Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis.

Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments.

Tributyltin induces apoptotic signaling in hepatocytes through pathways involving the endoplasmic reticulum and mitochondria.

Tri-n-butyltin is a widespread environmental toxicant, which accumulates in the liver. This study investigates whether tri-n-butyltin induces pro-apoptotic signaling in rat liver hepatocytes through pathways involving the endoplasmic reticulum and mitochondria. Tri-n-butyltin activated the endoplasmic reticulum pathway of apoptosis, which was demonstrated by the activation of the protease calpain, its translocation to the plasma membrane, followed by cleavage of the calpain substrates, cytoskeletal protein vinculin, and caspase-12.

Parathyroid hormone-related protein (PTHrP) inhibits mitochondrial-dependent apoptosis through CK2.

Over the past decade, parathyroid hormone-related protein (PTHrP) has been identified as a key survival factor for cells subjected to apoptotic stimuli. Its anti-apoptotic activity has been attributed to nuclear accumulation of the intact protein, or a synthetic peptide corresponding to its nuclear targeting sequence (NTS), which promotes rapid exit of nutrient deprived cells from the cell cycle. Intracellular PTHrP also inhibited apoptosis by blocking tumor necrosis factor alpha (TNFalpha)-induced apoptosis by blocking signaling from the "death receptor" and preventing damage to the mitochondrial membrane.

Parathyroid hormone related protein (PTHrP) inhibits TNFalpha-induced apoptosis by blocking the extrinsic and intrinsic pathways.

Parathyroid hormone related protein (PTHrP) is expressed at low levels in many fetal and adult tissues where it plays a central role in regulating cell proliferation, cell death, and tissue homeostasis. In vivo and in vitro, PTHrP has been shown to promote the survival of a variety of cells by regulating expression of the anti-apoptotic protein Bcl2. Additional work has shown that intra-nuclear accumulation of PTHrP in CFK2 (PTH1R positive) and 27m21 (PTH1R negative) condrogenic cells promotes their survival by closing down ribosome biogenesis and promoting quiescence.

Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes.

Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment.

Cadmium-induced apoptosis in rat hepatocytes does not necessarily involve caspase-dependent pathways.

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. Depending on the exposure conditions, Cd may cause necrosis or apoptosis. Oxidative stress is believed to participate in Cd toxicity but the molecular signaling responsible for Cd-induced apoptosis in non-malignant liver cells still needs to be clarified.

Multiple effects of mercury on cell volume regulation, plasma membrane permeability, and thiol content in the human intestinal cell line Caco-2.

In a previous study, we characterized Cd-Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation value of 0.

Kinetics of the early subcellular distribution of cadmium in rat hepatocytes.

The kinetics of the early subcellular distribution of cadmium (Cd) was characterized in primary cultures of rat hepatocytes exposed to 10, 50 and 100 microM Cd in a serum-free WME medium for 10, 30 or 60 min. Our results demonstrate a time- and concentration-dependent increase in Cd content with the highest metal concentration measured in the cytosol, whereas the lowest was observed in the mitochondria. With the exception of early localization in the plasma membrane, Cd concentrations in fractions were characterized by the following decreasing order of magnitude: cytosol > low density molecules approximately nuclei > lysosomes approximately mitochondria.

Cadmium uptake in rat hepatocytes in relation to speciation and to complexation with metallothionein and albumin.

Cadmium (Cd) uptake has been studied in primary cultures of rat hepatocytes focusing on the impact of inorganic and organic speciation. Uptake time-course studies over a 60-min exposure to 0.3 microM (109)Cd revealed a zero-time uptake and a slower process of accumulation which proceeds within minutes.

Involvement of mitochondrial and death receptor pathways in tributyltin-induced apoptosis in rat hepatocytes.

Tri-n-butyltin (TBT), a biocide, is known for its immunotoxicity and hepatotoxicity and is a well-characterised mitochondrial toxin. This report investigates the mechanisms involved in induction of apoptosis by TBT in primary cultures of rat hepatocytes. Release of cytochrome c from mitochondria into the cytosol was apparent after 15 min of exposure to 2.

Possible mechanisms underlying the mitogenic action of heptachlor in rat hepatocytes.

The worldwide use of the organochlorine pesticide heptachlor has led to widespread contamination in the environment. Like many other organochlorine pesticides, heptachlor is considered to pose a threat to human health. It has been shown that heptachlor is a tumor-promoting agent, but the mechanisms involved still remain unclear.

Cadmium-induced mitochondrial membrane-potential dissipation does not necessarily require cytosolic oxidative stress: studies using rhodamine-123 fluorescence unquenching.

The impact of cadmium on the cellular redox state and mitochondrial membrane potential (psi(m)) has been studied by monitoring dichlorofluorescein (DCF), CMXRos (dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine), and Rh-123 fluorescence in 5-day-old TC7 cells, a highly differentiated clone of the human intestinal Caco-2 cell line. Flow cytometry analyses, using DCFH oxidation to DCF, clearly revealed that a 30-min incubation to 50 microM cadmium (Cd) is sufficient to induce reactive oxygen species (ROS) formation; this effect was completely eliminated by the presence of 50 mM mannitol for the 30-min incubation period, but mannitol only partially scavenged ROS for the longer period of time studied. Imaging studies using fluorescence video microscopy revealed a parallel increase in (DCF) fluorescence in the nuclear and cytoplasmic regions as soon as Cd was added to the exposure medium.

Polyvinylalcohol three-dimensional matrices for improved long-term dynamic culture of hepatocytes.

Rat hepatocytes were seeded on three-dimensional highly porous polyvinylalcohol (PVA) and aminoethyl-modified polyvinylalcohol (AE-PVA) matrices. Hepatocytes were cultured under static and dynamic conditions. The three-dimensional matrices offered an improved extracellular microenvironment for long-term (5 days) maintenance of hepatocytes, compared to reference monolayer cultures on collagen.

Different transport mechanisms for cadmium and mercury in Caco-2 cells: inhibition of Cd uptake by Hg without evidence for reciprocal effects.

Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203Hg(II) and 109Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K(i) for Hg of 9.

Vitellogenin in tilapia male fishes exposed to organochlorine pesticides in Ouémé 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.

Resistance to Fas-induced apoptosis in hepatocytes: role of GSH depletion by cell isolation and culture.

The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies.

Assessing the estrogenic potential of organochlorine pesticides in primary cultures of male rainbow trout (Oncorhynchus mykiss) hepatocytes using vitellogenin as a biomarker.

Many organochlorine pesticides are suspected of impairing natural hormonal function in organisms by mimicking endogenous estrogen. The aim of this study was to assess the estrogenic activity of the organochlorine pesticides o,p'-DDT, dieldrin, aldrin, heptachlor, mirex and DDT in rainbow trout hepatocyte cultures using vitellogenin (Vtg) as the biomarker. A wide range of pesticide concentrations (0.

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).

Cadmium uptake by Caco-2 cells: effects of Cd complexation by chloride, glutathione, and phytochelatins.

Short-term cadmium uptake by the highly differentiated TC7 clone of enterocytic-like Caco-2 cells was studied as a function of Cd speciation. For low metal concentrations and with a constant free [Cd(2+)] = 43 nM, initial uptake rates of (109)Cd increased linearly as a function of increasing concentration of chlorocomplexes (Sigma[(109)CdCl(2-n)(n)]) over the range from 0 to 250 nM. When normalized as a function of the metal concentration, the absorption rate for the chlorocomplexes was less than that estimated for uptake of the free Cd(2+) cation.

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice.

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.

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.

Identification of a multidrug resistance-like system in Tetrahymena pyriformis: evidence for a new detoxication mechanism in freshwater ciliates.

The freshwater ciliate Tetrahymena pyriformis is an ubiquitous organism that is present in all aquatic ecosystems. This protozoan showed a clear resistance against some polycyclic aromatic hydrocarbons which can be attributed to an efflux pump probably of the multidrug resistance (MDR) type. Immunocytochemical detection showed a positive stain of ciliate cells using the monoclonal antibodies 4E3, raised against P-glycoprotein (P-gp).

Metallothionein induction attenuates the effects of glutathione depletors in rat hepatocytes.

Metallothionein (MT) is a protein involved in heavy metal homeostasis and detoxification. According to several studies, MT could be involved in the antioxidant defense system, in which glutathione (GSH) is an essential component. The aim of this study was to verify the implication of MT in the antioxidant defense system in isolated rat hepatocytes.

Evidence for an intracellular barrier to cadmium transport through Caco-2 cell monolayers.

109Cd transport was studied in the highly differentiated TC7 clone of the enterocytic-like Caco-2 cells grown on filters. Accumulation curves for 0.3 microM 109Cd over 12 h from the apical (AP) or the basal (BL) sides revealed a three-step mechanism involving: 1) a zero-time accumulation Ao; 2) a fast process Af(t1/2 < or = 10 min); and 3) a slow process of uptake As (5 h < or = t1/2 < or = 10 h) responsible for the major cellular levels of 109Cd.

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.