Toxicology, 76 (1992) 27-38 Elsevier Scientific Publishers Ireland Ltd.

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Hepatic chemiluminescence and lipid peroxidation in mild iron overload Monica Galleano and Susana Puntarulo Physical Chemistry Division, School of Pharmacy and Biochemistry, University of Buenos Aires (Argentina) (Received April 1lth, 1992; accepted July 23rd, 1992)

Summary The effect of iron-overload on both hepatic lipid peroxidation and chemiluminescence Was studied in early stages after iron-dextran injection. Total hepatic iron content was markedly elevated over control values 2-6 h after iron dose. A 4-fold increase in light emission was detected after 4-6 h after iron injection. Plasma GOT, GPT and LDH activities were not affected by the treatment suggesting that cell permeability was not affected by necrosis. Increases in the generation of thiobarbituric acid reactive substances (TBARS) and chemiluminescence in liver homogenates, were determined as a function of time after iron administration, in the presence of NADPH as cofactor, Under the same experimental conditions, microsomal cytochrome P-450 content was decreased by 40%, 2 h after iron treatment. To evaluate liver antioxidant defenses, catalase, superoxide dismutase and glutathione peroxidase activities were determined. Glutathione peroxidase activity in the homogenate was not affected by the treatment. Catalase and superoxide dismutase activities declined by 25 and 36%, respectively, compared with control values 4 h after the iron dose. Our data suggest that lipid peroxidation occurs after mild iron overload even though the liver remains functional.

Key words: Iron overload; Chemiluminescence; Lipid peroxidation; Antioxidant enzymes

Introduction Hepatotoxicity is the most common finding in patients with iron overload since the liver is the major recipient of the excess of iron. Although the toxicity of iron has been described clinically [1] the specific cytopathological mechanisms whereby hepatocytes are injured in iron overload remain to be elucidated [2-4]. Iron ions seem to play a major role in initiation and propagation reactions of intracellular lipid peroxidation [5]. Iron catalyzes the decomposition of stable hydroperoxides formed during the course of propagation reactions which reinitiate radical species [5]. In vitro studies showed that incubation of isolated liver organelles with ionic iron and a reductant, stimulated lipid peroxidation. Some of the wide ranging effects of iron-induced lipid peroxidation by Fe 2+ or Fe 3+ in vitro, included increasCorrespondence to: Dr. Susana Puntarulo, Catedra de Fisicoquimica, Facultad de Farmacia y Bioquimica, Junin 956, 1113, Buenos Aires, Argentina. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

28 ed lysosomal UDP-glucuronyl transferase, decreased lysosomal latency, formation of mitochondrial ghosts, mitochondrial uncoupling, reduced respiratory control ratio, decreased microsomai cytochrome P-450 content, decreased content of microsomal 20:4 and 22:6 fatty acids, etc. [6-8]. Studies of iron liver toxicity developed using models of in vivo iron overload have focused on abnormalities in lysosomal function [9], hepatic microsomal [7,10-13] and mitochondrial [14] TBARS production. In these experiments, only when the hepatic iron concentration was higher than 2400 #g/g wet weight was there an increase in lysosomal fragility [9]. At iron concentrations of -3000 #g/g wet weight evidence for hepatic microsomal lipid peroxidation was found [10]. However, at iron concentrations of - 1000 tzg/g wet weight no effects on mitochondrial lipid peroxidation, assessed by conjugated dienes formation, were detected [14]. On the other hand in vivo studies by Dillard and Tappel indicated an increase in ethane and pentane exhalation in rats receiving multiple injections of iron dextran (iron total dose of 4.6 g/kg body wt.) [15], but administrating a lower dose of iron-dextran (116 mg/kg body wt.), Younes et al. could not detect differences between iron supplemented and control rats [16]. Spontaneous organ chemiluminescence reflects the rate of lipid peroxidation reactions through the detection of the steady-state level of excited species. It is considered as a useful technique to evaluate oxidative stress 'in vivo' [17]. In this study we have investigated the early stages of the onset of oxidative stress due to a mild iron overload in rats. The results were correlated with changes in liver cell integrity (plasma GOT, GPT and LDH activities) and antioxidant enzyme activities. Materials and methods

Male Wistar rats (100-150 g) were injected i.p. with iron-dextran (500 mg/kg body wt.). Control animals were injected either with saline solution or dextran. Homogenates were prepared from livers that were excised immediately and homogenized using a Potter-Elvehjem Teflon glass homogenizer in 100 mM Tris-HCl (pH 7.4), at 4°C. Microsomes were prepared by differential centrifugation as previously described [18]. Hepatic total iron concentration was determined by atomic absorption after treating the livers with HNO3-HC104 (1:1) solution [19]. Control and treated rats were anesthetized by administration of 30% w/v urethane at a dose of 0.5 ml/100 g body weight. The liver surface was exposed by laparotomy and chemiluminescence was measured with a Johnson Foundation photon-counter (Johnson Research Foundation, University of Pennsylvania, Philadelphia, PA). The single-photon counting system contained a photomultiplier placed in a light-tight chamber with a thermoelectric cooler, a phototube EMI 9658 B with high sensitivity in the red adapted to detect singlet oxygen dimol emission, frequency counter, high voltage, recorder and a lucite rod placed near the exposed organs. The results were expressed in counts per second per cm 2 of liver surface (cps/cm 2) [20]. Homogenate chemiluminescence was measured in a Packard Tri-Carb model 3320 liquid scintillation counter in the out-of-coincidence mode. The basic reaction system for chemiluminescence consisted of 100 mM potassium phosphate buffer (pH 7.4), 1 mg/ml homogenate protein and 0.5 mM NADPH. The results were expressed as

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arbitrary units (area under emission curves) over 30 min. Homogenate lipid peroxidation was determined by assaying for the rate of production of TBARS (expressed as malondialdehyde equivalents). Reactions were carried out utilizing a system consisting of 100 mM Tris-HC1 buffer (pH 7.4) and 0.5 mM NADPH, 1.0 mg homogenate protein/ml ( - 8 . 0 mg liver/ml) in a final volume of 1 ml [21]. The content of microsomal cytochrome P-450 was determined by the method of Omura and Sato [22]. Superoxide dismutase activity in the homogenates was determined by measuring the inhibition of the rate of autocatalytic adrenochrome formation in a reaction medium containing 1 mM epinephrine and 50 mM NaOH-glycine (pH 9.6), [23]. Glutathione peroxidase activity was determined spectrophotometrically by measuring NADPH-dependent decrease in absorbance at 340 nm [24]. Catalase activity was measured according to the modified method of Aebi [25]. Plasma GOT (L-aspartate:2 oxoglutarate aminotransferase), GPT (L-alanine:2 oxoglutarate amino transferase) and LDH (lactic dehydrogenase) activities were assayed using commercially available assays kits (Boehringer, Mannheim). Ferric-ATP complex was prepared by dissolving ferric ammonium sulfate in 0.1 N HC1 and then diluting with the appropriate ATP solution to a final concentration ratio of 1:20. The buffers and the water used to prepare all solutions were passed through columns containing Chelex 100 resin to remove metal contaminants. Symbols and lines in the figures indicate mean values 4- S.E.M. of 3-7 samples. Significance was determined using t-test. Results

Total liver iron concentration was measured after acid digestion of liver samples obtained from rats after 2-16 h of iron-dextran administration. Increases of 5-14fold over the control values were detected in the period of 2-24 h after iron supplementation (Table I). Cytosols were prepared by differential centrifugation

TABLE I LIVER I R O N CONTENT Liver iron content in Fe-dextran treated rats is significantly increased over the 24 h time period (P < 0.01) as compared with the control group. Fe Content (~g/g dry liver) Control Iron loaded 2h 4 h 8 h 16 h 24 h

257 ±

11

871-~ 1475 ± 1837 ± 1808 ± 3447 ±

98 119 205 477 256

(t~g/g wet liver) 77 ± 262 443 552 544 1038

4

± 30 ± 36 ± 62 ± 144 ± 77

30 (700 x g, 11 000 x g and 105 000 × g) of the homogenates. Iron content in the cytosol was assayed after precipitation with 15% TCA. The following values were obtained: 31 ± l, 139 ± 5 and 99 ± 7 nmol/mg cytosolic protein, for control and samples from iron overloaded rats after 4 and 20 h, respectively. In spite of iron deposition, plasma GOT, G P T and L D H activities did not differ between control and iron overloaded rats (Table II). Liver water content, as an indication of cell edema and evaluated as weight loss after liver dessication, showed a variation of - 2 % (statistically not significant) between control and treated samples. These data suggest that liver necrosis was not developed during the early stages of iron overload. Spontaneous liver chemiluminescence was measured as an indicator of oxidative stress and lipid peroxidation in the in situ liver. Iron supplementation increased spontaneous chemiluminescence significantly 2 h after the iron-dextran dose. A maximum in emission was detected 6 h after iron administration. Control rats injected either with saline solution or dextran (identical mol. wt. to the one administrated in the iron-dextran complex) did not change emission values over the studied period (Fig. 1). Control rats injected with dextran were included to assess an eventual increase in chemiluminescence due to macrophage migration and activation to the liver, caused by dextran itself. The data indicate that under these conditions no chemiluminescence arising from macrophages interfered with the measurements. The hepatotoxic agent, carbon tetrachloride, was administrated to evaluate a possible potentiation with iron overload. Livers subjected to CCI 4 supplementation after iron-dextran dose showed higher emission than control livers, but no synergistic effect was detected (Table III). Essentially identical results were obtained when TBARS generation was measured in the presence of CC14 (data not shown). NADPH-dependent chemiluminescence was catalyzed by control liver homogenates (Table IV). Homogenates from livers excised 2 - 2 0 h after iron-dextran administration showed a significant increase in the emission compared to control values. The ability of the in vitro added iron ( F e - A T P ) to enhance chemiluminescence could be observed in assays performed with either control or

TABLE II PLASMA GOT, GPT AND LDH ACTIVITIES IN CONTROL AND IRON LOADED RATS No statistically significant differences (P > 0.05) were found in enzymeactivities in the iron overloaded rats as compared with the control animals.

Control Iron loaded 2h 6h 20 h

GOT (units/l)

GPT (units/l)

LDH (units/l)

42 4- 4

24 4- 5

224 4- 30

47 ± 5 444- 2 51 4- 10

19 ± 3 214-3 16 4- 4

1864-21 1924-16 236 4- 25

31

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E tJ

in Q. fj

60

LU U Z ul (J I/) U.I

40

20 ._i m

~r U.l .1(J

0

i 0

2

, 4 TIME

,

,

'

6

8

I0

!

h

II /.8

l

Fig. 1. Spontaneous rat liver chemiluminescence. In vivo chemiluminescence by the liver of rats injected either with saline solution or dextran (O) or 500 mg/kg iron-dextran (O). Chemiluminescence was measured as described under Materials and methods over a period of 40 h after iron-dextran injection Symbols indicate mean values from 4 rats and bars indicate S.E.M. t r e a t e d l i v e r s ( T a b l e IV). T h e s e r e s u l t s s u g g e s t t h a t t h e p r o p o r t i o n o f c a t a l y t i c a c t i v e i r o n i n c r e a s e d u p o n i r o n t r e a t m e n t a t a n o n - s a t u r a t i n g level. TBARS measurements were used as an assay for lipid pcroxidation in vitro. In the absence of added iron, control liver homogenates catalyzed a NADPH-dependent

TABLE III CCL 4 EFFECT ON SPONTANEOUS CHEMILUMINESCENCE IN CONTROL AND TREATED RATS Organ chemiluminescence was determined as indicated in the Materials and methods section. The numbers in parentheses refer to the increase in chemiluminescence as percentages of the control values 14 4- 2 cps/cm 2 and 26 ± 2 cps/cm 2, in the abscence or presence of CC14, respectively. Time indicates the period between iron-dextran injection and the measurement. Treated rats showed statistically significant differences (P < 0.01) as compared with the respective control group. Chemiluminescence (cps/cm 2)

Control Iron loaded 2h 4h 6h 8h

- CCI4

+ CCI4

14 ± 2

26 ± 2

28 42 52 32

4- 3 4- 2 ± 3 ± 4

(+100%) (+200%) (+271%) (+128%)

36 50 75 35

± 3 4- 4 ± 3 ± 2

(+38%) (+92%) (+188%) (+35%)

32 TABLE IV IN VITRO NADPH-DEPENDENT CHEMILUMINESCENCE FROM IRON-OVERLOADED LIVER HOMOGENATES Time indicates the period between iron-dextran injection and preparation of the homogenates. Salineinjected rats homogenates showed a NADPH-dependent chemiluminescence within the control values over the 20-h period. Where indicated, 50 t~M Fe-ATP (1:20) was added to the assay medium. Treated rats showed statistically significant differences(P < 0.01) as compared with the respectivecontrol group, except where indicated by an asterisk. Chemiluminescence (A.U./30 min per 0.5 mg prot.)

Control Iron loaded 2h 6h 20 h

No additions

+ Fe-ATP

0.10 ± 0.02

0.40 4- 0.03

0.15 4- 0.01" 0.30 4- 0.02 0.42 4- 0.03

0.48 4- 0.04* 0.55 4- 0.04 0.79 4- 0.03

production of TBARS. Homogenates from iron overloaded rats showed increased TBARS generation (Fig. 2). A significant increase in the production of TBARS by in vitro added iron ( F e - A T P ) was verified, suggesting that lipid peroxidation could be further increased upon iron supplementation. Iron catalytic activity is known to depend on the form of metal chelation [26]. Since homogenates from treated livers have an increased amount of iron, physiological chelators could be limiting. The generation of TBARS by homogenates was assayed in the presence of added ATP, a possible intracellular iron chelator. Control rate was not significantly affected by this addition, but homogenates from iron overloaded rats showed an increase in lipid peroxidation (Fig. 2). Maximum A T P effect was observed at a concentration of 500 #M (data not shown). TBARS generation was lower in the presence of N A D H compared with N A D P H , even in the presence of excess iron ( F e - A T P ) in vitro. TBARS production by either control or treated homogenates was slightly affected by A T P addition to the reaction medium (Table V). NADPH-dependent TBARS generation by both control and iron-overloaded homogenates, was sensitive to the addition of antioxidants such as 15 m M BHT (inhibition by 85 and 97%, respectively), or iron chelators such as I m M E D T A (inhibition by 71 and 94%, respectively) and 50/zM desferrioxamine (inhibition by 71 and 97%, respectively). However, lipid peroxidation in both control and treated homogenates was not affected by either superoxide dismutase or catalase addition to the reaction medium. The same pattern of effectiveness was observed in the presence of N A D H as the cofactor. The antioxidant enzyme status of the livers was estimated by determining glutathione peroxidase, superoxide dismutase and catalase activities. The specific activity of glutathione peroxidase was 60/~mol/min per mg in control homogenates and no decreases were determined in the homogenates from treated livers. Superoxide

33

~

0.6

| c X 0.2

0 0

1 2

I 4 Tilde

I 6

II

I 20

Ihl

Fig. 2. TBARS production by rat liver homogenates in the presence of NADPH. Time indicates the period between iron-dextran dose and removing of the liver. The in vitro assay was performed either under the conditions indicated in Materials and methods (O) or in the presence of 500/zM ATP (Z~) or 50/~M Fe-ATP (1:20) (0).

TABLE V NADH-DEPENDENT TBARS GENERATION IN CONTROL AND IRON LOADED RAT LIVER HOMOGENATES Time indicates the period between iron-dextran dose and preparation of the homogenates. No significant differences (P > 0.05) are shown as compared to the respective control group except where indicated by an asterisk (P < 0.01). TBARS (nmol MDA/min per mg prot.)

Control Iron loaded 2h 6h 20 h

No additions

+500 #M ATP

+50 #M Fe-ATP

0.04 4- 0.01

0.07 4- 0.03

0.22 4- 0.04

0.05 ± 0.02 O.11 ± 0.03* 0.12 4- 0.06

0.07 4- 0.03 0.15 4- 0.03* 0.15 ± 0.05

0.23 4- 0.04 0.25 4- 0.05 0.31 4- 0.08

34

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30

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Hepatic chemiluminescence and lipid peroxidation in mild iron overload.

The effect of iron-overload on both hepatic lipid peroxidation and chemiluminescence was studied in early stages after iron-dextran injection. Total h...
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