Human hepatoma cell line (Hep G2) cellular response to hypothermic stress with recovery: Induction of Hsp70, Hsp60 and Hsf1 expression / Respuesta celular de línea de hepatoma humano (HepG2) al estrés hipotérmico con recuperación: Inducción de la expresión de Hsp60, Hsp70, y Hsf1

The cell response of human HepG2 cells exposed to hypothermia with rewarming was analyzed. Ultrastructural findings in hypothermic stressed cells showed swollen mitochondria, dispersed chromatin, vacuoles and ring-shape nucleolar reorganization. These changes were coupled with significative differences in the induction of Hsp60, inducible Hsp70 and monomeric Hsf1 in all treated samples, but not in Hsc 70 expression. Cellular response to hypothermia could be associated with the synergistic induction of Hsp expression.

En este trabajo se analizó la respuesta celular de células HepG2 expuestas a hipotermia con posterior recuperación. Los hallazgos ultraestructurales en células sometidas a estrés hipotérmico incluyeron mitocondrias edematizadas, núcleos picnóticos, vacuolas y reorganización nucleolar en forma de anillo. Tales cambios están relacionados con diferencias significativas en la inducción de la expresión de Hsp60, Hsp70 inducible y Hsf 1 monomérico en todas las muestras tratadas, pero no de Hsc70. La respuesta celular a la hipotermia puede ser relacionada con la inducción sinergística de las Hsp.

Análisis cuantitativo del crecimiento y cambio morfométrico en poblaciones de Leishmania chagasi y Trypanosoma cruzi mantenidos en cultivos axénicos puros y mixtos / Quantitative approach to the analysis of the growth and morphometric change in populations of Leishmania chagasi and Trypanosoma cruzi maintained in pure and mixed axenic cultures

Las relaciones que se establecen entre géneros de la familia trypanosomatidae en condiciones de coexistencia en el mismo medioambiente pueden estar vinculadas a respuestas compensatorias inter-poblacionales que incluyen cambios morfológicos (diferentes estadios) y morfométricos (diferencias mensurables). El análisis cuantitativo de tales respuestas en cultivos axénicos puros de Leishmania chagasi y trypanosoma cruzi, así como en isomezclas axénicas de L. chagasi-T. cruzi mantenidas in vitro, no ha sido abordado, desconociéndose por lo tanto, particularidades biológicas. Muestras interdiarias de cultivo se fijaron, colorearon, observaron, digitalizaron y procesaron cuantitativamente. Además de cuantificar las densidades poblacionales, se registraron las magnitudes numéricas de variables morfométricas que, posteriormente, se analizaron con herramientas estadísticas. Los resultados indicaron cambios específicos en las variables investigadas, así como heterogeneidad morfométrica entre los mismos morfotipos de los mismos géneros al ser mantenidos en cultivos puros o mixtos. Los modelos de cambio morfométrico de L. chagasi y T. cruzi en cultivos puros difieren de los modelos de cambio morfométrico en los cultivos mixtos (L. chagasi-T. cruzi). Las metodologías biométricas discriminan, en términos morfométricos, poblaciones del mismo estadio (morfotipo) en ambientes diferentes.

The relations established among genera of the Trypanosomatidae family in coexisting conditions in the same environment may be linked to inter-population compensatory answers that include morphological (differences among stages) and morphometrical (measurable difference) changes. The quantitative analysis of these answers in Leishmania chagasi and Trypanosoma cruzi pure axenic cultures, as well as in L. chagasi - T. cruzi axenic iso-mixtures in vitro maintained has not been approached, and consequently, potentially useful biological particularities in the control of these important human parasites are unknown. Every other day culture samples were fixed, stained, observed, digitalized and quantitatively processed. In addition to quantify, the population densities and the appearance-disappearance stage (morphotypes) dynamics, the numeric magnitudes of the morphometric variables were recorded and later analyzed with multivariate statistical techniques. The results indicate specific changes in the investigated variables, as well as morphometric heterogeneity between the same morphotypes of the same genera when maintained in pure or mixed cultivation. The morphometric change models for L. chagasi and T. cruzi in pure culture differ from the models of morphometric change in mixed cultivation (L. chagasi-T. cruzi). The biometric methodologies discriminate in morphometric terms populations of the same stage (morfotype) in different environments.

Human hepatoma cell line (HepG2) cellular response to hypothermic stress with recovery. Induction of Hsp70, Hsp60 and Hsf1 expression.

The cell response of human HepG2 cells exposed to hypothermia with rewarming was analyzed. Ultrastructural findings in hypothermic stressed cells showed swollen mitochondria, dispersed chromatin, vacuoles and ring-shape nucleolar reorganization. These changes were coupled with significative differences in the induction of Hsp60, inducible Hsp70 and monomeric Hsfl in all treated samples, but not in Hsc 70 expression. Cellular response to hypothermia could be associated with the synergistic induction of Hsp expression.

Heat stress in rat adriamycin cardiomyopathy: heat shock protein 25 and Myosin accumulation.

In order to evaluate the effects of hyperthermia on adriamycin cardiomyopathy and its relationship with heat shock protein induction and myosin accumulation, female Sprague-Dawley rats (21-24 days) were randomized into four groups: the control, adriamycin, temperature and temperature-adriamycin groups. Adriamycin was injected i.v. at a dose of 27 mg/Kg (0.1 ml). The rats were exposed to a temperature of 45ºC for 35 min, followed by a recovery (1 h) at room temperature prior to adriamycin treatment. Body weight was recorded weekly. The thickness of the ventricular wall and percentage of cellular damage were biometrically and ultrastructurally evaluated, respectively. Heat shock protein 25 and myosin accumulation were determined through Western blot analysis. The determinations were carried out monthly until the third month after treatment. At eight and twelve weeks after treatment, the thickness of the ventricular wall seemed to decrease in the adriamycin-treated rats in relation to the other groups. An electron microscopic analysis of the adriamycin group's left ventricular wall samples, showed more sarcomeric changes and loss of myofibrils than the control, temperature and temperature-adriamycin groups. At 24 hours after treatment with adriamycin, higher levels of heat shock protein 25 and myosin were observed (week 0) in the temperature-adriamycin group than in the control and adriamycin groups (4, 8 and 12 weeks). Hyperthermia was confirmed by a multivariate approach to induce heat shock protein 25 and myosin, which would strengthen cardiac-sarcomeric myosin arrangement.

Skeletal Muscle for Endomyocardial Biopsy: Comparable Stress Response in Doxorubicin Cardio-myopathy.

In the present study, we compared the cell damage response in skeletal and cardiac muscle tissue when exposed to doxorubicin. This was carried out by means of a less invasive informative substitute to endomyocardiac biopsy based on Hsp70 immunodetection and a subcellular analysis of the nucleolus. Male Sprague Dawley rats (62 g body weight) were randomly distributed into 3 group, the control and doxorubicin I and doxorubicin II groups, in which 15 and 25 mg/kg body weight of doxorubicin (0.1 ml, i.v.) was administered, respectively. After 15, 30, 45 and 60 minutes, portions of the left and right ventricle wall and interventricle wall, together with skeletal muscle from the posterior and anterior member, were prepared for Hsp70 immunodetection by Western blot analysis and ultrastructural study using the thin cut technique. Differential cell response between the control and treated groups was observed in Hsp70 immunodetection and at the subcellular level. In the control group, the Hsp70 recognition levels and typical normal nucleolar morphology were similar, while the treated groups showed variable-dependent Hsp70 recognition and segregation of nucleolar components, forming ring-like figures of a variable-independent nature. Comparison of cardiac and skeletal muscle tissue cell response to doxorubicin toxic aggression revealed parallelism in terms of Hsp70 accumulation in certain regions of both tissues (15 mg/kg body weight of doxorubicin), which suggests that replacing endomyocardiac biopsy analysis with skeletal muscle analysis may be a safe option.

Differential expression of HSP70 and ultrastructure of heart and liver tissues of rats treated with adriamycin: protective role of L-carnitine.

The anticancer drug adriamycin has been associated to tissular oxidative stress. In this regard, the promotion of anti-stress protein synthesis by L-carnitine has been suggested in rat adriamycin-induced cardiomyopathy in the long-term. However, the citoprotective role of L-carnitine in cardiac and hepatic tissues after short-term adriamycin treatment is unknown. HSP70 in the supernatant of the homogenized cardiac and hepatic tissues after short-term adriamycin treatment was determined by Western blot analysis with and without L-carnitine protection and compared to the subcellular characteristics of both tissues by transmission electron microscopic analysis. Female Sprague-Dawley rats (n=6), body weight 40-60 g, were randomized into four groups: control, adriamycin, L-carnitine and L-carnitine-adriamycin. Saline, adriamycin (15 mg/kg body weight) and L-carnitine (20 mg before adriamycin) were given intravenously (0.1 mL). HSP70 accumulation was different between the control and the adriamycin samples of both tissues. HSP70 was higher in the liver than in the heart both with and without L-carnitine protection. The nuclei of heart cells, in the adriamycin group showed alterations including, form and irregular perinuclear cysternae with invaginations of different sizes that were not observed in the L-carnitine-adriamycin samples. Considering the differential expression of HSP70 between liver and heart, our results may be important for understanding the role of these proteins in the adriamycin-induced distinct levels of organ damage and dysfunction. We suggest that L-carnitine exogenous administration might enhance the relationship between the cellular energy state and the activation of heat shock response by an unknown mechanism. L-carnitine may enhance HSP70 in a cellular-type manner.

Hsp70 accumulation and ultrastructural features of lung and liver induced by ethanol treatment with and without L-carnitine protection in rats.

This study examined Hsp70 accumulation and the subcellular characteristics of liver and lung when exposed to ethanol (EtOH), with and without L-carnitine protection. Female Sprague-Dawley rats, 150-200 g body weight, were randomized into four groups: Control (CON), Alcohol (ALC), L-carnitine (CAR) and Alcohol-L-carnitine (ALC-CAR). EtOH was administered per os at a dose of 4 g/kg body weight (1 ml) daily for 4 weeks. Before alcohol intake, an oral dose of 500 mg/kg body weight of L-carnitine was also administered to the ALC-CAR group. The liver and lung samples were subjected to Hsp70 Western blot and ultrastructural analysis. The Hsp70 accumulation was higher in the liver than in the lung samples. Hepatic Hsp70 accumulation was similar for all groups in contrast to lung, where the Hsp70 accumulation depends on the group studied. The ultrastructural results showed lung but not liver alterations, evidencing a stressful condition and subsequent cellular injury for lung tissue but not for liver. The ALC-CAR group showed less lung damage than the non-protected group and resembles the general appearance of the CON and CAR groups. EtOH intoxication induced differential cellular response in liver and lung in a dose and tissue dependent manner. L-carnitine seems to reduce lung EtOH-induced subcellular damage. The promotion of heat shock or stress proteins might represent one of the mechanisms involved that need to be further investigated.

Is hypothermia a stress condition in HepG2 cells? Expression and localization of Hsp70 in human hepatoma cell line.

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.

Adriamycin toxic effect promotes embryonary cardiac vascular damage? / ¿La adriamicina promueve efectos tóxicos sobre el sistema cardiovascular embrionario?

The ultrastructural alterations in ventricular capillary endothelia of chick embryo heart treated with Adriamycin (ADRIA) are discussed. White-Leghorn chicken eggs on the third day of incubation (stage 18), were injected through the egg shell with 1 ml of ADRIA 5, ug/egg. Eggs were reincubated until day 15 (stage 41-42), at which time the embryos were sacrified and their hearts removed. Controls were injected with equal volume of saline solution. Unlike the controls, ADRIA treated embryonic ventricular capillary endothelia exhibited marked evidences of vascular damage. An apparent reduction of the cytoplasmic surface area of the abnormally dark and smooth endothelial cells was observed. Capillary endothelial alterations also included cell vacuolization, absence of cytoplasmic endothelial cell proyections and cytoplasmic blebs along capillar endothelial surface, most prominently along the free cell surface or lumen. The comparison of the ultrastructural characteristics between control and ADRIA-treated embryonic heart, may confirm ADRIA cardiotoxic effects during cardiogenesis. Capillary endothelium may be an alternative target of ADRIA and in appears likely that drug toxic effect may promote vascular damage