Induction of Apoptosis in Cells by Cadmium: Quantitative Negative Correlation between Basal or Induced Metallothionein Concentration and Apoptotic Rate

Ryuya Shimoda*,{dagger}, Takeaki Nagamine{ddagger}, Hitoshi Takagi*, Masatomo Mori* and Michael P. Waalkes{dagger},1

* First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan; {dagger} National Cancer Institute at the National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709; and {ddagger} Department of Health Science, Gunma University School of Medicine, Maebashi 371-8511, Japan

Received June 13, 2001; accepted September 12, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metallothionein (MT) often reduces the adverse effects of cadmium (Cd), but how it may alter Cd-induced apoptosis is unclear. The goal of this study was to define the role of MT in Cd-induced apoptosis using cell lines with widely varying sensitivity to Cd. Effects of Cd on growth of human hepatocellular carcinoma cell lines (HepG2 and PLC/PRF/5) were investigated and compared with Chang cells. These cells were cultured with 0, 5, 10, 20, 40, 80, and 120 µM of Cd for 3, 6, 12, and 24 h. Significant cytolethality was observed in HepG2 and PLC/PRF/5 cells in a time- and concentration-dependent manner, with LC50 values of 24 µM and 13 µM, respectively. However, Chang cells were much less sensitive to Cd-induced cytotoxicity (LC50, 64 µM). Apoptotic cell death occurring at cytolethal concentrations was demonstrated in all cell lines by DNA fragmentation on agarose gel electrophoresis or by ELISA. When MT was measured, there was a highly significant negative linear correlation between the basal cellular MT concentration or Cd-induced MT and the rate of apoptosis induced by Cd in these cell lines. Treating HepG2 cells with zinc (Zn) made the relatively sensitive HepG2 cell line resistant to Cd-induced apoptosis, likely due to Zn-induced MT. In fact, there was also a significant negative linear correlation between the amount of Zn-induced MT in HepG2 cells and the rate of Cd-induced apoptosis. These findings revealed that basal or induced MT perturbs Cd-induced apoptotic cell death in various cell lines, and a strong negative correlation exists between cellular MT content and the rate of apoptosis induced by Cd.

Key Words: cadmium; human; hepatoma; apoptosis; metallothionein; zinc; in vitro.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadmium (Cd), a toxic heavy metal, has hepatotoxic and nephrotoxic effects in human and rodents (Goering et al., 1994Go). Cd exposure is etiologically implicated in various other pathologic conditions, including testicular damage, hypertension, atherosclerosis, osteoporosis, anemia, and cancer (Goering et al., 1994Go; Waalkes et al., 1999aGo). In fact, Cd is a known human carcinogen, and there is a clear association between occupational Cd exposure and lung cancer in humans (IARC, 1993Go). Other organs, such as prostate, liver, kidney, stomach, and pancreas, are considered to be potential target sites of Cd carcinogenesis, although they are not as yet definitely established (IARC, 1993Go; Schwartz and Reis, 2000Go). The mode of action of Cd as a carcinogen is still not fully understood.

Recently, evidence has emerged that the process of carcinogenesis is often associated with alterations in apoptosis. In this regard, Cd induces apoptosis under some conditions (Achanzar et al., 2000Go; Habeebu et al., 1998Go; Hart et al., 1999Go) while it inhibits apoptosis in other cases (Shimada et al., 1998Go; von Zglinicki et al., 1992Go; Yuan et al., 2000Go). For instance, Cd inhibits apoptosis induced by chromium, actinomycin D, and hygromycin B (Shimada et al., 1998Go; Yuan et al., 2000Go). Cd acts as an effective inhibitor of caspase-3 (Yuan et al., 2000Go), a proapoptotic enzyme critical to dedication of cells to apoptosis (LaCasse et al., 1998Go). Likewise, zinc (Zn) can be highly effective as a caspase-3 inhibitor (Perry et al., 1997Go). In fact, it is suspected that free Zn may be a critical negative controlling factor for apoptosis (Chai et al., 1999Go). Inhibition of apoptosis may allow a greater portion of genetically damaged cells to survive and escape normal cell population control mechanisms to go on to form tumors. On the other hand, Cd is also an effective inducer of apoptosis in vitro in a variety of human or rodent cell lines (Achanzar et al., 2000Go; Hart et al., 1999Go) and there is evidence of Cd-induced apoptosis in certain tissues with in vivo exposure (Habeebu et al., 1998Go; Yan et al., 1997Go). So it appears Cd can be both pro- and antiapoptotic, depending on the conditions. Likewise Zn, although often antiapoptotic, can also induce apoptosis (Hamatake et al., 2000Go; Liang et al., 1999Go; Souza et al., 1999Go). For instance, Zn can induce apoptosis in human thyroid cell lines (Iitaka et al., 2001Go), colorectal carcinoma cells (Souza et al., 1999Go), Molt-4 cells (Hamatake et al., 2000Go), and in human prostate cells (Liang et al., 1999Go). So it is clear that both Cd and Zn can induce, as well as inhibit, apoptosis depending on the circumstances.

The metal-binding protein metallothionein (MT) is one important factor well known to alter Cd toxicity (Habeebu et al., 2000Go; Liu et al., 1995Go, 2000Go; Waalkes et al., 1999bGo). In this regard, it is thought that MT typically mitigates Cd toxicity by sequestration of the metal, thereby rendering it toxicologically inert (Waalkes and Goering, 1990Go). However, it is clear that, under most conditions, MT is a Zn-containing protein and that Cd binds to MT by the displacement of Zn (Waalkes, 1996Go). In this regard there is evidence that Cd may inhibit apoptosis through displacement of cellular stores of Zn (Yuan et al., 2000Go). It is also quite clear that MT-knockout animals show enhanced rates of apoptosis when exposed to Cd (Habeebu et al., 2000Go), and various studies have found MT to be generally antiapoptotic (Levadoux-Martin et al., 2001Go; Wang et al., 2001Go). On the other hand, exposure to the Cd-MT complex can, itself, induce apoptosis under some circumstances (Hamada et al., 1996Go; Ishido et al., 1998Go). High levels of MT can also be associated with stimulation of apoptosis under some conditions (Liu et al., 2001Go; Deng et al., 1998Go) and apoptotic rate increases with increasing tumor immunoreactivity for MT in some tumors (Zhang and Takenaka, 1998Go). Thus, MT appears to similarly have both pro- and antiapoptotic potential.

It is not clear how cellular MT levels would affect apoptosis induced by Cd, and there are several possible scenarios, including: (1) suppression of Cd-induced apoptosis by MT sequestration of Cd; (2) Cd displacement of Zn from MT, which in turn, stimulates or suppresses apoptosis; or (3) Cd-MT stimulation of apoptosis. We hypothesize that the presence of cellular MT will reduce Cd-induced apoptosis. Therefore, to test this hypothesis, the relationship between the amount of cellular MT and the toxic and apoptotic effects of Cd was investigated in 3 cell lines of varying relative endogenous MT levels, including Chang cells (high), HepG2 cells (intermediate) and PLC/PRF/5 cells (low). Effects of MT induction by Zn prior to Cd exposure were also studied.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Cadmium chloride (CdCl2) was purchased from Wako Chemicals Co. (Tokyo). MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was obtained from SIGMA (St. Louis, MO).

Cell lines and culture conditions.
Two human hepatoma cell lines (PLC/PRF/5 and HepG2) and the Chang cell line (likely a Hela cell-derived line) were used. Chang cells, PLC/PRF/5 cells and HepG2 cells were purchased from ATCC (Manassas, VA). These cell lines were selected because in preliminary testing they showed wide variability of sensitivity to Cd and corresponding differences in basal MT levels that showed a 4-fold range. Cells were maintained in a 5% CO2 atmosphere, and cultured as monolayer in Dulbecco's modified Eagle's medium (DMEM), GIBCO-Life Technologies (Gaithersburg, MD), supplemented with 10% fetal bovine serum, 31 U/ml penicillin G and 50 µg/ml streptomycin.

Measurement of cytolethality.
The effect of Cd on cell viability was assessed by an MTT assay, which measures viable cells by assessing metabolic integrity (Mosmann, 1983Go). Cells were seeded in 96-well microtiter plates (Nunc, Naperville, IL) at a cell density of 2 x 104 cells/well in unaltered media. After 24 h, the medium was replaced with media containing various concentration of Cd, (0, 5, 10, 20, 40, 80, and 120 µM). Cells were exposed to Cd for designated periods of time (3, 6, 12, and 24 h). Cell survival rates were then assayed.

MT quantitation.
MT concentrations were estimated by the Cd-hemoglobin radioassay method as described by Eaton and Toal (1982). MT was measured in untreated cells or in cells treated with Cd or Zn, at the designated concentrations, for 24 h prior to evaluation. Endogenous Zn and Cd content of MT was determined by using this assay in the absence of exogenously added Cd, thus allowing direct measurement of the Cd or Zn compliment in MT by using atomic absorption spectrometry.

Assessment of apoptosis by agarose gel electrophoresis.
Cells (4 x 106) were incubated with 0, 10, or 40 µM of Cd for 16 h. Attached and unattached cells were harvested and DNA was extracted with 0.5% triton-X100 cell lysis buffer. Small molecular weight DNA was obtained in the supernatant after centrifugation. DNA was electrophoresed on 2% agarose gel to detect the laddering characteristic of apoptotic DNA degradation.

Quantitation of apoptosis by DNA fragmentation detection assay (ELISA).
Quantitative determination of cytoplasmic histone-DNA fragments indicative of apoptosis was performed by enzyme-linked immunosorbent assay (ELISA) using the Cell Death Detection ELISA kit (Roche, Indianapolis, IN). The assay is based on a quantitative sandwich-enzyme-immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, respectively, which allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Cells were seeded in a 96-well plate (1 x 104/well) and treated with designated concentrations of Cd for 12 to 16 h. Floating cells were first pelleted by centrifugation, and then both adherent and floating cells were lysed and incubated with mouse monoclonal antihistone-biotin antibody and mouse monoclonal anti-DNA-peroxidase in streptavidin-coated microtiter plates. Unbound antibodies were washed out. The amount of nucleosome was determined quantitatively by evaluating peroxidase activity photometrically with 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) as substrate.

Statistics.
Data are given as the mean ± SEM unless otherwise noted. Results were analyzed by Student's t-test or Dunnett's t-test after ANOVA as appropriate. Correlation between apoptotic rates and basal or induced-MT concentrations was tested by calculating Pearson's correlation coefficient, r. The level of significance was set at p < 0.05 in all cases.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytolethal effects of Cd on the various cell lines are shown in Figures 1 and 2GoGo. Cd was clearly cytolethal in PLC/PRF/5 and HepG2 (Fig. 1Go). Cd-induced cytotoxicity in PLC/PRF/5 and HepG2 cells was both dose- and time-dependent. The Cd concentrations required for 50% cell death (lethal concentration 50%; LC50) were 13 µM for PLC/PRF/5 cells and 25 µM for HepG2 cells (Fig. 2Go). Incubation with 40 µM Cd for 24 h caused about 77.8% and 64.1% cell death for PLC/PRF/5 and HepG2 cells, respectively. On the other hand, Cd did not alter cell survival in Chang cells, even when treated with as high a concentration as 40 µM Cd. In fact, the LC50 for Cd in Chang cells was 64 µM. Morphological study confirmed that the hepatoma cells were much more sensitive to Cd than the Chang cells (not shown).



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FIG. 1. Cytolethality of human hepatoma cells induced by Cd. PLC/PRF/5 cells (A) and HepG2 cells (B) were treated with 5–120 µM of Cd for 3–24 h and cell survival rates were estimated by metabolic integrity. Values are the means ± SEM of 6 experimental samples of each group.

 


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FIG. 2. Differential sensitivity to Cd-induced cytolethality among Chang, PLC/PRF/5 and HepG2. Cells were cultured with 5–120 µM of Cd for 24 h and cell survival was estimated by metabolic integrity. Values are the means ± SEM of 6 samples for each group. Cytolethal effects did not occur in Chang cells even when treated with 40 µM of Cd, a concentration that killed more than 60% of the other cells.

 
The mechanism of cell death induced by Cd treatment in the various cell lines was investigated. Analysis of PLC/PRF/5 cells by DNA laddering on agarose gel electrophoresis (Fig. 3AGo) and the DNA fragmentation ELISA assay (Fig. 3BGo) demonstrated that apoptotic cell death was the primary form of cell elimination after Cd exposure. Similar results were observed for Cd-treatment and apoptosis in HepG2 cells and Chang cells, although Chang cells required significantly higher concentrations for Cd-induced apoptosis (data not shown).



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FIG. 3. Cd-induced apoptosis. Apoptosis was demonstrated by DNA laddering after agarose gel electrophoresis (A) and by DNA fragmentation as evaluated by ELISA (B). For DNA ladder formation, cells were incubated with 0, 10, or 40 µM Cd for 16 h and DNA was isolated and electrophoresed on 2% agarose gel. For DNA fragmentation by ELISA, cells were treated with 5–80 µM Cd for 14 h. Results shown are for PLC/PRF/5 cells. Similar results were observed for Cd treatment and apoptosis in HepG2 cells and Chang cells, although Chang cells required significantly higher concentrations for Cd-induced apoptosis (see Fig. 4Go).

 
Apoptotic rate was determined by ELISA after treatment with Cd (50 µM; 24 h) in the 3 cell lines (Fig. 4Go). Relative apoptotic rate in comparison to appropriate untreated control were 452, 324, and 97.4% in PLC/PRF/5, HepG2, and Chang cells, respectively.



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FIG. 4. Cd-induced apoptosis in PLC/PRF/5, HepG2, and Chang cells. Cells were incubated with 50 µM Cd for 16 h and apoptosis was determined by DNA fragmentation using the ELISA method. Apoptotic rates were increased 4.5- and 3.2-fold in PLC/PRF/5 and HepG2, respectively, in comparison with untreated control cells, while rates in Chang cells remained essentially unchanged. Values represent the mean ± SEM of 6 samples and are given as % relative to PLC/PRF/5 control rate as 100%.

 
To help define the role of MT in Cd-induced apoptosis, the amount of basal and Cd-induced (5 µM for 24 h) MT was determined in these cell lines (Fig. 5Go). There was a significant difference in both endogenous and induced levels of MT among the various cell lines. Both basal and Cd-induced MT concentrations were lowest in PLC/PRF/5 and highest in Chang cells. MT content was increased by Cd exposure in PLC/PRF/5, HepG2, and Chang cells by 6.8-, 4.2-, and 3.6-fold, respectively.



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FIG. 5. Basal and Cd-induced MT levels in cells. Chang, HepG2, and PLC/PRF/5 cells were treated with 5 µM of Cd for 24 h. MT was significantly increased relative to basal levels in each cell line after Cd treatment. There was also a significant difference among the 3 cell lines in both basal and Cd-induced MT levels. Superscript letters indicate significant differences. Values were expressed as means ± SEM of 7 samples in nmol MT/106 cells.

 
The correlation between the basal level of cellular MT and apoptotic rate upon Cd exposure was then evaluated (Fig. 6Go). This relationship was analyzed using the data on Cd-induced apoptotic rate together with basal MT level. We observed a strong negative correlation (r = –0.996) between basal MT concentration and the Cd-induced apoptotic rate.



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FIG. 6. Correlation between basal MT and Cd-induced apoptotic rates in the various cell lines. Cells were treated with 50 µM Cd for 16 h. A significant (r = –0.996; p < 0.0001) negative linear correlation occurred.

 
The relationship between the ability of the various cell lines to synthesize MT in response to Cd exposure and Cd-induced apoptotic rates was also analyzed (Fig. 7Go). As with basal MT, the ability to produce MT after Cd treatment showed a strong negative correlation (r = –0.975) with apoptosis after Cd exposure.



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FIG. 7. Correlation between ability to produce MT in response to Cd exposure and Cd-induced apoptosis in the various cell lines. A significant (r = –0.975; p < 0.001) negative, linear correlation occurred.

 
To determine if this negative correlation between cellular MT and susceptibility to Cd-induced apoptosis held, even after MT levels were markedly increased, HepG2 cells were pretreated with Zn at various concentrations and Cd-induced apoptosis was determined. There was a clear concentration-related increase in MT content, depending on the level of Zn exposure (Fig. 8AGo). Additionally, Zn-pretreatment protected HepG2 cells from Cd-induced apoptosis in a concentration-dependent manner (Fig. 8BGo). For instance, Zn pretreatment (100 µM; 24 h) increased cellular MT content up to 293% and decreased Cd-induced apoptotic cell death by 47% when compared with un-pretreated cells. A strong negative association (r = –0.964) between induced cellular MT content and Cd-induced apoptosis occurred in HepG2 cells after zinc induced stimulation of MT production (Fig. 9Go).



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FIG. 8. Effect of Zn pretreatment on MT levels in HepG2 cells (A) and inhibition of Cd-induced apoptosis after Zn pretreatment (B). Cells were pretreated with Zn (6.25–100 µM) for 24 h and then Cd (80 µM) for 16 h. Apoptosis was determined by ELISA and rates are based on untreated controls (no Zn or Cd) as 100%. Values are expressed as means ± SEM of 3 to 6 samples and an asterisk (*) indicates a significant difference from appropriate control.

 


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FIG. 9. Correlation between Zn-induced MT content and Cd-induced apoptosis in HepG2 cells. Data from Figures 8A and 8BGo were plotted together for regression analysis. There was a significant (r = -0.964, p < 0.00001) negative linear correlation between the induced amount of MT and the apoptotic rate.

 
To determine if Cd exposure resulted in displacement of MT-bound zinc, HepG2 cells were treated with Zn (100 µM for 24 h) and then briefly exposed to Cd (80 µM for 90 min). Measurement of the Zn content of MT showed that it was reduced by 68.6 ± 1.44 % after the Cd exposure.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of the present study show that apoptotic cell death occurs after Cd exposure in various cells, including human hepatocellular carcinoma cell lines and Chang cells. In addition, cellular susceptibility to Cd-induced apoptosis is clearly dependent on endogenous or induced MT levels. Indeed, strong negative correlations between endogenous, Cd-induced or Zn-induced cellular MT content and apoptosis induced by Cd treatment occurred. Thus, MT is protective against this biological effect of Cd as is the case with so many other aspects of Cd toxicity (Goering et al., 1994Go; Waalkes, 2000Go; Waalkes et al., 1999bGo). In this regard, MT is a cysteine rich, metal-binding protein and protects cells from cytotoxic heavy metals (Liu et al, 1995Go, 2000Go), such as Cd, by sequestration (Waalkes, 1990, 1996Go). Basal level of MT expression often dictates the sensitivity of a given cell or tissue to Cd toxicity (Liu et al., 1995Go, 2000Go; Waalkes et al., 1988Go) and increased MT gene expression induces resistance to Cd cytotoxicity (Waalkes et al., 1988Go). Transgenic mice that overexpress MT are protected from Cd lethality and hepatotoxocity (Liu et al., 1995Go). In the present study, the data confirm that this relationship holds true for Cd-induced apoptosis as basal levels of MT dictated sensitivity to apoptosis resulting from Cd exposure. Furthermore, at the point of initial Cd exposure, increasing the amount of MT by Zn pretreatment clearly mitigated Cd-induced apoptosis. Likewise, the capacity of a given cell line to induce MT in response to Cd also dictated sensitivity. Thus, relative sensitivity to apoptosis induced by Cd exposure appears to be determined by the amount of cellular MT at the time of exposure to the metal or by the ability of the cell to produce MT in response to Cd.

The sequestration of Cd by MT usually occurs through the displacement of bound Zn (Waalkes, 1996Go), and this was observed in the present study, at least in the HepG2 cells. Zn can be proapoptotic in many cases (Hamatake et al., 2000Go; Liang et al., 1999Go; Souza et al., 1999Go). Thus, MT sequestration of Cd and consequential Zn release might enhance apoptosis through Zn-mediated pathways. In fact, Liu et al. (2001) have shown that metal released from MT can go on to induce apoptosis, using copper-MT and a nitric oxide producing agent to release the MT bound metal, clearly showing the plausibility of such a scenario with Zn. However, the present results indicate this is probably not the case with released Zn, as the Zn released from MT during Cd sequestration did not appear to enhance apoptosis, at least not in the 3 cell lines used in this study. The role that Zn released from MT might play in stimulating apoptosis in other cell lines remains to be determined, but in the present series of experiments, release of Zn from this pool did not appear to substantially increase apoptosis.

Exposure to the Cd-MT complex can induce apoptosis in some instances (Hamatake et al., 1996; Ishido et al., 1998Go; 1999Go; Liu et al., 1998Go). In in vivo systems, Cd-MT exposure induces acute nephropathy and renal apoptosis (Ishido et al., 1998Go, 1999Go; Liu et al., 1998Go). However, it is thought that, in these cases, MT acts largely as a carrier for Cd that causes high levels to be deposited in specific renal cells (Liu et al., 1998Go). It appears the Cd-MT complex is taken up by renal cells, but it is then degraded to release locally high levels of Cd (Liu et al., 1998Go). It is the released Cd that likely induces the apoptosis, as exposure of renal cells to the Cd-MT complex in vitro does not produce apoptosis, pointing toward the Cd ion as the causative factor (Ishido et al., 1999Go). Indeed, the differential induction of Cd-MT seen in the cell lines used in the present study was not correlated with increased apoptosis. In fact, the inverse relationship occurred. Thus it appears likely that the intracellular Cd-MT complex does not stimulate apoptosis.

The availability of MT probably dictates Cd-induced apoptosis in a variety of circumstances. In this regard, hepatocellular tumors (Waalkes et al., 1991Go, 1993Go) and cell lines derived from hepatocellular tumors (present study) appear particularly sensitive to Cd. There appears to be a down-regulation of MT expression in human and murine liver tumors, while surrounding normal tissue has much higher MT levels (Ghoshal et al., 2000Go; Onosaka et al., 1986Go; Waalkes et al., 1996Go). Similar results are seen with murine lung tumors, which poorly express MT (Waalkes et al., 1991Go, 1993Go, 1996Go). The poor expression of MT appears to account for relative sensitivity of the hepatocellular carcinoma cell lines to Cd-induced apoptosis in the present study. In this regard, Cd can effectively and selectively destroy liver and lung tumors in mice (Waalkes et al., 1991Go, 1993Go), and inhibit growth and metastasis of human lung carcinoma xenografts implanted into mice (Waalkes and Diwan, 1999Go). Thus, the antitumor effects of Cd appear to be dictated by MT expression in tumors (Waalkes et al., 1991Go, 1993Go, 1996Go) and the present results show hepatoma-derived tumor cells appear more sensitive to Cd-induced apoptosis. Induction of tumor cell apoptosis is a very common mode of action for cancer chemotherapeutics (Martin and Green, 1994Go).

In summary, MT appears to mitigate Cd-induced apoptosis regardless of the precise circumstances. The relative expression of MT may well be predictive of the ability of Cd to kill cells, including tumor cells, through stimulation of apoptosis.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. Wei Qu and Tammy Dawson for their advice on MT quantitation. Thanks are due to Dr. Tomoyuki Kawada, Department of Public Health of Gunma University, for his skillful advice in statistical analysis.


    NOTES
 
1 To whom correspondence should be addressed at the Inorganic Carcinogenesis Section, NCI at NIEHS, PO Box 12233, Mail Drop F0-09, 111 Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 541–3970. E-mail: waalkes{at}niehs.nih.gov. Back


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