente con especies a las que se le asocia morfológicamente, como las aves domésticas, rumiantes, seudo rumiantes (camélidos) y algunos otros herbívoros.

An increased intraovarian synthesis of nerve growth factor and its low affinity receptor is a principal component of steroid-induced polycystic ovary in the rat.

A form of polycystic ovary (PCO) resembling some aspects of the human PCO syndrome can be induced in rats by a single injection of estradiol valerate (EV). An increase in sympathetic outflow to the ovary precedes, by several weeks, the appearance of cysts, suggesting the involvement of a neurogenic component in the pathology of this ovarian dysfunction. The present study was carried out to test the hypotheses that this change in sympathetic tone is related to an augmented production of ovarian nerve growth factor (NGF), and that this abnormally elevated production of NGF contributes to the formation of ovarian cysts induced by EV. Injection of the steroid resulted in increased intraovarian synthesis of NGF and its low affinity receptor, p75 NGFR. The increase was maximal 30 days after EV, coinciding with the elevation in sympathetic tone to the ovary and preceding the appearance of follicular cysts. Intraovarian injections of the retrograde tracer fluorogold combined with in situ hybridization to detect tyrosine hydroxylase (TH) messenger RNA-containing neurons in the celiac ganglion revealed that these changes in NGF/p75 NGFR synthesis are accompanied by selective activation of noradrenergic neurons projecting to the ovary. The levels of RBT2 messenger RNA, which encodes a beta-tubulin presumably involved in slow axonal transport, were markedly elevated, indicating that EV-induced formation of ovarian cysts is preceded by functional activation ofceliac ganglion neurons, including those innervating the ovary. Intraovarian administration of a neutralizing antiserum to NGF in conjunction with an antisense oligodeoxynucleotide to p75 NGFR, via Alzet osmotic minipumps, restored estrous cyclicity and ovulatory capacity in a majority of EV-treated rats. These functional changes were accompanied by restoration of the number of antral follicles per ovary that had been depleted by EV and a significant reduction in the number of both precystic follicles and follicular cysts. The results indicate that the hyperactivation of ovarian sympathetic nerves seen in EV-induced PCO is related to an overproduction of NGF and its low affinity receptor in the gland. They also suggest that activation of this neurotrophic-neurogenic regulatory loop is a component of the pathological process by which EV induces cyst formation and anovulation in rodents. The possibility exists that a similar alteration in neurotrophic input to the ovary contributes to the etiology and/or maintenance of the PCO syndrome in humans.

Intraovarian excess of nerve growth factor increases androgen secretion and disrupts estrous cyclicity in the rat.

A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.

Stress promotes development of ovarian cysts in rats: the possible role of sympathetic nerve activation.

Activation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found: 1. An increase in the content of norepinephrine (NE) in the celiac ganglion. 2. An increase in the release of NE from the ovary. 3. An unchanged NE uptake by the ovary. 4. An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

Monosialoganglioside attenuates the excitatory and behavioural sensitization effects of ethanol.

The effects of long-term monosialoganglioside GM1 treatment on the acute excitatory effects of ethanol and behavioural sensitization to this effect were studied, using locomotion frequency of mice observed in an open field as an experimental parameter. GM1 (30 mg/kg, once a day, for 21 days) did not modify mouse behaviour but decreased both the acute excitatory (1.8 g/kg) and the behavioural sensitization effects of ethanol (1.8 g/kg, once a day for 21 days, 30 min after GM1 injections). GM1 administered acutely 30 min or 24 h before ethanol did not modify the ethanol-induced increase in locomotion frequency. These results agree with previous reports in which ganglioside treatment modified both dopaminergic plasticity and other behavioural and biochemical effects of ethanol.

Ovarian steroidal response to gonadotropins and beta-adrenergic stimulation is enhanced in polycystic ovary syndrome: role of sympathetic innervation.

Experimental induction of a polycystic ovarian syndrome (PCOS) in rodents by the administration of a single dose of estradiol valerate (EV) results in activation of the peripheral sympathetic neurons that innervate the ovary. This activation is evidenced by an increased capacity of ovarian nerve terminals to incorporate and release norepinephrine (NE), an increase in ovarian NE content, and a decrease in ovarian beta-adrenergic receptor number in the ovarian compartments receiving catecholaminergic innervation. The present experiments were undertaken to examine the functional consequences of this enhanced sympathetic outflow to the ovary. The steroidal responses of the gland to beta-adrenergic receptor stimulation and hCG were examined in vitro 60 days after EV administration, i.e. at the time when follicular cysts are well established. EV-treated rats exhibited a remarkable increase in ovarian progesterone and androgen responses to isoproterenol, a beta-adrenergic receptor agonist, with no changes in estradiol responsiveness. Basal estradiol release was, however, 50-fold higher than the highest levels released from normal ovaries at any phase of the estrous cycle. The ovarian progesterone and androgen responses to hCG were enhanced in EV-treated rats, as were the responses to a combination of isoproterenol and hCG. Transection of the superior ovarian nerve (SON), which carries most of the catecholaminergic fibers innervating endocrine ovarian cells, dramatically reduced the exaggerated responses of all three steroids to both beta-adrenergic and gonadotropin stimulation. SON transection also reduced the elevated levels of ovarian NE resulting from EV treatment and caused up-regulation of beta-adrenoreceptors. Most importantly, SON transection restored estrous cyclicity and ovulatory capacity. The results indicate that the increased output of ovarian steroids in PCOS is at least in part due to an enhanced responsiveness of the gland to both catecholaminergic and gonadotropin stimulation. The ability of SON transection to restore a normal response indicates that the alteration in steroid output results from a deranged activation of selective components of the noradrenergic innervation to the ovary. These findings support the concept that an alteration in the neurogenic control of the ovary contributes to the etiology of PCOS.

O controle biologico laboratorial da exposicao de trabalhadores e substancias quimicas. A medicina do trabalho e a prevencao da intoxicacao. / Biological laboratorial control of exposure of workers to chemical substances. Occupational medicine and the prevention of poisoning

Neste trabalho os autores discutem a relacao entre Medicina do Trabalho e Toxicologia Ocupacional; informam sobre os meios de pesquisa utilizados a fim de obter o conhecimento toxicologico, para finalmente, discorrerem a respeito do importante tema do uso dos Indicadores Biologicos de Exposicao (IBEs) e dos Limites de Tolerancia Biologicos (LTBs) no controle biologico de exposicao de trabalhadores a substancias quimicas. E apresentada, ainda, uma extensa relacao de IBEs e LTBs propostos