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. Therefore we have studied apoptosis in primary cultures of rat hepatocytes incubated with low levels of Cd for short exposure times. Studies of nuclear morphology, chromatin condensation, and oligonucleosomal DNA fragmentation demonstrate that 1-5 microM Cd induces apoptosis as early as 6-12 h with minor effects on MTT activity. A concomitant time- and concentration-dependent increase in caspase-9 and -3 activities was observed, whereas Cd did not affect caspase-8 activity as much, suggesting a minor role of the death-receptor pathway. Significant release of cytochrome c into the cytosol demonstrated the involvement of a mitochondrial-dependent apoptotic pathway. However, cell pre-treatment with caspase inhibitors (Z-VAD-fmk or Ac-DEVD-CHO) did not prevent apoptosis. Increases in the cytosolic levels of the mitochondrial apoptosis-inducing factor (AIF) were also observed: kinetics of cytochrome c and AIF release were similar. These results show that Cd-induced apoptosis in rat hepatocytes is time- and concentration-dependent. The early apoptotic events involved mitochondrial-dependent pathways but not necessarily caspase-dependent signaling.

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 0.3 micromol/L (203)Hg was measured in the presence of 100 micromol/L unlabeled Hg in the serum-free exposure medium without modification in the initial uptake rate. This phenomenon was eliminated at 4 degrees C. Mercury induced an increase in tritiated water and [(3)H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 micromol/L Hg and 2 mmol/L N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects. Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport. During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and consequently cell capacity.

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. (109)Cd uptake showed saturation kinetics (K(m) = 3.5 +/- 0.8 microM), and was highly sensitive to inhibition by Zn and Hg. There was no evidence for sensitivity to the external pH nor for any preferential transport of the free cation Cd(2+) over CdCl(n) (2-n) chloro-complexes. According to the assumption that only inorganic metal species are available, metal uptake decreased upon albumin (BSA) addition to the exposure media. In contrast, higher levels of (109)Cd accumulation were obtained under optimal conditions for Cd complexation by MT. Comparison among uptake data obtained under inorganic and organic conditions revealed that Cd-MT would be taken up 0.4 times as rapidly as Cd(inorg). We conclude that uptake of Cd in rat hepatocytes involves specific transport mechanism(s) subjected to Zn or Hg interactions. Uptake of inorganic Cd is not proportional to the levels of free Cd(2+) and does not involve the divalent cation transporter DCT1 nor the co-transporter Fe(2+)-H(+) NRAMP2. We found Cd-MT but not Cd-BSA to be available for the liver cells, and have estimated a binding affinity four orders of magnitude higher for Cd complexation with MT compared to BSA; MT may have a significant role in Cd delivery to the liver.

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. Flow cytometry analyses monitoring CMXRos fluorescence clearly showed that Cd also leads to psi(m) disruption, but, contrary to what was observed for ROS formation, mannitol was completely inefficient in preventing this effect. Further investigation using fluorescence microscopy and Rh-123 fluorescence unquenching revealed that although mannitol did not protect against Cd-induced dissipation of psi(m), it considerably delayed the process. We found that Rh-123 unquenching, occurring during probe redistribution, is a suitable tool to monitor the decrease of psi(m). We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH(*), as well as the loss of psi(m). However, psi(m) dissipation does not necessarily require cellular OH(*) and may occur in the absence of apparent oxidative injury.

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. Metal absorption decreased upon organic ligand addition in the exposure media, but much less than predicted from the assumption that only inorganic metal species would be transported. Under exposure conditions where the concentration of each of the inorganic species was kept constant, (109)Cd uptake increased with increasing concentrations of cadmium glutathione ((109)Cd-GSH) or phytochelatin ((109)Cd-hmPC(3)) complexes. A specific system of very high affinity but low capacity has been characterized for (109)Cd-GSH transport, whereas accumulation data increased linearly with (109)Cd-hmPC(3) up to 6 microM. Comparison among uptake data for 0.3 microM inorganic (109)Cd, (109)Cd-GSH, or (109)Cd-hmPC(3) yields the following accumulation ratios: Cd-GSH/Cd(inorg) = 0.2; Cd-hmPC(3)/Cd(inorg) = 0.5. These results clearly show that Cd(2+) is not the exclusive metal species participating in Cd absorption, though, for comparable Cd concentrations, its contribution to transport would be more important than that of other species. Cadmium bound to thiol-containing peptides may be absorbed via transport systems that differ from those involved in absorption of the inorganic metal species.

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)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.

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.

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). The kinetics of P-gp expression were studied for control cultures and cultures treated with 15 microM benzo(a)pyrene. Western blot analysis using the Ab1, anti-P-gp polyclonal antibodies indicates the presence of two bands of 66 and 96 kDa of which the intensity increased with time in benzo(a)pyrene-treated ciliates. Uptake experiments with target compounds for the MDR pump, namely adriamycin, rhodamine 123 and two polycyclic aromatic hydrocarbons, benzo(a)pyrene and 7,12-dimethylbenzanthracene, were carried out by flow cytometry, in the presence or absence of cyclosporin (an inhibitor of the multidrug resistant pump). The data indicate that the accumulation of these compounds by ciliate cells is significantly enhanced in the presence of cyclosporin. This suggests that Tetrahymena is provided with a P-gp-like system that is functionally active in a way similar to that of the mammalian 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. For this purpose, hepatocyte cultures were exposed to treatments known to modify MT or GSH levels. Zinc (Zn) was used as an inducer of MT while diethyl maleate (DEM) and buthionine sulfoximine (BSO) were used as GSH depletors. GSH, MT, and antioxidant enzyme activities were measured under conditions of MT induction and GSH depletion. Induction of MT synthesis through an 18-hour exposure to Zn (20 microM), did not result in any significant change in GSH levels or in activities of the antioxidant enzymes, glutathione-peroxidase (GSH-Px), catalase, and superoxide dismutase (SOD). DEM caused GSH depletion in cells, whether they were exposed to Zn or not, that lasted one h; after that time, GSH rose back to basal levels. BSO also caused GSH-depletion in cells exposed or unexposed to Zn, and no recovery in GSH levels was detectable during the entire period of exposure (12 h). However, GSH depletion induced by both DEM or BSO was attenuated in Zn-treated hepatocytes. Moreover, DEM and BSO exposures led to a depletion of MT levels in Zn-treated hepatocytes, indicating a link between GSH and MT metabolism. In cells unexposed to either Zn, DEM or BSO, there was an increase in GSH-Px and SOD activities after 6 and 12 h of incubation, respectively. Under the same conditions, catalase activity was inhibited after 6 h of incubation and returned to the activity found at t = 0 after 12 h of incubation. DEM and BSO treatments had no significant effect on GSH-Px or SOD activities although they led to inhibition of catalase activity. Taken together, our data indicate that MT induction, which creates a new pool of thiol groups in the cell cytosol, can attenuate GSH depletion induced by DEM or BSO. It appears that catalase is most sensitive to oxidative stress and that MT induction can antagonize the deleterious effects of such stress on the enzyme. This study supports the view that MT is part of the hepatocyte antioxidant-defense-system.

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. The relative contribution of adsorption to total accumulation is greater for short exposure times (< or = 35%), but is no longer significant after the exposure times needed to reach equilibrium. Transepithelial transport was less than 4% of the cellular level at 12 h. A negligible but specific binding onto the BL surface of the filters was characterized. Saturable systems of accumulation with comparable affinities (Km = 2.5+/-0.5 and 5.4+/-0.4 microM) but distinct capacities (Vmax = 8.9+/-1.2 and 312+/-22 pmol/min/mg protein) were identified at the AP and BL cell membranes, respectively. Efflux studies revealed that Cd accumulation is only partially reversible, with an exclusive metal release at the same side. A 2-h exposure on both sides simultaneously failed to demonstrate any competition for cellular accumulation: uptake was additive relative to AP and BL uptake values. These data suggest that Af leads to an accumulation of loosely bound Cd, whereas As represents irreversible intracellular binding processes. We conclude that Cd transport occurs exclusively by a transcellular route and that saturation of the intracellular high-capacity binding sites is the rate-limiting step in Cd absorption.

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.

Lack of suppressive effects of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDS), polychlorinated dibenzofurans (PCDFS), and aroclor biphenyls (PCBS) on mixed lymphocyte reaction, phagocytic, and natural killer cell activities of rat leukocytes in vitro.

Rat splenocyte mixed leukocyte reaction (MLR), splenic natural killer (NK) cell activity, and phagocytic activities of splenic, peritoneal, and peripheral blood leukocytes (PBLs) were evaluated in vitro to determine the immunotoxicity of mixtures containing low levels of methylmercury (MeHg), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and Aroclor polychlorinated biphenyls (PCBs). The mixtures were based on the concentrations of the chemicals in fish flesh. Leukocytes from male Fischer rats were exposed to MeHg (0.1-2 microg/ml), PCDD/PCDF mixtures (1-15 pg/ml) of three PCDDs (2,3,7,8-tetrachlorodibenzo-p-dioxin, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin) and two PCDFs (2,3,7,8-tetrachlorodibenzofuran and 1,2,3,7,8-pentachlorodibenzofuran), three Aroclor PCB (Aroclor 1242, 1254, and 1260) mixtures (0.01-0.5 microg/ml), or combinations of MeHg/PCB/PCDD/PCDF mixtures for 24 or 72 h before immunological assays. Phagocytosis and NK cell cytotoxicity were evaluated with a flow cytometer, and MLR of Fischer rat responder splenocytes cultured with mitomycin C-treated Long-Evans splenocytes by [3H]thymidine uptake. Exposure to MeHg (2 microg/ml) alone or with PCB/ PCDD/PCDF resulted in significant cytolethality in rat splenocytes, peritoneal leukocytes, and PBLs at 24 h exposure. Treatment with Aroclor PCB mixtures, PCDD/PCDF mixtures, 0.1 microg MeHg/ml (noncytolethal), or PCB/PCDD/PCDF mixtures with 0.1 microg MeHg/ml caused no suppression of splenocyte MLR response, splenic NK cell-mediated lysis of Yac-l cells, or phagocytosis of fluorescent beads by splenic, peritoneal, and peripheral blood phagocytic cells. The results indicate that in vitro exposure of rat leukocytes to low levels of MeHg, Aroclor PCB mixtures, PCDD/PCDF mixtures, or MeHg/PCB/PCDD/PCDF mixtures had no suppressive effects on the immune functions assayed, and thus produced no additive immunotoxicity. However, in order to predict the potential risk of these chemical mixtures to the human immune system, in vivo animal studies with blood (tissue) levels compatible with the levels of MeHg, PCBs, and PCDDs/PCDFs in exposed human populations should be evaluated.

Caco-2 cell line used as an in vitro model to study cadmium accumulation in intestinal epithelial cells.

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 microM 109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t(1/2) = 17.3 +/- 1.3 min) with an apparent in-to-out distribution ratio (alphae) 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 alphae values were observed at equilibrium. In both clones, the t(1/2) and alphae 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 microM). 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.

Uptake and efflux of polycyclic aromatic hydrocarbons by Tetrahymena pyriformis: evidence for a resistance mechanism.

The action of benzo(a)pyrene (BP), 3-methylcholanthrene (3MC), benzanthracene (BA), and 7,12-dimethylbenzanthracene (DMBA), four polycyclic aromatic hydrocarbons (PAHs), was studied on the unicellular protozoan Tetrahymena pyriformis. This ciliate was exposed to the PAHs at 1, 15, and 37 microM for up to 6 h. BP and BA caused a slight inhibition of cell growth, whereas 3MC and DMBA showed no detectable effect. Cell viability remained unaffected by the PAHs at all concentrations and exposure times tested. Cellular accumulation of PAHs was studied using flow cytometry. The results show immediate accumulation followed by rapid elimination of the compounds. BP uptake was also studied in the presence of verapamil and cyclosporin, compounds known as inhibitors of the multidrug resistance (MDR) pump. In the presence of verapamil, BP was accumulated in larger amounts in cells. With cyclosporin, the accumulation of the PAH was several times higher than under control conditions. The results of GC/MS analysis show that PAH elimination was not linked to biotransformation. These results suggest that the resistance of Tetrahymena against PAH cytotoxicity may be attributed to the rapid efflux of these agents from the cells via an efflux pump probably of the MDR type.

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.

Rat hepatocytes with elevated metallothionein expression are resistant to N-methyl-N'-nitro-N-nitrosoguanidine cytotoxicity.

Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.

Induction of metallothionein gene expression by epidermal growth factor and its inhibition by transforming growth factor-beta and dexamethasone in rat hepatocytes.

Metallothionein (MT) is a small cysteine-rich protein thought to be mainly involved in metal regulation and detoxification. The implication of MT in cell growth and differentiation has also been suggested. This latter hypothesis was further investigated in adult rat hepatocytes induced to proliferate by epidermal growth factor (EGF). Exposure of hepatocytes to EGF resulted in significant increases (approximately twofold) in MT protein and MT-1 messenger RNA (mRNA) levels, which were maximal after 48 hours. As revealed by nuclear run-on analysis, these changes were the result of transcriptional activation. Increases of MT occurred concomitantly with stimulation of DNA synthesis (48 hours). Addition of ZnSO4 or dexamethasone (Dex) was also effective at inducing MT protein (approximately 3.6 to 3.3 times) and mRNA. Combined addition of Zn and EGF produced an additive increase in MT protein and MT-1 mRNA levels. When both Dex and EGF were present together, the EGF-induced MT protein and mRNA expression was lost, whereas it had only minor inhibitory effects on DNA synthesis. Transforming growth factor beta (TGF-beta), a known antagonist of EGF on hepatocytes, blocked the EGF-induced MT accumulation and stimulation of DNA synthesis. In addition, under the same conditions, the EGF-induced c-fos mRNA accumulation was blocked by Dex whereas TGF-beta had no effect. These results show that growth factors believed to play a role in liver regeneration can also modulate MT gene expression in vitro. This modulation does not strictly parallel that of DNA synthesis. The possibility that c-fos stimulation may play a role in MT induction by EGF cannot be ruled out.

Immunocytochemical evidence for a nuclear and a cytoplasmic O6-methylguanine repair mechanism in cultured rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-metG and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2-24 h to 5 mM N-nitrosodimethylamine (NDMA) or 0.1 mM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). In both cases, the O6-metG immunoreactive sites were concentrated in the nucleus and in the rough endoplasmic reticulum (RER) rich cytoplasmic regions. The highest gold labeling density measured was observed at 2 h of NDMA or MNNG treatment. However, after a 24-h exposure, very little labeling was observed in both the nuclear and the cytoplasmic compartments. The rate of disappearance of immunoreactive sites was faster in the cytoplasm than in the nucleus, Untreated control preparations showed no specific immunogold labeling. Furthermore, when cells were exposed first to NDMA and MNNG for a few hours and then to culture medium containing no genotoxin, and subsequently were reexposed to NDMA or MNNG for a few hours, very little labeling of both the nuclear and cytoplasmic compartments was observed. Control preparations without a second genotoxin exposure showed a normal labeling pattern. Control preparations without genotoxin showed no gold labeling. Our results provide evidence for the existence of a cytoplasmic O6-metG repair mechanism that behaves like its nuclear counterpart.

Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes: a flow cytometric analysis.

The effects of tri-n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca(2+)-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 microM TBT caused a rapid and sustained elevation of cytosolic free Ca2+ concentration [Ca2+]i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 microM TBT, there was a slight but not significant increase in [Ca2+]i and a significant reduction in intracellular pH (pHi) only after 60 min of exposure. A rise in [Ca2+]i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca(2+)-free medium suggest that TBT mainly mobilizes Ca2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.

O6-methylguanine-DNA adducts in rat lymphocytes after in vivo exposure to N-nitrosodimethylamine (NDMA).

A non-invasive approach in immunopathological risk assessment was applied for analysis of the in vivo formation of DNA adducts. DNA methylation was studied in peripheral blood lymphocytes (PBLs) collected from Sprague-Dawley rats exposed to a single dose (75 mg/kg b.w.) of N-nitrosodimethylamine (NDMA). Three different techniques were applied for characterization and quantification of DNA adducts: (i) colloidal gold ultraimmunocytochemical localization of O6-methylguanosine (O6-meG)-DNA adducts, using affinity-purified, polyclonal antibody directed against O6-meG, (ii) quantitative assay using enzyme-linked immunosorbent assay (ELISA), amplified by the avidin-biotin (AB) system, and (iii) high-performance liquid chromatography (HPLC). The O6-meG-immunoreactive sites in PBLs seem to be concentrated in the nucleus. However, significant immunolabelling was also noted in the cytoplasm of the in vivo NDMA-exposed PBLs. Control preparations showed no specific gold immunolabelling. The O6-meG-DNA adduct formation in PBLs and hepatocytes, at 2-24 h following the exposure to NDMA, was analogous for both types of cells. The data showed high correlation for the ELISA and HPLC analytical methods. The data suggest an efficient O6-metG-DNA repair mechanism in lymphocytes, possibly analogous to the enzymatic repair of DNA adducts in liver cells.