Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.

hbut//ehpnnl.nihs.nih.d/isl5. pthm The metallothioneins (MTs) are a family of low molecular weight, cysteine-rich intracellular proteins that bind transition metals, induding Cu2+, Zn2+, Ag2+, Hg2+, and CdP (1). MTs have been shown in numerous animal and human tissues and derived cell lines to be highly inducible by these metals and by a variety of other chemicals and stimuli (2). MTs have been proposed to play a role in metal detoxification (Cd2+, Hg2+, Ag+, and Pb2+), in essential metal homeostasis (Zn2+ and Cu2+), and in the protection of cells against damage induced by alkylating agents, oxygen radicals, and ionizing radiation (3). Of particular importance is the role MTs may play in mediating the adverse effects of the heavy metal pollutant CdP. As summarized by Misra et al. (4), the particular target site sensitive to the toxic and carcinogenic effects of cadmium often depends on the species, strain, age, or sex of the animal used as a test subject. Furthermore, there is evidence that these differences in susceptibility to the adverse effects of Cd> may be related, at least in part, to the expression patterns of the MTs. Oberdorster et al. (5) demonstrated that expression of the MTs may mediate the species-specific susceptibility to Cd>_ induced pulmonary carcinogenesis in rodents. Deficiencies in MT expression have been implicated in susceptibility to Cd> carcinogenesis in mammalian testes (6)(7)(8) and the rat ventral prostate (9,10Q. While the basis for the majority of species-and strain-related differences in Cd2+ carcinogenicity and toxicity remains undefined in animals, the above studies provide evidence suggesting that deficiencies in MT gene expression may also underlie susceptibilities in the human. Knowledge regarding the regulation of MT gene expression is even more limited in humans than in rodent models. This is partly due to the fact that the organization of the human MT gene family is more complex. In both mice and humans, there are four classes of MT genes, designated MT-1 through 4, based on sequence and charge characteristics. In the mouse, the genes encoding the MT-1 and MT-2 isoforms are both single copy genes and were the only known rodent MT genes prior to 1992. They are located approximately 6 kb apart on mouse chromosome 8, are coordinately regulated, and the proteins are thought to be functionally equivalent (11,12). Two additional single-gene members of the MT gene family have recently been identified and designated as MT-3 and MT-4; these are closely linked to, but not coordinately regulated with, the other MT genes on mouse chromosome 8 (13,14). The mouse has no known MT pseudogene sequences as determined by Southern analysis. In contrast, the human MTs are encoded by a family of genes consisting of 10 (13)(14)(15)(16).
To address the possible alterations in MT gene regulation in humans, we began an analysis of the metal-induced expression of the MT gene family in cultures of human proximal tubule (HPT) cells. We chose this cell culture system because the kidney, and the proximal tubule in particular, represent an organ and cell type that are critically affected by chronic Cd2+ exposure in both animals and humans (17)(18)(19)(20)(21). In the present report, the expression of MT protein and MT-2A mRNA is determined as a function of metal exposure and cell toxicity in cultured HPT cells. The MT-2A gene was chosen for initial examination because it appears to be expressed ubiquitously and induced by metals in a wide variety of cells and tissues (22).

Materials and Methods
Cell culture. Stock cultures of HPT cells were grown in 75-cm2 T-flasks using procedures recently detailed by this laboratory (23). The growth medium was a serum-free formulation, and the growth surface was treated with a collagen matrix to promote cell attachment and subculture. The cells were fed fresh growth medium every 3 days and were subcultured at confluence (normally 3-6 days post subculture) using trypsin (0.05%)-EDTA (0.02%). For use in experimental protocols, cells were subcultured at a 1:2 ratio, allowed to reach confluence (6 days following subculture), and exposed to medium containing the various concentrations of metals. HepG2 and Hela cells were obtained from the American Type Culture Collection (Manassis, VA) and grown according to instructions.
Preliminary experiments were performed to determine the approximate concentrations of metal salts resulting in HPT cell toxicity over a 16-day period of exposure. Three concentrations of each metal were chosen so that over a 16- All experiments were performed in triplicate. Statistical analysis was performed using SYSTAT software (Systat, Inc., Evanston, IL). Standard statistical tests included determining the mean and standard error of the mean. When multiple groups were compared, ANOVA was used to assess differences between groups using the Tukey HSD (honestly significance difference) test. Unless otherwise stated, differences were considered significant when a = 0.050. Cell viability. The effect of metal treatment on the viability of confluent cell monolayers was determined by the automated counting of 4',6-diamidino-2phenylindole (DAPI)-stained nuclei of cells fixed and processed at days 1, 4, 7, 10, 13, and 16 of metal exposure. In this procedure, we used the nuclear stain DAPI and Kontron KS 400 image analysis software (Kontron Elektronik, Newport Beach, CA) as described previously (24). At the indicated time points, wells containing the monolayers were rinsed with phosphate-buffered saline (PBS), fixed for 15 min in 70% ethanol, rehydrated with PBS, and stained with 10 pl DAPI (10 pg/ml in distilled water). For analysis, each well was examined under epifluorescent illumination at 40x magnification on a Zeiss Axiovert 35 (Carl Zeiss Inc., Thornwood, NY) linked to the computer with an Optronics DEI 470 CCD camera (Optronics, Goleta, CA). For each time point, a minimum of 20 fields per well and three wells per data point were determined. Both nuclear counts and total nuclear area were obtained from the program and yielded equivalent results.
RNA isolation and reverse transcriptasepolymerase chain reaction (RT-PCR). Total RNA was isolated according to the protocol supplied with TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) as described previously (25). We determined the concentration and purity ofthe RNA samples using spectrophotometer scan in the UV region and ethidium bromide (EtBr) visualization of intact 18S and 28S RNA bands following agarose gel electrophoresis. Total RNA (0.1 pg) was reverse transcribed using murine leukemia virus reverse transcriptase (50 units) in IX PCR buffer (50 mM KCI and 10 mM Tris-HCI, pH 8.3), 5 mM MgCl2 solution, 20 units RNase inhibitor, 1 mM each of the dNTPs, and 2.5 mM random hexanucleotide primers. The samples were reverse transcribed for 20 min at 42°C, followed by a 5 min denaturation step at 99C using a DNA thermocycler (Perkin-Elmer-Cetus 9600; Perkin-Elmer, Foster City, CA). The reverse transcribed product was then used for PCR amplification using the AmpliTaq DNA polymerase enzyme (2.5 units; Perkin-Elmer) and the specific upstream and downstream primers at a concentration of 0.075 pM each. The primers developed for analysis of each of the active MT genes have been previously described (25). The primers for analysis of MT-2A mRNA were upper 5'CCGACTCTAGC-CGCCTCTT3' and lower 5'GTG-GAAGTCGCGTTCTTTACA3', yielding a 259 bp product. Primers for the determination of glyceraldehyde 3-phosphate dehydrogenase (g3pdh) were obtained commercially (Clontech, Palo Alto, CA). The thermocycler was programmed to cycle at 95C for a 2-min initial step, at 95C for 15 sec, and at 68C for 30 sec, with a final elongation step at 68°C for 7 min. Controls for each PCR included a no-template control in which water was added instead of the RNA and a no-reverse transcriptase control in which water was added instead of the enzyme. Samples were removed at 25, 30, 35, and 40 PCR cycles to ensure that the reaction remained in the linear region. The final PCR products were electrophoresed on 2% agarose gels containing EtBr along with DNA markers.
Restriction enzyme digestion ofthe MT-2A RT-PCR product. Following RT-PCR (50 pl reaction volume), 25 pl of PCR product was purified using the QiaQuik PCR Purification system (Qiagen, Valencia, CA) according to the manufacturer's recommended protocol. DNA was eluted into 30 pl distilled H20. SgrAI Days of exposure 4 7 10 Days of exposure 13 16 Volume 106, Number 9, September 1998 * Environmental Health Perspectives Days of exposure restriction digestion of MT-2A was carried out by adding 15 pl DNA, 2 pl NE4 Buffer (New England Biolabs, Beverly, MA), distilled H20, and 20 units sgrAI enzyme (New England Biolabs) in a final reaction volume of 20 pl. Following incubation at 37°C for 1 hr, the reaction was terminated by adding 5 pl gel-loading buffer. Samples were electrophoresed on 2% agarose gels containing 0.5X TBE (0.045 M Tris-borate, 0.001 M EDTA) and 0.5 pg/ml EtBr. MTprotein determination. Cells were washed two times with PBS, harvested in buffer containing 10 mM Tris (pH 7.4) and 75 mM NaCl, and lysed by rapid freezing and thawing in liquid nitrogen. Protein concentration was determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL). For copper-induced samples, 1 mM dithiothreitol was added to the extract and total protein was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). MT protein was detected by immunoblotting using a mouse anti-horse antibody (DAKO-MT, E9; Dako, Carpinteria, CA) as the primary antibody following the procedure outlined by Mizzen et al. (26). Aliquots of 3 pg total protein were diluted to 75 pl in the above harvest buffer and mixed with 75 pl of 3% glutaraldehyde to enhance the detection of MT. One hundred microliters of this mixture (2 pg total protein) was applied to a dotblot apparatus with a polyvinylidine difluoride membrane. Samples were allowed to flow through the apparatus by gravity; this was followed by two PBS washes. After blocking with 10% skim milk in PBS, the membrane was soaked in primary antibody [1:100 dilution, 1% bovine serum albumin (BSA) in PBS] for 2 hr. The membrane was washed three times with PBS and anti-mouse alkaline phosphatase-conjugated secondary antibody (Promega, Madison, WI) was added (1:500 dilution, 0.4 % BSA in PBS). Antibody complexes were visualized using the alkaline phosphatase kit III (Vector, Burlingame, CA). Standard curves of rabbit liver Cd/Zn metallothionein-1 (Sigma, St. Louis, MO) were applied to each blot. MT protein was quantified by comparing the optical density of the dots to the standard MT curve using image analysis software (KS 400, Kontron). This assay has detection limits in the range of 0. 1-0.5 ng MT protein.

Results
Effect of metal exposure on HPT cell viability. A goal of the present study was to Days of exposure Figure 3. Metallothionein (MT) protein expression as a function of Cd exposure. Protein extracts were prepared from three independent HPT cell isolates (A,B,C) at the indicated time points and analyzed for MT protein by immunoblot. *Significantly different from control, a = 0.000; #significantly different from 9 pM concentration, a = 0.000.
Environmental Health Perspectives * Volume 106, Number  line the pattern of MT protein and were exposed over a 16-day time course to A mRNA expression in HPT cells three metal concentrations selected to yield ring exposure to metals, especially patterns of cell viability ranging from miniat both sublethal and lethal concen-mal cell death to a significant loss of cell is. Confluent cultures of HPT cells viability. Three independently derived HPT cell isolates were analyzed in the --~U~~A 20 e t CCl2 studies to control for the possibility of isolate-to-isolate variability. This was accomplished by exposure of HPT cell monolayers derived from three independent kidney isolates to 9, 27, and 45 pM concentrations of Cd2+ over a 16-day period ( Fig. 1). For all three HPT cell isolates, there was minimal cell death for the cells obtained when HPT cells were exposed to *Control * 501M A 100PM * 0>MAg2+ at 50, 100, and 150 pM concentrations. Like the case for Cd2 -exposed cells, exposure of the HPT cells to the lowest concentration of Ag2+ had little overall effect on cell viability, whereas the highest concentration elicited a complete loss of cell viability following 10 (Fig. 3). At loss of HPT cell viability over time, there was a dose-dependent increase in MT protein levels compared to the lower exposure level, but there was no marked alteration in the pattern of MT protein accumulation that would correlate specifically to the loss of cell viability. The level of MT protein at the highest Cd2+ concentration was only obtainable following 1 day of exposure due to cell death, and even at day 1 there was approximately 50% cell death. While these samples were analyzed for MT protein, the large standard errors associated with measurement (the errors were larger than the measured values) rendered these samples unsuitable for analysis. Overall, the results clearly demonstrated that Cd2+ was an effective inducer of MT protein accumulation in HPT cells well before any loss of cell viability was noted to occur.
The expression of MT protein was also increased due to exposure of the HPT cells to Zn2+, although the expression pattern was distinct from that of Cd2P at the lower levels of exposure (Fig. 4A). In contrast to Cd2+-exposed HPT cells, Zn2+-exposed cells demonstrated only a very modest induction of MT protein when exposed to the low and intermediate concentrations to occur late in the time course, the levels of MT protein were markedly induced, compared to both the lower concentrations and the control. Furthermore, the level of induction was comparable to that noted for HPT cells exposed to Cd2P. The expression of MT protein was also increased due to exposure of the HPT cells to toxic concentrations of Cu2+ (Fig. 4B). The increase in MT protein accumulation was both dosage dependent and comparable in magnitude to that noted previously for the Cd2+-and Zn2 -exposed cells. Similar to Zn2+ exposed HPT cells, MT protein accumulation was also blunted at the lowest level of Cu2+ exposure when compared to the increase obtained for Cd2+-exposed cells.
In marked contrast to the large induction of MT protein accumulation found for HPT cells exposed to Cd2+, Zn2+, or Cu2+, the expression of MT protein was only marginally increased, if at all, when the HPT cells were exposed to sublethal or lethal concentrations of Ag2+, Hg2+, or Pb2+ (Fig. 4C,D,E). For HPT cells exposed to Ag2+, there was no increase in MT protein at the lowest level of exposure and only a very modest increase at the intermediate level of exposure, an exposure level where Volume 106, Number 9, September 1998 * Environmental Health Perspectives loss of cell viability occurred late in the time course. The increase in MT protein in cells exposed to Ag2+ was less than 5% of that found for Cd +-exposed cells at similar time points and toxicities. For HPT cells exposed to Hg2+ (Fig. 4D), there was also only a very modest induction of MT protein compared to cells exposed to Cd + Zn2+, or Cu2+ at similar time points and toxicities. The very small increase in MT protein that did occur was not dosage dependent and the expression pattern demonstrated no obvious relationship to the loss of cell viability. For HPT cells exposed to Pb2+, there was no increase in MT protein regardless of exposure level or loss of cell viability (Fig. 4E).
Determination of MT-2A mRNA. Accumulation of MT-2A mRNA was determined using an RT-PCR strategy based on the sequence diversity present in the 5' and 3' untranslated regions of the active MT genes (25). This strategy was chosen over Northern analysis due to the high degree of sequence homology among the active MT genes. This high degree of similarity among the nucleotide sequences of the 10 active MT genes makes it difficult to confirm if an individual probe for Northern analysis is specific for measuring a given individual MT isoform. The advantage of the RT-PCR strategy is that the resultant PCR product itself can be analyzed to determine if the primer pairs chosen for analysis were indeed specific for the MT-2A mRNA. This was accomplished in the present study by determining the profile of restriction endonuclease cutting sites for all 10 active MT gene sequences and identifying a restriction site present only in the MT-2A sequence. For the MT-2A gene, a unique restriction site was identified as an sgrAI site (CR/CCGGYG) that would hypothetically cleave the 259 bp MT-2A RT-PCR product into two fragments (51 bp and 208 bp) if the reaction was specific for mRNA from the MT-2A gene. Using the noted MT-2A primers, this was tested on the RT-PCR products of the reaction initiated with 100 ng of total RNA obtained from normal human kidney tissue, from control HPT cells, and from HPT cells treated 24 hr with 45 jiM Cd2+ (Fig. 5). It was demonstrated that each RNA source resulted in reaction products that were cleaved by the sgrAI restriction endonuclease into fragments of approximately 200 bp and a smaller fragment of approximately 50 bp, consistent with cleavage of the 259-bp MT-2A reaction product into 208-and 51-bp products as predicted by sequence information. In all cases, sgrAI digestion resulted in the complete disappearance of the 259-bp reaction product, indicating that the reaction product was composed entirely of the MT-2A sequence and not partially contaminated with a product generated by false priming. The negative control (MT-3 cDNA), which has no sgrAl restriction site, was obtained from a previous study in which RT-PCR primers for MT-3 were shown to yield a legitimate MT-3 cDNA reaction product by a combination of both restriction endonuclease digestion by FokI and direct sequencing (27-    of total RNA isolated from HPT cells at identical time points and concentrations of metal exposures, as described above in the viability and MT protein expression studies. To ensure that MT-2A mRNA expression was determined in the linear region of the RT-PCR, reactions were stopped and samples removed for analysis at various time points. The need to analyze comparative RT-PCR in linear regions of the reaction cyde is illustrated effectively by the determination of MT-2A mRNA expression in control HPT cells unexposed to metals (Fig. 6). For 30 and 35; indicating that results at these cydes would be unsuitable for comparative analysis (Fig. 6A). In contrast, the reaction product bands at cyde 30 indicate that the reaction is in the linear region and would be suitable for use in comparative RT-PCR It was also confirmed for the other two HPT cell isolates that the PCR product at 30 cydes was in the linear range ofthe reaction (Fig. 6B,C). The effect of Cd2+ exposure on the expression of MT-2A mRNA was also determined at 30 reaction cycles using 0.1 pg of total RNA from the three independent isolates of HPT cells. A comparative analysis demonstrated that expression of MT-2A mRNA did not appear to increase beyond control levels in any of the three independent isolates of HPT cells regardless of Cd2+ concentration, time of exposure to Cd2P, or whether there was a CPinduced loss of cell viability (Fig. 7). The failure of metal treatment to elicit a large increase in the accumulation of MT-2A mRNA was also confirmed for HPT cells exposed to Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ (Fig. 8). Similar to the case for Cd2Pexposed cells, MT-2A mRNA levels were not increased over control by the other metals regardless of concentration, duration of exposure, or whether a given metal exposure resulted in loss of cell viability.
Because the finding that MT-2A mRNA was not increased in the metaltreated HPT cells was unexpected, a control was sought to validate the RT-PCR procedure. The HepG2 cell line was chosen because exposure to Cd2P and Zn2+ has been shown to result in 10to 20-fold elevations of MT-2A mRNA (22). HepG2 cells were grown to confluency and exposed to 9 pM Cd2P or 100 pM Zn2+, and total RNA was isolated following 8 and 24 hr of exposure. The results of this determination, using an input of 0.1 pg total RNA for RT-PCR, clearly demonstrated that both Cd2+ and Zn2+ were effective inducers of MT-2A mRNA expression (Fig. 9). Control cells demonstrated a reaction product for MT-2A mRNA beginning at 26 reaction cycles. Both the Cd2+and Zn2+-exposed cells demonstrated MT-2A reaction products beginning at 20 reaction cycles. These findings effectively demonstrate that the RT-PCR method used to determine MT-2A mRNA is valid and can detect an increase in MT-2A mRNA. A similar induction of MT-2A mRNA has also been reported for Hela cells (22). Hela cell MT-2A induction was also confirmed using the RT-PCR assay for MT-2A mRNA (data not shown).

Discussion
Many of the major features of renal MT protein expression expected from the accumulated knowledge gained from rodent animal models were demonstrated in cultures of HPT cells exposed to various metals. When differences were noted in the induction of MT protein expression between the cultured cells and the rodent animal models, these proved to be areas in which there was a limited database of rodent-based studies. In these instances, the results with cultured cells can be looked upon as extensions of the overall knowledge base of the renal effects of each given metal rather than defining large discrepancies between humans and animal models. A major shared feature of the rodent and human renal systems was clearly demonstrated in the capacity for Cd2+ exposure to induce a large increase in MT protein within the renal proximal tubule. In general, it has been shown in numerous rodent studies that Cd2+ is a potent inducer of the hepatic and renal MT-1 and MT-2 genes, which leads to large increases in MT protein accumulation (1,(28)(29)(30). In the present study, exposure of HPT cells to both lethal and sublethal Cd2+ concentrations elicited greater than 20-fold accumulations of MT protein in a dose-dependent fashion over the time course. In accord with rodent animal models, we demonstrated that Cd2+ was the strongest inducer of MT protein expression in HPT cells at concentrations below those eliciting cell death. In whole animals, Cd2+ has been shown to be the strongest inducer of the metallothionein genes and protein, followed by Zn2+, Ag2+, Hg2+, Bi2+, and Cu2+, with Ni2W, Ca2+, and Pb2+ being poor inducers (30). Thus, the pattern of MT protein expression for HPT cells exposed to Cd2+ was very similar to that expected from animal model studies.
The demonstration that HPT cells accumulated MT protein when exposed to Zn2+ is also in agreement with studies in   rodent models and derived cell cultures (1,2,28,29). The present studies with HPT cells also extended this observation. In HPT cells exposed to Zn2+, we have shown that the degree of MT protein accumulation was influenced by the level of metal exposure. While the MT protein content of HPT cells was elevated significantly above control at Zn2+ concentrations eliciting little or no cell death, this level of induction was only modest when compared to the MT protein levels obtained when the cells were exposed to Cd2+ at nonlethal concentrations. In contrast, when HPT cells were exposed to Zn2+ at concentrations that elicited cell death late in the time course, the increase in MT accumulation was equal to or greater than that found for HPT cells exposed to Cd2+. These findings are of interest in the human renal system because one of the functions ascribed to MT is to buffer intracellular Zn2+ concentrations and thereby attenuate toxicity and deficiency (31). Further evidence for this concept has come from recent studies with transgenic mice demonstrating that MT-1 and MT-2 protect against zinc deficiency and toxicity and that mice which overexpress MT are resistant to dietary zinc deficiency (32,33). Although fewer studies document the ability of Cu2+ to induce MT in the rodent renal system, existing studies demonstrate the induction of MT protein accumulation by Cu2+, although at levels 100% less effective than Zn2+ (34,35). As such, the finding that HPT cells accumulate MT protein as a result of Cu2+ exposure is consistent with current animal model studies. However, as noted for Zn2+-exposed HPT cells, the current studies also demonstrated that the degree of MT protein accumulation by HPT cells exposed to Cu2+ was clearly influenced by metal exposure levels, either at lethal or sublethal concentrations. The accumulation of MT protein by Cu2+exposed cells was highest at concentrations that elicited cell death. Cu2+-exposed HPT cells were also shown to accumulate similar quantities of MT protein as Cd2+and Zn2+-exposed cells. The current findings in HPT cells suggest that the human proximal tubule is capable of a significant increase in the accumulation of MT protein as Zn2+ or Cu2+ reach levels eliciting cell death. There were also some differences between animal model data and the HPT cells that involved an inability of HPT cells to accumulate MT protein upon metal exposure. Most striking was the marginal induction of MT protein accumulation when HPT cells were exposed to Hg2 . Several studies have shown that treatment of rats with either a single dose or repeated doses of inorganic Hg + causes the renal concentration of MT to increase (36)(37)(38). This increase occurs particularly in the outer cortex and outer stripe of the outer medulla and is presumably due to increased transcription of the MT genes (3,9). In contrast, an increase of this magnitude was not noted for HPT cells exposed to Hg2+ at either lethal or sublethal levels. A lack of induction of MT protein was also noted when the HPT cells were exposed to Ag2+ at both lethal and sublethal concentrations. Even though silver is classified as having minor toxicity, these findings are of interest because the intentional use of silver in medical and dental products increases potential exposure (40). However, the results regarding Ag2+ exposure are difficult to interpret, as there is only a very limited literature base detailing the response to Ag+ exposure in general, and the MT response in particular, for both animals and humans (41,42). The present studies using HPT cells demonstrate that both Hg2+ and Ag2+ elicit only marginal, if any, MT protein accumulation.
The exposure of HPT cells to Pb2+ was designed to serve as a negative control for MT protein induction since exposure to . MT-2A mRNA expression in HepG2 cells following exposure to zinc or cadmium. RT-PCR for MT-2A was performed on RNA from cultured HepG2 cells exposed to zinc (100 pM) or cadmium (9 pM). RT Pb2+ has been reported to induce hepatic but not renal MT in animals (34,(43)(44)(45)(46)(47). The findings with HPT cells were in agreement because no concentration of Pb2+, lethal or sublethal, was noted to increase MT accumulation. If one ranks the ability of the metals to induce the accumulation of MT protein in HPT cells at exposure levels near lethal conditions, Cd2 , Zn2+, and Cu2+ are potent inducers with very similar efficiencies, while Hg2+, Ag2+, and Pb2+ demonstrate little, if any, significant induction of MT protein accumulation.
Even though the results regarding MT protein accumulation by HPT cells exposed to metals were similar to those noted in animal models, the regulation of the MT-2A gene in HPT cells appears unique compared both to animal models and to other human systems. In rodents, overwhelming evidence suggests that the hepatic and renal MT-1 and MT-2 genes are widely expressed and co-regulated by many agents including metals, hormones, and xenobiotics (2,11). We could find no instances in the literature of studies using rodent models or rodent tissuederived cell cultures in which renal and hepatic MT-2 mRNA has not been demonstrated to be induced upon exposure to Zn2+ or Cd2P. This also appears to be true in the human system, although for obvious reasons the induction of MT-2A mRNA via metals has been tested only under cell culture conditions. The initial observation of the induction of the human MT-2A gene by metals demonstrated that in 15 human-derived cell cultures, Cd2+ treatment resulted in a 10-to 30-fold induction of MT-2A mRNA (22).
Induction of MT-2A mRNA by Cd2P treatment has also been demonstrated for lymphoid-derived cell lines (48). The potential significance of the expression and induction of MT-2A mRNA in the human is underscored by the observation that the ability to induce expression of MT-2A mRNA correlates with development of cisplatin resistance in three human squamous cell carcinoma cell lines (49). In this study, the pattern of gene expression was determined for the MT-2A, MT-lA, B, E, F, and G genes in three pairs of cell lines in which one member of each pair was resistant to cisplatin. Yang et al. (49) found that MT-2A mRNA was inducible by zinc, that the most highly overexpressed MT isoform in all three cisplatin resistant human carcinoma cell pairs was MT-2A, and that no other isoform was universally overexpressed. This finding was interpreted as being consistent with a role for the MT-2A isoform in mediating cisplatin resistance. To our knowledge, the finding that MT-2A mRNA accumulation was not increased by Cd2+ exposure in HPT cells is the first demonstration that the human MT-2A gene can be refractory to heavy metal treatment. Proof that RT-PCR was specific for MT-2A mRNA and that increased accumulation could be detected was provided by two procedures. First, an advantage provided by RT-PCR is the ability to recover the reaction product from the agarose gel and to subject this product to further analysis to confirm identity of a given gene. In the present study, this advantage was used to determine that the MT-2A RT-PCR product contained a sgrAl restriction endonuclease cleavage site, a site known from sequencing data to be present only in the MT-2A gene of the human MT family. The demonstration that sgrAl digestion completely cleaved the MT-2A reaction product into appropriate fragments provides a high degree of confidence that the primers developed for MT-2A mRNA and the RT-PCR were specific for the transcription product of the MT-2A gene. Proof that the RT-PCR protocol could detect an increased accumulation of MT-2A mRNA relied on previous studies which demonstrated that the HepG2 cell line, when exposed to either Cd2+ or Zn2+, displayed a 10to 20-fold accumulation of MT-2A mRNA (22). Utilizing the HepG2 cell line and exposures to Cd2+ and Zn2+, the RT-PCR protocol that we used demonstrated a large increase in the amount of accumulated MT-2A mRNA for treated versus control cells.
The present study demonstrates strong similarities between the human and rodent renal system in the ability to accumulate MT protein as a result of metal exposure. However, the studies also provide initial evidence that this accumulation of MT protein may be mediated through distinctly different patterns of gene regulation between humans and animals.