Cell Biology and Toxicology, Vol. 8, No. 4, 1992
277
CADMIUM-2-ACETYLAMINOFLUORENE INTERACTION IN ISOLATED RAT HEPATOCYTES PIERRE MOFFATT, MICHEL MARION, AND FRANCINE DENIZEAU Department of Chemistry and T O X E N Universit6 du Qu6bec ~ Montr6al Montr6al, Quebec, Canada
Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 pM) of 109Cd. This was followed by a 10 hr exposure to 1 IxM [3H]AAF. Cellular distribution of Cd and 3H was determined. Sephadex G75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of 3H was found in the low molecular weight components. Further analysis of lMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some 3H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of 3H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.
1. Address all correspondence to: Dr. F. Denizeau, Department of Chemistry, Universit6 du Qu6bec ~ Montr6al, C.P. 8888, Succ. 'A', Montr6al, Qu6bec, Canada H3C 3P8. 2. Key Words: Cadmium, 2-acetylaminofluorene, hepatocytes, DNA binding, metallothionein. 3. Abbreviations: AAF, 2-acetylaminofluorene; Cd, cadmium; DMSO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; MT, metallothionein; UDS, unscheduled DNA synthesis. Cell Biology and Copyright © 1992
Toxicology, Vol. 8, No. 4, pp. Princeton Scientific Publishing ISSN: 0742-2091
277-290 Co., Inc.
278
Moffatt et al.
INTRODUCTION Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. The perturbations caused by Cd in living organisms have been extensively studied (Foulkes, 1986). The liver, which plays a major role in detoxification processes, represents a critical target organ for Cd toxicity (Nordberg and Nordberg, 1988). In the liver or hepatic systems, Cd can induce many biochemical changes and, in particular, affect parameters which are of great importance in the response towards xenobiotics. Among the effects of Cd likely to alter the action of xenobiotics upon the cell, changes in mixed-function oxidase activity have been observed. Treatment of male rats with Cd leads to a significant inhibition of drug metabolism and to a reduction in cytochrome P-450 levels in microsomal subfractions obtained from the liver of these animals. Furthermore, in vitro addition of Cd to isolated microsomes produces inhibition of drug biotransformation by cytochrome P-450 (Hadley et al., 1974; Means et al., 1979; Eaton et al., 1980). Cd also interacts with glutathione (GSH), a tripeptide of prominent function in biochemical mechanisms of toxicity. GSH is believed to diminish the effects of many toxicants because of its radical scavenging properties and its capacity to bind reactive electrophiles through its sulfhydryl group. In addition to the fact that Cd-induced hepatotoxicity is modified by changes in GSH concentration, this metal can cause an increase in hepatic GSH in vivo (Dudley and Klaassen, 1984). Enhancement of GSH levels in cultured cells by Cd has similarly been reported (Bannai et al., 1991). Another endogenous molecule, which shares unique properties with GSH, is the low molecular weight protein metallothionein (MT). MT contains a high proportion of cysteine and has been postulated to be involved in protection mechanisms. Cd, along with other factors (Oh et al., 1978; Kotsonis and Klaassen, 1979; Karin et al. 1980; Klaassen and Lehman-McKeeman, 1989; Schroeder and Cousins, 1990), causes the induction of MT. This phenomenon has been associated with the development of resistance towards heavy metals, including Cd itself (Leber and Miya, 1976), ionizing radiations and alkylating agents (Tobey et al., 1982; Lohrer and Robson, 1989; Kelley et al., 1989; Renan and Dowman, 1989). It is of interest to note that Cd can produce anticarcinogenic effects, as recently observed in mouse liver, although the mechanisms still remain undefined (Waalkes et al., 1991). As described above, evidence now exists that Cd can alter the biochemical status of the cell and modulate the impact of further exposure to xenobiotics. However, there is a lack of information about the mechanisms that take place at the subcellular and molecular levels when Cd and other toxins are present. In view of this, the purpose of the present work was to examine the biochemical events pertaining to the interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF), in cells exposed to both agents. Isolated rat hepatocytes were used because they represent an established model which has been widely applied to the study of cellular effects of various hepatotoxins. The hepatocytes were incubated with 109Cd and [3H]AAF. Cellular uptake and cytosolic distribution of Cd and 3H were followed. The levels of MT and the binding of AAF to the DNA were measured. It was
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
279
found that treatment with Cd leads to reduced AAF-DNA adduct levels. Exposure to the metal could therefore protect against the genotoxicity of the carcinogen.
METHODS Chemicals Cell culture materials (Williams' Medium E (WME), Minimal Essential Medium (MEM), and fetal bovine serum (FBS)) were obtained from Gibco (Canada). 109CDC12 and [3H]thymidine were purchased from New England Nuclear Corp., and G-[3H]AAF from Chemsyn Science Laboratories. Proteinase K was from Boehringer Mannheim; Sephadex G-75 and DEAE A-25 were from Pharmacia; coUagenase type IV (EC 3.4.24.3) and rabbit MT were from Sigma Chemical Co. Hepatocyte preparation and culture Rat parenchymal hepatocytes were isolated from male Sprague-Dawley rats (-250 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, USA) by a two-step collagenase perfusion technique (Seglen, 1976) with slight modifications (Denizeau et al., 1985). Viability of isolated hepatocytes was >85%, as judged by the trypan blue exclusion test. Liver cells were suspended in WME supplemented with 10% FBS and 0.5% gentamicin. Aliquots containing 4.5 x 106 hepatocytes were seeded into 60 mm petal dishes and incubated for 2 hr at 37°C in an atmosphere of 95% 02 and 5% CO2 saturated with humidity, to permit attachment of viable cells. After this period, any dead or unattached cells were removed by washing the monolayer 3 times with serum-free MEM. The experiments were then initiated. Control medium consisted of MEM supplemented with 1% FBS and 0.5% gentamicin. In Cdpretreated samples, 109CDC12 (sp. act. 2.88 ~tCiAtg) was added to a nominal concentration of 0.22 ~tM and 3.17 ~tCi/ml. After a 10 hr incubation, the previous media were removed and replaced either with fresh medium containing 1 ktM [3H]AAF (sp. act. 4.15 ~tCi/l.tg and 0.925 ~tCi/ml) or control medium containing 0.5% DMSO. The incubation was pursued for an additional 10 hr. The incubation protocols are summarized in the following scheme:
Time 0
e (A) i ] R , O : T O (B) 'i C _,
10
109Cd
> ,.
i
10
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!/
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control
(h),
ontro ! 20
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t3AAF > !
280
Moffatt et al.
Assessment of cell viability and cytochrome P-450 activity Hepatocyte viability was monitored by measuring LDH activity in the extraceUular medium, according to Mold6us et al., (1978). P-450-dependent mixed-function oxidase activity was evaluated by the fluorometfic assay of 3-cyano-7-ethoxycoumarin O-deethylase (using a Perkin Elmer LS-50 fluorometer) according to White (1988). DNA binding Each exposure condition was tested in triplicate using the material from 2 petri dishes per replicate. After the exposure period, the cells were washed 3 times with HBSS and removed by scraping with a rubber policeman. The DNA was then extracted and purified according to procedures inspired by those of Sambrook et al. (1989). Briefly, the cells were pelleted and lysed with 20 mM Tris-HC1 buffer (pH 8.0), containing 150 mM NaC1, 1% sodium dodecyl sulfate, and 50 l.tg/ml proteinase K (previously autodigested at 55°C for 30 min). The samples were placed on a mobile plate and subjected to gentle agitation (35 inversions/min) for 3 hr at 37°C. The cellular material was then extracted with chloroform:isoamyl alcohol (24:1). The aqueous phase was reextracted with chloroform and the DNA was subsequently precipitated at 20°C with ice-cold absolute ethanol. The DNA pellets were washed with 70% ethanol and resuspended in Tris-HC1 buffer pH 7.9. The amount of DNA was determined by measuring the absorbance at 260 nm. Purity estimations of all samples gave A260 nm/A280 nm ratios >1.80, indicating the absence of significant protein contamination. Aliquots of the same samples were taken for 109Cd and [3H]AAF radioactivity measurements by liquid scintillation counting (LSC) (Packard, Minaxi-B tri-carb 4000 series). Cellular and cytosolic uptake of AAF and Cd For each determination, the material from 8 petri dishes was pooled. The cells were washed and detached using HBSS as described above. The pelleted cells were resuspended in 50 mM Tris-acetate buffer (pH 7.4) containing 250 mM sucrose, 5 mM 13-mercaptoethanol and 0.01% sodium azide. Aliquots of cell suspensions were taken for the analysis of 109Cd and 3H by LSC. The remaining cell suspension was sonicated at 4°C and the cytosol was prepared by ultracentrifugation at 107,000g for 90 min (4°C) (Beckman L8-M ultracentrifuge). Aliquots of cytosol preparations were also taken for the quantitation of 109Cd and 3H. Sephadex chromatography of cell cytosol The cytosol preparations were chromatographed on a Sephadex G-75 column (35 x 0.9 cm) under a nitrogen atmosphere at room temperature. The elution buffer consisted of 10 mM Trisacetate (pH 7.4) containing 2 mM 13-mercaptoethanol and 0.01% sodium azide. Fractions of 0.7 ml were collected at a flow rate of approximately 20 ml/hr and aliquots were taken for 109Cd and 3H determination. The fractions corresponding to MT (Ve/Vo = 1.8-2.3) were pooled and further resolved by anion-exchange chromatography using DEAE Sephadex A-25. The samples were applied to the column (18 x 0.9 cm) which had been pre-equilibrated with 10 mM Tris-acetate buffer (pH 7.4) at 4°C; it was then washed with 20 ml of the same buffer. Elution was carded out using a 60 ml linear gradient of 10-200 mM Tris-acetate (pH 7.4). Fractions of 2 ml were collected at a flow rate of-~20 ml/hr and were analysed for radioactivity as described above.
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
281
MT and protein determination The levels of cytosolic MT were determined by the silver (Ag)-saturafion assay (Scheuhammer and Cherian, 1986) except that non-radioactive Ag was used. Ag was analysed using a graphite furnace atomic absorption spectrophotometer with a continuous lamp background correction (Varian AA-1475, GTA-95). Levels of MT were evaluated using standard rabbit MT as calibration. Protein estimation was evaluated by the method of Bradford (1976) using bovine serum albumin as standard. Measurement of DNA repair activity Measurement of unscheduled DNA synthesis (UDS) was performed through incorporation of [3H]thymidine into the DNA, as previously described (Denizeau et al., 1985). Data collection and statistical analysis Each experiment was repeated at least three times using cell preparations obtained from different animals. Statistical analysis was performed using the Student's t test with a level of significance of P81%). It is noteworthy that more than 95% of the Cd initially added to the culture medium was accumulated by the cells within the 10 hr pretreatment period. For its part, [3H]AAF exhibited equal accumulation, as expressed by the ratio of 3H per mg protein, in both cellular and cytosolic compartments. It was also observed that Cd exposure had no effect on [3H]AAF uptake. Similarly, intracellular Cd was unaffected by AAF treatment. 12
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Ve/Vo FIGURE 3. Sephadex G-75 elution profile of cytosolic 3H and 109Cd for hepatocytes subjected to the Cd/AAF treatment (protocol B). The profile is separated into 3 distinct elution regions: Ve/Vo=[0-1.4], [1.4-2.6], and [2.6-5.0] corresponding respectively to high, intermediate and low molecular weight components (HMW, IMW, LMW). Ve: elution volume; Vo: column void volume. Standard rabbit MT eluted at Ve/Vo=[1.8-2.3]. The chromatogramis representative of results fxom at least 3 experiments.
284
Moffatt et al.
Cytosolic distribution of 3 H and 109Cd The cytosol was further analysed by Sephadex G-75 chromatography in order to determine the distribution of radioactivity from [3H]AAF and 109CDC12. The system used allows the separation of cytosolic content into high molecular weight (HMW), low molecular weight (LMW) and intermediate molecular weight (IMW) components. Fig. 3 shows the elution profile of both 3H and 109Cd. 3H was chiefly present in similar proportions in the HMW and LMW fractions. Cd was mainly associated with IMW components, which include endogenous MT, as demonstrated from the elution position of standard MT (Ve/Vo = 1.8-2.3). Cd was also bound, although to a lesser extent, to HMW proteins. As seen in Table I, preincubation with Cd did not seem to affect the distribution percentage of 3H among cytosolic HMW, IMW and LMW components. Similarly, Cd distribution was comparable whether or not AAF had been added to the incubation medium.
TABLE 1.
Distribution of cytosolic 3H and 109C d 3H ( % )a,b
109C d (%)a,b
Cd/AAF
control/AAF
Cd/control
Cd/AAF
HMW
39.1 _+ 2.4
38.2 + 1.7
8.6 + 0.9
9.1 _+ 1.0
IMW
11.0 _+ 0.4
11.1 + 0.3
80.1 + 2.9
80.2 _+ 2.9
LMW
48.9 + 3.1
49.1 + 2.6
10.4 + 1.7
9.9 + 1.6
aThe data are expressed as the percentage of 109Cd and 3H recovered in each of the high, intermediate and low molecular weight components (HMW, IMW, LMW) as fractionated by Sephadex G-75 chromatography. Radioactivity recoveries from the column runs were >98%. bData represent means + SE (4 experiments).
Further purification of IMW fractions was performed because of their low 3H levels which did not provide adequate resolution on Sephadex G-75. DEAE A-25 chromatography of the pooled IMW fractions from the Sephadex eluate was carried out. The separation profile obtained is presented in Fig. 4B. Two distinct peaks are observed. It is noteworthy that these peaks
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
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coincide with the elution positions of the two isoforms of MT. The peaks contain equal proportions of Cd. In addition, a detectable quantity of 3H also co-eluted with both peaks, but preferentially with the one corresponding to MT-I (Fig. 4B). Moreover, the silver-saturation assay for MT determination was applied to the cytosol. The results indicated that at the time when AAF was introduced (t=10 hr), Cd exposure had caused a 1.6-fold increase in the protein level as compared to that measured in the controls (Fig. 4A). Effect of Cd on binding of AAF to the DNA The genotoxic effects of AAF were evaluated as the amount of 3H bound to the DNA at the end of the experiment (t=20 hr) (Fig. 5). It was observed that the binding of 3H to the DNA was 17% lower when hepatocytes were pretreated with Cd. In parallel, the presence of 109Cd in the purified DNA fraction was also examined. The results show that the amounts of Cd were comparable, whether the hepatocytes were exposed to AAF or not: the values for 109Cd were 715 + 84 and 725 + 96 DPM/~tg DNA for the Cd/control samples (protocol A) and the Cd/AAF samples (protocol B) respectively (means + SE; n=3). In addition, unscheduled DNA synthesis ODDS) was used as a means to evaluate DNA repair activity (Williams, 1980) in AAF- and Cd/AAF-treated hepatocytes. Both series of samples yielded similar [3H]thymidine incorporation (5440 +635 and 5861 + 257 DPM/Ixg DNA respectively), suggesting maintenance of normal DNA repair upon exposure to Cd under the conditions of the experiments.
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277
CADMIUM-2-ACETYLAMINOFLUORENE INTERACTION IN ISOLATED RAT HEPATOCYTES PIERRE MOFFATT, MICHEL MARION, AND FRANCINE DENIZEAU Department of Chemistry and T O X E N Universit6 du Qu6bec ~ Montr6al Montr6al, Quebec, Canada
Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 pM) of 109Cd. This was followed by a 10 hr exposure to 1 IxM [3H]AAF. Cellular distribution of Cd and 3H was determined. Sephadex G75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of 3H was found in the low molecular weight components. Further analysis of lMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some 3H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of 3H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.
1. Address all correspondence to: Dr. F. Denizeau, Department of Chemistry, Universit6 du Qu6bec ~ Montr6al, C.P. 8888, Succ. 'A', Montr6al, Qu6bec, Canada H3C 3P8. 2. Key Words: Cadmium, 2-acetylaminofluorene, hepatocytes, DNA binding, metallothionein. 3. Abbreviations: AAF, 2-acetylaminofluorene; Cd, cadmium; DMSO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; MT, metallothionein; UDS, unscheduled DNA synthesis. Cell Biology and Copyright © 1992
Toxicology, Vol. 8, No. 4, pp. Princeton Scientific Publishing ISSN: 0742-2091
277-290 Co., Inc.
278
Moffatt et al.
INTRODUCTION Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. The perturbations caused by Cd in living organisms have been extensively studied (Foulkes, 1986). The liver, which plays a major role in detoxification processes, represents a critical target organ for Cd toxicity (Nordberg and Nordberg, 1988). In the liver or hepatic systems, Cd can induce many biochemical changes and, in particular, affect parameters which are of great importance in the response towards xenobiotics. Among the effects of Cd likely to alter the action of xenobiotics upon the cell, changes in mixed-function oxidase activity have been observed. Treatment of male rats with Cd leads to a significant inhibition of drug metabolism and to a reduction in cytochrome P-450 levels in microsomal subfractions obtained from the liver of these animals. Furthermore, in vitro addition of Cd to isolated microsomes produces inhibition of drug biotransformation by cytochrome P-450 (Hadley et al., 1974; Means et al., 1979; Eaton et al., 1980). Cd also interacts with glutathione (GSH), a tripeptide of prominent function in biochemical mechanisms of toxicity. GSH is believed to diminish the effects of many toxicants because of its radical scavenging properties and its capacity to bind reactive electrophiles through its sulfhydryl group. In addition to the fact that Cd-induced hepatotoxicity is modified by changes in GSH concentration, this metal can cause an increase in hepatic GSH in vivo (Dudley and Klaassen, 1984). Enhancement of GSH levels in cultured cells by Cd has similarly been reported (Bannai et al., 1991). Another endogenous molecule, which shares unique properties with GSH, is the low molecular weight protein metallothionein (MT). MT contains a high proportion of cysteine and has been postulated to be involved in protection mechanisms. Cd, along with other factors (Oh et al., 1978; Kotsonis and Klaassen, 1979; Karin et al. 1980; Klaassen and Lehman-McKeeman, 1989; Schroeder and Cousins, 1990), causes the induction of MT. This phenomenon has been associated with the development of resistance towards heavy metals, including Cd itself (Leber and Miya, 1976), ionizing radiations and alkylating agents (Tobey et al., 1982; Lohrer and Robson, 1989; Kelley et al., 1989; Renan and Dowman, 1989). It is of interest to note that Cd can produce anticarcinogenic effects, as recently observed in mouse liver, although the mechanisms still remain undefined (Waalkes et al., 1991). As described above, evidence now exists that Cd can alter the biochemical status of the cell and modulate the impact of further exposure to xenobiotics. However, there is a lack of information about the mechanisms that take place at the subcellular and molecular levels when Cd and other toxins are present. In view of this, the purpose of the present work was to examine the biochemical events pertaining to the interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF), in cells exposed to both agents. Isolated rat hepatocytes were used because they represent an established model which has been widely applied to the study of cellular effects of various hepatotoxins. The hepatocytes were incubated with 109Cd and [3H]AAF. Cellular uptake and cytosolic distribution of Cd and 3H were followed. The levels of MT and the binding of AAF to the DNA were measured. It was
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
279
found that treatment with Cd leads to reduced AAF-DNA adduct levels. Exposure to the metal could therefore protect against the genotoxicity of the carcinogen.
METHODS Chemicals Cell culture materials (Williams' Medium E (WME), Minimal Essential Medium (MEM), and fetal bovine serum (FBS)) were obtained from Gibco (Canada). 109CDC12 and [3H]thymidine were purchased from New England Nuclear Corp., and G-[3H]AAF from Chemsyn Science Laboratories. Proteinase K was from Boehringer Mannheim; Sephadex G-75 and DEAE A-25 were from Pharmacia; coUagenase type IV (EC 3.4.24.3) and rabbit MT were from Sigma Chemical Co. Hepatocyte preparation and culture Rat parenchymal hepatocytes were isolated from male Sprague-Dawley rats (-250 g) obtained from Harlan Sprague-Dawley Laboratories (Indianapolis, USA) by a two-step collagenase perfusion technique (Seglen, 1976) with slight modifications (Denizeau et al., 1985). Viability of isolated hepatocytes was >85%, as judged by the trypan blue exclusion test. Liver cells were suspended in WME supplemented with 10% FBS and 0.5% gentamicin. Aliquots containing 4.5 x 106 hepatocytes were seeded into 60 mm petal dishes and incubated for 2 hr at 37°C in an atmosphere of 95% 02 and 5% CO2 saturated with humidity, to permit attachment of viable cells. After this period, any dead or unattached cells were removed by washing the monolayer 3 times with serum-free MEM. The experiments were then initiated. Control medium consisted of MEM supplemented with 1% FBS and 0.5% gentamicin. In Cdpretreated samples, 109CDC12 (sp. act. 2.88 ~tCiAtg) was added to a nominal concentration of 0.22 ~tM and 3.17 ~tCi/ml. After a 10 hr incubation, the previous media were removed and replaced either with fresh medium containing 1 ktM [3H]AAF (sp. act. 4.15 ~tCi/l.tg and 0.925 ~tCi/ml) or control medium containing 0.5% DMSO. The incubation was pursued for an additional 10 hr. The incubation protocols are summarized in the following scheme:
Time 0
e (A) i ] R , O : T O (B) 'i C _,
10
109Cd
> ,.
i
10
"~
' i
9Cd
!/
O
L
(C)
"l
control
(h),
ontro ! 20
[3H]AAF
t3AAF > !
280
Moffatt et al.
Assessment of cell viability and cytochrome P-450 activity Hepatocyte viability was monitored by measuring LDH activity in the extraceUular medium, according to Mold6us et al., (1978). P-450-dependent mixed-function oxidase activity was evaluated by the fluorometfic assay of 3-cyano-7-ethoxycoumarin O-deethylase (using a Perkin Elmer LS-50 fluorometer) according to White (1988). DNA binding Each exposure condition was tested in triplicate using the material from 2 petri dishes per replicate. After the exposure period, the cells were washed 3 times with HBSS and removed by scraping with a rubber policeman. The DNA was then extracted and purified according to procedures inspired by those of Sambrook et al. (1989). Briefly, the cells were pelleted and lysed with 20 mM Tris-HC1 buffer (pH 8.0), containing 150 mM NaC1, 1% sodium dodecyl sulfate, and 50 l.tg/ml proteinase K (previously autodigested at 55°C for 30 min). The samples were placed on a mobile plate and subjected to gentle agitation (35 inversions/min) for 3 hr at 37°C. The cellular material was then extracted with chloroform:isoamyl alcohol (24:1). The aqueous phase was reextracted with chloroform and the DNA was subsequently precipitated at 20°C with ice-cold absolute ethanol. The DNA pellets were washed with 70% ethanol and resuspended in Tris-HC1 buffer pH 7.9. The amount of DNA was determined by measuring the absorbance at 260 nm. Purity estimations of all samples gave A260 nm/A280 nm ratios >1.80, indicating the absence of significant protein contamination. Aliquots of the same samples were taken for 109Cd and [3H]AAF radioactivity measurements by liquid scintillation counting (LSC) (Packard, Minaxi-B tri-carb 4000 series). Cellular and cytosolic uptake of AAF and Cd For each determination, the material from 8 petri dishes was pooled. The cells were washed and detached using HBSS as described above. The pelleted cells were resuspended in 50 mM Tris-acetate buffer (pH 7.4) containing 250 mM sucrose, 5 mM 13-mercaptoethanol and 0.01% sodium azide. Aliquots of cell suspensions were taken for the analysis of 109Cd and 3H by LSC. The remaining cell suspension was sonicated at 4°C and the cytosol was prepared by ultracentrifugation at 107,000g for 90 min (4°C) (Beckman L8-M ultracentrifuge). Aliquots of cytosol preparations were also taken for the quantitation of 109Cd and 3H. Sephadex chromatography of cell cytosol The cytosol preparations were chromatographed on a Sephadex G-75 column (35 x 0.9 cm) under a nitrogen atmosphere at room temperature. The elution buffer consisted of 10 mM Trisacetate (pH 7.4) containing 2 mM 13-mercaptoethanol and 0.01% sodium azide. Fractions of 0.7 ml were collected at a flow rate of approximately 20 ml/hr and aliquots were taken for 109Cd and 3H determination. The fractions corresponding to MT (Ve/Vo = 1.8-2.3) were pooled and further resolved by anion-exchange chromatography using DEAE Sephadex A-25. The samples were applied to the column (18 x 0.9 cm) which had been pre-equilibrated with 10 mM Tris-acetate buffer (pH 7.4) at 4°C; it was then washed with 20 ml of the same buffer. Elution was carded out using a 60 ml linear gradient of 10-200 mM Tris-acetate (pH 7.4). Fractions of 2 ml were collected at a flow rate of-~20 ml/hr and were analysed for radioactivity as described above.
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
281
MT and protein determination The levels of cytosolic MT were determined by the silver (Ag)-saturafion assay (Scheuhammer and Cherian, 1986) except that non-radioactive Ag was used. Ag was analysed using a graphite furnace atomic absorption spectrophotometer with a continuous lamp background correction (Varian AA-1475, GTA-95). Levels of MT were evaluated using standard rabbit MT as calibration. Protein estimation was evaluated by the method of Bradford (1976) using bovine serum albumin as standard. Measurement of DNA repair activity Measurement of unscheduled DNA synthesis (UDS) was performed through incorporation of [3H]thymidine into the DNA, as previously described (Denizeau et al., 1985). Data collection and statistical analysis Each experiment was repeated at least three times using cell preparations obtained from different animals. Statistical analysis was performed using the Student's t test with a level of significance of P81%). It is noteworthy that more than 95% of the Cd initially added to the culture medium was accumulated by the cells within the 10 hr pretreatment period. For its part, [3H]AAF exhibited equal accumulation, as expressed by the ratio of 3H per mg protein, in both cellular and cytosolic compartments. It was also observed that Cd exposure had no effect on [3H]AAF uptake. Similarly, intracellular Cd was unaffected by AAF treatment. 12
30
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Ve/Vo FIGURE 3. Sephadex G-75 elution profile of cytosolic 3H and 109Cd for hepatocytes subjected to the Cd/AAF treatment (protocol B). The profile is separated into 3 distinct elution regions: Ve/Vo=[0-1.4], [1.4-2.6], and [2.6-5.0] corresponding respectively to high, intermediate and low molecular weight components (HMW, IMW, LMW). Ve: elution volume; Vo: column void volume. Standard rabbit MT eluted at Ve/Vo=[1.8-2.3]. The chromatogramis representative of results fxom at least 3 experiments.
284
Moffatt et al.
Cytosolic distribution of 3 H and 109Cd The cytosol was further analysed by Sephadex G-75 chromatography in order to determine the distribution of radioactivity from [3H]AAF and 109CDC12. The system used allows the separation of cytosolic content into high molecular weight (HMW), low molecular weight (LMW) and intermediate molecular weight (IMW) components. Fig. 3 shows the elution profile of both 3H and 109Cd. 3H was chiefly present in similar proportions in the HMW and LMW fractions. Cd was mainly associated with IMW components, which include endogenous MT, as demonstrated from the elution position of standard MT (Ve/Vo = 1.8-2.3). Cd was also bound, although to a lesser extent, to HMW proteins. As seen in Table I, preincubation with Cd did not seem to affect the distribution percentage of 3H among cytosolic HMW, IMW and LMW components. Similarly, Cd distribution was comparable whether or not AAF had been added to the incubation medium.
TABLE 1.
Distribution of cytosolic 3H and 109C d 3H ( % )a,b
109C d (%)a,b
Cd/AAF
control/AAF
Cd/control
Cd/AAF
HMW
39.1 _+ 2.4
38.2 + 1.7
8.6 + 0.9
9.1 _+ 1.0
IMW
11.0 _+ 0.4
11.1 + 0.3
80.1 + 2.9
80.2 _+ 2.9
LMW
48.9 + 3.1
49.1 + 2.6
10.4 + 1.7
9.9 + 1.6
aThe data are expressed as the percentage of 109Cd and 3H recovered in each of the high, intermediate and low molecular weight components (HMW, IMW, LMW) as fractionated by Sephadex G-75 chromatography. Radioactivity recoveries from the column runs were >98%. bData represent means + SE (4 experiments).
Further purification of IMW fractions was performed because of their low 3H levels which did not provide adequate resolution on Sephadex G-75. DEAE A-25 chromatography of the pooled IMW fractions from the Sephadex eluate was carried out. The separation profile obtained is presented in Fig. 4B. Two distinct peaks are observed. It is noteworthy that these peaks
Cell Biology and Toxicology, Vol. 8, No. 4, 1992
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coincide with the elution positions of the two isoforms of MT. The peaks contain equal proportions of Cd. In addition, a detectable quantity of 3H also co-eluted with both peaks, but preferentially with the one corresponding to MT-I (Fig. 4B). Moreover, the silver-saturation assay for MT determination was applied to the cytosol. The results indicated that at the time when AAF was introduced (t=10 hr), Cd exposure had caused a 1.6-fold increase in the protein level as compared to that measured in the controls (Fig. 4A). Effect of Cd on binding of AAF to the DNA The genotoxic effects of AAF were evaluated as the amount of 3H bound to the DNA at the end of the experiment (t=20 hr) (Fig. 5). It was observed that the binding of 3H to the DNA was 17% lower when hepatocytes were pretreated with Cd. In parallel, the presence of 109Cd in the purified DNA fraction was also examined. The results show that the amounts of Cd were comparable, whether the hepatocytes were exposed to AAF or not: the values for 109Cd were 715 + 84 and 725 + 96 DPM/~tg DNA for the Cd/control samples (protocol A) and the Cd/AAF samples (protocol B) respectively (means + SE; n=3). In addition, unscheduled DNA synthesis ODDS) was used as a means to evaluate DNA repair activity (Williams, 1980) in AAF- and Cd/AAF-treated hepatocytes. Both series of samples yielded similar [3H]thymidine incorporation (5440 +635 and 5861 + 257 DPM/Ixg DNA respectively), suggesting maintenance of normal DNA repair upon exposure to Cd under the conditions of the experiments.
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