Cadmium-induced inhibition of hepatic drug oxidation in the rat: Time dependency of tolerance development and metallothionein synthesis

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

64,42-5

(1982)

1

Cadmium-Induced Inhibition of Hepatic Drug Oxidation in the Rat: Time Dependency of Tolerance Development and Metallothionein Synthesis A. ROBERTS’

STANLEY Department of Pharmacology University, West Lafayette, College of Pharmacy,

AND R. CRAIG SCHNELL'

and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue Indiana 47907: and Department of Pharmacodynamics and Toxicology, University of Nebraska Medical Center, Omaha, Nebraska 68105

Received

September

9, 1981;

accepted

January

.?I, 1982

Cadmium-Induced Inhibition of Hepatic Drug Oxidation in the Rat: Time Dependency of Tolerance Development and Metallothionein Synthesis. ROBERTS, S. A., AND SCHNELL, R. C. (1982). Tonicol. Appl. Pharmacol. 64, 42-51. Experiments were undertaken to determine if a correlation existed between the time-dependent synthesis of hepatic metallothionein and the development of tolerance to the inhibition of hepatic drug oxidation elicited by cadmium in the male rat. Maximal rates of metallothionein synthesis were achieved within 2 hr after administering cadmium in a 0.2 1 mg Cd/kg (ip) dose. The total hepatic concentration of metallothionein, as well as the cadmium binding capacity of this protein, also increased rapidly with maximal values being observed from 8 to 67 hr after cadmium administration. Despite this rapid increase in hepatic metallothionein levels, the tolerance to the inhibition of in vivo drug oxidation induced by a challenge cadmium (0.84 mg Cd/kg) dose did not develop until 16 hr after treatment with the 0.21 mg/kg cadmium dose. Furthermore, hepatic metallothionein levels decreased from a maximum at 67 hr to approximately control levels at 336 hr. Although the tolerance to the inhibition of drug oxidation also decreased from a maximum at 72 hr a modest degree of protection was maintained even at 336 hr. Pretreatment with the 0.21 mg Cd/kg dose increased the hepatic uptake of a ‘%d challenge dose (0.84 mg Cd/kg). This increase was associated with an increased ““Cd binding to metallothionein in the cadmium-pretreated animals. While these data are suggestive of a role for metallothionein in tolerance development, the lack of correlation of the time course of metallothionein synthesis with the development of the tolerance would suggest that factors in addition to metallothionein may also participate.

It has been demonstrated that prior cadmium (Cd) treatment diminishes the toxic effects of subsequently administered cadmium challenges. This tolerance exists for cadmium-induced testicular necrosis (Ito and Sawachi, 1966; Nordberg, 197 l), acute lethality (Leber and Miya, 1967; Webb and

Verschoyle, 1976; Probst et al., 1977b), and the inhibition of insulin secretion (Yau and Mennear, 1977). In addition, the prior administration of cadmium doses that are too low or alter drug metabolism have been shown to produce a tolerance to the cadmium-induced inhibition of oxidative xenobiotic metabolism (Roberts et al., 1976; Yoshida et al;., 1976). It has been suggested that the cadmium binding protein, metallothionein, serves a protective role in these tolerance phenomena by sequestering, and thereby, rendering the subsequent challenge cadmium dose biolog-

’ Present Address: Preclinical Safety Assessment, Sandoz, Incorporated, East Hanover, N.J. 07936. 2 To whom correspondence should be sent: Dr. R. Craig Schnell, Department of Pharmacodynamics and Toxicology, College of Pharmacy, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebr. 68 105. 0041-008X/82/070042-10$02.00/0 Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

42

CADMIUM-INDUCED

TOLERANCE

AND METALLOTHIONEIN

ically inert (Nordberg, 1971; Leber and Miya, 1976; Squibb et al., 1976; Probst et al., 1977b; Schnell et al., 1979). To examine this hypothesis we have conducted a series of experiments to determine if a correlation exists between the time-dependent synthesis of metallothionein and the development of the tolerance to the inhibition of oxidative drug metabolism produced by cadmium. METHODS Animals. Male, Sprague-Dawley-derived rats (230 to 280 g) were obtained from Laboratory Supply Company (Indianapolis, Ind.). The animals were housed in stainless-steel community cages for at least 7 days prior to use. Food (Wayne Lab Blox, Allied Feed Mills, Chicago, Ill.), and tap water were provided ad libitum. Chemicnls. All chemicals were of reagent grade. Cadmium and sodium acetates (Fisher Scientific, Fairlawn, N.J.), hexobarbital sodium (Winthrop Laboratories, New York, N.Y.), and cadmium-109 acetate (New England Nuclear, Boston, Mass.) were prepared in double-distilled, deionized water, and [ l-‘sS]cystine (New England Nuclear), was prepared to 0.1 N HCI. Pharmacological response. The duration of hypnosis induced by hexobarbital(100 mg/kg, ip) was measured as the time elapsing from the loss of the righting reflex until the animal could successfully right itself from a supine position twice within 30 sec. Drug biotransformation. Hepatic oxidative drug metabolism was assessed utilizing the 105,OOOg,,, microsomal pellet, (Means et al., 1979). Ethylmorphine-Ndemethylase was determined by measuring the formation of formaldehyde following the modified procedure of Anders and Mannering (1966). Aniline hydtoxylase was determined by measuring the formation of p-aminophenol according to the procedure of Imai et al. ( 1966). The metabolism of hexobarbital was determined according to the method of Brodie et al. (1953) by measuring the hexobarbital remaining after the incubation period. Sephadex chromatography paric metallothionein. The

and quantitation

of he-

hepatic cytosol(105,OOOg,,, supernatant fraction) was obtained by ultracentrifugation in a Beckman L5-65 ultracentrifuge utilizing a type 30 rotor (30,000 rpm fat 60 min. 4’C). A 5.0-ml aliquot of the cytosol was fractionated over 80 cm of Sephadex G-75 in a Pharmacia K26/100 column (Pharmacia, Piscataway, N.J.) at 4°C. The proteins were eluted with TBAN buffer (20 mM Tris-HCI, pH 8.6; 5 mM 2-mercaptoethanol; 0.02% sodium azide; 50 mM NaCI) under gravity Aow (40 cm) at a rate of 35 to 40 ml/hr.

SYNTHESIS

43

The column was calibrated with blue dextran (MW = 2,OOO,OOO,daltons), hemoglobin (64,500 daltons), chymotrypsinogen (25,000 daltons), ribonuclease ( 13,700 daltons), and insulin (5733 daltons) according to the method of Andrews (1965). Metallothionein concentrations were determined according to the procedures described by Probst et al. (1977a). Concentrations of the protein were determined indirectly by measuring the cadmium bound to the metallothionein. Cadmium in the chromatographic profile was determined by atomic absorption spectrometry in a Perkin-Elmer 2908 atomic absorption spectrometer (Norwalk, Conn.) calibrated with certified cadmium standards (Fisher Scientific Co.) Two measures of hepatic metallothionein, “nonsaturated cadmium metallothionein” and “total metallothionein,” were made. Following the in vivo induction of metallothionein with the 0.21 mg/kg cadmium dose, the metallothionein is only partially saturated with cadmium. The remaining metal binding sites are predominantly occupied by zinc (Probst er al., 1977a). These metallothionein concentrations are based upon cadmium content, and the values are referred to as “nonsaturated cadmium metallothionein.” “Total hepatic metallothionein” was estimated by adding 2.0 rmol of cadmium to a 6.0-ml aliquot of the cytosol sample (Probst et al., 1977a). Since cadmium can effectively displace zinc from the protein. the metal binding sites become cadmium saturated, and, therefore, indirectly represent total metallothionein concentration. The difference between the total metallothionein levels and the nonsaturated cadmium metallothionein levels represent the capacity of metallothionein to bind a challenge cadmium dose and is termed the “cadmium-binding capacity.” Determination sis. Male rats

of kinelics

of melallothionein

synthe-

received 0.21 mg Cd/kg at intervals from 0 to 336 hr prior to the administration of a dose of [I“Slcystine (500 &/kg; 41.24 Ci/nmol, ip). The control group of animals (designated as 0 hr) did not receive any cadmium treatment. Animals were killed by decapitation I5 min after receiving the [ l-“Sjcystine, and the livers were excised and homogenized (33%, w/v) in ice-cold sucrose buffer (250 mM sucrose; 20 mM TrisHCI, pH 8.6; 5 mM 2-mercaptoethanol). The cytosol (105,OOOg,., supernatant fraction) was stored at -20°C until chromatography. Preliminary experiments indicated the maximal uptake of [ 1-‘SS]cysteine into hepatic metallothionein occurred within 15 min. The chromatographic profile of ‘$S concentration was assessed by liquid scintillation spectrometry. (Packard C2425 liquid scintillation spectrometer). A 1.O-ml aliquot of each chromatographic fraction was transferred to a scintillation vial containing 0.3 ml of 70% perchloric acid. Following mixing by gentle swirling, 0.6 ml of 3tY% hydrogen peroxide was added. The vials were tightly capped and heated at 70°C for 5 hr. After cooling to room temperature, 10 ml of 2-ethoxyethanol and 10 ml

44

ROBERTS

AND SCHNELL

I

FIG. 1. Distribution of j5S activity in hepatic cytosol protein. Male rats received 0.21 mg Cd/kg (ip) 8 hr prior to the administration of [ l-35S]cystine (500 &i/ kg, ip). The animals were killed 15 min later and the livers were removed; the cytosol was obtained and fractionated utilizing Sephadex G-75 chromatography as described under Methods. The data are presented as dpm [35S]cysteine per 1.O-ml aliquot of each chromatographic fraction as described under Methods. The [ ‘5S]cysteine incorporated into metallothionein (VJ I’,, = 1.58 to 2.00) is represented.

counting efficiencies for the whole homogenates (85.4%), microsomes (97.4%), and cytosol (78.0%) were determined by internal standardization procedures. The data are expressed as nanograms of Cd/per milligram of protein. A 3.0-m] aliquot of hepatic cytosol was fractionated utilizing Sephadex G-75 chromatography. A 1.O-ml aliquot from each chromatographic fraction was wet oxidized using the perchloric acid-hydrogen peroxide technique with gentle heating. Vials were prepared and counted for total radioactivity. The apparent counting efficiency (89.4%) was determined by internal standardization. The data are expressed as nanograms of Cd bound per milligram of cytosol protein to high- (Ye/ V, = 0.92 to 1.25) or intermediate- (VJV, = 1.26 to 1.57) molecular-weight proteins or to metallothionein (V,/VO = 1.58 to 2.00). A representative chromatographic profile of the “‘Cd levels is presented in Fig. 2. Determination of protein concentrations. The protein concentrations of the tissue or subcellular fractions were determined by a modification of the procedure of Lowry et al. (1951) with cystalline bovine serum albumin as the standard. Statistical analysis. The data were analyzed by analysis of variance followed by application of the NewmanKeuls test where appropriate (Anderson and McLean,

of scintillation mixture (0.6% PO in toluene( were added to the samples. The apparent counting efficiency (50.4%) was determined by internal standardization. The decay of ?S was corrected according to the method of Wang and Willis (1965). The data are expressed as pmoles cysteine incorporated into metallothionein ( VJ V, = 1.58 to 2.00/mg cytosol protein). A representative chromatographic profile of the “S concentration is presented in Fig. 1. The recovery of ‘5S from the column was greater than 99%. Determination of ‘09Cd binding to hepatic microsomal and cysiosol subcellular fractions and to metallothionein. Male rats received either 1.23 mg sodium acetate/kg (ip) or 0.21 Cd/kg 72 hr prior to the ad-

ministration of a challenge 0.84 mg Cd (50 PCi ‘09Cd)/ kg dose. The animals were killed by decapitation at intervals of 1, 3, 6, 9, 12, or 72 hr after the challenge dose. The livers were rapidly excised and homogenates (33%) were prepared in sucrose-TKMB (250 mM sucrose; 20 mM Tris-HCI, pH 8.6; 5 mM KCI; 5 mM MgCL; 5 mM 2-mercaptoethanol) buffer. Washed microsomal and cytosolic samples, obtained by ultracentrifugation as previously described, were then stored at -20°C. The whole homogenate (0.1 ml), microsome (0.5 ml), or cytosol (0.1 ml) samples were wet oxidized as previously described using perchloric acid and hydrogen peroxide with gentle heating. Scintillation solution and 2-ethoxyethanol were added to the samples, and these were counted for total radioactivity. The apparent

0.92

1.25

1.58

2.00

ve/vo

FIG. 2. Cytosolic distribution of a ‘09Cd challenge dose (0.84 mg Cd/kg, 50 &i/kg, ip) 3 hr prior to death. The livers were removed and the cytosol was obtained and fractionated utilizing Sephadex G-75 chromatography as described under Methods. The data are expressed as ng jWCd per l.O-ml aliquot of each fraction as described under Methods. The binding of ‘09Cd to high ( V,/VO = 0.92 to 1.25) or intermediate- (VJV,, = 1.26 to 1.57) molecular weight fractions or to metallothionein (V./V,, = 1.58 to 2.00) is presented.

CADMIUM-INDUCED

TOLERANCE

AND

METALLOTHIONEIN

a,b I M

a& 7-

a,b.c T

a

a.b

0

r

144

240

a

0 r

INTERVAL

0

16

BETWEEN

ADMINISTRATION

I8

OF TOLERANCE

72

AND CHALLENGE

a

1

1

CONTROL

336

TOLERANCE

72

144

DOSES

(hr)

FIG. 3. Time-dependent development of Cd-induced tolerance to prolongation of hexobarbital Male rats received either 1.23 mg sodium acetate/kg (ip) or a 0.21 mg Cd/kg iip) tolerance the indicated intervals prior to the administration of a Cd challenge (0.84 mg/kg. ip) dose. The of hexobarbital (100 mg/kg, ip) hypnosis was determined 72 hr following the challenge. The presented as X + SEM for six to eight rats per group. Means that are significantly (p < 0.05) do not have any letters in common.

1974). The acceptable lished at p < 0.05).

level

of significance

was estab-

RESULTS The minimum time period required for a subthreshold cadmium (0.21 mg/kg) dose to produce tolerance to the potentiation of hexobarbital hypnosis induced by the challenge cadmium (0.84 mg/kg) dose was 14 hr (Fig. 3). Furthermore, the degree of tolerance produced was maximal at 72 hr. Although the tolerance appeared to decrease with time, a partial tolerance was still observed, even when the subthreshold cadmium dose was administered 336 hr prior to the challenge dose. The development and the duration of this tolerance were also evaluated by examining the time course for the tolerance to the inhibition of hepatic microsomal drug oxidation. Tolerance to the inhibition of hexobarbital, ethylmorphine, or aniline microsomal

45

SYNTHESIS

DOSE ALONE

hypnosis. dose at duration data are different

metabolism was not present at 12 hr but developed by I6 hr (Fig. 4). As previously observed with the in vivo drug response, tolerance was maximal at 72 hr but decreased thereafter (Fig. 5). The time-dependent synthesis of hepatic metallothionein was evaluated utilizing the incorporation of [35S]cysteine into the protein following the administration of a 0.21 mg Cd/kg dose (Table 1). The maximal rate (619% increase above control levels) of synthesis occurred within 2 hr. The rate of metallothionein synthesis was increased over the first 24 hr after Cd treatment and returned to approximately control levels at 39 hr. Total metallothionein concentrations and cadmium-binding capacity increased rapidly and were maximal from 8 to 67 hr but remained above control values from 67 to 240 hr. By 336 hr after cadmium administration, however, these levels returned to approximately control values. The hepatic distribution of a challenge

ROBERTS

AND SCHNELL

the “‘Cd was observed in the cadmium-pretreated, tolerant animals as compared to the nontolerant, control animals at all time periods. Microsomal levels of the “‘Cd increased to maximal values at 3 hr for both nontolerant and tolerant animals (Table 2); however, these levels were onty 15% higher in the nontolerant animals. A rapid decrease in the “‘Cd levels was observed from 3 to 6 hr with

b

c

r ‘1 d

b

(10001

b

b

f=l

0

(455)

(6371

(55

I)

(m4)

(6051

::

b.c WOOL CONTROL

0

8

12

INTERVAL BETWEEN ADMINISTRATION TOLERANCE AND CHALLENGE DOSES

OF (hr)

FIG. 4. Development of Cd-induced tolerance to inhibition of hepatic drug metabolism. Male rats received 1.23 mg sodium acetate/kg (ip) or a 0.21 mg Cd/kg (ip) challenge dose (0.84 mg/kg, ip). The animals were killed 72 hr after the challenge and the microsomal metabolism of the various substrates was determined as described. The data are presented as i It SEM for six rats per group. Means that are significantly (p < 0.05) different do not have any letters in common.

ro9Cd dose (0.84 mg Cd/kg; 50 &i/kg, ip) was examined in both nontolerant controls (1.23 mg NaAc/kg, 72 hr prior) and Cdpretreated (0.21 mg/kg, 72 hr prior) tolerant animals as a function of time. Hepatic uptake of the “‘%Zd challenge dose occurred rapidly with maximal concentrations achieved within 6 to 9 hr in the livers of both nontolerant and tolerant animals (Table 2). Hepatic levels of Cd were then observed to decline from 9 to 72 hr in both treatment groups. An increased uptake (10 to 30%) of

0 OONTROL

0

72

144

240

INTERVAL BETWEEN ADMINISTRATION TOLERANCE AND CHALLENGE DOSES

336 OF (hr)

FIG. 5. Duration of cadmium-induced tolerance to inhibition of hepatic drug metabolism. Male rats received either 1.23 mg sodium acetate/kg (ip) or a 0.21 mg Cd/kg (ip) tolerance dose at various intervals prior to the administration of a Cd challenge dose (0.84 mg/ kg, ip). The animals were killed 72 hr following the challenge and the microsomal metabolism of the various substrates was determined as described. The data are presented as i + SEM for six rats per group. Means that are significantly (,o < 0.05) different do not have any letters-in common

CADMIUM-INDUCED

TOLERANCE

AND

METALLOTHIONEIN

TABLE TEMPORAL

Hours after cadmium administration

KINETICS

3.7 23.1 12.6 10.8 7.7 8.3 12.1 4.1 4.1 3.7 4.7 2.5

0 2 4 8 12 16 24 39 67 144 240 336

1

OF CADMIUM-INDUCED

Rate of protein synthesis (pm01 [%]cysteine incorporated/mg cytosol protein)

HEPATIC

METALLOTHIONEIN

Non-Cd-saturated metallothionein (ng Cd/mg cytosol protein)

(loo) (619) (336) (289) (206) (221) (324) (109) (109) (99) (125) (67)

47

SYNTHESIS

SYNTHESIS Metallothionein cadmiumbinding capacity (ng Cd/mg cytosol protein) -_-

Metallothionien concentrations (ng Cd/m cytosol protein)

0 24 24 44 24 42 34 42 44 48 28 28

96 188 188 296 337 304 274 297 310 210 234 118

96 163 164 252 313 262 239 255 266 162 206 90

(100) (196) (197) (309) (352) (318) (286) (311) (324) (220) (245) (123)

( 100 I (171) (172) (263) (328) (274) (250) (2671 (278) (170) (217) (9.5)

Note. Male rats (four per group) received a 0.21 mg Cd/kg tolerance dose (ip) either 0 (no treatment 1 or at the indicated time intervals prior to the administration of [I-%]cystine (500 pCi/kg, ip). The animals were killed 15 min later and the hepatic cytosol was obtained, pooled, and fractionated by Sephadex G-75 chromatography. The incorporation of [%]cysteine into metallothionein, and total concentrations and Cd binding capacity of the protein were determined as described. Metallothionein Cd-binding capacity was determined as the difference between non-Cd-saturated metallothionein and Cd-saturated metallothionein. The percentage of 0-hr control values is presented in parentheses,

a lesser rate of decrease continuing to 72 hr in both the nontolerant and tolerant rats. Cytosolic levels of the ‘09Cd rapidly in-

creased in both the nontolerant and tolerant animals and were maximal in both groups within 6 hr (Table 2). At all time periods

TABLE SUBCELLULAR

DISTRIBUTION

OF ‘@‘Cd WITHIN

THE HEPATOCYTE

Total hepatic and subcellular Time after challenge (hrj I 3 6 9 12 12

Whole homogenate Nontolerant 60+ 96 f 94 k 101 t 87 ?I 86 IT

I” 9”.+’ I 10.6 12’J 5-Q 9E.b

Tolerant 84 It 12” 105* 8”” 103 rt 13”” 134 * 316 106 t 9a.b 93 + 7O.b

2

cadmium

IN TOLERANT concentration

AND NONTOLERANT

(ng Cd/mg

protein)

Microsomes Nontolerant 41 63 3, 21 20 14

-+ It + + 2 k

36 5 4b.=.d 30bC 04”’ 0”

RATS

Cytosol Tolerant 33 2 8C.d 43 jl 34 27 1 40.b.c 25 + 73’ 17 c 2.J 14 It 1”

Nontolerant 163 269 325 341 335 352

-+ + + 2 k k

9 13”,* 20**,’ IV, 1746’ 14’,“’

--Tolerant 248 339 357 382 409 325

t + * 2 5 +

37” 36’,“< 3Ob.C 2’ 29 lPb,’

Note. Male rats received ip administrations of either 1.23 mg sodium acetate/kg (nontolerant) or 0.21 mg Cd/kg (tolerant) 72 hr prior to the administration of 0.84 mg ‘“Cd/kg (50 &i/kg. ip). The animals were killed 1, 3. 6, 9, 12, or 72 hr later. the hepatic subcellular fractions isolated, and the “‘%Zd concentrations of each were determined as described under Methods. Five animals were utilized per group. The data are presented as 2 -+ SEM. Groups of means (within each subcellular fraction) that are significantly (p < 0.05) different do not have any letter in common.

48

ROBERTS

AND

SCHNELL

TABLE DISTRIBUTION

OF ‘@Cd WITHIN

THE HEPATIC

3

CYTOSOL

PROTEINS

Distribution of cadmium (% of Cd binding

Time after challenge 6-j 1 3 6 9 12 12

High

molecular

weight

Nontolerant

Tolerant

64.0 40.4 7.7 5.2 3.0 1.8

29.2 14.9 5.0 3.5 1.9 1.7

IN TOLERANT

binding in hepatic cytosol in cytosol protein fractions)

Intermediate molecular weight Nontolerant 5.4 4.3 1.0 0.9 0.1 0.6

AND NONTOLERANT

Tolerant 4.2 2.1 0.8 0.8 0.7 0.6

RATS

proteins

Metallothionein Nontolerant 30.6 55.2 91.3 94.0 96.2 97.6

Tolerant 66.6 83.0 94.1 95.7 97.4 91.7

Note. Male rats received an ip administration of either 1.23 ng sodium acetate/kg (nontolerant) or 0.21 mg Cd/kg (tolerant) 72 hr prior to the administration of 0.84 mg “‘%d/kg (50 rCi/kg ip). The animals were killed 1, 3, 6, 9, 12, or 72 hr later, and the hepatic cytosol was isolated and pooled, prior to fractionation with Sephadex G-75 chromatography and the ‘Yd concentrations were determined as described and expressed as a percentage within the cytosol is designated as Cd bound to high of Cd bound per total cytosol Cd. The distribution of “%d ( V,/V, = 0.92 to 1.25) or intermediate (VJ V,, = 1.26 to 1.57) molecular weight macromolecules or to metallothionein (V./V, = 1.58 to 2.00). These percentage values are derived from total cytosolic cadmium presented in Table 2.

examined, the total cytosolic cadmium was greater in the tolerant animals; in particular, the total uptake of the “‘Cd into the cytosol was 152 and 126% greater at I and 3 hr, respectively. Sephadex G-75 fractionation of the cytosol demonstrated that the increased cytosol uptake of the ‘09Cd dose in the tolerant animal was primarily the result of an increased metallothionein binding of the metal (Table 3). In the tolerant animal at 1 and 3 hr, 66.6 or 83.0%, respectively, of the total cytosol cadmium was bound to metallothionein; whereas, in the nontolerant animal, only 30.6 and 55.2% of the total cytosol cadmium was metallothionein bound. For the nontolerant animals, a majority of cytosolic cadmium was bound to high and intermediatemolecular-weight proteins at 1 hr following administration of the ‘09Cd. Thereafter, this cadmium was redistributed to metallothionein. After 6 hr, greater than 90% of the total cytosolic cadmium was bound to me-

tallothionein in both the tolerant tolerant animals.

and non-

DISCUSSION Although the biological function of metallothionein has not been fully established, several interesting speculations concerning the possible metabolic role(s) of the protein in the regulation of zinc metabolism (Richards and Cousins, 1975a, b; 1976) or in the circulatory transport of cadmium (Nordberg, 1972) have been proposed. It has also been suggested that metallothionein can function as an adaptive agent in ameliorating acute or chornic cadmium toxicity (Nordberg, 1971; Webb, 1972a; Leber and Miya, 1976; Squibb et al., 1976; Probst et al., 1977b; Schnell et al., 1979). Leber and Miya (1976) and Probst et al., (1977b) have demonstrated a tolerance to acute cadmium lethality as evidenced by an increased LD50 to cadmium in male mice pretreated with nonlethal cadmium doses.

CADMIUM-INDUCED

TOLERANCE

AND

This decrease in cadmium lethality was positively correlated with a dose-dependent increase in hepatic metallothionein levels (Probst et al., 1977b). Previous studies have demonstrated that the cadmium-induced tolerance to the inhibition of hepatic oxidative drug metabolism by a subsequent larger cadmium dose (Roberts et al., 1976; Yoshida et al., 1976; Roberts and Schnell, 198 1) is also correlated with increased levels of hepatic metallothionein (Roberts and Schnell, 198 I). Mechanistically, it has been proposed that metallothionein concentrations are increased by the initial subthreshold cadmium dose, and that the subsequent cadmium challenge dose is sequestered by the protein, thus reducing the availability of thecadmium challenge to critical sites and thereby reducing the toxicity of the metal (Nordberg, 1971; Webb, 1972b,; Leber and Miya, 1976; Squibb et al., 1976; Probst et al., 1977b). The data presented in this manuscript demonstrate that the time dependency of metallothionein synthesis is not completely consistent with the development of the tolerance phenomenon to the cadmium-induced inhibition of drug oxidation. The minimum time period required for the subthreshold cadmium (0.21 mg/kg) dose to produce a tolerance to the potentiation of hexobarbital hypnosis or to the inhibition of microsomal drug oxidation by cadmium was approximately 16 hr. However, the cadmium binding capacity of metallothionein was increased to 263% of control values by 8 hr. Furthermore, this tolerance was observed to decrease from the maximum at 72 hr even though the subthreshold cadmium dose still provided a partial protection against the inhibition of drug oxidation (both hexobarbital hypnosis and microsomal metabolism) when administered 336 hr prior to the challenge cadmium dose. However, hepatic levels of metallothionein were observed to decrease from the maximum at 67 hr to control levels by 336 hr.

METALLOTHIONEIN

SYNTHESIS

49

It has been previously reported that the tolerance to cadmium-induced lethality (Webb and Verschoyle, 1976; Yoshikawa, 1970) and the inhibition of drug metabolism (Yoshida et al., 1976) are diminished when the cadmium pretreated animals are challenged 7 or more days later. Webb and Verschoyle (1976) have reported that hepatic metallothionein concentrations are similar whether examined 1 or 10 days following cadmium pretreatment and have proposed that the metallothionein induced by this pretreatment, does not, per se, play a significant role in the tolerance to cadmium lethality. Instead, these investigators have suggested that the cadmium-pretreated animaI has a greater ability to synthesize new metallothionein in response to the challenge Cd dose. This new metallothionein, in turn, sequesters the cadmium challenge. However, to estimate quantitatively the total metallothionein levels by determining cadmium bound to the protein, it is essential that the protein be cadmium saturated (Probst et al., 1977a). Since Webb and Verschoyle (1976) have not reported that their metallothionein was cadmium saturated, their reported estimates of protein concentration may not be strictly quantitative and thus, interpretation is difficult. Administration of the subthreshold cadmium dose 72 hr prior to the Cd/kg challenge dose produced a trend of increased hepatic uptake of the challenge dose from 1 to 9 hr after the challenge dose. The administration of the subthreshold cadmium dose decreased microsomal uptake and increased cytosolic uptake of the challenge cadmium dose from 1 to 3 hr. One hr following administration of the challenge dose, the percentage of the total ‘“‘Cd binding to both high- and intermediate-molecularweight proteins was greater in nontolerant animals. However, as the ‘09Cd binding to those proteins decreased at 3 hr in the nontolerant animals, only 30.6 or 55.2% of the cytosolic cadmium was bound to metallo-

50

ROBERTS

AND SCHNELL

thionein. In contrast, metallothionein bound a major percentage (66.6 or 83.0%) of the total cytosolic “‘Cd at 4 and 3 hr after the challenge dose in tolerant animals. Within 6 hr, greater than 90% of all cytosolic cadmium was metallothionein bound. Thus, the ability of the cadmium-tolerant animals to increase the hepatic uptake of the challenge cadmium dose, decrease microsomal binding, and increase metallothionein binding of the challenge dose at 1 or 3 hr postchallenge would appear to be correlated with the tolerance phenomenon. Means et al. (1970) demonstrated that the in vivo inhibition of drug oxidation by cadmium was not the result of a direct inhibition of the cytochrome P-450 enzyme activity. Instead, cadmium apparently decreases the synthesis, as well as accelerates the degradation, of the heme protein. Therefore, the ability of prior cadmium exposure to produce a tolerance to subsequent cadmiumproduced decreased in cytochrome P-450 (Roberts and Schnell, 198 1) must depend upon a diversion of the challenge cadmium dose from the critical subcellular sites at the early time periods. Since the induction of metallothionein by a prior cadmium exposure clearly results in a redistribution of the challenge cadmium dose to the cytosol, these data are consistent with a causal role of metallothionein in the tolerance phenomenon. However, a failure of the time-dependent synthesis of metallothionein to correlate with the development of the tolerance phenomenon would suggest that factors in addition to metallothionein may play a role in the development of this tolerance.

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This investigation was supported by NIEHS Research Grant ES-00921 and NIGMS Pharmacology-Toxicology Training Grant T32-GM07095

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