Effect of antibiotics and NSAIDs on cyclooxygenase-2 in the enamel mineralization.

The objective of this study was to determine whether the use of the most commonly prescribed antibiotics and non-steroidal anti-inflammatory drugs in childhood could disturb enamel mineralization. Forty-two Swiss mice were divided into seven groups: controls; amoxicillin; amoxicillin/clavulanate; erythromycin; acetaminophen; ibuprofen and celecoxib, to inhibit cyclooxygenase 2 (COX2). SEM-EDX analysis was conducted on all cusps of the third molars. Calcium (Ca), phosphorus (P), aluminum, potassium, sodium, magnesium and chlorine were quantified. The stoichiometric Ca/P molar ratios were calculated. Immunohistochemical quantification of COX2 in incisors was carried out by image analysis using COX2-specific immunostaining. Groups treated with antibiotics showed no significant differences in the content of the chemical elements. Only acetaminophen and celecoxib showed a significant decrease in Ca and P compared with the control samples. Ca/P ratios showed no difference. Groups treated with amoxicillin, amoxicillin/clavulanate, erythromycin and acetaminophen showed significantly lower amounts of immunoreactive COX2 at the enamel organ maturation stage of the mouse incisors. Our results suggest that COX2 is involved in the maturation stage of the enamel organ and that its inhibition would appear to alter amelogenesis, producing hypomineralization.

Effect of atorvastatin and diet on non-alcoholic fatty liver disease activity score in hyperlipidemic chickens.

Non-alcoholic steatohepatitis (NASH) is part of the spectrum of non-alcoholic fatty liver disease (NAFLD), which includes from simple steatosis and steatohepatitis, to the most severe cirrhosis and carcinoma, which develops in the absence of excessive alcohol intake. NAFLD is the most common liver disorder in affluent societies. There is no proven treatment for NAFLD/NASH. One of the most frequent adverse effects of statins is an increase in hepatic aminotransferases. Studies that evaluate if the benefits of statins overcome the risks in NASH are lacking. The present study was conceived to explore the effect of both atorvastatin and diet on regression of steatohepatitis, using a chicken experimental model induced by a hyperlipidemic diet (HD). Plasma lipid levels, liver enzymes and hepatic histopathology, as well as image analysis were performed to determine changes in liver lipid deposits and inflammatory infiltration. Features of steatosis, cell-ballooning, and inflammation were scored to obtain the NAFLD activity score (NAS). A severe level of steatosis was found in animals fed on HD. Atorvastatin treated groups showed smaller size of lipid deposits and a lower level of inflammation than non-treated groups. Atorvastatin therapy induced a significant reduction of hepatocellular damage, even though in the animals which continuously received a hyperlipidemic diet. The combination of atorvastatin therapy and a standard diet produced the lowest decrease of NAS. Our results show that atorvastatin therapy not only decreased plasmatic levels of cholesterol and triglycerides, but also induced a reduction of liver steatosis, inflammation and hepatocellular damage, without increasing plasmatic aminotransferase levels.

Cytochemical and biochemical evidences for a complex tridimensional structure of the hamster zona pellucida.

Zona pellucida (ZP) is an extracellular matrix that surrounds eggs and pre-implantation embryos and is required for in vivo fertility. A key event in successful fertilization is sperm binding to the surface of the ZP. It has been previously described that the hamster sperm binds mainly the outer region of the ZP which corresponds to the porous region in contact with the cumulus cells. Using ultrastructural cytochemistry approaches with an antibody developed against porcine ZP, this study shows that the pig ZP shares epitopes with some rodent species like hamster, rat and mouse. In the hamster, these epitopes are located mainly in the outer region of the ZP of preovulatory and ovulated oocytes. By means of biochemical approaches it was demonstrated that 1) the antibody is specific for the native hamster ZP3, 2) four different bands with a molecular weight of 67, 60, 48 and 38 kDa after N-linked deglycosylation suggesting that the hamster ZP is formed by four proteins, and 3) the different composition observed in the outer region of the hamster ZP could be due to a specific supramolecular structure that makes some epitopes accessible for the antibodies. In summary, this study provides evidence that the different composition observed in the different regions of the ZP is mediated by a different organization of the components of the ZP produced during the oocyte maturation. This different organization could be responsible for the different sperm binding affinity observed for sperm to the outer region versus the inner region of the ZP.

Molecular and morphological changes in placenta and embryo development associated with the inhibition of polyamine synthesis during midpregnancy in mice.

Polyamines play an essential role in murine development, as demonstrated by both gene ablation in ornithine decarboxylase (ODC)-deficient embryos and pharmacological treatments of pregnant mice. However, the molecular and cellular mechanisms by which ODC inhibition affects embryonic development during critical periods of pregnancy are mostly unknown. Our present results demonstrate that the contragestational effect of alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ODC, when given at d 7-9 of pregnancy, is associated with embryo growth arrest and marked alterations in the development of yolk sac and placenta. Blood island formation as well as the transcript levels of embryonary globins alpha-like x chain and beta-like y-chain was markedly decreased in the yolk sac. At the placental level, abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer and a deficient development of the labyrinthine zone were evident. Real-time RT-PCR analysis showed that transcript levels of the steroidogenic genes steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase VI, and 17alpha-hydroxylase were markedly decreased by DFMO treatment in the developing placenta at d 9 and 10 of pregnancy. Plasma values of progesterone and androstenedione were also decreased by DFMO treatment. Transcriptomic analysis also detected changes in the expression of several genes involved in placentation and the differentiation of trophoblastic lineages. In conclusion, our results indicate that ODC inhibition at d 8 of pregnancy is related to alterations in yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steroidogenesis.

Intracellular retention of hepatic serpins caused by severe hyperlipidemia.

BACKGROUND: High levels of circulating lipids contribute to both the development of non-alcoholic liver steatosis (NALS) and peripheral arterial disease, leading to increased thrombotic risk. However, the effects of hyperlipidemia on hepatic proteins have barely been studied. Antithrombin is a hepatic serpin with anticoagulant and anti-inflammatory roles. The conformational flexibility of antithrombin renders it susceptible to both, genetic and posttranslational modifications. Thus, mutations and environmental factors have been shown to alter this molecule. METHODS: We used a chick model to assess the effects of hyperlipidemic diets (HD) on this conformationally sensitive molecule. We determined antithrombin activity in plasma and evaluated the histological and immunohistological features of livers from these animals. RESULTS: A HD for 6 months led to a significant intrahepatic retention and aggregation of antithrombin, which correlated with hepatic steatosis, as revealed by immunohistological analysis. Accordingly, a decrease in circulating antithrombin activity (48.71 +/- 6.35%) was observed. Other hepatic proteins, including heparin cofactor II, another anticoagulant serpin, also accumulated intracellularly. Atorvastatin and reversion to a normal diet after 3 months partially protected livers from these deleterious effects. CONCLUSIONS: Our results support that hyperlipidemia-induced NALS causes a significant intracellular aggregation of hemostatic serpins in liver, which determines a decrease in their circulating levels.

Effects of nifedipine, verapamil and diltiazem on serum biochemical parameters and aortic composition of atherosclerotic chickens.

Calcium appears to be involved in many of the cellular events, which are thought to be important in atherogenesis. In this study, we examine the effects of three calcium entry blockers (nifedipine, verapamil, and diltiazem at clinical and higher doses) on serum biochemical parameters and aortic calcium, cholesterol and triglyceride concentrations of atherosclerotic egg-fed chickens. All egg-fed chickens (treated and non-treated) showed an increase in serum total cholesterol, LDL-cholesterol and triglycerides without significant effect when calcium entry blockers were used. Increased HDL values were observed in clinical and high-dose nifedipine and clinical dose verapamil groups. The high-dose diltiazem group presented increased zinc values with respect to the clinical dose diltiazem and control groups. The sodium concentrations were significantly decreased in all the groups of animals treated with calcium entry blockers at high-doses and nifedipine at clinical doses. Measurements of aortic calcium concentration showed a significant decrease in the high-dose nifedipine and verapamil groups. Calcium channel blockers had a tendency to decrease total cholesterol in aortas. The values were statistically significant for the high-dose verapamil, and nifedipine groups. Only nifedipine showed a significant decrease for this parameter at clinical dosages. Triglyceride concentrations in aortas were significantly low in animals fed an atherogenic diet and treated with calcium channel blockers, without differences between drugs or dosages used in the experiment. In addition, the chicken atherosclerosis model has proved itself useful and very suitable for in vivo drug intervention studies.

Carbohydrate analysis of the zona pellucida and cortical granules of human oocytes by means of ultrastructural cytochemistry.

BACKGROUND: The zona pellucida (ZP), the mammalian oocyte coat, consists of a restricted number of highly glycosylated proteins. In vitro sperm binding studies suggest a higher binding affinity for the outer region of the ZP compared to its inner region in different species. However, the reason for this difference in binding distribution remains unresolved. Many studies suggest that the carbohydrate sequences linked to ZP glycoproteins act as ligands for sperm binding to this matrix. METHODS: Lectins and antibodies that recognize different carbohydrates were employed to perform an ultrastructural analysis of human ZP and cortical granule glycosylation. RESULTS: This study reveals variable glycosylation of the human ZP throughout its thickness, with pronounced differences between the most external and internal regions of this matrix. The binding studies also indicate that ZP glycoproteins express some carbohydrate sequences not previously detected in other species. Finally, cytochemical analysis of human cortical granules suggests similarities in glycosylation to ZP glycoproteins but not to cortical granules from other mammalian species. CONCLUSION: A heterogeneous carbohydrate composition was observed in the thickness of the human ZP that could be responsible for the different sperm binding affinity detected between the outer and inner regions of the ZP.

Effects of U-50488H and U-50488H withdrawal on c-fos expression in the rat paraventricular nucleus. Correlation with c-fos in brainstem catecholaminergic neurons.

1. In the present work, we have studied the expression of Fos during acute and chronic administration of the kappa-opioid receptor agonist U-50488H and after U-5088H withdrawal in the rat hypothalamic paraventricular nucleus (PVN). Fos production was also studied in brainstem regions that innervate the PVN: the A(2) cell group of the nucleus of solitary tract (NTS-A(2)) and the A(1) cell group of the ventrolateral medulla (VLM-A(1)), combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons after acute U-50488H administration. 2. For acute experiments, male rats were treated with saline i.p. for 4 days. On day 5, rats were given saline or U-50488H (15 mg x kg(-1), i.p.). Other groups of rats were rendered tolerant/dependent on U-50488H by injecting the drug twice daily (15 mg x kg(-1), i.p.) for 4 days. Control animals received saline i.p. on the same time schedule. On day 5, rats were treated with vehicle i.p., with U-50488H (15 mg x kg(-1)) or with the selective kappa opioid-receptor antagonist nor-binaltorphimine (Nor-BNI, 5 mg x kg(-1), i.p.). 3. Using immunohistochemical staining of Fos, present results indicate that acute administration of U-50488H produced an increase in Fos expression in the PVN and in the noradrenergic A(1) and A(2) cell groups. Moreover, when double-label immunohistochemistry was used to identify Fos and catecholaminergic-positive neurons in the brainstem, it was found that catecholaminergic-positive neurons in the NTS and VLM showed a significant increase in Fos expression in response to acute U-50488H injection. Chronic application of U-50488H leads to the development of tolerance towards their effects on Fos expression in the PVN as well as in the NTS and VLM. However, administration of Nor-BNI to U-50488H-dependent rats did not induce any changes in Fos immunoreactivity in the PVN or in the brainstem. 4. These findings demonstrate that acute activation of kappa-opioid receptors results in different altered patterns of immediate-early gene expression in the PVN, which occurs concurrently with an increased activity of their inputs from the brainstem. Interestingly in contrast to morphine withdrawal, present results demonstrate that rats withdrawn from U-50488H did show no changes in Fos-immunoreactivity in the PVN, NTS or VLM, indicating the absence of dependence on the kappa-agonist under the present experimental conditions.

Effects of morphine and morphine withdrawal on brainstem neurons innervating hypothalamic nuclei that control the pituitary-adrenocortical axis in rats.

Different data support a role for brainstem noradrenergic inputs to the hypothalamic paraventricular nucleus (PVN) in the control of hypothalamus - pituitary - adrenocortical (HPA) axis. However, little is known regarding the functional adaptive changes of noradrenergic afferent innervating the PVN and supraoptic nucleus (SON) during chronic opioid exposure and upon morphine withdrawal. Here we have studied the expression of Fos after administration of morphine and during morphine withdrawal in the rat hypothalamic PVN and SON. Fos production was also studied in brainstem regions that innervate hypothalamic nuclei: the nucleus of solitary tract (NTS - A2) and the ventrolateral medulla (VLM - A1) and combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons during morphine withdrawal. Male rats were implanted with s.c. placebo or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received an injection of saline i.p., morphine i.p., saline s.c. or naloxone s.c. Acute morphine administration produced an increase in Fos expression at hypothalamic nuclei and in the brainstem regions, and tolerance developed towards this effect. Precipitated morphine withdrawal induced marked Fos immunoreactivity within the PVN and SON. Concomitantly, numerous neurons in the brainstem were stimulated by morphine withdrawal. Moreover, catecholaminergic-positive neurons in the brainstem showed a significant increase in Fos expression in response to morphine withdrawal. These findings demonstrate that chronic activation of opioid receptors results in altered patterns of immediate-early genes (IEG) expression in the PVN and SON, which occurs concurrently with an increased activity of their inputs from the brainstem.