TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
112, 318-323 (1992)
Induction of Metallothionein by Cadmium-Metallothionein A Proposed Mechanism’ J. M. MCKIM, Department
of Pharmacology,
Toxicology,
in Rat Liver:
JR.,’ J. Lru, Y. P. LIU, AND C. D. KLAASSEN~ and Therapeutics,
University
of Kansas
Medical
Center.
Kansas
City, Kansas
66103
Received June 24, 199 1; accepted October 2, 199 1
with a high degree of homology (Andersen et al., 1986, 1983; Crawford et al., 1985; Durnam et al., 1980). Although a single physiologic function for MT has not been identified, the protein does appear to have a number macol. 112, 318-323. of roles in tissue. For example, MT has been implicated in Zn and Cu homeostasis and as a source of Zn for stabilizing Distribution of Cd to various organs following iv administration of CdC12 (3.5 mg Cd/kg) resulted in more than 43% of total biological membranes (Bettger and O’Dell, 1981). Its prestissue Cd accumulating in the liver. In contrast, after CdMT ence can decrease the hepatotoxic effects of cadmium (Goeradministration (0.5 mg Cd/kg), only 1% of the Cd was found in ing and Klaassen, 1984a,b,c), the cytotoxic and hepatotoxic liver. Rats administered CdC12 (1 .O mg Cd/kg) had hepatic MT effects of chemotherapeutic drugs such as cisplatin (Basu values 30-fold greater than controls and a hepatic Cd concen- and Lazo, 1990) and adriamycin (Satoh et al., 1988) scavtration of 17 pg/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than enge free radicals (Thornalley and Vasak, 1985; Abel and controls and a hepatic Cd concentration of 0.80 pg/g. However, Ruiter, 1989; Mello-Filho et al., 1988; Coppen et al., 1988), when hepatic MT levels were normalized to tissue Cd concen- and aid in the treatment of Wilson’s disease (Lee et al., 1989). On the basis of these possible functions, it is important to trations, induction of MT by CdMT was 5-fold greater than by CdC12. Northern and slot-blot analyses of mRNA showed that understand the molecular and cellular mechanisms that regulate MT production. The induction of MT by inorganic both CdCl, and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl, increase hepatic Cd is dose-dependent, and the amount of MT in tissues corMT by similar mechanisms. A dose-response increase in MT responds to tissue metal concentration. However, it appears produced by CdCl, indicated a biphasic response, with low doses that CdMT is more effective than CdC12 in inducing MT producing relatively more hepatic MT than higher doses. In ad- (Sendelbach and Klaassen, 1988; Koropatnick and Cherian, dition, the amount of MT produced per unit Cd after CdMT 1988). In the former study, MT was induced in a dose-detreatment wassimilar to those observed after low doses of CdC12 pendent manner after administration of CdC12 and CdMT. in the dose-response experiment. These data provide strong evHowever, when tissue MT levels were compared to tissue idence to support the conclusion that the apparent potency of Cd levels, CdMT appeared to be more potent than CdC& at CdMT observed here and in previous studies is most likely due inducing MT. In the later study, induction of hepatic MT to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concen- mRNA in mice was measured after CdC12 and CdMT adtrations. 0 1992 Academic Press, I~C. ministration. Induction of MT mRNA correlated well with cellular Cd concentrations following CdC12 ; however, CdMT appeared to produce peak mRNA levels independent of tissue metal concentration. These studies suggest that CdMT Since its discovery (Margoshes and Vallee, 1957) the may induce MT by a mechanism other than metal-stimustructure, function, and regulation of metallothionein (MT) lated transcription. have been extensively studied. MT is characterized by its Therefore, the purpose of the present study was to deterlow molecular weight (M, 6500-7000), absence of aromatic mine whether or not CdMT is a more potent inducer of MT amino acids or histidine, and high cysteine content (Hamer, than CdC12 in rat liver and to determine if the induction of 1986). In rodents, MTs comprise a gene family consisting hepatic MT can be attributed to the Cd associated with MT. of two coordinately regulated isoforrns (MT-I and MT-II) Induction of Metallothionein by Cadmium-Metallothionein in Rat Liver: A Proposed Mechanism. MCKIM, J. M., JR., LIU, J., LIU, Y. P., AND KLAASSEN, C. D. (1992). Toxicof. Appl. Phar-
MATERIALS
AND METHODS
’ This research was supported by U.S. Public Health Service Grant ES001142. Animals. Male Sprague-Dawley rats (300-400 g) were obtained from ’ Supported by U.S. Public Health Service Training Grant ES-07079. Sasco Inc. (Omaha. NE). All animals were housed in groups of three in 3 To whom all correspondence should be addressed. plastic cages and fed Ralston Purina pellet chow and water ad libitum for 318 0041-008X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
INDUCTION
319
OF METALLOTHIONEIN
I week. Lighting was maintained on a 12-m light/dark cycle. The temperature was maintained between 2 1 and 22°C. Chemicals. Cadmium chloride was obtained from Sigma Chemical Co. (St. Louis, MO). Carrier-free ‘@‘Cdwas obtained from New England Nuclear (Boston, MA). Sephadex G-75 and DEAE anion exchange gels were obtained from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ). RNAzol-B was obtained from TELTEST, Inc. (Friendswood, TX). Ultrapure formamide and chloroform were from International Biotechnologies, Inc. (New Haven, CT). All other reagents used for RNA analysis were molecular biology grade from Sigma Chemical Co. Terminal deoxytransferase, CoCl,, tailing buffer, and DNA G-25 quick-spin columns were from Boehringer-Mannheim, Inc. (Indianapolis, IN). Zeta-probe nylon membrane was from Bio-Rad, Inc. (Richmond, CA). Deoxyadenosine 5’-[cu-32P]triphosphate, 6000 Ci/mmol, was from Amersham Corp. (Arlington Heights, IL). The oligonucleotide probes were prepared by Genosys Biotechnologies, Inc. (Woodlands, TX). Preparation of CdMT. Five rats were injected SCwith CdClz (3.0 mg Cd/kg and 2.0 PCi ‘@?Zd/kg)once daily for 4 days. The rats were decapitated and their livers removed. The same protocol was used when preparing unlabeled MT, but without the 2.0 pCi “‘Cd/kg. Preparation of the liver and isolation of the CdMT was done as described previously in this laboratory (Sendelbach and Klaassen, 1988). Following separation of MT-I and MTII isoforms by DEAE anion-exchange chromatography, the two fractions were desalted using Bio-Rad P6-DG columns and dried by lyophilization. The protein was resolubilized in 5-10 ml of ultrapure sterile water and the Cd content analyzed using a Perkin-Elmer Model 2380 flame atomic absorption spectrophotometer. In order to significantly increase the radioactivity associated with MT, the purified isoforms were incubated with lwCd (8.0 &i/ml) for 12 hr. The preparation was then desalted again, to remove unincorporated lmCd, and lyophilized. The final preparations were aliquoted and stored at -20°C. CdMT-II was used in all experiments. Organ distribution of CdCI, and CdMT. ‘09CdC12(3.5 mg Cd/kg) and ‘09CdMT-II (0.5 mg Cd/kg) were administered as a single iv injection. Two hours later the animals were decapitated and their organs excised for analysis. ‘09Cd was measured using a Packard Auto-gamma 5000 scintillation spectrometer. Tissue Cd concentrations were expressed as pg Cd/g tissue and were determined using the calculated specific activity of the dosing solutions. Induction of MT in liver by CdCI, and CdMT-ZZ. The ability of each Cd form to induce MT was determined by administering a single iv dose of either CdCl, (1 .O mg Cd/kg) or CdMT-II (0.4 mg Cd/kg). After 24 hr the animals were decapitated and the livers removed for analysis of MT. Total MT was measured by the Cd-hemoglobin binding assayof Onosaka et al. ( 1978) as modified by Eaton and Toal (1982). Hepatic Cd content was determined by atomic absorption spectrophotometry after digesting 1.O g of tissue in concentrated nitric acid at 80-90°C until clear, when unlabeled Cd was administered, or by gamma spectrometry when “‘Cd was used. RNA isolation. Total RNA was isolated from rats treated with CdC&, CdMT-II, or saline, using RNAzol-B as previously described (Chomczynski and Sacchi. 1987). Briefly, approximately 250 mg liver was flash frozen in liquid nitrogen and stored at -80°C. The liver was homogenized in a glass homogenizer with 5 ml RNAzol-B. The homogenate was transferred to a sterile 15-ml Falcon tube and 0.5 ml chloroform added. The remaining steps were exactly as described by the manufacturers of RNAzol. All solutions were treated with 0.1% DEPC prior to use. RNA yield, purity, and integrity were determined by absorbance at 260nm, AZ60/A280 ratios (1.7 to 1.9) and agarose/formaldehyde electrophoresis. Northern analysis of RNA. Preparation of RNA for electrophoresis, agarose gel preparation, and Northern transfer were done essentially as described by Lehrach er al. (1977) and De et al. (1989). Briefly, RNA (8 pg) was denatured in 20 mM 3-(N-morpholino)propanesulfonic acid (Mops), 5 mM sodium acetate, 1 mM EDTA, pH 7.0, 50% deionized formamide, and 2.2 M formaldehyde. The mixture was heated at 65°C for 15 min, then cooled on ice prior to loading a 1.2% (5 X 7.5 cm) agarose gel. Following electrophoresis, the gel was washed in deionized water or sodium chloridesodium citrate ( I X SSC) buffer (0.15 M and 0.0 15 M, respectively) prior to capillary transfer of RNA in 10X SSC to zeta-probe nylon membrane. Ri-
bosomal RNA bands were visualized by soaking the membrane in 0.5 M sodium acetate, pH 5.2, and 0.04% methylene blue dye for 3-5 min. The membrane was destained by soaking in ultrapure diethyl pyrocarbonate (DEPC)-treated water. The 28 S and 18 S rRNAs appear as dark blue bands. Gel loading, transfer efficiency, and RNA integrity could then be monitored by comparing the staining intensity of the 28 S and 18 S bands. Prior to loading slot-blots, RNA from each exposure group was analyzed by Northern analysis. The 28 S and 18 S rRNA bands were compared to ensure that the RNA concentration obtained from A 2M)resulted in equal amounts of RNA loaded. Slot-blot analysis. Slot-blot analysis of RNA was done using a Bio-Rad slot-blot vacuum apparatus. RNA (8 pg) was denatured with 0.5 ml of 10 mM NaOH containing 1 mM EDTA just prior to loading. Labeling Metallothionein probes. Oligonucleotide probes (20 mers) were prepared from 3’-nontranslated regions of known rat MT cDNA sequences in order to obtain probes specific for MT-I (‘5GAGGGCAGCAGCACTGTTCG-3’) (Andersen et al., 1986) and MT-II (‘5-ACACCATTGTGAGGACGCCC-3’) (R. D. Andersen, personal communication). The probes were 3’-end labeled (Collins and Hunsaker, 1985) with deoxyadenosine[a-“P]triphosphate (6000 Ci/mmol) using a DNA tailing kit (Boehringer-Mannheim, Inc.). The tailing reaction (15 ~1) contained 3.0 ~1 tailing buffer, 4.5 ~1 CoCl,, 3.4 pmol of probe. 45 pmol deoxyadenosine triphosphate (270 &i on reference date), and 55 units terminal deoxytransferase. The reaction was initiated with addition of enzyme and allowed to incubate for 25 min at 37°C. The reaction was stopped by adding 10 ~1 of ice cold 0.5 M EDTA. Unincorporated label was removed using G-25 Sephadex quick-spin columns (Boehringer-Mannheim, Inc.). This procedure yielded a tail length of approximately 5 bases with a specific activity of 8.0 X lo9 dpms/rg DNA. Hybridization of labeled probes. Prehybridization and hybridization reactions were identical for Northerns and slot-blots. Prehybridization (20 mM sodium phosphate, pH 7.0, 7% SDS. 20% formamide, 5X SSC, 5X Denhardt’s solution. 100 p&ml salmon sperm DNA, 250 p&ml yeasttRNA, and 5.0 @g/ml d[pAls or d[pA],) was at 47°C for 8-12 hr. Hybridization incubations were similar, except that the Denhardt’s solution was IX and the incubation was 18-20 hr. The membranes were washed at 47°C in 200 ml of 3X SSC and 2% SDS for 30 min and then washed four times subsequently with decreasing salt and increasing temperature to final conditions of0.5X SSC, 2% SDS at 55°C. Total wash time was 2.5 hr. Autoradiography was accomplished by exposing x-ray film (XAR-05, Kodak, Rochester. NY) using a high-plus intensifying screen at -80°C for the required time. Dose-response induction of MT. Rats (IV = 3/dose) received single iv injections via the tail vein of CdClz with lmCd (0.5 &i/ml) in 0.9% saline at 0.008, 0.022, 0.072, 0.21. 0.75, 2.4 mg Cd/kg. After 24 hr the animals were decapitated and their livers excised for analysis. MT levels were measured by the Cd-hemoglobin assay (Eaton and Toal, 1982) and Cd tissue levels were determined by quantitating the lmCd in 1 g portions on a Packard Auto-gamma 5000 scintillation spectrometer. Stutistics. Statistical comparisons were done using analysis of variance followed by Duncan’s multiple range test. Differences between treatments were considered significant at p < 0.05.
RESULTS The distribution of Cd, administered as inorganic or organic cadmium, to several organs is shown in Fig. 1. Two hours after CdC12 (3.5 mg/kg) administration, Cd accumulation was greater in the liver (34 pg/g) than kidney (17 pg/g). Following CdMT administration (0.5 mg Cd/kg), Cd was distributed primarily to the kidney (23 pg/g) with less than 1 pg/g present in the liver. The ability of CdClz and CdMT to induce MT synthesis in rat liver is shown in Fig. 2. CdClz ( 1.O mg Cd/kg) increased
320
MCKIM
m
CdC12
1
ICdMT
FIG. 1. Organ distribution of Cd following CdC12 and CdMT-II administration. Rats (N = 3 or 4) received 3.5 mg Cd/kg as CdClz or 0.5 mg Cd/kg as CdMT iv. Two hours after administration, various tissues were analyzed for Cd content. Values represent means + SEM.
MT to 295 pg/g, a 30-fold increase over control values (9.0 pg/g), while CdMT (0.4 mg Cd/kg) increased MT to 68 pg/g, a 7-fold increase over controls (top). Hepatic Cd concentration after administration of CdC& was 17 pg/g and 0.80 pg/g after CdMT (middle). When tissue MT values (top) were normalized to tissue Cd values (middle), inorganic cadmium produced about 17 Fg MT/pg Cd, whereas CdMT produced about 85 pg MT/pg Cd (bottom). Thus, it appeared that CdMT was more potent than CdCl* at inducing MT in rat liver. Metals are the primary inducers of MT. Therefore, it was important to determine if increases in MT following CdMT exposure would also cause an increase in MT mRNA. Northern analyses were done to determine probe specificity, mRNA size, and appropriate stringency conditions. The data in Fig. 3 clearly show that both MT-I and MT-II mRNAs are significantly increased over controls following both treatments. In order to quantitate MT mRNA in control rats and rats administered either CdClz or CdMT, slot-blot analysis (Fig. 4) was used. The inductive effects of both forms of Cd on MT mRNA and protein are summarized in Table 1. MTI and MT-II mRNAs were increased after CdC& and CdMT exposure. As depicted in Table 1, CdC& produced a 40-fold induction of MT-I and a 30-fold increase in MT-II mRNA, while CdMT increased MT-I and MT-II mRNA 6-fold over controls. Both forms of Cd increased MT mRNA and protein coordinately and to approximately the same extent. In addition, when MT mRNA data are normalized to liver Cd concentrations, the results were similar to those obtained for MT protein in Fig. 2 (bottom). Because it appeared that CdMT may be more efficient at inducing MT than CdC12 (Fig. 2, bottom), a dose-response experiment was carried out to determine whether or not tissue Cd concentrations, similar to those found in liver after CdMT exposure, can account for the observed increase in MT. When MT concentration in liver was compared to the dose of CdQ administered, a biphasic response was observed
ET AL.
(Fig. 5, top), indicating that low dosages of Cd produce pronounced increases in MT. However, Cd concentrations increased linearly in liver with dose (Fig. 5, middle). When hepatic MT concentrations were normalized to tissue Cd levels (Fig. 5, bottom), it was apparent that low amounts of Cd produce relatively larger increases in hepatic MT while higher amounts of Cd are less effective at increasing hepatic MT. When the amount of MT produced in the liver was compared to the concentration of Cd in the liver after each dose of CdClz (Fig. 6), the biphasic nature of the curve obtained in Fig. 5, (top) was retained. Moreover, the concentration of Cd in the liver following CdMT administration induced MT in a manner quantitatively similar to that observed after low doses of CdCl* (Fig. 6, solid circle). Hepatic Cd and MT concentrations are compared (Table 2) after administration of CdCl* at doses that gave higher and lower concentrations of Cd in the liver than the dose of CdMT used. As can be seen, CdMT produced an increase in MT proportional to the concentration of Cd present. These data provide additional evidence that induction of MT by
CdCIP
FIG. 2. Induction of hepatic MT following CdClz (1.0 mg/kg) and CdMT-II (0.4 mg/kg), top. Accumulation of Cd metal in liver following each Cd exposure, middle. MT induction expressed as pg MT/pg Cd, bottom. Values represent the mean + SEM of 3 or 4 animals. Asterisks show significant difference from controls @ > 0.05). Due to low variance within samples, some standard error bars are not visible.
INDUCTION
28s
-
18s
-
DYE
-
MT-I
MT-II
123
456
321
OF METALLOTHIONEIN MT-I CdMT
CON
Cd’&
MT-II CON
CdMT
CdC12
i
FIG. 3. Northern analysis of MT-I and MT-II mRNA. RNA (8 pg) from control (saline, lanes 1,4), CdMT-II (0.4 mg Cd/kg, lanes 2, 5) and CdC12 (1 .O mg Cd/kg, lanes 3,6) was loaded onto an agarose (1.2%) formaldehyde (2.2 M) gel and electrophoresed for 3 hr at 35 mA. Following electrophoresis, RNA was transferred to zeta-probe membrane. The membrane was cut in half, with one half being hybridized with MT-I and the other with an MT-II-specific oligo probe. After washing, the two halves were placed next to each other for autoradiography.
either form of Cd is most likely dependent on tissue metal concentration and, more importantly, that the relatively low concentration of Cd observed following CdMT administration can account for the observed increase in hepatic MT. DISCUSSION There is a marked difference in the distribution of Cd when administered as CdC& or CdMT. In the present study, after administration of CdC&, approximately 43% of the total tissue Cd was in the liver of rats. In contrast, only 1% of the measured tissue Cd was in the liver of rats after CdMT administration. Because CdMT is a potent renal toxicant (Squibb et al., 1984; Squibb and Fowler, 1984), only small doses can be given without producing renal toxicity to the animals. The combination of low doses of CdMT as well as the poor hepatic uptake of CdMT results in extremely low tissue Cd concentrations following a single iv dose of CdMT. Because more Cd distributes to the liver after CdC& than after CdMT administration, one would expect to find higher levels of MT in the livers of rats that received the inorganic form, as opposed to those that received the organic form of Cd. As predicted, rats treated with CdQ produced about fourfold more MT than rats treated with CdMT (Fig. 2). However, when the hepatic MT concentrations were normalized to tissue Cd concentrations, CdMT-treated rats produced fivefold more MT per unit Cd than did animals treated with CdC12. This was also true when MT mRNA was nor-
FIG. 4. Slot-blot analysis of MT-I and MT-II mRNA. RNA (8 rg) was loaded onto the Bio-Rad slot-blot apparatus and applied to the zeta-probe membrane by vacuum. Columns represent the three exposure groups, while each row represents RNA from a separate animal.
malized to liver Cd. Thus, CdMT is a more potent inducer of MT in the liver of rats as has been reported previously for the mouse (Sendelbach and Klaassen, 1988; Koropatnick and Cherian, 1988). In order to better understand why CdMT is a more potent inducer of MT than CdCl*, the transcriptional response to both inorganic and organic Cd was examined. MT mRNA for both isoforms was measured and compared to increases
TABLE 1 Summary of Metallothionein mRNA and Protein Induction by Organic and Inorganic Cadmium MT mRNA (cpm)” Treatment
Dose (w Cd/k)
Saline
NA
CdMT
0.4
I 109 f 27 (lJb 640a
41
422
1.0
4080
f 277 (401
12
211
f
(1) 11
68k
(6) 1170
f 1301
1
9f
(1)
(6) CdC12
MT Protein Wg liver)
II
I
(7) 80
295
i
18
(30)
a cpm values were obtained from slot-blot data in Fig. 3. All table values represent the means of four to six animals f SEM. b Values in parenthesis represent fold increase over controls.
322
MCKIM
ET AL. 800
1
soo-
P
c 1$
400.
3
300
5i
200: 100. 093 0
o/o @/
6
10
15
Cd @g/g
20
25
D
liver)
FIG. 6. MT produced by different liver Cd concentrations. The doseresponse values in Fig. 5 for liver MT (top) and liver Cd (middle) were used to prepare Fig. 6. The open circles represent the values from the CdCl, doseresponse curve. The solid circle depicts the liver MT and Cd concentrations obtained after CdMT administration. All values are means + SEM. Due to low variation between samples, some standard error bars are not visible.
0 O* 0.0
0.6
1.0
1.5
2.0
2.5
Dose (mg Cd/ kg) FIG. 5. Dose-response induction of MT by cadmium. Rats (N = 3) received a single iv dose of Cd (0.008,0.022, 0.072,0.21, 0.75, 2.4 mg Cd/ kg) and 24 hr later their livers were removed and analyzed for Cd and MT content. The top shows the induction of MT at each dose. The middle depicts the change in hepatic Cd concentrations over the doses used. The bottom represents MT concentration normalized to Cd concentration. All values represent a mean + SEM. Due to low variation between samples, some standard error bars are not visible.
in MT protein. The results clearly show that both MT-I and MT-II mRNA are coordinately increased by both CdMT and CdC& and that the increase in mRNA corresponds well with the increase in MT protein (Table 1). This information provides evidence showing that MT protein concentrations following exposure to both Cd forms correlate with increased MT mRNA. This is especially plausible because it is generally accepted that the mechanism by which metals induce MT is via enhanced transcription (reviewed by Hamer, 1986 and Andrews, 1990). Thirefore, there now appears to be a connection between metal content and MT production following CdMT administration. The transcriptional information discussed above does not provide an explanation for the higher potency of CdMT than CdC12 to induce MT (Fig. 2). It is well known that inorganic Cd induces hepatic MT in a dose-dependent manner; however, these studies used relatively high doses of the metal (Sendelbach and Klaassen, 1988). In the present study, we
examined the ability of low doses of Cd to induce hepatic MT. Interestingly, a biphasic response was observed (Fig. 5). Production of MT at low doses (0.008 to 0.20 mg Cd/kg) was relatively greater than at higher doses (0.20 to 2.4 mg Cd/kg). It therefore appears that small amounts of Cd are comparatively more effective than large amounts at inducing MT. Moreover, it is clear from Fig. 6 and Table 2 that, following CdMT administration, both tissue Cd and MT concentrations fall within the linear portion of the curve established using CdC12. These data suggest that the Cd present in the liver after CdMT exposure would be sufficient to produce the observed levels of MT. The biphasic nature of the dose-response curve implies that metal-induced transcription of MT may be mediated by a cytosolic protein that binds Cd cooperatively and is saturable. This would enable the cell to produce a large amount of mRNA in response to a small amount of metal. A system of this type has been described for a metallothionein-like protein in the yeast Saccharomyces cerevisiae (Furst and Hamer, 1989). The existence of trans-acting proteins that mediate transcriptional activity of MT in higher eukaryotes, following
TABLE 2 Hepatic Cadmium Concentration Versus Metallothionein Concentration Treatment
Dose (m8 CWcs)
CdCIZb CdMT CdC12
0.20 0.40 0.02
Cd liver)”
MT (he/g liver)
4.0 ?I 0.22 0.8 f 0.05 0.4 f 0.04
164 + 15 68-1- 7 27+ 6
(&g
’ Values represent the mean f SEM of three to four animals. b Cd& values taken from a dose-response experiment (Fig. 5).
INDUCTION
OF METALLOTHIONEIN
Cd and Zn exposure, has been reported (reviewed by Andrews, 1990; Imbert et al., 1990). These studies provide strong evidence for the existence of cellular protein components that bind metals, become activated, and then bind to metal regulatory regions on the MT gene, thereby increasing transcription. It is possible that Cd carried to the liver by CdMT is passed to these metal-binding factors or that all or part of the CdMT peptide may serve as the carrier protein. The existence of this type of regulatory system could explain why, in the present study, extremely low concentrations of Cd had a more pronounced effect on MT production than did higher doses. The results of the present study strongly suggest that the induction of MT by CdMT is due to the metal associated with the protein. Further, these data indicate that the apparent potency of CdMT in inducing MT, when normalized to liver Cd, is most likely due to the low concentrations of Cd present in the tissue, which are relatively more efficient at inducing MT than higher concentrations. REFERENCES Abel, J., and Ruiter, N. (1989). Inhibition of hydroxyl-radical generated DNA degradation by metallothionein. Toxicol. Lett. 47, 19 1- 196. Andersen, R. D., Buren, B. W., Ganz, T., Piletz, J. E., and Herschman, H. R. (1983). Molecular cloning of the rat metallothionein-1 (MT-I) mRNA sequence. DNA 2, 15-22. Andersen, R. D., Buren, B. W., Taplitz, S. J., and Herschman, H. R. (1986). Rat metalIothionein-1 structural gene and three pseudogenes, one of which contains S-regulatory sequences. Mol. Cell. Biol. 6, 302-3 14. Andrews, G. K. (1990). Regulation of metallothionein gene expression. Prog. Food Nutri. Sci. 14, 193-258. Basu, A., and Lazo, J. S. (1990). A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drugs. Toxicol. Lett. 50, 123-l 35. Bettger, W. J., and O’Dell, B. L. (198 I). A critical physiological role of zinc in the structure and function of biomembranes. Life Sci. 28, 1425-l 438. Chomnynski, P., and Sac&i, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159. Collins, M. L., and Hunsaker, W. R. (1985). Improved hybridization assays employing tailed oligonucleotide probes: A direct comparison with 5’end-labeled oligonucleotide probes and nick-translated plasmid probes. Anal. Biochem El,21 l-224. Coppen, D. E., Richardson, D. E., and Cousins, R. J. (1988). Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole. Proc. Sot. Exp. Biol. Med. 189, lOO109. Crawford, B. D., Enger, M. D., Griffith, B. B., Griffith, J. K., Hanners, J. L., Longmire, J. L., Munk, A. C., Stall&s, R. L., Tesmer, J. G., Walters, R. A., and Hildebrand, C. E. (1985). Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: Implications for mechanisms regulating metallothionein gene expression. Mol. Cell. Biol. 5, 320-329.
323
De, S. K., McMaster, M. T., Dey, S. K., and Anclrews, G. K. (1989). Cellspecific metallothionein gene expression in mouse decidua and placentae. Development 107,6 1l-62 1. Dumam, D. M., Perrin, F., Gannon, F., and Palmiter, R. D. (1980). Isolation and characterization of the mouse metallothionein-I gene. Proc. Natl. Acad. Sci. USA 77,65 1 l-65 15. Eaton, D. L., and Toal, B. F. (1982). Evaluation of the Cd/hemoglobin affinity assayfor the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134-142. Furst, P., and Hamer, D. (1989). Cooperative activation of a eukaryotic transcription factor: Interaction between Cu(1) and yeast ACE1 protein. Proc. Natl. Acad. Sci. USA 86, 5267-527 1. Goering, P. L., and Klaassen, C. D. (1984a). Zinc-induced tolerance to cadmium hepatotoxicity. Toxicol. Appl. Pharmacol. 74,299-307. Goering, P. L., and Klaassen, C. D. (1984b). Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74,308-3 13. Goering, P. L., and Klaassen, C. D. (1984~). Tolerance to cadmium-induced toxicity depends on presynthesized metallothionein in liver. J. Toxicol. Environ. Health 14, 803-812. Hamer, D. H. (1986). Metallothionein. Ann. Rev. Biochem. S&913-951. Imbert, J., Culotta, V., Furs& P., Gedamu, L., and Hamer, D. (1990). Regulation of metallothionein gene transcription by metals. Adv. Inorg. Biochem. 8, 139- 164. Koropatnick, J., and Cherian, M. G. (1988). Exposure to different forms of cadmium in mice: Differences in metallothionein and alphafetoprotein mRNA induction in liver and kidney. J. Biochem. Toxicol. 3, 159- 172. Lee, D. Y., Brewer, G. J., and Wang, Y. (1989). Treatment of Wilson’s disease with zinc. VII. Protection of the liver from copper toxicity by zincinduced metallothionein in a rat model. J. Lab. Clin. Med. 114, 639645. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977). RNA molecular weight determination by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16,4743-475 1. Margoshes, M., and Vallee, B. L. (1957). A cadmium protein from equine kidney cortex. J. Am. Chem. Sot. 79,4813-4814. Mello-Filho, A. C., Chubatsu, L. S., and Meneghini, R. (1988). V79 chinesehamster cells rendered resistant to high cadmium concentration also become resistant to oxidative stress. Biochem. J. 256, 475-479. Onosaka, S., Keiichi, T., Doi, M., and Kunio, 0. (1978). A simplified procedure for determination of metallothionein in animal tissues. Eisei Kogaku 24, 128-131. Satoh, M., Naganuma, A., and Imura, N. (1988). Metallothionein induction prevents toxic side effectsof cisplatin and adriamycin used in combination. Cancer Chemother. Pharmacol. 21, 176-178. Sendelbach, L. E., and Klaassen, C. D. (1988). Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmiummetallothionein. Toxicol. Appl. Pharmacol. 92,95-102. Squibb, K. S., and Fowler, B. A. (1984). Intracellular metabolism and effects of circulating cadmium-metallothionein in the kidney. Environ. Health Perspect. 54, 31-35. Squibb, K. S., Pritchard, J. B., and Fowler, B. A. (1984). Cadmium-metallothionein nephropathy: Relationships between ultrastructural/biochemical alterations and intracellular cadmium binding. J. Pharmacol. Exp. Ther. 229,3 1 l-32 1. Thornalley, P. J., and Vasak, M. (1985). Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36-44.
AND
APPLIED
PHARMACOLOGY
112, 318-323 (1992)
Induction of Metallothionein by Cadmium-Metallothionein A Proposed Mechanism’ J. M. MCKIM, Department
of Pharmacology,
Toxicology,
in Rat Liver:
JR.,’ J. Lru, Y. P. LIU, AND C. D. KLAASSEN~ and Therapeutics,
University
of Kansas
Medical
Center.
Kansas
City, Kansas
66103
Received June 24, 199 1; accepted October 2, 199 1
with a high degree of homology (Andersen et al., 1986, 1983; Crawford et al., 1985; Durnam et al., 1980). Although a single physiologic function for MT has not been identified, the protein does appear to have a number macol. 112, 318-323. of roles in tissue. For example, MT has been implicated in Zn and Cu homeostasis and as a source of Zn for stabilizing Distribution of Cd to various organs following iv administration of CdC12 (3.5 mg Cd/kg) resulted in more than 43% of total biological membranes (Bettger and O’Dell, 1981). Its prestissue Cd accumulating in the liver. In contrast, after CdMT ence can decrease the hepatotoxic effects of cadmium (Goeradministration (0.5 mg Cd/kg), only 1% of the Cd was found in ing and Klaassen, 1984a,b,c), the cytotoxic and hepatotoxic liver. Rats administered CdC12 (1 .O mg Cd/kg) had hepatic MT effects of chemotherapeutic drugs such as cisplatin (Basu values 30-fold greater than controls and a hepatic Cd concen- and Lazo, 1990) and adriamycin (Satoh et al., 1988) scavtration of 17 pg/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than enge free radicals (Thornalley and Vasak, 1985; Abel and controls and a hepatic Cd concentration of 0.80 pg/g. However, Ruiter, 1989; Mello-Filho et al., 1988; Coppen et al., 1988), when hepatic MT levels were normalized to tissue Cd concen- and aid in the treatment of Wilson’s disease (Lee et al., 1989). On the basis of these possible functions, it is important to trations, induction of MT by CdMT was 5-fold greater than by CdC12. Northern and slot-blot analyses of mRNA showed that understand the molecular and cellular mechanisms that regulate MT production. The induction of MT by inorganic both CdCl, and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl, increase hepatic Cd is dose-dependent, and the amount of MT in tissues corMT by similar mechanisms. A dose-response increase in MT responds to tissue metal concentration. However, it appears produced by CdCl, indicated a biphasic response, with low doses that CdMT is more effective than CdC12 in inducing MT producing relatively more hepatic MT than higher doses. In ad- (Sendelbach and Klaassen, 1988; Koropatnick and Cherian, dition, the amount of MT produced per unit Cd after CdMT 1988). In the former study, MT was induced in a dose-detreatment wassimilar to those observed after low doses of CdC12 pendent manner after administration of CdC12 and CdMT. in the dose-response experiment. These data provide strong evHowever, when tissue MT levels were compared to tissue idence to support the conclusion that the apparent potency of Cd levels, CdMT appeared to be more potent than CdC& at CdMT observed here and in previous studies is most likely due inducing MT. In the later study, induction of hepatic MT to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concen- mRNA in mice was measured after CdC12 and CdMT adtrations. 0 1992 Academic Press, I~C. ministration. Induction of MT mRNA correlated well with cellular Cd concentrations following CdC12 ; however, CdMT appeared to produce peak mRNA levels independent of tissue metal concentration. These studies suggest that CdMT Since its discovery (Margoshes and Vallee, 1957) the may induce MT by a mechanism other than metal-stimustructure, function, and regulation of metallothionein (MT) lated transcription. have been extensively studied. MT is characterized by its Therefore, the purpose of the present study was to deterlow molecular weight (M, 6500-7000), absence of aromatic mine whether or not CdMT is a more potent inducer of MT amino acids or histidine, and high cysteine content (Hamer, than CdC12 in rat liver and to determine if the induction of 1986). In rodents, MTs comprise a gene family consisting hepatic MT can be attributed to the Cd associated with MT. of two coordinately regulated isoforrns (MT-I and MT-II) Induction of Metallothionein by Cadmium-Metallothionein in Rat Liver: A Proposed Mechanism. MCKIM, J. M., JR., LIU, J., LIU, Y. P., AND KLAASSEN, C. D. (1992). Toxicof. Appl. Phar-
MATERIALS
AND METHODS
’ This research was supported by U.S. Public Health Service Grant ES001142. Animals. Male Sprague-Dawley rats (300-400 g) were obtained from ’ Supported by U.S. Public Health Service Training Grant ES-07079. Sasco Inc. (Omaha. NE). All animals were housed in groups of three in 3 To whom all correspondence should be addressed. plastic cages and fed Ralston Purina pellet chow and water ad libitum for 318 0041-008X/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
INDUCTION
319
OF METALLOTHIONEIN
I week. Lighting was maintained on a 12-m light/dark cycle. The temperature was maintained between 2 1 and 22°C. Chemicals. Cadmium chloride was obtained from Sigma Chemical Co. (St. Louis, MO). Carrier-free ‘@‘Cdwas obtained from New England Nuclear (Boston, MA). Sephadex G-75 and DEAE anion exchange gels were obtained from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ). RNAzol-B was obtained from TELTEST, Inc. (Friendswood, TX). Ultrapure formamide and chloroform were from International Biotechnologies, Inc. (New Haven, CT). All other reagents used for RNA analysis were molecular biology grade from Sigma Chemical Co. Terminal deoxytransferase, CoCl,, tailing buffer, and DNA G-25 quick-spin columns were from Boehringer-Mannheim, Inc. (Indianapolis, IN). Zeta-probe nylon membrane was from Bio-Rad, Inc. (Richmond, CA). Deoxyadenosine 5’-[cu-32P]triphosphate, 6000 Ci/mmol, was from Amersham Corp. (Arlington Heights, IL). The oligonucleotide probes were prepared by Genosys Biotechnologies, Inc. (Woodlands, TX). Preparation of CdMT. Five rats were injected SCwith CdClz (3.0 mg Cd/kg and 2.0 PCi ‘@?Zd/kg)once daily for 4 days. The rats were decapitated and their livers removed. The same protocol was used when preparing unlabeled MT, but without the 2.0 pCi “‘Cd/kg. Preparation of the liver and isolation of the CdMT was done as described previously in this laboratory (Sendelbach and Klaassen, 1988). Following separation of MT-I and MTII isoforms by DEAE anion-exchange chromatography, the two fractions were desalted using Bio-Rad P6-DG columns and dried by lyophilization. The protein was resolubilized in 5-10 ml of ultrapure sterile water and the Cd content analyzed using a Perkin-Elmer Model 2380 flame atomic absorption spectrophotometer. In order to significantly increase the radioactivity associated with MT, the purified isoforms were incubated with lwCd (8.0 &i/ml) for 12 hr. The preparation was then desalted again, to remove unincorporated lmCd, and lyophilized. The final preparations were aliquoted and stored at -20°C. CdMT-II was used in all experiments. Organ distribution of CdCI, and CdMT. ‘09CdC12(3.5 mg Cd/kg) and ‘09CdMT-II (0.5 mg Cd/kg) were administered as a single iv injection. Two hours later the animals were decapitated and their organs excised for analysis. ‘09Cd was measured using a Packard Auto-gamma 5000 scintillation spectrometer. Tissue Cd concentrations were expressed as pg Cd/g tissue and were determined using the calculated specific activity of the dosing solutions. Induction of MT in liver by CdCI, and CdMT-ZZ. The ability of each Cd form to induce MT was determined by administering a single iv dose of either CdCl, (1 .O mg Cd/kg) or CdMT-II (0.4 mg Cd/kg). After 24 hr the animals were decapitated and the livers removed for analysis of MT. Total MT was measured by the Cd-hemoglobin binding assayof Onosaka et al. ( 1978) as modified by Eaton and Toal (1982). Hepatic Cd content was determined by atomic absorption spectrophotometry after digesting 1.O g of tissue in concentrated nitric acid at 80-90°C until clear, when unlabeled Cd was administered, or by gamma spectrometry when “‘Cd was used. RNA isolation. Total RNA was isolated from rats treated with CdC&, CdMT-II, or saline, using RNAzol-B as previously described (Chomczynski and Sacchi. 1987). Briefly, approximately 250 mg liver was flash frozen in liquid nitrogen and stored at -80°C. The liver was homogenized in a glass homogenizer with 5 ml RNAzol-B. The homogenate was transferred to a sterile 15-ml Falcon tube and 0.5 ml chloroform added. The remaining steps were exactly as described by the manufacturers of RNAzol. All solutions were treated with 0.1% DEPC prior to use. RNA yield, purity, and integrity were determined by absorbance at 260nm, AZ60/A280 ratios (1.7 to 1.9) and agarose/formaldehyde electrophoresis. Northern analysis of RNA. Preparation of RNA for electrophoresis, agarose gel preparation, and Northern transfer were done essentially as described by Lehrach er al. (1977) and De et al. (1989). Briefly, RNA (8 pg) was denatured in 20 mM 3-(N-morpholino)propanesulfonic acid (Mops), 5 mM sodium acetate, 1 mM EDTA, pH 7.0, 50% deionized formamide, and 2.2 M formaldehyde. The mixture was heated at 65°C for 15 min, then cooled on ice prior to loading a 1.2% (5 X 7.5 cm) agarose gel. Following electrophoresis, the gel was washed in deionized water or sodium chloridesodium citrate ( I X SSC) buffer (0.15 M and 0.0 15 M, respectively) prior to capillary transfer of RNA in 10X SSC to zeta-probe nylon membrane. Ri-
bosomal RNA bands were visualized by soaking the membrane in 0.5 M sodium acetate, pH 5.2, and 0.04% methylene blue dye for 3-5 min. The membrane was destained by soaking in ultrapure diethyl pyrocarbonate (DEPC)-treated water. The 28 S and 18 S rRNAs appear as dark blue bands. Gel loading, transfer efficiency, and RNA integrity could then be monitored by comparing the staining intensity of the 28 S and 18 S bands. Prior to loading slot-blots, RNA from each exposure group was analyzed by Northern analysis. The 28 S and 18 S rRNA bands were compared to ensure that the RNA concentration obtained from A 2M)resulted in equal amounts of RNA loaded. Slot-blot analysis. Slot-blot analysis of RNA was done using a Bio-Rad slot-blot vacuum apparatus. RNA (8 pg) was denatured with 0.5 ml of 10 mM NaOH containing 1 mM EDTA just prior to loading. Labeling Metallothionein probes. Oligonucleotide probes (20 mers) were prepared from 3’-nontranslated regions of known rat MT cDNA sequences in order to obtain probes specific for MT-I (‘5GAGGGCAGCAGCACTGTTCG-3’) (Andersen et al., 1986) and MT-II (‘5-ACACCATTGTGAGGACGCCC-3’) (R. D. Andersen, personal communication). The probes were 3’-end labeled (Collins and Hunsaker, 1985) with deoxyadenosine[a-“P]triphosphate (6000 Ci/mmol) using a DNA tailing kit (Boehringer-Mannheim, Inc.). The tailing reaction (15 ~1) contained 3.0 ~1 tailing buffer, 4.5 ~1 CoCl,, 3.4 pmol of probe. 45 pmol deoxyadenosine triphosphate (270 &i on reference date), and 55 units terminal deoxytransferase. The reaction was initiated with addition of enzyme and allowed to incubate for 25 min at 37°C. The reaction was stopped by adding 10 ~1 of ice cold 0.5 M EDTA. Unincorporated label was removed using G-25 Sephadex quick-spin columns (Boehringer-Mannheim, Inc.). This procedure yielded a tail length of approximately 5 bases with a specific activity of 8.0 X lo9 dpms/rg DNA. Hybridization of labeled probes. Prehybridization and hybridization reactions were identical for Northerns and slot-blots. Prehybridization (20 mM sodium phosphate, pH 7.0, 7% SDS. 20% formamide, 5X SSC, 5X Denhardt’s solution. 100 p&ml salmon sperm DNA, 250 p&ml yeasttRNA, and 5.0 @g/ml d[pAls or d[pA],) was at 47°C for 8-12 hr. Hybridization incubations were similar, except that the Denhardt’s solution was IX and the incubation was 18-20 hr. The membranes were washed at 47°C in 200 ml of 3X SSC and 2% SDS for 30 min and then washed four times subsequently with decreasing salt and increasing temperature to final conditions of0.5X SSC, 2% SDS at 55°C. Total wash time was 2.5 hr. Autoradiography was accomplished by exposing x-ray film (XAR-05, Kodak, Rochester. NY) using a high-plus intensifying screen at -80°C for the required time. Dose-response induction of MT. Rats (IV = 3/dose) received single iv injections via the tail vein of CdClz with lmCd (0.5 &i/ml) in 0.9% saline at 0.008, 0.022, 0.072, 0.21. 0.75, 2.4 mg Cd/kg. After 24 hr the animals were decapitated and their livers excised for analysis. MT levels were measured by the Cd-hemoglobin assay (Eaton and Toal, 1982) and Cd tissue levels were determined by quantitating the lmCd in 1 g portions on a Packard Auto-gamma 5000 scintillation spectrometer. Stutistics. Statistical comparisons were done using analysis of variance followed by Duncan’s multiple range test. Differences between treatments were considered significant at p < 0.05.
RESULTS The distribution of Cd, administered as inorganic or organic cadmium, to several organs is shown in Fig. 1. Two hours after CdC12 (3.5 mg/kg) administration, Cd accumulation was greater in the liver (34 pg/g) than kidney (17 pg/g). Following CdMT administration (0.5 mg Cd/kg), Cd was distributed primarily to the kidney (23 pg/g) with less than 1 pg/g present in the liver. The ability of CdClz and CdMT to induce MT synthesis in rat liver is shown in Fig. 2. CdClz ( 1.O mg Cd/kg) increased
320
MCKIM
m
CdC12
1
ICdMT
FIG. 1. Organ distribution of Cd following CdC12 and CdMT-II administration. Rats (N = 3 or 4) received 3.5 mg Cd/kg as CdClz or 0.5 mg Cd/kg as CdMT iv. Two hours after administration, various tissues were analyzed for Cd content. Values represent means + SEM.
MT to 295 pg/g, a 30-fold increase over control values (9.0 pg/g), while CdMT (0.4 mg Cd/kg) increased MT to 68 pg/g, a 7-fold increase over controls (top). Hepatic Cd concentration after administration of CdC& was 17 pg/g and 0.80 pg/g after CdMT (middle). When tissue MT values (top) were normalized to tissue Cd values (middle), inorganic cadmium produced about 17 Fg MT/pg Cd, whereas CdMT produced about 85 pg MT/pg Cd (bottom). Thus, it appeared that CdMT was more potent than CdCl* at inducing MT in rat liver. Metals are the primary inducers of MT. Therefore, it was important to determine if increases in MT following CdMT exposure would also cause an increase in MT mRNA. Northern analyses were done to determine probe specificity, mRNA size, and appropriate stringency conditions. The data in Fig. 3 clearly show that both MT-I and MT-II mRNAs are significantly increased over controls following both treatments. In order to quantitate MT mRNA in control rats and rats administered either CdClz or CdMT, slot-blot analysis (Fig. 4) was used. The inductive effects of both forms of Cd on MT mRNA and protein are summarized in Table 1. MTI and MT-II mRNAs were increased after CdC& and CdMT exposure. As depicted in Table 1, CdC& produced a 40-fold induction of MT-I and a 30-fold increase in MT-II mRNA, while CdMT increased MT-I and MT-II mRNA 6-fold over controls. Both forms of Cd increased MT mRNA and protein coordinately and to approximately the same extent. In addition, when MT mRNA data are normalized to liver Cd concentrations, the results were similar to those obtained for MT protein in Fig. 2 (bottom). Because it appeared that CdMT may be more efficient at inducing MT than CdC12 (Fig. 2, bottom), a dose-response experiment was carried out to determine whether or not tissue Cd concentrations, similar to those found in liver after CdMT exposure, can account for the observed increase in MT. When MT concentration in liver was compared to the dose of CdQ administered, a biphasic response was observed
ET AL.
(Fig. 5, top), indicating that low dosages of Cd produce pronounced increases in MT. However, Cd concentrations increased linearly in liver with dose (Fig. 5, middle). When hepatic MT concentrations were normalized to tissue Cd levels (Fig. 5, bottom), it was apparent that low amounts of Cd produce relatively larger increases in hepatic MT while higher amounts of Cd are less effective at increasing hepatic MT. When the amount of MT produced in the liver was compared to the concentration of Cd in the liver after each dose of CdClz (Fig. 6), the biphasic nature of the curve obtained in Fig. 5, (top) was retained. Moreover, the concentration of Cd in the liver following CdMT administration induced MT in a manner quantitatively similar to that observed after low doses of CdCl* (Fig. 6, solid circle). Hepatic Cd and MT concentrations are compared (Table 2) after administration of CdCl* at doses that gave higher and lower concentrations of Cd in the liver than the dose of CdMT used. As can be seen, CdMT produced an increase in MT proportional to the concentration of Cd present. These data provide additional evidence that induction of MT by
CdCIP
FIG. 2. Induction of hepatic MT following CdClz (1.0 mg/kg) and CdMT-II (0.4 mg/kg), top. Accumulation of Cd metal in liver following each Cd exposure, middle. MT induction expressed as pg MT/pg Cd, bottom. Values represent the mean + SEM of 3 or 4 animals. Asterisks show significant difference from controls @ > 0.05). Due to low variance within samples, some standard error bars are not visible.
INDUCTION
28s
-
18s
-
DYE
-
MT-I
MT-II
123
456
321
OF METALLOTHIONEIN MT-I CdMT
CON
Cd’&
MT-II CON
CdMT
CdC12
i
FIG. 3. Northern analysis of MT-I and MT-II mRNA. RNA (8 pg) from control (saline, lanes 1,4), CdMT-II (0.4 mg Cd/kg, lanes 2, 5) and CdC12 (1 .O mg Cd/kg, lanes 3,6) was loaded onto an agarose (1.2%) formaldehyde (2.2 M) gel and electrophoresed for 3 hr at 35 mA. Following electrophoresis, RNA was transferred to zeta-probe membrane. The membrane was cut in half, with one half being hybridized with MT-I and the other with an MT-II-specific oligo probe. After washing, the two halves were placed next to each other for autoradiography.
either form of Cd is most likely dependent on tissue metal concentration and, more importantly, that the relatively low concentration of Cd observed following CdMT administration can account for the observed increase in hepatic MT. DISCUSSION There is a marked difference in the distribution of Cd when administered as CdC& or CdMT. In the present study, after administration of CdC&, approximately 43% of the total tissue Cd was in the liver of rats. In contrast, only 1% of the measured tissue Cd was in the liver of rats after CdMT administration. Because CdMT is a potent renal toxicant (Squibb et al., 1984; Squibb and Fowler, 1984), only small doses can be given without producing renal toxicity to the animals. The combination of low doses of CdMT as well as the poor hepatic uptake of CdMT results in extremely low tissue Cd concentrations following a single iv dose of CdMT. Because more Cd distributes to the liver after CdC& than after CdMT administration, one would expect to find higher levels of MT in the livers of rats that received the inorganic form, as opposed to those that received the organic form of Cd. As predicted, rats treated with CdQ produced about fourfold more MT than rats treated with CdMT (Fig. 2). However, when the hepatic MT concentrations were normalized to tissue Cd concentrations, CdMT-treated rats produced fivefold more MT per unit Cd than did animals treated with CdC12. This was also true when MT mRNA was nor-
FIG. 4. Slot-blot analysis of MT-I and MT-II mRNA. RNA (8 rg) was loaded onto the Bio-Rad slot-blot apparatus and applied to the zeta-probe membrane by vacuum. Columns represent the three exposure groups, while each row represents RNA from a separate animal.
malized to liver Cd. Thus, CdMT is a more potent inducer of MT in the liver of rats as has been reported previously for the mouse (Sendelbach and Klaassen, 1988; Koropatnick and Cherian, 1988). In order to better understand why CdMT is a more potent inducer of MT than CdCl*, the transcriptional response to both inorganic and organic Cd was examined. MT mRNA for both isoforms was measured and compared to increases
TABLE 1 Summary of Metallothionein mRNA and Protein Induction by Organic and Inorganic Cadmium MT mRNA (cpm)” Treatment
Dose (w Cd/k)
Saline
NA
CdMT
0.4
I 109 f 27 (lJb 640a
41
422
1.0
4080
f 277 (401
12
211
f
(1) 11
68k
(6) 1170
f 1301
1
9f
(1)
(6) CdC12
MT Protein Wg liver)
II
I
(7) 80
295
i
18
(30)
a cpm values were obtained from slot-blot data in Fig. 3. All table values represent the means of four to six animals f SEM. b Values in parenthesis represent fold increase over controls.
322
MCKIM
ET AL. 800
1
soo-
P
c 1$
400.
3
300
5i
200: 100. 093 0
o/o @/
6
10
15
Cd @g/g
20
25
D
liver)
FIG. 6. MT produced by different liver Cd concentrations. The doseresponse values in Fig. 5 for liver MT (top) and liver Cd (middle) were used to prepare Fig. 6. The open circles represent the values from the CdCl, doseresponse curve. The solid circle depicts the liver MT and Cd concentrations obtained after CdMT administration. All values are means + SEM. Due to low variation between samples, some standard error bars are not visible.
0 O* 0.0
0.6
1.0
1.5
2.0
2.5
Dose (mg Cd/ kg) FIG. 5. Dose-response induction of MT by cadmium. Rats (N = 3) received a single iv dose of Cd (0.008,0.022, 0.072,0.21, 0.75, 2.4 mg Cd/ kg) and 24 hr later their livers were removed and analyzed for Cd and MT content. The top shows the induction of MT at each dose. The middle depicts the change in hepatic Cd concentrations over the doses used. The bottom represents MT concentration normalized to Cd concentration. All values represent a mean + SEM. Due to low variation between samples, some standard error bars are not visible.
in MT protein. The results clearly show that both MT-I and MT-II mRNA are coordinately increased by both CdMT and CdC& and that the increase in mRNA corresponds well with the increase in MT protein (Table 1). This information provides evidence showing that MT protein concentrations following exposure to both Cd forms correlate with increased MT mRNA. This is especially plausible because it is generally accepted that the mechanism by which metals induce MT is via enhanced transcription (reviewed by Hamer, 1986 and Andrews, 1990). Thirefore, there now appears to be a connection between metal content and MT production following CdMT administration. The transcriptional information discussed above does not provide an explanation for the higher potency of CdMT than CdC12 to induce MT (Fig. 2). It is well known that inorganic Cd induces hepatic MT in a dose-dependent manner; however, these studies used relatively high doses of the metal (Sendelbach and Klaassen, 1988). In the present study, we
examined the ability of low doses of Cd to induce hepatic MT. Interestingly, a biphasic response was observed (Fig. 5). Production of MT at low doses (0.008 to 0.20 mg Cd/kg) was relatively greater than at higher doses (0.20 to 2.4 mg Cd/kg). It therefore appears that small amounts of Cd are comparatively more effective than large amounts at inducing MT. Moreover, it is clear from Fig. 6 and Table 2 that, following CdMT administration, both tissue Cd and MT concentrations fall within the linear portion of the curve established using CdC12. These data suggest that the Cd present in the liver after CdMT exposure would be sufficient to produce the observed levels of MT. The biphasic nature of the dose-response curve implies that metal-induced transcription of MT may be mediated by a cytosolic protein that binds Cd cooperatively and is saturable. This would enable the cell to produce a large amount of mRNA in response to a small amount of metal. A system of this type has been described for a metallothionein-like protein in the yeast Saccharomyces cerevisiae (Furst and Hamer, 1989). The existence of trans-acting proteins that mediate transcriptional activity of MT in higher eukaryotes, following
TABLE 2 Hepatic Cadmium Concentration Versus Metallothionein Concentration Treatment
Dose (m8 CWcs)
CdCIZb CdMT CdC12
0.20 0.40 0.02
Cd liver)”
MT (he/g liver)
4.0 ?I 0.22 0.8 f 0.05 0.4 f 0.04
164 + 15 68-1- 7 27+ 6
(&g
’ Values represent the mean f SEM of three to four animals. b Cd& values taken from a dose-response experiment (Fig. 5).
INDUCTION
OF METALLOTHIONEIN
Cd and Zn exposure, has been reported (reviewed by Andrews, 1990; Imbert et al., 1990). These studies provide strong evidence for the existence of cellular protein components that bind metals, become activated, and then bind to metal regulatory regions on the MT gene, thereby increasing transcription. It is possible that Cd carried to the liver by CdMT is passed to these metal-binding factors or that all or part of the CdMT peptide may serve as the carrier protein. The existence of this type of regulatory system could explain why, in the present study, extremely low concentrations of Cd had a more pronounced effect on MT production than did higher doses. The results of the present study strongly suggest that the induction of MT by CdMT is due to the metal associated with the protein. Further, these data indicate that the apparent potency of CdMT in inducing MT, when normalized to liver Cd, is most likely due to the low concentrations of Cd present in the tissue, which are relatively more efficient at inducing MT than higher concentrations. REFERENCES Abel, J., and Ruiter, N. (1989). Inhibition of hydroxyl-radical generated DNA degradation by metallothionein. Toxicol. Lett. 47, 19 1- 196. Andersen, R. D., Buren, B. W., Ganz, T., Piletz, J. E., and Herschman, H. R. (1983). Molecular cloning of the rat metallothionein-1 (MT-I) mRNA sequence. DNA 2, 15-22. Andersen, R. D., Buren, B. W., Taplitz, S. J., and Herschman, H. R. (1986). Rat metalIothionein-1 structural gene and three pseudogenes, one of which contains S-regulatory sequences. Mol. Cell. Biol. 6, 302-3 14. Andrews, G. K. (1990). Regulation of metallothionein gene expression. Prog. Food Nutri. Sci. 14, 193-258. Basu, A., and Lazo, J. S. (1990). A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drugs. Toxicol. Lett. 50, 123-l 35. Bettger, W. J., and O’Dell, B. L. (198 I). A critical physiological role of zinc in the structure and function of biomembranes. Life Sci. 28, 1425-l 438. Chomnynski, P., and Sac&i, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159. Collins, M. L., and Hunsaker, W. R. (1985). Improved hybridization assays employing tailed oligonucleotide probes: A direct comparison with 5’end-labeled oligonucleotide probes and nick-translated plasmid probes. Anal. Biochem El,21 l-224. Coppen, D. E., Richardson, D. E., and Cousins, R. J. (1988). Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole. Proc. Sot. Exp. Biol. Med. 189, lOO109. Crawford, B. D., Enger, M. D., Griffith, B. B., Griffith, J. K., Hanners, J. L., Longmire, J. L., Munk, A. C., Stall&s, R. L., Tesmer, J. G., Walters, R. A., and Hildebrand, C. E. (1985). Coordinate amplification of metallothionein I and II genes in cadmium-resistant Chinese hamster cells: Implications for mechanisms regulating metallothionein gene expression. Mol. Cell. Biol. 5, 320-329.
323
De, S. K., McMaster, M. T., Dey, S. K., and Anclrews, G. K. (1989). Cellspecific metallothionein gene expression in mouse decidua and placentae. Development 107,6 1l-62 1. Dumam, D. M., Perrin, F., Gannon, F., and Palmiter, R. D. (1980). Isolation and characterization of the mouse metallothionein-I gene. Proc. Natl. Acad. Sci. USA 77,65 1 l-65 15. Eaton, D. L., and Toal, B. F. (1982). Evaluation of the Cd/hemoglobin affinity assayfor the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66, 134-142. Furst, P., and Hamer, D. (1989). Cooperative activation of a eukaryotic transcription factor: Interaction between Cu(1) and yeast ACE1 protein. Proc. Natl. Acad. Sci. USA 86, 5267-527 1. Goering, P. L., and Klaassen, C. D. (1984a). Zinc-induced tolerance to cadmium hepatotoxicity. Toxicol. Appl. Pharmacol. 74,299-307. Goering, P. L., and Klaassen, C. D. (1984b). Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74,308-3 13. Goering, P. L., and Klaassen, C. D. (1984~). Tolerance to cadmium-induced toxicity depends on presynthesized metallothionein in liver. J. Toxicol. Environ. Health 14, 803-812. Hamer, D. H. (1986). Metallothionein. Ann. Rev. Biochem. S&913-951. Imbert, J., Culotta, V., Furs& P., Gedamu, L., and Hamer, D. (1990). Regulation of metallothionein gene transcription by metals. Adv. Inorg. Biochem. 8, 139- 164. Koropatnick, J., and Cherian, M. G. (1988). Exposure to different forms of cadmium in mice: Differences in metallothionein and alphafetoprotein mRNA induction in liver and kidney. J. Biochem. Toxicol. 3, 159- 172. Lee, D. Y., Brewer, G. J., and Wang, Y. (1989). Treatment of Wilson’s disease with zinc. VII. Protection of the liver from copper toxicity by zincinduced metallothionein in a rat model. J. Lab. Clin. Med. 114, 639645. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977). RNA molecular weight determination by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16,4743-475 1. Margoshes, M., and Vallee, B. L. (1957). A cadmium protein from equine kidney cortex. J. Am. Chem. Sot. 79,4813-4814. Mello-Filho, A. C., Chubatsu, L. S., and Meneghini, R. (1988). V79 chinesehamster cells rendered resistant to high cadmium concentration also become resistant to oxidative stress. Biochem. J. 256, 475-479. Onosaka, S., Keiichi, T., Doi, M., and Kunio, 0. (1978). A simplified procedure for determination of metallothionein in animal tissues. Eisei Kogaku 24, 128-131. Satoh, M., Naganuma, A., and Imura, N. (1988). Metallothionein induction prevents toxic side effectsof cisplatin and adriamycin used in combination. Cancer Chemother. Pharmacol. 21, 176-178. Sendelbach, L. E., and Klaassen, C. D. (1988). Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmiummetallothionein. Toxicol. Appl. Pharmacol. 92,95-102. Squibb, K. S., and Fowler, B. A. (1984). Intracellular metabolism and effects of circulating cadmium-metallothionein in the kidney. Environ. Health Perspect. 54, 31-35. Squibb, K. S., Pritchard, J. B., and Fowler, B. A. (1984). Cadmium-metallothionein nephropathy: Relationships between ultrastructural/biochemical alterations and intracellular cadmium binding. J. Pharmacol. Exp. Ther. 229,3 1 l-32 1. Thornalley, P. J., and Vasak, M. (1985). Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36-44.
