Exploring the presence of pollutants at sea: Monitoring heavy metals and pesticides in loggerhead turtles (Caretta caretta) from the western Mediterranean.

Marine turtles are considered good sentinel species for environmental assessment because of their long lifespan, feeding ecology, habitat use and migratory nature. In the present study, we assessed presence of cadmium, lead and mercury, together with organic pollutants, both in fat and muscle tissue of 25 stranded loggerhead turtles (Caretta caretta) stranded along the Valencian Community coast (East Spain) (43.7±13.5cm). Mean concentrations of Cd, Pb and Hg were 0.04µg/g w.w., 0.09µg/g w.w. and 0.03µg/g w.w. in fat and 0.05µg/g, 0.08µg/g and 0.04µg/g in muscle, respectively. These measures indicate a relatively low mean heavy metal concentration, which may be explained by juvenile size and developmental stage of the turtles analysed. A preliminary non-targeted analysis (using time-of-flight (TOF) technology), made for the first time in marine turtles, allowed to detect 39 different pesticides, most of them previously undetected in this species. Most of the organic substances detected are used in agricultural activities, and the use of 15 of them (38.5%) is not approved in the European Union. Our sample did not show any trend on pollutant contents in relation to turtle size or stranding location, probably because of the high diversity of pollutants found. However, the potential for a positive latitudinal gradient should be explored in future studies due to riverine inputs and high agricultural and industrial activities in the area. Despite the high diversity of pollutants found here, comparative studies of pollutants in other matrices at sea are needed to ascertain whether the loggerhead turtle is a good sentinel of chemical pollution in the western Mediterranean.

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.

Origin and evolution of binucleate cells in cultures of HEp-2 cells.

The origin and evolution of binucleate cells in cultures of HEp-2 cells have been studied by means of interval photography and time-lapse video-recording. Binucleate cells most frequently formed by the fusion of two sister cells born in a previous mitosis. The study of binucleate cells has shown that they are a cellular type able to successfully undergo mitosis. However, the mitosis may be bipolar, tripolar or multipolar. The daughter cells arising from these divisions do not follow a clear pattern in the number of nuclei they have, instead showing a wide range of possibilities.

Ultrastructural localization of cisplatin in Ehrlich ascites tumor cells.

The incorporation of cis-diammine Dichloro Platinum (II) (cisplatin) on the Ehrlich ascites carcinoma (EAT) cells has been studied in this paper. Ultrastructural study of cells treated 'in vivo' with cisplatin showed that a new treatment with this substance after fixation, blocks uranyl acetate staining with the consequent lack of heterochromatin contrast.

Carboplatin treatment induces dose-dependent increases in the frequency of micronuclei in Ehrlich ascites tumor cells.

The effect of various doses (75-300 mg/kg b.w.) of carboplatin on the induction of micronuclei in Ehrlich ascites tumor (EAT) cells was studied. Carboplatin treatment resulted in a dose-dependent increase in the frequency of micronucleated EAT cells. Carboplatin treatment also decreased the mitotic index. The dose-effect curves were linear-quadratic for all the parameters studied. The maximal effect of the drug was obtained in all the treatments after 48 h.

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study.

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.

Possible relationship between micronucleated and binucleated cells induced by cisplatin in cultured CHO cells.

Chinese hamster ovary (CHO) cells were treated with a single dose (10 micrograms/ml) of cis-diamminodichloroplatinum (II) (cisplatin) for 1 h and the effect of the drug on the kinetics of proliferation of the cultures was studied. It was found that the drug produces a delay in the proliferation rates of the treated cultures. The induction of micronuclei and binucleated cells (BC) at different times after treatment have also been studied, and the ability of these cells to undergo DNA synthesis (measured as the ability to incorporate [3H]thymidine) is shown. It was also found that cisplatin induced a particular type of BC that contains one or more micronuclei rather than a pure population of BC. The results obtained show a possible relationship between micronuclei and BC. The possibility that some of the micronucleated cells evolve in subsequent cell divisions to BC with micronuclei is suggested.

A comparison of the effects of bilateral efferent duct ligation and of partial epididymectomy on the testes of rats.

Testis weight as a percentage of body weight did not change following bilateral ligation of the efferent ducts (EDL) close to the epididymis, whereas following removal of part of the epididymis between the site of ligation and a point close to the junction between the caput and corpus (PCE), testis weight first rose linearly until Day 4 and then showed an exponential decrease between Days 4 and 28. After EDL, the perimeter of the seminiferous tubules rose for the first 7 days and then remained elevated, whereas after PCE, there was a linear decrease between Days 4 and 28. Following EDL, the percentage of altered and degenerated tubular cross-sections rose to about 30% and 10%, respectively, during the first 7 days after operation and then remained constant; after PCE, the percentage of altered tubules reached a maximum of 54% by 4 days and then fell, whereas the percentage of degenerated tubules continued to rise to 95% by 28 days. It would appear that all the effects of removal of a portion of the epididymis cannot be explained by blockage of the excurrent ducts, and a specific endocrine effect of the epididymis on the testis is proposed.

Oral administration of pharmacological doses of vitamins C and E reduces reproductive fitness and impairs the ovarian and uterine functions of female mice.

This study aims to ascertain whether oral administration of pharmacological doses of Vitamins C and E has any detrimental effect on reproductive fitness of female mice. We fed hybrid female mice from the first day of weaning a standard diet supplemented or not supplemented with pharmacological doses of Vitamins C and E. At the age of 28 weeks, we individually caged females with a male for the rest of their reproductive life. We performed a series of mating experiments to ascertain the number of oocytes ovulated and the potential for embryo development in vitro to the blastocyst stage and in vivo to Day 12 of gestation. The antioxidant diet decreased the frequency of litters, litter size, total number of offspring born and survival of male pups to weaning. This effect was associated with lower number of corpora lutea in the left ovary, decreased percentage of viable fetuses, and higher number of fetal resorptions in the left uterine horn when compared to the control group. The strategy of supplementing the diet with antioxidant vitamins to prevent the age associated decrease in reproductive potential should not be implemented in human beings until a safe and efficient diet is designed.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: a proteomic approach.

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.

[Computerized models of the variation of the different stages of the cell cycle in a hypertriploid Ehrlich ascites tumor in the early days after implantation]. / Modelos computerizados de la variación de los distintos tiempos del ciclo celular de un tumor ascítico de Ehrlich hipertriploide, en los primeros días de implantación. I. Variaciones del índice mitótico.

The duration of the cell cycle of Ehrlich ascites cancer cells changes with the age of the tumor. Mitotic index shows the minimal values between the third and the fourth days. The authors propose a computerized model to analyze the mitotic rhythm of cell populations.

Binucleate cells in the Ehrlich ascites tumor. Action of 5-fluorouracil.

Time-dependent frequency distribution of binucleate cells (BC) was studied in Ehrlich ascites tumor (EAT) growing in mice. In animals that received no further treatment, the number of BC increased slowly from 2.6% to 16.5% of total cells within 8 days. In animals that were treated with different doses of 5-fluorouracil (FU) we found clearly higher numbers of BC. The number of BC increased with tumor age. The increase observed after treatment was reached more quickly in animals that had received the highest FU dose. The final number of BC was also dependent on the age of the tumor at the time of FU injection.

Monte carlo simulation of 3-D buffered Ca(2+) diffusion in neuroendocrine cells.

Buffered Ca(2+) diffusion in the cytosol of neuroendocrine cells is a plausible explanation for the slowness and latency in the secretion of hormones. We have developed a Monte Carlo simulation to treat the problem of 3-D diffusion and kinetic reactions of ions and buffers. The 3-D diffusion is modeled as a random walk process that follows the path of each ion and buffer molecule, combined locally with a stochastic treatment of the first-order kinetic reactions involved. Such modeling is able to predict [Ca(2+)] and buffer concentration time courses regardless of how low the calcium influx is, and it is therefore a convenient method for dealing with physiological calcium currents and concentrations. We study the effects of the diffusional and kinetic parameters of the model on the concentration time courses as well as on the local equilibrium of buffers with calcium. An in-mobile and fast endogenous buffer as described by, Biophys. J. 72:674-690) was able to reach local equilibrium with calcium; however, the exogenous buffers considered are displaced drastically from equilibrium at the start of the calcium pulse, particularly below the pores. The versatility of the method also allows the effect of different arrangements of calcium channels on submembrane gradients to be studied, including random distribution of calcium channels and channel clusters. The simulation shows how the particular distribution of channels or clusters can be of relevance for secretion in the case where the distribution of release granules is correlated with the channels or clusters.

Binucleate cells in the Ehrlich ascites tumor. Autoradiographic labeling.

An autoradiographic study was performed on binucleate and mitotic cells in the Ehrlich ascites tumor (EAT) untreated and after treatment with 5-fluorouracil (FU). The number of binucleate cells was greater in the treated tumor than in the controls. It was also observed that the number of labeled mitoses was greater in the Fu-treated tumor. Autoradiographic labeling showed that the cells that proved to be binucleate had previously passed through S-phase; thus, these cells belonged to the proliferative compartment.

Morphological and quantitative study of multinucleated bodies appearing in rat seminiferous tubules after bilateral caput epididymectomy.

Adult Wistar rats were bilaterally caput epididymectomized and the effects on testicular germinal epithelium and formation of multinucleated bodies were studied and quantified at 2, 4, 7, 14, 21, and 28 days after surgery. Sham-operated and bilaterally efferentectomized animals served as controls. No alterations were found in sham-operated animals. Efferentectomized animals showed a progressive alteration of the seminiferous tubule epithelium and a (very occasional) presence of multinucleated bodies. Epididymectomized animals presented a progressive degeneration of the germinal epithelium, which was almost complete at 28 days. This epithelial degeneration was accompanied by the formation of multinucleated bodies from germinal cells, whose number and characteristics varied with the experimental interval. The multinucleated bodies described here resemble the multinucleated cells mentioned by other authors. They do not seem to be cellular; instead, they appear to be debris, since electron microscopic observations do not reveal a plasma membrane.

Regulation of pancreatic beta-cell electrical activity and insulin release by physiological amino acid concentrations.

The mutual enhancement of insulin release by glucose and amino acids is not clearly understood. In this study, the effects on electrical activity and insulin release of a mixture of amino acids and glucose at concentrations found in fed (aaFD) and fasted (aaFT) animals were determined using freshly isolated mouse islets. Islets perifused with aaFD mixture showed an oscillatory pattern of electrical activity at lower glucose concentrations (5 mmol/l) than in islets perifused with the aaFT mixture and with glucose (G) alone (10 mmol/l). The concentration/response curve for the fraction of time spent by the membrane potential in the active phase in aaFD-stimulated islets was found to be significantly shifted to the left and had a smaller slope than that for glucose-stimulated islets. Insulin release followed the same pattern. This resulted in a concentration/response curve for glucose that was closer to that recorded "in vivo". We have also found that four amino acids (leucine, isoleucine, alanine and arginine) are largely responsible for the observed effects and that there is a non-linear enhancement of insulin release as a consequence of the combined effect of amino acids and glucose. This effect was more pronounced in the second phase of insulin release and was dependent on intracellular Ca2+. These findings indicate that amino acids account for most of the left-ward shift in the concentration/response curve for glucose and that a reduction in the threshold for the glucose-induced oscillatory electrical activity response and in the generation of Ca2+ spikes accounts for the triggering of insulin release at lower glucose concentrations. Nevertheless, the effects on insulin release at high glucose concentrations cannot be explained solely by the increase in glucose-induced electrical activity.

Effects of calcium buffering on glucose-induced insulin release in mouse pancreatic islets: an approximation to the calcium sensor.

1. The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. 2. Slow Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 microm), their effects being observed in the innermost shells dependent on Ca2+ affinity. 3. In contrast, fast Ca2+ buffers (around 1 mmol l-1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50 % inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. 4. These data are consistent with the existence in pancreatic beta-cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) 'primed' vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) 'reserved pool' vesicles, not colocalized and responsible for the second phase.

Testicular changes in adult rat following bilateral partial (caput) epididymectomy.

Adult Wistar rats were either partial (caput) and bilaterally epididymectomized or bilaterally efferentectomized, as controls of duct obstruction. The effects on testicular germinal epithelium were studied at 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 days after surgery. No abnormalities were detected in sham-operated animals. Epididymectomized animals showed different levels of alterations with progressive disruption of the seminiferous epithelium, emergence of multinucleated bodies and some tubules obliterated by degenerated cells and cellular debris. Half way through the experiment there were tubules lacking their epithelia, as well as the Sertoli cells. On the 25th day degeneration was so important that is affected not only the epithelium (missing in almost all tubules) but also the tubular morphology. Eventually efferentectomized animals showed a progressive alteration, but its level was much lower than that observed after partial epididymectomy, indicating a possible specific function of the caput epididymidis in the control of testicular function.

High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells.

Glucose-induced insulin secretion from hyperglycemic 90% pancreatectomized rats is markedly impaired, possibly because of loss of beta cell differentiation. Association of these changes with beta cell hypertrophy, increased mRNA levels of the transcription factor c-Myc, and their complete normalization by phlorizin treatment suggested a link between chronic hyperglycemia, increased c-Myc expression, and altered beta cell function. In this study, we tested the effect of hyperglycemia on rat pancreatic islet c-Myc expression both in vivo and in vitro. Elevation of plasma glucose for 1-4 days (glucose infusion/clamp) was followed by parallel increases in islet mRNA levels (relative to TATA-binding protein) of c-Myc and two of its target genes, ornithine decarboxylase and lactate dehydrogenase A. Similar changes were observed in vitro upon stimulation of cultured islets or purified beta cells with 20 and 30 mmol.liter(-1) glucose for 18 h. These effects of high glucose were reproduced by high potassium-induced depolarization or dibutyryl-cAMP and were inhibited by agents decreasing cytosolic Ca(2+) or cAMP concentrations. In conclusion, the expression of the early response gene c-Myc in rat pancreatic beta cells is stimulated by high glucose in a Ca(2+)-dependent manner and by cAMP. c-Myc could therefore participate to the regulation of beta cell growth, apoptosis, and differentiation under physiological or pathophysiological conditions.