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Effects of diethyldithiocarbamate and nickel chloride on glutathione and trace metal concentrations in rat liver
Toxicology, 32 (1984) 11-21 Elsevier Scientific Publishers Ireland
Ltd.
EFFECTS OF DIETHYLDITHIOCARBAMATE AND NICKEL CHLORIDE ON GLUTATHIONE AND TRACE METAL CONCENTRATIONS IN RAT LIVER*
F.W. SUNDERMAN Jr.a, 0. ZAHARIAa, O’LEARY Jr.b and H. GRIFFINC
M.C.
REIDa,
J.F.
BELLIVEAUb,
G.P.
aDepartments of Laboratory Medicine and Pharmacology, University of Connecticut School of Medicine, Fannington, CT; bDepartments of Biology and Chemistry, ProviTexas Instruments, Inc., dence College, Providence, RI; and ‘Ch em&try Laboratory, Attleboro, MA (U.S.A.) (Received (Accepted
January 3rd, 1984) March 21st, 1984)
SUMMARY
Concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) and 4 trace metals (Ni, Cu, Mn, Zn) were measured in livers from rats treated with sodium diethyldithiocarbamate (DDC, 0.67 or 1.33 mmol/ kg, i.m.) and NiClz (0.25 or 0.50 mmol/kg, s.c.), singly or in combination. In rats treated with DDC or NiCl*, singly, hepatic GSH was diminished at 4 h and returned to control levels (or slightly above) at 17 h. In rats that received DDC plus NiCl*, hepatic GSH was not diminished at 4 h and was increased 1.4-1.Sfold at 17 h. Hepatic GSSG was diminished at 4 h after NiC& treatment and returned to control values at 17 h; hepatic GSSG did not differ from control values at 4 h or 17 h after treatment with DDC, alone or combined with Ni&. Hepatic Ni was below the detection limit (-20 nmol/g) in control and DDC-treated rats; hepatic Ni was increased to 53 + 26 (SD.) nmol/g at 17 h after treatment with NiC12alone, and was increased 6-fold (308 + 63 nmol/g) in rats that received Ni plus DDC. Under the same conditions, hepatic Zn was increased 33% or 41%, respectively, *Supported by Grant No. ES-01337 from the National Institute of Environmental Health Sciences, Grant No. EV-03140 from the U.S. Department of Energy, and Providence College Fund for Faculty Research. Address all correspondence to: F. William Sunderman Jr., M.D., Prof. of Laboratory Medicine and Pharmacology, Univ. of Connecticut School of Medicine, 263 Farmington Ave., Farmington, CT 06032, U.S.A. Abbreviations: DDC, sodium diethyldithiocarbamate; GSH, glutathione; GSSH, oxidised glutathione; NEM, N-ethylmaleimide. 0300-483X/84/$03.00 o 1984 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
11
in rats that received NiClz or DDC, singly, and was not further increased by combined treatment; hepatic Cu and Mn concentrations were unaffected by NiClz or DDC, singly, but were diminished in rats that received NiClz and DDC. This study suggests: (a) that increased hepatic uptake of Ni is largely responsible for the synergistic induction of heme oxygenase activity in rats treated with NiClz and DDC; and (b) that increased hepatic uptake of Zn contributes to the induction of hepatic metallothionein by NiClz and DDC.
Key words: Copper (hepatic); Diethyldithiocarbamate (sodium); Glutathione (hepatic); Manganese (hepatic); Nickel chloride; Nickel (hepatic); Zinc (hepatic)
INTRODUCTION
Sunder-man et al. [l-3] observed that combined treatment of rats with sodium diethyldithiocarbamate (DDC) and nickel chloride (NiCl*) causes synergistic induction of heme oxygenase activity in hepatic microsomes and additive increases of metallothionein concentrations in hepatic cytosol. In order to probe the toxicological interactions of DDC and NiCl*, we have measured the concentrations of glutathione and 4 trace metals (Ni, Cu, Mn, Zn) in livers from rats treated with DDC and NiC12, singly and in combination. The rationale for this study derives from reports that hepatic glutathione and trace metal concentrations modulate heme oxygenase and metallothionein levels in rat liver [4-6]. The present investigation utilizes the same dosages of DDC and NiClz and identical experimental conditions as the previous studies of hepatic heme oxygenase activity and metallothionein concentrations [l-3]. MATERIALS
AND METHODS
The test substances were sodium diethyldithiocarbamate (Sigma Chemical Co., St. Louis, MO), recrystallized according to Baselt et al. [7], and ultrapure nickel chloride (NiQ, Ventron Corp., Beverly, MA). Analytical reagents were purchased from Sigma Chemical Co. The experimental animals were 128 male rats of the Fischer-344 strain (170-260 g, Charles River Breeding Laboratories, Inc., Wilmington, MA), housed in polypropylene cages in a laminar-flow hood and fed Purina rat chow and water ad libitum. There were 19 experimental groups, containing 4-11 rats. Rats in the control groups received injections of NaCl vehicle solution (0.15 mol/l, 0.3-0.5 ml/rat) by the same routes and at the same times that rats in the treated groups received injections of test substances. The dosages of DDC were 0.67 or 1.33 mmol/kg, i.m.; the dosages of NiCl, were 0.25 or 0.50 12
mmol/kg, S.C. Rats in one control group were not fasted; alI of the other rats were fasted for 17 h before they were decapitated by guilIotine and exsanguinated by draining. To avoid the possible influence of circadian fluctuations, each step of the protocol was performed at a constant time-of-day, as follows: (a) food was removed from the cages at 5 p.m.; (b) injections of NiClz and DDC were performed either at 5 p.m. or 6 a.m.; and (c) the rats were killed at 10 a.m. The time interval between the NiClz and DDC injections was
Concentrations of Ni, Cu, Mn, and Zn in liver homogenates were measured by plasma emission spectroscopy, as described by Belliveau et al. [ 121, Liver (1.5 g) was homogenized in 6 ml of ultrapure water* by use of a PotterElvehjem homogenizer. Duplicate 3-ml samples of the homogenate were added to 3 ml of mixed acid solution (concentrated HN03, HzS04, and HC104, 3: 1 :l by vol.)**; the samples were digested in a temperaturecontrolled heating block (1 h at 110°C; 2 h at 140°C; 30 min at 190°C; 30 min at 300°C). The liver digests were diluted to 4 ml with ultrapure HCl (1.2 mol/l), and nebulized into the plasma source of a d-c plasma emission spectrometer (Spectrascan III, Spectrometrics, Inc., Haverhill, MA). Emission intensities for Ni (361.9 nm), Cu (327.2 nm), Mn (260.5 nm), and Zn (206.2 nm) were compared to calibration curves prepared by analysis of standard solutions. The Mann-Whitney U-test was performed according to Sokol and Rohlf [ 131. Synergism was considered to occur when the effect of 2 compounds given together was greater than the sum of the effects of the same dosages given separately [2]. An index of synergism (\cI) was computed by the following equation:
IL=
[Ea,
iEta, + b,)-Eel -Eel
+ [Eb, -Eel
where E, = effect observed in vehicle controls; E, = effect produced by compound a at dosage a, ; Eb, = effect produced by compound b at dosage b,; and E(a, + b,) = effect produced by the 2 compounds, a and b, given together at dosages a, and b 1. RESULTS
A pilot experiment indicated that food consumption was reduced during the night following administration of DDC or NiClz to rats; to avoid this source of variation, all rats that received DDC and/or NiClz treatments were fasted for 17 h before sacrifice. Two sets of control rats, fed (Group A) and fasted (Group B), were tested to assess the effects of fasting (Table I). Hepatic GSH concentrations in the fed controls (Group A) agreed with values previously observed in male Fischer rats by Sasame and Boyd [14] (mean GSH = 5.75 mmol/g liver, S.E. f 0.18). Hepatic GSH concentrations in fasted controls (Group B) were significantly lower than in the fed controls. The proportion of GSSG, expressed as a percentage of total glutathione (GSH + GSSG), was increased in fasted controls (Table I). In rats treated with DDC or NiCl*, singly, hepatic GSH concentrations *Ultrapure water was prepared by deionization and distillation in an all-glass still. **Ultrapure nitric, sulfuric, and perchloric acids were purchased from Baker Chemical Co., Phillipsburg, NJ.
14
+ cn
1.33 0.67 1.33 1.33 0.67 1.33 1.33 0.67 1.33
0.25 0.25 0.50 0.50 0.25 0.25 0.25 0.50 0.50 0.50
A(fed) B (fasted) C D E F G H I J K L M N 0
6 15d 5 5 5 11 5 5 7 5 5 6 5 5 5
4 17 4 17 4 17 17 4 17 17 4 17 17
No. of rats
Hours, injections to death 58 +12 59f19 31 flog 60 f21 29 +13s 46 +17 56 +25 50 f12 45 +10 54 f 16 52 -I 13 57 f19 52flB 60f14 52+ 3
* 0.5 * 0.5 e + 0.7 s f 0.7 -I 0.5 s + 0.7 s f 0.4 f f 1.2 f 0.6 + 0.4 +1.2a + 0.6 s + 0.3 * 1.3s + 0.7 g
5.9 4.6 3.6 4.6 3.9 5.6 4.0 5.1 5.1 4.3 6.6 6.5 4.6 6.8 8.2
GSSG flmol/g (B)
GSH + GSSG mmol/g (A)b 5.8 4.5 3.5 4.5 3.8 5.5 3.8 5.0 5.0 4.2 6.5 6.4 4.5 6.7 8.1
f 0.4 f 0.5 f 0.6 f 0.7 f 0.7 * 0.7 f 0.4 f 1.2 + 0.6 f 0.4 z!z1.2 + 0.6 f 0.3 + 1.3 ? 0.7
GSH mmol/g (A-2B)C
g g
s s
f s s
e s
1.9 2.6 1.7 2.6 1.6 1.7 2.5 2.1 1.9 2.5 1.6 1.8 2.3 1.8 1.3
(TX
f 0.3 + 0.8 * 0.4 f 0.8 f 0.8 kO.8 + 1.1 f 1.1 + 0.4 + 0.7 + 0.4 + 0.6 + 0.6 +0.6 *0.1
f s
s s
s s
e a
100)
GSSG (%)
(GSH + GSSH), REDUCED GLUTA-
a Control rats in Group A were not fasted; controis in Group B and all rats that received NiCl, and/or DDC (groups C to 0) were fasted for 17 h before sacrifice. b GSSG in total glutathione (GSH/GSSG) is expressed in terms of GSH. Results are given as mean f S.D. c 1 mol of GSSG corresponds to 2 mol of GSH. d Since no significant difference was found between results in 6 fasted controls killed 4 h after vehicle injections and 9 controls killed 17 h after vehicle injections, the results in these controls were pooled. e P < 0.01 vs. fed controls (Group A). f P < 0.05 vs. fasted controls (Group B). a P < 0.01 vs. fasted controls (Group B).
DDC mmol/ kg, i.m.
NiCl, mmol/ kg, S.C.
Groupa
EFFECTS OF DIETHYLDITHIOCARBAMATE (DDC) AND NiCl, ON TOTAL GLUTATHIONE THIONE (GSH), AND OXIDIZED GLUTATHIONE (GSSG) IN RAT LIVER
TABLE I
were diminished at 4 h after treatment and returned to control levels (or slightly above) at 17 h. Treatment of rats with DDC plus NiClz prevented the initial diminution of hepatic GSH concentrations and enhanced the subsequent elevation of GSH concentrations. At 17 h after combined administration of NiClz (0.50 mmol/kg) and DDC (1.33 mmol/kg), hepatic GSH averaged 8.1 + 0.7 mmol/g (Group 0), compared to 4.5 f 0.5 in fasted controls (Group B), 5.5 ? 0.7 mmol/g in rats that received NiClz alone (Group F), and 5.0 + 0.6 mmol/g in rats that received DDC alone (Group I). At this combination of NiClz and DDC dosages, the index of synergism (J/ ) was 2.4; in groups K, L, and N, the values ranged from 1.5-4.0. Thus, synergistic effects of DDC and NiClz on hepatic GSH concentrations were observed at all of the specified dosage combinations. Oxidized glutathione (GSSG) concentrations were diminished in rat liver at 4 h after administration of NiClz and returned to control values at 17 h. Hepatic GSSG concentrations did not differ significantly from control values at 4 h or 17 h after administration of DDC, alone or in combination with NiCl* (Groups G to 0). Since hepatic GSH was markedly elevated in Groups K, L, N, and 0, relative depletion of hepatic GSSG was observed in these groups, when GSSG was expressed as a percentage of total glutathione (GSH + GSSG) (Table I). Concentrations of 4 trace metals in rat liver at 17 h after treatment with DDC and/or NiC12 are summarized in Table II. Hepatic Ni concentrations were below the analytical detection limit in control rats (Group P) and in rats that received DDC alone (Group R). Hepatic Ni concentrations were increased to 53 f 26 nmol/g at 17 h after administration of NiClz alone (Group Q), and were synergistically increased 6-fold in rats that received NiC& plus DDC (Group S). Hepatic Cu and Mn concentrations were unaffected by NiCl, or DDC, individually, but were depleted in rats that received NiClz plus DDC. Hepatic Zn concentration was increased 33% or 41% respectively in rats that received NiClz or DDC, singly, and was not further increased by combined treatment with NiClz and DDC. DISCUSSION
Administration of DDC to rats in combination with NiClz caused sub stantial (6-fold) increase of hepatic Ni concentrations at 17 h, compared to rats that received NiClz alone. This finding corroborates observations of Oskarsson and Tjalve [12], who administered DDC (4.1 mmol/kg, i.p.) to mice 10 min before ‘j3NiC1*(0.1 pmol/kg, i.v.); at 4 h, livers of DDC-treated mice contained 16-times more 63Ni than livers of mice treated only with 63NiC12.Baselt and Hanson [13] administered DDC (5.8 mmol/kg, p.o.) to rats 10 min after inhalation of 63Ni(CO)4 (0.2 mmol/l/l5 min); at 24 h, livers of DDC-treated rats contained 3.5-times more 63Ni than livers of rats treated only with 63Ni(C0)4. Thus, the present findings and the results of previous studies indicate that combined administration of DDC and nickel compounds to rodents causes synergistic enhancement of hepatic Ni concentrations. 16
II
1.33 1.33 P). P).
0.25 0.25
a P < 0.05 vs. controls (Group b P < 0.01 vs. controls (Group c P < 0.01 vs. Group Q.
R S
Q
P
DDC mmol/ kg, i.m.
DIETHYLDITHIOCARBAMATE
NiCl, mmol/ kg, S.C.
OF
Group
EFFECTS
TABLE
17 17 17
Hours, injections to death
(DDC)
AND
8 4 8 4
No. of rats
NiCl,
Zn
<-20 53 f 26b <-20 308 f 63c
Ni 64 69 60 53
cu f 8 + 9 -f: 8 f 3a
45+ 4 47 f13 43f 2 29 f 6b
442 590 622 613
Zn
IN RAT mean f S.D.)
Mn
(nmol/g,
CONCENTRATIONS
Trace metal concentrations
ON Ni, Cu, Mn, AND
f + f +
32 62b 63b 54b
LIVER
Treatment of rats with NiClz or DDC increased hepatic Zn concentrations at 17 h, but did not significantly affect Cu and Mn concentrations. Combined administration of DDC and NiClz did not exert any additive or synergistic effect on hepatic Zn concentrations, but caused synergistic reduction of Cu and Mn concentrations. Although strictly comparable studies have not been reported, 6 previous investigations deserve mention. Maitani and Suzuki [ 171 found a 35% increase of hepatic Zn concentrations and no significant change of hepatic Cu concentrations in mice killed 24 h after treatment with nickel acetate (0.15 mmol/kg, i.p.). Chmielnicka et al. [ 181 reported a 2.5-fold increase of hepatic Zn concentrations and a 1.3-fold increase of hepatic Cu concentrations in rats following repeated administration of NiClz (8.5 pmoljkg, s.c., 7 injections, every 2 days for 2 weeks). Koutensky et al. [19] measured 64Cu retention in mice at 24 h and 48 h after injection of 64CuC12(5.6 pmol/kg, i.v.), alone, or combined with DDC (0.15 mmol/kg, i.p.); the DDC treatment did not affect 64Cu retention in liver, but it enhanced 64Cu uptake in kidney, heart, and brain. Bertram et al. [20] administered DDC (1.5 mmol/kg, p.o., for 10 days) to phenobarbitaltreated rats (0.1% PB in drinking water for 14 days); under these conditions, DDC did not affect hepatic Zn, Cu, or Mn concentrations. Marselos et al. [21] noted that treatment of rats with DDC (1.8 mmol/kg, p.o., for 3 days) caused a 24% reduction of hepatic Zn concentrations and a 21% reduction of hepatic Cu concentrations. Tandon et al. [22] reported that hepatic Zn concentrations were increased 1.6-1.8-fold in rats killed 24 h after treatment with DDC (0.5 mmol/kg, i.p., daily for l-6 days); hepatic Cu concentrations were not significantly affected by DDC treatment. In the present study, hepatic GSH concentrations were reduced 16% at 4 h following administration of DDC. For comparison, Goldstein et al. [23] reported that hepatic non-protein sulhydryl levels were reduced 35% in rats at 4 h and 26% at 6 h after DDC treatment (7.0 mmol/kg, i.p.); Siegers et al. [24] found that hepatic GSH concentrations were reduced 16% in rats at 6 h after DDC treatment (5.8 mmol/kg, i.p.). Miller et al. [25] reported that hepatic GSH concentrations were normal in rats at 1 h or 8 h following DDC treatment (2.9 mmol/kg, i.p.). Thus, the available data suggest that transient diminution of hepatic GSH concentrations occurs in rats at 4-6 h after administration of DDC. The present study demonstrated 16-22s reductions of hepatic GSH concentrations at 4 h after NiClz injection; by 17 h, hepatic GSH concentrations returned to control values at the lower dosage of NiCl* (0.25 mmol/kg) and increased 22% above control values at the higher dosage (0.50 mmol/kg). Maines and Kappas [4,26] reported a 30% diminution of hepatic GSH levels 6 h after NiClz injection (0.25 mmol/kg, s.c.), followed by 2-3-fold increase at 12-24 h. Sasame and Boyd [14] did not detect any disturbance of hepatic GSH concentrations in rats killed 8 h or 24 h after NiClz treatment (0.25 mmol/kg, s.c.), and Eaton et al. [5] did not observe any change of hepatic GSH concentrations in rats that received 2 injections of nickel acetate (0.12 mmol/kg, i.p.) at 12 h and 36 h before death. These findings suggest that a transient diminution of hepatic GSH occurs in rats at 18
4-6 h after Ni(I1) treatment, and that a delayed increase may ensue, depending upon age, strain, and nutritional status. The transient diminution of hepatic GSH concentration after Ni(I1) treatment may reflect enhanced biliary excretion of Ni-GSH complexes, diffusion of GSH out of the liver, and/or depletion of the hepatic GSH pool, owing to induction of hepatic metallothionein. The latter suggestion is based on reports of Wong and Klaassen [27] and Kawata and Suzuki [28] that catabolism of glutathione can provide cysteine required for biosynthesis of metallothionein. Combined treatment of rats with DDC and NiClz did not potentiate the depletion of hepatic GSH at 4 h; instead, the combined treatment ameliorated the depletion at 4 h and synergistically increased hepatic GSH concentrations at 17 h. The present results are consistent with the proposal of Sunder-man et al. [2] that enhanced hepatic uptake of Ni is primarily responsible for synergistic induction of hepatic heme oxygenase activity by DDC and NiC12. Oskarsson and Tjalve [ 151 showed that DDC and Ni(I1) react in vivo to form a lipophilic complex (nickel bis-diethyldithiocarbamate), which facilitates the passage of nickel across cell membranes. Sunderman et al. [l] showed that hepatic heme oxygenase activity is stimulated by Ni(I1) in a dose-related fashion. Kikuchi and Yoshida [ 291 showed that metals induce hepatic heme oxygenase activity by stimulating the synthesis of mRNA for the enzyme. This chain of evidence suggests that the synergistic effect of DDC and NiClz to increase the hepatic Ni concentrations may lead to proportionally increased synthesis of mRNA for heme oxygenase. The present measurements of hepatic GSH concentrations do not support a second hypothesis, proposed by Sunderman et al. [ 21, that synergistic depletion of hepatic GSH may potentiate the induction of heme oxygenase activity. This hypothesis was suggested by reports that diethyl maleate, a compound that profoundly depresses hepatic GSH content, increases hepatic heme oxygenase activity and enhances the induction of the enzyme by NiClz [26,29-311. Sunder-man et al. [2] also postulated that the mechanism for synergistic induction of heme oxygenase activity by DDC and NiClz may involve increased intracellular concentrations of heme. In support of this hypothesis, DDC and Ni(I1) enhance the degradation of hepatic cytochrome P-450, releasing heme [14,21,25,32,33], which activates a second pathway for inducing hepatic synthesis of m RNA for heme oxygenase [ 291. The present observations that administration of DDC or NiCl?, individually, increases hepatic Zn concentrations is consistent with the proposal that Zn uptake into the liver mediates the induction of metallothionein [6]. This does not appear to be.the entire answer, however, since hepatic Zn concentrations were no higher in rats treated with DDC plus NiClz than in rats that received either substance alone. It seems likely that elevated levels of plasma glucagon may contribute to the increased hepatic MT concentrations in rats treated with DDC plus NiClz [3,6,34]. Alternatively, increased hepatic Ni(I1) concentrations may stimulate transcription of the MT-gene.
19
ACKNOWLEDGEMENTS
The following students at Providence College assisted in trace metal analyses: Mary Hillstrom, Elizabeth Doll, Lisa Lauder, and Mary Roque; the following research assistants at the University of Connecticut assisted in glutathione analyses: Connie Liber and Cristina Crisostomo. REFERENCES 1 F.W. Sunderman Jr., M.C. Reid, L.M. microsomal heme oxygenase activity
Bibeau and J.V. Linden, Nickel induction in rodents. Toxicol. Appl. Pharmacol.,
of 68
(1983) 87. 2 F.W. Sunderman Jr., L.M. Bibeau and M.C. Reid, Synergistic induction of microsomal heme oxygenase activity in rat liver and kidney by diethyldithiocarbamate and NiCl,. Toxicol. Appl. Pharmacol., 71 (1983) 436. 3 F.W. Sunderman Jr. and C.B. Fraser, Effects of nickel chloride and diethyldithiocarbamate on metallothionein in rat liver and kidney. Ann. Clin. Lab. Sci., 13 (1983) 489. 4 M.D. Maines and A. Kappas, Regulation of heme pathway enzymes and cellular glutathione content by metals that do not chelate with tetrapyrroles: Blockade of metal effects by thiols. Proc. Natl. Acad. Sci. USA, 74 (1977) 1875. 5 D.L. Eaton, N.H. Stacey, K-L Wong and C.D. Klaasen, Dose-response effects of various metal ions on rat liver metallothionein, glutathione, heme oxygenase, and cytochrome P-450. Toxicol. Appl. Pharmacol., 55 (1980) 393. 6 R.J. Cousins, Relationship of metallothionein synthesis and degradation to intracellular zinc metabolism, in E.C. Foulkes (Ed.), Biological Roles of Metallothionein, Elsevier/North Holland Biomedical Press, Amsterdam, 1982, pp. 251-260. 7 R.C. Baselt, F.W. Sunderman Jr., J. Mitchell and E. Horak, Comparisons of antidotal efficacy of sodium diethyldithiocarbamate, D-pencillamine, and triethylenetramine upon acute toxicity of nickel carbonyl in rats. Res. Commun. Chem. Pathol. Pharmacol., 18 (1977) 677. 8 F. Tietze, Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem., 27 (1969) 502. 9 J.E. Brehe and H.B. Burch, Enzymatic assay of glutathione. Anal. Biochem., 74 (1976) 189. 10 G.A. Hazelton and C.A. Lang, Glutathione contents of tissues in the aging mouse. Biochem. J., 188 (1980) 25. 11 T.P.M. Akerboom and H. Sies, Assay of glutathione, glutathione disulfide, and glutathione-mixed disulfides in biological samples. Methods Enzymol., 77 (1981) 373. H. Griffin and A. 12 J.F. Belliveau, G.P. O’Leary Jr., M. Lalor, E. Doll, M. Hillstrom, Savolainen, Multi-element d-c plasma emission spectroscopic analysis of human and animal tissues. Providence College Biological Notes, 5 (1981) 1. 13 R.R. Sokol and F.J. Rohlf, Biometry: The Principles and Practice of Statistics in Biological Research, W.H. Freeman Co., San Francisco, 1969, pp. 299-342. 14 H.A. Sasame and M.R. Boyd, Paradoxical effects of cobaltous chloride and salts of other divalent metals on tissue levels of reduced glutathione and microsomal mixedfunction oxidase components. J. Pharmacol. Exp. Ther., 205 (1978) 718. 15 A. Oskarsson and H. Tjalve, Effects of diethyldithiocarbamate and penicillamine on the tissue distribution of 63NiC1, in mice. Arch. Toxicol., 45 (1980) 45. 16 R.C. Baselt and V.H. Hanson, Efficacy of orally-administered chelating agents for nickel carbonyl toxicity in rats. Res. Commun. Chem. Pathol. Pharmacol., 38 (1982) 113.
20
17 T. Maitani and K.T. Suzuki, Extents of hepatic zinc-thionein induction in mice given an equimolar dose of various heavy metals. Chem. Pharm. Bull., 30 (1982) 4164. 18 J. Chmielnicka, J.A. Szymanska and J. Tyfa, Disturbances in the metabolism of endogenous metals (Zn and Cu) in nickel-exposed rats. Environ. Res., 27 (1982) 216. I9 J. Koutensky, V. Eybl, M. Koutenska, J. Sykora and F. Mertl, Influence of sodium diethyldithiocarbamate on the toxicity and distribution of copper in mice. Eur. J. Pharmacol., 14 (i971) 389. 20 B. Bertram, J. Schuhmacher, E. Frei, N. Frank and M. Wiessler, Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats. Biochem. Pharmacol., 31 (1982) 3613. 21 M. Marselos, P. Alakuijala, M. Lang and R. Torronen, Studies on the mechanism by which disulfiram and diethyldithiocarbamate affect drug oxidation, in V. Ullrich, I. Roots, A. Hildebrand, R.W. Estabrook and A.H. Conney (Eds.), Microsomes and Drug Oxidations, Pergamon Press, Oxford, 1977, pp. 589-596. 22 S.K. Tandon, J.R. Behari and M. Ashquin, Effects of thiol chelators on trace metal levels. Res. Commun. Chem. Pathol. Pharmacol., 42 (1983) 501. 23 B.D. Goldstein, M.G. Rozen, J.C. Quintavalla and M.A. Amoruso, Decrease in mouse lung and liver glutathione peroxidase activity and potentiation of the lethal effects of ozone and paraquat by the superoxide dismutase inhibitor, diethyldithiocarbamate. Biochem. Pharmacol., 28 (1979) 27. 24 C.P. Siegers, M. Younes and G. Schmitt, Effects of dithiocarb and (+ )-cyanidanol-3 on the hepatoxicity and metabolism of vinylidine chloride in rata. Toxicology, 15 (1979) 55. 25 G.E. Miller, M.A. Zemaitis and F.E. Greene, Mechanisms of diethyldithiocarbamateinduced loss of cytochrome P-450 from rat liver. Biochem. Pharmacol., 32 (1983) 2433. 26 M.D. Maines and A. Kappas, Nickel-mediated alterations in the activity of hepatic and renal enzymes of heme metabolism and heme dependent cellular activities, in S.S. Brown (Ed.), Clinical Chemistry and Chemical Toxicology of Metals, Elsevier/North Holland Biomedical Press, Amsterdam, 1977, pp. 75-81. 27 K-L Wong and C.D. Klaassen, Relationship between liver and kidney levels of glutathione and metallothionein in rata. Toxicology, 19 (1981) 39. 28 M. Kawata and K.T. Suzuki, Relation between metal and glutathione concentrations in mouse liver after cadmium, zinc or copper loading. Toxicol. Lett., 15 (1983) 131. 29 G. Kikuchi and T. Yoshida, Function and induction of microsomal heme oxygenase. Mol. Cell. Biochem., 53 (1983) 163. 30 R.F. Burk and M.A. Correia, Stimulation of rat hepatic microsomal heme oxygenase by diethyl maleate. Res. Commun. Chem. Pathol. Pharmacol. 24 (1979) 205. 31 M.D. Maines, Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney. Arch. Biochem. Biophys., 216 (1982) 17. 32 R.A. Neal, Microsomal enzymes and the toxicity of thiono-sulfur compounds, in M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien (Eds.), Microsomes, Drug Oxidations and Chemical Carcinogenesis, Vol. 2, Academic Press, New York, 1980, pp. 791-799. 33 M.A. Zemaitis and F.E. Greene, In uiuo and in vitro effects of thiuram disulfides and dithiocarbamates on hepatic microsomal drug metabolism in the rat. Toxicol. Appl. Pharmacol., 48 (1979) 343. 34 T. Maitani and K.T. Suzuki, Dose-dependent induction of metallothionein in kidneys of mice injected with indium and nickel ions. Chem. Pharm. Bull., 31 (1983) 979.
21
Ltd.
EFFECTS OF DIETHYLDITHIOCARBAMATE AND NICKEL CHLORIDE ON GLUTATHIONE AND TRACE METAL CONCENTRATIONS IN RAT LIVER*
F.W. SUNDERMAN Jr.a, 0. ZAHARIAa, O’LEARY Jr.b and H. GRIFFINC
M.C.
REIDa,
J.F.
BELLIVEAUb,
G.P.
aDepartments of Laboratory Medicine and Pharmacology, University of Connecticut School of Medicine, Fannington, CT; bDepartments of Biology and Chemistry, ProviTexas Instruments, Inc., dence College, Providence, RI; and ‘Ch em&try Laboratory, Attleboro, MA (U.S.A.) (Received (Accepted
January 3rd, 1984) March 21st, 1984)
SUMMARY
Concentrations of reduced glutathione (GSH) and oxidized glutathione (GSSG) and 4 trace metals (Ni, Cu, Mn, Zn) were measured in livers from rats treated with sodium diethyldithiocarbamate (DDC, 0.67 or 1.33 mmol/ kg, i.m.) and NiClz (0.25 or 0.50 mmol/kg, s.c.), singly or in combination. In rats treated with DDC or NiCl*, singly, hepatic GSH was diminished at 4 h and returned to control levels (or slightly above) at 17 h. In rats that received DDC plus NiCl*, hepatic GSH was not diminished at 4 h and was increased 1.4-1.Sfold at 17 h. Hepatic GSSG was diminished at 4 h after NiC& treatment and returned to control values at 17 h; hepatic GSSG did not differ from control values at 4 h or 17 h after treatment with DDC, alone or combined with Ni&. Hepatic Ni was below the detection limit (-20 nmol/g) in control and DDC-treated rats; hepatic Ni was increased to 53 + 26 (SD.) nmol/g at 17 h after treatment with NiC12alone, and was increased 6-fold (308 + 63 nmol/g) in rats that received Ni plus DDC. Under the same conditions, hepatic Zn was increased 33% or 41%, respectively, *Supported by Grant No. ES-01337 from the National Institute of Environmental Health Sciences, Grant No. EV-03140 from the U.S. Department of Energy, and Providence College Fund for Faculty Research. Address all correspondence to: F. William Sunderman Jr., M.D., Prof. of Laboratory Medicine and Pharmacology, Univ. of Connecticut School of Medicine, 263 Farmington Ave., Farmington, CT 06032, U.S.A. Abbreviations: DDC, sodium diethyldithiocarbamate; GSH, glutathione; GSSH, oxidised glutathione; NEM, N-ethylmaleimide. 0300-483X/84/$03.00 o 1984 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
11
in rats that received NiClz or DDC, singly, and was not further increased by combined treatment; hepatic Cu and Mn concentrations were unaffected by NiClz or DDC, singly, but were diminished in rats that received NiClz and DDC. This study suggests: (a) that increased hepatic uptake of Ni is largely responsible for the synergistic induction of heme oxygenase activity in rats treated with NiClz and DDC; and (b) that increased hepatic uptake of Zn contributes to the induction of hepatic metallothionein by NiClz and DDC.
Key words: Copper (hepatic); Diethyldithiocarbamate (sodium); Glutathione (hepatic); Manganese (hepatic); Nickel chloride; Nickel (hepatic); Zinc (hepatic)
INTRODUCTION
Sunder-man et al. [l-3] observed that combined treatment of rats with sodium diethyldithiocarbamate (DDC) and nickel chloride (NiCl*) causes synergistic induction of heme oxygenase activity in hepatic microsomes and additive increases of metallothionein concentrations in hepatic cytosol. In order to probe the toxicological interactions of DDC and NiCl*, we have measured the concentrations of glutathione and 4 trace metals (Ni, Cu, Mn, Zn) in livers from rats treated with DDC and NiC12, singly and in combination. The rationale for this study derives from reports that hepatic glutathione and trace metal concentrations modulate heme oxygenase and metallothionein levels in rat liver [4-6]. The present investigation utilizes the same dosages of DDC and NiClz and identical experimental conditions as the previous studies of hepatic heme oxygenase activity and metallothionein concentrations [l-3]. MATERIALS
AND METHODS
The test substances were sodium diethyldithiocarbamate (Sigma Chemical Co., St. Louis, MO), recrystallized according to Baselt et al. [7], and ultrapure nickel chloride (NiQ, Ventron Corp., Beverly, MA). Analytical reagents were purchased from Sigma Chemical Co. The experimental animals were 128 male rats of the Fischer-344 strain (170-260 g, Charles River Breeding Laboratories, Inc., Wilmington, MA), housed in polypropylene cages in a laminar-flow hood and fed Purina rat chow and water ad libitum. There were 19 experimental groups, containing 4-11 rats. Rats in the control groups received injections of NaCl vehicle solution (0.15 mol/l, 0.3-0.5 ml/rat) by the same routes and at the same times that rats in the treated groups received injections of test substances. The dosages of DDC were 0.67 or 1.33 mmol/kg, i.m.; the dosages of NiCl, were 0.25 or 0.50 12
mmol/kg, S.C. Rats in one control group were not fasted; alI of the other rats were fasted for 17 h before they were decapitated by guilIotine and exsanguinated by draining. To avoid the possible influence of circadian fluctuations, each step of the protocol was performed at a constant time-of-day, as follows: (a) food was removed from the cages at 5 p.m.; (b) injections of NiClz and DDC were performed either at 5 p.m. or 6 a.m.; and (c) the rats were killed at 10 a.m. The time interval between the NiClz and DDC injections was
Concentrations of Ni, Cu, Mn, and Zn in liver homogenates were measured by plasma emission spectroscopy, as described by Belliveau et al. [ 121, Liver (1.5 g) was homogenized in 6 ml of ultrapure water* by use of a PotterElvehjem homogenizer. Duplicate 3-ml samples of the homogenate were added to 3 ml of mixed acid solution (concentrated HN03, HzS04, and HC104, 3: 1 :l by vol.)**; the samples were digested in a temperaturecontrolled heating block (1 h at 110°C; 2 h at 140°C; 30 min at 190°C; 30 min at 300°C). The liver digests were diluted to 4 ml with ultrapure HCl (1.2 mol/l), and nebulized into the plasma source of a d-c plasma emission spectrometer (Spectrascan III, Spectrometrics, Inc., Haverhill, MA). Emission intensities for Ni (361.9 nm), Cu (327.2 nm), Mn (260.5 nm), and Zn (206.2 nm) were compared to calibration curves prepared by analysis of standard solutions. The Mann-Whitney U-test was performed according to Sokol and Rohlf [ 131. Synergism was considered to occur when the effect of 2 compounds given together was greater than the sum of the effects of the same dosages given separately [2]. An index of synergism (\cI) was computed by the following equation:
IL=
[Ea,
iEta, + b,)-Eel -Eel
+ [Eb, -Eel
where E, = effect observed in vehicle controls; E, = effect produced by compound a at dosage a, ; Eb, = effect produced by compound b at dosage b,; and E(a, + b,) = effect produced by the 2 compounds, a and b, given together at dosages a, and b 1. RESULTS
A pilot experiment indicated that food consumption was reduced during the night following administration of DDC or NiClz to rats; to avoid this source of variation, all rats that received DDC and/or NiClz treatments were fasted for 17 h before sacrifice. Two sets of control rats, fed (Group A) and fasted (Group B), were tested to assess the effects of fasting (Table I). Hepatic GSH concentrations in the fed controls (Group A) agreed with values previously observed in male Fischer rats by Sasame and Boyd [14] (mean GSH = 5.75 mmol/g liver, S.E. f 0.18). Hepatic GSH concentrations in fasted controls (Group B) were significantly lower than in the fed controls. The proportion of GSSG, expressed as a percentage of total glutathione (GSH + GSSG), was increased in fasted controls (Table I). In rats treated with DDC or NiCl*, singly, hepatic GSH concentrations *Ultrapure water was prepared by deionization and distillation in an all-glass still. **Ultrapure nitric, sulfuric, and perchloric acids were purchased from Baker Chemical Co., Phillipsburg, NJ.
14
+ cn
1.33 0.67 1.33 1.33 0.67 1.33 1.33 0.67 1.33
0.25 0.25 0.50 0.50 0.25 0.25 0.25 0.50 0.50 0.50
A(fed) B (fasted) C D E F G H I J K L M N 0
6 15d 5 5 5 11 5 5 7 5 5 6 5 5 5
4 17 4 17 4 17 17 4 17 17 4 17 17
No. of rats
Hours, injections to death 58 +12 59f19 31 flog 60 f21 29 +13s 46 +17 56 +25 50 f12 45 +10 54 f 16 52 -I 13 57 f19 52flB 60f14 52+ 3
* 0.5 * 0.5 e + 0.7 s f 0.7 -I 0.5 s + 0.7 s f 0.4 f f 1.2 f 0.6 + 0.4 +1.2a + 0.6 s + 0.3 * 1.3s + 0.7 g
5.9 4.6 3.6 4.6 3.9 5.6 4.0 5.1 5.1 4.3 6.6 6.5 4.6 6.8 8.2
GSSG flmol/g (B)
GSH + GSSG mmol/g (A)b 5.8 4.5 3.5 4.5 3.8 5.5 3.8 5.0 5.0 4.2 6.5 6.4 4.5 6.7 8.1
f 0.4 f 0.5 f 0.6 f 0.7 f 0.7 * 0.7 f 0.4 f 1.2 + 0.6 f 0.4 z!z1.2 + 0.6 f 0.3 + 1.3 ? 0.7
GSH mmol/g (A-2B)C
g g
s s
f s s
e s
1.9 2.6 1.7 2.6 1.6 1.7 2.5 2.1 1.9 2.5 1.6 1.8 2.3 1.8 1.3
(TX
f 0.3 + 0.8 * 0.4 f 0.8 f 0.8 kO.8 + 1.1 f 1.1 + 0.4 + 0.7 + 0.4 + 0.6 + 0.6 +0.6 *0.1
f s
s s
s s
e a
100)
GSSG (%)
(GSH + GSSH), REDUCED GLUTA-
a Control rats in Group A were not fasted; controis in Group B and all rats that received NiCl, and/or DDC (groups C to 0) were fasted for 17 h before sacrifice. b GSSG in total glutathione (GSH/GSSG) is expressed in terms of GSH. Results are given as mean f S.D. c 1 mol of GSSG corresponds to 2 mol of GSH. d Since no significant difference was found between results in 6 fasted controls killed 4 h after vehicle injections and 9 controls killed 17 h after vehicle injections, the results in these controls were pooled. e P < 0.01 vs. fed controls (Group A). f P < 0.05 vs. fasted controls (Group B). a P < 0.01 vs. fasted controls (Group B).
DDC mmol/ kg, i.m.
NiCl, mmol/ kg, S.C.
Groupa
EFFECTS OF DIETHYLDITHIOCARBAMATE (DDC) AND NiCl, ON TOTAL GLUTATHIONE THIONE (GSH), AND OXIDIZED GLUTATHIONE (GSSG) IN RAT LIVER
TABLE I
were diminished at 4 h after treatment and returned to control levels (or slightly above) at 17 h. Treatment of rats with DDC plus NiClz prevented the initial diminution of hepatic GSH concentrations and enhanced the subsequent elevation of GSH concentrations. At 17 h after combined administration of NiClz (0.50 mmol/kg) and DDC (1.33 mmol/kg), hepatic GSH averaged 8.1 + 0.7 mmol/g (Group 0), compared to 4.5 f 0.5 in fasted controls (Group B), 5.5 ? 0.7 mmol/g in rats that received NiClz alone (Group F), and 5.0 + 0.6 mmol/g in rats that received DDC alone (Group I). At this combination of NiClz and DDC dosages, the index of synergism (J/ ) was 2.4; in groups K, L, and N, the values ranged from 1.5-4.0. Thus, synergistic effects of DDC and NiClz on hepatic GSH concentrations were observed at all of the specified dosage combinations. Oxidized glutathione (GSSG) concentrations were diminished in rat liver at 4 h after administration of NiClz and returned to control values at 17 h. Hepatic GSSG concentrations did not differ significantly from control values at 4 h or 17 h after administration of DDC, alone or in combination with NiCl* (Groups G to 0). Since hepatic GSH was markedly elevated in Groups K, L, N, and 0, relative depletion of hepatic GSSG was observed in these groups, when GSSG was expressed as a percentage of total glutathione (GSH + GSSG) (Table I). Concentrations of 4 trace metals in rat liver at 17 h after treatment with DDC and/or NiC12 are summarized in Table II. Hepatic Ni concentrations were below the analytical detection limit in control rats (Group P) and in rats that received DDC alone (Group R). Hepatic Ni concentrations were increased to 53 f 26 nmol/g at 17 h after administration of NiClz alone (Group Q), and were synergistically increased 6-fold in rats that received NiC& plus DDC (Group S). Hepatic Cu and Mn concentrations were unaffected by NiCl, or DDC, individually, but were depleted in rats that received NiClz plus DDC. Hepatic Zn concentration was increased 33% or 41% respectively in rats that received NiClz or DDC, singly, and was not further increased by combined treatment with NiClz and DDC. DISCUSSION
Administration of DDC to rats in combination with NiClz caused sub stantial (6-fold) increase of hepatic Ni concentrations at 17 h, compared to rats that received NiClz alone. This finding corroborates observations of Oskarsson and Tjalve [12], who administered DDC (4.1 mmol/kg, i.p.) to mice 10 min before ‘j3NiC1*(0.1 pmol/kg, i.v.); at 4 h, livers of DDC-treated mice contained 16-times more 63Ni than livers of mice treated only with 63NiC12.Baselt and Hanson [13] administered DDC (5.8 mmol/kg, p.o.) to rats 10 min after inhalation of 63Ni(CO)4 (0.2 mmol/l/l5 min); at 24 h, livers of DDC-treated rats contained 3.5-times more 63Ni than livers of rats treated only with 63Ni(C0)4. Thus, the present findings and the results of previous studies indicate that combined administration of DDC and nickel compounds to rodents causes synergistic enhancement of hepatic Ni concentrations. 16
II
1.33 1.33 P). P).
0.25 0.25
a P < 0.05 vs. controls (Group b P < 0.01 vs. controls (Group c P < 0.01 vs. Group Q.
R S
Q
P
DDC mmol/ kg, i.m.
DIETHYLDITHIOCARBAMATE
NiCl, mmol/ kg, S.C.
OF
Group
EFFECTS
TABLE
17 17 17
Hours, injections to death
(DDC)
AND
8 4 8 4
No. of rats
NiCl,
Zn
<-20 53 f 26b <-20 308 f 63c
Ni 64 69 60 53
cu f 8 + 9 -f: 8 f 3a
45+ 4 47 f13 43f 2 29 f 6b
442 590 622 613
Zn
IN RAT mean f S.D.)
Mn
(nmol/g,
CONCENTRATIONS
Trace metal concentrations
ON Ni, Cu, Mn, AND
f + f +
32 62b 63b 54b
LIVER
Treatment of rats with NiClz or DDC increased hepatic Zn concentrations at 17 h, but did not significantly affect Cu and Mn concentrations. Combined administration of DDC and NiClz did not exert any additive or synergistic effect on hepatic Zn concentrations, but caused synergistic reduction of Cu and Mn concentrations. Although strictly comparable studies have not been reported, 6 previous investigations deserve mention. Maitani and Suzuki [ 171 found a 35% increase of hepatic Zn concentrations and no significant change of hepatic Cu concentrations in mice killed 24 h after treatment with nickel acetate (0.15 mmol/kg, i.p.). Chmielnicka et al. [ 181 reported a 2.5-fold increase of hepatic Zn concentrations and a 1.3-fold increase of hepatic Cu concentrations in rats following repeated administration of NiClz (8.5 pmoljkg, s.c., 7 injections, every 2 days for 2 weeks). Koutensky et al. [19] measured 64Cu retention in mice at 24 h and 48 h after injection of 64CuC12(5.6 pmol/kg, i.v.), alone, or combined with DDC (0.15 mmol/kg, i.p.); the DDC treatment did not affect 64Cu retention in liver, but it enhanced 64Cu uptake in kidney, heart, and brain. Bertram et al. [20] administered DDC (1.5 mmol/kg, p.o., for 10 days) to phenobarbitaltreated rats (0.1% PB in drinking water for 14 days); under these conditions, DDC did not affect hepatic Zn, Cu, or Mn concentrations. Marselos et al. [21] noted that treatment of rats with DDC (1.8 mmol/kg, p.o., for 3 days) caused a 24% reduction of hepatic Zn concentrations and a 21% reduction of hepatic Cu concentrations. Tandon et al. [22] reported that hepatic Zn concentrations were increased 1.6-1.8-fold in rats killed 24 h after treatment with DDC (0.5 mmol/kg, i.p., daily for l-6 days); hepatic Cu concentrations were not significantly affected by DDC treatment. In the present study, hepatic GSH concentrations were reduced 16% at 4 h following administration of DDC. For comparison, Goldstein et al. [23] reported that hepatic non-protein sulhydryl levels were reduced 35% in rats at 4 h and 26% at 6 h after DDC treatment (7.0 mmol/kg, i.p.); Siegers et al. [24] found that hepatic GSH concentrations were reduced 16% in rats at 6 h after DDC treatment (5.8 mmol/kg, i.p.). Miller et al. [25] reported that hepatic GSH concentrations were normal in rats at 1 h or 8 h following DDC treatment (2.9 mmol/kg, i.p.). Thus, the available data suggest that transient diminution of hepatic GSH concentrations occurs in rats at 4-6 h after administration of DDC. The present study demonstrated 16-22s reductions of hepatic GSH concentrations at 4 h after NiClz injection; by 17 h, hepatic GSH concentrations returned to control values at the lower dosage of NiCl* (0.25 mmol/kg) and increased 22% above control values at the higher dosage (0.50 mmol/kg). Maines and Kappas [4,26] reported a 30% diminution of hepatic GSH levels 6 h after NiClz injection (0.25 mmol/kg, s.c.), followed by 2-3-fold increase at 12-24 h. Sasame and Boyd [14] did not detect any disturbance of hepatic GSH concentrations in rats killed 8 h or 24 h after NiClz treatment (0.25 mmol/kg, s.c.), and Eaton et al. [5] did not observe any change of hepatic GSH concentrations in rats that received 2 injections of nickel acetate (0.12 mmol/kg, i.p.) at 12 h and 36 h before death. These findings suggest that a transient diminution of hepatic GSH occurs in rats at 18
4-6 h after Ni(I1) treatment, and that a delayed increase may ensue, depending upon age, strain, and nutritional status. The transient diminution of hepatic GSH concentration after Ni(I1) treatment may reflect enhanced biliary excretion of Ni-GSH complexes, diffusion of GSH out of the liver, and/or depletion of the hepatic GSH pool, owing to induction of hepatic metallothionein. The latter suggestion is based on reports of Wong and Klaassen [27] and Kawata and Suzuki [28] that catabolism of glutathione can provide cysteine required for biosynthesis of metallothionein. Combined treatment of rats with DDC and NiClz did not potentiate the depletion of hepatic GSH at 4 h; instead, the combined treatment ameliorated the depletion at 4 h and synergistically increased hepatic GSH concentrations at 17 h. The present results are consistent with the proposal of Sunder-man et al. [2] that enhanced hepatic uptake of Ni is primarily responsible for synergistic induction of hepatic heme oxygenase activity by DDC and NiC12. Oskarsson and Tjalve [ 151 showed that DDC and Ni(I1) react in vivo to form a lipophilic complex (nickel bis-diethyldithiocarbamate), which facilitates the passage of nickel across cell membranes. Sunderman et al. [l] showed that hepatic heme oxygenase activity is stimulated by Ni(I1) in a dose-related fashion. Kikuchi and Yoshida [ 291 showed that metals induce hepatic heme oxygenase activity by stimulating the synthesis of mRNA for the enzyme. This chain of evidence suggests that the synergistic effect of DDC and NiClz to increase the hepatic Ni concentrations may lead to proportionally increased synthesis of mRNA for heme oxygenase. The present measurements of hepatic GSH concentrations do not support a second hypothesis, proposed by Sunderman et al. [ 21, that synergistic depletion of hepatic GSH may potentiate the induction of heme oxygenase activity. This hypothesis was suggested by reports that diethyl maleate, a compound that profoundly depresses hepatic GSH content, increases hepatic heme oxygenase activity and enhances the induction of the enzyme by NiClz [26,29-311. Sunder-man et al. [2] also postulated that the mechanism for synergistic induction of heme oxygenase activity by DDC and NiClz may involve increased intracellular concentrations of heme. In support of this hypothesis, DDC and Ni(I1) enhance the degradation of hepatic cytochrome P-450, releasing heme [14,21,25,32,33], which activates a second pathway for inducing hepatic synthesis of m RNA for heme oxygenase [ 291. The present observations that administration of DDC or NiCl?, individually, increases hepatic Zn concentrations is consistent with the proposal that Zn uptake into the liver mediates the induction of metallothionein [6]. This does not appear to be.the entire answer, however, since hepatic Zn concentrations were no higher in rats treated with DDC plus NiClz than in rats that received either substance alone. It seems likely that elevated levels of plasma glucagon may contribute to the increased hepatic MT concentrations in rats treated with DDC plus NiClz [3,6,34]. Alternatively, increased hepatic Ni(I1) concentrations may stimulate transcription of the MT-gene.
19
ACKNOWLEDGEMENTS
The following students at Providence College assisted in trace metal analyses: Mary Hillstrom, Elizabeth Doll, Lisa Lauder, and Mary Roque; the following research assistants at the University of Connecticut assisted in glutathione analyses: Connie Liber and Cristina Crisostomo. REFERENCES 1 F.W. Sunderman Jr., M.C. Reid, L.M. microsomal heme oxygenase activity
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17 T. Maitani and K.T. Suzuki, Extents of hepatic zinc-thionein induction in mice given an equimolar dose of various heavy metals. Chem. Pharm. Bull., 30 (1982) 4164. 18 J. Chmielnicka, J.A. Szymanska and J. Tyfa, Disturbances in the metabolism of endogenous metals (Zn and Cu) in nickel-exposed rats. Environ. Res., 27 (1982) 216. I9 J. Koutensky, V. Eybl, M. Koutenska, J. Sykora and F. Mertl, Influence of sodium diethyldithiocarbamate on the toxicity and distribution of copper in mice. Eur. J. Pharmacol., 14 (i971) 389. 20 B. Bertram, J. Schuhmacher, E. Frei, N. Frank and M. Wiessler, Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats. Biochem. Pharmacol., 31 (1982) 3613. 21 M. Marselos, P. Alakuijala, M. Lang and R. Torronen, Studies on the mechanism by which disulfiram and diethyldithiocarbamate affect drug oxidation, in V. Ullrich, I. Roots, A. Hildebrand, R.W. Estabrook and A.H. Conney (Eds.), Microsomes and Drug Oxidations, Pergamon Press, Oxford, 1977, pp. 589-596. 22 S.K. Tandon, J.R. Behari and M. Ashquin, Effects of thiol chelators on trace metal levels. Res. Commun. Chem. Pathol. Pharmacol., 42 (1983) 501. 23 B.D. Goldstein, M.G. Rozen, J.C. Quintavalla and M.A. Amoruso, Decrease in mouse lung and liver glutathione peroxidase activity and potentiation of the lethal effects of ozone and paraquat by the superoxide dismutase inhibitor, diethyldithiocarbamate. Biochem. Pharmacol., 28 (1979) 27. 24 C.P. Siegers, M. Younes and G. Schmitt, Effects of dithiocarb and (+ )-cyanidanol-3 on the hepatoxicity and metabolism of vinylidine chloride in rata. Toxicology, 15 (1979) 55. 25 G.E. Miller, M.A. Zemaitis and F.E. Greene, Mechanisms of diethyldithiocarbamateinduced loss of cytochrome P-450 from rat liver. Biochem. Pharmacol., 32 (1983) 2433. 26 M.D. Maines and A. Kappas, Nickel-mediated alterations in the activity of hepatic and renal enzymes of heme metabolism and heme dependent cellular activities, in S.S. Brown (Ed.), Clinical Chemistry and Chemical Toxicology of Metals, Elsevier/North Holland Biomedical Press, Amsterdam, 1977, pp. 75-81. 27 K-L Wong and C.D. Klaassen, Relationship between liver and kidney levels of glutathione and metallothionein in rata. Toxicology, 19 (1981) 39. 28 M. Kawata and K.T. Suzuki, Relation between metal and glutathione concentrations in mouse liver after cadmium, zinc or copper loading. Toxicol. Lett., 15 (1983) 131. 29 G. Kikuchi and T. Yoshida, Function and induction of microsomal heme oxygenase. Mol. Cell. Biochem., 53 (1983) 163. 30 R.F. Burk and M.A. Correia, Stimulation of rat hepatic microsomal heme oxygenase by diethyl maleate. Res. Commun. Chem. Pathol. Pharmacol. 24 (1979) 205. 31 M.D. Maines, Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney. Arch. Biochem. Biophys., 216 (1982) 17. 32 R.A. Neal, Microsomal enzymes and the toxicity of thiono-sulfur compounds, in M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brien (Eds.), Microsomes, Drug Oxidations and Chemical Carcinogenesis, Vol. 2, Academic Press, New York, 1980, pp. 791-799. 33 M.A. Zemaitis and F.E. Greene, In uiuo and in vitro effects of thiuram disulfides and dithiocarbamates on hepatic microsomal drug metabolism in the rat. Toxicol. Appl. Pharmacol., 48 (1979) 343. 34 T. Maitani and K.T. Suzuki, Dose-dependent induction of metallothionein in kidneys of mice injected with indium and nickel ions. Chem. Pharm. Bull., 31 (1983) 979.
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