Genotoxic bioactivation of constituents of a diesel exhaust particle extract by the human lung.

The ability of the human lung to catalyze genotoxic bioactivation of constituents of diesel exhaust particle (DEP) extract (DEPE) and the identity of the lung enzymes involved in the bioactivation were investigated using human lung tissues obtained from surgical resections. Genotoxicity was determined by lung S9-catalyzed mutagenicity of DEPE constituents to Salmonella typhimurium TA98NR in the Ames test and by DEPE-induced pneumocyte DNA damage response as determined by γH2Ax expression in ex vivo tissues. S9 was prepared from lung explants treated ex vivo with either DEPE to induce pulmonary enzymes (DEPE-S9) or vehicle only (CON-S9). TA98NR served as the tester strain for the purpose of enhancing and minimizing the contribution of lung S9 and Salmonella, respectively, to DEPE bioactivation. DEPE-S9 was 2.2-fold more active than CON-S9 or rat liver S9 in DEPE bioactivation and the bioactivation was inhibited 58, 45, 22, and 16% by α-naphthoflavone, dicumarol, ketoconazole, and ticlopidine, respectively. Alveolar S9 was less active than bronchioalveolar S9 in DEPE bioactivation. DEPE and diesel exhaust particles (DEP) induced γ-pH2Ax expression in pulmonary cells. Pulmonary CYP1A1 and NQO1 were induced by DEPE treatment, with the constitutive and induced CYP1A1 distributed throughout all peripheral lung regions, whereas NQO1 was limited in distribution to bronchiolar epithelium. The results show that the human lung is highly active in catalyzing genotoxic bioactivation of diesel emission constituents and that CYP1A and NQO1 play major roles in the reaction. The findings underscore the usefulness of human lung tissues in studies of the pneumotoxicity potential of chemicals to humans.

Cytochromes P4501 (CYP1): catalytic activities and inducibility by diesel exhaust particle extract and benzo[a]pyrene in intact human lung ex vivo.

Catalytic activities of CYP1A1, CYP1A2 and CYP1B1, and inducibility of the activities were studied in intact human lung samples (from 23 human subjects) ex vivo. The activities [as measured by ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and 3-cyano-7-ethoxycoumarin O-deethylase (CECOD)] were present in the lungs and were induced by benzo[a]pyrene (B[a]P) and diesel exhaust particle extract (DEE) but with extensive inter-subject variability. All three activities were substantially inhibited (>or=75%) by the CYP1 inhibitor alpha-naphthoflavone, whereas only MROD and CECOD were substantially inhibited by the CYP1A2-preferential inhibitor fluvoxamine. None of the three activities was substantially inhibited by the CYP1B1-preferential inhibitor tetramethoxystilbene, indicating lack of involvement of CYP1B1 in the activities in the intact lung. CYP1A1 and CYP1A2 proteins were present in the lungs (by western blot analysis), also with extensive inter-subject variability, and were induced by B[a]P or DEE more extensively than by the combination of B[a]P and DEE (B[a]P+DEE). CYP1B1 was also present in the lungs and its level varied extensively between subjects. In contrast with CYP1A and CYP1A2 levels, CYP1B1 level was not significantly altered by B[a]P or DEE treatment and was diminished more extensively by treatment with B[a]P+DEE. The findings point to the potential usefulness of the intact lung for assessing in situ xenobiotic biotransformation reactions as well as the CYP1 specificity of the reactions ex vivo. The findings also suggest MROD and CECOD as potential markers of CYP1A2 activity in the intact lung.

Effect of n-hexane on the disposition and toxicity of the 1,3-butadiene metabolite 3-butene-1,2-diol.

3-Butene-1,2-diol (butenediol), a major metabolite of 1,3-butadiene (butadiene), can undergo either detoxification or biotransformation to potentially toxic metabolites, including 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone (HMVK). Butadiene exposure can occur concomitantly with hexanes, which share common biotransformation pathways with butadiene. To determine the potential influence of hexane co-exposure on butadiene toxicity, the present study examined the effect of n-hexane on butenediol disposition [as measured by urinary excretion of (N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine) (MI level)] and genotoxicity (as measured by the frequency of bone marrow micronucleated erythrocytes) and acute toxicity (as measured by body weight changes) in the rat. The results show that butenediol was not genotoxic to adult or immature rats but was acutely toxic to adult but not immature rats. The results also suggest that n-hexane co-exposure may attenuate the acute toxicity by butenediol in adult rats and that immature rats may be less sensitive than adults to the acute toxicity.

Differential inducibility of rat pulmonary CYP1A1 by cigarette smoke and wood smoke.

Mainstream cigarette smoke (CS) and wood smoke (WS) were compared in terms of their pulmonary CYP1A1 inducibility. The inducibility was assessed in pulmonary microsomes from rats exposed to freshly generated CS or WS and in rat lung explants treated with extracts of CS or WS total particulate matter (TPM). Mutagenicity in Salmonella typhimurium TA98 and TA100, an effect established for CS and WS in previous studies, was also examined as a test of the biological activity of the smoke samples in the present study. Pulmonary microsomal CYP1A1 activity (as measured by ethoxyresorufin O-deethylase), was induced 4.4-fold and 8.3-fold following exposure of rats to smoke from a single cigarette and three cigarettes, respectively, relative to the activity in control rats. The induction was paralleled by elevated CYP1A1 mRNA level (by northern blot analysis). WS, in contrast to CS, induced neither pulmonary CYP1A1 activity nor mRNA in exposed rats. CYP1A1 protein (by western blot analysis) was induced in cultured rat lung explants by extracts of CS TPM or by a high concentration (496 nM) of benzo[a]pyrene (B[a]P) but not by extracts of WS TPM or a low concentration (0.110 nM) of B[a]P. The induction by high B[a]P concentration was inhibited by extracts of CS or WS TPM, with the inhibition by extracts of WS TPM (75%) being greater than that by extracts of CS TPM (31%). Extracts of CS TPM were as mutagenic as extracts of WS TPM to Salmonella typhimurium TA98 but were more mutagenic than extracts of WS TPM to Salmonella typhimurium TA100. The results show that CS and WS are mutagenic but that WS differs from CS in its inability to induce pulmonary CYP1A1.

Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Determination of nicotine metabolism/pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. METHODS: We developed a sensitive method for analysis of nicotine and five major nicotine metabolites, including cotinine, trans-3'-hydroxycotinine, nicotine-N'-oxide, cotinine-N-oxide, and nornicotine, in human urine by liquid chromatography coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (LC/MS/MS). Urine samples to which deuterium-labeled internal standards had been added were extracted with a simple solid-phase extraction procedure. Anabasine, a minor tobacco alkaloid, was also included. RESULTS: The quantification limits of the method were 0.1-0.2 microg/L, except for nicotine (1 microg/L). Cotinine-N-oxide, trans-3'-hydroxycotinine, nicotine, and anabasine in urine were almost completely recovered by the solid-phase extraction, whereas the mean extraction recoveries of nicotine-N'-oxide, cotinine, and nornicotine were 51.4%, 78.6%, and 78.8%, respectively. This procedure provided a linearity of three to four orders of magnitude for the target analytes: 0.2-400 microg/L for nicotine-N'-oxide, cotinine-N-oxide, and anabasine; 0.2-4000 microg/L for cotinine, nornicotine, and trans-3'-hydroxycotinine; and 1.0-4000 microg/L for nicotine. The overall interday method imprecision and recovery were 2.5-18% and 92-109%, respectively. CONCLUSIONS: This sensitive LC/MS/MS procedure can be used to determine nicotine metabolism profiles of smokers, people during nicotine replacement therapy, and passively exposed nonsmokers. This method avoids the need for a time-consuming and labor-intensive sample enrichment step and thus allows for high-throughput sample preparation and automation.

Constitutive and inducible levels of CYP1A1 and CYP1A2 in rat cerebral cortex and cerebellum.

We examined the constitutive and inducible levels of microsomal cytochromes P450 1A1 and 1A2 (CYP1A) in rat cerebral cortex and cerebellum at the level of proteins by western blot analysis, and by catalytic activities via ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD). In the cerebral cortex, cytochrome P450 1A1 (CYP1A1) protein was more abundant than cytochrome P450 1A2 (CYP1A2) protein. Treatment with beta-naphthoflavone (beta-NF) caused a slight decrease in the level of the former but induced the latter 5.8-fold. In the cerebellum, in contrast to the cerebral cortex, CYP1A1 protein was less abundant than CYP1A2 protein in untreated rats, and while beta-NF treatment caused a 3.3-fold induction of CYP1A1 protein, it resulted in a 10-fold decrease in CYP1A2 protein. The CYP1A-preferential activity EROD was 2.3-fold higher in the cerebellum than in the cerebral cortex, and was induced 1.5-fold and 1.9-fold in the cerebellum and cerebral cortex, respectively, by beta-NF treatment. The CYP1A2-preferential activity MROD was 3-fold higher in the cerebellum than in the cerebral cortex, and was repressed 2.2-fold in the cerebellum but induced 3.7-fold in the cerebral cortex following beta-NF treatment. The results show that CYP1A1 and CYP1A2 proteins and catalytic activities are constitutively expressed in brain but are differentially inducible in the rat cerebral cortex and cerebellum.

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.

Induction of CYP1A1 and CYP1A2 expressions by prototypic and atypical inducers in the human lung.

The inducibility of cytochrome P4501A1 gene (CYP1A1) expression was examined in human lung samples from 27 subjects, using an explant culture system and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. CYP1A1 transcripts were present in all of the lung specimens and were induced by the prototypic inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (B[a]P), and by the atypical inducers pyridine, nicotine, and omeprazole. 2-Hydroxypyridine was a better inducer than pyridine, implicating metabolites in CYP1A1 induction by the parent compound. The prototypical inducers were the most effective inducers in many samples but were ineffective in some samples in which the atypical compounds were effective inducers. Cytochrome P4501A2 (CYP1A2) transcripts were also detected in most of the lung specimens and were inducible in some specimens. The results show the suitability of the explant culture system for examining the inducibility of human pulmonary CYP1A1 and CYP1A2, indicate the heterogeneity in individual sensitivity to the induction, and underscore the need to include atypical inducers in studies of CYP1A inducibility in humans.