Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa

Chen, Ying; Liu, Yi Qing; Su, Ting; Ren, Xiang; Li, Shi; Liu, Dazi; Gu, Jun; Zhang, Qing Yu; Ding, Xinxin

doi:10.1016/s0006-2952(03)00476-3

Cited by 34 publications

(41 citation statements)

References 26 publications

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“…A densitometric analysis of the data shown in Fig. 2B (and similar data from additional experiments that are not presented) suggested that the content of CYP2A13 protein in mouse NM microsomes was ϳ100 pmol/mg protein, a level similar to that of CYP2A5 protein in mouse NM microsomes determined in a previous study (Gu et al, 1998) and much higher than the levels detected in human fetal or adult NM microsomes (Ͻ5 pmol/mg protein) (Chen et al, 2003;Wong et al, 2005).…”

Section: Resultssupporting

confidence: 83%

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Wei

Lei

et al. 2012

Drug Metab Dispos

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ABSTRACT:CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (ϳ100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (ϳ0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10-and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (ϳ0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

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Section: Resultssupporting

confidence: 83%

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Wei

Lei

et al. 2012

Drug Metab Dispos

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“…Bovine serum albumin (BSA), thiourea, and dithiothreitol (DTT) were ordered from Sigma-Aldrich. IsoGel low EEO agarose was ordered from BMA (Rockland, ME), 14 C-1-NN was synthesized as previously described (13). Rhinohide was purchased from Molecular Probes (Eugene, OR).…”

Section: Chemicalsmentioning

confidence: 99%

“…Transitional epithelium (from maxilloturbinates), respiratory epithelium (from nonolfactory septal epithelium), and olfactory epithelium (from septum) was isolated through microdissection from rats exposed to 14 C-labeled 1-NN (Table 1). Samples were homogenized in lysis buffer (2 M thiourea, 7 M urea, 4% wt/vol CHAPS, 0.5% wt/vol Triton X-100, 1% wt/vol [65 mM] DTT, and 2% vol/vol protease inhibitor cocktail III) as previously described (6).…”

Section: Determination Of Total 14 C-1-nn Protein Bindingmentioning

confidence: 99%

“…Alterations in the adduction of specific protein species due to prior long-term ozone exposure were primarily observed in the NTE epithelium (Figure 6), but not in OE and RE. A total of 10 specific protein spots adducted by 14 C-1-NN metabolites were discernible on the storage phosphorimages. The location of six of these spots ( Figure 6, spots 1-6) corresponded with proteins previously shown to be adducted both in target tissue (airway epithelium) and nontarget tissue (liver) (6).…”

Section: -Nn Metabolite Bindingmentioning

confidence: 99%

“…Human nasal mucosa also shows P450 expression and activity (10)(11)(12)(13)(14), implying that nasal mucosa may be a major site of in situ 1-NN metabolism and toxicity also in humans (15). Activities of CYP2B1, reportedly the main isoform responsible for 1-NN activation (16), have been shown to be 20% higher in rat nasal mucosa than in lung parenchyma, and 2-fold higher than in liver (15).…”

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confidence: 99%

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Long-Term Ozone Exposure Attenuates 1-Nitronaphthalene–Induced Cytotoxicity in Nasal Mucosa

Lee

Wheelock

Boland

et al. 2008

Am J Respir Cell Mol Biol

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1-Nitronaphthalene (1-NN) and ozone are cytotoxic air pollutants commonly found as components of photochemical smog. The mechanism of toxicity for 1-NN involves bioactivation by cytochrome P450s and subsequent adduction to proteins. Previous studies have shown that 1-NN toxicity in the lung is considerably higher in rats after long-term exposure to ozone compared with the corresponding filtered air-exposed control rats. The aim of the present study was to establish whether long-term exposure to ozone alters the susceptibility of nasal mucosa to the bioactivated toxicant, 1-NN. Adult male Sprague-Dawley rats were exposed to filtered air or 0.8 ppm ozone for 8 hours per day for 90 days, followed by a single treatment with 0, 12.5, or 50.0 mg/kg 1-NN by intraperitoneal injection. The results of the histopathologic analyses show that the nasal mucosa of rats is a target of systemic 1-NN, and that long-term ozone exposure markedly lessens the severity of injury, as well as the protein adduct formation by reactive 1-NN metabolites. The antagonistic effects were primarily seen in the nasal transitional epithelium, which corresponds to the main site of histologic changes attributed to ozone exposure (goblet cell metaplasia and hyperplasia). Long-term ozone exposure did not appear to alter susceptibility to 1-NN injury in other nasal regions. This study shows that longterm ozone exposure has a protective effect on the susceptibility of nasal transitional epithelium to subsequent 1-NN, a result that clearly contrasts with the synergistic toxicological effect observed in pulmonary airway epithelium in response to the same exposure regimen.

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Olfactory Mucosa: Composition, Enzymatic Localization, and Metabolism

Ding

Xie

2015

Handbook of Olfaction and Gustation

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Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa

Cited by 34 publications

References 26 publications

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Generation and Characterization of aCYP2A13/2B6/2F1-Transgenic Mouse Model

Long-Term Ozone Exposure Attenuates 1-Nitronaphthalene–Induced Cytotoxicity in Nasal Mucosa

Olfactory Mucosa: Composition, Enzymatic Localization, and Metabolism

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