ARTICLE |
Correspondence to: Shigeru Saito, Dept. of Preventive Medicine, St Marianna University School of Medicine, Kawasaki 216-8511, Japan.
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Summary |
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To clarify the relationships between DNA damage and Cu-MT and between DNA damage and Cu in kidneys of rats injected with Au, we examined the histochemical localization of DNA damage, metallothionein (MT), and the accumulated Cu in the kidneys of rats injected with Au, Cu, or Cu-MT. The immunoreactivity of MT was observed predominantly in the outer stripe of the outer medulla and the inner cortex of the Au-injected rat, and the signals of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) were observed in the cortex. Cu detected by Timm's method was mainly distributed in the cortex of the Au-injected rat. These results indicated that DNA damage could be caused by free Cu in the cortex but not by the Cu bound to MT in the outer stripe of the outer medulla. This consideration was supported by the data from rats injected with Cu and Cu-MT. Furthermore, we determined the Cu contents in three fractions (cytosol, organelle, and precipitate-containing nuclei) of the kidneys. Interestingly, most of the Cu content in the kidney of the rat injected with Au or Cu-MT was detected in the cytosol, whereas most of the Cu content in the kidney of the rat injected with Cu was detected in the nuclei-containing precipitate. These findings suggest that the DNA damage in the kidneys of rats injected with Au may be associated with Cu-binding proteins but not with Cu-MT. (J Histochem Cytochem 50:12631271, 2002)
Key Words: gold, copper, Cu-MT, kidney, TUNEL, metallothionein, DNA damage
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Introduction |
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GOLD COMPLEXES are among the more effective medications available for rheumatoid arthritis (RA), although many authors have reported serious renal damage during chrysotherapy (
In a previous study (
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Materials and Methods |
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Chemicals
Biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-16-dUTP), blocking reagent, and proteinase K were purchased from Boehringer Mannheim (Mannhein, Germany). Recombinant terminal deoxynucleotidyl transferase (rTdT) was from Life Technologies (Birmingham, AL). Propidium iodide, 4-chloro-1-naphthol, phosphate-citrate buffer with sodium perborate capsules, H2O2diaminobenzidine, avidinbiotinalkaline phosphatase, and avidinperoxidase were from Sigma Chemical (St Louis, MO). Fluorescein isothiocyanate (FITC)-labeled streptavidin and species-specific biotinylated sheep anti-mouse IgG (second antibody) were obtained from Amersham International (Poole, UK). We prepared Cu12-MT from commercially available rabbit Cd5,Zn2-MT (Sigma) by the method described previously (
Animals
Male SpragueDawley rats (Japan SLC; Tokyo, Japan) weighing 120145 g were housed at a constant temperature of 21.5 ± 1.5C on a 12-hr light/12-hr dark cycle for 1 week before starting the experiments. They were provided with a commercial diet (Clea Japan; Tokyo, Japan) and water ad libitum. Rats were injected daily for 5 days with an IP dose of 1 mg Au/kg as sodium tetrachloroaurate(III)dihydrate [Na(AuCl4)·2H2O] in 0.9% NaCl solution. Rats for control were injected with equal volumes of 0.9% NaCl solution. To compare with Au-injected rats, Cu or Cu-MT was administered to rats daily for 3 days by an IP dose of 1.5 mg of Cu/kg bw as CuCl2 in 0.9% NaCl or 6 mg Cu-MT/kg bw in 10 mM Tris-HCl, pH 8.1, containing 100 mM NaCl. All rats were injected with an overdose of pentobarbital (60 mg/kg bw, IP) 24 hr after the final injection and transcardially perfused with 40 mM Tris-HCl containing 152 mM NaCl, pH 8.1 (500 ml/kg). The kidneys were removed, frozen with liquid nitrogen, and immediately stored at -80C until use.
Immunohistochemistry of MT
All IHC procedures were carried out according to the method of
TdT-mediated dUTPBiotin Nick End Labeling (TUNEL) Assay
DNA damage in the kidney was measured by the TUNEL assay described by
Confocal Laser Microscopy
The fluorescent signals of dUTP in the renal sections were imaged by a confocal laser microscopy (Biorad MRC-1024; Hercules, CA) with an upright compound microscope (Zeiss, numerical aperture 0.75). All fluorescent images were obtained by 488 nm 15 mW kryptonargon ion laser excitation with filter blocks T1 and T2A.
Cu Staining
Cu staining with the modified Timm's method, which includes trichloroacetic acid (TCA) treatment (
Gel Filtration Chromatography
To examine the Cu concentrations of the MT in kidneys of rats injected with Au, Cu, or Cu-MT, 0.5 g of frozen kidney was thawed at 4C, cut into pieces, and homogenized (10:1 v:w) in ice-cold 50 mM Tris-HCl, pH 8.1, with a polytron model PT 10-35 (Kinematica; Basel, Switzerland) three times for 30-sec intervals. The homogenate was centrifugated at 10,000 x g for 30 min at 4C with a Kubota centrifuge, model KR/200B. An aliquot (4 ml) of the supernatant was applied to a Sephadex G-75 column (1.0 x 100 cm) equilibrated with 10 mM Tris-HCl, pH 8.1 and eluted with the same solution at 4C. The eluent was collected in 1.5-ml fractions and assayed for Au, Cu, and Zn with a Hitachi atomic absorption spectrophotometer, model 180-30.
Cu Content in Three Fractions of Kidneys
The kidneys were divided into three fractions (cytosol, organelle, and precipitate containing the nuclei). To prepare three fractions, 0.5 g of frozen kidneys from three rats was thawed at 4C, cut into pieces, and homogenized in 10 volumes (v/w) of ice-cold 50 mM Tris-HCl, pH 8.1, with the polytron three times for 30-sec intervals. The homogenate was centrifuged at 10,000 x g for 30 min at 4C. To prepare the cytosol fraction, the supernatant was recentrifuged at 110,000 x g for 60 min at 4C using a Hitachi ultracentrifuge, model 70P-72. After centrifugation, the precipitate was digested with mixed acids (2 ml concentrated HClO4 and 4 ml concentrated HNO3). The supernatant, cytosol, and digested precipitate were assayed for Cu with the Hitachi atomic absorption spectrophotometer.
Statistics
The data were statistically analyzed using the Statview II program on a Macintosh computer. The data were compared by an unpaired Student's t-test and a probability value of p<0.05 was accepted as significant.
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Results |
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The immunoreactivity of MT was observed predominantly in the outer stripe of the outer medulla and the inner cortex in the kidneys of Au-injected rats (Fig 1A2). Faint immunoreactivity for MT was found in the inner cortex in the kidneys of control rats (Fig 1A1). To determine whether DNA damage was induced by Au, Cu, or Cu-MT injection, we examined the histochemical localization of DNA damage by the TUNEL assay (Fig 1B11B16). Fig 1B5 and 1B6 show that nuclei and TUNEL signals were co-localized in the renal cortex of the Au-injected rat, suggesting that DNA damage was observed in the nuclei of the renal cortex, whereas TUNEL-positive cells were not observed in the outer stripe of the outer medulla of the Au-injected rat (Fig 1B8) and the kidneys of the control rat (Fig 1B2 and 1B4). The results indicated that the DNA damage occurred in the cortex of the Au-injected rat. Fig 1B91B16 show the localization of nuclei and TUNEL signals in the kidneys of the rats injected with Cu and Cu-MT by confocal laser fluorescence microscopy, respectively. TUNEL-positive cells were observed in the cortex (Fig 1B10) and the outer stripe of the outer medulla (Fig 1B12) of rats injected with Cu, whereas they were scarcely detected in the renal cortex (Fig 1B14) and the outer stripe of the outer medulla (Fig 1B16) in rats injected with Cu-MT. To clarify why the DNA damage occurred in the kidney in rats injected with Au and Cu, we examined the localization of Cu staining in the kidney using Timm's method (Fig 2). Cu staining was not detected in the cortex (Fig 2a) and the outer stripe of the outer medulla (Fig 2b) of the control rat by Timm's method. Faint staining of Cu was mainly distributed in the cortex (Fig 2c) but not the outer stripe of the outer medulla (Fig 2d) of the Au-injected rat. In addition, intense staining of Cu was distributed in the cortex (Fig 2e) and in the outer stripe of the outer medulla (Fig 2f) in rats injected with Cu, whereas the staining was scarcely detected in the cortex (Fig 2g) and in the outer stripe of the outer medulla (Fig 2h) in rats injected with Cu-MT. The detailed localization of the Cu staining was observed predominantly in the nuclei of the kidneys of rats injected with Au and Cu (Fig 2c, Fig 2e, and Fig 2f), whereas faint staining of Cu was found in the cytoplasm. To determine the relationship between the Cu concentration in the MT fraction and DNA damage, we performed gel filtration of the cytosol obtained from the kidneys of the rats injected with Cu-MT, Au, and Cu. The distribution profiles of the renal cytosol of the control rat, and the rats injected with Au, Cu, and Cu-MT on a Sephadex G-75 column are shown in Fig 3. Peak I, with an elution volume of 3342 ml, comprised high molecular weight proteins and peak II, with an elution volume of 5470.5 ml, was identified as MT (
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Discussion |
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The present study investigated the relationships between induced Cu-MT and DNA damage and between accumulated Cu and DNA damage in the kidneys of rats injected with Au, Cu, and Cu-MT. The effects observed on each renal section of rats injected with Au, Cu and Cu-MT are summarized in Table 2. Immunoreactivity for MT was observed in the outer stripe of the outer medulla and the inner cortex in the kidneys of Au-injected rats. However, unlike the immunoreactivity for MT, the autofluorescent signals of the Cu-MT and the staining of MT mRNA were detected predominantly in the outer stripe of the outer medulla in the rat kidneys (
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In this study, it was interesting that the Cu was distributed in the renal cortex of the Au-injected rat (Fig 2c), whereas the autofluorescent signals of the Cu-MT and the staining of the MT mRNA were detected predominantly in the outer stripe of the outer medulla in the kidneys of Au-injected rats (
The localization of the MT from the Au-injected rats was not identical to that of the MT of kidneys in rats injected with Cu or Cu-MT revealed by our previous study (
Interestingly, the intense staining of Cu was distributed in the cortex and the outer stripe of the outer medulla of rats injected with Cu (Fig 2e and Fig 2f). The localization of Cu staining in kidneys of rats injected with Cu-MT or Cu was not identical to that of Cu-MT in kidneys of rats injected with Cu-MT or Cu (
In this study, the localization of DNA damage in the kidneys of the rats injected with Cu and Cu-MT was identical to that of Cu staining but not of Cu-MT. DNA damage was caused by Cu injection but not by Cu-MT injection. In rats injected with Au, the Cu staining was located in the cortex. On the basis of these results it was thought that DNA damage in the kidneys was associated with free Cu but not with Cu-MT.
The amount of Cu-MT (Fig 3) was believed not to correlate with the strength of DNA damage (Fig 1B). These results were contrary to those of the study by
In this study approximately 65% of the Cu content in the kidney of rats injected with Cu was detected in the nuclei-containing precipitate (Table 1B), while approximately 30% of the Cu content in the rats injected with Cu-MT and Au was observed in the nuclei-containing precipitate (Table 1A and Table 1B). The DNA damage in the kidney after Cu injection was stronger than that after Cu-MT and Au injection (Fig 1B51B16). These results suggested that DNA damage might be caused by the accumulated Cu from Cu-binding proteins except for Cu-MT in the nuclei-containing fractions.
In conclusion, our results indicate that the degree of DNA damage in kidneys of rats injected with Au, Cu, or Cu-MT correlated with the strength of Cu staining but not with the amount or the location of Cu-MT. These findings suggest that the DNA damage in the kidneys of rats injected with Au may associate with Cu-binding proteins except Cu-MT, but not with Cu-MT.
The reasons for the high Cu accumulation in the kidney induced by Au injection and the discrepancy in the localization of the induced MT in the kidneys of Au, Cu, or Cu-MT are still unclear. However, in a previous study (
Received for publication June 25, 2001; accepted March 6, 2002.
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