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Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden (A.F., M.L., E.B.B.); and Department of Environmental Toxicology, Uppsala University, Uppsala, Sweden (C.C., V.H.)
(Received July 1, 2005; accepted October 7, 2005)
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Abstract |
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; Piras et al., 2003; Ling et al., 2004). The P450-mediated metabolism is generally regarded as a protection mechanism, and the high expression of drug-metabolizing P450s is most likely related to the clearance of odorants and other airborne chemicals. These enzymes may also protect the central nervous system against inhaled toxicants. P450 enzymes are, however, also central for the metabolic activation of foreign compounds into reactive, toxic metabolites, and olfactory P450s have been suggested to play a key role in the tissue-selective toxicity of drugs and chemicals in this tissue (Gaskell, 1990; Dahl and Hadley, 1991; Reed, 1993; Brittebo, 1997; Ding and Kaminsky, 2003). A decreased olfactory toxicity has been demonstrated after pretreatment with various P450 inhibitors (Brandt et al., 1990; Genter et al., 1994; Bahrami et al., 2000b). Many olfactory toxicants and carcinogens, including dichlobenil, coumarin, hexamethylphosphoramide, and the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone are preferentially bioactivated to reactive intermediates by the predominantly expressed mouse CYP2A5 (Negishi et al., 1989; Liu et al., 1996; Thornton-Manning et al., 1997; Gu et al., 1998; Zhuo et al., 1999; Felicia et al., 2000).
2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) (Fig. 1) is a potent olfactory toxicant in rodents, and similar 2,6-dichlorobenzene derivatives with a large, polar, and strong electron-withdrawing substituent in the primary position have recently been reported as potential olfactory toxicants in mice (Brandt et al., 1990; Bahrami et al., 1999, 2000a; Carlsson et al., 2004). The 2,6-diClPh-MeSO2-induced olfactory toxicity is markedly reduced in animals pretreated with the broad-spectrum P450 inhibitor metyrapone, and this inhibitor also abolishes the extensive bioactivation of 2,6-diClPh-MeSO2 to a reactive, tissue-binding metabolite in rat olfactory microsomes (Bahrami et al., 2000b, Franzen et al., 2003). The role of specific P450 forms in the bioactivation of this toxicant has not been examined. The covalent binding in rat liver microsomes is very low, suggesting involvement of P450 forms preferentially expressed in the olfactory mucosa. Light microscopic autoradiography revealed that the reactive 2,6-diClPh-MeSO2 intermediate is selectively bound to the mucus-secreting Bowman's glands in the olfactory mucosa. The studies also demonstrated that glutathione (GSH) depletion potentiates covalent binding and toxicity in the mouse olfactory mucosa in vivo (Bahrami et al., 2000b). We have recently reported an extensive up-regulation of the ER stress protein GRP78 in the Bowman's glands following a single injection of 2,6-diClPh-MeSO2 in mice. In addition, there is an early-onset activation of the ER-situated initiator caspase 12 and the downstream effector caspase 3 in these glands; both caspases are known to play a critical role in the signaling mechanisms of apoptosis (Franzen and Brittebo, 2005). However, light microscopic histopathological studies revealed that the Bowman's glands are markedly swollen and their mucus content is lost 4 h after a single injection of 2,6-diClPh-MeSO2, indicating necrotic cell death (Bahrami et al., 2000b; Franzen and Brittebo, 2005).
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The aim of the present study was to examine P450-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). Covalent binding studies were conducted using mouse olfactory and liver S9 fractions, hepatic microsomes from mice treated with the P450-inducing agent pyrazole, known to increase the hepatic expression of CYP2A4, 2A5, 2E1, and 2J (Juvonen et al., 1987; Su et al., 1998; Xie et al., 2000), and microsomes from recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 (Juvonen et al., 1987; Su et al., 1998; Xie et al., 2000). The effects of this olfactory toxicant on the NP-SH levels were examined in the olfactory mucosa and liver. Finally, the early ultrastructural changes in the dorsomedial part of the olfactory region following a single i.p. injection of 2,6-diClPh-MeSO2 in mice were investigated using transmission electron microscopy.
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Materials and Methods |
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Chemicals. 2,6-diClPh-MeSO2 was prepared according to published procedures by Professor Åke Bergman and Dr. Christina Larsson (Stockholm University, Stockholm) (Bergman and Wachtmeister, 1987). Chemical purity (>99.5% pure) of the substances was assessed using gas chromatography and gas chromatography-mass spectrometry. 2,6-Dichlorophenyl-14C-methylsulfone (2,6-diClPh-14C-MeSO2, specific activity, 57 mCi/mmol) was synthesized from 14C-methyl iodide and 2,6-dichlorobenzenethiol according to the method of Bergman and Wachtmeister (1987). 5,5'-Dithio-bis(2-nitrobenzoic acid) was obtained from Sigma-Aldrich (Stockholm, Sweden). Glucose 6-phosphate, glucose-6-phosphate dehydrogenase, NADP+, glutathione, coumarin, and metyrapone were obtained from Sigma-Aldrich (Stockholm, Sweden), and 2,6-dichlorobenzonitrile (dichlobenil) was obtained from Sigma-Aldrich Chemie (Steinheim, Germany), and chlorzoxazone was obtained from Acros Organics (Geel, Belgium). The epoxy embedding resin, TAAB 812, was purchased from TAAB Laboratories Equipment (Aldermaston, UK). Zymolyase 100-T was obtained from MP Biomedicals (Irvine, CA), and yeast nitrogen base without amino acids was obtained from Sigma-Aldrich (Stockholm, Sweden).
Preparation of S9 Fractions and Microsomes. NMRI mice were anesthetized with gaseous carbon dioxide and exsanguinated. The olfactory mucosa and pieces of the livers were dissected, pooled, homogenized in ice-cold KCl (1.15%), and centrifuged at 9000g for 10 min. The pellets were dissolved in ice-cold 0.5 M Tris, pH 7.5, sampled for protein determination, and stored at –70°C until use. DBA/2J mice were given three consecutive daily i.p. injections with pyrazole (200 mg/kg; dissolved in saline) or vehicle (saline). The mice were killed 24 h after the last injection, and the livers were dissected, pooled, and homogenized in ice-cold KCl buffer. The homogenates were centrifuged at 12,000g for 20 min, and the resulting supernatants were collected and centrifuged at 100,000g for 60 min. The pellets were dissolved in ice-cold potassium phosphate buffer, pH 7.4, sampled for protein determination, and stored at –70°C until use. The protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Yeast Cells (Saccharomyces cerevisiae). Recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 and wild-type (wt) yeast cells (Pelkonen et al., 1994) were grown in cell medium containing yeast nitrogen base without amino acids, glucose, and histidine. After full growth, the cells were centrifuged, suspended in cell medium containing 20% glycerol, and stored at –70°C until use. The expression of CYP2A5 and CYP2A6 had been confirmed previously (Pelkonen et al., 1994) and was monitored by addition of the CYP2A5/6 substrate coumarin and determination of the fluorescent metabolite 7-hydroxycoumarin.
The pellets from the yeast cell culture were thawed and incubated for 90 min in 30°C in zymolyase buffer and zymolyase 100-T. After zymolyase digestion, the cells were centrifuged, rinsed in zymolyase buffer, sonicated five times for 1 min each and centrifuged at 10,000g for 20 min. The resulting supernatant was collected and centrifuged at 100,000g for 60 min. The pellets were resuspended in potassium phosphate buffer, pH 7.4, containing 20% glycerol, sampled for protein determination, and stored at –70°C until use. The protein concentration was determined as described above.
Determination of Covalent Binding in Vitro. Covalent protein binding was determined according to the method of Wallin et al. (1981) with a few modifications, as follows. Incubations were made in capped tubes containing 50 to 100 µg protein of NMRI mouse liver and olfactory mucosa S9 fraction, pyrazole- or vehicle-pretreated DBA/2J mice liver microsomes, or yeast cell microsomes, a NADPH-generating system (5 mM MgCl2, 5 mM glucose 6-phosphate, 0.06 U glucose-6-phosphate dehydrogenase, and 1 mM NADP+), 100 mM Tris buffer (pH 7.5), or 100 mM phosphate buffer (pH 7.4), and 2,6-diClPh-14C-MeSO2 (1.3–750 µM) dissolved in 2.5 µl of dimethyl sulfoxide (DMSO) in a final volume of 100 µl. The effects of the broad-spectrum P450 inhibitor metyrapone (100 µM), the CYP2E1 substrate chlorzoxazone (100 µM), and the CYP2A5/CYP2G1 substrates coumarin (100 µM) and dichlobenil (100 µM) on the covalent binding of 2,6-diClPh-14C-MeSO2 (1.3–5.4 µM) were investigated. Results were analyzed by two-tailed, unpaired t test, and a value of p < 0.05 was considered significant. The S9 fractions were preincubated with the substrates/inhibitors (dissolved in DMSO, final concentration 2%) or vehicle for 10 min at 37°C before 2,6-diClPh-14C-MeSO2 was added. DMSO was selected as vehicle based on a previous study on the inhibitory effects of various organic solvents on the metabolism of this compound (Franzen et al., 2003). Vials kept on ice were used as negative controls. The incubations were stopped by placing the vials on ice and transferring 80 µl of the incubation mixture to a glass microfiber filter paper (Whatman GF/C, diameter 25 mm). To remove unbound parent 2,6-diClPh-14C-MeSO2 and metabolites, each filter paper was stepwise extracted in 10 ml of solvent with occasional agitations, once in 95% ethanol, twice in methanol, twice in acetone, and once in n-heptane. Each extraction lasted for 10 min. The filters were then dried at room temperature. Ultima Gold (PerkinElmer Life and Analytical Sciences, Boston, MA) was added and the emittance of radioactivity was measured in a liquid scintillator analyzer (Tri-Carb 1900CA; PerkinElmer Life and Analytical Sciences).
Analysis of Kinetic Data. Apparent Km and Vmax values for covalent binding were estimated by fitting the observed velocity (pmol/min/mg protein) versus the 2,6-diClPh-MeSO2 concentrations (1.3–750 µM) to the Michaelis-Menten equation. The kinetic parameters were determined for olfactory mucosa S9 fractions, pyrazole-pretreated liver microsomes, and CYP2A5 yeast cell microsomes using the software program GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA).
Determination of NP-SH. Groups of mice (n = 6/group) were given a single i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg body weight) or vehicle (corn oil, 10 ml/kg body weight). The animals were killed 1 or 4 h after injection by exposure to gaseous carbon dioxide and decapitated. The olfactory mucosa and samples of the liver were rapidly excised. Tissues from two animals were pooled and homogenized in ice-cold Na2-EDTA. NP-SH concentrations were then determined by the method of Sedlak and Lindsay (1968). Protein concentrations were measured as described above. Results were analyzed by two-tailed, unpaired t test, and a value of p < 0.05 was considered significant. The study was repeated once.
Electron Microscopy. Mice (n = 3/group) were injected i.p. with 2,6-diClPh-MeSO2 (32 mg/kg body weight) or vehicle (corn oil, 10 ml/kg body weight). One and 4 h after administration, the mice were killed by exposure to gaseous carbon dioxide and quickly decapitated. The entire nasal region was dissected, gently flushed through the nasopharyngeal duct, and fixed overnight in ice-cold phosphate-buffered formaldehyde/glutaraldehyde (1.5%/1.5%) (pH 7.4). The fixed nasal regions were decalcified with a formaldehyde/glutaraldehyde solution containing Na2-EDTA (5.5%) for 1 week. After decalcification, a thin slice was cut at the second palatal ridge (level 3) according to the method of Young (1981). The slices were rinsed in three changes of phosphate-buffered formaldehyde/glutaraldehyde (pH 7.4) for a total of 30 min. In addition, the slices were treated with osmium tetroxide (1%, 2 h at 4°C), rinsed in phosphate-buffered saline buffer, dehydrated in series with ethanol, treated with acetone, and finally embedded on silicon plates in TAAB 812 resin. Sections (2 µm) were cut and stained with methylene blue to determine the area of examination (the dorsal meatus) in a light microscope. Ultrathin sections (50 nm) were then cut with a diamond knife. The sections were placed on copper grids, counterstained with uranyl acetate (4%, 30 min) and lead citrate (0.1 M, 5 min), and examined in a Philips CM10 transmission electron microscope at 60 keV (Philips, Eindhaven, the Netherlands).
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There was also a high concentration-dependent covalent binding of 2,6-diClPh-14C-MeSO2 to microsomes from yeast cells transfected with CYP2A5 as compared with microsomes from wt yeast. The predicted Km and Vmax values were 163.6 ± 20.7 µM and 11.4 ± 0.8 pmol/min/mg protein, respectively. The covalent binding of 2,6-diClPh-14C-MeSO2 to recombinant CYP2A6 yeast microsomes was less pronounced but higher than that in microsomes from wt yeast (Fig. 2D).
Determination of NP-SH
The NP-SH levels in the olfactory mucosa of 2,6-diClPh-MeSO2-treated mice (32 mg/kg) were reduced to 61 and 45% of the vehicle-treated control at 1 and 4 h after a single injection (1 h, p < 0.05; 4 h, p < 0.01). In contrast, no statistically significant change of the NP-SH levels was observed in the liver, 84 and 104%, respectively, as compared with vehicle-treated controls. The level of NP-SH in vehicle-treated mice was higher in the liver than in the olfactory mucosa, 982 ± 151 and 209 ± 70 nmol of NP-SH/mg of protein, respectively.
Electron Microscopy
Vehicle-Treated Control Mice. The ultrastructural features of the olfactory mucosa were similar to those previously reported (Frisch, 1967; Bergstrom et al., 2003). In the lamina propria, numerous Bowman's glands, blood vessels, and axon bundles were present. The pyramidal cells of Bowman's glands were characterized by large electron-lucent secretory granules, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and mitochondria throughout the cytoplasm (Fig. 3A; see Fig. 5D). Excretory ducts of the Bowman's glands were crossing the basement membrane passing through the neuroepithelium to the luminal surface. The duct cells close to the basement membrane contained electron-lucent secretory granules, mitochondria, SER, and RER. The apical duct cells were flat with no secretory granules. The flat duct cells had microvilli and a thin layer of electron-dense cytoplasm containing SER and RER.
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2,6-diClPh-MeSO2-Treated Mice. Bowman's Glands and Excretory Ducts. One hour after administration, there were vacuoles in intraepithelial excretory duct cells above the basement membrane (Fig. 4A). Some mitochondria in these cells were swollen and contained flocculent densities. In acinar cells in the lamina propria the secretory granule membranes were ruptured, resulting in dispersed granules throughout the whole cytoplasm. The mitochondria were swollen and contained flocculent densities (Figs. 4C and 5E). The cytoplasm was vacuolated, the SER was severely swollen, and both undamaged and swollen RER were present (Fig. 5E). After 4 h, the size of the vacuoles had increased in the intraepithelial excretory duct cells (Fig. 3B), and most cytoplasmic organelles including mitochondria, SER, and RER were severely swollen. In the lamina propria the individual acinar cells were difficult to distinguish and the radial cell organization was seldom discernible in the acini. Secretory granule content was dispersed throughout the cytoplasm (Figs. 3B and 5G). The mitochondria were severely swollen and electron-lucent with some flocculent densities. The cytoplasm was vacuolated, the SER was severely swollen, and both undamaged and swollen RER were present. The acinar cell nuclei were irregular, and the nuclear chromatin was aggregated and attached to the nuclear membrane (Fig. 5, F and H).
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Sustentacular Cells. After 1 h, a few mitochondria in the apical part of the sustentacular cells were swollen, were less electron-dense, and contained flocculent densities. Vacuoles and swelling of some SER and RER were also observed in the apical part (Figs. 4B and 5B). After 4 h, most of the mitochondria in the apical part of the cells were severely swollen, were less electron-dense, and contained flocculent densities. Both RER and SER were swollen (Fig. 5C). The cell nuclei were, first, irregular (1 h) and, later on, regular and swollen (4 h), and the cytoplasm was more electron-lucent. Large vacuoles appeared in the apical parts. At both survival times, many severely swollen mitochondria and large vacuoles occurred in the basal part of the neuroepithelium, but it was not possible to elucidate whether these organelles belonged to the foot processes of the sustentacular cells or not. No apoptotic bodies were observed among these cells.
Neurons. Most neurons had an intact appearance, and no marked changes were observed 1 and 4 h after administration. An expansion of the intercellular space between the columnar rows of neurons could, however, be observed. Scattered apoptotic bodies were also present.
Basal Cells. After 1 h, some HBCs had an electron-lucent cytoplasm (Fig. 4A). After 4 h, most of the HBCs showed an electron-lucent cytoplasm and cell nuclei (Fig. 3B). Most of the GBCs had an intact appearance.
Other Tissue Structures. The blood vessels were dilated 1 and 4 h after administration. The axon bundles appeared intact at both survival times.
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; Piras et al., 2003; Ling et al., 2004). The present results also demonstrated that a single injection of a toxic dose of 2,6-diClPh-MeSO2 induced a tissue-selective depletion of olfactory NP-SH levels and early swelling of ER and mitochondria, as well as rupture of secretory granules in non-neuronal cell types in the dorsomedial part of the mouse olfactory region. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2.
The initial 2,6-diClPh-MeSO2-induced damage preferentially occurs in the rodent olfactory region, whereas no damage can be noted in the liver (Bahrami et al., 1999). The results of the present study confirm that the bioactivation of 2,6-diClPh-MeSO2 is very low in the mouse liver. To investigate the role of specific P450s in the bioactivation of 2,6-diClPh-MeSO2, covalent binding studies were conducted using hepatic microsomes from pyrazole-treated mice. Pyrazole is known to increase the hepatic expression of CYP2A4, 2A5, 2E1 and 2J (Juvonen et al., 1987; Su et al., 1998; Xie et al., 2000). There was a significantly increased level of covalent binding of 2,6-diClPh-MeSO2 to hepatic microsomes of pyrazole-treated mice demonstrating that pyrazole-inducible hepatic P450s can bioactivate this toxicant. Addition of the CYP2E1 substrate chlorzoxazone markedly decreased the covalent binding to microsomes of pyrazole-treated mice, suggesting that CYP2E1 also may bioactivate 2,6-diClPh-MeSO2. However, since chlorzoxazone did not significantly change the covalent binding of 2,6-diClPh-MeSO2 to olfactory S9 fractions, the contribution of CYP2E1 in the olfactory metabolism of 2,6-diClPh-MeSO2 appears to be negligible. Furthermore, the liver normally contains relatively high levels of CYP2E1, and if this P450 form plays a major role in the toxicity of 2,6-diClPh-MeSO2, hepatic damage would be expected. However, no morphological lesion in the liver could be observed.
To further clarify the role of CYP2A5 in the bioactivation of 2,6-diClPh-MeSO2, microsomes from recombinant yeast cells expressing CYP2A5 were used. The marked covalent binding detected confirmed that this enzyme bioactivates 2,6-diClPh-MeSO2 to a reactive intermediate that binds covalently to protein in ER. In recombinant yeast cell microsomes expressing human CYP2A6, the bioactivation of 2,6-diClPh-MeSO2 was less prominent but higher than that in wt yeast microsomes. The toxicity of 2,6-diClPh-MeSO2 in CYP2A6-expressing human tissue has not been examined. The marked metabolic activation demonstrated in olfactory S9 fractions was significantly reduced by the CYP2A5/2G1 substrates dichlobenil and coumarin, supporting the involvement of at least one of these enzymes in the bioactivation of the compound.
The present study also revealed a significant depletion of NP-SH in the olfactory mucosa following a single dose of 2,6-diClPh-MeSO2 in mice. No similar reduction was observed in liver, confirming a tissue-selective effect. This is in analogy with previous results showing that addition of GSH, but not methyl-GSH, significantly reduces the covalent binding of 2,6-diClPh-MeSO2 to rat olfactory microsomes (Franzen et al., 2003). The lack of effect of methyl-GSH implies that the putative electrophilic 2,6-diClPh-MeSO2 intermediate has affinity for cysteinyl thiols.
We have recently reported that a single dose of 2,6-diClPh-MeSO2 induces an up-regulation of the ER stress protein GRP78, and activation of the initiator caspase 12 and the downstream effector caspase 3 in the Bowman's glands of mice (Franzen and Brittebo, 2005). These results imply an early activation of apoptotic pathways. The present study revealed that a single dose of 2,6-diClPh-MeSO2 induced early and severe swelling of both ER and mitochondria in the mucus producing Bowman's glands. The swelling of these organelles was not accompanied by an extensive formation of apoptotic bodies, although scattered cells with condensed nuclei were found. On the contrary, most of the Bowman's gland cells rapidly became severely swollen, the cell membrane integrity was ruptured, and 4 h after dosing, the radial organization of the Bowman's gland acini was completely lost. The extensive formation of reactive intermediates of 2,6-diClPh-MeSO2 in ER, i.e., at the principal site of P450 localization, may be of importance for the early changes in this organelle. Notably, CYP2A5 has also been detected in the mitochondrial fraction of rodent liver tissue (Honkakoski et al., 1988). The observed ultrastructural changes are consistent with previous light microscopic findings showing a severe swelling of Bowman's gland acini and suggest that the terminal phase of apoptosis was not completed. Increased levels of cytosolic Ca2+ from damaged ER stores and/or pertubation of cellular sulfhydryls will lead to insufficient ATP levels to execute the apoptotic pathway of cell death (Nicotera and Melino, 2004). In addition, caspases may be inactivated by electrophilic compounds with an affinity to thiols, allowing caspase-independent pathways of cell death to prevail (Finkelstein et al., 2001).
The Bowman's glands contain a large amount of secretory granules and contribute to the composition of the mucus covering the luminal surface of the neuroepithelium. Electron microscopy revealed an early loss of secretory granules in the Bowman's glands, demonstrating that these glands are sensitive targets for 2,6-diClPh-MeSO2-induced toxicity. The secretory granule membranes were severely ruptured and the granule contents were dispersed in the cytosol. These results confirm previous light microscopic reports suggesting that loss of secretory granule contents is an early marker for a disturbed function in this cell type (Brandt et al., 1990; Bahrami et al., 2000b; Franzen and Brittebo, 2005).
No early changes were observed in the olfactory neurons except for an increased intercellular space between the columnar rows of neurons following exposure to 2,6-diClPh-MeSO2. In addition, large vacuoles in the basal part of the neuroepithelium were evident, probably due to degeneration of excretory ducts of Bowman's glands and/or foot processes of sustentacular cells.
The sustentacular cells in the neuroepithelium also showed early-onset changes such as severely swollen ER and mitochondria after injection of 2,6-diClPh-MeSO2. In addition, the apical cell membrane showed early and extensive damage. Similar results, indicating that both the sustentacular cells and the Bowman's glands are early target cells in the olfactory mucosa, have been observed after systemic exposure to other olfactory toxicants such as 3-methylindole and a phosphodiesterase inhibitor (Pino et al., 1999; Miller and O'Bryan, 2003). The earliest ultrastructural change detected in these cells is dilatation of ER.
No apoptotic bodies were observed in the sustentacular cells following exposure to 2,6-diClPh-MeSO2. This is consistent with our previous data demonstrating that there is no early up-regulation of ER stress protein or activation of caspases in the sustentacular cells and indicate that other cell death pathways occur in this cell type (Franzen and Brittebo, 2005). The severe damage in the sustentacular cells may be secondary to the deleterious changes in the mucus-producing Bowman's glands. Changed properties of the protective mucus may lead to increased exposure of the sustentacular cells to inhaled air, and changes of apical cell membrane integrity may lead to perturbations of ion homeostasis and organelle swelling. There is a high expression of CYP2A5 in the apical part of the sustentacular cells (Piras et al., 2003). Notably, however, there is no accumulation of protein adducts in the sustentacular cells after injection of 2,6-diClPh-MeSO2 in mice and rats, although there are high levels of adducts in the Bowman's glands (Bahrami et al., 2000b; Franzen et al., 2003). The lack of 2,6-diClPh-MeSO2 adducts in the sustentacular cells suggests that 2,6-diClPh-MeSO2 is rapidly detoxified by other xenobiotic-metabolizing enzymes or that reactive intermediates in these cells are rapidly detoxified by, for instance, GSH.
In conclusion, the present study demonstrates that the olfactory toxicant 2,6-diClPh-MeSO2 was bioactivated by recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 and that CYP2A5/2G1 substrates but not a CYP2E1 substrate reduced the bioactivation in mouse olfactory S9 fraction. The results also show that a single injection of this toxicant in mice induced a tissue-selective depletion of olfactory NP-SH levels and an early-onset swelling in the ER and mitochondria of the Bowman's glands and the sustentacular cells in the olfactory epithelium. Secretory granule membranes were also identified as early targets in the Bowman's glands. We propose that the extensive olfactory lesions induced by 2,6-diClPh-MeSO2 were initiated by a P450-mediated bioactivation and adduct formation in the Bowman's glands accompanied by a depletion of NP-SH, resulting in perturbations of ion homeostasis, organelle swelling, and cell death. The predominant expression of CYP2A5 in the mouse olfactory mucosa is suggested to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2 at this site.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: P450, cytochrome P450; 2,6-diClPh-MeSO2, 2,6-dichlorophenyl-methylsulfone; DMSO, dimethyl sulfoxide; ER, endoplasmic reticulum; GBC, globose basal cell; GSH, glutathione; HBC, horizontal basal cell; NP-SH, nonprotein sulfhydryl; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; wt, wild type.
1 Current affiliation: Swedish Medical Products Agency, Uppsala, Sweden. 
Address correspondence to: Eva B. Brittebo, Department of Pharmaceutical Biosciences, Uppsala University, Box 594, SE-751 24 Uppsala, Sweden. E-mail: Eva.Brittebo{at}farmbio.uu.se
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