J. Dent. 1991; 19: 319-321
Copper-zinc superoxide dismutase human and animal dental pulp G. B. Grossi, S. Borrello*, Institutes
M. Giuliani, T. Galeotti*
of Clinical Dentistry
and C. Miani
Pathology, Catholic University,
ABSTRACT The activity of the enzyme superoxide dismutase, scavenger of the superoxide anion (0; ), was assayed in the normal dental pulp of man, dog. rabbit and pig. Very low levels of Cu-Zn-containing superoxide dismutase found in the pulpal tissue investigated support the hypothesis of a relationship between the reactivity of the dental pulp to inflammatory processes and a low protection against oxy-radicals. KEY WORDS: J. Dent.
Pulp, Enzymes, Inflammation 19:
(Received 28 January
accepted 3 May
1991) Correspondence should be addressed to: Dr T. Galeotti, Institute of General Pathology, Catholic University, 00168 Roma. Italy.
INTRODUCTION Many studies have been reported concerning the role of free radicals in various pathological processes (for reviews see Pryor, 1976-1984). Among the reactive chemical species which have been shown to be cytotoxic, oxyradicals, produced by subcellular organelles and by enzymic cytosolic reactions, are considered to be strongly involved in different forms of tissue damage. These have been implicated in cardiovascular diseases, cancer, ageing and inflammation (Fridovich, 1976; Freeman and Crapo, 1982; Del Maestro, 1984; Halliwell and Gutteridge, 1984; Halliwell and Grootveld, 1987). The cellular targets are proteins, DNA and lipids. but the toxic effects are normally controlled by enzymic and non-enzymic defenses found in aerobic cells (Fridovich. 1976; Freeman and Crapo, 1982: Sies, 1986). The enzymic defenses of aerobic cells include superoxide dismutase (SOD), glutathione peroxidase, catalase, DT-diaphorase, glutathione reductase, methionine sulphoxide reductase, NADH semidehydroascorbate reductase, DNA repair enzymes: non-enzymic defenses are vitamin E, glutathione, p-carotene, ascorbate, ubiquinone, uric acid, coeruloplasmin and transferrin. The damage to cellular and extracellular structures is determined only by an increased production or a decreased removal of oxy-radicals. During inflammation, for instance, activated phagocytes produce high levels of oxy-radicals, mainly (Oj ) (Babior et al., 1973), which, while displaying their microbicidal activity. can be
@ 1991 Butterworth-Heinemann 0300-5712/91/050319-03
deleterious to the phagocytes and the surrounding tissue (Weiss and LoBuglio, 1982; Del Maestro, 1984). In this respect, the enzyme SOD-which exists in animal cells in three forms designated Cu-Zn SOD (cytosolic) (McCord and Fridovich, 1969), Mn SOD (mitochondrial) (Weisiger and Fridovich, 1973) and EC-SOD (extracellular) (Marklund et al.. 1982), principally to eliminate superoxide radicals (0: ) by catalysing the reaction: of great biological 20: + 2H+ + H,O, + O,-is importance as an anti-inflammatory component of the cellular defense system (Flohk, 1986). In addition, 0; radicals scavenging may minimize the iron-catalysed Haber-Weiss reaction (Haber and Weiss, 1934) which generates the very toxic hydroxyl radicals (OH’) according to the following scheme: 0; + Fe3+ + Fez+ + 0, Fe% + H z0 ,+Fe3++OH-+OH* This is considered to be the first report on the determination of Cu-Zn SOD content in normal dental Pulp.
Normal dental pulps were obtained from man, dog, rabbit and pig. The samples, weighing 0.3-3.7 g, were washed twice in 0.15M NaCl, finely minced with scissors and homogenized with an Ultra-Turrax (Janke and Kunkel KG Staufen. Germany) in 10 vol. of 0.05 M K,HPO,,
J. Dent. 1991; 19: No. 5
Table 1. Superoxide dismutase content of dental pulp from man and different animal species
Sources Man Dog Rabbit Pig
: ._ .? 9 f z
Cu-Zn SOD (1-199 wet m-‘l 31.5* 18.1* 18.9 (2)* 11.3 + 1.4 (3)t
*The amount of enzyme was calculated from a standard curve of purified bovine blood SOD (generousgift from ProfessorG. Rotilio, Rome, Italy). Fifty per cent inhibition was attained by 65 ng ml-’ of
SOD. tPurified bovine blood SOD (SIGMA Chemie) was used for the standard curve. Fifty per cent inhibition was attained by 46 ng ml-’ of SOD.
Tissue extract (~1)
Fig. 7. Inhibition curves of the autoxidation of adrenaline to adrenochrome, obtained with different aliquots of tissue extracts from normal dental pulp of man (a), dog (b), rabbit (c) and pig (d). Adrenaline autoxidation was followed at 480 nm in a reaction mixture containing 50 m/W sodium carbonate, 0.1 m/W EDTA and 0.4 m/W adrenaline (pH 10.2). The temperature was kept at 30°C. 0.1 mM ethylenediaminetetracetate (EDTA), pH 7.8, for eight periods of 15 s each, at intervals of 30 s. The Cu-Zn SOD extraction procedure was essentially that reported by McCord and Fridovich (1969), with the omission of the DE 32 column (McCord and Fridovich, 1968), as suggested by Sykes et al. (1978). SOD content in the dental pulp was determined by testing the inhibition of increasing volumes of tissue extract on the autoxidation of adrenaline to adrenochrome, according to Misra and Fridovich (1972). The 50 per cent inhibition values obtained from such curves were compared with those of two standard curves of bovine blood SOD where 65 ng/ml and 46 ng/ml respectively gave 50 per cent inhibition.
RESULTS AND DISCUSSION The inhibition curves of adrenaline autoxidation obtained with tissue extracts prepared from normal dental pulp of man, dog, rabbit and pig are shown in Fig. 1. In Table I the Cu-Zn SOD content of the samples investigated is reported. The Cu-Zn-containing enzyme has either been detected quantitatively or its activity measured in a large range of human and animal tissues (Halliwell and Gutteridge, 1986). The tissues possessing the highest amount of this enzyme have been shown to be liver, brain and kidney, both in humans and animals, presumably in relation to the rate of 0’ production under physiological conditions. Other tissues which are less endowed with this protein, although equally well protected under normal conditions,
may become susceptible to oxidative damage under particular pathological conditions when overproduction of 0: radicals occurs. Dental pulp may belong to this category. Indeed, the data obtained in this study are indicative of a markedly low enzymic 0: dismutation capacity, presumably also related to the limited cell content of this tissue. Owing to its catalytic activity as a superoxide radical scavenger, SOD can be considered an anti-inflammatory enzyme. Indeed it is involved in the elimination of superoxide anions generated by phagocytic cells which play a very important role in the process. We selected the normal dental pulp as a model of a tissue in which inflammation initiates very easily and lasts for long periods of time. The observation of a low content of SOD in this tissue, which is very prone to inflammation, strengthens the hypothesis of a relationship between low SOD activity and high susceptibility to phlogistic processes.
Acknowledgements This investigation was in part supported by a grant from Minister0 dell’ Universita e della Ricerco Scientifica e Tecnologica.
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