Cancer Letters,
58 (1991)
49
49-56
Elsevier Scientific Publishers Ireland Ltd.
The effects of exogenous glutathione and cysteine on oxidative stress induced by ferric nitrilotriacetate T. Umemura, Division
K. Sai, A. Takagi,
of Toxicology,
National
R. Hasegawa
Institute of Hygienic
Sciences,
and Y. Kurokawa I -18-1,
Kamiyogo,
Setagayaku.
158,
Tokyo
(Japan)
(Received 12 December 1990) (Revision received 4 March 1991) (Accepted 7 March 1991)
Summary
Introduction
The effects of the antioxidants, glutathione (GSH) and its precursor cysteine (Cys) on oxidative damage induced by ferric nitrilotriacetate (Fe-NTA) were examined. Fe-NTAassociated oxidatioe stress caused the depletion of renal cellular GSH content. Administration of exogenous GSH and Cys suppressed 8-hydroxydeoxyguanosine (8-OH-dG) formation, an indicator of oxidatiue DNA damage and nephrotoxicity following Fe-NTA treatment. This suggests that generation of free radicals may be causally involved in oxidatiue lesion generation. Since lipid peroxidation was found to be inhibited only by GSH and not Cys treatment, this suggests that this effect and the DNA damage might be mediated by different
Iron complexed nitrilotriacetate (Fe-NTA) , which has been widely used as a substitute for polyphosphates in household detergents, is a severely nephrotoxic compound which also induces high incidences of renal cell carcinomas in rats [20] despite being a non-mutagenic chemical by the Ames test [ 151. We previously demonstrated that formation of &hydroxydeoxyguanosine (8-OH-dG), considered a suitable marker for oxidative DNA damage, can be observed after Fe-NTA treatment [26]. Other workers have shown that lipid peroxidation increased in kidneys of rats following administration of Fe-NTA [7,21]. In another study our laboratory found that Fe-NTA caused oxidative damage to rat kidney DNA and at the same time induced peroxidation of renal tubular cell membrane [27]. It is well known that glutathione (GSH) can play a protective role in cells against various types of oxidative stress by acting as a hydrogen donor for the reduction of free radicals or as a substrate for enzymatic reduction of peroxides [3,14]. And the rate of GSH synthesis in cells is dependent upon the supply of cystein (Cys) which is one of the constituent amino acids of GSH [24]. Therefore investigation of whether exogenous GSH and Cys exerts protective effects on oxidative stimulation
pathways.
Fe-NTA-associated
in renal tubular both intracellular
oxidatiue
Keywords: glutathione; cysteine; stress; %hydroxydeoxyguanosine; nitrilotriacetate
Correspondence Toxicology, Valhalla,
NY.
to: T. Umemura,
American 10595,
0304-3835/91/$03.50
stress
cells might thus operate via and external space modes.
Health
0
Division of Pathology
Foundation,
U.S.A.
1991
Published and Printed in Ireland
oxidative ferric
One
Dana
and
Road,
Elsevier Scientific Publishers Ireland Ltd
50
by Fe-NTA appeared of interest with regard to Fe-NTA carcinogenesis. In the present study, 8-OH-dG levels were examined in kidneys of Fe-NTA-treated rats after intraperitoneal treatment with GSH or Cys along with lipid peroxidation levels, some serum biochemical parameters and histopathological changes. Materials
and Methods
Animals and housing conditions Five-week-old male Wistar rats (specific pathogen free, Shizuoka Laboratory Animal Center, Japan) were given F-Z pellet basal diet (Funabashi Farm Co., Japan) and tap water freely; and were used after 1 week of acclimation. Compounds Ferric nitrate (Fe(NO,),) and nitrilotriacetic acid disodium salt (NazNTA) were purchased from Wako Chemical Industry, Japan and dissolved in distilled water before use. GSH in reduced form, L-cysteine in free form and alkaline phosphatase were obtained from Sigma Chemical Co. Nuclease P1 was obtained from Yamasa Shoyu Co. Ltd., Japan. GSH and Cys were dissolved in saline before use. Fe-NTA solution was prepared by the method of Awai et al. [Z]. Fe(NO& was mixed in a 4-fold molar excess of Na,NTA, and the pH was adjusted to 7.4 with NaHC03 (Wako Chem., Japan). Animal treatments Treatments were as follows (Table I). All doses were given to ten rats in each group intraperitoneally. Group 2,4 and 6 animals each received Fe-NTA at a dose of 15 mgFe/kg. Group 1, 3 and 5 animals each received saline at a dose of 6 ml/kg. These animals were also given GSH (Groups 3 and 4), Cys (Groups 5 and 6) or saline (Groups 1 and 2) at doses of 800 mg/kg, 400 mg/kg or 6 ml/kg, respectively, 30 min before and after the above treatments. Five animals of each group were killed 1 h after the Fe-NTA (or saline) treatment and the kidneys were immediately excis-
Table 1. Treatment
schedules.
Group
Pretreatment
Treatment
Post-treatment
1 2 3 4 5 6
Saline Saline GSH GSH
Saline Fe-NTA Saline Fe-NTA Saline Fe-NTA
Saline Saline GSH GSH
CYS CYS
CYS CYS
Animals were sacrificed 1 or 6 h after treatment. Saline: 6 ml/kg, GSH: 800 mg/kg, Cys: 400 mg/kg, Fe-NTA: 15 mg Fe/kg.
ed for measurement of lipid peroxidation (LPO) and non-protein SH (NPSH) levels. The remaining animals of each group were anesthetized with ether 6 h after the Fe-NTA (or saline) treatment, and blood was cbllected from the jugular vein for measurement of blood urea nitrogen (BUN) and creatinine (CRN). The kidneys were weighed and used for measurement of 8-OH-dG levels. Determination of BUN and CRN was carried out using commercially available kits with a Hitachi, Automatic Analyzer 7150. For histological examination, kidney slices were fixed in 10% buffered formalin and stained with hematoxylin and eosin after routine histological processing. Measurement of B-OH-dG Kidney DNA, isolated immediately by the method of Kasai et al. [12], was digested to deoxynucleotides by treatment with nuclease P1 and alkaline phosphatase. 8-OH-dG levels were analyzed by HPLC using an electrochemical detection system (SPD-6A, Shimadzu Co. Ltd., Japan; Coulochem Model5100A, ESA, U.S.A.). Estimation of IVPSH and LPO Aliquots of tissue samples were homogenized with 6% trichloroacetic acid or 1.15% KCI solution for measurement of NPSH and LPO, respectively. Supernatants after centrifugation
51
at 3000 rev./min for 10 min were used to measure NPSH and LPO by the methods of Ellman [4] and Uchiyama et al. [25], respectively. Statistical
analysis
The significance results was evaluated t-test.
of differences between by applying the Cochran
Results 1
Results for non-protein sulfhydryl (NPSH) levels in the kidneys of treated rats are illustrated in Fig. 1, values for saline-treated rats given GSH (Group 3) and Fe-NTA-treated rats given GSH (Group 4) being significantly increased as compared to those for salinecontrol rats (Group 1). There were also significant differences between Groups 3 and 4, between Group 1 and the Fe-NTA-treated rats (Group 2) and between Group 2 and the FeNTA-treated rats given Cys (Group 6). Figure 2 illustrates the results for levels of thiobarbituric acid-reactive substances, measured as an index of lipid peroxidation, in the kidneys of treated rats. While LPO levels in Fe-
?’ S E IL $ 5
2
3
4
5
6
Experimental groups
Fig. 2. Kidney levels of lipid peroxidation (LPO). Values represent the mean f S.D. of data for five rats. A A P < 0.01 as compared with saline control rats (Group 1). A A P < 0.01 as compared with Fe-NTAtreated rats (Group 2).
NTA-treated rats given saline (Group 2)) GSH (Group 4) and Cys (Group 6) were all significantly higher than those in saline-treated rats (Group l), statistically significant differences were also found between Groups 2 and 4, but not between Groups 2 and 6. Figure 3 shows 8-OH-dG levels in the
6.0 8 “0 5. ’ 6 cb
4.0
2.0
3.0
2.0
1 .o
1
2
3
4
5
6
Experimental groups
Fig. 1. Kidney levels of non-protein SH (NPSH). Values represent the mean f S.D. of data for five rats. A , A A P < 0.05, 0.01 as compared with saline control rats (Group 1). A P < 0.05 as compared with salinetreated rats given GSH (Group 3). ‘P C 0.01 as compared with Fe-NTA-treated rats (Group 2).
1
2
3
4
5
6
Experimental groups
Fi2. 3. Kidney levels of &OH-dG. Values represent the mean f S.D. of data for five rats. A, A A P < 0.05, 0.01 as compared with saline control rats (Group 1). A P < 0.05 as compared with Fe-NTA-treated rats (Group 2).
52
kidneys of treated rats, values for the Fe-NTAtreated group given saline (Group Z), GSH (Group 4) and Cys (Group 6) being significantly elevated when compared to saline-treated rats (Group 1). However, the levels in both Groups 4 and 6 were significantly lower than those of Group 2. As shown in Fig. 4, similar changes to those observed for 8-OH-dG levels were also apparent regarding relative kidney weights and serum biochemical parameters measured as indicators of nephrotoxicity. Namely, while significant increases of relative kidney weights,
BUN and CRN were found in Fe-NTA-treated rats given saline (Group 2), GSH (Group 4) and Cys (Group 6) when compared to salinetreated rats (Group l), these were less prominent in the Group 4 and 6 animals. Microscopically, extensive renal tubular necrosis was observed in Fe-NTA-treated rats (Group 2). Though pyknotic changes and dilatation of renal tubules were also observed in Fe-NTA treated rats given GSH or Cys (Groups 4 and 6), the severity of these renal lesions was to a much lesser degree than that in Group 2 (Fig. 5).
0.0 2
1
3
4
5
6
Experimentalgroups 80
1
2
3
4
Experimental groups
5
6
1
2
3
4
5
6
Experimental groups
Fig. 4. Findings for (A) relative kidney weights (B) BUN (C) CRN. Values represent the mean f S.D. of data for five rats. A , A A P < 0.05,0.01 ascompared with saline control rats (Group 1). A , A A P < 0.05, 0.01 as compared with Fe-NTA-treated rats (Group 2).
53
Fig. 5. extensive
Histopathological findings in the kidneys of Fe-NTA-treated rats given (A) saline (B) GSH (C) Cys. (A) Note renal tubular necrosis. (B) and (C) note relatively weak injuries of renal proximal tubules. (H.E. stain X 240).
54
Discussion It has been reported that Fe-NTA is an effective iron-chelator inducing iron-mediated oxidative stress [8], injected Fe-NTA being proposed to initially act on the brush border membrane on excretion into the lumen of the renal proximal tubules, resulting in lipid peroxidation [9,16]. In addition, we showed earlier that the oxidative stress generated by Fe-NTA results in formation of S-OH-dG in kidney DNA [26]. Furthermore, we showed that a peak of lipid peroxidation level was found 1 h after the Fe-NTA treatment and the highest level of 8-OH-dG was observed after 6 h in a similar dose-dependent manner to the appearance of nephrotoxic responses [27]. In this experiment, we examined the effects of exogenous GSH and one of its precursor, Cys, on the lipid peroxidation at 1 h after the treatment and the formation of 8-OH-dG together with the nephrotoxic responses at 6 h. From our results, it can be seen that administration of exogenous GSH inhibited both lipid peroxidation and the formation of 8-OHdG induced by Fe-NTA, while exogenous Cys inhibited only the 8-OH-dG formation (see Figs. 2 and 3). Our present data showing decrease of NPSH after Fe-NTA treatment further suggest that the oxidative stress causes depletion of renal GSH content (see Fig. 1). GSH synthesized in the liver is translocated to blood plasma and removed mainly by the kidney, where the constituent amino acids produced by the degradation are recirculated to the liver and partially absorbed into renal cells for the re-synthesis of GSH in the kidney 151. Since the luminal space of renal brush border has a high activity of gamma-glutamyl transpeptidase, turnover of GSH in the kidney is very rapid (a half-life of 29 min) [22]. And when GSH is injected intravenously to rats, it disappears from the blood-stream within 30 min after injection [6]. As mentioned above, intact GSH filtered into the luminal space of renal tubules does not enter the cells [5,6]. Thus with regard to lipid peroxidation caused by Fe-NTA, GSH might exert protective action
in the extracellular space. However, the increase of 8-OH-dG levels which is a result of intracellular oxidative damage could be inhibited by both GSH and Cys treatments. Therefore adequate amounts of GSH for protection against Fe-NTA-associated intracellular oxidative stress were presumably generated by uptake of constituent amino acid resulting from degradation of the administered GSH at the luminal surface of the brush border membrane [lO,ll]. Since Cys is a GSH precursor, which is rate-limiting in cellular GSH synthesis [17,24], it can be considered that the inhibition of Fe-NTA-mediated GSH depletion as a result of the supplement of Cys was attributable to the acceleration of GSH synthesis [18,19], leading to the suppression of 8-OHdG formation (see Figs. 1 and 3). On the other hand, Cys itself might have the potential for the generation of some oxygen radicals during the rapid oxidation of Cys to the corresponding disulfide [28]. Moreover, it has also been reported that Fe-NTA is reduced specifically by Cys in renal brush border which actually promotes the peroxidation of the membrane [9]. These facts suggest that exogenous Cys may enhance Fe-NTA-promoted lipid peroxidation. Hydroxyl radicals generated by the reaction of Fe-NTA with H202 [l] could be the cause of the oxidative DNA damage. The fact that administered Cys protected only against oxidative DNA damage therefore indicates the possibility that Fe-NTAdependent oxidative stress in renal tubular cells may be mediated in two ways: both internally and via the external space. If the intracellular oxidative damage was caused by lipid peroxidation induced via the extracellular space, a direct correlation between the occurrence of lipid peroxidation and 8-OH-dG formation would have been expected. The effects of GSH and Cys on Fe-NTAinduced toxic renal lesions did, however, closely relate to formation of 8-OH-dG (see Fig. 4). Along with our previous confirmation in a dose-response study of correlation between 8-OH-dG levels and renal toxicity in FeNTA-treated rats [27], the present results seem
to suggest that intracellular oxidative stress plays an important role in the pathogenesis of nephrotoxicity caused by Fe-NTA. To date, several studies have clearly demonstrated a close link between formation of 8-OH-dG and carcinogenesis involving agents associated with active oxygen radicals [X2,13,23]. Further studies appear warranted to elucidate possible relation between carcinogenesis and renal injuries following oxidative DNA damage generated by Fe-NTA.
10
11
12
13
Acknowledgments This work was supported by a grant-in-aid from the Ministry of Health and Welfare of Japan.
14
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Role of glutathione. In: Oxand Medicine. Basic Life Editors: M.G. Simic, K.A. Sonntag.
Ellman, G.L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82, 70-77. Griffith, O.W. and Meister. A. (1979) Giutathione: fnterorgan translocation, turnover, and metabolism. Proc. Natl. Acad. Sci. U.S.A., 76, 5606-5610. Hahn, R., Wendel, A. and Flohe. L. (1978) The fate of extracellular glutathione in the rat. Biochim. Biophys. Acta, 539, 324-337.
326-331. Kasai, H., Nishimura, S.. Kurokawa, Y. and Hayashi, Y. (1987) Oral administration of the renal carcinogen. potassium bromate, specifically produces 8-hydroxydeoxyguanosine in rat target organ DNA. Carcinogenesis, 8. 1959-1961. Kasai, H., Okada, Y., Nishimura. S., Rao. H.S. and Reddy, J.K. (1989) Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator. Cancer Res., 49, 2603-2605. Ketterer, B.. Meyer, D.J. and Tan, K.H. (1988) The role of glutathione transferase in the detoxication and repair of lipid and DNA hydroperoxides. In. Oxygen Radicals in Biology and Medicine. Basic Life Sciences, pp. 669-675. Editors: M.G. Simic, K.A. Taylor, J.F. Ward and C.V. Sonntag.
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Li. J.-L., Okada, S., Hamazaki, S.. Ebina. Y. and Midorikawa. S. (1987) Subacute nephrotoxicity and induction of renal ceil carcinoma in mice treated with ferric nitrilotriacetate. Cancer Res., 47. 1867- 1869.
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Li, J.-L., Okada. S., Hamazaki. S Deng. I.-L. and Midorikawa. 0. (1988) Sex differences m ferric nitrilotriacetate-induced lipid peroxidation and nephrotoxicity in mice. Biochim. Biophys. Acta. 963, 82-87.
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Mcgowan, C. and Donaldson, W.E. (1987) Effect of lead toxicity on the organ concentration of glutathione-related free amino acids in the chick. Toxicol. Lett.. 38, 265-270.
18
Miranda, C.L., Reed, R.L.. Cheeke. P.R. and Buhler. D.R. (1981) Protective effects of butylated hydroxyanisole against the acute toxicity of monocrotaline in mice Toxicol. Appl. Pharmacol., 59. 424-430.
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Numan, I.T., Hassan, M.Q. and Stohs. S.J. (19901 Protective effects of antioxidants against endrin-induced lipid peroxidation, glutathione depletion. and lethality in rats. Arch. Environ. Contam. Toxicol.. 19. 302-306.
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Okada. S.. Hamazaki, S., Ebina. Y., Fujioka, M. and Midorikawa. 0. (1983) Nephrotoxicity and induction of the renal adenocarcinoma by ferric nitrilotriacetate (FeNTA) in rats. In: Structure and Function of Iron Storage and Transport Proteins. Editors: I. Urushizaki. I. Litowsky and J.W. Drysdale. Elsevier, New York, pp. 473-478. Preece, N.E.. Hall, D.E., Howarth, J.A., King, L.J. and Parke, D.V. (1988) The induction of autoxidative tissue
Hamazaki, S., Okada, S., Ebina, Y., Li, J.-L. and Midorikawa, 0. (1988) Effect of dietary vitamin E on ferric nitrilotriacetate-induced nephrotoxicity in rats. Toxicol. Appl. Pharmacol.. 92, 500-506. Okada, S., Li, J.-L., Toyokuni, S. and (1989) Oxygen reduction and lipid peroxchelates with special reference to ferric Arch. Biochem. Biophys.. 272, lo- 17.
21
Hamazaki, S.. Okada, S., Toyokuni. S. and Midorikawa. 0. (1989) Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate. Arch. Biochem. Biophys., 274, 348-354.
22
Hamazaki, S., Midorikawa. 0. idation by iron nitrilotriacetate.
Hassan, M.Q., Stohs, S.J. and Murray, W.J. (1985) Inhibition of TCDD-induced lipid peroxidation, glutathione peroxidase activity and toxicity by BHA and glutathione. Bull. Environ. Contam. Toxicol., 34, 787-796. moue, M. and Morino, Y. (1985) Direct evidence for role of membrane potential in glutathione transport by renal Chem.. 260, brush-border membranes. J. Biol
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damage by iron nitrilotriacetate in rats and mice. Toxicol. Appl. Pharmacol., 93, 89-100. Sekure, R. and Meister, A. (1974) Glutathione turnover in the kidney; Considerations relating to the gamma-glutamyl cycle and the transport of amino acids Proc. Natl. Acad. Sci. U.S.A., 71, 2969-2972. Takagi, A., Sai, K.. Umemura, T., Hasegawa, R. and
56
Kurokawa,
Y. (1990)
oxyguanosine
posure to the peroxisome phthalate
and
proliferators
di@ethylhexyl)adipate.
di(2-ethylhexybJpn.
J.
Cancer
Res., 81, 213-215. 24
Thor, H., Moldeus, activation
and
acetylcysteine, Arch. Biochem.
26
27 P. and Orrenius,
hepatotoxicity;
S. (1979)
Effect
of
Metabolic
cysteine,
toxicity
Biophys.,
in isolated
rat hepatocytes. 28
192, 405-413.
administration
(Fe-NTA).
Carcinogenesis, T.Y.,
Kurokawa,
Y. (1990)
nitrilotriacetate
Lett.,
54, 95-100. R. and Krebs, H.A.
Umemura,
Takagi,
Hasegawa,
R. and
A.,
Hasegawa,
R. and
G.T.,
induced in the rat kidney after
J.,
on isolated
after in-
nitrilotriacetate
11, 345-347.
(Fe-NTA)
Hems, 39-44.
ferric
8-hydroxydeDNA
Oxidative DNA damage, lipid perox-
Vina,
oxidation
Saez,
of
Sai, K., Takagi,
ferric
of
in rat kidney
Umemura,
86, 271-278. A.,
Formation
traperitoneal
id test. Anal. Biochem., Sai, K.,
(1990) (SOH-dG)
Uchiyama, M. and Mihara, M. (1978) Determination of malonaldehyde precursor in tissue by the thiobarbituric acT.,
Y.
oxyguanosine
idation and nephrotoxicity
N-
and methionine on glutathione biosynthesis
and bromobenzene 25
Kurokawa,
Significant increase of 8-hydroxyde-
in liver DNA of rats following short-term ex-
administration.
Wiggins, (1983)
hepatocytes.
D.,
Roberts,
Cancer A.F.C.,
The effect of cysteine Biochem.
J.,
212,
58 (1991)
49
49-56
Elsevier Scientific Publishers Ireland Ltd.
The effects of exogenous glutathione and cysteine on oxidative stress induced by ferric nitrilotriacetate T. Umemura, Division
K. Sai, A. Takagi,
of Toxicology,
National
R. Hasegawa
Institute of Hygienic
Sciences,
and Y. Kurokawa I -18-1,
Kamiyogo,
Setagayaku.
158,
Tokyo
(Japan)
(Received 12 December 1990) (Revision received 4 March 1991) (Accepted 7 March 1991)
Summary
Introduction
The effects of the antioxidants, glutathione (GSH) and its precursor cysteine (Cys) on oxidative damage induced by ferric nitrilotriacetate (Fe-NTA) were examined. Fe-NTAassociated oxidatioe stress caused the depletion of renal cellular GSH content. Administration of exogenous GSH and Cys suppressed 8-hydroxydeoxyguanosine (8-OH-dG) formation, an indicator of oxidatiue DNA damage and nephrotoxicity following Fe-NTA treatment. This suggests that generation of free radicals may be causally involved in oxidatiue lesion generation. Since lipid peroxidation was found to be inhibited only by GSH and not Cys treatment, this suggests that this effect and the DNA damage might be mediated by different
Iron complexed nitrilotriacetate (Fe-NTA) , which has been widely used as a substitute for polyphosphates in household detergents, is a severely nephrotoxic compound which also induces high incidences of renal cell carcinomas in rats [20] despite being a non-mutagenic chemical by the Ames test [ 151. We previously demonstrated that formation of &hydroxydeoxyguanosine (8-OH-dG), considered a suitable marker for oxidative DNA damage, can be observed after Fe-NTA treatment [26]. Other workers have shown that lipid peroxidation increased in kidneys of rats following administration of Fe-NTA [7,21]. In another study our laboratory found that Fe-NTA caused oxidative damage to rat kidney DNA and at the same time induced peroxidation of renal tubular cell membrane [27]. It is well known that glutathione (GSH) can play a protective role in cells against various types of oxidative stress by acting as a hydrogen donor for the reduction of free radicals or as a substrate for enzymatic reduction of peroxides [3,14]. And the rate of GSH synthesis in cells is dependent upon the supply of cystein (Cys) which is one of the constituent amino acids of GSH [24]. Therefore investigation of whether exogenous GSH and Cys exerts protective effects on oxidative stimulation
pathways.
Fe-NTA-associated
in renal tubular both intracellular
oxidatiue
Keywords: glutathione; cysteine; stress; %hydroxydeoxyguanosine; nitrilotriacetate
Correspondence Toxicology, Valhalla,
NY.
to: T. Umemura,
American 10595,
0304-3835/91/$03.50
stress
cells might thus operate via and external space modes.
Health
0
Division of Pathology
Foundation,
U.S.A.
1991
Published and Printed in Ireland
oxidative ferric
One
Dana
and
Road,
Elsevier Scientific Publishers Ireland Ltd
50
by Fe-NTA appeared of interest with regard to Fe-NTA carcinogenesis. In the present study, 8-OH-dG levels were examined in kidneys of Fe-NTA-treated rats after intraperitoneal treatment with GSH or Cys along with lipid peroxidation levels, some serum biochemical parameters and histopathological changes. Materials
and Methods
Animals and housing conditions Five-week-old male Wistar rats (specific pathogen free, Shizuoka Laboratory Animal Center, Japan) were given F-Z pellet basal diet (Funabashi Farm Co., Japan) and tap water freely; and were used after 1 week of acclimation. Compounds Ferric nitrate (Fe(NO,),) and nitrilotriacetic acid disodium salt (NazNTA) were purchased from Wako Chemical Industry, Japan and dissolved in distilled water before use. GSH in reduced form, L-cysteine in free form and alkaline phosphatase were obtained from Sigma Chemical Co. Nuclease P1 was obtained from Yamasa Shoyu Co. Ltd., Japan. GSH and Cys were dissolved in saline before use. Fe-NTA solution was prepared by the method of Awai et al. [Z]. Fe(NO& was mixed in a 4-fold molar excess of Na,NTA, and the pH was adjusted to 7.4 with NaHC03 (Wako Chem., Japan). Animal treatments Treatments were as follows (Table I). All doses were given to ten rats in each group intraperitoneally. Group 2,4 and 6 animals each received Fe-NTA at a dose of 15 mgFe/kg. Group 1, 3 and 5 animals each received saline at a dose of 6 ml/kg. These animals were also given GSH (Groups 3 and 4), Cys (Groups 5 and 6) or saline (Groups 1 and 2) at doses of 800 mg/kg, 400 mg/kg or 6 ml/kg, respectively, 30 min before and after the above treatments. Five animals of each group were killed 1 h after the Fe-NTA (or saline) treatment and the kidneys were immediately excis-
Table 1. Treatment
schedules.
Group
Pretreatment
Treatment
Post-treatment
1 2 3 4 5 6
Saline Saline GSH GSH
Saline Fe-NTA Saline Fe-NTA Saline Fe-NTA
Saline Saline GSH GSH
CYS CYS
CYS CYS
Animals were sacrificed 1 or 6 h after treatment. Saline: 6 ml/kg, GSH: 800 mg/kg, Cys: 400 mg/kg, Fe-NTA: 15 mg Fe/kg.
ed for measurement of lipid peroxidation (LPO) and non-protein SH (NPSH) levels. The remaining animals of each group were anesthetized with ether 6 h after the Fe-NTA (or saline) treatment, and blood was cbllected from the jugular vein for measurement of blood urea nitrogen (BUN) and creatinine (CRN). The kidneys were weighed and used for measurement of 8-OH-dG levels. Determination of BUN and CRN was carried out using commercially available kits with a Hitachi, Automatic Analyzer 7150. For histological examination, kidney slices were fixed in 10% buffered formalin and stained with hematoxylin and eosin after routine histological processing. Measurement of B-OH-dG Kidney DNA, isolated immediately by the method of Kasai et al. [12], was digested to deoxynucleotides by treatment with nuclease P1 and alkaline phosphatase. 8-OH-dG levels were analyzed by HPLC using an electrochemical detection system (SPD-6A, Shimadzu Co. Ltd., Japan; Coulochem Model5100A, ESA, U.S.A.). Estimation of IVPSH and LPO Aliquots of tissue samples were homogenized with 6% trichloroacetic acid or 1.15% KCI solution for measurement of NPSH and LPO, respectively. Supernatants after centrifugation
51
at 3000 rev./min for 10 min were used to measure NPSH and LPO by the methods of Ellman [4] and Uchiyama et al. [25], respectively. Statistical
analysis
The significance results was evaluated t-test.
of differences between by applying the Cochran
Results 1
Results for non-protein sulfhydryl (NPSH) levels in the kidneys of treated rats are illustrated in Fig. 1, values for saline-treated rats given GSH (Group 3) and Fe-NTA-treated rats given GSH (Group 4) being significantly increased as compared to those for salinecontrol rats (Group 1). There were also significant differences between Groups 3 and 4, between Group 1 and the Fe-NTA-treated rats (Group 2) and between Group 2 and the FeNTA-treated rats given Cys (Group 6). Figure 2 illustrates the results for levels of thiobarbituric acid-reactive substances, measured as an index of lipid peroxidation, in the kidneys of treated rats. While LPO levels in Fe-
?’ S E IL $ 5
2
3
4
5
6
Experimental groups
Fig. 2. Kidney levels of lipid peroxidation (LPO). Values represent the mean f S.D. of data for five rats. A A P < 0.01 as compared with saline control rats (Group 1). A A P < 0.01 as compared with Fe-NTAtreated rats (Group 2).
NTA-treated rats given saline (Group 2)) GSH (Group 4) and Cys (Group 6) were all significantly higher than those in saline-treated rats (Group l), statistically significant differences were also found between Groups 2 and 4, but not between Groups 2 and 6. Figure 3 shows 8-OH-dG levels in the
6.0 8 “0 5. ’ 6 cb
4.0
2.0
3.0
2.0
1 .o
1
2
3
4
5
6
Experimental groups
Fig. 1. Kidney levels of non-protein SH (NPSH). Values represent the mean f S.D. of data for five rats. A , A A P < 0.05, 0.01 as compared with saline control rats (Group 1). A P < 0.05 as compared with salinetreated rats given GSH (Group 3). ‘P C 0.01 as compared with Fe-NTA-treated rats (Group 2).
1
2
3
4
5
6
Experimental groups
Fi2. 3. Kidney levels of &OH-dG. Values represent the mean f S.D. of data for five rats. A, A A P < 0.05, 0.01 as compared with saline control rats (Group 1). A P < 0.05 as compared with Fe-NTA-treated rats (Group 2).
52
kidneys of treated rats, values for the Fe-NTAtreated group given saline (Group Z), GSH (Group 4) and Cys (Group 6) being significantly elevated when compared to saline-treated rats (Group 1). However, the levels in both Groups 4 and 6 were significantly lower than those of Group 2. As shown in Fig. 4, similar changes to those observed for 8-OH-dG levels were also apparent regarding relative kidney weights and serum biochemical parameters measured as indicators of nephrotoxicity. Namely, while significant increases of relative kidney weights,
BUN and CRN were found in Fe-NTA-treated rats given saline (Group 2), GSH (Group 4) and Cys (Group 6) when compared to salinetreated rats (Group l), these were less prominent in the Group 4 and 6 animals. Microscopically, extensive renal tubular necrosis was observed in Fe-NTA-treated rats (Group 2). Though pyknotic changes and dilatation of renal tubules were also observed in Fe-NTA treated rats given GSH or Cys (Groups 4 and 6), the severity of these renal lesions was to a much lesser degree than that in Group 2 (Fig. 5).
0.0 2
1
3
4
5
6
Experimentalgroups 80
1
2
3
4
Experimental groups
5
6
1
2
3
4
5
6
Experimental groups
Fig. 4. Findings for (A) relative kidney weights (B) BUN (C) CRN. Values represent the mean f S.D. of data for five rats. A , A A P < 0.05,0.01 ascompared with saline control rats (Group 1). A , A A P < 0.05, 0.01 as compared with Fe-NTA-treated rats (Group 2).
53
Fig. 5. extensive
Histopathological findings in the kidneys of Fe-NTA-treated rats given (A) saline (B) GSH (C) Cys. (A) Note renal tubular necrosis. (B) and (C) note relatively weak injuries of renal proximal tubules. (H.E. stain X 240).
54
Discussion It has been reported that Fe-NTA is an effective iron-chelator inducing iron-mediated oxidative stress [8], injected Fe-NTA being proposed to initially act on the brush border membrane on excretion into the lumen of the renal proximal tubules, resulting in lipid peroxidation [9,16]. In addition, we showed earlier that the oxidative stress generated by Fe-NTA results in formation of S-OH-dG in kidney DNA [26]. Furthermore, we showed that a peak of lipid peroxidation level was found 1 h after the Fe-NTA treatment and the highest level of 8-OH-dG was observed after 6 h in a similar dose-dependent manner to the appearance of nephrotoxic responses [27]. In this experiment, we examined the effects of exogenous GSH and one of its precursor, Cys, on the lipid peroxidation at 1 h after the treatment and the formation of 8-OH-dG together with the nephrotoxic responses at 6 h. From our results, it can be seen that administration of exogenous GSH inhibited both lipid peroxidation and the formation of 8-OHdG induced by Fe-NTA, while exogenous Cys inhibited only the 8-OH-dG formation (see Figs. 2 and 3). Our present data showing decrease of NPSH after Fe-NTA treatment further suggest that the oxidative stress causes depletion of renal GSH content (see Fig. 1). GSH synthesized in the liver is translocated to blood plasma and removed mainly by the kidney, where the constituent amino acids produced by the degradation are recirculated to the liver and partially absorbed into renal cells for the re-synthesis of GSH in the kidney 151. Since the luminal space of renal brush border has a high activity of gamma-glutamyl transpeptidase, turnover of GSH in the kidney is very rapid (a half-life of 29 min) [22]. And when GSH is injected intravenously to rats, it disappears from the blood-stream within 30 min after injection [6]. As mentioned above, intact GSH filtered into the luminal space of renal tubules does not enter the cells [5,6]. Thus with regard to lipid peroxidation caused by Fe-NTA, GSH might exert protective action
in the extracellular space. However, the increase of 8-OH-dG levels which is a result of intracellular oxidative damage could be inhibited by both GSH and Cys treatments. Therefore adequate amounts of GSH for protection against Fe-NTA-associated intracellular oxidative stress were presumably generated by uptake of constituent amino acid resulting from degradation of the administered GSH at the luminal surface of the brush border membrane [lO,ll]. Since Cys is a GSH precursor, which is rate-limiting in cellular GSH synthesis [17,24], it can be considered that the inhibition of Fe-NTA-mediated GSH depletion as a result of the supplement of Cys was attributable to the acceleration of GSH synthesis [18,19], leading to the suppression of 8-OHdG formation (see Figs. 1 and 3). On the other hand, Cys itself might have the potential for the generation of some oxygen radicals during the rapid oxidation of Cys to the corresponding disulfide [28]. Moreover, it has also been reported that Fe-NTA is reduced specifically by Cys in renal brush border which actually promotes the peroxidation of the membrane [9]. These facts suggest that exogenous Cys may enhance Fe-NTA-promoted lipid peroxidation. Hydroxyl radicals generated by the reaction of Fe-NTA with H202 [l] could be the cause of the oxidative DNA damage. The fact that administered Cys protected only against oxidative DNA damage therefore indicates the possibility that Fe-NTAdependent oxidative stress in renal tubular cells may be mediated in two ways: both internally and via the external space. If the intracellular oxidative damage was caused by lipid peroxidation induced via the extracellular space, a direct correlation between the occurrence of lipid peroxidation and 8-OH-dG formation would have been expected. The effects of GSH and Cys on Fe-NTAinduced toxic renal lesions did, however, closely relate to formation of 8-OH-dG (see Fig. 4). Along with our previous confirmation in a dose-response study of correlation between 8-OH-dG levels and renal toxicity in FeNTA-treated rats [27], the present results seem
to suggest that intracellular oxidative stress plays an important role in the pathogenesis of nephrotoxicity caused by Fe-NTA. To date, several studies have clearly demonstrated a close link between formation of 8-OH-dG and carcinogenesis involving agents associated with active oxygen radicals [X2,13,23]. Further studies appear warranted to elucidate possible relation between carcinogenesis and renal injuries following oxidative DNA damage generated by Fe-NTA.
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Acknowledgments This work was supported by a grant-in-aid from the Ministry of Health and Welfare of Japan.
14
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