Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein Jie Liu , William C. Kershaw & Curtis D. Klaassen To cite this article: Jie Liu , William C. Kershaw & Curtis D. Klaassen (1992) Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein, Journal of Toxicology and Environmental Health, 35:1, 51-62, DOI: 10.1080/15287399209531593 To link to this article: http://dx.doi.org/10.1080/15287399209531593
Published online: 15 Oct 2009.
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Date: 06 November 2015, At: 23:46
PROTECTIVE EFFECTS OF ZINC O N CULTURED RAT PRIMARY HEPATOCYTES TO METALS WITH LOW AFFINITY FOR METALLOTHIONEIN Jie Liu, William C. Kershaw, Curtis D. Klaassen
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Environmental Health & Occupational Medicine Center, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCI2 (100 µM) for 24 h; which increased the MT concentration 15-fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn-, Cr-, and V-induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr-, Mn-, Fe-, Se-, or V-induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn-induced thiol or membrane stabilization and/or other biological changes produced by Zn.
INTRODUCTION Zinc is an essential trace element and protects against hepatotoxicity produced by a variety of chemicals. For example, Zn pretreatment has been shown to protect against toxicity of carbon tetrachloride (Cagen and Klaassen, 1979; Clarke and Lui, 1987), bromobenzene (McMillan and Schnell, 1984; Szymañska et al., 1991), ethanol (Dar et al., 1986), acetaminophen (Chengelis et al., 1986), and pyrrolizidine alkaloid (Miranda et al., 1982), as well as Cd and Hg (Goering and Klaassen, 1984; Fukino et al., 1986). The mechanism(s) of Zn protection has been attributed to the following: (1) stabilization of lysosomal, mitochondrial, and microsomal membranes, resulting in a resistance of lipid peroxidation in these membranes (Chvapil et al., 1972a, 1972b; Ludwig and Chvapil, 1980; Thomas et al., 1986); (2) inhibition of P-450-dependent monooxyThis work supported by USPHS Grant ES-01142; W.C. Kershaw was supported by USPHS Training Grant ES-07079. Requests for reprints should be sent to Curtis D. Klaassen, Ph.D., Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66103. 51 Journal of Toxicology and Environmental Health, 35:51-62, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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genäse activity, thus reducing the amount of toxic metabolites available for covalent binding to critical organdíes or proteins (McMillan and Schnell, 1984; Pour et al., 1986); (3) stabilization of cellular thiols and the activation of glutathione-associated enzymes (Cho and Fong, 1990; Floersheim and Floersheim, 1986; Fukino et al., 1986); and (4) sequestration of reactive moieties, free radicals, and metal ions by the Zninduced metallothionein (MT) (Cagen and Klaassen, 1979; Dunn et al., 1987; Goering and Klaassen, 1984). Metallothionein, a cysteine-rich, metal-binding protein, has been proposed to function in metal ion detoxification (for reviews, see Dunn et al., 1987; Webb, 1987). We observed that Zn pretreatment of cultured hepatocytes induced MT more than 15 times over control cells, and Zninduced MT appeared to be responsible for the protection against cytotoxicity of metals with a high affinity for this protein (Liu et al., 1990, 1991). However, whether metals with low or no affinity for MT can be detoxified by Zn pretreatment remains to be examined. Therefore, the primary purpose of the present study was to determine whether Zn pretreatment protects against Mn-, Se-, V-, Cr-, and Fe-induced hepatotoxicity in rat primary hepatocyte cultures. These metals were chosen because they have little or no affinity for MT (Waalkes et al., 1984) and do not induce MT in cultured hepatocytes (Bracken and Klaassen, 1987). Initial experiments indicated that Zn pretreatment had a cytoprotective effect for all metals tested. Subsequent studies were performed to determine if the mechanism of protection involved altered metal uptake, subcellular distribution of metals, or changes in glutathione (GSH) homeostasis.
MATERIALS AND METHODS Chemicals
CrCl3, FeCI3, and ZnCI2 were obtained from Fisher Scientific Co. (Fair Lawn, N.J.), MnCI2 and Na3VO4 from Sigma Chemical Co. (St. Louis, Mo.), and Na3SeO3 from J.T. Baker Chemical Co. (Phillipsburg, N.J.). 54 Mn (510 mCi/mg) and 75Se (64.8 mCi/mg) were purchased from Amersham Corp. (Arlington Heights, III.) and New England Nuclear Corp. (Boston, Mass.), respectively. All other chemicals were of the highest purity commercially available. Animals Male Sprague-Dawley rats (Sasco Inc., Omaha, Neb.) weighing 250350 g were housed in animal facilities maintained at 22 ± 2°C with a 12h light/dark cycle. Purina Laboratory Rodent Chow (St. Louis, Mo.) and tap water were provided ad libitum.
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Preparation and Culture of Hepatocytes
Hepatocytes were prepared by a two-stage single-pass perfusion method (Berry and Friend, 1969) as modified by Bissell and Guzelian (1980). Briefly, an oxygenated Hanks balanced salt solution (Ca2+, Mg 2+ free) supplemented with Tris-HCI (25 mM) and EGTA (0.5 mM) was perfused through the liver via the portal vein for 15 min at 37°C. The liver was then perfused with 200 ml serum-free media containing 0.05% collagenase type II (Worthington Biochemical Co., Freehold, N.J.) at a flow rate of 12-15 ml/min. Following enzymatic digestion, the liver was removed, minced, and filtered through four layers of sterile gauze. Hepatocytes were separated from nonparenchymal cells and debris by centrifugation (50 x g, 40 s) and the cell pellet washed three times with media. Cell viability ranged from 85 to 92% as determined by trypan blue dye exclusion. Primary cultures were established by seeding 3 x 106 cells onto 60mm culture dishes coated with collagen, obtained from Collagen Corp. (Palo Alto, Calif.) and incubated at 37°C (5% CO2 and 95% humidity) for 60 min. Media was changed after cell attachment and the cultures were incubated in fresh media for an additional 22 h before treatment. The culture media was a modified Waymouth 75Vi serum-free media as previously described (Bracken and Klaassen, 1987). Insulin (1 fiM), ornithine (20 mg/l), ascorbic acid (0.3 mM), methionine (0.5 mM), and cysteine (0.5 mM) were added. Experimental Design Twenty-four hours after hepatocyte isolation, dishes were treated with ZnCI2 (100 fiM) for 24 h. This pretreatment schedule was previously shown to increase MT concentration in hepatocytes about 15-fold (Liu et al., 1990) from 0.45 ± 0.029 to 7.04 ± 0.319 /¿g/mg protein in the Zn pretreated cells. Subsequently, the Zn-containing media was removed and fresh media without Zn was added. At this point, the cultures were treated with metals for an additional 24 h. All treatments were 1% volume of media. Metallothionein Assay MT concentrations were estimated by the Cd-hemoglobin radioassay method as previously described by Eaton and Toal (1982). Concentration of total cellular protein was measured by the method of Bradford (1976), using bovine serum albumin as standard. Evaluation of Cytotoxicity Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity in the culture media and intracellular potassium (K ) content. For the measurement of LDH, media was collected, centrifugated
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(1000 x g, 5 min), and the supernatant analyzed for LDH activity as described by Henry et al. (1960), using a DU-8 spectrophotometer (Beckman Instruments, Inc., Palo Alto, Calif.). For the measurement of K+ content, hepatocytes were rinsed with isotonic saline three times and sonicated in 2 ml Tris-HCI buffer (10 mM, pH 7.4) with an Ultrasonics model W-10 cell disrupter (Plainview, N.Y.). Aliquots of sonicated cell suspensions were centrifugated (10,000 x g, 3 min) and 100 ¡A supernatant was analyzed for K+ by flame photometry (Instrument Lab., Inc., Lexington, Mass.). GSH Assay Hepatocellular GSH content was determined using the enzymatic method of Tietze (1969). A 250-/J aliquot of sonicated cell suspension was mixed with 250 ¡x\ of 4% (w/v) sulfosalicylic acid at 4°C and stored overnight at this temperature. Samples were centrifuged (3000 x g, 10 min) the next day and a 100-fil aliquot of the supernatant was mixed with 700 fi\ of the incubation buffer (0.1 M NaH2PO4 and 0.5 mM EDTA), 50 (i\ of 10 mM 5,5'-dithiobis (2-nitrobenzoic acid), 50 /¿I GSH reductase (10 U/ml), and 100 /¿I NADPH (2 mg/ml), and the change in absorbance at 412 nm was determined with a Beckman model 25 spectrophotometer. Hepatocellular Uptake and Accumulation of Mn or Se Following Zn treatment, fresh media containing 300 ¡xM MnCI 2 + 0.3 /¿Ci ^Mn/dish or 20 fiM Na2SeO3 + 0.3 ¿iCi 7SSe/dish were added to cultures and incubated for 1, 2, 4, 8, 12, or 24 h. At the end of each incubation, radioactive media was removed, and cells were washed twice with ice-cold buffered saline (10 mM Hepes, 154 mM NaCI, 10>nM EDTA, pH 7.4) and once with Hepes buffered Hank's salt solution (Failla et al., 1979). Cells were collected and sonicated in 1 ml Tris buffer. Metal content was determined by gamma scintillation spectrometry (Packard, Downers Grove, III.). Total cellular protein was determined as described above. Subcellular Distribution of Mn and Se Radioactive media was removed 12 h following incubation with Mn or Se, and dishes were washed as described above. Cells from six dishes were scraped, pooled in a total of 8 ml homogenizing buffer (0.25 M sucrose, 10 mM Tris-acetate, pH 7.4), and disrupted with a motor-driven Teflon pestle glass homogenizer (10 up-and-down strokes). The various fractions were prepared by differential centrifugation (Goering and Klaassen, 1984), and the resultant pellets were defined as nuclear (600 x g, 10 min), mitochondria (10,000 x g, 10 min), microsomes (100,000 x g, 60 min), and cytosol (100,000 x g supernatant). The distribution of Mn and Se in the cytosol was determined by
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Sephedex G-75 gel filtration chromatography. The samples were eluted from the column (1.5 x 60 cm) with 10 miW Tris acetate (pH 7.4) at a rate of 30 ml/h at 4°C. Fractions of 5.0 ml were collected and metal content was determined as described above. Statistical Analysis
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Comparison between control and treatment was made by the Student's í test. The .05 level of probability was used as the criterion of significance. RESULTS The effect of Zn pretreatment on the toxicity to cultured hepatocytes produced by five metals that have little or no affinity for MT are shown in Figures 1 and 2. Following Zn treatment of cultured hepatocytes for 24 h to increase MT concentrations, five concentrations of 2000 Control •O Zn-pretreated 1600-. 1200800400H
1
1
I-
H
1
1
1
1
H-
1512963-
0 300 Mn
900
1500
30
0 10
50
Se
FIGURE 1. Effect of Zn pretreatment on the toxicity of Se and Mn in rat primary hepatocyte cultures. Monolayers were pretreated with ZnCI 2 (100 fiM) for 24 h, then exposed to metals for an additional 24 h. Each value represents the mean ± SE of four dishes from one of three rats. Asterisk indicates values significantly different from controls (p < .05).
J. LIU ET AL.
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1200
Control O—O Zn-prelreated
900
600
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300
8-
\
0 400
1Z00 2000
0 300
B00
1500
0 300
BOO 1500
FIGURE 2. Effect of Zn pretreatment on the toxicity of Fe, V, and Cr in rat primary hepatocyte cultures. Monolayers were pretreated with ZnCI 2 (100 ¡iM) for 24 h, then exposed to metals for an additional 24 h. Each value represents the mean ±SE of four dishes from one of three rats. Asterisk indicates values significantly different from controls (p < .05).
each metal were added to the monolayers and the toxicity quantitated 24 h later. Figure 1 shows the effect of Zn pretreatment on Mn and Se toxicity. A much higher concentration of Mn (300 (iM) was necessary to produce toxicity than Se (30 fiM), as indicated by LDH leakage and cellular K+ loss. Zn-pretreated cells were more resistant to Mn and Se than were the control cells. The effects of Zn pretreatment on Cr-, V-, and Feinduced hepatotoxicity are shown in Figure 2. All three of these metals were relatively nontoxic to the hepatocytes, as 300, 900, and 400 ¡xM of Cr, V, and Fe, respectively, were required to produce toxicity. Although higher concentrations of Fe appear to inhibit LDH activity, Fe produced a dose-dependent release of K+ release from hepatocytes. Zn pretreatment of the cultured hepatocytes did decrease the hepatotoxicity produced by all three metals. Of the five metals examined in Figures 1 and 2, Zn pretreatment seems to be more effective against Se and V than Fe, Mn, or Cr. In order to examine possible mechanisms for the protective effect that Zn has on the toxicity of metals that have little or no affinity for MT, we examined the hepatocellular uptake and distribution of two of the tested metals, namely, Se and Mn. Figure 3 shows the hepatocellular uptake of these two metals. Zn pretreatment had no appreciable effect
HEPATOPROTECTIVE EFFECTS OF ZINC
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400
• — • Control
O—O Zn-pretreated £
300-
Cu
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a
S
12
IS
20
24
TIME (hr) FIGURE 3. Effect of Zn pretreatment on the uptake of 75Se and M M n into cultured hepatocytes. Monolayers were pretreated with ZnCI 2 (100 /tM) for 24 h, then exposed to 20 ¡iM (0.3 /iCi) Se or 300 ¡iM (0.3 /id) Mn for various times. Each value represents the mean ±SE of four dishes from one of three rats.
on the uptake of either metal over the entire 24-h period. Within 8 h of exposure, the intracellular concentrations of both metals have attained an apparent steady-state condition. There was a marked difference in the subcellular distribution of Mn and Se (Table 1). About 70% of Mn taken up by the cells was distributed to the cytosol, in contrast to 12% of Se. Zn pretreatment had minimal TABLE 1. Subcellular Distribution of Mn and Se in Hepatocyte Cultures
Croup
Nuclei (%)
Mitochondria (%)
Microsomes (%)
Cytosol (%)
4.6 ± 0.2 3.0 + 0.1 3
67.2 ± 4.1 72.2 ± 5.5
7.2 ± 0.2 5.5 ± 0.2a
11.8 ± 1.7 11.5 ± 0.7
Manganese Control Zn-pretreated
18.1 ± 0.5 18.3 ± 1.4
Control Zn-pretreated
46.2 ± 3.0 50.7 ± 3.3
9.8 ± 0.3 6.8 ± 0.1 a Selenium 34.8 ± 1 . 6 32.8 ± 2.4
Note. Values are means ±SE of three cultures from one of three rats. a Significantly different from controls, p < .05.
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effects on the subcellular distribution of Mn and Se; however, a small decrease in the amount of Mn in the mitochondrial and microsomal fractions and a small decrease in the amount of Se in the microsomal fraction were noted in the Zn-pretreated hepatocytes. The distribution of Mn and Se in the cytosol of control and Znpretreated hepatocytes is shown in Figure 4. MT elutes between fractions 45 and 55 (data not shown). From these profiles there is no evidence that either metal is associated with MT. Instead, the metals mainly appeared in molecular weight fractions (65-85), smaller than MT, and a small amount of Se appeared in high-molecular-weight fractions (15-30). There was no difference in the distribution of either Se or Mn within the cytosol of Zn-pretreated cells and control cells. The effect of Zn-pretreatment on cellular GSH concentration is shown in Figure 5. Mn (500 pM), Cr (500 pM), V (1500 /xM), and Fe (2000 fiM) all decreased the concentration of GSH in the hepatocytes. Although Se at 40 pM was cytotoxic to cultured hepatocytes (Fig. 1 and Table 2), it did not decrease GSH concentrations, but increased GSH levels. Zn pretreatment had no direct effect on cellular GSH levels of control hepatocytes, but markedly decreased the GSH-depleting effects produced by Mn, V, and Cr. There was no difference in GSH concentration between control and Zn-pretreated cells exposed to Se or Fe. DISCUSSION All five metals employed in the present study are cytotoxic in a concentration-dependent manner to cultured hepatocytes as indicated by intracellular K+ loss and extracellular LDH leakage. Se is the most toxic of these metals, as only 30 fiM produced injury, whereas with the other metals 300 /xM or more was required to produce cytotoxicity. More importantly, the present study has shown that Zn pretreatment protects against all these five metals, which have little or no affinity for MT. Table 2 compares the effectiveness of Zn pretreatment to decrease the toxicity of the five metals. It appears that Zn has a lesser protective effect on Mn and Cr than the other three metals. One possible mechanism by which Zn might produce the protective effect is by decreasing the uptake of metals into the hepatocytes. As shown in Figure 3, Zn pretreatment had no appreciable effect on Se or Mn uptake and accumulation. Therefore, Zn-induced protection against these two metals does not appear to be due to a decreased uptake into the hepatocytes. It is known that Zn-induced protection against Cd hepatoxicity in the intact animal (Goering and Klaassen, 1984) as well as in isolated hepatocytes (Din and Frazier, 1985; Liu et al., 1990) is due to MT that is induced by the Zn pretreatment. The MT binds the metals in the cytosol and thus alters the subcellular distribution of Cd. Similar alterations
59
HEPATOPROTECTIVE EFFECTS OF ZINC
1 s
o. Ji,
4000
• — • Control o—o Zn-pretreated 3000-
•z
o
2000-
1000-
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o
1500-
fe? 1000500.
0
10
20
30
40
50
60
70
B0 SO
FRACTION 75
FIGURE 4. G-75 gel filtration profiles of Se and 54 Mn from cultured hepatocyte cytosol when administered 20 ¡xM (0.3 ftCi) Se or 300 ¡iM (0.3 ¡iCt) Mn in control and Zn-pretreated cells 12 h prior to harvest. Samples were eluted from the column with 10 mM Tris buffer at a rate 30 ml/h. Fractions of each 5.0 ml were collected and analyzed. 400
M Control CXI Zn-pretreated
.S Ü o
300-
B
200-
« m
100
O
Con
J Mn
II I Cr
Se
Fe
FIGURE 5. Effect of Zn pretreatment on cellular GSH levels in rat hepatocyte cultures exposed to five metals. Hepatocytes were pretreated with ZnCI 2 (100 ¡iM) for 24 h, then exposed to metals (Mn 500 fiM, Cr 500 /tM, V 1500 ¿«M, Se 40 pM, Fe 200 ¡iM) for an additional 24 h. Each value represents the mean ±SE of four dishes. Asterisk indicates values are significantly different from the corresponding control (p < .05).
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TABLE 2. Comparison of Protection of Zn Pretreatment on Metal Toxicity in Rat Primary Hepatocyte Cultures
Metal
Cone. (liM)
V
1200
Se
40
Fe
2000
Mn
900
Cr
1200
Group
Extracellular LDH (U/l)
Control Zn-treated Control Zn-treated Control Zn-treated Control Zn-treated Control Zn-treated
682 ± 55 386 ± 57a 896 ± 161 512 ± 141a 359 ± 42 150 ± 35a 1385 ± 182 1047 ± 91 a 911 ± 69 733 ± 80a
Percent of control
57 57 42 76 80
Intracellular K + (nmol/mg protein) 8.20 12.55 6.89 9.40 7.84 10.45 10.07 11.04 6.26 8.02
± ± ± ± ± ± ± ± ± ±
0.49 0.78a 0.26 0.86a 0.45 0.43a 1.29 1.25a 0.65 0.87a
Percent of control
153 136 133 110 128
"Significant at p < .05.
in the subcellular distribution and toxicity with other metals that have a high affinity for MT have also been shown (Liu et al., 1991). However, in the present study, five metals that have little or no affinity for MT (Waalkes et al., 1984) were examined, yet protection was observed. As one would anticipate, in the present study there was no indication that the metals bound to MT in the cytosol (Figure 4), nor was there a marked increase in the subcellular distribution of metals to the cytosol as observed with metals that have a high affinity for MT (Liu et al., 1990, 1991). Therefore, the Zn-induced protection for these metals does not appear to be due to the well-known sequestering role of MT for metals. Zn-induced protection against some organic toxicants such as acetaminophen (Chengelis et al., 1986) and alkylating agents (Tobey et al., 1982) also does not appear to be due to MT. There appears to be at least one additional protective mechanism produced by Zn. It has been proposed that Zn may protect against the toxicity of chemicals by inhibiting lipid peroxidation (Chvapil et al., 1972b; Thomas et al., 1986; Willson, 1987). It has been shown previously (Stacey and Klaassen, 1981) that of the five metals examined in this study, two (Cr and Mn) do not produce any lipid peroxidation and Se produces only a minimal amount. Thus, it seems unlikely that Zn has a universal action of protection against the toxicity of metals by inhibiting lipid peroxidation. Another mechanism for the protection provided by Zn may involve the stabilization of tissue thiols or the activation of GSH-associated enzymes (Cho and Fong, 1990; Fukino et al., 1986; Ploersheim and Floersheim, 1986; Szymariska et al., 1991). In the present study, Zn pretreatment did not increase cellular GSH concentration in cultured isolated hepatocytes as has been previously reported for the intact animal (Chengelis et al., 1986; Wong and Klaassen, 1981), but it did prevent the GSH depletion caused by Mn, Cr, and V. Because cellular GSH has an important role in protection against toxic chemicals, this protection
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against GSH depletion may in part explain Zn-induced protection for these three metals. Se and Fe did not deplete GSH at cytotoxic concentrations; therefore, the protection for these two metals must be due to another mechanism. Zn pretreatment can also induce other biological changes such as stabilization of lysosomes and cellular membranes (Dar et al., 1986; Chvapil et al., 1972; Ludwig and Chvapil, 1980; Pfeiffer et al., 1980). This stabilization might also play a role in the protection Zn provides against the toxicity of these metals. In conclusion, Zn pretreatment of cultured hepatocytes protects against Se-, V-, Mn-, Fe-, and Cr-induced cytotoxicity. This protection does not appear to be due to decreasing cellular metal uptake, altering subcellular distribution of metals, nor the binding of metals to MT. The mechanism of Zn-induced protection against the cytotoxicity of metals is not known but may be related to the stabilization of cellular membranes. REFERENCES Berry, M. N. and Friend, D.S. 1969. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43:506-520. Bissell, D. M., and Guzelian, P, S. 1980. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann. NX Acad. Sci. 349:85-98. Bracken, W. M., and Klaassen, C. D. 1987. Induction of metallothionein in rat primary hepatocyte cultures: Evidence for direct and indirect induction. J. Toxicol. Environ. Health 22:163-174. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Cagen, S. Z., and Klaassen, C. D. 1979. Protection of carbon tetrachloride-induced hepatotoxicity by zinc: Role of metallothionein. Toxicol. Appl. Pharmacol. 51:107-116. Chengelis, C. P., Dodd, D. C., Means, J. R., and Kotsonis, F. N. 1986. Protection by zinc against acetaminophen induced hepatotoxicity in mice. Fundam. Appl. Toxicol. 6:278-284. Cho, C. H., and Fong, L. Y. Y. 1990. The interaction of ethanol and zinc on hepatic glutathione and glutathione transferase activity in mice. Agents Actions 29:382-385. Chvapil, M., Ryan, J. N., and Zukoski, C. F. 1972a. The effect of zinc and other metals on the stability of lysosomes. Proc. Soc. Exp. Biol. Med. 140:642-646. Chvapil, M., Ryan, J. N. and Zukoski, C. F. 1972b. Effect of zinc on lipid peroxidation in liver microsome and mitochondria. Proc. Soc. Exp. Biol. Med. 141:150-153. Clarke, I. S., and Lui, E. M. K. 1986. Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol. 64:1104-1110. Dar, M. S., Townsend, S. M. and Wooles, W.R. 1986. Protective effect of zinc against ethanol toxicity in mice. J. Toxicol. Environ. Health. 18:41-48. Din, W. S., and Frazier, J. M. 1985. Protective effect of metallothionein on Cd toxicity in isolated rat hepatocytes. Biochem. J. 230:395-402. Dunn, M. A., Blalock, T. L., and Cousins, R. J. 1987. Metallothionein. Proc. Soc. Exp. Biol. Med. 185:107-119. Eaton, D. L., and Toal, B. F. 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66:134-142. Failla, M. L., Cousins, R. J., and Mascenik, M. J. 1979. Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture. Biochim. Biophys. Acta 583:63-72. Floersheim, C. L., and Floersheim, P. 1986. Protection against ionizing radiation and synergism with thiols by zinc aspartate. Br. J. Radiol. 59:597-602. Fukino, H., Hirai, M., Hsueh, Y. M., Moriyasu, S., and Yamane, Y. 1986. Mechanism of protection by
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zinc against mercuric chloride toxicity in rats: Effects of zinc and mercury on glutathione metabolism. J. Toxicol. Environ. Health 19:75-89. Goering, P. L., and Klaassen, C. D. 1984. Zinc-incluced tolerance to cadmium hepatotoxicity. Toxicol. Appl. Pharmacol. 74:299-307. Henry, R. T., Chiamori, N., Golub, O. J., and Berkman, S. 1960. Spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and lactic dehydrogenase. Am. J. Clin. Pathol. 34:381-398. Liu, J., Kershaw, W. C., and Klaassen, C. D. 1990. Rat primary hepatocyte cultures are a good model for examining metallothionein-induced tolerance to cadmium toxicity. In Vitro Cell Dev. Biol. 26:75-79. Liu, J., Kershaw, W. C., and Klaassen, C. D. 1991. The protective effects of metallothionein on the toxicity of various metals in rat primary hepatocyte culture. Toxicol. Appl. Pharmacol. 107:2734. Ludwig, J. C., and Chvapil, M. 1980. Reversible stabilization of liver lysosomes by zinc ions. J. Nutr. 110:945-953. McMillan, D. A., and Schnell, R. C. 1984. Zinc protection against bromobenzene induced hepatotoxicity in the rat. Toxicologist 4:451. Miranda, C. L., Henderson, M. C., Reed, R. L., Schmitze, J. A., and Buhler, D. R. 1982. Protective action of zinc against pyrrolizidine alkaloid-induced hepatotoxicity in rats. J. Toxicol. Environ. Health 9:359-366. Pfeiffer, C. J., Cho, C. H., Cheema, A. and Saltman, D. 1980. Reserpine-induced gastric ulcers: Protection by lysosomal stabilization due to zinc. J. Pharmacol. 61:347-358. Pour, A. M., Davies, M. H., Blacker, A., Weir, S. A., and Schnell, R. C. 1986. Effect of zinc on acetaminophen biotransformation in male rats. Toxicologist 6:269. Stacey, N. H., and Klaassen, C. D. 1981. Comparison of the effects of metals on cellular injury and lipid peroxidation in isolated rat hepatocytes. J. Toxicol. Environ. Health. 7:139-147. Szymariska, J. A., Świetlicka, E. A., and Piotrowski, J. K. 1991. Protective effect of zinc in the hepatotoxicity of bromobenzene and acetaminophen. Toxicology 66:81-91. Thomas, J. P., Bachowski, C. J., and Girotti, A. W. 1986. Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins. Biochim. Biophys. Acta. 884:448-461. Tietze, F. 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27:502-522. Tobey, R. A., Enger, M. D., Griffith, J. K., and Hildebrand, C. E. 1982. Zinc-induced resistance to alkylating agents: Lack of correlation between cell survival and metallothionein content. Toxicol. Appl. Pharmacol. 64:72-78. Waalkes, M. P., Harvey, M. J., and Klaassen, C. D. 1984. Relative in vitro affinity of hepatic metallothionein for metals. Toxicol. Lett. 20:33-39. Webb, M. 1987. Toxicological significance of metallothionein. Experientia (Suppl.) 52:109-134. Willson, R. L. 1987. Vitamin, selenium, zinc and copper interactions in free radical protection against ill-placed iron. Proc. Nutr. Soc. 46:27-34. Wong, K, L., and Klaassen, C. D. 1981. Relationship between liver and kidney levels of glutathione and metallothionein in rats. Toxicology 19:39-47. Received November 16, 1990 Accepted August 12, 1991
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Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein Jie Liu , William C. Kershaw & Curtis D. Klaassen To cite this article: Jie Liu , William C. Kershaw & Curtis D. Klaassen (1992) Protective effects of zinc on cultured rat primary hepatocytes to metals with low affinity for metallothionein, Journal of Toxicology and Environmental Health, 35:1, 51-62, DOI: 10.1080/15287399209531593 To link to this article: http://dx.doi.org/10.1080/15287399209531593
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Date: 06 November 2015, At: 23:46
PROTECTIVE EFFECTS OF ZINC O N CULTURED RAT PRIMARY HEPATOCYTES TO METALS WITH LOW AFFINITY FOR METALLOTHIONEIN Jie Liu, William C. Kershaw, Curtis D. Klaassen
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Environmental Health & Occupational Medicine Center, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
The purpose of this study was to determine if Zn pretreatment could protect rat primary hepatocyte cultures from the cytotoxicity of five metals that have little or no affinity for metallothionein (MT). Hepatocytes were grown in monolayer cultures for 22 h and subsequently treated with ZnCI2 (100 µM) for 24 h; which increased the MT concentration 15-fold. Following Zn pretreatment, hepatocytes were exposed to various concentrations of Mn, V, Cr, Se, or Fe for an additional 24 h. Cytotoxicity was assessed by enzyme leakage and loss of intracellular K+. The toxicity of all five metals was significantly reduced in the Zn-pretreated cells. Zn pretreatment had no appreciable effect on the hepatocellular uptake (1-24 h) of Mn or Se. Zn pretreatment also did not increase the distribution of Mn or Se to the cytosol and neither metal was bound to MT, suggesting the protection was not due to their binding to MT. However, Zn pretreatment significantly decreased Mn-, Cr-, and V-induced cellular glutathione depletion. In summary, Zn pretreatment of rat primary hepatocyte cultures protects against Cr-, Mn-, Fe-, Se-, or V-induced hepatotoxicity. This protection does not appear to be related to MT induction but may be due to Zn-induced thiol or membrane stabilization and/or other biological changes produced by Zn.
INTRODUCTION Zinc is an essential trace element and protects against hepatotoxicity produced by a variety of chemicals. For example, Zn pretreatment has been shown to protect against toxicity of carbon tetrachloride (Cagen and Klaassen, 1979; Clarke and Lui, 1987), bromobenzene (McMillan and Schnell, 1984; Szymañska et al., 1991), ethanol (Dar et al., 1986), acetaminophen (Chengelis et al., 1986), and pyrrolizidine alkaloid (Miranda et al., 1982), as well as Cd and Hg (Goering and Klaassen, 1984; Fukino et al., 1986). The mechanism(s) of Zn protection has been attributed to the following: (1) stabilization of lysosomal, mitochondrial, and microsomal membranes, resulting in a resistance of lipid peroxidation in these membranes (Chvapil et al., 1972a, 1972b; Ludwig and Chvapil, 1980; Thomas et al., 1986); (2) inhibition of P-450-dependent monooxyThis work supported by USPHS Grant ES-01142; W.C. Kershaw was supported by USPHS Training Grant ES-07079. Requests for reprints should be sent to Curtis D. Klaassen, Ph.D., Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66103. 51 Journal of Toxicology and Environmental Health, 35:51-62, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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genäse activity, thus reducing the amount of toxic metabolites available for covalent binding to critical organdíes or proteins (McMillan and Schnell, 1984; Pour et al., 1986); (3) stabilization of cellular thiols and the activation of glutathione-associated enzymes (Cho and Fong, 1990; Floersheim and Floersheim, 1986; Fukino et al., 1986); and (4) sequestration of reactive moieties, free radicals, and metal ions by the Zninduced metallothionein (MT) (Cagen and Klaassen, 1979; Dunn et al., 1987; Goering and Klaassen, 1984). Metallothionein, a cysteine-rich, metal-binding protein, has been proposed to function in metal ion detoxification (for reviews, see Dunn et al., 1987; Webb, 1987). We observed that Zn pretreatment of cultured hepatocytes induced MT more than 15 times over control cells, and Zninduced MT appeared to be responsible for the protection against cytotoxicity of metals with a high affinity for this protein (Liu et al., 1990, 1991). However, whether metals with low or no affinity for MT can be detoxified by Zn pretreatment remains to be examined. Therefore, the primary purpose of the present study was to determine whether Zn pretreatment protects against Mn-, Se-, V-, Cr-, and Fe-induced hepatotoxicity in rat primary hepatocyte cultures. These metals were chosen because they have little or no affinity for MT (Waalkes et al., 1984) and do not induce MT in cultured hepatocytes (Bracken and Klaassen, 1987). Initial experiments indicated that Zn pretreatment had a cytoprotective effect for all metals tested. Subsequent studies were performed to determine if the mechanism of protection involved altered metal uptake, subcellular distribution of metals, or changes in glutathione (GSH) homeostasis.
MATERIALS AND METHODS Chemicals
CrCl3, FeCI3, and ZnCI2 were obtained from Fisher Scientific Co. (Fair Lawn, N.J.), MnCI2 and Na3VO4 from Sigma Chemical Co. (St. Louis, Mo.), and Na3SeO3 from J.T. Baker Chemical Co. (Phillipsburg, N.J.). 54 Mn (510 mCi/mg) and 75Se (64.8 mCi/mg) were purchased from Amersham Corp. (Arlington Heights, III.) and New England Nuclear Corp. (Boston, Mass.), respectively. All other chemicals were of the highest purity commercially available. Animals Male Sprague-Dawley rats (Sasco Inc., Omaha, Neb.) weighing 250350 g were housed in animal facilities maintained at 22 ± 2°C with a 12h light/dark cycle. Purina Laboratory Rodent Chow (St. Louis, Mo.) and tap water were provided ad libitum.
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Preparation and Culture of Hepatocytes
Hepatocytes were prepared by a two-stage single-pass perfusion method (Berry and Friend, 1969) as modified by Bissell and Guzelian (1980). Briefly, an oxygenated Hanks balanced salt solution (Ca2+, Mg 2+ free) supplemented with Tris-HCI (25 mM) and EGTA (0.5 mM) was perfused through the liver via the portal vein for 15 min at 37°C. The liver was then perfused with 200 ml serum-free media containing 0.05% collagenase type II (Worthington Biochemical Co., Freehold, N.J.) at a flow rate of 12-15 ml/min. Following enzymatic digestion, the liver was removed, minced, and filtered through four layers of sterile gauze. Hepatocytes were separated from nonparenchymal cells and debris by centrifugation (50 x g, 40 s) and the cell pellet washed three times with media. Cell viability ranged from 85 to 92% as determined by trypan blue dye exclusion. Primary cultures were established by seeding 3 x 106 cells onto 60mm culture dishes coated with collagen, obtained from Collagen Corp. (Palo Alto, Calif.) and incubated at 37°C (5% CO2 and 95% humidity) for 60 min. Media was changed after cell attachment and the cultures were incubated in fresh media for an additional 22 h before treatment. The culture media was a modified Waymouth 75Vi serum-free media as previously described (Bracken and Klaassen, 1987). Insulin (1 fiM), ornithine (20 mg/l), ascorbic acid (0.3 mM), methionine (0.5 mM), and cysteine (0.5 mM) were added. Experimental Design Twenty-four hours after hepatocyte isolation, dishes were treated with ZnCI2 (100 fiM) for 24 h. This pretreatment schedule was previously shown to increase MT concentration in hepatocytes about 15-fold (Liu et al., 1990) from 0.45 ± 0.029 to 7.04 ± 0.319 /¿g/mg protein in the Zn pretreated cells. Subsequently, the Zn-containing media was removed and fresh media without Zn was added. At this point, the cultures were treated with metals for an additional 24 h. All treatments were 1% volume of media. Metallothionein Assay MT concentrations were estimated by the Cd-hemoglobin radioassay method as previously described by Eaton and Toal (1982). Concentration of total cellular protein was measured by the method of Bradford (1976), using bovine serum albumin as standard. Evaluation of Cytotoxicity Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) activity in the culture media and intracellular potassium (K ) content. For the measurement of LDH, media was collected, centrifugated
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(1000 x g, 5 min), and the supernatant analyzed for LDH activity as described by Henry et al. (1960), using a DU-8 spectrophotometer (Beckman Instruments, Inc., Palo Alto, Calif.). For the measurement of K+ content, hepatocytes were rinsed with isotonic saline three times and sonicated in 2 ml Tris-HCI buffer (10 mM, pH 7.4) with an Ultrasonics model W-10 cell disrupter (Plainview, N.Y.). Aliquots of sonicated cell suspensions were centrifugated (10,000 x g, 3 min) and 100 ¡A supernatant was analyzed for K+ by flame photometry (Instrument Lab., Inc., Lexington, Mass.). GSH Assay Hepatocellular GSH content was determined using the enzymatic method of Tietze (1969). A 250-/J aliquot of sonicated cell suspension was mixed with 250 ¡x\ of 4% (w/v) sulfosalicylic acid at 4°C and stored overnight at this temperature. Samples were centrifuged (3000 x g, 10 min) the next day and a 100-fil aliquot of the supernatant was mixed with 700 fi\ of the incubation buffer (0.1 M NaH2PO4 and 0.5 mM EDTA), 50 (i\ of 10 mM 5,5'-dithiobis (2-nitrobenzoic acid), 50 /¿I GSH reductase (10 U/ml), and 100 /¿I NADPH (2 mg/ml), and the change in absorbance at 412 nm was determined with a Beckman model 25 spectrophotometer. Hepatocellular Uptake and Accumulation of Mn or Se Following Zn treatment, fresh media containing 300 ¡xM MnCI 2 + 0.3 /¿Ci ^Mn/dish or 20 fiM Na2SeO3 + 0.3 ¿iCi 7SSe/dish were added to cultures and incubated for 1, 2, 4, 8, 12, or 24 h. At the end of each incubation, radioactive media was removed, and cells were washed twice with ice-cold buffered saline (10 mM Hepes, 154 mM NaCI, 10>nM EDTA, pH 7.4) and once with Hepes buffered Hank's salt solution (Failla et al., 1979). Cells were collected and sonicated in 1 ml Tris buffer. Metal content was determined by gamma scintillation spectrometry (Packard, Downers Grove, III.). Total cellular protein was determined as described above. Subcellular Distribution of Mn and Se Radioactive media was removed 12 h following incubation with Mn or Se, and dishes were washed as described above. Cells from six dishes were scraped, pooled in a total of 8 ml homogenizing buffer (0.25 M sucrose, 10 mM Tris-acetate, pH 7.4), and disrupted with a motor-driven Teflon pestle glass homogenizer (10 up-and-down strokes). The various fractions were prepared by differential centrifugation (Goering and Klaassen, 1984), and the resultant pellets were defined as nuclear (600 x g, 10 min), mitochondria (10,000 x g, 10 min), microsomes (100,000 x g, 60 min), and cytosol (100,000 x g supernatant). The distribution of Mn and Se in the cytosol was determined by
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Sephedex G-75 gel filtration chromatography. The samples were eluted from the column (1.5 x 60 cm) with 10 miW Tris acetate (pH 7.4) at a rate of 30 ml/h at 4°C. Fractions of 5.0 ml were collected and metal content was determined as described above. Statistical Analysis
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Comparison between control and treatment was made by the Student's í test. The .05 level of probability was used as the criterion of significance. RESULTS The effect of Zn pretreatment on the toxicity to cultured hepatocytes produced by five metals that have little or no affinity for MT are shown in Figures 1 and 2. Following Zn treatment of cultured hepatocytes for 24 h to increase MT concentrations, five concentrations of 2000 Control •O Zn-pretreated 1600-. 1200800400H
1
1
I-
H
1
1
1
1
H-
1512963-
0 300 Mn
900
1500
30
0 10
50
Se
FIGURE 1. Effect of Zn pretreatment on the toxicity of Se and Mn in rat primary hepatocyte cultures. Monolayers were pretreated with ZnCI 2 (100 fiM) for 24 h, then exposed to metals for an additional 24 h. Each value represents the mean ± SE of four dishes from one of three rats. Asterisk indicates values significantly different from controls (p < .05).
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1200
Control O—O Zn-prelreated
900
600
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300
8-
\
0 400
1Z00 2000
0 300
B00
1500
0 300
BOO 1500
FIGURE 2. Effect of Zn pretreatment on the toxicity of Fe, V, and Cr in rat primary hepatocyte cultures. Monolayers were pretreated with ZnCI 2 (100 ¡iM) for 24 h, then exposed to metals for an additional 24 h. Each value represents the mean ±SE of four dishes from one of three rats. Asterisk indicates values significantly different from controls (p < .05).
each metal were added to the monolayers and the toxicity quantitated 24 h later. Figure 1 shows the effect of Zn pretreatment on Mn and Se toxicity. A much higher concentration of Mn (300 (iM) was necessary to produce toxicity than Se (30 fiM), as indicated by LDH leakage and cellular K+ loss. Zn-pretreated cells were more resistant to Mn and Se than were the control cells. The effects of Zn pretreatment on Cr-, V-, and Feinduced hepatotoxicity are shown in Figure 2. All three of these metals were relatively nontoxic to the hepatocytes, as 300, 900, and 400 ¡xM of Cr, V, and Fe, respectively, were required to produce toxicity. Although higher concentrations of Fe appear to inhibit LDH activity, Fe produced a dose-dependent release of K+ release from hepatocytes. Zn pretreatment of the cultured hepatocytes did decrease the hepatotoxicity produced by all three metals. Of the five metals examined in Figures 1 and 2, Zn pretreatment seems to be more effective against Se and V than Fe, Mn, or Cr. In order to examine possible mechanisms for the protective effect that Zn has on the toxicity of metals that have little or no affinity for MT, we examined the hepatocellular uptake and distribution of two of the tested metals, namely, Se and Mn. Figure 3 shows the hepatocellular uptake of these two metals. Zn pretreatment had no appreciable effect
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400
• — • Control
O—O Zn-pretreated £
300-
Cu
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a
S
12
IS
20
24
TIME (hr) FIGURE 3. Effect of Zn pretreatment on the uptake of 75Se and M M n into cultured hepatocytes. Monolayers were pretreated with ZnCI 2 (100 /tM) for 24 h, then exposed to 20 ¡iM (0.3 /iCi) Se or 300 ¡iM (0.3 /id) Mn for various times. Each value represents the mean ±SE of four dishes from one of three rats.
on the uptake of either metal over the entire 24-h period. Within 8 h of exposure, the intracellular concentrations of both metals have attained an apparent steady-state condition. There was a marked difference in the subcellular distribution of Mn and Se (Table 1). About 70% of Mn taken up by the cells was distributed to the cytosol, in contrast to 12% of Se. Zn pretreatment had minimal TABLE 1. Subcellular Distribution of Mn and Se in Hepatocyte Cultures
Croup
Nuclei (%)
Mitochondria (%)
Microsomes (%)
Cytosol (%)
4.6 ± 0.2 3.0 + 0.1 3
67.2 ± 4.1 72.2 ± 5.5
7.2 ± 0.2 5.5 ± 0.2a
11.8 ± 1.7 11.5 ± 0.7
Manganese Control Zn-pretreated
18.1 ± 0.5 18.3 ± 1.4
Control Zn-pretreated
46.2 ± 3.0 50.7 ± 3.3
9.8 ± 0.3 6.8 ± 0.1 a Selenium 34.8 ± 1 . 6 32.8 ± 2.4
Note. Values are means ±SE of three cultures from one of three rats. a Significantly different from controls, p < .05.
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effects on the subcellular distribution of Mn and Se; however, a small decrease in the amount of Mn in the mitochondrial and microsomal fractions and a small decrease in the amount of Se in the microsomal fraction were noted in the Zn-pretreated hepatocytes. The distribution of Mn and Se in the cytosol of control and Znpretreated hepatocytes is shown in Figure 4. MT elutes between fractions 45 and 55 (data not shown). From these profiles there is no evidence that either metal is associated with MT. Instead, the metals mainly appeared in molecular weight fractions (65-85), smaller than MT, and a small amount of Se appeared in high-molecular-weight fractions (15-30). There was no difference in the distribution of either Se or Mn within the cytosol of Zn-pretreated cells and control cells. The effect of Zn-pretreatment on cellular GSH concentration is shown in Figure 5. Mn (500 pM), Cr (500 pM), V (1500 /xM), and Fe (2000 fiM) all decreased the concentration of GSH in the hepatocytes. Although Se at 40 pM was cytotoxic to cultured hepatocytes (Fig. 1 and Table 2), it did not decrease GSH concentrations, but increased GSH levels. Zn pretreatment had no direct effect on cellular GSH levels of control hepatocytes, but markedly decreased the GSH-depleting effects produced by Mn, V, and Cr. There was no difference in GSH concentration between control and Zn-pretreated cells exposed to Se or Fe. DISCUSSION All five metals employed in the present study are cytotoxic in a concentration-dependent manner to cultured hepatocytes as indicated by intracellular K+ loss and extracellular LDH leakage. Se is the most toxic of these metals, as only 30 fiM produced injury, whereas with the other metals 300 /xM or more was required to produce cytotoxicity. More importantly, the present study has shown that Zn pretreatment protects against all these five metals, which have little or no affinity for MT. Table 2 compares the effectiveness of Zn pretreatment to decrease the toxicity of the five metals. It appears that Zn has a lesser protective effect on Mn and Cr than the other three metals. One possible mechanism by which Zn might produce the protective effect is by decreasing the uptake of metals into the hepatocytes. As shown in Figure 3, Zn pretreatment had no appreciable effect on Se or Mn uptake and accumulation. Therefore, Zn-induced protection against these two metals does not appear to be due to a decreased uptake into the hepatocytes. It is known that Zn-induced protection against Cd hepatoxicity in the intact animal (Goering and Klaassen, 1984) as well as in isolated hepatocytes (Din and Frazier, 1985; Liu et al., 1990) is due to MT that is induced by the Zn pretreatment. The MT binds the metals in the cytosol and thus alters the subcellular distribution of Cd. Similar alterations
59
HEPATOPROTECTIVE EFFECTS OF ZINC
1 s
o. Ji,
4000
• — • Control o—o Zn-pretreated 3000-
•z
o
2000-
1000-
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o
1500-
fe? 1000500.
0
10
20
30
40
50
60
70
B0 SO
FRACTION 75
FIGURE 4. G-75 gel filtration profiles of Se and 54 Mn from cultured hepatocyte cytosol when administered 20 ¡xM (0.3 ftCi) Se or 300 ¡iM (0.3 ¡iCt) Mn in control and Zn-pretreated cells 12 h prior to harvest. Samples were eluted from the column with 10 mM Tris buffer at a rate 30 ml/h. Fractions of each 5.0 ml were collected and analyzed. 400
M Control CXI Zn-pretreated
.S Ü o
300-
B
200-
« m
100
O
Con
J Mn
II I Cr
Se
Fe
FIGURE 5. Effect of Zn pretreatment on cellular GSH levels in rat hepatocyte cultures exposed to five metals. Hepatocytes were pretreated with ZnCI 2 (100 ¡iM) for 24 h, then exposed to metals (Mn 500 fiM, Cr 500 /tM, V 1500 ¿«M, Se 40 pM, Fe 200 ¡iM) for an additional 24 h. Each value represents the mean ±SE of four dishes. Asterisk indicates values are significantly different from the corresponding control (p < .05).
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TABLE 2. Comparison of Protection of Zn Pretreatment on Metal Toxicity in Rat Primary Hepatocyte Cultures
Metal
Cone. (liM)
V
1200
Se
40
Fe
2000
Mn
900
Cr
1200
Group
Extracellular LDH (U/l)
Control Zn-treated Control Zn-treated Control Zn-treated Control Zn-treated Control Zn-treated
682 ± 55 386 ± 57a 896 ± 161 512 ± 141a 359 ± 42 150 ± 35a 1385 ± 182 1047 ± 91 a 911 ± 69 733 ± 80a
Percent of control
57 57 42 76 80
Intracellular K + (nmol/mg protein) 8.20 12.55 6.89 9.40 7.84 10.45 10.07 11.04 6.26 8.02
± ± ± ± ± ± ± ± ± ±
0.49 0.78a 0.26 0.86a 0.45 0.43a 1.29 1.25a 0.65 0.87a
Percent of control
153 136 133 110 128
"Significant at p < .05.
in the subcellular distribution and toxicity with other metals that have a high affinity for MT have also been shown (Liu et al., 1991). However, in the present study, five metals that have little or no affinity for MT (Waalkes et al., 1984) were examined, yet protection was observed. As one would anticipate, in the present study there was no indication that the metals bound to MT in the cytosol (Figure 4), nor was there a marked increase in the subcellular distribution of metals to the cytosol as observed with metals that have a high affinity for MT (Liu et al., 1990, 1991). Therefore, the Zn-induced protection for these metals does not appear to be due to the well-known sequestering role of MT for metals. Zn-induced protection against some organic toxicants such as acetaminophen (Chengelis et al., 1986) and alkylating agents (Tobey et al., 1982) also does not appear to be due to MT. There appears to be at least one additional protective mechanism produced by Zn. It has been proposed that Zn may protect against the toxicity of chemicals by inhibiting lipid peroxidation (Chvapil et al., 1972b; Thomas et al., 1986; Willson, 1987). It has been shown previously (Stacey and Klaassen, 1981) that of the five metals examined in this study, two (Cr and Mn) do not produce any lipid peroxidation and Se produces only a minimal amount. Thus, it seems unlikely that Zn has a universal action of protection against the toxicity of metals by inhibiting lipid peroxidation. Another mechanism for the protection provided by Zn may involve the stabilization of tissue thiols or the activation of GSH-associated enzymes (Cho and Fong, 1990; Fukino et al., 1986; Ploersheim and Floersheim, 1986; Szymariska et al., 1991). In the present study, Zn pretreatment did not increase cellular GSH concentration in cultured isolated hepatocytes as has been previously reported for the intact animal (Chengelis et al., 1986; Wong and Klaassen, 1981), but it did prevent the GSH depletion caused by Mn, Cr, and V. Because cellular GSH has an important role in protection against toxic chemicals, this protection
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against GSH depletion may in part explain Zn-induced protection for these three metals. Se and Fe did not deplete GSH at cytotoxic concentrations; therefore, the protection for these two metals must be due to another mechanism. Zn pretreatment can also induce other biological changes such as stabilization of lysosomes and cellular membranes (Dar et al., 1986; Chvapil et al., 1972; Ludwig and Chvapil, 1980; Pfeiffer et al., 1980). This stabilization might also play a role in the protection Zn provides against the toxicity of these metals. In conclusion, Zn pretreatment of cultured hepatocytes protects against Se-, V-, Mn-, Fe-, and Cr-induced cytotoxicity. This protection does not appear to be due to decreasing cellular metal uptake, altering subcellular distribution of metals, nor the binding of metals to MT. The mechanism of Zn-induced protection against the cytotoxicity of metals is not known but may be related to the stabilization of cellular membranes. REFERENCES Berry, M. N. and Friend, D.S. 1969. High yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43:506-520. Bissell, D. M., and Guzelian, P, S. 1980. Phenotypic stability of adult rat hepatocytes in primary monolayer culture. Ann. NX Acad. Sci. 349:85-98. Bracken, W. M., and Klaassen, C. D. 1987. Induction of metallothionein in rat primary hepatocyte cultures: Evidence for direct and indirect induction. J. Toxicol. Environ. Health 22:163-174. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Cagen, S. Z., and Klaassen, C. D. 1979. Protection of carbon tetrachloride-induced hepatotoxicity by zinc: Role of metallothionein. Toxicol. Appl. Pharmacol. 51:107-116. Chengelis, C. P., Dodd, D. C., Means, J. R., and Kotsonis, F. N. 1986. Protection by zinc against acetaminophen induced hepatotoxicity in mice. Fundam. Appl. Toxicol. 6:278-284. Cho, C. H., and Fong, L. Y. Y. 1990. The interaction of ethanol and zinc on hepatic glutathione and glutathione transferase activity in mice. Agents Actions 29:382-385. Chvapil, M., Ryan, J. N., and Zukoski, C. F. 1972a. The effect of zinc and other metals on the stability of lysosomes. Proc. Soc. Exp. Biol. Med. 140:642-646. Chvapil, M., Ryan, J. N. and Zukoski, C. F. 1972b. Effect of zinc on lipid peroxidation in liver microsome and mitochondria. Proc. Soc. Exp. Biol. Med. 141:150-153. Clarke, I. S., and Lui, E. M. K. 1986. Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepatotoxicity. Can. J. Physiol. Pharmacol. 64:1104-1110. Dar, M. S., Townsend, S. M. and Wooles, W.R. 1986. Protective effect of zinc against ethanol toxicity in mice. J. Toxicol. Environ. Health. 18:41-48. Din, W. S., and Frazier, J. M. 1985. Protective effect of metallothionein on Cd toxicity in isolated rat hepatocytes. Biochem. J. 230:395-402. Dunn, M. A., Blalock, T. L., and Cousins, R. J. 1987. Metallothionein. Proc. Soc. Exp. Biol. Med. 185:107-119. Eaton, D. L., and Toal, B. F. 1982. Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66:134-142. Failla, M. L., Cousins, R. J., and Mascenik, M. J. 1979. Cadmium accumulation and metabolism by rat liver parenchymal cells in primary monolayer culture. Biochim. Biophys. Acta 583:63-72. Floersheim, C. L., and Floersheim, P. 1986. Protection against ionizing radiation and synergism with thiols by zinc aspartate. Br. J. Radiol. 59:597-602. Fukino, H., Hirai, M., Hsueh, Y. M., Moriyasu, S., and Yamane, Y. 1986. Mechanism of protection by
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