Hepatic isometallothioneins in mice: Induction in adults and postnatal ontogeny

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

104,267-275

(1990)

Hepatic lsometallothioneins in Mice: Induction in Adults and Postnatal Ontogeny’ WILLIAMC.KERSHAW,~ Department

LOISD. LEHMAN-MCKEEMAN,~

ofPharmacology, Toxicology 39th Street and Rainbow

Received

September

ANDCURTIS

D. KLAASSEN

and Therapeutics, University of Kansas Medical Boulevard, Kansas City, Kansas 66103

25, 1989; accepted January

Center,

29, 1990

Hepatic Isometallothioneins in Mice: Induction in Adults and Postnatal Ontogeny. KERW. C., LEHMAN-MCKEEMAN, L. D., AND KLAASSEN, C. D. (1990). Toxicol. Appl. Pharmacol. 104, 267-275. The purpose of this study was to quantitate hepatic metallothionein-I (MT-I) and metallothionein-II (MT-II) in adult mice pretreated with various dosages of selected inorganic and organic compounds and in nonchemically treated neonatal mice. Male CF- 1 mice received Zn (0.38-6.0 mmol/kg, SC),Cd (5-80 nmol/kg, SC),dexamethasone (10-1000 rmol/ kg, SC),or ethanol (60- 180 mmol/kg, PO). Liver cytosol was prepared 24 hr after the administration of each compound. In another experiment, liver cytosols were prepared from male and female neonates 1 to 35 days after parturition. MT-I and MT-II in liver cytosols were isolated by high-performance anion-exchange chromatography and quantitated by atomic absorption spectrometry. Hepatic MT-I and MT-II concentrations in adult controls were 5.1 + 1.3 and 3.7 -C 1.O &g liver, respectively. All compounds increased hepatic MT levels in a dose-dependent manner over a narrow range of dosages. The lowest dosages of Zn, Cd, dexamethasone, and ethanol that produced a significant increase in total MT content (MT-I plus MT-II) were 0.38, 0.005,0.3, and 90 mmol/kg, respectively. Maximal induction of total MT following the highest dosages of Zn, Cd, ethanol, and dexamethasone was 58, 34,24, and 13 times the control value (8.8 f 2.4 pg total MT/g liver), respectively. The relationship between dose and hepatic MT content was linear following ethanol administration and log-linear following Zn, Cd, and dexamethasone administration. The ratio of MT-I/MT-II was approximately 2.4 following all dosages of metals. Following low and high dosages of organic compounds, the ratio of MT-I/MT-II was approximately 1.O and 1.5, respectively. Total MT concentration in livers of I- to lCdayold mice was approximately 40 times that observed in adult liver (5.5 + 1.6 pg total MT/g liver) and returned toward adult levels 2 1 days after parturition. The ratio of MT-I/MT-II was approximately 1.8 during Postpartum Days 1 through 14 and thereafter decreased to approximately 1.O.These results indicate that MT-I is more abundant than MT-II in mouse liver following chemical exposure and during neonatal development. o 1990 Academic PKS, IIIC. SHAW,

Metallothionein (MT) is a cytosolic, cysteinerich, metal-binding protein present in most animal tissues and has been implicated in the

maintenance of Zn and Cu homeostasis as well as in the detoxication of Cd (for reviews see Webb, 1987; Kagi and Kojima, 1987; Bremner, 1987). In mammals, MT exists as a group of isoforms that differ slightly in amino acid sequence and net negative charge. Two major isoforms have been isolated in rodent tissues and are referred to as MT-I and MT-II on the basis of their elution positions during anion-exchange chromatography. MTs are

’ Supported by U.S. Public Health Grant ES-01 142. 2 Supported by U.S. Public Health Training Grant ES07079. Current address: Pfizer Central Research, Groton, CT. 3 Current address: Miami Valley Laboratories, Procter & Gamble Co., Cincinnati, OH. 267

004 1-008X/90

$3.00

Copyright 0 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.

268

KERSHAW,

LEHMAN-McKEEMAN,

inducible proteins as demonstrated by dramatic increases in rat liver MT content following the administration of metals (Bremner and Davis, 1975, Shaikh and Smith, 1976) or organic compounds such as ethanol (Bracken and Klaassen, 1987) and dexamethasone (Quinones and Cousins, 1984). High levels of MT have also been detected in liver of neonatal rats (Wong and Klaassen, 1979). Previous characterization of MT induction has been performed predominately in rats by means of analytical techniques that do not measure the concentration of individual isoproteins. Consequently, little information exists concerning the induction of MT isoproteins in mice. Recently, a method for the rapid quantitation of MT-I and MT-II was developed (Lehman and Klaassen, 1986) and employed to assess iso-MT levels in rats. These recent investigations demonstrated that hepatic MT-II is more abundant than MT-I in nontreated adults, adults treated with Zn or dexamethasone (LehmanMcKeeman and Klaassen, 1987; LehmanMcKeeman et al., 1988a), and nontreated neonates (Lehman-McKeeman et al., 1988b). In mice, only mRNA levels of MT-I and MTII have been quantitated and indicate that the relative amounts of MT isoproteins in mice and rats differ substantially. Specifically, MTI mRNA is more abundant than MT-II mRNA in the livers of metal- or glucocorticoid-treated mice (Searle et al., 1984) as well as in cultured mouse Hepa cells treated with the same class of compounds (Yagle and Palmiter, 1985). Thus, it appears that MT-I predominates in mouse whereas MT-II is more abundant in rat. These observations suggest significant qualitative differences in the regulation of iso-MT induction between mice and rats. However, this conclusion is tentative as the concentrations of MT isoproteins in mice have not been reported. The major objective of this study was to determine the hepatic concentrations of MT-I and MT-II isoproteins in mice under conditions associated with elevated MT levels such

AND KLAASSEN

as chemical exposure and postnatal development. In this manner, mouse MT isoprotein levels can be compared with information concerning the molecular regulation of mouse MT genes. The method of Lehman and Klaassen (1986) was employed to determine the concentration of each isoprotein. However, this method was developed originally for the quantitation of MTs in rat tissue. Accordingly, the appropriateness of this method for measuring mouse MT-I and MTII was also examined. METHODS Chemicals. Chloride salts of Zn and Cd were purchased from Fisher Scientific Co. (Fairlawn, NJ). Ethanol and dexamethasone sodium phosphate were obtained from Aaper Alcohol and Chemical Co. (Shelbyville, KY) and Merck Sharpe & Dohme Research Laboratories (West Point, PA), respectively. Carrier-free lwCd (1 Ci/mg) was obtained from New England Nuclear (Boston, MA). Tris(hydroxymethyl)aminomethane (free base) was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were ofthe highest purity commercially available. Animals and treatments. Male and female CF-I mice (20-25 g) and male Sprague-Dawley rats (225-250 g) were purchased from Sasco, Inc. (Omaha, NE), and were housed in hanging plastic cages containing corncob bedding (Paxton Processing Co., Paxton, IL). Animal facilities were maintained at 25 + 2°C with a 12-hr light cycle (0600- 1800). Animals had free access to tap water and Rodent Laboratory Chow No. 5 100 (Ralston Purina Co., St. Louis, MO) and were acclimated to housing facilities for 1 week prior to use. For the purpose of breeding, male and female mice were housed together (1 male:3 females) for 1 week and pregnant mice were allowed to go to parturition. Dams and pups were housed together throughout the 35day experiment. In the dose-response studies, Zn (0.38-6.0 mmol/kg), Cd (5-80 pmol/kg), or dexamethasone (IO-1000 pmol/ kg) was administered SCto male mice (25-30 g) as solutions in 0.90% saline. Additional male mice weregavaged with ethanol (60- 180 mmol/kg) dissolved in distilled water. Control animals received either 5 ml 0.90% saline/ kg, sc or 10 ml distilled water/kg, po. Liver cytosol was prepared 24 hr after experimental or vehicle treatment. In another experiment, liver cytosol of male and female offspring was prepared 1,4,7, 10, 14,2 1,28, and 35 days after partutition. Tissue preparation for HPLC analysis. Livers were excised from mature and immature mice after cervical dislocation and decapitation, respectively, and placed into

HEPATIC

ISOMETALLOTHIONEINS

ice-cold 10 mM Tris-HCl (pH 7.4 at room temperature). Livers were homogenized in 2 vol of the same buffer using a glass-Teflon tissue grinder. Homogenates were centrifuged (lO,OOOg for 10 min) and the resulting supematants centrifuged (100,OOOg for 1 hr) to obtain cytosol. Livers of I - to 1O-day-old mice were pooled from two to seven pups and cytosol was prepared. Cytosol from mice 14 days of age and older was prepared from livers of individual animals. Analysis of MT-Z and MT-II by HPLC coupled with atomic absorption spectrometry (AAS). MT-I and MT-II were quantitated in liver with the HPLC-AAS method of Lehman and Klaassen (1986). Chromatography was performed on an anion-exchange column (DEAE-5PW, 7.5 cm X 7.5 mm; Waters Associates, Milford, MA). MTI and MT-II were eluted with a linear gradient of TrisHCI mobile phase (solvent A: 10 mM Tris-HCl, pH 7.4; solvent B: 200 mM Tris-HCl, pH 7.4) from 0 to 40% solvent B in 12 min at a flow rate of 1 ml/min. Elution of proteins was monitored by measuring the uv absorbance at 2 14 nm. The atomic absorption of Cd was determined in Cd-saturated MT-I and MT-II by connecting the outlet of the ultraviolet detector ofthe HPLC system directly to the nebulizer uptake capillary of an atomic absorption spectrophotometer (Perkin-Elmer, Model 2380, Norwalk, CT). Integration of Cd-containing peak areas was performed with an IBM computer (Model 9000) and the chromatography applications program (Version 1.3, Danbury, CT). Total MT concentration was determined by summation of MT-I and MT-II values. Purified MT-I and MT-II for use as standards were ob tained from livers of Cd-treated adult rats by a modilication of the method of Lehman and Klaassen (I 986). The modified method is described below under validation of methods. Freeze-dried MT-I and MT-II were reconstituted in distilled water and the protein concentration of the solutions was determined by the Kjeldahl method for the determination of nitrogen as described by Lang (1958). MT standards were prepared for HPLC analysis by the addition of 50 ~1 Cd (1000 ygCd/ml) to 950 pl MT solutions. In the dose-response studies, MT solutions contained 346 pg MT-I and 377 pg MT-II/ml, whereas in the ontogeny studies, MT solutions contained 379 pg MT-I and 369 pg MT-II/ml Mouse liver cytosol was incubated with 35 ppm Cd (final concentration) at room temperature for 15 min, placed in a boiling water bath for I mitt, and then centrifuged (10,OOOg for 3 min). Dilutions (1:3, 1:6, 1:12, 1:25, and 1:50) of Cd-saturated MT standards (100 ~1) or Cd-saturated, heat-treated mouse liver cytosol(25 to 500 ~1) were injected onto the anion-exchange column. MT-I and MT-II were eluted and detected as described above. Validation of the HPLC-AAS methodfor the analysis ofMT-Z and MT-II in mice. For validation purposes, the chromatographic and Cd-binding properties of rat and mouse MT-I and MT-II were compared. Purified hepatic MT-I and MT-II were prepared from male rats and mice

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by a modification of the method described by Lehman and Klaassen (1986). Animals were injected with a solution of Cd& (3 mg Cd/kg, SC) 1,2, and 3 days prior to sample collection. In addition, each animal received a single ip injection of “?d (1 &i/kg, ip) 12 hr prior to sample collection. Liver homogenates were prepared in 10 mM Tris-HCl, pH 7.4 (1: 1, w/v), and centrifuged (lO,OOOg for 10 min). The resulting supematants were heated for 5 min at 80°C and centrifuged (30,OOOg for 20 min). Heat-treated 30,OOOgsupematants (20 ml) were applied to Sephadex G-75 (Sigma) gel-filtration columns (60 X 2.6 cm) equilibrated with 10 mM Tris-acetate buffer. Fractions of 5 ml were collected and assayed for radioactivity using an auto-gamma scintillation counter (Packard, Model 5130, Downers Grove, IL). The Cdcontaining peak with a retention coefficient of 2.0 to 2.2 was considered to represent total MT (I&assert, 1978). Rat and mouse total MTs obtained by gel-filtration chromatography were applied to Sephadex A-25 (Sigma) anion-exchange columns (40 X 2.6 cm) and MT-I and MTII eluted with Tris-acetate (pH 7.4) using a linear gradient of 10 to 240 mM. Gel-filtration and anion-exchange chromatographies were performed at 4°C with a flow rate of 30 ml/hr. Solutions (2.5 ml) of MT-I and MT-II were applied to Sephadex G-25 columns (PD-10 column, Pharmacia Fine Chemical Co., Piscataway, NJ) and eluted with 3.0 ml of 10 mM Tris-HCI (pH 7.4). Desalted MT solutions were lyophilized and stored at -70°C. Reconstituted freeze-dried MT-I and MT-II standards from rats and mice were subsequently assayed for protein content by the Kjeldahl method and analyzed by the HPLCAAS method. The amount of Cd required to fully saturate MT-I and MT-II contained in mouse liver cytosol was also assessed. Cytosols were prepared 24 hr alter Zn treatment (3000 rmol Zn/kg, SC),incubated with Cd at various concentrations, and analyzed by the HPLC-AAS method as described previously except that the atomic absorption of Cd and Zn was measured. Stafistics. The 0.05 level of probability was used as the criterion of significance. Hepatic concentrations of MTI and MT-II were compared by the Student t test. Dunnett’s t test was utilized to determine whether total MT concentration in control adult liver was significantly different from hepatic MT values observed in neonates or chemical-treated adults (Steel and Torrie, 1960).

RESULTS

Validation of the HPLC-AAS method for the analysis of MT-I and MT-II in mice. Chromatographic properties of rat and mouse hepatic MT (total and &forms) were identical when each protein was isolated on

KERSHAW.

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50

100

ELUTION

150

200

250

VOLUME

(ml)

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FIG. 1. Chromatographic properties of rat and mouse Cd-induced MT. All animals received 3 mg Cd/kg, sc once daily for 3 consecutive days prior to sample collection. In addition, each animal was injected with radiolabeled Cd (I pCi/kg, ip) 12 hr prior to sample collection. Liver cytosol was prepared and applied to gel-filtration columns as described under Materials and Methods. Total MT and its isoforms were identified as peaks containing radioactive Cd. Total MT from rat (A) and mouse (B) eluted in 95 to 130 ml 10 mM Tris-acetate (pH 7.4). Fractions containing total MT were pooled and applied to glass anion-exchange columns and iso-MTs were separated using a linear gradient of 10 to 240 mM Tris-acetate (pH 7.4). Rat (C) and mouse (D) MT-I eluted in 85 to 110 ml buffer. Rat and mouse MT-II eluted in 170 to 200 ml buffer.

gel-filtration (Figs. 1A and B) and anion-exchange columns (Figs. 1C and D). Furthermore, retention times of Cd-saturated, purified MT-I and MT-II standards on the HPLC anion-exchange column were 7.5 and 10.4 min, respectively, regardless of whether mouse or rat liver was the source of the protein. The Cd-containing peak areas of mouse MT-I and MT-II standards measured during HPLC-AAS analysis were similar to those produced by rat MT-I and MT-II (Fig. 2). These results demonstrate the extensive homology between rat and mouse MTs.

AND KLAASSEN

Cd saturation of MTs in standards and samples is a prerequisite for the HPLC-AAS analysis. Saturation. of Zn-induced MTs in samples occurred at a final concentration of 35 ppm Cd as demonstrated by the complete displacement of Zn from MT-I and MT-II (Fig. 3). Final concentrations of Cd greater than 35 ppm did not increase the Cd-containing peak areas of either isoprotein (data not shown), which provides additional evidence that Cd saturation of MTs in samples occurs at 35 ppm. Induction of hepatic MT-I and MT-II in mice. Induction of MTs by Zn, Cd, ethanol, and dexamethasone in mature male mice was studied. Only the highest dosage of each compound was lethal, as approximately 25% of the exposed population died by 24 hr after treatment. Hepatic MT-I and MT-II concentrations of saline-treated mice did not differ from those noted in water-treated mice. Consequently, both control groups (N = 16) were employed to estimate constitutive levels of MT-I (5.1 * 1.3 pg/g wet liver) and MT-II (3.7 + 1.0 fig/g wet liver) and total MT (8.8 + 2.4 pg/g wet liver). The lowest dosages of Zn, Cd, and dexamethasone at which total

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HEPATIC

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FIG. 3. Disappearance of Zn from MT-I and MT-II following Cd saturation in vitro. Pooled liver cytosol was prepared from five adult male mice 24 hr after the administration of 3000 rmol Zn/kg, sc. Cytosols were incubated with various concentrations of Cd for 15 min at room temperature, heated for 1 min in boiling water, and centrifuged (10,OOOg X 3 min). The resulting supematants containing 7.9 pg MT-I and 4.0 pg MT-II were injected onto the anion-exchange column of the HPLCAAS apparatus. Aspiration of Zn standard (0.5 ppm Zn) into the atomic absorption spectrometer resulted in an absorbance units full scale value of 0.05.

hepatic MT concentration was significantly greater than the control value were 380, 5, and 300 pmol/kg, respectively (Fig. 4) whereas a much higher dosage of ethanol (90 mmol/kg) was required to significantly increase total MT concentration. Maximum induction of total MT concentration following the highest dosages of Zn, Cd, ethanol, and dexamethasone was 58, 34, 24 and 13 times the control value, respectively. The administration of each compound increased hepatic MT-I and MT-II concentrations in a dose-dependent manner over a narrow range of dosages (Fig. 5). The dose-response relationship was linear following ethanol administration and log-linear following Zn, Cd, and dexamethasone administration. Following administration of Zn or Cd, the concentration of MT-I was approximately 2.4 times greater than that of MT-II at all dosages. Mean MT-I/MT-II ratios ranged in value from 2.0 to 2.2 and from 2.4 to 3.3 in Zn- and Cd-treated mice, respectively. Ethanol and

271

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dexamethasone administration increased hepatic MT-I and MT-II contents to similar extents following all but the highest dosages where approximately 1.5 times more MT-I was noted. In control animals, the hepatic concentration of MT-I was not statistically different from that of MT-II. In a separate experiment, constitutive levels of MT-I and MT-II in livers of l- to 35day-old male and female mice were measured. No sex differences in MT levels were observed (data not shown). Consequently, in this experiment mean iso-MT values as well as total MT values were calculated from data combined from both sexes. As indicated in Fig. 6A, total hepatic MT concentration during the first 10 days following parturition was approximately 40 times greater than that observed in the adult mouse (35 days of age). Three weeks after parturition total MT content in liver approached the low levels noted in the adult (5.5 -t 1.6 pg/g wet liver). During the first 14 days after parturition the hepatic concentration of MT-I was approximately I .8 times greater than that of MT-II; thereaf-

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FIG. 4. Concentration oftotal MT in livers ofneonates, adults, and chemically treated adult mice. Total MT concentration in livers of chemical and vehicle-treated mice was estimated by the summation of MT-I and MT-II values presented in Fig. 5. Hepatic concentration of total MT in neonates was calculated from mean values noted on Postpartum Days 1, 4, 7, and 10 in Fig. 6. Standard errors not shown were too small for graphic representation. Asterisks indicate that total MT concentration is significantly different from that measured in adult mouse liver (p < 0.05).

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FIG. 5. Chemical-induced elevation of MT-I and MTII in mouse liver. Adult male mice received Zn (0.38-6.0 mmol/kg, SC),Cd (5-80 pmol/kg, SC),ethanol (60-180 mmol/kg, PO), or dexamethasone (lo- 1000 pmol/kg, SC) and MT levels were determined 24 hr thereafter. Results represent the means f SE of four to eight mice. Standard errors not shown were too small for graphic representation. Asterisks indicate dosages at which concentrations of MT-I and MT-II were significantly different (p < 0.05).

ter, the concentrations of MT-I and MT-II were not significantly different (Fig. 6B). Mean MT-I/MT-II ratios ranged in value from 1.5 to 2.0 on Days 1 to 14.

AND IUAASSEN

Lehman and Klaassen (1986) was employed to measure MTs and was validated prior to use since it was used originally to measure MTs in rat tissue. Quantitation of MT-I and MT-II by the HPLC-AAS method is dependent on the chromatographic and Cd-binding properties of each isoprotein. There were no fundamental differences between mouse MT-I and rat MT-I or between mouse MT-II and rat MTII with respect to these properties. This was evident by the elution volume of each protein during purification (Fig. 1) as well as the retention times and Cd-containing peak areas of purified standards on the HPLC-AAS system (Fig. 2). The observation that the chromatographic and Cd-binding properties of MT are identical between mice and rats was not unexpected, as the amino acid sequences of the two isoproteins are highly homologous in

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The primary objective of this investigation was to evaluate the induction of MT-I and MT-II isoproteins in mouse liver. It has been shown previously that metal and glucocorticoid pretreatment causes MT-I mRNA to a0 cumulate to a greater extent than MT-II mRNA in the livers of mice (Searle et al., 1984) as well as in cultured mouse cells (Yagle and Palmiter, 1985). However, a thorough and quantitative evaluation of mouse MT isoprotein induction has not been reported to date. The HPLC-AAS method of

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DAYS FIG. 6. Concentrations of total MT and the isoforms of MT in mourn liver during postnatal development. Liver cytosol of male and female mice was prepared on selected days, 1 to 35 days after parturition as described under Materials and Methods. Results represent the means f SE of six to seven pooled or individual samples. Standard errors not shown were too small for graphic representation. Asterisks indicate the age at which hepatic MT-I and MT-II levels were significantly different (p < 0.05).

HEPATIC

ISOMETALLOTHIONEINS

these species (Winge et al., 1984, Huang et al., 1977 and 198 1). The few amino acid substitutions that occur do not involve residues (cysteine) that directly participate in the binding of metals. Thus, these validation studies indicate that the HPLC-AAS method of Lehman and Klaassen (1986) is directly applicable to the measurement of MT-I and MT-II in mouse liver. The results of the present study indicate that in mice, MT-I is the more abundant MT isoform. Hepatic MT-I content was significantly greater than MT-II in immature mice (Fig. 6B), in adult mice treated with various dosages of Zn and Cd, and in adults treated with high dosages of dexamethasone and ethanol (Figs. 5A-D). However, hepatic levels of MT-I and MT-II were not significantly different in untreated adult mice or in adults treated with low dosages of ethanol or dexamethasone. Thus, despite the high degree of homology between the isoforms in mice and rats, the abundance of and the ratio of MT-I and MT-II in mice differ dramatically from those noted in rats. Specifically, MT-II is more abundant than MT-I in the livers of Znand dexamethasone-treated adult rats as well as control adults and neonates, whereas the hepatic concentrations of MT-I and MT-II are equivalent in Cd-treated rats (LehmanMcKeeman and Klaassen, 1987; LehmanMcKeeman et al., 1988a,b). Marked differences in the relative amounts of MT-I and MT-II in mice and rats are particularly surprising considering the genetic similarities between these species. Why are the relative concentrations of MT-I and MT-II in mice and rats so different? In an attempt to answer this question it must be recognized that the concentration of a nonsecreted protein, such as MT, is controlled by the rates at which it is synthesized and degraded. These regulatory processes have been examined in Zn-treated rats where preferential accumulation of hepatic MT-II resulted from a higher rate of synthesis and a lower rate of degradation relative to that of MT-I (Lehman-McKeeman et al., 1988~).

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When maximal MT synthesis occurred (6 to 9 hr post-Zn injection), MT-II mRNA was two times more abundant than MT-I mRNA. Consequently, the high rate of MT-II synthesis in rat (relative to MT-I) can be partially explained by higher levels of translatable mRNA (MT-II mRNA versus MT-I mRNA). Since neither synthesis nor degradation rates of MT-I and MT-II have been measured in mice, cogent comparisons between species cannot be conducted. However, it seems reasonable to speculate that the abundance of MT-I in mice results from a higher rate of synthesis in view of the relative abundance of translatable MT-I mRNA in mouse tissues. The discrepancy between mice and rats with respect to relative &o-MT mRNA levels is particularly significant because it suggests that regulatory processes prior to translation of MT differ markedly in these species. Relative efficacies and potencies of selected inorganic and organic compounds to induce total MT in mouse liver can be assessedin the present investigation. Samples were collected 24 hr following compound administration to allow each treatment to produce maximum induction of MTs (Etzel et al., 1979; Waalkes et al., 1984; Olafson, 198 1). The rank order of efficacy (magnitude of MT induction) was Zn > Cd = ethanol > dexamethasone and the rank order of potency (lowest dosage associated with MT induction) was Cd % dexamethasone = Zn $ ethanol (Fig. 4). These same rank orders apparently hold true for chemical-induced accumulation of total MT in rat liver although the data are from different laboratories and were obtained using different methodologies (Bremner and Davis, 1975; Shaikh and Smith, 1976, Quinones and Cousins, 1984; Bracken and Klaassen, 1987). Therefore, chemical-induced accumulation of total MT in liver appears to be quite similar in mice and rats, despite the fact that the proportions of MT-I and MT-II differ dramatically. Livers of neonatal mice contained about 40 times more total MT than that noted in adult mice, and as previously mentioned the

274

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predominant isoform was MT-I. The observation concerning total MT elevation is consistent with the finding that MT mRNA levels in neonatal mouse liver are considerably higher than those found in the adult mouse (Ouellette, 1982; Andrews et al., 1984; Quaife et al., 1986). Hepatic MT content in neonatal rats is also much higher than that in adult rats (Wong and Klaassen, 1979), with MT-II being the most prevalent isoform in developing rat liver (Lehman-McKeeman et al., 1988b). Thus, the differences in the relative proportions of MT isoproteins observed in chemically treated adult mice and rats are also seen in developing animals. Another significant difference between mouse and rat MTs concerns the total amount that accumulates in developing liver. Peak levels of hepatic MTs in developing rats are approximately 1000 pg MT/g liver on Postpartum Day 7 (Lehman-McKeeman et al., 1988b). In contrast, peak levels of MTs synthesized in developing mouse liver are much lower at approximately 275 pg MT/g liver. We are unable to provide an adequate explanation for this phenomenon. In summary, the HPLC-AAS method of Lehman and Klaassen (1986) is directly applicable to the quantitation of mouse MT-I and MT-II. MT-I is more abundant than MT-II in mouse liver following chemical exposure and during neonatal development. Despite the extensive homology between mouse and rat MTs, the induction pattern identified in mouse is clearly different from that in rats, in which MT-II is the predominant isoform. ACKNOWLEDGMENT The authors thank Ms. Deepa Satsangi for technical assistance.

AND KLAASSEN

BRACKEN, W. M., AND KLAASSEN, C. D. (1987). Induction of hepatic metallothionein by alcohols: Evidence for an indirect mechanism. Toxicol. Appl. Pharmacol. 81,257-263.

BREMNER. 1. (1987). Nutritional and physiological significance of metallothionein. In Experientia Supplementurn. Vol. 52. Metallolhionein (J. H. R. Kagi and Y. Kojima, Eds.), pp. 8 I- 107, Birkhauser Verlag, Basel. BREMNER, I., AND DAVIES, N. T. (1975). The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem. J. 149,733-738. ETZEL, K. R., SHAPIRO, S. G.. AND COUSINS. R. J. (1979). Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone. Biochem. Biophys. Res. Commun. 89,11201126. HUANG, I.-Y., KIMURA, M., HATA, A., TSUNOO, H., AND YOSHIDA, A. (198 1). Complete amino acid sequence of mouse liver metallothionein-II. J. Biochem. 89,1839-1845.

HUANG, I.-Y., YOSHIDA, A., TSUNOO, H., AND NAKAJIMA, H. (1977). Mouse liver metallothioneins: Complete amino acid sequence of metallothionein-I. J. Biol. Chem. 252,82 17-822 1. MAGI. J. H. R., AND KOJIMA. Y. (1987). Chemistry and Biochemistry of Metallothionein. In Experientia Supplementum. Vol. 52, Metallothionein (J. H. R. Kagi and Y. Kojima, Eds.), pp. 25-61, Birkhauser Verlag, Basel. KLAASSEN, C. D. (1978). Effect of metallothionein on hepatic disposition of metals. Amer. J. Physiol. 234, E47-E53. LANG, C. A. ( 1958). Simple microdetermination of Kjeldahl nitrogen in biological materials. Anal. Chem. 30, 1692-1694.

LEHMAN, L. D., AND KLAASSEN, C. D. (I 986). Separation and quantitation of metallothioneins by high performance liquid chromatography coupled with atomic absorption spectrometry. Anal. Biochem. 236, 610614.

LEHMAN-MCKEEMAN, L. D., AND KLAASSEN, C. D. ( 1987). Induction of metallothionein-I and metallothionein-II in rats by cadmium and zinc. Toxicol. Appi. Pharmacol.

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