- Email: [email protected]
Antioxidant status of the rat nasal cavity
Free Radical Biology & Medicine, Vol. 34, No. 5, pp. 607– 615, 2003 Copyright © 2003 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter
doi:10.1016/S0891-5849(02)01367-9
Original Contribution ANTIOXIDANT STATUS OF THE RAT NASAL CAVITY CELIA J. REED,* DARREN A. ROBINSON,*
and
EDWARD A. LOCK†
*School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, England; and †Syngenta Central Toxicology Laboratory, Cheshire, England (Received 25 July 2002; Revised 11 November 2002; Accepted 21 November 2002)
Abstract—Despite extensive interest in the rodent nasal cavity as a target organ for toxicity, there is very limited information regarding nasal defenses against oxidative stress and xenobiotic-derived oxidants. Using immunohistochemistry, we have examined the distribution of Cu,Zn and Mn superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase, and DT-diaphorase in rat nasal tissues. In addition, we have determined the concentrations of ascorbate and ␣-tocopherol and the activities of SOD (combined Cu,Zn and Mn forms), catalase, GSH peroxidase, GSH reductase, and DT-diaphorase in nasal respiratory epithelium (RE), olfactory epithelium (OE), and in lung. Immunohistochemistry demonstrated that all four enzymes were similarly distributed, with the greatest staining intensity in dorsal-medial regions of the nasal cavity. In respiratory epithelium, ciliated columnar cells and subepithelial glands stained positively, while in olfactory tissue the enzymes were detected in the sustentacular cells and Bowman’s glands. With the exception of SOD, enzyme activities were higher in RE than OE, while concentrations of ascorbate and ␣-tocopherol were higher in OE than RE. With the exception of catalase, nasal activities were either higher than or comparable to those of the lung. Thus, the rat nasal cavity appears to be well protected against oxidative damage. © 2003 Elsevier Science Inc. Keywords—Olfactory, Nasal, Superoxide dismutase, Glutathione reductase, Glutathione peroxidase, Catalase, DTdiaphorase, Ascorbate, ␣-Tocopherol, Free radicals
INTRODUCTION
ents of cigarette smoke) are themselves potent oxidants. Secondly, chemicals such as carbon tetrachloride undergo cytochrome P450-dependent metabolism within olfactory epithelium (OE), resulting in the generation of free radicals. Finally, compounds may compromise the endogenous antioxidants of the nasal cavity and render it susceptible to oxidative damage. For example, methyl iodide is conjugated with glutathione and results in a rapid and extensive depletion of this thiol within the nasal cavity [12], and diethyldithiocarbamate (DDTC) chelates metal ions and, thereby, inhibits copper/zinc superoxide dismutase (Cu,ZnSOD, [13]) and glutathione (GSH) peroxidase [14]. To defend themselves against oxidative stress, all cells contain an endogenous antioxidant system that maintains the cells’ normal redox potential. This system comprises a variety of antioxidant enzymes including SOD, catalase, GSH peroxidase, GSH reductase, and NAD(P)H:quinone oxidoreductase (DT-diaphorase), as well as low molecular weight components such as glutathione, ascorbate, and ␣-tocopherol (vitamin E). There
The tissues of the respiratory tract are some of the main targets for the effects of inhaled environmental pollutants and industrial chemicals. Furthermore, the abundant blood supply to the respiratory system ensures delivery of potential toxins to respiratory tract cells following systemic exposure. Thus, in recent years it has become apparent that the rodent nasal cavity is an important target organ for toxicity, and a wide variety of compounds including environmental contaminants [1–3], drugs [4,5], herbicides [6,7], and industrial chemicals [8 –11] have been shown to cause nasal lesions or tumors in experimental animals. Oxidative damage may be involved in the development of nasal toxicity in a number of ways. Firstly, many environmental pollutants (e.g., ozone and the constituAddress correspondence to: Dr. Celia J. Reed, Liverpool John Moores University, School of Biomolecular Sciences, Byrom Street, Liverpool L3 3AF, UK; Tel: ⫹44 151-231-2187; Fax: ⫹44 151-2312982; E-Mail: [email protected]. 607
608
C. J. REED et al.
has been only limited examination of the intracellular antioxidant systems of the nasal cavity. Both manganese SOD (MnSOD) and Cu,ZnSOD have been localized within human [15], rat [16], and mouse [17,18] nasal mucosae, and a novel peroxiredoxin with antioxidant properties has recently been isolated from rat OE [19 – 21]. GSH peroxidase and GSH reductase activities have been determined in rat nasal epithelia [22,23], and several groups have measured GSH (or nonprotein sulphydryls) and its turnover within the nasal cavity [12,22,24,25]. One of the striking characteristics of nasal toxins is their ability to target specific regions within the nasal cavity. That is, methyl iodide [10], DDTC [26], dichlobenil [6], and methyl methacrylate [27] all cause lesions in the OE of the posterior cavity, ozone affects only the transitional epithelium (TE) [2,23], and formaldehyde is toxic to the respiratory epithelium (RE) of the anterior nasal cavity [22,23]. This site selectivity is thought to be partly due to tissue-specific susceptibility, in particular the regional distribution of biotransformation enzymes. However, we have recently postulated that methyl iodide toxicity may result from GSH depletion, rendering tissues susceptible to methylation and/or oxidative stress [28]; and, Ravi et al. [26] suggested that the olfactory toxicity of DDTC may be due to the ability of this agent to chelate metal ions and, thus, compromise the antioxidant systems of the OE. The differential susceptibilities of the nasal epithelia may relate to the activities and concentrations of their antioxidant defense mechanisms. The aim of this study was to carry out a systematic investigation of nasal intracellular antioxidants. As such, several antioxidant enzymes have been localized within the nasal cavity and their activities measured in RE and OE, and the concentrations of ascorbate and ␣-tocopherol also have been determined in these tissues. MATERIALS AND METHODS
Materials Commercially available antibodies (sheep antihuman) were obtained from the following sources: SOD (Cu,Zn and Mn) and catalase, The Binding Site (Birmingham, England); and GSH peroxidase, Biogenesis (Poole, England). The antibody against DT-diaphorase was a kind gift from David Siegel and David Ross of the University of Colorado Health Sciences Center (Boulder, CO, USA). Animals Adult male, specific pathogen-free, Alpk:APfsD (Wistar-derived) albino rats, were supplied from the colony maintained at Alderley Park, Cheshire, England.
Rats were housed in cages suspended over collecting trays lined with absorbent paper at a density of up to five per cage. Animal rooms were maintained at a temperature of 21 ⫾ 2°C, a relative humidity of 50 ⫾ 15%, with a 12 h light/dark cycle (lights on at 6 a.m.). Water from an automatic system and pelleted Porton Combined Diet (Special Diet Services, Witham, Essex, England) were provided ad libitum. Rats were killed by anesthetic overdose. Biochemical analysis For determination of GSH reductase, GSH peroxidase, catalase, and SOD activities, heads were dissected and naso- and maxilloturbinates (RE) and ethmoturbinates (OE) from two to three animals were pooled and placed in 1 ml of ice-cold sucrose-EDTA-Tris buffer (SET, 0.25M sucrose, 5.4 mM EDTA, 20 mM Tris, pH 7.4). Lungs were perfused with 0.9% saline; the caudal lobe was removed, weighed, and a sample placed in ice-cold SET (1 g in 4 ml). Tissues were homogenized and then centrifuged at 800 ⫻ g at 4°C for 10 min. Triton X-100 was added to the supernatant to give a final concentration of 0.1% detergent (v/v), and the mixture was then sonicated on ice at 300 W for 3 ⫻ 10 s and centrifuged at 20,000 ⫻ g at 4°C for 10 min. The activities of GSH peroxidase [29], GSH reductase [30], and catalase [30] were determined using t-butyl hydroperoxide, oxidized GSH, and hydrogen peroxide, respectively, as substrates. Total SOD activity [30] was determined as the inhibition of the autoxidation of adrenaline to adrenochrome, 1 U of activity resulting in 50% inhibition. For determination of DT-diaphorase activity, tissues from individual animals were removed as described above, weighed, and homogenized in ice-cold 0.32 M sucrose (caudal lobe of lung, 4 vol; OE, 10 vol; RE, 1.0 ml) using an ultraturrax homogenizer. After centrifugation at 100,000 ⫻ g for 60 min at 4°C, the supernatants were carefully decanted and stored at ⫺70°C. DT-diaphorase activity was determined according to the method of Ernster [31], using 2,6-dichlorophenolindophenol as the electron acceptor and NADPH. DT-diaphorase activity was measured as the dicoumarol- (10 M) sensitive oxidation of NADPH. For determination of ascorbate, tissues were dissected as described above, weighed, and homogenized in icecold 0.15 M phosphoric acid (OE and caudal lobe of lung, 0.25 g in 1 ml; RE, 0.15 g in 1 ml); tissues were then placed on ice for 10 min and, subsequently, centrifuged at 15,000 ⫻ g at 4°C for 10 min. Aliquots of supernatant were diluted 1 in 10 in distilled water and filtered (0.22 m) before analysis by HPLC [32]. Pellets
Rat nasal antioxidants
609
were resuspended in 1 M sodium hydroxide (OE and lung, 1 ml; RE, 0.5 ml) for protein analysis. For determination of ␣-tocopherol, tissues were dissected as described above, with OE and RE pooled from two animals. Tissues were weighed and then homogenized in the dark in ice-cold phosphate-buffered saline (caudal lobe of lung, 25% (w/v); OE and RE, 1.2 ml); next, tissues were prepared and analyzed as described by Nierenberg and Lester [33] using a Luna 5u C18(2) column (Phenomenex, Cheshire, England; 50 ⫻ 4.60 mm). Protein concentrations were determined by the method of Lowry [34] using bovine serum albumin as standard. Immunohistochemistry For immunohistochemical studies, heads were fixed in neutral buffered formal saline and decalcified in neutral EDTA, as described previously [35]. Heads were trimmed to produce levels 1– 4 as described by Young [36], processed to paraffin blocks, and sections (5 m) cut. Localization of catalase, glutathione peroxidase, Cu,ZnSOD, MnSOD, and DT-diaphorase was carried out, as described previously [35], using a 1/50 dilution of the primary antibodies for all the enzymes except DTdiaphorase, which was diluted to 1/200. Statistics All results are means ⫾ SD of at least four determinations. Results were analyzed by ANOVA followed by the Bonferroni test, and a value of p ⬍ .05 was considered statistically significant.
Fig. 1. Antioxidant enzyme activities in rat RE, OE, and lung (■ RE; OE; lung). GR ⫽ GSH reductase, nmol/min/mg protein; GP ⫽ GSH peroxidase, nmol/min/mg protein; catalase ⫽ catalase, mK/mg protein; SOD ⫽ total superoxide dismutase, U/mg protein; DT-D ⫽ DT-diaphorase, 10⫺4 mol/min/mg protein. a ⫽ RE significantly different from OE, p ⬍ .05; b ⫽ RE significantly different from lung, p ⬍ .05; c ⫽ OE significantly different from lung, p ⬍ .05.
In transitional epithelium (TE), the staining appeared diffuse with some cells staining moderately positive and others being completely negative (Fig. 3a). In RE, the ciliated columnar cells stained positive while both the goblet cells and nonciliated columnar cells stained negative (Fig. 3b). The glands underlying the RE (seromucus tubular and acinar) all stained positive, but the tubu-
RESULTS
Biochemical analysis With the exception of SOD, antioxidant enzyme activity was significantly higher (1.5- to 3.3-fold) in RE than in OE (Fig. 1). Olfactory SOD activity (all forms) was approximately 3-fold greater than that of RE. Compared to the nasal cavity, activities in the lung were lower for GSH reductase, SOD, and DT-diaphorase, higher for catalase, and comparable for GSH peroxidase (Fig. 1). The concentrations of ascorbate and ␣-tocopherol were higher in OE compared to RE (4.2- and 1.4-fold, respectively). Alpha-tocopherol concentrations were higher in lung than in the nasal cavity, while pulmonary ascorbate was comparable to that of the OE (Fig. 2). Immunohistochemical analysis In general, all of the enzymes were localized to the same cell types within the different nasal epithelia. In squamous epithelium (SE), there was very weak staining in the apical layer (stratum corneum, results not shown).
Fig. 2. Concentrations of ascorbate and ␣-tocopherol in rat RE, OE, and lung (■ RE; OE; lung). Ascorbate is given in g/mg protein and ␣-tocopherol (vitamin E) is given in ng/mg protein.a ⫽ RE significantly different from OE, p ⬍ .05; b ⫽ RE significantly different from lung, p ⬍ .05; c ⫽ OE significantly different from lung, p ⬍ .05.
610
C. J. REED et al.
Fig. 3. Immunohistochemical localization of (a) DT-diaphorase in TE and (b) GSH peroxidase in RE. (a) TE lining the maxilloturbinate at level 1 of the rat nasal cavity; microscope magnification is ⫻400. (b) RE lining the lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. AG ⫽ acinar gland; CCC ⫽ ciliated columnar cell; NCC ⫽ nonciliated columnar cell; TG ⫽ tubular gland.
Fig. 4. Immunohistochemical localization of (a) catalase, (b) catalase, (c) Cu,ZnSOD, and (d) GSH peroxidase in rat OE. (a) OE on the tip of the third ethmoturbinate at level 3 of the rat nasal cavity; microscope magnification is ⫻400. (b) OE from the lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. (c) OE on the fourth ethmoturbinate at level 4 of the rat nasal cavity; microscope magnification is ⫻400. (d) OE of the dorsal lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. AC ⫽ apical cytoplasm; BG ⫽ Bowman’s gland; NB ⫽ neural bundle; SN ⫽ sustentacular cell nuclei.
Rat nasal antioxidants
611
Fig. 5. Diagrammatic representations of levels 1– 4 of the rat nasal cavity identifying some of the major structures. DM ⫽ dorsal meatus; ETs ⫽ ethmoturbinates; NT ⫽ nasoturbinate; MT ⫽ maxilloturbinate; S ⫽ septum.
lar glands stained more intensely for all five of the enzymes than did the acinal glands (Fig. 3b). In OE, the sustentacular cells and Bowman’s glands stained positive while the olfactory neurons and basal cells consistently failed to stain (Figs. 4a– 4d). With catalase (Fig. 4a), DT-diaphorase, and MnSOD, sustentacular cell staining was confined to the apical cytoplasm. In isolated patches of OE, GSH peroxidase and Cu,ZnSOD also stained the nuclei of the sustentacular cells (Figs. 4c and 4d). In all areas the neural bundles stained positive, even those on the septum at level 2 (Figs. 4a– 4d). We have mapped the distribution of these enzymes at the four levels of the nasal cavity described by Young [36] (Fig. 5), and we have demonstrated that they have similar localization patterns (Fig. 6). In general, staining intensity was greatest in the dorsal-medial regions of the nasal cavity. Thus, at levels 1 and 2, the dorsal meatus, parts of the septum, and the medial surfaces of the naso-
and maxilloturbinates showed the greatest staining intensity. At level 3, the dorsal meatus, the medial tips of the ethmoturbinates, and the septum were stained, while at level 4 the enzymes were generally confined to the medial aspects of the ethmoturbinates. With the exception of catalase at levels 3 and 4, no staining was detected in the mucosa lining the lateral walls. DISCUSSION
In this study, we have determined the activities of five important antioxidant enzymes and the concentrations of ascorbate and ␣-tocopherol within the nasal cavity and lung. The caudal lobe of the lung was explored primarily as a reference respiratory tract tissue, and antioxidants measured in the lung were of a similar magnitude to those reported previously [37,38]. With the exception of catalase, nasal enzyme activities were found to be greater
612
C. J. REED et al.
Fig. 6. Diagrammatic representations of levels 1– 4 of the rat nasal cavity showing the distribution of staining for antioxidant enzymes (■ intense staining; moderate staining). All other areas stained weakly for the enzymes.
Rat nasal antioxidants
than or similar to those of the lung (Fig. 1). Catalase is an exceptionally specific enzyme, catalyzing the breakdown of hydrogen peroxide alone. Hydrogen peroxide is produced during the respiratory burst of phagocytic cells, and the higher catalase activity in the lung compared to the nasal epithelia may reflect the presence of alveolar macrophages and a need to protect pulmonary tissue from hydrogen peroxide. Alpha-tocopherol concentrations in the nasal tissues were less than those of the lung (Fig. 2), while ascorbate concentrations were low in RE but comparable in OE and lung (Fig. 2). Alpha-tocopherol ideally should be expressed as nmol/mg lipid, as it is a very lipid-soluble vitamin. However, we have no lipid data for either the RE or OE and we have, therefore, expressed our results per mg protein. This may explain why higher concentrations of ␣-tocopherol were found in the lung, which would be expected to have a higher lipid to protein content. Thus, no consistent pattern of the nasal tissues having either higher or lower antioxidant capacity than the lung emerged. Similarly, comparison of RE and OE revealed no obvious trend: RE had higher GSH reductase, GSH peroxidase, catalase, and DT-diaphorase activities, but lower total SOD activity and lower ascorbate and ␣-tocopherol concentrations. These data must be interpreted with some caution, as nasal turbinates are cartilaginous structures covered in epithelia of varying thickness and cellular heterogeneity. During homogenization, the activity/concentration of cells rich in antioxidants will be diluted by the contribution of the cartilage and those cells that do not contain the antioxidants. This may occur to a greater extent in OE than in RE, as there are very marked differences in the distributions of the antioxidant enzymes within the former tissue (Fig. 6). Homogenization and pooling of all the OE results in a mean value that may not accurately reflect the antioxidant status of individual ethmoturbinates. However, it can be concluded that the antioxidant defenses of the nasal cavity appear to be comparable to those of the lung. Immunohistochemical localization of the antioxidant enzymes in the nasal cavity revealed that they are all expressed in the glands and ciliated columnar cells of RE and the Bowman’s glands and sustentacular cells of OE. In some areas of OE, GSH peroxidase and Cu,ZnSOD were also detected in the nuclei of the sustentacular cells. Ashihara et al. [17] reported a similar cellular and intracellular distribution for Cu,ZnSOD in murine nasal tissues, while Kulkarni-Narla et al. also detected Cu,Zn and MnSOD in olfactory neuronal cell bodies of both rats and humans [15,16]. The latter group concluded that the lack of neuronal cell body staining reported by Ashihara et al. [17] was due to a loss of immunoreactivity during fixation in paraformaldehyde. In the present study, the
613
tissues were fixed in formalin, which may have resulted in a similar artefact. However, localization of a wide variety of enzymes has been examined in OE and a lack of expression/activity in neuronal cell bodies has been reported following fixation of the tissue in Bouin’s solution [39], acetic acid in ethanol [40], as well as formaldehyde [35,41]. Therefore, we assert that the apparent lack of expression of the antioxidant enzymes within the olfactory neurons is unlikely to be due to the fixation method employed. Mapping of the distribution of the enzymes at four levels of the nasal cavity revealed that all the enzymes were predominantly expressed in the dorsal meatus and on the tips of the turbinates projecting into the medial meatus (Figs. 5 and 6). Computer simulation of inspiratory airflow in the rat nasal cavity has identified the major airflow streams [42], and those areas with high antioxidant enzyme expression are generally associated with the regions of high airflow. The epithelia in these areas will be exposed to comparatively high concentrations of inhaled toxins, and the dorsal meatus, the medial septum, and the tips of the ethmoturbinates projecting into the dorsal and medial airways are frequently targeted by nasal toxins [43–50]. Thus, it is not surprising that these tissues also contain relatively high concentrations of protective agents. However, no further conclusions regarding the mechanisms of olfactory toxicity of methyl iodide and DDTC can be drawn following our investigations. The antioxidant capacity of OE is not appreciably different from that of RE, and those regions damaged by methyl iodide [12] and DDTC [50] are ones with relatively high staining for the antioxidant enzymes. At this stage, it is premature to comment on how effective these antioxidants are in protecting the nose against oxidative damage. Suffice it to say, both the RE and OE have oxidant defenses comparable to the lung.
REFERENCES [1] Turk, M. A. M.; Henk, W. G.; Flory, W. 3-Methylindole-induced nasal mucosal damage in mice. Vet. Pathol. 24:400 – 403; 1987. [2] Johnson, N. F.; Hotchkiss, J. A.; Harkema, J. R.; Henderson, R. F. Proliferative responses of rat nasal epithelia to ozone. Toxicol. Appl. Pharmacol. 103:143–155; 1990. [3] Bascom, R.; Kesavanathan, J.; Fitzgerald, T. K.; Cheng, K. H.; Swift, D. L. Sidestream tobacco smoke exposure acutely alters human nasal mucociliary clearance. Environ. Health Perspect. 103:1026 –1030; 1995. [4] Johansson, S. L. Carcinogenicity of analgesics: long-term treatment of Sprague-Dawley rats with phenacetin, phenazone, caffeine, and paracetamol (acetaminophen). Int. J. Cancer 27:521– 529; 1981. [5] Hart, S. G. E.; Cartun, R. W.; Wyand, D. S.; Khairallah, E. A.; Cohen, S. D. Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: correspondence of covalent binding with toxicity. Fundam. Appl. Toxicol. 24:260 – 274; 1995. [6] Brandt, I.; Brittebo, E. B.; Feil, V. J.; Bakke, J. E. Irreversible
614
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
C. J. REED et al. binding and toxicity of the herbicide dichlobenil (2,6-dichlorobenzonitrile) in the olfactory mucosa of mice. Toxicol. Appl. Pharmacol. 103:491–501; 1990. Morgan, K. T.; Gross, E. A.; Joyner, D. R.; Ishmael, J.; Thake, D. Proliferative nasal lesions induced in rats by alachlor, acetochlor, and butachlor originate in specific regions of the olfactory mucosa. Toxicologist 36:112(abstract 575); 1997. Gaskell, B. A.; Hext, P. M.; Pigott, G. H.; Hodge, M. C. H.; Tinston, D. J. Olfactory and hepatic changes following inhalation of 3-trifluoromethyl pyridine in rats. Toxicology 50:57– 68; 1988. Keenan, C. M.; Kelly, D. P.; Bogdanffy, M. S. Degeneration and recovery of rat olfactory epithelium following inhalation of dibasic esters. Fundam. Appl. Toxicol. 15:381–393; 1990. Reed, C. J.; Gaskell, B. A.; Banger, K. K.; Lock, E. A. Olfactory toxicity of methyl iodide in the rat. Arch. Toxicol. 70:51–56; 1995. Morgan, K. T. A brief review of formaldehyde carcinogenesis in relation to rat nasal pathology and human health risk assessment. Toxicol. Pathol. 25:291–307; 1997. Chamberlain, M. P.; Lock, E. A.; Gaskell, B. A.; Reed, C. J. The role of glutathione S-transferase and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat. Arch. Toxicol. 72:420 – 428; 1998. Heikkila, R. E.; Cabbat, F. S.; Cohen, G. In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J. Biol. Chem. 251:2182–2185; 1976. Trombetta, L. D.; Toulon, M.; Jamall, S. Protective effect of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: a possible mechanism. Toxicol. Appl. Pharmacol. 93:154 –164; 1988. Kulkarni-Narla, A.; Getchell, T. V.; Schmitt, F. A.; Getchell, M. L. Manganese and copper-zinc superoxide dismutases in the human olfactory mucosa: increased immunoreactivity in Alzheimer’s disease. Exp. Neurol. 140:115–125; 1996. Kulkarni-Narla, A.; Getchell, T. V.; Getchell, M. L. Differential expression of manganese and copper-zinc superoxide dismutases in the olfactory and vomeronasal olfactory receptor neurons of rats during ontogeny. J. Comp. Neurol. 381:31– 40; 1997. Ashihara, M.; Ohmori, T.; Nishimura, T.; Sakai, M.; Nagatsu, I. Immunohistochemical study of superoxide dismutase in the olfactory mucosa. Acta Otolaryngol. Suppl. 506:34 –36; 1993. Sawada, T.; Nishimura, T.; Sakai, M.; Nagatsu, I. Immunohistochemical examination of NOS and SOD in nasal mucosa. Acta Otolaryngol. Suppl. 539:83– 86; 1998. Novoselov, S. V.; Peshenko, I. V.; Popov, V. I.; Novoselov, V. I.; Bystrova, M. F.; Evdokimov, V. J.; Kamzalov, S. S.; Merkulova, M. I.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Localization of 28 kDa peroxiredoxin in rat epithelial tissues and its antioxidant properties. Cell Tissue Res. 298:471– 480; 1999. Peshenko, I. V.; Novoselov, V. I.; Evdokimov, V. A.; Nikolaev, Y. V.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Novel 28 kDa secretory protein from rat olfactory epithelium. FEBS Lett. 381:12–14; 1996. Peshenko, I. V.; Novoselov, V. I.; Evdokimov, V. A.; Nikolaev, Y. V.; Kamzalov, S. S.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Identification of a 28 kDa secretory protein from rat olfactory epithelium as a thiol-specific antioxidant. Free Radic. Biol. Med. 25:654 – 659; 1998. Cassee, F. R.; Groten, J. P.; Feron, V. J. Changes in the nasal epithelium of rats exposed by inhalation to mixtures of formaldehyde, acetaldehyde, and acrolein. Fundam. Appl. Toxicol. 29: 208 –218; 1996. Cassee, F. R.; Feron, V. J. Biochemical and histopathological changes in the nasal epithelium of rats after 3 day intermittent exposure to formaldehyde and ozone alone or in combination. Toxicol. Lett. 72:257–268; 1994. Maples, K. R.; Nikula, K. J.; Chen, B. T.; Finch, G. L.; Griffith, W. C. Jr.; Harkema, J. R. Effects of cigarette smoke on the glutathione status of the upper and lower respiratory tract of rats. Inhal. Toxicol. 5:389 – 401; 1993. Potter, D. W.; Finch, L.; Udinsky, J. R. Glutathione content and
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36] [37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
turnover in rat nasal epithelia. Toxicol. Appl. Pharmacol. 135: 185–191; 1995. Ravi, R.; Krall, A.; Rybak, L. P.; Struble, R. G. Olfactory mucosal lesions following subcutaneous diethyldithiocarbamate (DDTC). Neurotoxicology 18:123–128; 1997. Hext, P. M.; Pinto, P. J.; Gaskell, B. A. Methyl methacrylate toxicity in rat nasal epithelium: investigation of the time course of lesion development and recovery from short-term vapor inhalation. Toxicology 156:119 –128; 2001. Chamberlain, M. P.; Sturgess, N. C.; Lock, E. A.; Reed, C. J. Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione. Toxicology 139:27–37; 1999. Ruiz-Torres, P.; Lucio, P.; Gonzalez-Rubio, M.; RodriguezPuyol, M.; Rodriguez-Puyol, D. Oxidant/antioxidant balance in isolated glomeruli and cultured mesangial cells. Free Radic. Biol. Med. 22:49 –56; 1997. Jung, K.; Kuhler, S.; Klotzek, S.; Becker, S.; Henke, W. Effect of storage temperature on the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase in rat liver and kidney homogenates. Enzyme Protein 47:149 –155; 1993. Ernster, L. DT-diaphorase. Methods Enzymol. X:309 –317; 1967. Riederer, P.; Sofic, E.; Rausch, W. D.; Schmidt, B.; Reynolds, G. P.; Jellinger, K.; Youdim, M. B. Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 52:515–520; 1989. Nierenberg, D. W.; Lester, D. C. Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance liquid chromatography. J. Chromatog. 345: 275–284; 1985. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenl reagent. J. Biol. Chem. 193:265–275; 1951. Banger, K. K.; Foster, J. R.; Lock, E. A.; Reed, C. J. Immunohistochemical localization of six glutathione S-transferases within the nasal cavity of the rat. Arch. Toxicol. 69:91–98; 1994. Young, J. T. Histopathological examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1:309 –312; 1981. Duncan, K.; Harris, S.; Ardies, C. M. Running exercise may reduce risk for lung and liver cancer by inducing activity of antioxidant and phase II enzymes. Cancer Lett. 116:151–158; 1997. Slade, R.; Stead, A. G.; Graham, J. A.; Hatch, G. E. Comparison of lung antioxidant levels in humans and laboratory animals. Am. Rev. Respir. Dis. 131:742–746; 1985. Walters, E.; Buchheit, K.; Maruniak, J. A. Olfactory cytochrome P450 immunoreactivity in mice is altered by dichlobenil but preserved by metyrapone. Toxicology 81:113–122; 1993. Voigt, J. M.; Guengerich, F. P.; Baron, J. Localization and induction of cytochrome P450 1A1 and aryl hydrocarbon hydroxylase activity in rat nasal mucosa. J. Histochem. Cytochem. 41:877– 885; 1993. Thornton-Manning, J. R.; Nikula, K. J.; Hotchkiss, J. A.; Avila, K. J.; Rohrbacher, K. D.; Ding, X.; Dahl, A. R. Nasal cytochrome P450 2A: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen. Toxicol. Appl. Pharmacol. 142:22–30; 1997. Kimbell, J. S.; Godo, M. N.; Gross, E. A.; Joyner, D. R.; Richardson, R. B.; Morgan, K. T. Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages. Toxicol. Appl. Pharmacol. 145:388 –398; 1997. Pino, M. V.; Valerio, M. G.; Miller, G. K.; Larson, J. L.; Rosolia, D. L.; Jayyosi, Z.; Crouch, C. N.; Trojanowski, J. Q.; Geiger, L. E. Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP73401 on the nasal olfactory tissue in rats. Toxicol. Pathol. 27:383–394; 1999. Irwin, R. D.; Chou, B. J.; Mellick, P. W.; Miller, R. A.; Mahler, J.; Roycroft, J. Toxicity of furfuryl alcohol to F344 rats and B6C3F1 mice exposed by inhalation. J. Appl. Toxicol. 17:159 – 169; 1997. Melnick, R. L.; Elwell, M. R.; Roycroft, J. H.; Chou, B. J.; Ragan,
Rat nasal antioxidants H. A.; Miller, R. A. Toxicity of inhaled chloroprene (2-chloro1,3-butadiene) in F344 rats and B6C3F1 mice. Toxicology 108: 79 –91; 1996. [46] Nikula, K. J.; Lewis, J. L. Olfactory mucosal lesions in F344 rats following inhalation exposure to pyridine at threshold limit value concentrations. Fundam. Appl. Toxicol. 23:510 –517; 1994. [47] Brenneman, K. A.; James, R. A.; Gross, E. A.; Dorman, D. C. Olfactory neuron loss in adult male CD rats following subchronic inhalation exposure to hydrogen sulfide. Toxicol. Pathol. 28:326 – 333; 2000. [48] Sun, J. D.; Dahl, A. R.; Gillett, N. A.; Barr, E. B.; Crews, M. L.; Eidson, A. F.; Bechtold, W. E.; Burt, D. G.; Dieter, M. P.; Hobbs,
615
C. H. Two-week, repeated inhalation exposure of F344/N rats and B6C3F1 mice to ferrocene. Fundam. Appl. Toxicol. 17:150 –158; 1991. [49] Genter, M. B.; Deamer, N. J.; Blake, B. L.; Wesley, D. S.; Levi, P. E. Olfactory toxicity of methamizole: dose response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa. Toxicol. Pathol. 23:477– 486; 1995. [50] Deamer, N. J.; Genter, M. B. Olfactory toxicity of diethyldithiocarbamate (DDTC) and disulfiram and the protective effect of DDTC against the olfactory toxicity of dichlobenil. Chem. Biol. Interact. 95:215–226; 1995.
doi:10.1016/S0891-5849(02)01367-9
Original Contribution ANTIOXIDANT STATUS OF THE RAT NASAL CAVITY CELIA J. REED,* DARREN A. ROBINSON,*
and
EDWARD A. LOCK†
*School of Biomolecular Sciences, Liverpool John Moores University, Liverpool, England; and †Syngenta Central Toxicology Laboratory, Cheshire, England (Received 25 July 2002; Revised 11 November 2002; Accepted 21 November 2002)
Abstract—Despite extensive interest in the rodent nasal cavity as a target organ for toxicity, there is very limited information regarding nasal defenses against oxidative stress and xenobiotic-derived oxidants. Using immunohistochemistry, we have examined the distribution of Cu,Zn and Mn superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase, and DT-diaphorase in rat nasal tissues. In addition, we have determined the concentrations of ascorbate and ␣-tocopherol and the activities of SOD (combined Cu,Zn and Mn forms), catalase, GSH peroxidase, GSH reductase, and DT-diaphorase in nasal respiratory epithelium (RE), olfactory epithelium (OE), and in lung. Immunohistochemistry demonstrated that all four enzymes were similarly distributed, with the greatest staining intensity in dorsal-medial regions of the nasal cavity. In respiratory epithelium, ciliated columnar cells and subepithelial glands stained positively, while in olfactory tissue the enzymes were detected in the sustentacular cells and Bowman’s glands. With the exception of SOD, enzyme activities were higher in RE than OE, while concentrations of ascorbate and ␣-tocopherol were higher in OE than RE. With the exception of catalase, nasal activities were either higher than or comparable to those of the lung. Thus, the rat nasal cavity appears to be well protected against oxidative damage. © 2003 Elsevier Science Inc. Keywords—Olfactory, Nasal, Superoxide dismutase, Glutathione reductase, Glutathione peroxidase, Catalase, DTdiaphorase, Ascorbate, ␣-Tocopherol, Free radicals
INTRODUCTION
ents of cigarette smoke) are themselves potent oxidants. Secondly, chemicals such as carbon tetrachloride undergo cytochrome P450-dependent metabolism within olfactory epithelium (OE), resulting in the generation of free radicals. Finally, compounds may compromise the endogenous antioxidants of the nasal cavity and render it susceptible to oxidative damage. For example, methyl iodide is conjugated with glutathione and results in a rapid and extensive depletion of this thiol within the nasal cavity [12], and diethyldithiocarbamate (DDTC) chelates metal ions and, thereby, inhibits copper/zinc superoxide dismutase (Cu,ZnSOD, [13]) and glutathione (GSH) peroxidase [14]. To defend themselves against oxidative stress, all cells contain an endogenous antioxidant system that maintains the cells’ normal redox potential. This system comprises a variety of antioxidant enzymes including SOD, catalase, GSH peroxidase, GSH reductase, and NAD(P)H:quinone oxidoreductase (DT-diaphorase), as well as low molecular weight components such as glutathione, ascorbate, and ␣-tocopherol (vitamin E). There
The tissues of the respiratory tract are some of the main targets for the effects of inhaled environmental pollutants and industrial chemicals. Furthermore, the abundant blood supply to the respiratory system ensures delivery of potential toxins to respiratory tract cells following systemic exposure. Thus, in recent years it has become apparent that the rodent nasal cavity is an important target organ for toxicity, and a wide variety of compounds including environmental contaminants [1–3], drugs [4,5], herbicides [6,7], and industrial chemicals [8 –11] have been shown to cause nasal lesions or tumors in experimental animals. Oxidative damage may be involved in the development of nasal toxicity in a number of ways. Firstly, many environmental pollutants (e.g., ozone and the constituAddress correspondence to: Dr. Celia J. Reed, Liverpool John Moores University, School of Biomolecular Sciences, Byrom Street, Liverpool L3 3AF, UK; Tel: ⫹44 151-231-2187; Fax: ⫹44 151-2312982; E-Mail: [email protected]. 607
608
C. J. REED et al.
has been only limited examination of the intracellular antioxidant systems of the nasal cavity. Both manganese SOD (MnSOD) and Cu,ZnSOD have been localized within human [15], rat [16], and mouse [17,18] nasal mucosae, and a novel peroxiredoxin with antioxidant properties has recently been isolated from rat OE [19 – 21]. GSH peroxidase and GSH reductase activities have been determined in rat nasal epithelia [22,23], and several groups have measured GSH (or nonprotein sulphydryls) and its turnover within the nasal cavity [12,22,24,25]. One of the striking characteristics of nasal toxins is their ability to target specific regions within the nasal cavity. That is, methyl iodide [10], DDTC [26], dichlobenil [6], and methyl methacrylate [27] all cause lesions in the OE of the posterior cavity, ozone affects only the transitional epithelium (TE) [2,23], and formaldehyde is toxic to the respiratory epithelium (RE) of the anterior nasal cavity [22,23]. This site selectivity is thought to be partly due to tissue-specific susceptibility, in particular the regional distribution of biotransformation enzymes. However, we have recently postulated that methyl iodide toxicity may result from GSH depletion, rendering tissues susceptible to methylation and/or oxidative stress [28]; and, Ravi et al. [26] suggested that the olfactory toxicity of DDTC may be due to the ability of this agent to chelate metal ions and, thus, compromise the antioxidant systems of the OE. The differential susceptibilities of the nasal epithelia may relate to the activities and concentrations of their antioxidant defense mechanisms. The aim of this study was to carry out a systematic investigation of nasal intracellular antioxidants. As such, several antioxidant enzymes have been localized within the nasal cavity and their activities measured in RE and OE, and the concentrations of ascorbate and ␣-tocopherol also have been determined in these tissues. MATERIALS AND METHODS
Materials Commercially available antibodies (sheep antihuman) were obtained from the following sources: SOD (Cu,Zn and Mn) and catalase, The Binding Site (Birmingham, England); and GSH peroxidase, Biogenesis (Poole, England). The antibody against DT-diaphorase was a kind gift from David Siegel and David Ross of the University of Colorado Health Sciences Center (Boulder, CO, USA). Animals Adult male, specific pathogen-free, Alpk:APfsD (Wistar-derived) albino rats, were supplied from the colony maintained at Alderley Park, Cheshire, England.
Rats were housed in cages suspended over collecting trays lined with absorbent paper at a density of up to five per cage. Animal rooms were maintained at a temperature of 21 ⫾ 2°C, a relative humidity of 50 ⫾ 15%, with a 12 h light/dark cycle (lights on at 6 a.m.). Water from an automatic system and pelleted Porton Combined Diet (Special Diet Services, Witham, Essex, England) were provided ad libitum. Rats were killed by anesthetic overdose. Biochemical analysis For determination of GSH reductase, GSH peroxidase, catalase, and SOD activities, heads were dissected and naso- and maxilloturbinates (RE) and ethmoturbinates (OE) from two to three animals were pooled and placed in 1 ml of ice-cold sucrose-EDTA-Tris buffer (SET, 0.25M sucrose, 5.4 mM EDTA, 20 mM Tris, pH 7.4). Lungs were perfused with 0.9% saline; the caudal lobe was removed, weighed, and a sample placed in ice-cold SET (1 g in 4 ml). Tissues were homogenized and then centrifuged at 800 ⫻ g at 4°C for 10 min. Triton X-100 was added to the supernatant to give a final concentration of 0.1% detergent (v/v), and the mixture was then sonicated on ice at 300 W for 3 ⫻ 10 s and centrifuged at 20,000 ⫻ g at 4°C for 10 min. The activities of GSH peroxidase [29], GSH reductase [30], and catalase [30] were determined using t-butyl hydroperoxide, oxidized GSH, and hydrogen peroxide, respectively, as substrates. Total SOD activity [30] was determined as the inhibition of the autoxidation of adrenaline to adrenochrome, 1 U of activity resulting in 50% inhibition. For determination of DT-diaphorase activity, tissues from individual animals were removed as described above, weighed, and homogenized in ice-cold 0.32 M sucrose (caudal lobe of lung, 4 vol; OE, 10 vol; RE, 1.0 ml) using an ultraturrax homogenizer. After centrifugation at 100,000 ⫻ g for 60 min at 4°C, the supernatants were carefully decanted and stored at ⫺70°C. DT-diaphorase activity was determined according to the method of Ernster [31], using 2,6-dichlorophenolindophenol as the electron acceptor and NADPH. DT-diaphorase activity was measured as the dicoumarol- (10 M) sensitive oxidation of NADPH. For determination of ascorbate, tissues were dissected as described above, weighed, and homogenized in icecold 0.15 M phosphoric acid (OE and caudal lobe of lung, 0.25 g in 1 ml; RE, 0.15 g in 1 ml); tissues were then placed on ice for 10 min and, subsequently, centrifuged at 15,000 ⫻ g at 4°C for 10 min. Aliquots of supernatant were diluted 1 in 10 in distilled water and filtered (0.22 m) before analysis by HPLC [32]. Pellets
Rat nasal antioxidants
609
were resuspended in 1 M sodium hydroxide (OE and lung, 1 ml; RE, 0.5 ml) for protein analysis. For determination of ␣-tocopherol, tissues were dissected as described above, with OE and RE pooled from two animals. Tissues were weighed and then homogenized in the dark in ice-cold phosphate-buffered saline (caudal lobe of lung, 25% (w/v); OE and RE, 1.2 ml); next, tissues were prepared and analyzed as described by Nierenberg and Lester [33] using a Luna 5u C18(2) column (Phenomenex, Cheshire, England; 50 ⫻ 4.60 mm). Protein concentrations were determined by the method of Lowry [34] using bovine serum albumin as standard. Immunohistochemistry For immunohistochemical studies, heads were fixed in neutral buffered formal saline and decalcified in neutral EDTA, as described previously [35]. Heads were trimmed to produce levels 1– 4 as described by Young [36], processed to paraffin blocks, and sections (5 m) cut. Localization of catalase, glutathione peroxidase, Cu,ZnSOD, MnSOD, and DT-diaphorase was carried out, as described previously [35], using a 1/50 dilution of the primary antibodies for all the enzymes except DTdiaphorase, which was diluted to 1/200. Statistics All results are means ⫾ SD of at least four determinations. Results were analyzed by ANOVA followed by the Bonferroni test, and a value of p ⬍ .05 was considered statistically significant.
Fig. 1. Antioxidant enzyme activities in rat RE, OE, and lung (■ RE; OE; lung). GR ⫽ GSH reductase, nmol/min/mg protein; GP ⫽ GSH peroxidase, nmol/min/mg protein; catalase ⫽ catalase, mK/mg protein; SOD ⫽ total superoxide dismutase, U/mg protein; DT-D ⫽ DT-diaphorase, 10⫺4 mol/min/mg protein. a ⫽ RE significantly different from OE, p ⬍ .05; b ⫽ RE significantly different from lung, p ⬍ .05; c ⫽ OE significantly different from lung, p ⬍ .05.
In transitional epithelium (TE), the staining appeared diffuse with some cells staining moderately positive and others being completely negative (Fig. 3a). In RE, the ciliated columnar cells stained positive while both the goblet cells and nonciliated columnar cells stained negative (Fig. 3b). The glands underlying the RE (seromucus tubular and acinar) all stained positive, but the tubu-
RESULTS
Biochemical analysis With the exception of SOD, antioxidant enzyme activity was significantly higher (1.5- to 3.3-fold) in RE than in OE (Fig. 1). Olfactory SOD activity (all forms) was approximately 3-fold greater than that of RE. Compared to the nasal cavity, activities in the lung were lower for GSH reductase, SOD, and DT-diaphorase, higher for catalase, and comparable for GSH peroxidase (Fig. 1). The concentrations of ascorbate and ␣-tocopherol were higher in OE compared to RE (4.2- and 1.4-fold, respectively). Alpha-tocopherol concentrations were higher in lung than in the nasal cavity, while pulmonary ascorbate was comparable to that of the OE (Fig. 2). Immunohistochemical analysis In general, all of the enzymes were localized to the same cell types within the different nasal epithelia. In squamous epithelium (SE), there was very weak staining in the apical layer (stratum corneum, results not shown).
Fig. 2. Concentrations of ascorbate and ␣-tocopherol in rat RE, OE, and lung (■ RE; OE; lung). Ascorbate is given in g/mg protein and ␣-tocopherol (vitamin E) is given in ng/mg protein.a ⫽ RE significantly different from OE, p ⬍ .05; b ⫽ RE significantly different from lung, p ⬍ .05; c ⫽ OE significantly different from lung, p ⬍ .05.
610
C. J. REED et al.
Fig. 3. Immunohistochemical localization of (a) DT-diaphorase in TE and (b) GSH peroxidase in RE. (a) TE lining the maxilloturbinate at level 1 of the rat nasal cavity; microscope magnification is ⫻400. (b) RE lining the lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. AG ⫽ acinar gland; CCC ⫽ ciliated columnar cell; NCC ⫽ nonciliated columnar cell; TG ⫽ tubular gland.
Fig. 4. Immunohistochemical localization of (a) catalase, (b) catalase, (c) Cu,ZnSOD, and (d) GSH peroxidase in rat OE. (a) OE on the tip of the third ethmoturbinate at level 3 of the rat nasal cavity; microscope magnification is ⫻400. (b) OE from the lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. (c) OE on the fourth ethmoturbinate at level 4 of the rat nasal cavity; microscope magnification is ⫻400. (d) OE of the dorsal lateral wall at level 3 of the rat nasal cavity; microscope magnification is ⫻400. AC ⫽ apical cytoplasm; BG ⫽ Bowman’s gland; NB ⫽ neural bundle; SN ⫽ sustentacular cell nuclei.
Rat nasal antioxidants
611
Fig. 5. Diagrammatic representations of levels 1– 4 of the rat nasal cavity identifying some of the major structures. DM ⫽ dorsal meatus; ETs ⫽ ethmoturbinates; NT ⫽ nasoturbinate; MT ⫽ maxilloturbinate; S ⫽ septum.
lar glands stained more intensely for all five of the enzymes than did the acinal glands (Fig. 3b). In OE, the sustentacular cells and Bowman’s glands stained positive while the olfactory neurons and basal cells consistently failed to stain (Figs. 4a– 4d). With catalase (Fig. 4a), DT-diaphorase, and MnSOD, sustentacular cell staining was confined to the apical cytoplasm. In isolated patches of OE, GSH peroxidase and Cu,ZnSOD also stained the nuclei of the sustentacular cells (Figs. 4c and 4d). In all areas the neural bundles stained positive, even those on the septum at level 2 (Figs. 4a– 4d). We have mapped the distribution of these enzymes at the four levels of the nasal cavity described by Young [36] (Fig. 5), and we have demonstrated that they have similar localization patterns (Fig. 6). In general, staining intensity was greatest in the dorsal-medial regions of the nasal cavity. Thus, at levels 1 and 2, the dorsal meatus, parts of the septum, and the medial surfaces of the naso-
and maxilloturbinates showed the greatest staining intensity. At level 3, the dorsal meatus, the medial tips of the ethmoturbinates, and the septum were stained, while at level 4 the enzymes were generally confined to the medial aspects of the ethmoturbinates. With the exception of catalase at levels 3 and 4, no staining was detected in the mucosa lining the lateral walls. DISCUSSION
In this study, we have determined the activities of five important antioxidant enzymes and the concentrations of ascorbate and ␣-tocopherol within the nasal cavity and lung. The caudal lobe of the lung was explored primarily as a reference respiratory tract tissue, and antioxidants measured in the lung were of a similar magnitude to those reported previously [37,38]. With the exception of catalase, nasal enzyme activities were found to be greater
612
C. J. REED et al.
Fig. 6. Diagrammatic representations of levels 1– 4 of the rat nasal cavity showing the distribution of staining for antioxidant enzymes (■ intense staining; moderate staining). All other areas stained weakly for the enzymes.
Rat nasal antioxidants
than or similar to those of the lung (Fig. 1). Catalase is an exceptionally specific enzyme, catalyzing the breakdown of hydrogen peroxide alone. Hydrogen peroxide is produced during the respiratory burst of phagocytic cells, and the higher catalase activity in the lung compared to the nasal epithelia may reflect the presence of alveolar macrophages and a need to protect pulmonary tissue from hydrogen peroxide. Alpha-tocopherol concentrations in the nasal tissues were less than those of the lung (Fig. 2), while ascorbate concentrations were low in RE but comparable in OE and lung (Fig. 2). Alpha-tocopherol ideally should be expressed as nmol/mg lipid, as it is a very lipid-soluble vitamin. However, we have no lipid data for either the RE or OE and we have, therefore, expressed our results per mg protein. This may explain why higher concentrations of ␣-tocopherol were found in the lung, which would be expected to have a higher lipid to protein content. Thus, no consistent pattern of the nasal tissues having either higher or lower antioxidant capacity than the lung emerged. Similarly, comparison of RE and OE revealed no obvious trend: RE had higher GSH reductase, GSH peroxidase, catalase, and DT-diaphorase activities, but lower total SOD activity and lower ascorbate and ␣-tocopherol concentrations. These data must be interpreted with some caution, as nasal turbinates are cartilaginous structures covered in epithelia of varying thickness and cellular heterogeneity. During homogenization, the activity/concentration of cells rich in antioxidants will be diluted by the contribution of the cartilage and those cells that do not contain the antioxidants. This may occur to a greater extent in OE than in RE, as there are very marked differences in the distributions of the antioxidant enzymes within the former tissue (Fig. 6). Homogenization and pooling of all the OE results in a mean value that may not accurately reflect the antioxidant status of individual ethmoturbinates. However, it can be concluded that the antioxidant defenses of the nasal cavity appear to be comparable to those of the lung. Immunohistochemical localization of the antioxidant enzymes in the nasal cavity revealed that they are all expressed in the glands and ciliated columnar cells of RE and the Bowman’s glands and sustentacular cells of OE. In some areas of OE, GSH peroxidase and Cu,ZnSOD were also detected in the nuclei of the sustentacular cells. Ashihara et al. [17] reported a similar cellular and intracellular distribution for Cu,ZnSOD in murine nasal tissues, while Kulkarni-Narla et al. also detected Cu,Zn and MnSOD in olfactory neuronal cell bodies of both rats and humans [15,16]. The latter group concluded that the lack of neuronal cell body staining reported by Ashihara et al. [17] was due to a loss of immunoreactivity during fixation in paraformaldehyde. In the present study, the
613
tissues were fixed in formalin, which may have resulted in a similar artefact. However, localization of a wide variety of enzymes has been examined in OE and a lack of expression/activity in neuronal cell bodies has been reported following fixation of the tissue in Bouin’s solution [39], acetic acid in ethanol [40], as well as formaldehyde [35,41]. Therefore, we assert that the apparent lack of expression of the antioxidant enzymes within the olfactory neurons is unlikely to be due to the fixation method employed. Mapping of the distribution of the enzymes at four levels of the nasal cavity revealed that all the enzymes were predominantly expressed in the dorsal meatus and on the tips of the turbinates projecting into the medial meatus (Figs. 5 and 6). Computer simulation of inspiratory airflow in the rat nasal cavity has identified the major airflow streams [42], and those areas with high antioxidant enzyme expression are generally associated with the regions of high airflow. The epithelia in these areas will be exposed to comparatively high concentrations of inhaled toxins, and the dorsal meatus, the medial septum, and the tips of the ethmoturbinates projecting into the dorsal and medial airways are frequently targeted by nasal toxins [43–50]. Thus, it is not surprising that these tissues also contain relatively high concentrations of protective agents. However, no further conclusions regarding the mechanisms of olfactory toxicity of methyl iodide and DDTC can be drawn following our investigations. The antioxidant capacity of OE is not appreciably different from that of RE, and those regions damaged by methyl iodide [12] and DDTC [50] are ones with relatively high staining for the antioxidant enzymes. At this stage, it is premature to comment on how effective these antioxidants are in protecting the nose against oxidative damage. Suffice it to say, both the RE and OE have oxidant defenses comparable to the lung.
REFERENCES [1] Turk, M. A. M.; Henk, W. G.; Flory, W. 3-Methylindole-induced nasal mucosal damage in mice. Vet. Pathol. 24:400 – 403; 1987. [2] Johnson, N. F.; Hotchkiss, J. A.; Harkema, J. R.; Henderson, R. F. Proliferative responses of rat nasal epithelia to ozone. Toxicol. Appl. Pharmacol. 103:143–155; 1990. [3] Bascom, R.; Kesavanathan, J.; Fitzgerald, T. K.; Cheng, K. H.; Swift, D. L. Sidestream tobacco smoke exposure acutely alters human nasal mucociliary clearance. Environ. Health Perspect. 103:1026 –1030; 1995. [4] Johansson, S. L. Carcinogenicity of analgesics: long-term treatment of Sprague-Dawley rats with phenacetin, phenazone, caffeine, and paracetamol (acetaminophen). Int. J. Cancer 27:521– 529; 1981. [5] Hart, S. G. E.; Cartun, R. W.; Wyand, D. S.; Khairallah, E. A.; Cohen, S. D. Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: correspondence of covalent binding with toxicity. Fundam. Appl. Toxicol. 24:260 – 274; 1995. [6] Brandt, I.; Brittebo, E. B.; Feil, V. J.; Bakke, J. E. Irreversible
614
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
C. J. REED et al. binding and toxicity of the herbicide dichlobenil (2,6-dichlorobenzonitrile) in the olfactory mucosa of mice. Toxicol. Appl. Pharmacol. 103:491–501; 1990. Morgan, K. T.; Gross, E. A.; Joyner, D. R.; Ishmael, J.; Thake, D. Proliferative nasal lesions induced in rats by alachlor, acetochlor, and butachlor originate in specific regions of the olfactory mucosa. Toxicologist 36:112(abstract 575); 1997. Gaskell, B. A.; Hext, P. M.; Pigott, G. H.; Hodge, M. C. H.; Tinston, D. J. Olfactory and hepatic changes following inhalation of 3-trifluoromethyl pyridine in rats. Toxicology 50:57– 68; 1988. Keenan, C. M.; Kelly, D. P.; Bogdanffy, M. S. Degeneration and recovery of rat olfactory epithelium following inhalation of dibasic esters. Fundam. Appl. Toxicol. 15:381–393; 1990. Reed, C. J.; Gaskell, B. A.; Banger, K. K.; Lock, E. A. Olfactory toxicity of methyl iodide in the rat. Arch. Toxicol. 70:51–56; 1995. Morgan, K. T. A brief review of formaldehyde carcinogenesis in relation to rat nasal pathology and human health risk assessment. Toxicol. Pathol. 25:291–307; 1997. Chamberlain, M. P.; Lock, E. A.; Gaskell, B. A.; Reed, C. J. The role of glutathione S-transferase and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat. Arch. Toxicol. 72:420 – 428; 1998. Heikkila, R. E.; Cabbat, F. S.; Cohen, G. In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J. Biol. Chem. 251:2182–2185; 1976. Trombetta, L. D.; Toulon, M.; Jamall, S. Protective effect of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: a possible mechanism. Toxicol. Appl. Pharmacol. 93:154 –164; 1988. Kulkarni-Narla, A.; Getchell, T. V.; Schmitt, F. A.; Getchell, M. L. Manganese and copper-zinc superoxide dismutases in the human olfactory mucosa: increased immunoreactivity in Alzheimer’s disease. Exp. Neurol. 140:115–125; 1996. Kulkarni-Narla, A.; Getchell, T. V.; Getchell, M. L. Differential expression of manganese and copper-zinc superoxide dismutases in the olfactory and vomeronasal olfactory receptor neurons of rats during ontogeny. J. Comp. Neurol. 381:31– 40; 1997. Ashihara, M.; Ohmori, T.; Nishimura, T.; Sakai, M.; Nagatsu, I. Immunohistochemical study of superoxide dismutase in the olfactory mucosa. Acta Otolaryngol. Suppl. 506:34 –36; 1993. Sawada, T.; Nishimura, T.; Sakai, M.; Nagatsu, I. Immunohistochemical examination of NOS and SOD in nasal mucosa. Acta Otolaryngol. Suppl. 539:83– 86; 1998. Novoselov, S. V.; Peshenko, I. V.; Popov, V. I.; Novoselov, V. I.; Bystrova, M. F.; Evdokimov, V. J.; Kamzalov, S. S.; Merkulova, M. I.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Localization of 28 kDa peroxiredoxin in rat epithelial tissues and its antioxidant properties. Cell Tissue Res. 298:471– 480; 1999. Peshenko, I. V.; Novoselov, V. I.; Evdokimov, V. A.; Nikolaev, Y. V.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Novel 28 kDa secretory protein from rat olfactory epithelium. FEBS Lett. 381:12–14; 1996. Peshenko, I. V.; Novoselov, V. I.; Evdokimov, V. A.; Nikolaev, Y. V.; Kamzalov, S. S.; Shuvaeva, T. M.; Lipkin, V. M.; Fesenko, E. E. Identification of a 28 kDa secretory protein from rat olfactory epithelium as a thiol-specific antioxidant. Free Radic. Biol. Med. 25:654 – 659; 1998. Cassee, F. R.; Groten, J. P.; Feron, V. J. Changes in the nasal epithelium of rats exposed by inhalation to mixtures of formaldehyde, acetaldehyde, and acrolein. Fundam. Appl. Toxicol. 29: 208 –218; 1996. Cassee, F. R.; Feron, V. J. Biochemical and histopathological changes in the nasal epithelium of rats after 3 day intermittent exposure to formaldehyde and ozone alone or in combination. Toxicol. Lett. 72:257–268; 1994. Maples, K. R.; Nikula, K. J.; Chen, B. T.; Finch, G. L.; Griffith, W. C. Jr.; Harkema, J. R. Effects of cigarette smoke on the glutathione status of the upper and lower respiratory tract of rats. Inhal. Toxicol. 5:389 – 401; 1993. Potter, D. W.; Finch, L.; Udinsky, J. R. Glutathione content and
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36] [37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
turnover in rat nasal epithelia. Toxicol. Appl. Pharmacol. 135: 185–191; 1995. Ravi, R.; Krall, A.; Rybak, L. P.; Struble, R. G. Olfactory mucosal lesions following subcutaneous diethyldithiocarbamate (DDTC). Neurotoxicology 18:123–128; 1997. Hext, P. M.; Pinto, P. J.; Gaskell, B. A. Methyl methacrylate toxicity in rat nasal epithelium: investigation of the time course of lesion development and recovery from short-term vapor inhalation. Toxicology 156:119 –128; 2001. Chamberlain, M. P.; Sturgess, N. C.; Lock, E. A.; Reed, C. J. Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione. Toxicology 139:27–37; 1999. Ruiz-Torres, P.; Lucio, P.; Gonzalez-Rubio, M.; RodriguezPuyol, M.; Rodriguez-Puyol, D. Oxidant/antioxidant balance in isolated glomeruli and cultured mesangial cells. Free Radic. Biol. Med. 22:49 –56; 1997. Jung, K.; Kuhler, S.; Klotzek, S.; Becker, S.; Henke, W. Effect of storage temperature on the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase in rat liver and kidney homogenates. Enzyme Protein 47:149 –155; 1993. Ernster, L. DT-diaphorase. Methods Enzymol. X:309 –317; 1967. Riederer, P.; Sofic, E.; Rausch, W. D.; Schmidt, B.; Reynolds, G. P.; Jellinger, K.; Youdim, M. B. Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 52:515–520; 1989. Nierenberg, D. W.; Lester, D. C. Determination of vitamins A and E in serum and plasma using a simplified clarification method and high-performance liquid chromatography. J. Chromatog. 345: 275–284; 1985. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenl reagent. J. Biol. Chem. 193:265–275; 1951. Banger, K. K.; Foster, J. R.; Lock, E. A.; Reed, C. J. Immunohistochemical localization of six glutathione S-transferases within the nasal cavity of the rat. Arch. Toxicol. 69:91–98; 1994. Young, J. T. Histopathological examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1:309 –312; 1981. Duncan, K.; Harris, S.; Ardies, C. M. Running exercise may reduce risk for lung and liver cancer by inducing activity of antioxidant and phase II enzymes. Cancer Lett. 116:151–158; 1997. Slade, R.; Stead, A. G.; Graham, J. A.; Hatch, G. E. Comparison of lung antioxidant levels in humans and laboratory animals. Am. Rev. Respir. Dis. 131:742–746; 1985. Walters, E.; Buchheit, K.; Maruniak, J. A. Olfactory cytochrome P450 immunoreactivity in mice is altered by dichlobenil but preserved by metyrapone. Toxicology 81:113–122; 1993. Voigt, J. M.; Guengerich, F. P.; Baron, J. Localization and induction of cytochrome P450 1A1 and aryl hydrocarbon hydroxylase activity in rat nasal mucosa. J. Histochem. Cytochem. 41:877– 885; 1993. Thornton-Manning, J. R.; Nikula, K. J.; Hotchkiss, J. A.; Avila, K. J.; Rohrbacher, K. D.; Ding, X.; Dahl, A. R. Nasal cytochrome P450 2A: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen. Toxicol. Appl. Pharmacol. 142:22–30; 1997. Kimbell, J. S.; Godo, M. N.; Gross, E. A.; Joyner, D. R.; Richardson, R. B.; Morgan, K. T. Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages. Toxicol. Appl. Pharmacol. 145:388 –398; 1997. Pino, M. V.; Valerio, M. G.; Miller, G. K.; Larson, J. L.; Rosolia, D. L.; Jayyosi, Z.; Crouch, C. N.; Trojanowski, J. Q.; Geiger, L. E. Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP73401 on the nasal olfactory tissue in rats. Toxicol. Pathol. 27:383–394; 1999. Irwin, R. D.; Chou, B. J.; Mellick, P. W.; Miller, R. A.; Mahler, J.; Roycroft, J. Toxicity of furfuryl alcohol to F344 rats and B6C3F1 mice exposed by inhalation. J. Appl. Toxicol. 17:159 – 169; 1997. Melnick, R. L.; Elwell, M. R.; Roycroft, J. H.; Chou, B. J.; Ragan,
Rat nasal antioxidants H. A.; Miller, R. A. Toxicity of inhaled chloroprene (2-chloro1,3-butadiene) in F344 rats and B6C3F1 mice. Toxicology 108: 79 –91; 1996. [46] Nikula, K. J.; Lewis, J. L. Olfactory mucosal lesions in F344 rats following inhalation exposure to pyridine at threshold limit value concentrations. Fundam. Appl. Toxicol. 23:510 –517; 1994. [47] Brenneman, K. A.; James, R. A.; Gross, E. A.; Dorman, D. C. Olfactory neuron loss in adult male CD rats following subchronic inhalation exposure to hydrogen sulfide. Toxicol. Pathol. 28:326 – 333; 2000. [48] Sun, J. D.; Dahl, A. R.; Gillett, N. A.; Barr, E. B.; Crews, M. L.; Eidson, A. F.; Bechtold, W. E.; Burt, D. G.; Dieter, M. P.; Hobbs,
615
C. H. Two-week, repeated inhalation exposure of F344/N rats and B6C3F1 mice to ferrocene. Fundam. Appl. Toxicol. 17:150 –158; 1991. [49] Genter, M. B.; Deamer, N. J.; Blake, B. L.; Wesley, D. S.; Levi, P. E. Olfactory toxicity of methamizole: dose response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa. Toxicol. Pathol. 23:477– 486; 1995. [50] Deamer, N. J.; Genter, M. B. Olfactory toxicity of diethyldithiocarbamate (DDTC) and disulfiram and the protective effect of DDTC against the olfactory toxicity of dichlobenil. Chem. Biol. Interact. 95:215–226; 1995.