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Intratracheal injection of nickel chloride and copper -zinc superoxide dismutase activity in lung of rats

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ETHELL. B. NOVELLI AND NEYL. RODRIGUES Deprtaments de Quimica, Instituto de Biocitncias, Universidade Estudual Paulista, UNESP, 18610 Botucatu, Sdo Puulo, Brasil

B A R ~ L O0. MRIBAS ~ Instituto de Salmrd Carlos PII, Ministerio de Sunidad y Consurno, Madrid, Spain AND

PAULOR. CURI Servip de Estatisticta da ficuldade de Medicincn Veterinciria e Zootecnia, UNESP, Botucatu, Sdo Paulo, Brasil Received June 27, 1991 NOVBLEI,E. E. B., ROBRIGUES, N. L., RIBAS,B. O., and CURI,P. R. 1392. Intratracheal injection of nickel chloride and copper -zinc superoxide dismutase activity in lung of rats. Can. J. Physiol. Pharmacol. 70: 709-71 1. Superoxide radical (8;) is a free radical that may be involved in various toxic processes. Cu -Zn superoxide dismutase catalyses the dismutation of the superoxide free radical and protects cells from oxidative damage, and it has been used clinically. The concentration of Ni2+ and Cu - Zn superoxide dismutase activity were measured in lungs of rats at time intervals of 5, 12, 19, 26, 33, and 40 days following an intratrached injection of 127 nmol of NiCl,. Nickel chloride increased nickel content and resulted in a significant increase of Cu-Zn superoxide dismutase activity in lungs. This elevation of Cu-Zn superoxide dismutase activity was highest on the 12th day (approximately threefold) and is at levels comparable to controls rats on day 40 onwards. Since Cu-Zn superoxide dismutase activity was increased in lung throughout our experimental period without corresponding increases of Cu2+ and Zn2+, we speculate that the elevation of Cu -Zn superoxide dismutase activity might be due to an increased half-life of the enzyme, induced by nickel. Key words: nickel chloride, intratrached , Cu -Zn superoxide dismutase, lung. NOVELLI,E. L. B., WBRIGUES,N. L., RIBAS,B. O., et Cum, P. R. 1992. Intratracheal injection of nickel chloride and copper -zinc superoxide dismutase activity in lung of rats. Can. J. Physiol. Pharmacol. 70 : 709 -71 1. Le radical superoxyde (85) est un radical libre qui pourrait &re irnpliquk dans divers processus toxiques. La superoxyde dismutase de type Cu-Zn, qui cablyse la dismutation du radical Iibre superoxyde et protkge les cellules d'une altCration oxydative, a CtC cliniquement utiliske. On a dtteminC la concentration de l'activitt de peroxyde dismutase de type Cu-Zn et Ni2+ dans le poumon de rat B des intervdles de 5, 12, 19, 26, 33 et 40 jours, aprbs une injection intratrachkale de 127 nmol de NiCI,. Le chlorure de nickel a augment6 la teneur en nickel et provoquk une hausse significative de l'activitt de superoxyde dismutase de type Cu-Zn dans les poumons. Cette Clkvation de lqactivitCde peroxyde dismutase de type Cu-Zn a CtC trbs forte le 12e jour (d'un facteur trois approximativement) et se situe B des taux comparables B ceux des rats tCmoins au jour 40 et plus. L'activitC de peroxyde dismutase de type Cu-Zn ayant CtC augmentte dans le poumon pendant toute la durke de notre expbimentation, sans qu'il y ait eu d'augmentations correspondantes de Cu2+et de Zn2+, nous SUPposons que l'kltvation de l'activite de peroxyde dismutase de type Cu -Zn pourrait &re due B une augmentation, induite par le nickel, de la demi-vie de I'emyme. Mots cids : chlorure de nickel, intratrachtal, superoxyde dismutase de type Cu-Zn, poumon. [Traduit par la rkdaction]

Introduction appears to play an important role Superoxide radical (05) in adult lung oxygen toxicity (Hass and Massaro 1987), inflammation, radiation-induced damage (Yarnamoto et al . 1989), carcinogenicity (Eze et al. 1998), aging (Vani et al. 1990), bone resorption (Garret et d. 1990), vasoconstriction of rat thoracic aortic rings (Lawson et al. 1990), and oxidative skeletal muscle fatigue (Barclay and Hansel 1991). Apparently the toxicity of the superoxide radical may be due to the fact that it can inactivate sulfhydryl enzymes in the cell, induce DNA damage, and destroy lipid membranes by lipid peroxidation. Hn cancer tissues, several laboratories have found very little or no enzymatic activity of Cu -Zn superoxide dismutase (SOD; EC 1.15.1.1) (Westman and Marklund 1981). It has been reported that SOD protects various organs from damage induced by reactive oxygen species and SOD has been used clinically (Chamber et al. 1985). Printed in Canada I Imprim6 au Canada

Since nickel-bearing compounds are widely distributed in the atmosphere in urban centers, in foods, dental restorations alloys, and industrial exposure, we decided to study the effect of nickel chloride (PJiCl,) on Cu-Zn superoxide dismutase activity in lung.

Materials and methods Male Wistar rats weighing 180-200 g (120 animals) were used. Food ( h r i n a Ltd., 3074 SIF) and water were provided ad libitum. The animals were treated with a single dose of NiC1, (Merck; 127 nmol; 60 rats) by intratrached injection in a volume of 8.1 mL of 0.9 % saline (pH 7.4). This dose was previously used by Willians et al. (1980) to study the distributional kinetics of Ni2+ in the rat lung. Controls (60 rats) received 8.1 mL of saline. Each ether-anesthetized rat was suspended dorsally on a slant board by its upper incisors. Light was focused down the spoon-shaped portion of the speculum, which was inserted into the rat's mouth and acted as a tongue depressor during injection. The device offend visualization

CAN. J . PHYSIOL.

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TABLE1. Nickel, copper, zinc, and total protein concentrations of lung in control rats and following an intratracheal injection of NiCl, (127 nmol)

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Biochemical determinations

Days following treatment 5

12

19

26

33

48

Nickel (pglg tissue) Control NiCl, Copper (pglg tissue) Control NiCI, Zinc (pglg tissue) Control NiCl, Total protein (glg tissue) Control NiC1, *Values are significantly different from those s f the control group by the Student's t-test ( p

of the vocal cords and upper trachea so that injections, made through a blunted and curved 8-cm 18-gauge hypodermic needle, were performed quicldy and without undue trauma. Rats were sacrificed (5 for each time point) by decapitation at time intervals of 5 . 12, 19, 26, 33, and 48 days after treatment. The lung was excised and washed in saline to remove excess connective tissues and red blood cells. TWOsamples of lung were taken. One part was weighed and homogenized in five volumes of phosphate buffer solution (8.1 hid, pH 7.49 with a motor-driven Teflon-glass tissue homogenizer. The komogenate was centrifuged at 12Wg for 15 min. The supernatant was used for protein assay (Lowry et d.1951) and determination of Cu-Zn superoxide dismutase activity, based on the ability of the enzyme to inhibit the reduction of nitro blue tetrazoliurn (NBT; Sigma Chemical Co., St. Louis, Mo.), which was generated by hydroxylamine in alkaline solution (Crouch et al. 1982). Hydroxylamine (37.5 mM) (Carlo Erba) was utilized to generate a superoxide flux. The assay was performed in 8.5 M sodium carbonate (pH 10.2) with 0.1 M EDTFB. The reduction of NBT by 8;to blue formazan was measured spectrophotometrically at 560 nm. The rate of NBT reduction in the absence of tissue was used as the reference rate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition. All data were expressed in units of SOB per milligram of protein. The mitochondrial Mn superoxide dismutase was inactivated by treatment with mixtures of chloroform and ethanol (Weisiger and Fridovich 1973). Another part of lung was used for nickel, copper, and zinc detemination by atomic spectrophotometry with a model M-1475-Kntralab atomic absorption spectrometer, which was calibrated with standard solutions of salts of these metals. Tissues were ashed in muffle furnaces at 75Z0K; in siIica dishes, after drying to constant weight at 230°F. The ash was acidified and dissolved with 3 mL of 2 M HC1 (Merck) and stored at room temperature in polyethylene bottles until analysis. All experiments were carried out in duplicate. Values are presented as means f SD. Significance of difference was tested by Student's t-test.

Results Table 1 summarizes measurements of nickel, copper, zinc, and totd protein in the lung. The rate of removal of Ni2+ from the airways is time related and appears to require at least 40 days. The concentrations of Cu2+ m d Zn2+ in the lung were significantly increased only at 5 days following an intratracheal injection of NiC12. The data show (Table 1) that the zinc and copper contents are high on the 5th day after Ni2+ injection and are at levels comparable to control rats from day 12

< 8.05).

onwards. Nickel chloride treatment resulted in a significant increase (approximately threefold) of Cu - Zn superoxide dismutase activity of lung. This increase of SOB activity was highest on the 12th day and decreased to near control levels from day 40 onwards (Fig. 1).

Discussion Given the current lack of knowledge concerning the oxidative state of nickel in the atmosphere, Ni2+ serves as a good model for inhalation studies about the toxicity of nickelbearing compounds. Note (Table I) that intratracheal injection of NiC12, a water-soluble compound, increased nickel, copper, and zinc contents and the activity of Cu -Zn superoxide dismutase from lungs of rats (Fig. I), and considerable evidence has been accumulated in recent years that the physiological fbnction of SOB is a protective one. A relationship between SOD and anti-inflammatory activity became obvious with the observation by Babior et al. (1973) that phagocytosing polymorphonuclear leukocytes, effector cells of the acute inflammatory response, release large amounts of superoxide that apparently contributes to the killing of ingested bacteria. There are two types of dismutases in eukaryotic cells, one containing Cu2+ and Zn2+ and the other Mn, that are essential cofactors. They differ substantidly in their localization, primary structure, and in some other characteristics including the induction mechanism (Konstantinova and Russanov 1988). The activity of Cu-Zn SOD increases or decreases upon andogous changes in the levels of the cofactors of the enzyme (Lyutakora et d. 1984). Koppenol (1976) observed that the reduction of the couple SOB-Cu2+ to SOD -Cu+ and O2 formation was the enzymatic step that detoxified the superoxide radical. Ciriolo et d. (1990) point to a role of zinc in giving the adjacent copperbinding site the geometry that is typical of the native holoenzyme. However, as shown in Table 1, both Cu2+ and Zn2+ ions were significantly increased only at 5 days after NiCB2 treatment, while SOD activity and Ni2+ content were increased throughout the experimental period (Table 1, Fig. 1). It is clear that activity of SOD increased in lungs without corresponding increases of Cu2+ and Zn2+. Freundt and Ibrahim (1991) found that Cu2+ and Znu sdts showed very little or no effect on the activity of dismutases in liver of rats.

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Acknowledgements We thank Mr. Guerino S. Bianchi Filho for his help. This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgico (CNPq).

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0

71 1

k

5

I

12

I

19

1

26

8

33

1 48

BAYS FOLLOWING THE TREATMENT

FIG. 1 . Cu-Zn superoxide dismutase activity in lungs of control rats (G)and 5, 12, 19,26, 33, and 40 days following an intratrached injection of NiCl, (@).

The observation that Cu - Zn SOD activity increases (Fig. 1) while total protein remained constant is not inconsistent with increased SOD synthesis. SOD constitutes a very small percentage of total lung protein, so a sizable synthesis could occur without significantly altering total protein concentration. In general there seems to be a balance between the substrate superoxide and the activity of the SOB. In this paper, hydroxylmine (47.5 nM) was used to generate superoxide flux and was found to produce sufficient superoxide to effectively saturate the enzyme. Hass and Massaro (1987) demonstrated that the increase of SOB activity, which takes place from birth to adulthood, is brought about by a rate of SOD synthesis that slightly exceeds its rate of degradation. These authors found the enzyme's resistance to inactivation by heat and copper chelation correlates with its calculated half-lives, suggesting the possibility that ligands or non-covalent binding proteins may be involved in limiting the enzyme's degradation. Based on these observations and since SOD activity was increased in lungs throughout our experimental period without corresponding increases of Cu2+ and Zn2+ (Table I), we speculate that the elevation in SOD activity (Fig. 1) might be due to an increase of the half-life of SOD, perhaps related to decreased susceptibility to proteolysis. In view of these observations, the nickel effect on SOD activity may be due to a direct action on the enzyme. Increased stabilization of enzyme conformation might account for an increased half-life of the SOD, in lungs of rats treated with NiC12. Since NiC12 increased activity of SOD in lungs of rats, we could suggest the possibility that Ni2+ has potential benefit in the protection of cells against the various disorders caused by 0 5 and might have anti-inflammatory activity.

Babior, B. M., Kipnes, R. S., and Curnutte, 9. T. 1973. Biological defense mechanisms. The production of leukocytes of superoxide, a potent bactericidal agent. J. Clin. Invest. 52: 741 -744. Barclay, J. K., and Hansel, M. 1991. Free radicals may contribute to oxidative skeletal muscle fatigue. Can. J . Physi~1.Phamacol . 69: 279-284. Chamber, B. E., Parkers, D. A . , and Patterson, G. 1985. Xanthine oidase as the source of free radical h a g e in myocardial ischemia. J. Mol. Cell. Cardiol. 17: 145 - 152. Ciriolo, M. R., Desideri, A., Paci, M.. and Rotilio, G. 1990. Reconstitution of Cu, Zn-superoxide dismutase by the Cu(I) -glutathione complex. J. Biol. Chem. 264: 5598 -5605. Crouch, R.,Gandy, S. E., Kinsey, G., et al. 1982. The inhibition of islet superoxide dismutase by diabetogenic drugs. Diabetes, 30: 235-241. Eze, M. O.,Hunting, D. J., and Ogan, A. U. 1990. Reactive oxygen production against malaria - a potential cancer risk factor. Med. Hypotheses, 32: 121 - 123. Freundt, K. J . , and Ibrahim, H. A. 1991. influence of Pb, Cd, Zn, Mn, Cu, Hg, or Be salts on the glutathione s-transferase of the rat liver. Bull. Environ. Contam. Toxicol. 46: 618 - 624. Garret, R., Boyce, B. F., Oreffo, R. 0. C., d al. 1990. Oxygen derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J. Clin. Invest. 85: 632-639. Hass, M. A., and Massaro, D. 1987. Development regulation of ratlung Cu, Zn superoxide dismutase. J. Clin. Invest. 246: 697 - 783. Konstantinova, S. G., and Russanov, E. M. 1988. Effect of pregnancy and fetal development on sheep liver superoxide dismutase activity. Res. Vet. Sci. 45: 287-290. Koppenol, W. H. 1975. Reactions involving singlet oxygen and the superoxide anion. Nature (London), 262: 420 -42 1 . Lawson, B.L., Mehta, J. L., Nichols, W. W., at al. 1990. Superoxide radical mediated endothelial injury and vasoconstriction of rat thoracic aortic rings. J. Lab. Clin. Med. 115: 541 -548. Lowry, D. H., Rosenbrough, N. L., Farr, A. L., and Randall, R. J. 195 1 . Protein measurement with Folin -phenol reagent. J. Biol. Chem. 193: 265-275. Lyutakora, S. G., Russanov, E. M., and Liochev, S. I. 1984. Cu-Zn superoxide dismutase and the cofactors of the enzyme. Arch. Biochem. Biophys. 235: 636 -643. Vani, M., Reddy, G. P . , Reddy, G. R., et a&.1990. Glutathione-stransferase, superoxide dismutase, xanthine oxidase, catalase, glutathione peroxidase and lipid peroxidation in liver of exercised rats. Biochem. Int. 21: 17-26. Weisiger, R. A., and Fridovich, I. 1973. Superoxide dismutases. Organelle specificity. J. Biol. Chem. 248: 3582 -3592. Westman, N. G., and MarHund, S. E. 1981 . Copper and zinc containing superoxide dismubse in human tissue and malignant tumors. Cancer Res. 41: 2952 -2956. Willians, S. J., Holden, K. M.,Kabransky, M., and Menzel, D. B. 1980. The distributional kinetics of Ni2+ in the rat lung. Toxicol. Appl. Pharmacol. 55: 85-93. Yamamoto, H.,Mashino, T o ,Nagano, T., and Hirobe, M. 1989. The reactivity of superoxide: a potent oxidant generated in situ from superoxide and CO,. Tetrahedron Lett. 30: 4 133-4136.

Intratracheal injection of nickel chloride and copper-zinc superoxide dismutase activity in lung of rats.

Superoxide radical (O2-) is a free radical that may be involved in various toxic processes. Cu--Zn superoxide dismutase catalyses the dismutation of t...
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