Michel Marion

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.

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.

The modulation by metallothionein of cadmium-induced cytotoxicity in primary hepatocyte cultures

The cytotoxicity of CdCl2 and 2 isoforms of hepatic cadmium-metallothionein (CdMT I and II), was investigated using primary cultures of rat hepatocytes. The cell cultures were exposed to cadmium as CdCl2 or as either isoform of CdMT for a 20-h period at concentrations ranging from 50 to 500 ng Cd X ml-1. Cytotoxicity was assessed by determining the amount of lactic dehydrogenase released from the cells into the incubation medium and the incorporation of [3H] arginine into cell protein. The uptake of Cd by the cells was also measured. Cadmium chloride and both isoforms of CdMT were found to be toxic to hepatocytes although partial protection was afforded by the binding of cadmium to metallothionein (MT). At the higher exposure concentrations and in accordance with the toxicity data, the cells exposed to CdCl2 were found to accumulate more cadmium than those exposed to CdMT. The distribution of cadmium in the culture medium was examined using Sephadex G-75 chromatography. The speciation of cadmium is discussed in relation to its cytotoxicity.

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. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.

Mercuric chloride affects protein secretion in rat primary hepatocyte cultures: A biochemical ultrastructural, and gold immunocytochemical study

The toxicity of mercury on hepatocytes was studied at the ultrastructural, biochemical, and immunocytochemical levels. Albumin metabolism was examined because it is a representative liver-specific function. A novel cytochemical method using the protein A-gold technique for the in situ localization of albumin in hepatocyte cultures was applied. Primary rat hepatocyte cultures were exposed to increasing HgCl2 concentrations. Cytotoxicity was assessed by measuring the release of lactic dehydrogenase from the cells. At the highest exposure concentration tested (50 microM), Hg was found to be significantly cytotoxic in contrast to what occurred at 5.0 and 0.5 microM. The level of albumin secreted, as measured by ELISA, was decreased by approximately 38% at 5.0 microM HgCl2 and was found not to be different from that of controls at lower concentrations. The ultrastructural analysis showed that hepatocytes treated with 5.0 microM HgCl2 undergo drastic morphological changes such as a decreased number of ribosomes associated with the rough endoplasmic reticulum, and the disappearance of the latter organelle, proliferation of the smooth endoplasmic reticulum, and dilatation of both the Golgi apparatus and the biliary canaliculus-like structures. Immunocytochemical detection of albumin-immunoreactive sites using protein A-gold labeling further revealed that these were less abundant in hepatocytes treated with 5.0 microM HgCl2 (-64%) as compared to control preparations. These results suggest that one of the effects of mercury on hepatocytes is to affect liver-specific functions such as albumin production, possibly through interference with ribosomal function. This study also demonstrates for the first time the applicability of the high-resolution protein A-gold technique for toxicological investigations on hepatocytes in vitro.

The cytotoxic effects of cadmium chloride and mercuric chloride mixtures in rat primary hepatocyte cultures

The toxic effects of Cd and Hg mixtures were studied using primary monolayer cultures of rat hepatocytes. Cytotoxicity was assessed by measuring the release of lactic dehydrogenase from the cells. Cytotoxic and non-cytotoxic metal levels were used. At the higher exposure concentrations (0.2 micrograms Cd.ml-1 and 2.0 micrograms Hg.ml-1), Cd was very toxic to hepatocytes whereas Hg was only marginally toxic. The combination of Cd and Hg was more toxic than predicted by summation of the individual metal toxicities. The incorporation of [35S]cysteine into protein of the cytosol and insoluble cell fraction was increased in response to Cd or Hg exposure and was directly related to cell 35S accumulation. Combinations of Cd and Hg significantly increased the proportion of total 35S which was incorporated in cell protein, an effect that was attributed to the accumulation of protein in the insoluble cell fraction. Cd uptake by hepatocytes was related to exposure concentration but was lower when Hg was also present in the incubation medium. Gel chromatography of the cytosol from Cd-exposed cells showed 3 Cd containing fractions which corresponded to the elution positions of high Mr proteins, metallothionein (MT) and low Mr molecules. When hepatocytes were exposed to Hg in combination with Cd, the MT-like fraction was no longer evident and Cd in the low Mr fraction was greatly reduced. Regardless of the presence or absence of Cd in the exposure medium, 98% of cytosol Hg in Hg-exposed cells was found to elute after the low Mr fraction, at a position equivalent to inorganic salts. This indicates that the enhanced cytotoxicity of Cd and Hg may be related to a decrease in the MT-like protein in the cytosol and not due to a direct competitive binding interaction in relation to the protein.

Genotoxic effects of heavy metals in rat hepatocytes

The genotoxic interaction of metals, which are common environmental contaminants, was studied in cultured hepatocytes. Freshly isolated rat hepatocytes were exposed to concentrations of cadmium, copper, silver and lead salts ranging from non-cytotoxic to moderately cytotoxic (as determined by LDH release), and the incorporation of [3H]thymidine into the DNA, as a measure of repair synthesis, was followed. In addition, the uptake of metals by the nuclear fraction was determined using Inductively Coupled Plasma/Mass Spectrometry or atomic absorption spectrophotometry. The evaluation of binding of 109Cd to the DNA in situ was also attempted. It was observed that after a 20 h exposure period, all the metals investigated were found in the nuclear fraction of hepatocytes, with Ag apparently being accumulated less efficiently. In parallel, Cd (0.18 to 1.8 µM) and Cu (7.9 to 78.5 µM) consistently produced a statistically significant stimulation of [3H]thymidine incorporation into the DNA, in the presence or absence of hydroxyurea while Ag was active only at the highest concentration tested (18.5 µM). In contrast, Pb failed to induce a UDS response at the levels used. Moreover, exposure of hepatocytes to 1.8 µM 109CdCl2 for 20 h led to a DNA binding ratio of 0.98 ± 0.23 ng Cd/ µg DNA. The present results support the view that the nucleus may be an important target organelle for metal toxicity.

Absence of genotoxic effects of nonasbestos mineral fibers

The biological activity of natural and synthetic mineral fibers has been examined. Natural attapulgite [(Mg, Al)2Si4O10(OH).4H20], synthetic xonotlite [Ca3Si3O8(OH)2] and natural sepiolite [Mg2Si3O8.2H2O] were selected. Genotoxic effects were investigated by means of a well established cellular model based upon the measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. The intrinsic capacity of the fibers (1 and 10 µ/ml) to induce UDS was first tested. None of the fiber types showed detectable UDS-eliciting activity. Also, the possible modulation of the cellular response to genotoxic agents by the materials was examined by exposing the cells to mixtures of 2-acetylaminofluorene (AAF) (0.05 and 0.25 µg/ml) and fibers (1 and 10 µg/ml). In these experiments, the UDS response was significantly diminished in the presence of xonotlite. This phenomenon may reflect changes in the uptake and/or metabolism of AAF or may result from an inhibition of DNA repair processes, the latter suggesting a possible cocarcinogenic potential for this synthetic silicate. These results point to the immediate necessity of studying more extensively the biological effects of fibrous materials that can be used as substitutes for asbestos.