Oral methylmercury intoxication aggravates cardiovascular risk factors and accelerates atherosclerosis lesion development in ApoE knockout and C57BL/6 mice.

Methylmercury (MeHg) intoxication is associated with hypertension, hypercholesterolemia, and atherosclerosis by mechanisms that are not yet fully understood. We investigated the effects of MeHg intoxication in atherosclerosis-prone (ApoE-KO) and resistant C57BL/6 mice. Mice were submitted to carotid stenosis surgery (to induce atherosclerosis faster) and received water or MeHg solution (20 mg/L) for 15 days. Tail plethysmography was performed before and after MeHg exposure. Food and MeHg solution intakes were monitored weekly. On the 15th day, mice were submitted to intravital fluorescence microscopy of mesenteric vasculature to observe in vivo leukocyte rolling and adhesion. Results showed that despite the high hair and liver Hg concentrations in the MeHg group, food and water (or MeHg solution) consumption and liver function marker levels were similar to those in controls. MeHg exposure increased total cholesterol, the atherogenic (non-HDL) fraction and systolic and diastolic blood pressure. MeHg exposure also induced inflammation, as seen by the increased rolling and adhered leukocytes in the mesenteric vasculature. Atherosclerosis lesions were more extensive in the aorta and carotid sites of MeHg-ApoE knockout mice. Surprisingly, MeHg exposure also induced atherosclerosis lesions in C57BL/6 mice, which are resistant to atherosclerosis formation. We concluded that MeHg intoxication might represent a risk for cardiovascular diseases since it accelerates atherogenesis by exacerbating several independent risk factors.

Ethanol binge drinking during pregnancy and its effects on salivary glands of offspring rats: oxidative stress, morphometric changes and salivary function impairments.

OBJECTIVES: To investigate the biochemical and morphological effects of ethanol (EtOH) binge drinking during pregnancy on parotid glands (PG), submandibular glands (SMG), and saliva of offspring rats. METHODS: Pregnant Wistar rats (n = 8) were exposed to EtOH consumption (3 g/kg/day - 20 % w/v) for three consecutive days. The saliva of 40-day-old offspring rats was collected to determine amylase activity and total protein concentration. PG and SMG were collected to performe oxidative biochemistry, morphometric and immunohistochemistry analyses (Student's t-test, p < .05). RESULTS: EtOH consumption during pregnancy significantly decreased the total protein concentration and decreased amylase activity. In the PG, the EtOH group showed increased lipid peroxidation and decreased antioxidant capacity against peroxyl. In the SMG, the EtOH group showed increased lipid peroxidation and NOx metabolite levels. PG exposed to EtOH showed a decrease of acini, ducts, and total parenchymal area. SMG exposed to EtOH showed an increase in the total stromal area. The expression of CK-19 and Vimentin were found not different between groups. CONCLUSIONS: For the first time, a three-day EtOH binge-drinking protocol during pregnancy is associated with oxidative stress and morphometric alterations in the salivary glands of offspring rats and with the functional reduction of the main salivary enzyme (amylase). CLINICAL RELEVANCE: EtOH consumption during pregnancy altered the morphology and physiology of the salivary glands of offspring rats.

Genetic Susceptibility to Neurodegeneration in Amazon: Apolipoprotein E Genotyping in Vulnerable Populations Exposed to Mercury.

Human exposure to mercury is a serious problem of public health in Amazon. As in other vulnerable populations throughout the world, Amazonian riverine populations are chronically exposed to this metal and some symptoms of mercury intoxication were already detected in these populations. However, studies on the genetic susceptibility to mercury toxicity in the Amazon are scarce, and they tested a limited number of individuals. In this context, apolipoprotein E gene (APOE) is a key element with a well-established association among their alleles and the neurodegenerative consequences of mercury intoxication. However, no studies have addressed APOE genotyping in Amazonian exposed populations. Additionally, epidemiological studies with APOE genotyping in Amazon have been restricted to indigenous populations. Therefore, this work analyzed for the first time the genotypic and allelic profiles of APOE in Amazonian riverine populations chronically exposed to mercury. Eight hundred and twenty three individuals were enrolled in our study donating blood (794) and/or hair (757). APOE genotyping was analyzed by real-time PCR. Total mercury and mercury species were quantified by ICP-MS and GC-pyro-AFS, respectively. Genomic ancestry markers were evaluated by multiplex-PCR reaction, separated by capillary electrophoresis on the ABI 3130 Genetic Analyzer instrument and analyzed on GeneMapper ID v3.2. The 𝜀3 and 𝜀3/𝜀3 were the most frequent allele and genotype, respectively, followed by 𝜀4 allele and 𝜀3/𝜀4 genotype. Only 𝜀2/𝜀2 genotype was not found, suggesting that the absence of this genotype is a generalized phenomenon in Amazon. Also, our data supported an association between the presence of APOE4 and the Amerindian origin in these populations. Fifty-nine individuals were identified at maximum risk with levels of mercury above 10 µg/g and the presence of APOE4. Interestingly, among individuals with high mercury content, APOE4-carriers had high mercury levels than APOE2-carriers, pointing to a different heavy metal accumulation according to the APOE allele. These data suggest that APOE4, in addition to a possible pharmacodynamic effect, may influence pharmacokinetically the mercury exposure causing its higher accumulation and leading to worse deleterious consequences. Our results may aid in the development of prevention strategies and health policy decision-making regarding these at-risk vulnerable populations.

Exposure to Inorganic Mercury Causes Oxidative Stress, Cell Death, and Functional Deficits in the Motor Cortex.

Mercury is a toxic metal that can be found in the environment in three different forms - elemental, organic and inorganic. Inorganic mercury has a lower liposolubility, which results in a lower organism absorption and reduced passage through the blood-brain barrier. For this reason, exposure models that use inorganic mercury in rats in order to evaluate its effects on the central nervous system are rare, especially in adult subjects. This study investigated if a chronic exposure to low doses of mercury chloride (HgCl2), an inorganic form of mercury, is capable of promoting motor alterations and neurodegenerative in the motor cortex of adult rats. Forty animals were exposed to a dose of 0.375 mg/kg/day, for 45 days. They were then submitted to motor evaluation and euthanized to collect the motor cortex. Measurement of mercury deposited in the brain parenchyma, evaluation of oxidative balance, quantification of cellular cytotoxicity and apoptosis and density of mature neurons and astrocytes of the motor cortex were performed. It was observed that chronic exposure to inorganic mercury caused a decrease in balance and fine motor coordination, formation of mercury deposits and oxidative stress verified by the increase of lipoperoxidation and nitrite concentration and a decrease of the total antioxidant capacity. In addition, we found that this model of exposure to inorganic mercury caused cell death by cytotoxicity and induction of apoptosis with a decreased number of neurons and astrocytes in the motor cortex. Our results provide evidence that exposure to inorganic mercury in low doses, even in spite of its poor ability to cross biological barriers, is still capable of inducing motor deficits, cell death by cytotoxicity and apoptosis, and oxidative stress in the motor cortex of adult rats.

Chronic ethanol exposure during adolescence through early adulthood in female rats induces emotional and memory deficits associated with morphological and molecular alterations in hippocampus.

There is increasing evidence that heavy ethanol exposure in early life may produce long-lasting neurobehavioral consequences, since brain structural maturation continues until adolescence. It is well established that females are more susceptible to alcohol-induced neurotoxicity and that ethanol consumption is increasing among women, especially during adolescence. In the present study, we investigated whether chronic ethanol exposure during adolescence through early adulthood in female rats may induce hippocampal histological damage and neurobehavioral impairments. Female rats were treated with distilled water or ethanol (6.5 g/kg/day, 22.5% w/v) by gavage from the 35(th)-90(th) day of life. Ethanol-exposed animals displayed reduced exploration of the central area and increased number of fecal boluses in the open field test indicative of anxiogenic responses. Moreover, chronic high ethanol exposure during adolescence induced marked impairments on short-term memory of female rats addressed on social recognition and step-down inhibitory avoidance tasks. These neurobehavioral deficits induced by ethanol exposure during adolescence through early adulthood were accompanied by the reduction of hippocampal formation volume as well as the loss of neurons, astrocytes and microglia cells in the hippocampus. These results indicate that chronic high ethanol exposure during adolescence through early adulthood in female rats induces long-lasting emotional and memory deficits associated with morphological and molecular alterations in the hippocampus.

Local NO synthase inhibition produces histological and functional recovery in Achilles tendon of rats after tenotomy: tendon repair and local NOS inhibition.

Repair of injured tendon is a very slow process and involves the release of many molecules, including nitric oxide. We investigate the influence of local nitrergic inhibition in histological and functional recovery of injured Achilles tendon. A standard murine model of tendon injury by rupture was used. The animals were divided into three experimental groups: control, injury + vehicle (normal saline) and injury + Nω-nitro-L-arginine methyl ester (L-NAME). The products were injected into the paratendinous region every 2 days and body weight gain and Achilles functional index (AFI) were evaluated on days 0, 7, 14 and 21 after tendon injury. On day 21 post-injury, the animals were killed to evaluate nitric oxide production and tissue organization. We observed that tendon surgical division led to increased tissue nitrite levels, which were reduced in L-NAME-treated rats. The AFI revealed functional recovery of L-NAME-treated animals on day 21 post-injury, which was not observed in the saline-treated group. Microscopic analysis of hematoxylin-eosin staining and collagen autofluorescence showed that L-NAME-treated rats had more aligned areas of collagen fibers and that the diameter of newly organized collagen in this group was also greater than that in the vehicle-treated one. We demonstrate that local treatment with L-NAME significantly improves the functional parameters and accelerates histomorphological recovery.

Modes of reproduction of the hybridogenetic fish Squalius alburnoides in the Tejo and Guadiana rivers: an approach with microsatellites.

The Squalius alburnoides complex was produced by hybridization between female S. pyrenaicus (PP genome) and an hypothetical paternal ancestor related with Anaecypris hispanica (AA genome). This study examined a diversity of mating types and found that there is the potential for considerable gene exchange among diploid, triploid and tetraploid hybrids. Using microsatellites, genomes were attributed to Squalius pyrenaicus (P) or reconstituted "nuclear non-hybrid"S. alburnoides (A), and subsequently confirmed in hybrids. Recombination of AA genomes in the "nuclear non-hybrid males" and recombination of the homogametic genomes (AA or PP) after exclusion of the heterogametic genome in triploid females (PAA) were observed by analysing parents and progeny of breeding experiments. Reproduction of tetraploids, generating a symmetric tetraploid genotype (PPAA) in the progeny, suggests a process that could potentially lead to the formation of a new bisexual species. Present results also support: (i) previously hypothesized pathways, in which PPA S. alburnoides females exclude the A genome, exhibit meiotic recombination between the P genomes and generate haploid eggs; (ii) reconstitution of the diploid maternal ancestor genome (PP) as well as of the unknown paternal ancestor (AA); (iii) the occurrence of the same genomic reproductive mechanisms when Anaecypris hispanica is involved; and (iv) the existence of an A. hispanica-like ancestor as the paternal ancestor of S. alburnoides.