Superoxide Dismutase in Human Adrenal and its Disorders: A Correlation with Development and Neoplastic Changes
Hironobu Sasano, MD, PHD1 Aki Mizorogi, Bs1 Michiko Sato,Bs1 Hisayoshi Nakazumi, MD, PHD2 and Takashi Suzuki, MD, PHD1
Abstract
In adrenal glands, oxidative free radicals are synthesized in the course of hormonal pro- duction, and enzyme superoxide dismutase (SOD) is considered to scavenge these harm- ful superoxide radicals and, subsequently, to protect the cells. We studied immunohistochemical localization of Mn (manganese)-SOD and Cu,Zn (copper-zinc)-SOD in human adrenal and its disorders from fetus to adult obtained from autopsy or surgery in order to examine the possible biological significance of these two enzymes. In fetal adrenal (n = 4), Cu,Zn- SOD and Mn-SOD were detected only in the fetal cortex. In adrenal glands from children (n = 21) to adults (n = 15), Mn-SOD immunoreactivity was exclusively detected in adrenal medulla, whereas Cu,Zn-SOD immunoreactivity was present only in adrenocortical pa- renchymal cells, weakly in the zona glomerulosa, and markedly in the zona reticularis. There were no differences in relative immunointensity and/or patterns of immuno- localization of these two SODs among different age groups. Both Cu,Zn-SOD and Mn- SOD immunoreactivity were detected in compact tumor cells of adrenocortical adenoma (n = 16). Marked immunoreactivity of both Cu,Zn-SOD and Mn-SOD was detected in adrenocortical carcinoma (n = 11) and pheochromocytoma (n = 5). These results indicate that Cu,Zn-SOD and Mu-SOD may play different roles as a scavenger or antioxidants in normal human adrenal glands, i.e., Cu,Zn-SOD as a scavenger of toxic superoxide radicals generated during steroidogenesis and Mn-SOD during catecholamine production. Cu,Zn- SOD and Mn-SOD immunoreactivities detected in adrenal neoplasms are also considered to represent altered expression of these enzymes associated with neoplastic transforma- tion, as reported in other human malignancies.
Key Words: Adrenal; superoxide dismutase; neoplasms; human; immunohistochemistry.
Address correspondence to Hironobu Sasano, M.D. Department of Pathology Tohoku University School of Medicine 2-1 Seiryou-machi, Aoba-ku Sendai-shi, Miyagi-ken, Japan 980-8575 E-mail: hsasano@ patholo2.med.tohoku.ac.jp
Endocrine Pathology, vol. 10, no. 4, 325-333, Winter 1999 @ Copyright 1999 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/99/10:325-333/ $12.50
Introduction
Reactive oxygen species have been well- known to cause cell damage and apoptosis, a form of physiological cell death [1]. The generation of oxidative free radicals occurs not only in pathological conditions, includ- ing inflammation [2] and cancer [3], but also in all cells as a consequence of normal cellular metabolism and lipid membrane
turnover [4,5]. Enzyme superoxide dismutase (SOD) scavenges the harmful superoxide radicals and protect cells from the damag- ing effects of these reactive oxygen species. In mammals, two types of cellular SOD have been identified: cytoplasmic copper- zinc SOD (Cu,Zn-SOD) [6] and mito- chondrial manganese SOD (Mn-SOD) [7]. Both SODs belong to the first enzy-
matic step that protects cells against toxic radicals, and therefore these enzymes are very important in maintaining cell survival.
In adrenal, Yamakura et al. first reported the presence of both cyanide-sensitive and -insensitive SOD activity in bovine adreno- cortical cells [8]. Azhar et al. subsequently reported that SOD activity in adrenal decreased with age in rats [9]. These SODs are considered to play important roles in preventing cellular damage in adrenal glands, especially in adrenal cortex, because the cortical cells use molecular oxygen for steroid biosynthesis, and all interactions of the cellular cytochrome P450 enzymes with their lipid substrates and products are major sources of this free radical forma- tion [10,11], which results in increased risk of damage from lipid peroxidation in adrenal glands. In the rodent adrenal cor- tex, these lipid products, combined with the naturally high tissue content of unsat- urated lipids, increase the potential for cell damage and death from autooxidation events [12,13]. Despite these important roles of SODs in adrenal glands, the analy- sis of the enzyme has not been reported in human adrenal glands. Therefore, in the present study, we examined the immuno- histochemical distribution of Mn-SOD and Cu,Zn-SOD in various ages of human adrenals from fetus to adult, and adrenal disorders including adrenocortical adenoma, adrenocortical carcinoma, and pheochro- mocytoma in order to study the possible biological significance of these SODs in human adrenal glands, especially with relation to development and neoplastic change.
Materials and Methods
Human Adrenals
Nonpathologic adrenal glands from fetuses of 21, 23, 36, and 38 gestational
weeks (GW), children of 1- to 13-yr-old (n = 21), and adults of ages 27- to 87-yr- old (n = 15) were retrieved from autopsy or surgical pathology files of Tohoku Uni- versity Hospital, Sendai, Japan. Specimens of adrenocortical adenomas (n = 16; aldosteronoma; 6, Cushing’s adenoma; 5, nonfunctioning adenoma; 5), adrenocor- tical carcinoma (n = 11), and pheochro- mocytoma (n = 5) were also retrieved from surgical pathology files of Tohoku Univer- sity Hospital. They all had been fixed in 10% formalin for 24-48 h at room tem- perature and embedded in paraffin. Adrenocortical carcinomas and adenomas were histologically diagnosed using the criteria of Weiss [14].
Antibodies
The generation and characterization of primary monoclonal antibodies for Cu,Zn- SOD and Mn-SOD have been described previously [15,16], and application of these antibodies to immunohistochemistry was also reported previously [17,18]. The antibodies were generously provided from Fukuyama Medical Laboratory, Hiroshima, Japan.
Immunohistochemistry
Immunohistochemical analysis was per- formed employing the streptavidin-biotin amplification method using a Histofine Kit (Nichirei, Tokyo, Japan), and was des- cribed in detail previously [19,20]. After deparaffinization, slides were heated in an autoclave at 120°℃ for 5 min in citric acid buffer (2 mM citric acid and 9 mM triso- dium citrate dehydrate, pH 6.0) for Mn-SOD immunostaining. The dilutions of primary antibodies used in our study were as fol- lows: Mn-SOD, 1/100 and Cu,Zn-SOD, 1/50. The antigen-antibody complex was visualized with 3,3’-diaminobenizidine
| Cu,Zn-SOD | Mn-SOD | |||||||
|---|---|---|---|---|---|---|---|---|
| PC | FC | M | PC | FC | M | |||
| (A) Fetus | ||||||||
| 21 GW | - | + | - | - | ± | - | ||
| 23 GW | - | + | - | - | 土 | - | ||
| 36 GW | - | + | - | - | ± | ± | ||
| 38 GW | - | + | - | - | + | ± | ||
| (B) Children | ||||||||
| ZG | ZF | ZR | M | ZG | ZF | ZR | M | |
| 1-4-yr-old | 土 | + | + | - | - | - | - | + |
| (n = 5) | ||||||||
| 5-7-yr-old | 士 | + | + | - | - | - | - | + |
| (n = 8) | ||||||||
| 8-13-yr-old | + | + | ++ | - | - | - | - | + |
| (n = 8) | ||||||||
| (C) Adult | ||||||||
| 27-40-yr-old (n = 3) | ± | + | ++ | - | - | - | - | + |
| 40-60-yr-old | ± | + | ++ | - | - | - | - | + |
| (n = 6) | ||||||||
| 60-87-yr-old | + | + | ++ | - | - | - | - | + |
| (n = 6) | ||||||||
Cu,Zn-SOD: cytoplasmic copper-zinc superoxide dismutase; Mn-SOD: mitochondrial manganese superoxide dismutase ; PC: permanent cortex; FC: fetal cortex; M: medulla; ZG: zona glomerulosa; ZF: zona fasciculata; ZR: zona reticularis; GW: gestational weeks; -: negative; ±: (B: weakly positive; +: positive; ++: markedly positive).
(DAB) solution (1 mM DAB, 50 mM Tris- HCI buffer, pH 7.6, and 0.006% H2O2), and counterstained with methyl green. For negative controls, normal mouse IgG was used instead of the primary antibodies, and no specific immunoreactivity was detected in these tissue sections.
Results
Results are summarized in Tables 1 and 2.
Nonpathological Adrenal
Fetal Adrenal
Cu,Zn-SOD immunoreactivity was detected only in fetal cortex but not in per-
manent cortex and adrenal medulla. Weak Mn-SOD immunoreactivity was detected in fetal cortex but not in permanent cor- tex. Mn-SOD immunoreactivity was not detected in the medulla of 21 and 23 GW adrenal but weak immunoreactivity was present in the medulla of 36 and 38 GW adrenal.
Pediatric Adrenal
Cu,Zn-SOD immunoreactivity was not detected in adrenal medulla, and weakly in the zona glomerulosa from a 1 yr old. Both of the zona fasciculata and reticularis demonstrated Cu,Zn-SOD immunoreac- tivity in all pediatric age groups examined. Cu,Zn-SOD immunoreactivity in the zona
| Table 2. Summary of Cu,Zn-SOD and Mn-SOD in Human Adrenal Disorders | ||||
|---|---|---|---|---|
| Cu,Zn-SOD | Mn-SOD | |||
| Adrenocortical adenoma | Compact cells | Clear cells | Compact cells | Clear cells |
| (n = 16) | ||||
| (30-74, 48.5± 9.5) | ||||
| Aldosteronoma | + | - | + | - |
| (n = 6) | ||||
| Cushing's adenoma (n = 5) | + | - | + | - |
| Nonfunctioning adenoma (n = 5) | + | - | + | - |
| Adrenocortical | ++ | ++ | ||
| carcinoma | ||||
| (n= 11) | ||||
| (4-69, 41.5±B5.5) | ||||
| Pheochromocytoma | ++ | ++ | ||
| (n = 5) | ||||
| (25-51, 39.5±B 8.2) | ||||
Cu,Zn-SOD : cytoplasmic copper-zinc superoxide dismutase; Mn-SOD: mitochondrial manganese superoxide dismutase; -; negative; +: weakly positive; +: positive; ++: markedly positive.
reticularis became more pronounced from an 8 yr old. Mn-SOD immunoreactivity was detected only in adrenal medulla, but not in adrenal cortex.
Adult Adrenal
Cu,Zn-SOD immunoreactivity was detected only in adrenocortical parenchy- mal cells, weakly in the zona glomerulosa, and markedly in the zona reticularis but not in adrenal medulla (Fig. 1A). Mn-SOD immunoreactivity was exclusively detected in adrenal medulla but not in adrenocorti- cal parenchymal cells (Fig. 1B). There were no differences in relative immunointensity and/or patterns of immunolocalization of Cu,Zn-SOD and Mn-SOD among the patients examined.
Adrenal Disorders
Adrenocortical Adenoma
Cu,Zn-SOD and Mn-SOD immunore- activity was predominantly detected in com- pact tumor cells. There were no differences of relative immunointensity or patterns of
immunolocalization of Cu,Zn-SOD and Mn-SOD among aldosteronoma, Cushing’s adenoma, and nonfunctioning adrenocor- tical adenoma, and among different age groups of the patients.
Adrenocortical Carcinoma
Marked immunoreactivity of both Cu,Zn-SOD (Fig. 2A) and Mn-SOD (Fig. 2B) was detected in adrenocortical carcinoma cells. There was a marked het- erogeneity of Cu,Zn-SOD immunointen- sity in adrenocortical carcinoma cases. There were no differences of relative immu- nointensity or patterns of localization of Cu, Zn-SOD and Mn-SOD among dif- ferent age groups of the patients.
Pheochromocytoma
Marked immunoreactivity of both Cu,Zn-SOD (Fig. 3) and Mn-SOD was detected in tumor cells. There were no diffrences of relative immunointensity or patterns of localization of Cu, Zn-SOD and Mn-SOD among different age groups of the patients.
A
A
R
C
S
B
M
S
C
B
R
C
M
Discussion
Mammalian cells are equipped with antioxidant systems to deal with free radi- cal damage [21-23]. SOD is one of the
most important antioxidant components in these cells. In our study, there was a difference of intraadrenal distribution between the two major SODs in mamma-
lian cells, i.e., Cu,Zn-SOD and Mn-SOD. In nonpathologic adrenal, Mn-SOD was exclusively present in adrenal medulla except for weak immunoreactivity in fetal cortex. On the other hand, Cu,Zn-SOD was exclusively immunolocalized in adren- ocortical parenchymal cells, not in adrenal medulla. In other steroidogenic tissues, Sugino et al. recently reported the presence of both Cu,Zn-SOD and Mn-SOD mRNA in the corpus luteum of rat ovary [24,25]. We also recently demonstrated that both Cu,Zn-SOD and Mn-SOD immunore- activity was present in steroidogenic cells of the normal cycling human ovary using the same monoclonal antibodies employed in this study (Suzuki et al., unpublished observations). Therefore, Cu,Zn-SOD and Mn-SOD are considered to play different roles as an antioxidant in steroidogenesis between human ovary and adrenal. Super- oxide radicals have been reported to inhibit progesterone production among the path- way of steroidogenesis [26,27]. In the pro- cess of steroidogenesis, progesterone is formed by dehydrogenation of preg- nenolone by 3B-hydroxysteroid dehy- drogenase, which requires NAD+ and the hydrogen acceptor. NAD+ is supplied by
the oxidation of NADH by ascorbic acid radicals through a free radical system [28]. 3ß-Hydro-xysteroid dehydrogenase is localized in endoplasmic reticulum [29,30]. Therefore, the fact that Cu,Zn- SOD is located in the cytosol, while Mn- SOD is in the mitochondria, suggests that Cu,Zn-SOD plays an important role as a scavenger of toxic superoxide radicals gen- erated during active steroidogenesis, espe- cially in the dehydration process of human adrenocortical steroidogenesis. On the other hand, Mn-SOD is considered to be involved in prevention of cellular damage by superoxide free radical possibly pro- duced during catecholamine production and secretion. However, further investiga- tions for clarification are needed.
The amount of Cu,Zn-SOD expression in adrenal cortex is considered to be cor- related with an overall corticosteroid pro- duction and secretion of the cells, consid- ering the possible roles of the enzyme above. Relatively weak immunoreactivity of Cu,Zn-SOD in the zona glomerulosa of nonpathological adrenals and Cu,Zn- SOD immunoreactivity in compact tumor cells but not in clear tumor cells are con- sistent with the overall capacity of these cells to produce corticosteroids [29,31]. However, in normal human adrenal, maxi- mum corticosteroid production and out- put generally occurs in the zona fasciculata [29,31] but Cu,Zn-SOD immunoreactiv- ity was more pronounced in the zona reticularis. Cu,Zn-SOD in the zona reticularis is therefore considered not only to be involved in corticosteroidogenesis but also in protection against toxic radicals gen- erated in the process of degradation and/ or metabolism of lipids and others by vari- ous enzymes present in lysozymes.
Azhar et al. reported that aging in rats results in oxidative changes in adrenal tis- sue that appear linked to a reduction in efficiency of the normally protective anti-
oxidant defense system [9]. They also reported that these changes in the life of the rat may correlate with the age-related loss of corticosterone production in adre- nal gland [9]. It is well known that aging results in decreased corticosteroid produc- tion in human and oxygen-derived free radicals and the accumulation of unrepaired oxidant-damaged cellular products have been implicated in the aging process [32-34]. However, in our study, there were no differ- ences of relative immunointensity and/or pat- terns of intraadrenal immunolocalization of Cu,Zn-SOD among the patients of differ- ent age groups examined, with a possible exception of more pronounced Cu,Zn-SOD immunoreactivity in the zona reticularis after 8 yr old. The antioxidants consist of nonenzymatic substances, such as vitamins A, C, E, and other small-molecular-weight compounds such as glutathione [21-23] and three enzymes including SOD, cata- lase and glutathione peroxidase [35]. Therefore, the analysis of these compounds other than SOD that are also involved in protection against free radicals are neces- sary to clarify the possible correlation between the age-related reduction in oxi- dative mechanism, which may result in cel- lular damage and decreased corticosteroid production in the aging process of human adrenal.
Results of our study demonstrated that distribution of SODs in adrenal neoplasms was different from that of human adrenal. Mn-SOD immunoreactivity was not detected in nonneoplastic adrenocortical cells but present in compact cells of adreno- cortical adenoma and carcinoma cells. On the other hand, Cu,Zn-SOD immunore- activity was not present in adrenal medulla of nonpathologic adrenal but detected in pheochromocytoma. Altered SOD activi- ties have been reported in various human neoplastic tissues [36]. However, biologi- cal and/or clinical significance of these
altered expressions of SODs in human neo- plastic tissues still remains unknown. Especially, the enzyme SOD may represent a manifestation of overreactivity of the adrenal gland, and further investigations including the analysis of adrenocortical hyperplasia are required for clarification of these changes of SODs in human adrenal neoplasms.
Acknowledgments
This work is in part supported by The Grant-In-Aid for Cancer Research 7-1 from The Ministry of Health and Welfare, Japan, a grant-in-aid for scientific research area on priority area (A-11137301) from The Ministry of Education, Science and Culture, Japan, a grant-In-Aid for Scien- tific Reseach (B-11470047) from Japan Society for the Promotion of Science and a grant from The Naitou Foundation and Suzukenn Memorial Foundation.
Reference
1. Lynch RE, Fridovichi I. Effects of superoxide on the erythrocyte membrane. J Biol Chem 253:1838-1845, 1978.
2. McCord JM. Free radicals and inflammation: Protection of synovical fluid by superoxide dismutase. Science 185:529-531, 1974.
3. Ishikawa M, Yaginuma Y, Hayashi H, et al. Reactivity of a monoclonal to manganese superoxide dismutase with human ovarian car- cinoma. Cancer Res 50:2538-2542, 1990.
4. McCord JM, Day ED. Superoxide-dependant production of hydroxyl radical catalyzed by an iron-EDTA complex. FEBS Lett 86:139- 142, 1978.
5. Fridovich I. Biological effects of the superox- ide radical. Arch Biochem Biophys 247:1-11, 1986.
6. McCord JM, Fridovichi I. Superoxide Dis- mutase, an enzymic function for erythro- cuprein. J Biol Chem 244:6049-6055, 1969.
7. Barra D, Schnina M, Simmaco M, et al. The primary structure of human liver manganese
superoxide dismutase. J Biol Chem 259:12595- 12601, 1984.
8. Yamakura F, Ono Y, Ohmori D, Suzuki K. Localization, isolation and characterization of Mn-superoxide dismutase in bovine adreno- cortical cells. Comp Biochem Physiol - B: Comp Biochem 79:33-39, 1984.
9. Azhar S, Cao L, Reaven E. Alteration of the adrenal antioxidant defense system during aging in rats. J Clin Invest 96:141-1424, 1995.
10. Hornsby PJ, Crivello JF. The role of lipid peroxidation and biological antioxidant in the function of the adrenal cortex. Part 2. Mol Cell Endocrinol 30:123-147, 1983.
11. Hornsby PJ. Steroid and xenobiotic effects on the adrenal cortex: mediation by oxidative and other mechanisms. Free Radical Biol Med 6:103-115, 1989.
12. Hornsby PJ, Crivello JF. The role of lipid peroxidation and biological antioxidant in the function of the adrenal cortex. Part 1: back- ground review. Mol Cell Endocrinol 31:1-20, 1983.
13. Cheng B, Kowal J. Analysis of adrenal cholesteryl esters by reversed phase high per- formance liquid chromatography. J Lipid Res. 35:1115-1121, 1994.
14. Weiss LM. Comparative histologic study of 43 metastasizing and non metastasizing adrenocortical tumors. Am J Surg Pathol 8:163-169, 1984.
15. Oka S, Ogino K, Matsuura S Human serum immuno-reactive copper, zinc-superoxide dismutase assayed with an enzyme monoclonal immunosorbent patients with digestive can- cer. Clinica Chimica Acta. 182:209-219, 1989.
16. Kawaguchi T, Suzuki K, Matsuda Y. Serum- manganese-superoxide dismutase: normal val- ues and increased levels in patients with acute myocardial infarction and several malignant dis- eases determined by an enzyme-linked immu- nosorbent assay using a monoclonal antibody. J Immunol Methods 127:249-254, 1990.
17. Oka S, Ogino K, Houbara T. An immunohis- tochemical study of copper, zinc-containing superoxide dismutase detected by a monoclonal antibody gastric mucosa and gastric cancer. His- topathology 17:231-236, 1990.
18. Sugino N, Shimamura K, Takiguchi S, Tamura H, Ono M, Nakata M, Nakamura Y, Ogino K, Uda T, Kato K. Changes in activity of super-
oxide dismutase in the human endometrium throughout the menstrual cycle and in early pregnancy. Hum Reprod 11: 1073-1078, 1996.
19. Sasano H, Frost AR, Saitoh R, Harada N, Poutanen M, Vihko R, Bulun SE, Silverberg SG, Nagura H. Aromatase and 17 beta-hydro- xysteroid dehydrogenase type! in human breast carcinoma. J Clin Endocrinol Metab 81:4042-4046, 1996.
20. Sasano H, Kimura M, Shizawa S, Kimura N, Nagura H. Aromatase and steroid receptors in gynecomastia and male breast carcinoma: an immunohistochemical study. J Clin Endocrinol Metab 81:3063-3067, 1996.
21. Harris ED. Regulation of antioxidant enzymes. FASEB (Fed Am Soc Exp Biol) J 6:2675- 2683, 1992.
22. Beyer RE. The role of ascorbate in antioxi- dant protection of biomembranes: interaction with Vitamin E and coenzyme Q. Bioenerg Biomembr 26:349-358, 1994.
23. DiMascio P, Murphy ME, Sies H. Antioxi- dant defense systems: the role of carotenoids, tocopherols and thiols. Am J Clin Nutr 53:194S-200S, 1991.
24. Sugino N, Carlos, MT, Gibori T. Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase in rat corpus luteum: induction of manganese superoxide dismutase messenger ribonucleic acid by inflammatory cytokines. Biol Reprod 59:208-215, 1998.
25. Sugino N, Hirosawa-Takamori M, Zhong L. Hormonal regulation of copper zinc superox- ide dismutase and manganese superoxide dis- mutase messenger ribonucleic acid in the rat cor- pus luteum: induction by prolactin and placental lactogens. Biol Reprod 59:599-605, 1998.
26. Sugino N, Nakamura Y , Okuno N. Effects of ovarian ischemia-reperfusion on luteal function in pregnant rats. Biol Reprod 49:354-358, 1993.
27. Behrman HR, Preston SL. Luteolytic actions of peroxide in rat ovarian cells. Endocrinol- ogy 124:2895-2900, 1989.
28. Agrawal P, Laloraya MM. Induction of peroxi- dase in corpora lutea of rat ovary by luteinizing hormone. Biochem J 166:205-208, 1977.
29. Sasano H. Localization of steroidogenic enzymes in adrenal cortex and its disorders. Endocr J 41:471-482, 1994.
30. Sasano H, Mori T, Sasano N, et al. Immunolo- calization of 3b-hydroxysteroid dehydroge-
nase in human ovary. J Reprod Fertil 89:743- 751, 1990.
31. Sasano H, Mason JI, Sasano N. Immunohis- tochemical study of cytochrome P-45017 alpha in human adrenocortical disorders. Hum Pathol 20:113-117, 1989.
32. Pacifici RE, Davies KJA. Protein, lipid and DNA repair systems in oxidative stress. The free-radical theory of aging revisited. Geron- tology 37:166-180, 1991.
33. Nohl H. Involvement of free radicals in age- ing: a consequence or cause of senescence. Br Med Bull 49:653-667, 1993.
34. Shigenaga MK, Hagen TM, Ames BN. Oxi- dative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 91:10771- 10778, 1994.
35. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev 74:139- 162, 1994.
36. Iizuka S, Taniguchi N, Makita A. Enzyme- linked immunosorbent assay for human man- ganese-containing superoxide dismutase and its content in lung cancer. J Natl Cancer Inst 72:1043-1049, 1984.