* Départementes de Chimie,
des Sciences Biologiques, Centre TOXEN, Université du Québec à Montréal, C.P. 8888, Succ. centre-ville, Montréal, Québec, Canada H3C 3P8
Received June 10, 2003; accepted October 6, 2003
ABSTRACT
The impact of cadmium on the cellular redox state and mitochondrial membrane potential (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 µM 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
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
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
m. We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH, as well as the loss of
m. However,
m dissipation does not necessarily require cellular OH and may occur in the absence of apparent oxidative injury.
Key Words: cadmium; dichlorodihydrofluorescein diacetate (DCF), chloromethyl-X-rosamine CMXRos, rhodamine-123; reactive oxygen species (ROS); mitochondrial membrane potential; intestinal Caco-2 cells.
For many years, it has been known that cadmium (Cd), a ubiquitous heavy metal and an environmental pollutant, is highly cytotoxic. A number of studies have shown that it may induce either necrosis or apoptosis, depending on the exposure conditions and the model used (Galan et al., 2001; Ishido et al., 2002
). Many investigators have studied the ability of Cd to affect cellular function, homeostasis, as well as the cellular structural components. For many years, the redox state of the cell, as revealed by the measurement of cellular glutathione (GSH) levels, the activity of antioxidant enzymes (catalase, superoxide dismutase), or lipid peroxidation (malondialdehyde assay) have been the most widely studied parameters (for review, see Pinot et al., 2000
; Stohs and Bagchi, 1995
). More recently, studies have focused on the effect of Cd on gene expression and cellular signalling, with emphasis on the mitochondrial membrane potential (
m) (for review see Beyersmann and Hechtenberg, 1997
). Some results have demonstrated a relationship between metal-induced oxidative stress and apoptosis (Bagchi et al., 2000; Hart et al., 1999
), but very few studies have investigated the direct relationship between cellular redox state and
m. It is not clear whether the early event in the Cd-induced apoptosis is reactive oxygen species (ROS) formation or mitochondrial membrane depolarization. It has been suggested that intracellular sulfhydryl groups depletion by Cd2+ is the prerequisite for either ROS formation or
m disruption, and there is an increasing body of evidence for Cd-induced intramitochondrial ROS formation leading to
m collapse (Pourahmad and OBrien, 2000
; Yang et al., 1997
).
To date, one of the most straightforward ways of assessing redox state variations in living cells requires fluorescent reporter molecules. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most commonly used intracellular fluorescent probes, although several others have been developed in recent years [dihydrorhodamine 123, 5(and 6)-carboxy -2',7'-dichlorodihydrofluorescein diacetate, dihydroethidine]. DCFH-DA diffuses rapidly through cell membranes and becomes deacetylated by intracellular esterases. The nonfluorescent DCFH is then trapped in the cell and may serve as a sensitive cytosolic marker of oxidative stress upon oxidation to dichlorofluorescein (DCF). More recently, the use of DCFH-DA has been questioned on many aspects: it has been shown that aortic endothelial cells are unable to retain either DCFH or DCF (Ferrari et al., 1998); the cellular distribution of the probe has also been the subject of much debate and it is now believed that its intracellular localization varies with tissue or cell phenotype (Crow, 1997
; Gabriel et al., 1997
; Sarvazyan, 1996
; Swift and Sarvazyan, 2000
). Furthermore, the assumption that DCFH detects specific oxidizing species, such as H2O2, is being increasingly challenged. It has been proposed that DCFH, like the other ROS-specific probes, detects a broad range of oxidizing reactions occurring during intracellular oxidant stress (Hempel et al., 1999
).
Studies on apoptosis-related variations in m have also been conducted using a wide range of fluorescent probes. Numerous investigators have addressed the question of which mitochondrion-specific fluorochromes has the best sensitivity and accuracy to evaluate the state of
m. Although no consensus exists on which probe is best, some investigators show strong preference for JC-1 (Salvioli et al., 1997
) or DiOC6 (Rottenberg and Wu, 1999), while the use of rhodamine-123 (Rh-123) is being seriously questioned. Some studies have clearly demonstrated the inconsistent and unreliable behaviour of Rh-123 using flow cytometry (Salvioli et al., 1997
), and Métivier et al. (1998)
have hypothesized that excess Rh-123 may cause self-quenching of the probe and that the reported increase in Rh-123 would be related instead, at least in part, to unquenching following depolarization of the mitochondrial membrane. This property of the probe has even been used to detect
m variation in mitochondria isolated from rat or mouse liver by fluorometry (Zamzami et al., 2001
).
The aim of the present study was to investigate the relationship between cellular redox state, namely equilibrium between the pro-oxidant and antioxidant systems, and m in Caco-2 human enterocytic-like cells. We have previously demonstrated that Cd transport through Caco-2 cell monolayers occurs mainly via the trans-cellular pathways, with saturation of intracellular binding capacity as the rate-limiting step in the overall process of trans-epithelial transport (Jumarie et al., 1999
). Because oral absorption represents a major route of exposure to Cd for humans, a better understanding of effects of Cd on intestinal cells is of primary interest. Not only does the intestinal epithelium represent the first barrier to be crossed by the ingested metal, but it also represents the first target organ under oral exposure conditions. For example, we have provided evidence for Cd2+ transport through the cotransporter Fe2+-H+ NRAMP2 (Elisma and Jumarie, 2001
) supplying an explanation for the reported Cd-induced anemia in some people (Noda et al., 1991
). These studies show how Cd may affect absorptive functions of the intestinal epithelium. We have pursued our investigation on the impact of Cd-interactions with enterocytes, focusing on redox state and
m potential. We show that the unquenching property of Rh-123 can be used to easily detect disruption of
m in living cells, in parallel with the monitoring of cell redox state using DCFH-DA. This inexpensive and straightforward technique allows the study of the relationship between ROS formation and
m variation and provides new insights into the cellular mechanisms responsible for the cytotoxic effects of a wide range of xenobiotics including toxic metals.
MATERIALS AND METHODS
Cell culture.
The TC7 clone, isolated from late passage of the Caco-2 cell line (Chantret et al., 1994), was kindly supplied by Dr. A. Zweinbaum (INSERM U178, Villejuif, France). Stock cultures were grown in 75-cm2 plastic flasks in Dubelccos modified Eagle essential (minimum) medium (DMEM) containing 25 mM glucose and supplemented with 15% inactivated FBS, 0.1 mM nonessential amino acids, and 50 units/ml penicillin + 50 µg/ml streptomycin. Cultures were maintained at 37°C in a humidified 5% CO2air atmosphere and were passaged by trypsinization (0.05% trypsin/0.53 mM EDTA) every fortnight. For all experiments, cells were seeded at a density of 12 x 103 cells/cm2, either in standard tissue culture dishes (35 x 10 mm) for flow cytometry measurements, or on 19-mm-diameter glass coverslips. prewashed with nitric acid, and allowed to grow for 5 days. Cells were used between passages 55 and 65.
Flow cytometry.
For DCF fluorescence determination (ex: 495,
em: 515), cells were incubated in nitrate medium: 137 mM NaNO3, 5.9 mM KNO3, 2.5 mM CaNO3, 1.2 mM MgSO4, 4 mM D-glucose and 10 mM HEPES, buffered to pH 7.4 with NaOH. This medium was chosen because of the much lower complexity of Cd by NO3- as compared with Cl-, allowing the relative percentage of total Cd recovered as the free metal ion Cd2+ to increase from 14 to 80% (Jumarie et al., 2001
). The Cd2+ species is expected to be responsible for the cytotoxic effect of Cd, and we have shown that Cd2+ is taken up 2.6 times faster compared with chlorocomplexes CdCln2-n (Jumarie et al., 2001
). In some experiments, 50 mM mannitol, used as a ROS scavenger (especially for hydroxyl radical OH) was added to the nitrate medium.
Cells were washed 3 times with nitrate Cd-free medium for 30 min prior to incubation with 2 µM DCFH-DA at 37°C (5% CO2air:atmosphere) in a dark environment, and Cd (final concentration 50 µM) was added to the exposure medium either at the beginning of the experiment (1-h exposure time) or after a 30-min preincubation period with the probe alone (30-min exposure time). Note that none of these treatments significantly affected cell viability as revealed by lactate dehydrogenase leakage measurements or the iodide propidium exclusion assay. Cells were then washed three times with the Cd-free nitrate buffer, trypsinized, centrifuged, and resuspended in the NO3- buffer. DCF fluorescence was measured on the FL1 channel of a flow cytometer (FACS Scan, Beckton-Dickinson, San Jose, CA). Analysis was done using the WinMDI (Windows Multiple Document Interface for Flow Cytometry) shareware (V. 2.8, Joseph Trotter, http://facs.scripps.edu).
For m monitoring, cells were first exposed to Cd prior to incubation with 100 nM CMXRos (
ex = 488,
em = 575) for 15 min. Cells were then washed, harvested, and processed as described above, and fluorescence was measured on the FL2 channel of the FACS Scan.
Fluorescence microscopy.
Variation in probe fluorescence was monitored using a TE-300 inverted microscope (Nikon Canada, Inc., Mississauga, ON) equipped for epifluorescence video imaging with a 12-bit digital cooled camera (CoolSnap-Fx, Roper Scientific, Trenton, NJ). Images were acquired at room temperature using a 60 X oil immersion objective (N.A. 1.4, Nikon), a 515-longpass emission cube, and a 460-nm excitation filter (Canberra Packard Canada, Concord, ON). Cells were washed as described above for flow cytometry measurements and were then incubated either with 5 µM DCFH-DA for 30 min or with 10 µM Rh-123 (ex = 460,
em = 520) for 30 min. Cells were then washed three times with probe-free nitrate buffer. To avoid the reported photo-reduction of DCFH leading to superoxide formation and excessive oxidation of DCFH to DCF (
ex = 495,
em = 520) (Marchesi et al., 1999
), we used minimal light exposure, keeping the exposure time and time lapse to 50 ms and 30 s, respectively, compared with 100 ms every 10 s for Rh-123.
Statistical analyses were performed with the Turkey-Kramer multiple comparisons test using InStat software (GraphPad Software). Statistical significance was assessed at the p < 0.05 level.
Chemicals.
All culture ware (Falcon) was obtained from VWR Scientific (Toronto, ON) whereas Dulbeccos Modified Eagle essential minimum medium (DMEM), penicillin, streptomycin, and trypsin were purchased from Gibco Laboratories (Grand Island, NY). Fetal bovine serum (FBS) was obtained from Immunocorp (Montréal, QC) and was inactivated at 52°C for 30 min. 2',7'-Dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine (CMXRos), and rhodamine-123 were purchased from Molecular Probes Corp. (Eugene, OR). Anhydrous dimethyl sulfoxide (DMSO), carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and CdCl2 were all purchased from Sigma Chemicals (St. Louis, MO).
RESULTS
Flow Cytometry Measurements
The effect of Cd on mitochondrial membrane permeability and cell redox state was first tested using flow cytometry. As shown in Figure 1A, high levels of DCFH oxidation to DCF were detected in DCFH-DA-treated cells unexposed to Cd, suggesting the presence of basal oxidative activities in control cells. This basal oxidative activity was lowered in the presence of 50 mM mannitol but increased almost 3-fold in the presence of H2O2. A 30- and 60-min incubation with 50 µM Cd increased the DCF fluorescence level. Interestingly, the presence of mannitol completely prevented Cd-induced DCFH oxidation following a 30-min incubation period, whereas it did not have any significant effect after a 60-min incubation. At 60 min, coexposure to Cd and mannitol led to a significantly higher DCF fluorescence as compared with the mannitol conditions. Cadmium was also found to affect
m: 10, and 20% reductions in CMXRos fluorescence were recorded following 30- and 60-min exposures to 50 µM Cd, respectively (Fig. 1B
). For both exposure times, the effect of Cd was unchanged by the presence of mannitol. CCCP, a well-known
m uncoupler, has been used to confirm the validity of CMXRos as a probe to assess
m.
|
|
Figure 3 depicts the relative variations in Rh-123 fluorescence under various experimental conditions. The possibility of an outward-directed diffusion of intracellular Rh-123 (a process that is dependent on
p) to the extracellular medium, after removal of the probe from the incubation medium, had to be verified. Indeed, under optimal experimental conditions, cell incubation with a concentration of a lipophilic cation such as Rh-123 would lead to a 10-fold accumulation of the probe in the cytoplasm and a 1,000- to 10,000-fold accumulation in mitochondria at equilibrium of distribution (Chen, 1989
). However, because our experimental conditions never reached distribution equilibrium, the diffusion parameter of the probe out of the cells must be estimated. In agreement with the reported low cell-membrane permeability for Rh-123, we found the Rh-123 diffusion rate to be fairly weak and constant in the Caco-2 cell model (Nicholls and Ward, 2000
). Figure 3A
illustrates the typical diffusion rate observed with Rh-123 in Caco-2 cells. We averaged the rate of fluorescence diminution from 3 independent experiments and subtracted it from all the subsequent measurements for both regions. As expected, the addition of 50 µM CCCP to the exposure nitrate medium led to a significant and very rapid redistribution in Rh-123. Indeed, within 10 s, a 40% transient increase in nuclear fluorescence was noted, whereas a parallel decrease of 20% was recorded for cytoplasmic fluorescence (Fig. 3B
). Thereafter, fluorescence in both regions reached the steady-state level.
|
DISCUSSION
In toxicology studies, cell viability can be assessed using a number of techniques: cell membrane integrity testing (iodide propidium or Trypan Blue exclusion, lactate dehydrogenase leaking) for necrosis and DNA fragmentation (Tunnel and Comet assays) for apoptosis. All these methods constitute end-point measurements, which do not provide information on the time course of the intoxication process. In that context, the development of techniques allowing real-time observation of the cellular response to xenobiotics, using living samples, is becoming of prime importance. In this study, we have measured the time course of Cd effect on two critical parameters for the cellular function, which may, upon disruption, lead to cell death: the cell redox state and m, the mitochondrial membrane potential.
Cadmium Effect on Cellular Redox State and m
Numerous studies have shown that Cd, even in the nanomolar concentration range, may affect cell functions. However, studies investigating the short-term (<12 h) effect of Cd on the cell redox state are generally conducted with Cd concentrations in the micromolar range without significant loss in cell viability (Al-Nasser, 2000; Pourahmad and OBrien, 2000; Szuster-Ciesielska et al., 2000; Thévenod et al., 2000). In our study, the use of 50 µM Cd was required to see significant variations in probe fluorescence. Such a requirement, and the fact that exposure to micromolar levels of Cd for "short" periods of time does not lead to significant cell necrosis, is consistent with the general high intracellular-binding capacity of the cell. However, variation in sensitivity to Cd-induced necrosis or apoptosis among cell types has also been reported (Szuster-Ciesielska et al., 2000).
Cadmium has been shown to affect both cell redox state and m in Caco-2 cells (Cable et al., 1993
). Using flow cytometry, we were able to detect an increase in DCFH oxidation to the fluorescent product DCF as early as 30 min following exposure to 50 µM Cd (Fig. 1A
). Because of the excessive photo-oxidation of DCFH to DCF, we had to limit the number of data acquisitions, i.e., the amount of exposure to light, in order to study the effect of Cd over a longer period of exposure. Under these conditions, DCF fluorescence proved to be a good tool for evaluating the oxidative activities of Caco-2 cells.
In parallel with the increase in DCFH oxidation, Cd was found to lower m (Fig. 1B
). To gain insights into the possible direct relationship between ROS formation and
m disturbance, we used mannitol, a well-known scavenger of the OH radical, to test whether it could protect against one or both of the observed Cd-induced effects. Mannitol indeed prevented Cd-induced DCFH oxidation for the 30-min incubation period. Considering that DCFH detects a broad range of oxidizing reactions, this result first shows that, for the 30-min period of exposure, OH radicals are mainly responsible for the increase in DCFH oxidation; i.e., Cd-induced ROS would mainly be the OH radical. The fact that mannitol failed to completely eliminate DCFH oxidation during the 60-min period suggests that the capacity of mannitol may be insufficient or that ROS, other than the OH radical, started to be generated. The main point is that mannitol did not prevent
m disruption for both exposure times. Whether the level of ROS has an impact on
m needs further investigation, but at this point, the results do not support the hypothesis of OH formation as a prerequisite to
m disruption.
The Use of Rh-123 Fluorescence Unquenching to Detect Dissipation of m
To gain insights into the initial modification in the redox cell state and m, we used the fluorescence-microscopy technique, which allows the observation of very rapid events. Fluorescence video imaging was used instead of confocal microscopy to optimize light sensitivity and quantification range over image resolution, which is less critical for these types of experiments. Rhodamine-123 was used because of its peculiar fluorescence property to self-quench at high probe concentrations (Nicholls and Ward, 2000
), a property we used successfully to detect early
m dissipation. Indeed, as
m is lowered, the Rh-123 probe is no longer compartmentalized in the mitochondria, and it diffuses to reach a new subcellular distribution according to the new electrochemical gradients. Self-quenching is then reduced, resulting in a general increase in fluorescence. While others have used this technique with cortical neurons (Sensi et al., 2002
), the novelty of our approach resides in its capacity to discriminate variations of Rh-123 fluorescence between the cytoplasmic and nuclear regions of the cells. Monitoring increases in nuclear fluorescence allows a better estimation of Rh-123 unquenching in addition to its outward diffusion. This latter phenomenon may represent the main disadvantage of this new approach. However, it can be quantified and subtracted from the total fluorescence signal. The present experiments were carried out over a relatively short period of time (20 min) because of the decrease in the signal-to-noise ratio as a function of time (background fluorescence augmentation) and of Rh-123 photo-induced cytotoxicity (Nicholls and Ward, 2000
).
Relationship between Cd-Induced m Dissipation and ROS Formation
Using Rh-123 fluorescence unquenching, we were able to further investigate the time-course of Cd-induced m disruption in relation to the cellular redox state. Our results, obtained with cells coincubated with Cd and mannitol, show that the Cd-induced dissipation of
m was delayed in the presence of mannitol but became apparent after 10 min (Fig. 3D
); at that time, mannitol did protect the cells from oxidative stress (Fig. 1A
). Also, it is noteworthy that Cd increased DCFH oxidation over the control values only after 10 min (Fig. 2A
), whereas it induced redistribution of Rh-123 as early as 3 min (Fig. 3C
). Therefore, Cd decreases
m faster than it generates ROS, and it can affect
m under conditions of efficient ROS scavenging. Similar results have been obtained by others: antioxidants protected from Cd-induced lipid peroxidation were inefficient in preventing the observed increase in mitochondrial membrane permeability (Li et al. (2000
); Strubelt et al., 1996
), and have reported that Cd may induce loss of
m without any evidence of ROS formation. Now, how a ROS scavenger could protect only transiently against
m disruption needs to be further studied, but our results suggest that Cd may dissipate
m in the absence of a detectable level of OH radical and in the absence of apparent oxidative stress.
Cadmium is suspected of acting on the redox state of the cell and on m through different mechanisms. Cadmium is well known to deplete cellular sulfhydryl groups, which induces GSH, HSP, and MT syntheses in various cell models (Gaubin et al., 2000
; Klaassen et al., 1999
; Shukla et al., 2000). Furthermore, it has been demonstrated that de novo synthesis of these peptides and proteins does protect the cell from Cd toxicity, especially from oxidative stress (Casalino et al., 2000
; Frank et al., 2000
). It is generally accepted that Cd-induced cellular thiol depletion may cause imbalance between pro-oxidant and antioxidant systems leading to oxidative stress. In turn, the oxidative stress affects a number of cellular functions, regulation or signal transduction mechanisms: DNA synthesis, cell-cycle regulation, activation of transcription factors, mitochondrial permeability transition (MPT), and apoptosis (Rikans and Yamano, 2000
). A different mechanism of toxicity targeting
m would not be directly linked to the oxidative stress. Numerous studies have suggested that Cd may act directly on the mitochondria. Studies on the subcellular distribution of Cd in rat hepatocytes have revealed that, in mitochondria, Cd preferentially binds to the inner membrane (Muller, 1986
). It has been shown that Cd may affect mitochondrial inner-membrane permeability to H+ (Palmeira et al., 1994
) and K+ (Rasheed et al., 1984
), and inhibit enzymes involved in the electron transport chain as well as the succinate dehydrogenase complex (Miccadei and Floridi, 1993
). It has also been suggested that Cd may induce apoptosis through a loss of
m, cleavage of Bid and Bcl-XL, and an increase in Bcl-XS, leading to cytochrome c release and the subsequent activation of the effectors caspases (Li et al., 2000
). Note that ROS formation as a prerequisite to
m dissipation cannot be excluded since Cd may affect, in some way, the mitochondria redox state. It has been suggested that mitochondrial ROS, independently of cytosolic ROS, could be responsible for the Cd-induced
m collapse (Pourahmad and OBrien, 2000
).
This work clearly shows that Rh-123 fluorescence unquenching, occurring during probe redistribution, may be used to assess the decrease in m. We found that Cd rapidly increases oxidative activity in the Caco-2 cells and that the OH radical accounts for most of the ROS generated, at least for the first 30 min. Cadmium also decreases
m even faster than it modifies the cell redox state and in the absence of detectable ROS. We conclude that Cd-induced
m dissipation does not necessarily require oxidative injury. The possibility that
m dissipation occurs as a result of mitochondrial redox state perturbation cannot be excluded, but cytosolic oxidation would not be the first step.
ACKNOWLEDGMENTS
This research was supported by the Natural Sciences and Engineering Research Council of Canada, NSERC (C.J: grant 203202-02), as well as the Canadian Network of Toxicology Centres (C.J., and F.D.). The authors thank Denis Flipo (TOXEN, département des sciences biologiques, UQAM) and Dr. Jean-Louis Schwartz (GEPROM, Département de physiologie, Université de Montréal) for discussion and advice.
NOTES
1 To whom correspondence should be addressed at Dépt. des Sciences biologiques, Université du Québec à Montréal, C.P. 8888, Succ. centre-ville, Montréal (Qué.) Canada H3C 3P8. Fax: (514) 987-4647. E-mail: jumarie.catherine{at}uqam.ca.
REFERENCES
Barchi, D., Joshi, S. S., Bagchi, M., Balmoori, J., Benner, E. J., Kuszynski, C. A., and Stohs, S. J. (2000). Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. J. Biochem. Mol. Toxicol. 14, 3341.[CrossRef][ISI][Medline]
Beyersmann, D., and Hechtenberg, S. (1997). Cadmium, gene regulation, and cellular signaling in mammalian cells. Toxicol. Appl. Pharmacol. 144, 247261.[CrossRef][ISI][Medline]
Cable, J. W., Cable, E. E., and Bonkovsky, H. L. (1993). Induction of heme oxygenase in intestinal epithelial cells: Studies in Caco-2 cell cultures. Molec. Cell. Biochem. 129, 9398.[ISI][Medline]
Casalino, E., Calzaretti, G., Sblano, C., and Landriscina, C. (2000). Cadmium-dependent enzyme activity alteration is not imputable to lipid peroxidation. Arch. Biochem. Biophys. 383, 288295.[CrossRef][ISI][Medline]
Chantret, I., Rodolosse, A., Barbat, A., Dussaulx, E., Brot-Laroche, E., Zweibaum, A., and Rousset, M. (1994). Differential expression of sucrase-isomaltase in clones from early and late passages of the cell line Caco-2: Evidence for glucose-dependent negative regulation. J. Cell Sci. 107, 213225.
Chen, L. B. (1989). Fluorescent labeling of mitochondria. Methods Cell Biol. 29, 103123.[ISI][Medline]
Crow, J. P. (1997). Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: Implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1, 145157.[CrossRef][ISI][Medline]
Elisma, F., Jumarie, C. (2001). Evidence for cadmium uptake through NRAMP2: metal speciation studies with Caco-2 cells. Biochem. Biophys. Res. Commun. 285, 662668.[CrossRef][ISI][Medline]
Ferrari, R., Agnoletti, L., Comini, L., Gaia, G., Bachetti, T., Cargnoni, A., Ceconi, C., Curello, S., and Visioli, O. (1998). Oxidative stress during myocardial ischaemia and heart failure. Eur. Heart J. 19, B211.[ISI][Medline]
Frank, J., Pompella, A., and Biesalski, H. K. (2000). Histochemical visualization of oxidant stress. Free Radic. Biol. Med. 29, 109610105.[CrossRef][ISI][Medline]
Gabriel, C., Camins, A., Sureda, F. X., Aquirre, L., Escubedo, E., Pallas, M., and Camarasa, J. (1997). Determination of nitric oxide generation in mammalian neurons using dichlorofluorescein diacetate and flow cytometry. J. Pharmacol. Toxicol. Methods 38, 9398.[CrossRef][ISI][Medline]
Galan, A., Garcia-Bermejo, L., Troyano, A., Vilaboa, N. E., Fernandez, C., de Blas, E., and Aller, P. (2001). The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-ray). Eur. J. Cell. Biol. 80, 312320.[ISI][Medline]
Gaubin, Y., Vaissade, F., Croute, F., Beau, B., Soleilhavoup, J.-P., and Murat, J.-C. (2000). Implication of free radicals and glutathione in the mechanism of cadmium-induced expression of stress proteins in the A549 human lung-cell line. Biochim. Biophys. Acta 1495, 413.[CrossRef][ISI][Medline]
Hart, B. A., Lee, C. H., Shukla, A., Osier, M., Eneman, J. D., and Chiu, J. F. (1999). Characterization of cadmium-induced apoptosis in rat lung epithelial cells: Evidence for the participation of oxidant stress. Toxicology 133, 4358.[CrossRef][ISI][Medline]
Hempel, S. L., Buettner, G. R., OMalley, Y. Q., Wessels, D. A., and Flaherty, D. M. (1999). Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: Comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic. Biol. Med. 27, 146159.[CrossRef][ISI][Medline]
Ishido, M., Ohtsubo, R., Adachi, T., and Kunimoto, M. (2002). Attenuation of both apoptotic and necrotic actions of cadmium by Bcl-2. Environ. Health Perspect. 110, 3742.[ISI][Medline]
Jumarie, C. M., Campbell, P. G. C., and Denizeau, F. (1999). Evidence for an intracellular barrier to cadmium transport through Caco-2 cell monolayers. J. Cell. Physiol. 180, 285297.[CrossRef][ISI][Medline]
Jumarie, C., Fortin, C., Houde, M., Campbell, P. G. C., and Denizeau, F. (2001). Cadmium uptake by Caco-2 cells: Effects of Cd complexness by chloride, glutathione, and phytochelatins. Toxicol. Appl. Pharmacol. 170, 2938.[CrossRef][ISI][Medline]
Klaassen, C. D., Liu, J., and Choudhuri, S. (1999). Metallothionein: An intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39, 267294.[CrossRef][ISI][Medline]
Li, M., Kondo, T., Zhao, Q. L., Li, F. J., Tanabe, K., Arai, Y., Zhou, Z. C., and Kasuya, M. (2000). Apoptosis induced by cadmium in human lymphoma U937 cells through Ca2+ -calpain- and caspase mitochondria-dependent pathways. J. Biol. Chem. 275, 3970239709.
Marchesi, E., Rota, C., Fann, Y. C., Chignell, C. F., and Mason, R. P. (1999). Photo-reduction of the fluorescent dye 2'-7'-dichlorofluorescein: A spin trapping and direct electron spin-resonance study with implications for oxidative stress measurements, Free Radic. Biol. Med. 26, 148161.[CrossRef][ISI][Medline]
Metivier, D., Dallaporta, B., Zamzami, N., Larochette, N., Susin, S. A., Marzo, I., and Kroemer, G. (1998). Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. Immunol. Lett. 61, 157163.[CrossRef][ISI][Medline]
Miccadei, S., and Floridi, A. (1993). Sites of inhibition of mitochondrial electron transport by cadmium. Chem. Biol. Interact. 242, 11151122.
Muller, L. (1986). Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40, 285295.[CrossRef][ISI][Medline]
Nicholls, D. G., and Ward, M. W. (2000). Mitochondrial membrane potential and neuronal glutamate excitotoxicity: Mortality and minivolts. Trends Neurosci. 23, 166174.[CrossRef][ISI][Medline]
Noda, M., Yasuda, M., and Kitagawa, M. (1991). Iron as a possible aggravating factor for osteopathy in Itai-Itai disease, a disease associated with chronic cadmium intoxication. J. Bone Miner. Res. 6, 245255.[ISI][Medline]
Palmeira, C. M., Moreno, A. J., and Madeira, V. M. C. (1994). Interactions of herbicides 2,4-D and Dinoseb with liver mitochondrial bioenergetics. Toxicol. Appl. Pharmacol. 127, 5057.[CrossRef][ISI][Medline]
Pinot, F., Kreps, S. E., Bachelet, M., Hainaut, P., Bakonyi, M., and Polla, B. S. (2000). Cadmium in the environment: Sources, mechanisms of biotoxicity, and biomarkers. Rev. Environ. Health 15, 299323.[Medline]
Pourahmad, J., and OBrien, P. J. (2000). A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology 143, 263273.[CrossRef][ISI][Medline]
Rasheed, B. K. A., Diwan, J. J., and Sanadi, D. R. (1984). Activation of potassium transport in mitochondria by cadmium ion. Eur. J. Biochem. 144, 643647.[Abstract]
Rikans, L. E., and Yamano, T. (2000). Mechanisms of cadmium-mediated acute hepatotoxicity. J. Biochem. Mol. Toxicol. 14, 110117.[CrossRef][ISI][Medline]
Rottenberg, H., and Wu, S. (1998). Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1404, 393404.[CrossRef][ISI][Medline]
Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. (1997). JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 7782.[CrossRef][ISI][Medline]
Sarvazyan, N. (1996). Visualization of doxorubicin-induced oxidative stress in isolated cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol. 271, H20792085.
Sensi, S. L., Ton-That, D., and Weiss, J. H. (2002). Mitochondrial sequestration and Ca2+- dependent release of cytosolic Zn2+ loads in the cortical neurone. Neurobiol. Dis. 10, 100108.[CrossRef][ISI][Medline]
Shukla, G. S., Shukla, A., Potts, R. J., Osier, M., Hart, B. A., and Chiu, J.-F. (2002). Cadmium-mediated oxidative stress in alveolar epithelial cells induces the expression of -glutamylcysteine synthetase catalytic subunit and glutathione S-transferase
and ß isoforms: Potential role of activator protein-1. Cell Biol. Toxicol. 16, 347362.[CrossRef][ISI]
Stohs, A. J., and Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Rad. Biol. Med. 18, 321336.[CrossRef][ISI][Medline]
Strubelt, O., Kremer, J., Tisle, A., Keogh, J., Pentz, R., and Younes, M. (1996). Comparative studies on the toxicity of mercury, cadmium, and copper toward the isolated perfused rat liver. J. Toxicol. Environ. Health 47, 267283.[CrossRef][ISI][Medline]
Swift, L. M., and Sarvazyan, N. (2000). Localization of dichlorofluorescein in cardiac myocytes: Implications for assessment of oxidative stress. Am. J. Physiol. Heart Circ. Physiol. 278, H982990
Yang, C. F., Shen, H. M., Shen, Y., Zhuang, Z. X., and Ong, C. N. (1997). Cadmium-induced oxidative cellular damage in human fetal lung fibroblasts. Environ. Health Perspect. 105, 712716.[ISI][Medline]
Zamzami, N., Maisse, C., Métivier, D., and Kroemer, G. (2001). Measurement of membrane permeability and permeability transition of mitochondria. Methods Cell Biol. 65, 147158.[ISI][Medline]
|