Francine Denizeau

METALLOTHIONEIN IN PHYSIOLOGICAL AND PHYSIOPATHOLOGICAL PROCESSES

The multipurpose nature of MT that we have presented in this review has drawn attention from many different fields of research: biochemistry, molecular biology, toxicology, pharmacology, etc. In recent years, considerable advances have been made concerning the regulation of MT genes by metals. Little, however, is known at the molecular level about the mechanisms of MT induction by nonmetallic inducers such as growth factors. This is of particular interest since MT is highly expressed during liver regeneration, an event orchestrated by a series of growth stimulators and inhibitors. The significance of the nuclear distribution of MT in growing cells and what controls its translocation are questions that remain unanswered at the present time. The possibility that MT could participate in a DNA synthesis-related process through donation or abstraction of Zn to and from transcription factors has been inferred from in vitro studies. Such transfer mechanisms, however, have yet to be confirmed in vivo. Overexpression of MT is often accompanied by increased resistance towards a variety of alkylating agents and chemotherapeutic drugs. The mechanisms by which MT protects cells against these agents may depend on their distinct mode of toxic action. For some, MT cysteines can be the target of the direct attack from the parent compound. For others such as N-methyl-N-nitroso compounds, MT cysteines may serve as a sink for the reactive oxygen species now known to be derived from their metabolism. In either case, a primary consequence of such interactions is the release of the metals initially bound to MT. Therefore, the metal composition of MT appears to be an important factor to consider in determining the overall effect of MT in the resistance process.

Tributyltin triggers apoptosis in trout hepatocytes: the role of Ca2+, protein kinase C and proteases.

The purpose of the present study was to study the mechanisms involved in the induction of apoptosis and by tributyltin (TBT) in rainbow trout hepatocytes, and to examine the role of intracellular Ca2+, protein kinase C (PKC) and proteases in the apoptotic process. The intracellular Ca2+ chelator BAPTA-AM has a suppressive effect on TBT-mediated apoptosis. However, exposure to the ionophore A23187 is not sufficient to induce apoptosis in trout hepatocytes. The results obtained also show that TBT stimulates PKC gamma and delta translocation from cytosol to the plasma membrane in trout hepatocytes after 30 min of exposure. However, PKC gamma translocation is down-regulated after 90 min of treatment. The addition of protein kinase inhibitors (staurosporine and H-7) not only fails to inhibit apoptosis induced by TBT, but also leads to enhancement of DNA fragmentation. These inhibitors also afford a remarkable protection against the loss of plasma membrane integrity caused by TBT exposure. PMA, a direct activator of PKC, fails to stimulate DNA fragmentation. In addition, Z-VAD.FMK is an extremely potent inhibitor of TBT-induced apoptosis in trout hepatocytes, indicating that the activation of ICE-like proteases is a key event in this process. The cysteine protease inhibitor N-ethylmaleimide also prevented TBT-induced DNA fragmentation. Taken together, these data allow for the first time to suggest a mechanistic model of TBT-induced apoptosis. We propose that TBT could trigger apoptosis through a step involving Ca2+ efflux from the endoplasmic reticulum or other intracellular pools and by mechanisms involving cysteine proteases, such as calpains, as well as the phosphorylation status of apoptotic proteins such as Bcl-2 homologues.

Coupling the use of computer chemical speciation models and culture techniques in laboratory investigations of trace metal toxicity

Abstract The bioavailability and toxicity of a dissolved metal are closely linked to the metal’s chemical speciation in solution. A variety of inorganic and organic ligands are often used in laboratory toxicity tests to control the concentration of labile trace metal in solution. Computerised chemical speciation models based on thermodynamic principles can be used to estimate metal speciation under such experimental conditions. However, these models are sensitive to the quality of their thermodynamic databases. Detailed protocols for the incorporation of reliable equilibrium formation constants into widely available computer chemical speciation programs (e.g., MINEQL+ and MINTEQ) are provided. The examples demonstrate both the benefits and the potential pitfalls involved in the use of chemical speciation models. The application of chemical speciation modelling to metal toxicity studies is discussed and guidelines are proposed for its proper use. Both defined media and chemical speciation programs have co-existed for two decades but the combined use of these techniques has been reserved for those possessing in-depth knowledge of both chemistry and biology. The techniques presented should enable an investigator with basic biological, chemical and computing skills to design an aqueous medium and incorporate correct thermodynamic constants into a computer chemical speciation program, starting from a standardised database, thereby providing a sound framework for critically assessing the biological response of a particular test organism to a given metal.

Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum

The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4-0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.

Caco-2 Cell Line Used as an In Vitro Model to Study Cadmium Accumulation in Intestinal Epithelial Cells

Abstract.109Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 μm109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t½= 17.3 ± 1.3 min) with an apparent in-to-out distribution ratio (αe) of 11.6 ± 0.8. The amplitude of the rapid phase (U0) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable αe values were observed at equilibrium. In both clones, the t½ and αe values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, 109Cd uptake at 1 min (U1) was unaffected by Ca concentrations four order of magnitude in excess, but both U0 and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U0 disappeared when EDTA was present in the wash solutions. U1 showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (Km= 3.8 ± 0.7 μm). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of 109Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption.

Flow cytometric analysis of the effects of tri-n-butyltin chloride on cytosolic free calcium and thiol levels in isolated rainbow trout hepatocytes

The toxic effects of tri-n-butyltin chloride (TBT) were investigated on isolated trout hepatocytes by flow cytometry (FCM). We developed a procedure permitting the study of cytosolic free calcium in these cells using the new fluorescent probe Fura Red. In parallel, changes in thiol levels upon exposure to TBT were also followed by FCM with the probe 5-chloromethylfluorescein-diacetate. Cell viability was monitored through FCM analysis using propidium iodide. Treatment of hepatocytes with TBT caused a time- and concentration-dependent loss of viability. The results show that TBT induced a sustained elevation of cytosolic free calcium in isolated trout hepatocytes before loss of viability was detectable. Data for the viable cells remaining after incubation with TBT were selected after appropriate gating with the flow cytometer. When this was performed, the data revealed that changes in cytosolic free calcium were not dependent upon TBT concentration and duration of exposure. Moreover, TBT induced a rapid and important depletion of thiols in cells which survived TBT exposure. The present results suggest that alterations in calcium homeostasis and intracellular thiols are involved in the mechanism of trout hepatocyte injury by TBT. FCM is a powerful tool to study metabolic disturbances caused by toxic agents on cells at an individual level.

Effect of cadmium on membrane potential in isolated rat hepatocytes.

The effect of cadmium (Cd) on rat hepatocytes upon short term exposure was studied by focusing on the integrity of mitochondria and on the possible consequences of its disturbance, such as alterations in plasma membrane potential and loss of cell viability. Changes in the potential of mitochondrion and plasma membranes were monitored using [3H]triphenylmethylphosphonium (TPMP+) and [14C]SCN- probes, respectively. Isolated rat hepatocytes were exposed to increasing CdCl2 concentrations for short time periods (30-120 min). Cd measurement by atomic absorption showed that the cells efficiently accumulated Cd, as did mitochondria in situ. In CdCl2-treated cultures, it was observed that the release of TPMP+, which revealed a drop in the mitochondrial membrane potential, was time- and concentration-dependent, and that the first significant efflux was caused by a 30-min exposure to 89 microM CdCl2. No significant change in plasma membrane potential, as judged from the increase in the uptake of SCN-, was detected after 30 min, suggesting the greater precocity of the mitochondrial attack. Finally, the release of lactate dehydrogenase (LDH) occurred only after 2 h of exposure, reflecting ultimate stages of cell injury induced by Cd. These results suggest that Cd induces an alteration in mitochondrial function in hepatocytes which may lead to the loss of plasma membrane potential and cell viability. The study therefore adds further evidence of the role of mitochondria as primary targets in Cd-induced cytotoxicity.

Immunosuppression in mice fed on diets containing beluga whale blubber from the St Lawrence Estuary and the Arctic populations

In order to assess the immunotoxic potential of naturally relevant mixtures of PCBs and other organohalogens, C57Bl/6 mice were fed on diets in which lipids were replaced by blubber of beluga whales from the highly contaminated population of the Saint-Lawrence River, and the less contaminated population from the Arctic. Different ratios of blubber from both sources were mixed in order to allow a dose-response study. Mice were fed for a period of 90 days at the end of which their immunological status was monitored. For general parameters such as body weight, weight of the spleen and the thymus no significant effect of diets were observed. The immunological endpoints such as the blastic transformation of splenocytes and the spleen NK cell activity were not significantly affected by any of the diets compared to control diets. While the different cell subpopulations of peripheral blood and thymus were not affected by the diets, a significant decrease was noted in the CD8+ T cell population in the spleen of mice fed with most of the diets containing beluga blubber. Moreover, the ability of splenic cells to elicit humoral response against sheep red blood cells as well as the potential of peritoneal macrophages to perform phagocytosis were suppressed by all diets containing beluga blubbers. In summary, there was no differences between the groups fed with a blubber diet with low and high organochlorine contamination. However, a clear immunosuppression was demonstrated when these groups were compared to the group fed with beef oil. Despite the fact that we cannot exclude a possible contribution of the fatty acid composition of the beluga blubber to the immunosupression, these results suggest the sensitivity of mouse immune system towards organohalogens, and point out the toxic potential of contaminant mixtures as found in the less contaminated Arctic population.

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes☆

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.

VITELLOGENIN IN TILAPIA MALE FISHES EXPOSED TO ORGANOCHLORINE PESTICIDES IN OUEME RIVER IN REPUBLIC OF BENIN

In many African countries, the economy largely depends on agriculture. Pesticides are therefore likely to represent an important source of xenoestrogens in contaminated rivers and lagoons. The largely uncontrolled use of diverse pesticides led us to hypothesize that these agents, and particularly organochlorine compounds, may pose a serious problem in the Republic of Benin. To verify our hypothesis, tilapia (Sarotherodon melanotheron) from five sites in the southern part of the main Ouémé River were analyzed. Ouémé River drains the southern region of the country. Vitellogenin (Vtg) was used as an indicator of contaminated sites. This approach has its limitations, because there are a wide variety of man-made chemicals present in the aquatic environment likely to induce Vtg in male fish. Therefore, in this study this approach allows us to define potential contaminated target sites. In order to determine whether the presence of Vtg could be attributable to pesticides, organochlorine pesticides in the flesh of tilapia were also analyzed. Significant amounts of Vtg in fish from contaminated sites were detected, and were correlated with organochlorine pesticide levels in tissue. These results indicate that organochlorine pesticides are present in the Ouémé River and that these compounds can act as endocrine modulators in this ecosystem. Eating fish from contaminated rivers, such as the Ouémé River, may contribute to the accumulation of high concentrations of these pesticides in the body, leading to exposure to their negative effects.