Jacqueline Fung

Induction of pulmonary CYP1A1 by nicotine

1. We have examined the catalytic activities (7-ethoxyresorufin O-deethylase [EROD] and 7-methoxyresorufin O-demethylase [MROD]), protein levels (Western blot analysis) and mRNA levels (Northern blot analysis) of cytochrome P4501A (CYP1A1 and CYP1A2) in the lung, liver and kidney following a single 2.5 mg/kg (15.4 micromol/kg) subcutaneous dose of nicotine to the female Sprague-Dawley rat. 2. Only in lung microsomes was EROD activity significantly induced by nicotine treatment. The activity increased 4.4-fold at 6 h after treatment relative to controls, peaked at 12 h at 14.7-fold the control activity and returned to near control level at 24 h. 3. In parallel with EROD activity, CYP1A1 immunoreactive protein abundance was altered significantly by nicotine treatment only in the lung, peaking at 12 h and decreasing towards control levels thereafter. 4. Following subcutaneous nicotine treatment, CYP1A1 mRNA was detectable in the lung at 6 and 12 h but not at 24 h, was slightly elevated in the kidney at 12 h and was detectable in the liver only at the 12-h point. CYP1A2 immunoreactive protein and its mRNA were detectable only in the liver, and their levels were not affected significantly by nicotine pretreatment. 5. Nicotine affected the binding of Hepa 1c1c7 cytosolic protein to a CYP1A1 xenobiotic response element in a gel mobility shift assay, suggesting involvement of the aryl hydrocarbon receptor and transcriptional activation in CYP1A1 induction by the chemical. 6. Inhaled nicotine also induced pulmonary EROD activity, and the induction by either inhaled or injected nicotine was more pronounced in the male than in the female rat. 7. The findings show that nicotine is a potent, rapid but transient inducer of CYP1A1 in the rat lung and suggest that the alkaloid is a likely contributor to CYP1A1 induction by cigarette smoke.

Constitutive and induced expression by pyridine and β-naphthoflavone of rat CYP1A is sexually dimorphic

Abstract Adult male and female Sprague-Dawley rats were compared in terms of the constitutive levels and inducibility of CYP1A1 and CYP1A2 (CYP1A) in lung, kidney, and liver. CYP1A were induced by i.p. treatment with pyridine (75 mg/kg per day) or β-naphthoflavone (βNF; 25 mg/kg per day) for two consecutive days and analyzed catalytically (via O-dealkylation of resorufin ethers), at the protein level (by Western blot analysis) and at the mRNA level (by Northern blot analysis). In untreated rats, CYP1A1 protein and its mRNA were detectable only in the lung and kidney of females but not males, whereas CYP1A2 protein and its mRNA were detectable only in the liver in either gender. Pyridine treatment upregulated CYP1A1 mRNA and its protein in the lung, kidney and liver in female rats, and upregulated the mRNA but not the protein in the lung and liver in male rats. Conversely, pyridine induced both CYP1A2 mRNA and protein in the liver in female rats, whereas it induced the protein but not its mRNA in the liver in male rats. No gender difference was observed in the plasma elimination rate of administered pyridine. βNF, in contrast to pyridine, induced CYP1A proteins, activities, and mRNA to higher levels in male than in female rats. The results show that the constitutive as well as inducible expression of CYP1A is sexually dimorphic in the Sprague-Dawley rat, with females being more responsive than males to induction by pyridine but with males being more responsive than females to induction by βNF. The findings support the involvement of different mechanisms in CYP1A induction by pyridine and βNF.

Coordinate up-regulation of CYP1A1 and heme oxygenase-1 (HO-1) expression and modulation of δ-aminolevulinic acid synthase and tryptophan pyrrolase activities in pyridine-treated rats

To determine the changes in heme metabolism associated with induction of cytochrome P450 expression by pyridine, we compared the time course of CYP1A expression with the time course of (i) expression of heme oxygenase-1 (HO-1) (EC 1.14.99.3), (ii) activity of delta-aminolevulinic acid synthetase (ALAS) (EC 2.3.1.37), and (iii) heme saturation of tryptophan pyrrolase (TPO) (EC 1.13.11.11) in tissues of rats administered a single 100 or 150 mg/kg i.p. dose of pyridine. Both mRNA and protein of HO-1 and CYP1A1 were induced in the liver, kidney, and lung, with the induction of HO-1 mRNA preceding and paralleling that of CYP1A1 mRNA in the liver and lung but not kidney. Induction of CYP1A1 mRNA expression peaked within 9-12 hr and returned to control levels by 24 hr in all tissues examined, whereas induction of HO-1 mRNA expression was sustained for 48 hr in the lung and liver. In contrast to the transient up-regulation of CYP1A1 mRNA, increased microsomal CYP1A1 protein was sustained in all three tissues. Similar to the induction of HO-1 expression, lipid peroxidation was stimulated by pyridine treatment in the kidney, lung, and liver, but with the stimulation being more persistent in the liver and lung than in the kidney. Increased hepatic CYP1A1 or CYP1A2 activity was preceded by increased activities of HO-1 and ALAS. Pyridine treatment negatively modulated heme saturation of hepatic TPO. The findings indicate that pyridine stimulates the synthesis, utilization, and degradation of heme in a coordinate manner, and suggest that these alterations in heme metabolism may contribute to CYP1A1 induction by pyridine.

Cytochrome P450 1A1 in rat peripheral blood lymphocytes: inducibility in vivo and bioactivation of benzo[a]pyrene in the Salmonella typhimurium mutagenicity assay in vitro

The presence and inducibility of CYP1A1 in freshly isolated peripheral blood lymphocytes was examined in untreated rats and in rats pretreated with agents known to induce the enzyme in other tissues, as well as dexamethasone [CAS #50-02-2], which is not commonly associated with CYP1A1 induction. CYP1A1 but not CYP1A2 was detected by Western blot analysis of lymphocytes from untreated rats and was induced in lymphocytes from rats treated with the known CYP1A inducers beta-naphthoflavone [CAS #6051-87-2] or 3-methylcholanthrene [CAS #56-49-5] (7.3-fold), cigarette smoke (2. 8-fold), and pyridine [CAS #108-86-1] (2.6-fold). CYP1A1 was also induced in lymphocytes from rats treated with the nonprototypic inducer dexamethasone (17.7-fold) or bromobenzene [CAS #108-86-1] (3. 9-fold). Lymphocyte homogenate from rats treated with the inducers also catalyzed NADPH-dependent bioactivation of benzo[a]pyrene [CAS #50-32-8] to mutagens. The benzo(a)pyrene mutagenicity was detected using Salmonella typhimurium TA100 in the Ames test, and correlated positively with lymphocyte CYP1A1 content. The data show that CYP1A1 is present in rat peripheral blood lymphocytes in vivo, and is inducible by prototypic, as well as nonprototypic, inducers of the enzyme.

CYP1A1 induction by pyridine and its metabolites in HepG2 cells.

Pyridine and its metabolites have been shown in previous studies to induce cytochrome P4501A1 (CYP1A1) expression in vivo in the rat and in vitro in cultured human lung explants. In this study, we assessed the role of the metabolites in CYP1A1 induction by the parent compound. This was accomplished by comparing pyridine, 2-hydroxypyridine, 3-hydroxypyridine, pyridine N-oxide, and N-methylpyridinium in terms of the induction of CYP1A1 mRNA, CYP1A1 catalytic activity, and a xenobiotic response element-directed chloramphenicol acetyltransferase reporter gene, using HepG2 cells as the experimental system. We also assessed the effect of expression of the pyridine-metabolizing enzyme cytochrome P4502E1 on CYP1A1 induction by the parent pyridine. Only 2-hydroxypyridine significantly induced the CYP1A1 mRNA expression and CYP1A1-preferential activity ethoxyresorufin O-deethylase in wild-type HepG2 cells. Similarly, only 2-hydroxypyridine induced the expression of a xenobiotic response element-directed reporter gene in transfected HepG2 cells. Pyridine elevated CYP1A1 mRNA abundance 4.6-fold in HepG2 cells transfected with a human CYP2E1 expression vector relative to the abundance of the transcript in empty vector-transfected (control) HepG2 cells; the elevation was inhibited by the CYP2E1 inhibitor dimethyl sulfoxide. The results indicate that CYP1A1 induction by pyridine is mediated largely by metabolites, the formation of which may be catalyzed by CYP2E1.

Effect of gestational and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on the level and catalytic activities of hepatic microsomal CYP1A in prepubertal and adult rats

We determined the inducibility, as well as the persistence of the induction, of hepatic microsomal CYP1A1 and CYP1A2 (by western blot analysis), and their catalytic activities (as measured by resorufin ether O-dealkylation) in prepubertal (25-day-old) and adult (120-day-old) offspring of timed-pregnant Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD treatment was subcutaneous, at a low dose of 0.1 microg/kg, on gestational days 7, 14, and 20, and on lactational days 7 and 14. CYP1A1 protein was induced significantly (23-fold) in prepubertal but not in adult offspring of TCDD-exposed dams, whereas ethoxyresorufin O-deethylase (EROD) activity, which is CYP1A1-preferential, was induced less extensively (5-fold) and slightly (1.7-fold) in the prepubertal and adult offspring, respectively. Benzyloxyresorufin O-debenzylase (BROD) activity, which is CYP2B-preferential but has been reported to be catalyzed by CYP1A1, was also induced 5- and 6-fold in prepubertal and adult offspring, respectively, of TCDD-exposed dams. However, the induced BROD activity was neither inhibited by antibody against CYP1A1 nor accompanied by an elevated level of microsomal CYP2B. CYP1A2 was induced slightly only in prepubertal offspring of TCDD-treated dams. There was suggestive evidence of enhanced lipid peroxidation in hepatic microsomes from prepubertal but not adult offspring of TCDD-treated dams. These data showed that in utero plus lactational TCDD exposure effected transient induction of hepatic microsomal CYP1A1 but sustained induction of BROD activity, which may be catalyzed by enzymes other than CYP1A or CYP2B.

Induction of pulmonary cytochrome P4501A1: interactive effects of nicotine and mecamylamine.

The effect of the nicotinic receptor antagonist mecamylamine on nicotine-mediated convulsions and induction of pulmonary cytochrome P4501A1 (CYP1A1) was examined in the rat. Mecamylamine blocked the convulsions and inhibited CYP1A1 induction by nicotine at the level of CYP1A1 activity (93%) and protein (97%), but independently induced the enzyme also at the level of activity and protein. The results show that mecamylamine antagonizes both the CYP1A1 induction and convulsions by nicotine but, independently, is an inducer of the enzyme. The results indicate that CYP1A1 induction is not a consequence of the convulsant effects of nicotine.

Pulmonary Cyp1A1 and CYP1A2 levels and activities in adult male and female offspring of rats exposed during gestation and lactation to 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

The levels and activities of pulmonary microsomal CYP1A1 and CYP1A2 in 40-day-old male and female, and 120-day-old male offspring of pregnant rats treated with five weekly 0.1 microg/kg doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during gestation and lactation were compared with those in age-matched offspring of untreated dams. The CYP1A1-preferential activity, ethoxyresorufin O-deethylase (EROD), was comparably induced 5.3- and 6.4-fold in 40-day-old male and female offspring, respectively, but was not induced in 120-day-old male offspring, of TCDD-treated dams. Similarly, CYP1A1 protein was induced in 40-day-old female or male offspring of untreated dams but was undetectable in 120-day-old offspring of untreated or treated dams. CYP1A2 activity, as measured by the bioactivation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to mutagens in the Ames assay, was elevated 11.1- and 5.5-fold in 40-day-old female and male offspring, respectively, of TCDD-treated dams, but was unaffected by TCDD exposure in 120-day-old offspring. CYP1A2 protein was undetectable in 40-day-old male or female offspring of untreated dams or in 120-day-old male offspring of treated or untreated dams; it was detected in 40-day-old offspring of treated dams, at a level that was higher in females than in males. The results show that gestational and lactational exposure to TCDD causes long-lasting and gender-preferential induction of CYP1A1 as well as CYPIA2 in the lungs of rat offspring.