Santiago Hernández Torres

Evaluation of acute and chronic hepatotoxic effects exerted by anabolic-androgenic steroid stanozolol in adult male rats.

Abstract Stanozolol (ST) is a 17α-alkyl anabolic-androgenic steroid (17α-AAS) often misused by athletes and bodybuilders. The use of anabolic-steroids by sportsmen and teenagers has increased dramatically, thus raising the question about their hepatotoxicity, specially those such as ST which are orally administered. Previously, we have reported diverse in vivo effects exerted by this steroid and published the existence of a highly specific ST-binding site in male rat liver microsomes. The existence of this binding site, the reported hepatic effects exerted in humans, and the very limited information about its potential hepatotoxicity led us to treat adult male rats acutely and chronically with ST and study different parameters that could indicate liver damage: serum levels of transaminases, concentration of monooxygenase enzymes in liver, liver membrane lipid peroxidation products, liver histopathology, and cell cycle/ploidy status of liver cells. In our study, no changes in serum transaminases or lipid peroxidation levels were obtained. However, acute stanozolol treatment significantly decreased the levels of cytochrome P450 (Cyt. P450) and cytochrome b5 (Cyt. b5) during the first 48 h of treatment, while subsequently, at 72 and 96 h, these microsomal enzymes underwent a significant increase in their levels. In sharp contrast with this response to acute treatment, the content of these two enzymes during chronic treatment showed an important decrease. Interestingly, acutely and chronically ST-treated livers showed slight to moderate inflammatory or degenerative lesions in centrilobular hepatocytes. Flow cytometric analysis demonstrated that both acute and chronic ST treatment were capable of increasing the percentage of S-phase fraction (%SPF) of liver cells. These findings taken together clearly show that this steroid is capable of altering the liver capacity for metabolizing xenobiotics and indicate that high doses of ST could exert a proliferative effect on liver cells. Such data should be considered in risk evaluations for this compound.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER‐2/neu oncogene status and total cellular p185HER‐2 content were simultaneously analyzed in 415 invasive breast‐cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut‐off value of 1.7 for the ratio between the intensity of the HER‐2/neu gene band and the reference gene band, to consider the HER‐2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER‐2 over‐expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER‐2/neu gene amplification. Of these tumors, 87% showed over‐expression of the p185HER‐2. Of the remaining 352 specimens that did not display HER‐2/neu gene amplification, 97% showed no p185HER‐2 over‐expression (p < 0.0001). In 40 selected samples with a p185HER‐2 level lower than 260 fmol/mg protein, the degree of p185HER‐2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER‐2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER‐2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER‐2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER‐2/neu oncogene is amplified or p185HER‐2 is over‐expressed.

Evaluation of acute hepatotoxic effects exerted by environmental estrogens nonylphenol and 4-octylphenol in immature male rats

Nonylphenol (NP) and 4-Octylphenol (4OP) have shown estrogenic properties both in vivo and in vitro. Researchers have known for years that estrogens induce a wide number of hepatotoxic actions in rodents. In order to study the acute hepatic effects exerted by NP and 4OP on rat liver the following endpoints were evaluated: relative liver weight (RLW), morphology, cell cycle and ploidy status, monooxygenase enzymes content and levels of both, cytosolic estrogen receptor (cER) and microsomal binding sites for estrogens (mEBS). Immature male Sprague-Dawley rats were injected intraperitoneally (i.p.) with 60 mg/kg of NP or 4OP for 1, 5 or 10 days. Despite the fact that RLW of the animals was not modified but any treatment, the histopathological study revealed the presence of an increase in the percentage of both, mitotic activity and Ki-67-labeling index (LI) in the livers from animals treated with alkylphenols in absence of any degenerative lesion. Furthermore, all the livers from alkylphenols-treated groups showed the presence of abnormal mitosis and c-mitosis. Although the levels of both, cER and cytochrome P450 (Cyt. P450) were not affected by any treatment, concentration of the mEBS was decreased after 10 days of treatment with alkylphenols. These findings taken together suggest that the exposition to alkylphenols induce cell proliferation and spindle disturbances and that these compounds are capable of modulating the expression of putative membrane receptors for estrogens.

Quantitative analysis of p185HER‐2/neu protein in breast cancer and its association with other prognostic factors

The total cellular p185HER‐2/neu protein (p185) content was measured by ELISA in 346 invasive primary breast cancers, and the results were compared with those of estrogen (ER) and progesterone (PR) receptors, pS2 and Cathepsin D (Cat D) content. At a cut‐off level of 260 fmol/mg protein, 53 of the 346 tumors (15%) were p185‐positive. A significant positive correlation was observed between p185 levels and those of Cat D, and a weaker, though significant, positive correlation with ER, and pS2 levels, but not with those of PR. However, when only the 293 p185‐negative tumors were considered, the correlation between p185 and ER improved substantially, and statistical significance was reached for PR. p185‐positive tumors exhibited lower ER and PR content and higher Cat D content than p185‐negative tumors. The pS2 content, in contrast, did not undergo significant variation. Tumors considered to be p185‐positive were significantly more frequently positive for Cat D at the cut‐off of 45 pmol/mg protein, and were more frequently negative for ER and/or PR, but only significant at the cut‐off of 15 fmol/mg or higher for both steroid receptors. Finally, p185 status was not associated with menopausal status, tumor size, axillary‐lymph‐node invasiveness or distant metastases. These results suggest that 260 fmol/mg protein as the cut‐off for p185 allows the identification of a tumoral sub‐population with a more aggresive phenotype.Int. J. Cancer 74:175–179, 1997.

Prevention of Dexamethasone-Induced Lymphocytic Apoptosis in the Intestine and in Peyer Patches by Enteral Nutrition

BACKGROUND Apoptosis is a programmed cell death, genetically controlled, that can be activated by physiological and pathophysiological events and by the administration of several drugs, including the exogenous administration of corticosteroids. The aim of this study was to develop a rat model of intestinal lymphocytic apoptosis induced by dexamethasone to determine if apoptosis could be prevented by enteral or parenteral nutrition. METHODS Male Sprague-Dawley rats were used. On day 0, the right internal jugular vein was catheterized for parenteral nutrition, and a silicone tube was inserted into the duodenum for enteral feeding. Animals (n = 6/group) were randomly assigned to one of the following 3 feeding regimens: an immune-enhancing enteral diet; its placebo (the same formula without immunonutrients); and isocaloric isonitrogenous parenteral nutrition. On the seventh day, 200 microg of dexamethasone or vehicle were administered by i.v. bolus, and the diets were continued for 1 more day. Intestinal Peyer patches and thymic lymphocytic apoptosis were determined both by flow-cytometry and immunohistochemistry. RESULTS A single dexamethasone dose (200 microg/rat) administered to surgically treated rats fasted for 18 hours, 24 hours later, caused massive intestinal and Peyer patches lymphocytic apoptosis (96 +/- 2% and 85 +/- 5%, respectively; p < .0001 versus vehicle in both kinds of tissue). Lymphocytic apoptosis was reduced to almost indetectable levels in intestinal and lamina propia lymphocytes (from 96 +/- 2% to <0.6%; p < .0001). Peyer patches lymphocytic apoptosis was reduced as well (from 85 +/- 5% to 15 +/- 7.1%; p < .001) in animals prefed the 2 enteral nutrition formulas. Those prefed parenteral nutrition only showed a partial decrease of lymphocytic apoptosis in the intestine (from 96 +/- 2% to 53 +/- 23%; p < .001). Nutrition had no effect on the dexamethasone-induced thymus involution. CONCLUSIONS Enteral nutrition prevents intestinal intraepithelial and lamina propia lymphocytic apoptosis due to dexamethasone. These findings support the use of early enteral nutrition in critically ill patients treated with corticosteroids.

El impacto del turismo de masas en las Islas Canarias en el contexto de las reservas mundiales de la biosfera

Canarias es uno de los destinos turisticos principales de Europa. El Archipielago recibio en 2015 mas de 13 millones de turistas y todo ello en un territorio muy limitado (7.4 mil km2 y una poblacion de 2.1 millones). Las dimensiones de estas cifras adquieren relevancia en un contexto donde el 63 % de la superficie de las Islas ha sido declarada Reserva Mundial de la Biosfera. Se estudia aqui pues, el impacto del turismo –asi como algunas alternativas en aras de paliar dicho impacto–, en las cinco islas que han sido declaradas en su integridad como tales, partiendo de sus particularidades.