Gene expression profiling in respiratory tissues from rats exposed to mainstream cigarette smoke
Stephan Gebel1,
,
Bernhard Gerstmayer2,
Andreas Bosio2,
Hans-Jürgen Haussmann1,
,
Erik Van Miert3,
and
Thomas Müller1,4,
1 Philip Morris Research Laboratories GmbH, Fuggerstrasse 3, D-51149 Köln, Germany, 2 Memorec Stoffel GmbH, Stöckheimer Weg 1, D-50829 Köln, Germany,+ and 3 Philip Morris Research Laboratories bvba, Grauwmeer 14, B-3001 Leuven, Belgium
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Abstract
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Cigarette smoke (CS) is known to cause cancer and other diseases, but little is known about the global molecular and cellular changes that occur prior to the appearance of clinically detectable symptoms. Using DNA microarrays covering 2031 cDNA probes, we investigated differential gene expression in tissues of the rat respiratory tract, i.e. respiratory nasal epithelium (RNE) and lungs of rats exposed either acutely (3 h) or subchronically (3 h/day, 5 days/week, 3 weeks) to mainstream CS with death either immediately or at 20 h after exposure. Differential gene expression was most evident in RNE of rats exposed once and was characterized by strong up-regulation of genes encoding oxidative stress-responsive and Phase II drug-metabolizing enzymes, such as haem oxygenase-1 and NAD(P)H:quinone oxidoreductase, which are all, at least in part, transcriptionally regulated by NF-E2-related factor 2 (Nrf2). After 3 weeks of exposure, the strength of expression of this class of genes was markedly reduced, pointing to an adaptive response. The generally lower response in the lungs of exposed rats is indicative of a deposition gradient of active smoke constituents from the upper to the lower respiratory tract. In sharp contrast to the CS-induced expression of oxidative stress and Phase II-responsive genes, induction of the genes encoding the Phase I drug-metabolizing enzymes cytochrome P450 (CYP)1A1 and aldehyde dehydrogenase-3 was not reduced after 3 weeks of exposure and was similarly high in lungs and RNE. Gene expression patterns in rats allowed to recover for 20 h showed that the CS-induced transcriptional changes observed immediately after exposure returned almost completely to normal, even after 3 weeks of repeated CS exposure. In general, these results demonstrate that CS induces a specific differential gene expression pattern in vivo, which may be instrumental in identifying the molecular mechanisms leading to the onset of inflammatory and/or morphological changes.
Abbreviations: Ah, aryl hydrocarbon; ALDH3, cytosolic class-3 aldehyde dehydrogenase; ARE, antioxidant response element; aRNA, amplified RNA; B[a]P, benzo[a]pyrene; CS, cigarette smoke; CYP, cytochrome P450; DBP, D-site-binding protein; FIZZ, found in inflammatory zone; GSH, glutathione; HO-1, haem oxygenase-1; HSP, heat shock protein; IL, Interleukin; MT, metallothionein; NQO-1, NAD(P)H:quinone oxireductase; Nrf2, NF-E2-related factor 2; PAHs, polycyclic aromatic hydrocarbons; RELM, resistin-like molecule; RNE, respiratory nasal epithelium; SATT, neutral amino acid transporter A; TPM, total particulate matter; UGT, UDP-glucuronosyltransferase;
GCS,
-glutamyl-cysteine-synthetase;
GT1,
-glutamyl-transpeptidase.
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Introduction
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There is compelling evidence that cigarette smoking causes cancer and other diseases, such as cardiovascular disease and chronic obstructive pulmonary disease (13). However, little is known about the molecular and cellular processes that occur prior to the appearance of clinically detectable symptoms. Previous studies (mainly in vitro) revealed that cigarette smoke (CS) induces significant differential changes in the expression of stress-responsive genes, such as haem oxygenase-1 (ho-1), c-myc, c-jun, c-fos and genes encoding heat shock proteins (HSPs) in cells of various cell lines (47).
In particular, the expression of the proto-oncogene c-fos as a paradigmatic stress reporter gene has been used to evaluate in mechanistic terms the stress signalling activities inherent to aqueous solutions bubbled with CS (5). As demonstrated in cultured murine Swiss 3T3 fibroblasts exposed to subcytotoxic doses of aqueous extracts of CS, c-fos becomes expressed because of the CS-dependent formation of the strong oxidant peroxynitrite (8), which is the reaction product of nitric oxide (NO) and superoxide (O2-). This conclusion was drawn from experiments in which the preparation of aqueous extracts of CS was performed in the presence of either the nitric oxide scavenger oxyhaemoglobin or the superoxide-inactivating enzyme superoxide dismutase, both of which specifically prevented c-fos mRNA formation in exposed cells (8). Because the calculated peroxynitrite concentration potentially formed by CS in aqueous solution was not sufficient to explain the c-fos inducing effects, CS-related aldehydes, such as formaldehyde, acetaldehyde and acrolein, were identified as agonists in the CS-dependent c-fos expression by peroxynitrite. These aldehydes were shown to share peroxynitrite's propensity to efficiently deplete the intracellular glutathione (GSH) concentration (9), which is obviously a prerequisite for enabling peroxynitrite to interfere with specific target molecules in the activation of stress signal transduction and gene expression in CS-treated cells, at least in vitro.
Although these investigations have improved our understanding of the potential contribution of smoke-related chemical compounds in CS-induced cellular stress, they do not provide any information on overall changes in gene expression in exposed cells, which is subject to different signalling pathways addressing various sets of target genes. In fact, our knowledge is especially fragmentary regarding the global changes of gene expression patterns in cells of target tissues and organs exposed to CS, global changes which probably represent the first and most probably fully reversible step in the development of CS-related diseases. This knowledge gap was recently addressed experimentally by applying DNA chip technology to cultured murine fibroblasts exposed to subcytotoxic doses of aqueous extracts of CS (10). Results from this study showed the orchestrated, fine-tuned up-regulation of genes encoding proteins known to combat oxidative stress. This includes genes such as those encoding HO-1, HSPs and metallothioneins (MTs) as well as genes whose protein products have been implicated in the transcriptional regulation of oxidative stress-response genes, e.g. CAAT/enhancer binding protein-ß and JunB. In addition, this study showed the differential up-regulation of genes coding for proteins bearing cell cycle regulatory functions, such as Growth Arrest and DNA Damage (GADD) 34 and GADD 45. A special feature of this experiment was the significant up-regulation of genes described as mediators of an inflammatory/immune regulatory response, e.g. st2, kc and id3, thus demonstrating the pro-inflammatory qualities of CS in an in vitro cell culture system.
In this publication, we report on the extension of our gene expression studies of CS-exposed cells to the in vivo situation. Male SpragueDawley rats were exposed to 100 µg total particulate matter (TPM)/l smoke from the University of Kentucky Reference Cigarette 2R1 for either a single exposure 3 h (acute) or repeated exposure over 3 weeks (3 h/day, 5 days/week; subchronic) with and without a 20-h recovery period before death. Gene expression profiling using microarrays carrying >2000 cDNA probes revealed a distinct pattern of differentially expressed genes in the tissues investigated, i.e. respiratory nasal epithelium (RNE) and lung, from rats killed immediately after final exposure. Special features of this pattern are an apparent adaptive response for genes encoding oxidative stress-related and Phase II drug-metabolizing enzymes and the paramount up-regulation of cytochrome P4501A1 (cyp1a1) in cells of these tissues from exposed rats. In contrast, tissues from rats killed after 20-h recovery showed that nearly all changes observed immediately after exposure had reversed to control levels. In addition, in the course of these investigations, we identified a novel gene strongly related to RELM
/FIZZ1 (resistin like molecule
/found in inflammatory zone), which was originally found to be expressed mainly in lung (11). Notably, this novel gene, termed RELM
, which is characterized in detail elsewhere (12), was identified as the strongest repressed gene in the nose of exposed animals. Altogether, this acute/subchronic study shows that the analysis of differential gene expression in vivo is useful to detect CS-specific stress responses prior to the onset of inflammatory and/or morphological changes.
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Material and methods
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Cigarettes and cigarette smoke generation
The Reference Cigarette 2R1 was obtained from the Tobacco and Health Institute at the University of Kentucky. The cigarettes were conditioned according to ISO standard 3402 (13) and smoked in basic conformity with ISO standard 3308 (14). Mainstream smoke was generated on a 30-port INBIFO smoking machine (Type SM85, Philip Morris Research Laboratories GmbH, Germany) equipped with a 4-piston pump (15).
Mainstream smoke was diluted with filtered, conditioned air to a target concentration of 100 µg TPM/l. To monitor the stability and reproducibility of smoke generation, TPM (100 ± 6 µg/l), CO (51 ± 4 p.p.m.), nicotine (5.93 ± 0.70 µg/l), aldehydes (formaldehyde: 0.16 ± 0.06 µg/l; acetaldehyde: 0.50 ± 0.10 µg/l; acrolein: 4.18 ± 0.57 µg/l) and particle size distribution (mean mass aerodynamic diameter: 0.55 µm; geometric standard deviation: 1.66) as well as temperature and relative humidity in the exposure chambers (within standard limits) were determined (16).
Animals and exposure
Care and use of the rats was in conformity with the American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals (17). Outbred male SpragueDawley rats (Crl:CDBR) were obtained from Charles River Germany (Sulzfeld). The rats were
5 weeks old at arrival and were acclimatized for 5 days prior to exposure. Histopathological evaluation and serological screening confirmed the good health status of the rats at the beginning of the study (16).
Four rats per group were exposed in whole-body chambers to diluted mainstream smoke (100 µg TPM/l) or to conditioned fresh air (control) either once (3 h) or for 3 weeks (3 h/day, 5 days/week) and killed either immediately or after a 20-h recovery period. No significant body weight effects were seen during the 3-week inhalation period.
RNA extraction from respiratory tissues
Lung tissue was frozen in liquid nitrogen immediately after the dissection and stored at -70°C. For total RNA preparation,
100 mg frozen tissue was homogenized in RLT buffer with ß-mercaptoethanol (ß-ME) (Qiagen, Hilden, Germany). RNE was homogenized immediately after the preparation in RLT buffer with ß-ME. Further purification of total RNA from both tissues was done according to the manufacturer's manual (RNAeasy-Kit, Qiagen). Equal amounts of RNA from the four animals per group were pooled, DNase I digested and extracted with phenolchloroform. Qualitative integrity tests and quantitative measurements of purified total RNA were done with a spectrophotometer and capillary electrophoresis using the Bioanalyser 2100 system (Agilent Technologies, Palo Alto, CA).
RNA amplification and labelling
Linear amplification of RNA was done using a modified protocol of a method described previously (10,18). Amplified RNA (aRNA) samples were quantified by spectrophotometry and quality was checked by gel electrophoresis (Bioanalyser 2001, Agilent). Two micrograms of aRNA from CS-exposed and unexposed rat tissues were labelled by reverse transcription with Cy5 and Cy3 fluorescence, respectively, and subjected to a customized PIQORTM microarray consisting of 2031 stress-relevant rat cDNAs.
cDNA array production
cDNA array production was done essentially as described previously (10,12). In short, defined 200400 bp fragments (19) of selected cDNAs were generated by RTPCR (SuperscriptTMII, Invitrogen, Groningen, The Netherlands), cloned into pGEM®-T Vector (Promega, Mannheim, Germany) and sequence-verified (see Supplementary material under http://www.memorec.com for a list of all genes). Amplified inserts (Taq PCR Master Mix, Qiagen) were purified (Qiaquick 96 PCR BioRobot Kit, Qiagen), checked on an agarose gel, and spotted two times each (0.2 ng) on treated glass slides (20).
Array hybridization and data analysis
Hybridization, scanning and data analysis were performed as described in detail (10,12). Briefly, image capture and signal quantification of hybridized PIQORTM cDNA arrays were done with the ScanArray3000 (GSI Lumonics, Watertown, MA) and ImaGene software version 4.1 (BioDiscovery, Los Angeles, CA). Normalized ratios are shown as Cy5 signal intensity divided by Cy3 signal intensity. For linear scaling, ratios with values <1 were treated in the following way: (1/ratio) x -1. Therefore, -1 and +1 values are considered equal. Cluster analysis was carried out using Spotfire® software (Göteborg, Sweden).
Real-time RTPCR
Two micrograms of aRNA were reverse transcribed and 4 ng of the RT-reaction product were used as template for further analysis. Transcript levels were measured by real-time quantitative RTPCR using ABI 7000 SDS (PE Applied Biosystems, Foster City, CA) as described earlier (12). The sequences of forward and reverse primers as designed by Primer Express (PE Applied Biosystems) using the region of the corresponding array cDNA fragment as a template were: cytosolic class-3 aldehyde dehydrogenase (aldh3) forward 5'-CTCTGTTGAATGAAGAAGCTCACAA-3' and reverse 5'-TAAGCCAGGATGAGGCTCCAT-3'; cyp1a1 forward 5'-AGGCTCAACTGTCTTC CAACATG-3' and reverse 5'-TGCAAGGACAAGGAGACCTTGT-3'; ho-1 forward 5'-AACACAAAGACCAGAGTCCCTCAC-3' and reverse 5'-GATGAACTAGTGCTGATCTGGGATT-3'; NAD(P)H:quinone oxireductase (nqo-1) 5'-AAATGGCATCCAATCCTCCA-3' and reverse 5'-AAGTTAGTCCCTCAGCCATTGTTT-3'. Real-time PCR experiments were performed in triplicate with at least two independently isolated RNA samples. As microarray analysis and triplicate Taqman assays using independent RNAs confirmed that transcript levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not vary significantly, GAPDH was used to normalize TaqMan data.
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Results and discussion
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Concept of the study and general overview of expression profiles
Male SpragueDawley rats were exposed to mainstream CS (100 µg TPM/l; University of Kentucky Reference Cigarette 2R1) by applying an acute/subchronic CS inhalation study design in order to closely compare the emerging data with those obtained from a previous microarray study performed in vitro using CS-exposed 3T3 fibroblasts (10). The rats were either exposed once for 3 h (acute), or for 3 weeks (3 h/day, 5 days/week; subchronic) and were killed immediately after the exposure. In order to get a rough insight into the duration of differential gene expression induced by CS exposure in vivo, another two groups of rats were treated in the same way but were killed after a recovery period of 20 h after the final exposure.
For quantification, fluorescence derived from microarrays hybridized in parallel to control (Cy3-labelled) and exposed (Cy5-labelled) cDNA samples were overlayed and analysed (see Materials and methods) identifying up-regulated genes by red fluorescence, while green fluorescence reflects genes that become suppressed by CS exposure. The mean number of genes yielding a distinct signal during analysis was 873, differing between 756 and 1077 (Table I), with no detectable differences between samples generated from rats with and without a recovery period. However, the number of genes differentially expressed by >2-fold was considerably higher in the groups of rats killed immediately after final exposure. The most strongly differentially expressed gene identified was cyp1a1, which was induced 176-fold in the lung and 142-fold in the RNE of rats exposed for 3 weeks with no recovery. For a complete overview of differential gene expression in all tissues analysed, see Supplementary material under http://www.memorec.com.
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Table I. General quantitative overview of the expression profiles in RNE and lungs of rats exposed to CS under different conditions
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Cluster analysis, using a hierarchical clustering algorithm method to group genes on the basis of similarity in the pattern with which their expression varied over all groups of exposed rats (21) revealed a dendogram, which on its horizontal axis shows a clear separation into two subgroups, identifying the recovery period as the most influencing experimental parameter, i.e. genes can be clearly allocated to tissues from exposed rats with recovery (right-hand side) or without recovery (left-hand side) (the most relevant section of the dendogram comprising most of the Phase I and Phase II drug-metabolizing genes is shown in Figure 1; the complete dendogram with gene annotations is part of Supplementary material). Tissues of exposed rats without recovery generally show a similar pattern of induced genes, whereas the corresponding genes in the tissues of exposed rats with recovery are mostly unchanged compared with controls. This finding clearly indicates that exposure over 3 weeks is not sufficient to induce persistent alterations in the gene expression pattern of CS-exposed rats.

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Fig. 1. Section of a two-dimensional cluster analysis over two different tissues under four different sets of exposure conditions using genes with a 2-fold difference in expression in at least one of the eight samples. Red, increased expression; green, repressed expression; black, unaltered gene expression; grey, no signal detection.
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On the vertical axis of the dendogram (Figure 1), the intensity of fluorescence reflects the strength of gene expression within the different tissues. In this respect, RNE from rats treated once for 3 h without recovery showed the strongest differential gene expression rates, followed by the RNE and lung tissue from rats exposed for 3 weeks without recovery. However, it is worth noting that the lung tissue shows a broader overall spectrum of differentially expressed genes on the basis of a 2-fold induction/repression evaluation (Table I).
In order to check the validity of the current microarray experiment, four genes found to be differentially expressed in lung and RNE, i.e. aldh3, cyp1a1, ho-1 and nqo1 were selected and their expression analysed by real-time RTPCR (Figure 2). The specificity of the corresponding RTPCR products was documented by high-resolution gel electrophoresis and melting curve analysis. The product-specific melting curves showed only single peaks and no primer-dimer peaks (data not shown). In quantitative terms, microarray data correlated with the results obtained from the real-time RTPCR experiment with the only exception of nqo-1 expression after 3 weeks of repeated exposure (Figure 2).

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Fig. 2. Comparison of expression data from selected candidate genes as evaluated by microarray and by real-time RTPCR (Taqman). Black, real-time RTPCR analysis; grey, microarray analysis.
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Effects of CS inhalation on the expression of genes encoding oxidative stress-related and Phase II drug-metabolizing enzymes in tissues of the respiratory tract
Our previous cDNA microarray analysis on gene expression in CS-exposed cultured 3T3 fibroblasts showed a strong antioxidant response including the up-regulation of oxidative stress defensive genes encoding HO-1, HSPs and MT I/II (10). In the present in vivo study, a distinct pattern of antioxidant gene expression was also observed, predominantly in RNE, although this response was confined to the groups with no recovery (Table II, Figure 3). As seen with CS-exposed 3T3 fibroblasts (10), the antioxidant response in vivo was hallmarked by the up-regulation of ho-1, which was found to be significantly induced in both tissues from CS-exposed rats without recovery (Table II, Figure 3). HO-1 catalyses the initial steps in haem catabolism, eventually leading to the formation of biliverdin and bilirubin, both of which are powerful antioxidants (22,23). The HO-1 pathway, which represents a prime defence tool in protecting the cell from stress-dependent adverse effects mainly induced by reactive oxygen and/or nitrogen species, requires the parallel expression of ferritin, in order to protect the cell from oxidative stress due to the HO-1-dependent release of free iron (24). In fact, a slight but obvious induction of ferritin was observed in both exposed tissues (see Supplementary material), which is consistent with data reported for CS-exposed cells in vitro (10). With regard to the considerably lower expression of ferritin in comparison with ho-1 expression, it should be noted that every ferritin molecule might sequester up to 4500 iron atoms (25).
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Table II. Differential gene expression (fold induction/repression) of selected genes coding for oxidative stress-related and Phase II drug-metabolizing enzymes in respiratory tissues of CS-treated rats
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Fig. 3. CS inhalation in rats induces oxidative stress/Phase II-related genes along a potential deposition gradient from the upper to the lower respiratory tract; in comparison with a single 3 h exposure, induction factors of expressed genes are significantly lower after 3 weeks of repeated exposure indicating an adaptive response. Differential gene expression of selected genes in RNE and lungs of CS-treated rats is shown. Value at 0, not detectable.
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From the data shown in Figure 3, it is evident that the strength of ho-1 expression differs enormously between the various tissues: up-regulation of this gene in RNE was found to be
20-fold greater in comparison with the lung. As this effect was consistently observed for almost all other genes found to be differentially expressed, with the remarkable exception of genes coding for Phase I-metabolizing enzymes (see below), this phenomenon is a strong indication that there is a deposition gradient of (stress) gene-inducing CS compounds from the upper to the lower respiratory tract. Although the identity of these compounds remains to be determined, possible candidates might be aldehydes and (poly-)phenolic components of the TPM fraction, such as hydroquinone and catechol, which are known to release reactive oxygen species in aqueous solution (26). However, it should be stressed that the apparent deposition gradient is effectively cleared within 20 h, even in rats repeatedly exposed for 3 weeks, as evidenced by the fact that the differential gene expression seen for ho-1 and nearly all other genes in CS-exposed rats without recovery, was not seen in exposed rats with 20-h recovery (see Tables II and III and Supplementary material).
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Table III. Differential gene expression (fold induction/repression) of selected genes coding for Phase I drug-metabolizing enzymes in RNE and lungs of CS-treated rats
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Although the ho-1 promoter is equipped with multiple cis-elements addressed by various types of transcription factors such as AP-1, STAT3 and NF-
B (for review, see ref. 27), the (oxidative) stress-induced expression of ho-1 (28) and also of ferritin (29) has been demonstrated to be dependent mainly on the transcriptional activation mediated by the interaction of the transcription factor NF-E2-related factor 2 (Nrf2) and the antioxidant response element (ARE) in the promoter of the enzyme's gene. In fact, 3T3 fibroblasts stably transfected with a dominant negative mutant of nrf2 lose most of their ho-1 inducibility when exposed to aqueous extracts of CS (S.Gebel and T.Müller, in preparation). In the non-induced state, Nrf2 is sequestered in the cytosol by the actin-binding cysteine-rich protein Keap1 (30). Upon activation by a mechanism that is not yet completely understood, Nrf2 translocates to the nucleus and, by binding to ARE, induces the expression of a whole plethora of stress-responsive genes including those coding for Phase II-detoxifying enzymes (for review, see ref. 31). Thus, it is not surprising that in addition to ho-1, the Phase II-related genes nqo1,
-glutamyl-cysteine-synthetase (
gcs) and genes encoding various isozymes of UDP-glucuronosyltransferases (UGTs) also become significantly activated at least in the RNE of CS-exposed rats (Table II). The nrf2 gene itself was found to be only slightly affected in both CS-exposed tissues (Table II) indicating that Nrf2 is sufficiently present in target cells. This interpretation is conceivable because Nrf2 as a stress signal-transducing transcription factor needs to be available for immediate function.
Phase II-related genes such as
gcs and nqo1 are induced by (poly-)phenolic compounds and electrophiles via the Nrf2 signaling pathway (31,32), suggesting that CS-dependent radicals derived from catechol and/or hydroquinone via Fenton chemistry (33) are strong candidates as triggers for a Phase II response in CS-exposed tissues. Enzymes encoded by
gcs and nqo1 are involved in the detoxification of (poly-)phenolic compounds and harmful by-products of oxidative stress, such as lipid and DNA base hydroperoxides as well as quinones (26). In particular, the induction of
GCS, which is the rate-limiting enzyme in GSH synthesis, results in the replenishment of a decreased GSH pool, which has been reported to appear immediately following CS exposure both in vitro (9) and in vivo (34). Although
gcs and nqo1 were not included on the DNA chip used to evaluate the transcriptional response in CS-exposed 3T3 cells (10), the dramatic decrease in intracellular GSH followed by a rapid increase in the amount of this endogenous antioxidant molecule (9) shows that
gcs must also have become strongly induced in these cells. This interpretation is corroborated by a strong increase in
gcs expression in these cells upon exposure to TPM (S.Gebel and W.Schlage, unpublished results). Other genes coding for enzymes known to be involved in GSH-dependent detoxification reactions, e.g. GSH-S-transferases, were also induced, mainly in RNE, although to a lesser extent than
gcs (see Supplementary material). The trend seen for an increased nasal GSH-conjugating activity is further reflected by the up-regulation of genes, known to be involved in GSH homeostasis, such as neutral amino acid transporter A (satt) and
-glutamyl-transpeptidase (
gt1) (Table II).
An interesting aspect of the present study is that in the lungs of CS-exposed rats without recovery, nqo1 is the most strongly induced Phase II-related gene showing even stronger expression rates than ho-1 (Table II, Figure 3). Intriguingly, beyond its enzymatic function to catalyse the obligatory 2-electron reductions of quinones to hydroquinones, which protects cells from damage induced by redox cycling (for review, see ref. 35), NQO1 has been reported to stabilize the tumour suppressor protein p53, especially under induction of oxidative stress (36,37). Thus, in addition to its basic antioxidant function, the rather strong activation of nqo1 in the lungs of CS-exposed rats may be instrumental in the efficient stabilization of p53, which has been suggested to play a crucial role in the prevention of CS-dependent lung cancer (38).
Other Phase II-related genes which were found to be significantly up-regulated in the nose by CS inhalation are three members of the UGT enzyme family (for review, see ref. 39), i.e. genes encoding the isoforms 1A6, 1A7 and 2B12 (Table II). In general, genes coding for UGTs are also controlled, at least in part, by Nrf2 (40,41). UGTs are efficient tools for clearing the cell of many lipophilic xenobiotics and endobiotics by catalysing the glucuronidation of the glycosyl group of a nucleotide sugar to an acceptor compound at a nucleophilic functional group resulting in the formation of a ß-D-glucuronide. This reaction gives rise to a more water-soluble compound, which can be eliminated more easily (39).
A critical issue emerging from the current study is related to the phenomenon that all CS-induced oxidative stress- and Phase II-related genes, which are presumptively regulated by Nrf2, show significantly (25-fold) decreased expression rates after multiple exposures over 3 weeks when compared to the corresponding expression rates observed immediately after a single 3-h exposure (Table II). Since all genes in this group are efficiently down-regulated after 20-h recovery, regardless of whether there was a single 3-h exposure or repeated exposures over 3 weeks, this effect strongly points to an adaptive response, which, in mechanistic terms, may be caused by a steadily decreasing sensitivity of Nrf2 to become activated by CS-dependent stressors over prolonged exposure periods. Consequently, this effect may gradually compromise the ability of the respiratory tract cells to adequately respond to CS-dependent (oxidative) stress during chronic exposure, and may therefore be critically involved in CS-dependent carcinogenesis or other diseases. Alternatively, this effect may be explained by an acute strong up-regulation of these genes followed by an intermediate level of up-regulation high enough to maintain a steady-state level of defence.
Finally, when the oxidative stress response observed in the current in vivo study is compared with the corresponding data from the previous in vitro study (10), it seems that only a small subset of stress-responsive genes were detected in both studies. However, this is mainly due to the fact that the DNA chip used in the in vitro study was configured with a considerably smaller number of genes (i.e. several probes were missing, such as those for
gcs, nqo1 and ugts). In fact, both studies clearly show that CS-treatment triggers a strong (oxidative) stress response, presumptively because of the efficient activation of the transcription factor Nrf2 both in vitro and in vivo. This is exemplified by the strong up-regulation of ho-1 in both studies, which is mostly lost by the expression of a dominant-negative mutant of Nrf2 in vitro (S.Gebel and T.Müller, in preparation) and by the efficient replenishment of GSH in vitro (9), which requires the expression of the Nrf2-regulated gene
gcs. On the other hand, the lack of expression of MTI/II and HSPs in vivo may be caused by kinetic specificities inherent to the expression of these particular genes, which in the in vitro study were all found to become up-regulated only after 4 h of exposure (10). Other explanations for this apparent discrepancy may be that the CS dose and/or the duration used in the subchronic study was not sufficient to induce these genes in vivo, or that the inducing effect in a few cell types may not be recognizable in a whole tissue homogenate.
Effects of CS inhalation on the expression of genes encoding Phase I drug-metabolizing enzymes
The inducibility of Phase I-related genes in the respiratory tract by CS, especially those encoding cytochrome P450 isozymes, has been described extensively (42). One major route suggested for CS-induced lung cancer is the cytochrome P450 (CYP)1A1-dependent activation of CS-related polycyclic aromatic hydrocarbons (PAHs), especially benzo[a]pyrene (B[a]P), to biologically reactive intermediates, which by interfering with crucial loci on the DNA could finally result in cancer-initiating mutations (e.g. ref. 43). Potential crucial target genes include p53 and ras, for which distinct mutational signatures have been described upon interference with B[a]P diol epoxide, the CYP1A1-activated derivative of B[a]P (for review, see refs 38,44). cyp1a1 itself is highly inducible by a broad spectrum of xenobiotics, mostly substrates of the CYP1A1 enzyme, which bind to and thus activate the aryl hydrocarbon (Ah) receptor, the most prominent transcription factor of Phase I-related genes (for review, see ref. 45).
As could be expected from previous studies in vivo (for review, see ref. 42 and references cited therein), cyp1a1 was also found to be transcriptionally activated by CS inhalation in the present study. In fact, the induction rates detected for this gene were the highest observed in all tissues when RNA sampling was performed immediately after CS exposure (no recovery period). Within this group, the lungs of rats repeatedly exposed for 3 weeks showed a 176-fold induction of cyp1a1, the highest induction rate observed for any gene during the study (Table III). However, similar high expression rates were also observed in the RNE of exposed rats, showing that, unlike Phase II-related genes, cyp1a1 expression as well as the expression of the other Phase I-related gene found to be activated in this study, i.e. aldh3 (see below), is obviously not subject to a deposition gradient of active CS constituents. Instead, these findings clearly indicate that, in comparison with Phase II-related genes, the Phase I response is mediated by a different mechanism of activation and, consequently, by different CS-related compound(s). This interpretation of the data is further corroborated by the finding that, in contrast to the CS-dependent regulation of Phase II-related genes, no indication for an adaptive response is discernible within the group of Phase I up-regulated genes as indicated by the following observations: In RNE, induction factors for cyp1a1 and aldh3 were almost in the same range, independent of whether the rats were exposed once or for 3 weeks, while a 23-fold increase in the expression rates of these genes was seen in the lungs of rats exposed for 3 weeks in comparison to a single 3-h exposure (Table III, Figure 4).

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Fig. 4. Expression of Phase I-related genes induced by CS inhalation in rats is similar in RNE and lung tissues after a single exposure and after 3 weeks of repeated exposures. (A) Differential gene regulation of selected genes in respiratory tissues of CS-treated rats. (B) Higher resolution of the differential gene expression of cyp1b1 and cyp2b1/2. Value at 0, not detectable.
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The data reported here for CS-dependent cyp1a1 expression agree almost completely with previous investigations reported by Wardlaw et al. (46). These authors reported strong activation of CYP1A1 on the protein level in various tissues including those of the respiratory tract using immunodetection methods in F344 rats exposed to CS for similar periods of time as in this study. In particular, CYP1A1 was found to be induced in the nasal respiratory and olfactory epithelia and the lungs of exposed rats as assessed by western analysis. To the surprise of Wardlaw et al. (46), the enzyme activity often used to assess CYP1A1 function, i.e. ethoxyresorufin O-deethylase (EROD) activity, was induced in the lungs but was repressed in the nasal epithelia. The repression is consistent with a lack of a remarkable inducibility of B[a]P metabolism in the nasal respiratory and olfactory epithelia compared with lung tissue (47). In addition, further data reported by Wardlaw et al. (46) are consistent with the expression rates of other cyp genes detected in the present study since only minor effects of CS inhalation were observed by these authors in tissues of the respiratory tract on CYP1A2 activity while the activity of CYP2B1/2 (see below) was decreased.
As mentioned above, PAHs, especially B[a]P, are believed to play a role in the CS-dependent expression of cyp1a1, cyp1a2 and possibly cyp2e1 (for review, see ref. 48). This interpretation is supported by the observation that only the TPM fraction of CS harbours this induction potential in rats and mice (49,50); a potential which can be mimicked by administering individual PAHs to laboratory rodents (51).
Apart from the paramount up-regulation of cyp1a1 in all tissues of the respiratory tract, it is worth noting that genes encoding other isoforms of cytochrome P450 drug-metabolizing enzymes are not responsive or are much less responsive to CS. Out of 31 probes corresponding to individual cyp genes (including those coding for enzymes involved in endogenous substrate metabolism), only the expression of cyp1b1 and cyp2b1/2 was also found to be differentially regulated by CS inhalation (see Table III and Supplementary material). Up-regulation of cyp1b1 was clearly detectable in RNE, although not to the same extent as seen for cyp1a1, independent of whether the rats were exposed once or repeatedly over 3 weeks (Table III). Like cyp1a1, cyp1b1 is also controlled by the Ah-receptor, while its expression is subject to strict tissue-specific regulation (52). In this context, it is notable that cyp1b1 was only induced in the RNE of CS-exposed rats while it was almost completely silent in the lung (Table III). In fact, to our knowledge, this is the first report linking cyp1b1 expression in RNE to CS-exposure. While the meaning of this tissue specificity remains to be elucidated, recent investigations have revealed that CYP1B1 is responsible for the activation of 7,12-dimethylbenz[a]anthracene (53).
In contrast to cyp1a1, cyp2b1/2 was found to be down-regulated
5-fold in the RNE of rats exposed once for 3 h and 2-fold in the same tissue of rats exposed repeatedly for 3 weeks (Table III). In fact, a repression of pulmonary CYP2B1/2 has frequently been reported under conditions, which lead to the induction of CYP1A1. As mentioned above, Wardlaw et al. (46) reported a decrease of CYP2B1/2 protein in the lungs and in the respiratory and olfactory epithelium of the nose by CS inhalation. Likewise, the model enzyme activity of pentoxy-resorufin depentylase (PROD) was reduced. In our study, repression of cyp2b1/2 was confined to the RNE, although the cyp1a1-inducing effect was similar in the nose and in the lungs (Table III). In rats exposed to smoke concentrations of up to 250 µg TPM/l, PROD in lungs was decreased while EROD and CYP1A1 were induced (54). Currently, we are not able to explain this discrepancy. However, in this context it is notable that Foy et al. (55) suggested that B[a]P, after metabolic activation by CYP1A1, would be detoxified by CYP2B1. Accordingly, the ratio of CYP1A1 over CYP2B1 would serve as a surrogate for a measure of toxification versus detoxification of B[a]P in a given tissue.
Among the family of cyp-related genes, the lack of cyp2e1 expression in the present study seems especially noteworthy, in particular because of the very effective induction of this gene by CS inhalation in NMRI mice (5658). During these latter studies, the induction of cyp2e1 was remarkably more pronounced than the comparatively weak induction of cyp1a1. This is in contrast to the observation in A/J mice chronically exposed to cigarette sidestream smoke, where pulmonary CYP1A1 was induced (in capillary endothelial cells) but no effect was seen on 2E1 (59). Rat pulmonary CYP2E1-dependent enzyme activity was shown to be inducible by inhalation of CS constituents, i.e. styrene alone or in combination with ethylmethylketone (60). Acute acetone administration induced cyp2e1 mRNA and protein in nasal tissues and the lungs (61). However, in rats exposed at smoke concentrations of up to 250 µg TPM/l, no induction of the CYP2E1-dependent p-nitrophenol hydroxylation was seen (54). Since Villard et al. (5658) give no indications on the dosing, a possible explanation for the missing cyp2e1 expression as observed in the present study may be that, at these smoke concentrations, neither an acute nor a 3-week subchronic exposure are sufficient for the effective induction of this gene in tissues of the rat respiratory tract. On the other hand, simple species and strain differences controlling the pulmonary transcription of cyp2e1 or different morphological changes upon prolonged exposure to CS may also be responsible to a certain degree for the different outcomes. Nevertheless, the fate of respiratory tract CYP2E1 with CS inhalation is rather important, because a number of small, unsaturated hydrocarbons, such as 1,3-butadiene (62) and benzene (63), which are classified as human or animal carcinogens, can be activated by CYP2E1 at low concentrations. There are no data available on the potential induction of cyp2e1 transcription in the lungs of human smokers.
To our knowledge, this is the first report of CS inhalation-dependent up-regulation of aldh3 in tissues of the respiratory tract. Although not induced to the same extent as cyp1a1, the pattern of aldh3 expression parallels that observed for cyp1a1 in both tissues (Figure 4), indicating a similar mechanism of transcriptional activation. In fact, aldh3 expression is induced by xenobiotics via activation of the Ah-receptor in tissues, which do not constitutively express this gene (for review, see ref. 64). In the context of CS exposure, it is notable that experiments in vitro showed that aldh3 becomes induced by the polyphenol catechol (65) as well as that ALDH3 is critically involved in the detoxification of aldehydes derived from lipid peroxides (66).
As is the case for almost all other genes found to be differentially regulated by CS inhalation, the Phase I-related genes returned to normal expression levels when the rats were allowed to recover for 20 h (Table III). This is another striking indication that even after repeated exposures over 3 weeks, a period of 20 h is sufficient to clear exposed tissues also from CS-dependent constituents potentially able to activate Phase I-related genes.
Repressed genes
In addition to the repression of cyp2b1/2 in the RNE of CS-exposed rats discussed above, 3 more genes, i.e. relm
, kc/scyb1 and D-site-binding protein (dbp), become considerably repressed in RNE by CS inhalation. Strikingly, relm
, which was characterized during these investigations as a novel member of the resistin-like molecule/found in inflammatory zone family of proteins (RELM/FIZZ) (12), is the only gene that was found to be significantly differentially regulated after 20 h of recovery (Table IV). In fact, the repression of relm
observed directly after exposure was even enforced
4-fold after 20 h recovery, independent of whether the rats were exposed once or repeatedly for 3 weeks. Although relm
is closely related to relm
/fizz1, these two genes are clearly expressed in a highly tissue- and species-specific context (12). For example, in the rat relm
is maximally expressed in haemotopoietic tissues suggesting a cytokine-like role for RELM
, while relm
/fizz1 is found mainly in lung and white adipose tissue of mice. Nevertheless, based on the high homology of these two genes, it can be speculated that their proteins are related in function, at least to some extent. Therefore, since relm
/fizz1 has been shown to become markedly expressed during pulmonary inflammation (11), the strict repression of relm
in the nasal tissue of CS-exposed rats, which increases strongly during smoke-free periods, may point to an anti-inflammatory response in the RNE of CS-exposed rats. This interpretation is supported by a 4-fold repression of kc/scyb1, a putative functional homologue of human IL-8 (67), in the same tissue after 3 weeks, whereas kc/scyb1 was found to be up-regulated >4-fold in the lungs of rats exposed over 3 weeks and thus may indicate the beginning of a pro-inflammatory reaction in this tissue (Table IV).
Finally, repression of dbp was observed only in RNE from CS-exposed rats (Table IV). This gene encodes a member of the proline- and acid-rich domain subfamily of basic/leucine zipper proteins, which are involved in transcriptional regulation in the liver (68). Since expression of DBP is confined to the adult liver, although dbp mRNA is ubiquitously expressed indicating a post-transcriptional regulatory mechanism, the meaning, if any, of its transcriptional repression in the RNE of CS-exposed rats remains obscure.
 |
Conclusions
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---|
Using microarray techniques we have evaluated differential gene expression in tissues of the respiratory tract of rats exposed to CS either once for 3 h or repeatedly over 3 weeks, with and without a recovery period of 20 h. As could be expected, CS inhalation induced the expression of oxidative stress- and Phase II-responsive genes as well as the expression of Phase I-related genes. Almost all changes in gene expression returned to normal after 20 h in a CS-free environment. Some genes not previously linked to CS-inhalation, such as aldh3, were found to be up-regulated, while others, such as cyp1b1, were found to be regulated in a striking tissue-specific context. With regard to the induction of oxidative stress- and Phase II-responsive genes, the detection of a potential deposition gradient of active CS constituent(s), which is formed during each single exposure, as well as the phenomenon of an adaptive response are especially intriguing. Further studies will focus on the corroboration of the current findings in a dose-response study, the identification of potential CS-related inducers and on the question of whether the decreasing inducibility of these genes after repeated CS exposure is critically involved in CS-dependent carcinogenesis. As the former issue refers mainly to the expression of genes controlled, at least in part, by the transcription factor Nrf2, these studies may be performed in vitro, since, as discussed above, a similar broad expression of potentially Nrf2-regulated genes is expected to occur in CS-exposed cultured fibroblasts. Regarding the impact of an obvious adaptive response to CS-induced carcinogenesis, a longer subchronic inhalation study with intermediate dissections using a cDNA microarray that covers a broader spectrum of cancer-related genes is being investigated to monitor the gradual impairment of the oxidative stress/Phase II response versus the appearance of pre-cancerous lesions. This approach will also address the striking issue of the apparent reversibility of expression changes during the recovery period, even after 3 weeks of repeated exposures. In addition, the impact of inflammatory events on the gene expression pattern of target cells is being investigated, a first sign of which could be indicated by the slight up-regulation of kc/scyb1 in the lungs of rats repeatedly exposed to CS for 3 weeks. Finally, as no deposition gradient was detected for the expression of Phase I-related genes, the question of whether these genes are in fact exclusively induced by PAHs, which are part of the TPM fraction and, therefore, expected to be deposited as a gradient from the upper to the lower respiratory tract, needs to be re-addressed.
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Notes
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4 To whom correspondence should be addressed Email: thomas.mueller{at}pmintl.com 
Declaration of interest: S.Gebel, H.-J.Haussmann and T.Müller are employees of Philip Morris Research Laboratories GmbH, E. Van Miert is an employee of Philip Morris Research Laboratories bvba. Both institutes are part of Philip Morris International. 
+ Declarations of interest: Memorec Stoffel GmbH has received payments from Philip Morris Research Laboratories GmbH for production of the customized PIQORTM cDNA arrays and execution of the hybridization experiments. 
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Acknowledgments
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We thank L.Conroy (PM Research Laboratories) for expert editorial support and V.Böhm (PM Research Laboratories), G.Großhauser, D.Küsters (memorec) for skilful technical assistance.
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References
|
---|
- Peto,R., Lopez,A.D., Boreham,J., Thun,M. and Heath,C.,Jr (1994) Mortality from Smoking in Developed Countries, 19502000. Indirect Estimates from National Vital Statistics. Oxford University Press, Oxford, New York.
- US Department of Health and Human Services (1989) Coronary heart disease. In US Department of Health and Human Services (eds), Reducing the Health Consequences of Smoking: 25 Years of Progress. A Report of the Surgeon General. Public Health Service, Centers for Disease Control, Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. DHHS Publication No. (CDC) 89-8411, Rockville, MD, pp. 5865.
- US Department of Health and Human Services (1989) Chronic obstructive pulmonary disease. In U.S. Department of Health and Human Services (eds), Reducing the Health Consequences of Smoking: 25 Years of Progress. A Report of the Surgeon General. Public Health Service, Centers for Disease Control, Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health. DHHS Publication No. (CDC) 89-8411, Rockville, MD, pp. 6668.
- Müller,T. and Gebel,S. (1994) Heme oxygenase expression in Swiss 3T3 cells following exposure to aqueous cigarette smoke fractions. Carcinogenesis, 15, 6772.[Abstract]
- Müller,T. (1995) Expression of c-fos in quiescent Swiss 3T3 cells exposed to aqueous cigarette smoke fractions. Cancer Res., 55, 19271932.[Abstract]
- Vayssier,M., Favatier,F., Pinot,F., Bachelet,M. and Polla,B.S. (1998) Tobacco smoke induces coordinate activation of HSF and inhibition of NF
B in human monocytes: effects of TNF
release. Biochem. Biophys. Res. Commun., 252, 249256.[CrossRef][ISI][Medline]
- Gebel,S. and Müller,T. (2001) The activity of NF-
B in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke is dependent on thioredoxin. Toxicol. Sci., 59, 7581.[Abstract/Free Full Text]
- Müller,T., Haussmann,H.-J. and Schepers,G. (1997) Evidence for peroxynitrite as an oxidative stress-inducing compound of aqueous cigarette smoke fractions. Carcinogenesis, 18, 295301.[Abstract]
- Müller,T. and Gebel,S. (1998) The cellular stress response induced by aqueous extracts of cigarette smoke is critically dependent on the intracellular glutathione concentration. Carcinogenesis, 19, 797801.[Abstract]
- Bosio,A., Knörr,C., Janssen,U., Gebel,S., Haussmann,H.-J. and Müller,T. (2002) Kinetics of gene expression profiling in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke. Carcinogenesis, 23, 741748.[Abstract/Free Full Text]
- Holcomb,I.H., Kabakoff,R.C., Chan,B. et al. (2000) FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J., 29, 40464055.[CrossRef]
- Gerstmayer,B., Küsters,D., Gebel,S., Müller,T., Van Miert,E., Hofmann,K. and Bosio,A. (2003) Identification of RELM
, a novel resistin-like molecule with a distinct expression pattern. Genomics, 81, 588595.[CrossRef][ISI][Medline]
- ISO (1991) International Organization for Standardization: International Standard ISO 3402: Tobacco and Tobacco ProductsAtmosphere for Conditioning and Testing, 3rd Edition.
- ISO (1991) International Organization for Standardization: International Standard ISO 3308: Routine Analytical Cigarette-smoking MachineDefinitions and Standard Conditions, 3rd Edition.
- Vanscheeuwijk,P.M., Teredesai,A., Terpstra,P.M., Verbeeck,J., Kuhl,P., Gerstenberg,B., Gebel,S. and Carmines,E.L. (2002) Evaluation of the potential effects of ingredients added to cigarettes. Part 4: subchronic inhalation toxicity. Food Chem. Toxicol., 40, 113131.[CrossRef][ISI][Medline]
- Haussmann,H.-J., Anskeit,E., Becker,D., Kuhl,P., Stinn,W., Teredesai,A., Voncken,P. and Walk,R.A. (1998) Comparison of fresh and room-aged cigarette sidestream smoke in a subchronic inhalation study on rats. Toxicol. Sci., 41, 100116.[Abstract]
- AALAS (1991) American Association for Laboratory Animal Science Policy on the Humane Care and Use of Laboratory Animals. Lab. Animal Sci., 41, 91.
- Eberwine,J. (1996) Amplification of mRNA populations using aRNA generated from immobilized oligo(dT)-T7 primed cDNA. Biotechniques, 20, 584591.[ISI][Medline]
- Tomiuk,S. and Hofmann,K. (2001) Microarray probe selection strategies. Brief Bioinform., 2, 329340.[Medline]
- Bosio,A., Stoffel,W. and Stoffel,M. (1999) Support for the parallel identification and establishment of transcription profiles of polynucleic acids. WO9964623: MEMOREC Stoffel GmbH.
- Eisen,M.B., Spellman,P.T., Brown,P.O. and Botstein,D. (1998) Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA, 95, 1486314868.[Abstract/Free Full Text]
- Maines,M.D. (1988) Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J., 2, 25572568.[Abstract/Free Full Text]
- Stocker,R.Y., Yamamoto,Y., McDonagh,A.F., Glazer,A.N. and Ames,B.N. (1987) Bilirubin is an antioxidant of possible physiological importance. Science, 235, 10431046.[ISI][Medline]
- Tyrrell,R. (1999) Redox regulation and oxidant activation of heme oxygenase-1. Free Rad. Res., 31, 335340.[ISI][Medline]
- Richter,G.W. (1978) The iron-loaded cellthe cytopathology of iron storage. Am. J. Pathol., 91, 363404.
- Pryor,W.A. and Stone,K. (1993) Oxidants in cigarette smoke: radicals, hydrogen peroxide, peroxynitrate, and peroxynitrite. Ann. N.Y. Acad. Sci., 686, 1228.[ISI][Medline]
- Choi,A.M.K. and Alam,J. (1996) Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am. J. Respir. Cell Mol. Biol., 15, 919.[Abstract]
- Alam,J., Stewart,D., Touchard,C., Boinapally,S., Choi,A.M.K. and Cook,J.L. (1999) Nrf2, a cap'n'collar transcription factor, regulates induction of heme oxygenase-1 gene. J. Biol. Chem., 274, 2607126078.[Abstract/Free Full Text]
- Pietsch,E.C., Chan,J.Y., Torti,F.M. and Torti,S.V. (2003) Nrf2 mediates the induction of ferritin H in response to xenobiotics and cancer chemopreventive dithiolethiones. J. Biol. Chem., 278, 23612369.[Abstract/Free Full Text]
- Dinkova-Kostova,A.T., Holtzclaw,W.D., Cole,R.N., Itoh,K., Wakabayashi,N., Kat,Y., Yamamoto,M. and Talalay,P. (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc. Natl Acad. Sci. USA, 99, 1190811913.[Abstract/Free Full Text]
- Nguyen,T., Sherratt,P.J. and Pickett,C.B. (2003) Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol., 42, 233260.[CrossRef]
- Ishii,T., Itoh,K., Takahashi,S., Sato,H., Yanagawa,T., Katoh,Y., Bannai,S. and Yamamoto,M. (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J. Biol. Chem., 275, 1602316029.[Abstract/Free Full Text]
- Imlay,J.A., Chin,S.M. and Linn,S. (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science, 240, 640642.[ISI][Medline]
- Bilimoria,M.H. and Ecobichon,D.J. (1992) Protective antioxidant mechanisms in rat and guinea pig tissues challenged by acute exposure to cigarette smoke. Toxicology, 72, 131144.[CrossRef][ISI][Medline]
- Dinkova-Kostova,A.T. and Talalay,P. (2000) Persuasive evidence that quinone reductase type 1 (DT diaphorase) protects cells against the toxicity of electrophiles and reactive forms of oxygen. Free Rad. Biol. Med., 29, 231240.[CrossRef][ISI][Medline]
- Asher,G., Lotem,J., Kama,R., Sachs,L. and Shaul,Y. (2002) NQO1 stabilizes p53 through a distinct pathway. Proc. Natl Acad. Sci. USA, 99, 30993104.[Abstract/Free Full Text]
- Anwar,A., Dehn,D., Siegel,D., Kepa,J.K., Tang,L.J., Pietenpol,J.A. and Ross,D. (2003) Interaction of human NAD(P)H:quinone oxidoreductase 1 (NQO1) with the tumor suppressor protein p53 in cells and cell-free systems. J. Biol. Chem., 278, 1036810373.[Abstract/Free Full Text]
- Robles,A.I., Linke,S.P. and Harris,C.C. (2002) The p53 network in lung carcinogenesis. Oncogene, 21, 68986907.[CrossRef][ISI][Medline]
- King,C.D., Rios,G.R., Green,M.D. and Tephly,T.R. (2003) UDP-Glucoronosyl-transferases. Curr. Drug Metabol., 1, 143161.
- Cho,H-Y., Jedlicka,A.E., Reddy,S.P.M., Kensler,T.W., Yamamoto,M., Zhang,L.-Y. and Kleeberger,S.R. (2002) Role of NRF2 in protection against hyperoxic lung injury in mice. Am. J. Respir. Cell. Mol. Biol., 26, 175182.[Abstract/Free Full Text]
- Thimmulappa,R.K., Mai,K.H., Srisuma,S., Kensler,T.W., Yamamoto,M. and Biswal,S. (2002) Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res., 62, 51965203.[Abstract/Free Full Text]
- Ding,X. and Kaminsky,L.S. (2003) Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu. Rev. Pharmacol. Toxicol., 43, 149173.[CrossRef][ISI][Medline]
- Bartsch,H., Petruzzelli,S., De Flora,S., Hietanen,E., Camus,A.-M., Castegnaro,M., Alexandrov,K., Rojas,M., Saracci,R. and Giuntini,C. (1992) Carcinogen and metabolism in human lung tissues and the effect of tobacco smoking: results from a case-control multicenter study of lung cancer patients. Environ. Health Perspect., 98, 119124.[ISI][Medline]
- Hecht,S.S. (1999) Tobacco smoke carcinogens and lung cancer. J. Natl Cancer Inst., 91, 11941210.[Abstract/Free Full Text]
- Denison,M.S. and Nagy,S.R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol., 43, 309334.[CrossRef][ISI][Medline]
- Wardlaw,S.A., Nikula,K.J., Kracko,D.A., Finch,G.L., Thornton-Manning,J.R. and Dahl,A.R. (1998) Effect of cigarette smoke on CYP1A1, CYP1A2 and CYP2B1/2 of nasal mucosae in F344 rats. Carcinogenesis, 19, 655662.[Abstract]
- Bond,J.A. (1983) Some biotransformation enzymes responsible for polycyclic aromatic hydrocarbon metabolism in rat nasal turbinates: effects on enzyme activities of in vitro modifiers and intraperitoneal and inhalation exposure of rats to inducing agents. Cancer Res., 43, 48054811.[Abstract]
- Zevin,S. and Benowitz,N.L. (1999) Drug interactions with tobacco smoking. An update. Clin. Pharmacokinet., 36, 425438.[ISI][Medline]
- Gebremichael,A., Chang,A.M., Buckpitt,A.R., Plopper,C.G. and Pinkerton,K.E. (1995) Postnatal development of cytochrome P4501A1 and 2B1 in rat lung and liver: effect of aged and diluted sidestream cigarette smoke. Toxicol. Appl. Pharmacol., 135, 246253.[CrossRef][ISI][Medline]
- Witschi,H. (1998) Tobacco smoke as a mouse lung carcinogen. Exp. Lung Res., 24, 385394.[ISI][Medline]
- Rylander,R., Haussmann,H.-J. and Tewes,F.-J. (1989) Lung cancer risk by oral exposure. In Bieva,C.J., Courtois,Y. and Govaerts,M. (eds), Present and Future of Indoor Quality. Elsevier Science Publishers, Amsterdam, pp. 91100.
- Savas,U., Bhattacharyya,K.K., Christou,M., Alexander,D.L. and Jefcoate,C.R. (1994) Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450 s: cloning, sequence determination, and tissue expression. J. Biol. Chem., 269, 1490514911.[Abstract/Free Full Text]
- Heidel,S.M., MacWilliams,P.S., Baird,W.M,, Dashwood,W.M., Buters,J.T., Gonzalez,F.J., Larsen,M.C., Czuprynski,C.J. and Jefcoate,C.R. (2000) Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene. Cancer Res., 60, 34543460.[Abstract/Free Full Text]
- Roos,P.H., Schlage,W., Schepers,G., van Miert,E., Weisensee,D. and Haussmann,H.-J. (2003) Modulation of the cytochrome P450-profile in rat lungs by cigarette smoke inhalation. Arch. Pharmacol., 367 (suppl. 1), R139.
- Foy,J.W., Silverman,D.M. and Schatz,R.A. (1996) Low-level m-Xylene inhalation alters pulmonary and hepatic cytochrome P-450 activity in the rat. J. Toxicol. Environ. Health, 47, 135144.[CrossRef][ISI][Medline]
- Villard,P.H., Seree,E., Lacarelle,B., Therene-Fenoglio,M.C., Barray,Y., Attolini,L., Bruguerole,B., Durand,A. and Catalin,J. (1994) Effect of cigarette smoke on hepatic and pulmonary cytochromes P450 in mouse: Evidence for CYP2E1 induction in lung. Biochem. Biophys. Res. Commun., 202, 17311737.[CrossRef][ISI][Medline]
- Villard,P.H., Seree,E.M., Re,J.L., de Meo,M., Barra,Y., Attolini,L., Dumenil,G., Catalin,J., Durand,A. and Lacarelle,B. (1998a) Effects of tobacco smoke on the gene expression of the CYP1A, Cyp2B, CYP2E, and CYP3A subfamilies in mouse liver and lung: relation to single strand breaks of DNA. Toxicol. Appl. Pharmacol., 148, 195204.[CrossRef][ISI][Medline]
- Villard,P.H., Herber,R., Seree,E.M., Attolini,L., Magdalou,J. and Lacarelle,B. (1998b) Effect of cigarette smoke on UDP-glucuronosyltransferase activity and cytochrome P450 content in liver lung and kidney microsomes in mice. Pharmacol. Toxicol., 82, 7479.[ISI][Medline]
- Pinkerton,K.E., Peake,J.L., Espiritu,I., Goldsmith,M. and Witschi,H. (1996) Quantitative histology and cytochrome P-450 immunocytochemistry of the lung parenchyma following 6 months of exposure of strain A/J mice to cigarette mainstream smoke. Inhal. Toxicol., 8, 927945.[ISI]
- Nedelcheva,V. (1996) Interaction of styrene and ethylmethylketone in the induction of cytochrome P450 enzymes in rat lung, kidney and liver after separate and combined inhalation exposure. Cent. Eur. J. Public Health, 4, 115118.[Medline]
- Wang,H., Chanas,B. and Ghanayem,B.I. (2002) Effect of methacrylonitrile on cytochrome P-450 2E1 (CYP2E1) expression in male F344 rats. J. Toxicol. Environ. Health, 65, 523537.[CrossRef][ISI]
- Jackson,T.E., Lilly,P.D., Recio,L., Schlosser,P.M. and Medinsky,M.A. (2000) Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene. Toxicol. Sci., 55, 266273.[Abstract/Free Full Text]
- Powley,M.W. and Carlson,G.P. (2000) Cytochrome P450 involved with benzene metabolism in hepatic and pulmonary microsomes. J. Biochem. Mol. Toxicol., 14, 303309.[CrossRef][ISI][Medline]
- Burton,M., Reisdorph,R., Prough,R. and Lindahl,R. (1999) Modulation of class 3 aldehyde dehydrogenase gene expression. Adv. Exp. Med. Biol., 463, 165170.[ISI][Medline]
- Sreerama,L., Rekha,G.K. and Sladek,N.E. (1995) Phenolic antioxidant-induced overexpression of class-3 aldehyde dehydrogenase and oxazaphosphorine-specific resistance. Biochem. Pharmacol., 49, 669675.[CrossRef][ISI][Medline]
- Canuto,R.A., Muzio,G., Ferro,M., Maggiora,M., Federa,R., Bassi,A.M., Lindahl,R. and Dianzani,M.U. (1999) Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines. Free Radic. Biol. Med., 26, 333340.[CrossRef][ISI][Medline]
- Bozic,C.R., Gerard,N.P., von Uexkull-Guldenbrand,C., Kolakowski,L.F.,Jr, Conklyn,M.J., Breslow,R., Showell,H.J. and Gerard,C. (1994) The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J. Biol. Chem., 269, 2935529358.[Abstract/Free Full Text]
- Mueller,C.R., Maire,P. and Schibler,U. (1990) DBP, a liver-enriched transcriptional activator, is expressed late in ontogeny and its tissue specificity is determined posttranscriptionally. Cell, 61, 279291.[ISI][Medline]
Received June 11, 2003;
revised September 26, 2003;
accepted October 7, 2003.