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J Appl Physiol 102: 1185-1192, 2007. First published November 9, 2006; doi:10.1152/japplphysiol.00796.2006 Free Article
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Ozone induces clear cellular and molecular responses in the mouse lung independently of the transcription-coupled repair status

Ingeborg M. Kooter,1 Jeroen L. A. Pennings,2 Paul H. B. Fokkens,1 Daan L. A. C. Leseman,1 A. John F. Boere,1 Miriam E. Gerlofs-Nijland,1 Flemming R. Cassee,1 Johanna A. C. Schalk,3 Tom J. H. Orzechowski,3 Mirjam M. Schaap,2,4 Timo M. Breit,4 Jan A. M. A. Dormans,2 Conny T. M. van Oostrom,2 Annemieke de Vries,2 and Harry van Steeg2

1Centre for Environmental Health Research, 2Laboratory of Toxicology, Pathology and Genetics, and 3Centre for Biological Medicines and Medical Technology, National Institute for Public Health and the Environment, Bilthoven; and 4Micro-Array Department, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

Submitted 18 July 2006 ; accepted in final form 4 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
The oxidant ozone is a well-known air pollutant, inhalation of which is associated with respiratory tract inflammation and functional alterations of the lung. It is well established as an inducer of intracellular oxidative stress. We investigated whether Cockayne syndrome B, transcription-coupled, repair-deficient mice (Csb–/–), known to be sensitive to oxidative stressors, respond differently to ozone than repair-proficient controls (Csb+/–). Mice were exposed to 0.8 parts/million ozone for 8 h, and we examined a wide range of biological parameters in the lung at the gene expression, protein, and cellular level 4 h after the ozone exposure. Relevant biological responses to ozone for both repair-deficient Csb–/– and repair-proficient Csb+/– mice, as determined by biochemical analysis of bronchoalveolar lavage fluid (e.g., increases of polymorphonuclear neutrophils, alkaline phosphatase, macrophage-inflammatory protein-2, and tumor necrosis factor-{alpha}), pathological examinations, and gene expression (upregulation of oxidative-stress-related genes) analyses were observed. The bronchoalveolar lavage fluid showed significantly more tumor necrosis factor-{alpha} in repair-deficient Csb–/– mice than in repair-proficient Csb+/– mice after ozone exposure. In addition, a clear trend was observed toward fewer differentially expressed genes with a lower fold ratio in repair-deficient Csb–/– mice than in repair-proficient Csb+/– mice. However, repair-deficient Csb–/– mice do not respond significantly more sensitively to ozone compared with repair-proficient Csb+/– mice at the level of gene expression. We conclude that, under the conditions employed here, although small differences at the transcriptional level exist between repair-proficient Csb+/– mice and transcription-coupled repair defective Csb–/– mice, these do not have a significant effect on the ozone-induced lung injury.

oxidative stress; Cockayne syndrome B; microarray

OZONE is a primary component of photochemical smog. The action of sunlight on hydrocarbons and nitrogen oxides in vehicle exhaust produces a complex oxidant mixture, of which ozone is a main constituent. Ozone-induced pulmonary injury has been thoroughly investigated in the last three decades. It is characterized by epithelial cell damage, inflammation, impaired lung function, and airway hyperresponsiveness (35). These effects have been extensively examined in humans (20, 23), as well in laboratory animals (3, 16). Ozone reacts directly with the fluid components of the epithelial lining and with the membranes of the epithelial cells (32). In addition, alveolar macrophages, acting as scavenger cells in the lung, are injured and secrete a whole array of bioactive messengers, including proinflammatory cytokines. Tumor necrosis factor-{alpha} (TNF-{alpha}) is among the early-response cytokines that are released by target cells at sites of inflammation. The production of TNF-{alpha} by alveolar macrophages initiates the inflammatory response by promoting the production of chemotactic cytokines from a variety of cellular sources, all of which contribute to the recruitment of specific leukocyte subpopulations to sites of injury. The response is characterized by a significant increase of polymorphonuclear neutrophils (PMNs) in the bronchoalveolar lavage fluid (BALF) (1, 23), an increased production of inflammatory cytokines, chemokines (18), antioxidant protein expression (19), and perturbations of the epithelial tight junctions, which allow serum proteins and albumin into the air spaces (2).

At certain levels, exposure to ozone will result in oxidative stress, either via the direct action of this powerful oxidant with its target macromolecules (such as lipids, proteins, or DNA) or via free radicals formed by peroxidation of lipids, and/or action of macrophages and PMNs due to induction of inflammation processes. The accompanying effects, such as an increase of lipid peroxidation products (8), induction of stress proteins (37), depletion of antioxidants (37), and increased antioxidant enzyme activities, can be used to study the susceptibility to ozone.

The lung susceptibility to acute exposure to ozone is remarkably strain dependent. The relatively high susceptibility of the inbred mouse strain C57BL/6J to ozone exposure has been particularly well documented (21, 42). However, we wanted to test the susceptibility of even more sensitive strains. To accomplish this, we used Cockayne syndrome group B (Csb–/–) mice (in a C57BL/6 genetic background) that had a severe defect in a main DNA repair defense system, i.e., transcription-coupled repair (TCR). Patients with CSB gene defect develop Cockayne syndrome, a rare autosomal recessive disorder characterized by symptoms of premature aging, ultraviolet (UV) sensitivity abnormalities, severe neurological abnormalities, and progeriod symptoms (4, 29). The CSB protein is probably involved in the repair of all kinds of oxidative DNA lesions present in actively transcribed genes. The involvement of the CSB protein in both nucleotide excision repair (NER) and base excision repair (BER) has been documented in great detail (28, 30, 31, 36). As a consequence of a defect in TCR/NER and/or TCR/BER, Csb–/– mice are sensitive to oxidative stressors such as paraquat and gamma radiation (10, 11). Therefore, a predominant question and purpose of our present study is: Is the TCR pathway important in ozone-induced lung injury through oxidative stress? This important issue is addressed by studying the Csb–/– mouse model.

This study aims to investigate acute lung injury caused by ozone in repair-proficient Csb+/– mice and repair-deficient Csb–/– mice by studying BALF indicators, the relevant pathology, and gene expression profiles. More precisely, we tested whether the repair-deficient Csb–/– mice were more sensitive to the oxidative stress inducer ozone than the repair-proficient control mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal Housing and Ozone Exposure

Male and female repair-proficient (heterozygous counterparts, Csb+/–) and repair-deficient Csb–/– mice (10–14 wk old, 20–30 g) were bred at the National Institute for Public Health and the Environment (RIVM). The mice had a pure C57BL/6J genetic background, to which they were backcrossed. Csb+/– mice can be considered wild type (38, 43).

During the acclimatization period of at least 4 days, as well as during exposure and the recovery period of 4 h, the animals were housed in 0.2-m3 stainless steel and Lexan inhalation chambers in the RIVM inhalation facilities (27). Repair-proficient Csb+/– (n = 10) and repair-deficient Csb–/– mice (n = 10) were exposed once for 8 h to either 0.8 parts/million (ppm) ozone or clean air during the animals' dark cycle. For practical reasons, the exposure took place on two separate days with equal numbers of animals in each group (air Csb+/–, O3 Csb+/–, air Csb–/–, O3 Csb–/– mice) on each day. Necropsy was performed 4 h after exposure. Activated charcoal, a permanganate, and a very efficient particle (HEPA) filter was used to purify air, which was conditioned at a temperature of 21 ± 2 °C and 40–70% relative humidity. An airflow of 6 m3/h was maintained through each chamber. The animals had unlimited access to tap water and pelleted standard laboratory diet [SRM-A pellets (10 mm), Hope Farms, Woerden, the Netherlands]. The animals were maintained on a 12:12-h light-dark cycle (lights on at 9 AM). For ozone exposure, an ozone-oxygen mixture, generated by irradiation of oxygen with UV light, was metered into the inlet air stream with stainless steel mass flow controllers at a rate of ~30 ml/min at a target concentration of 0.8 ppm (1,600 µg/m3). An exposure control program running on a personal computer interfaced to the exposure equipment provided automatic exposure. Concentrations in the chambers were measured at 2-min intervals with Monitor Labs 8810 O3 analyzers (San Diego, CA), and adjustments of the flow controllers were made to maintain concentration at the desired level. The analyzers were checked against zero-air and a reference ozone-air mixture generated by a Thermo Instruments Systems model 49PS calibrator (Hopkinton, MA). Experiments were approved by the Ethical Review Committee of the National Institute for Public Health and the Environment.

Necropsy

On the basis of previous findings, necropsy was performed 4 h after exposure (22). At necropsy, animals were weighed and anesthetized with a mixture of ketamine (100 mg/ml, Aesculaap, Boxtel, the Netherlands) and xylazine (20 mg/ml, Bayer, Leverkusen, Germany) and saline in a ratio of 10:4:14. The anesthetic was injected intraperitoneally (2 ml/kg body wt), resulting in a dose of 70 mg/kg body wt for ketamine and 6 mg/kg body wt for xylazine. The animals were killed by exsanguination via the abdominal aorta. The lungs were perfused with saline via the right cardiac ventricle to remove blood. The lungs were lavaged (3 in and out lavages with the same fluid) with a volume of saline corresponding with 40 ml/kg body wt at 37°C to obtain BALF. They were washed a second time with an amount of saline that corresponded to the recovered volume of the first lavage. The recovered BALF was placed on ice until further processing. The right lung was dissected, weighed, and fixed for 1 h at a constant pressure of 20 cmH2O with 10% phosphate-buffered formalin for histopathology. The left lung was used for RNA isolation.

Analysis of BALF

The BALF from each animal was centrifuged at 400 g and 4°C for 10 min. The cell-free fluid from the lavage was used for measurements of cellular toxicity, inflammation, and oxidative stress. The pellet from the lavage was resuspended in 250 µl of saline and used for preparation of cytospins for differential cell counts.

Cell differentiation.   Cytospin slides were made in duplicate for differential cell counts and stained with the May-Grünwald and Giemsa method. With a total of 400 cells/exposure, we counted 200 cells/cytospin slide and calculated the percentage of each cell type (macrophages, PMNs, eosinophilic granulocytes, and lymphocytes).

Biochemical characterization.   We determined lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) concentrations with a commercial reagent kit (Roche Nederland, Almere, the Netherlands). All chemicals used for glutathione measurement were obtained from Sigma-Aldrich Chemie (Steinheim, Germany). Glutathione (GSH and GSSG) in BALF samples was measured as described previously (6).

The LDH was measured as a marker of cytotoxicity and ALP as a marker of type II cell damage. In addition, the antioxidant GSH was measured as a marker of oxidative stress.

Cytokines.   We determined the inflammatory mediators TNF-{alpha} and macrophage inflammatory protein-2 (MIP-2) with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Biosource, Etten-Leur, the Netherlands).

Pathology

The right lung was embedded in paraplast, and 5-µm-thick sections were stained with hematoxylin-eosin. The pathological lesions (influx of centriacinar alveolar macrophages, hypertrophy of bronchiolar epithelium, perivascular infiltrate, peribronchiolar infiltrate, and influx of bronchiolar macrophages) were scored semiquantitatively and blindly by light microscopy (as minimal, slight, moderate, marked, or strong).

Gene Expression Profiling

Trizol reagent (Invitrogen, Breda, the Netherlands) and RNeasy Mini Kit (Qiagen Benelux, Venlo, the Netherlands) were used to isolate RNA from the frozen, weighed, left lung lobes (stored in RNAlater RNA Stabilization Reagent, Qiagen) according to the manufacturer's instructions. Total RNA was treated with RNase-free DNase (Qiagen) to eliminate contaminating genomic DNA. For a common reference, total RNA of the lungs of BALB/c mice, which underwent intratracheal instillation with either saline or particulate matter (PM) suspended in saline, were pooled. Microarray slides containing 21,997 oligos from the Sigma-Compugen Mouse oligonucleotide library were spotted at the microarray department of University of Amsterdam. For microarray experiments, we amplified 1 µg of total lung RNA with the Amino Allyl MessageAmp aRNA kit (Ambion, Cambridgeshire, UK) and labeled it by using Cy3 for samples and Cy5 for the common reference. Six biological replicates were used for each group. However, after a quality check, one array of each sham-exposed group was dismissed from further analysis.

We log-normalized the microarray signal data with global loess and then analyzed them with R-language and Microsoft Excel. A difference in gene expression with a P value <0.001 (one-way ANOVA) and a minimal fold ratio (FR; ratio of maximum/minimum) of 1.5 or more was considered sufficiently important for further evaluation. The FR is considered negative if gene expression of the control group is greater than the gene expression of the ozone group. Database for Annotation Visualization and Integrated Discovery (DAVID; at www.apps1.niaid.nih.gov/david) was used for the Expression Analysis Systematic Explorer (EASE). The data from this study are publicly available at Array Express (http://www.ebi.ac.uk/arrayexpress, accession number E-TABM-137).

Quantitative Gene Expression

Quantitative real-time polymerase chain reactions (Q-PCRs) were run with the same RNA samples as were used for the microarray hybridizations. The total RNA (1 µg) was reverse-transcribed in a total volume of 20 µl with 200 ng random hexanucleotide primers (Roche Diagnostics, Mannheim, Germany), 0.5 mM dNTPs (Promega, Madison, WI), 20 mM DTT, 50 mM Tris·HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 40 U of RNasin (Promega), and 200 U of SuperScript II Rnase H-reverse transcriptase (Invitrogen, Carlsbad, CA) at 42°C for 50 min. Subsequently, 10 ng of reverse-transcribed total RNA was used for Q-PCR with the LightCycler (Roche Diagnostics). The Q-PCR was performed with the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics) according to the manufacturer's instructions with 0.5 µM of each PCR primer. The PCR program consisted of a 10-min hot start at 95°C to activate the Taq DNA polymerase, 40 cycles of 10 s denaturation at 95°C, 10 s annealing at 60°C, 10-s extensions at 72°C for metallothionein-1 (Mt-1), hypoxanthine guanine phosphoribosyl transferase-1 (Hprt-1), and amphiregulin (Areg), and 4-s extensions at 72°C for coagulation factor III (FIII), heme oxygenase-1 (HO-1), and thioredoxin reductase-1 (Txnrd-1). Melting curve analysis took place at the end of the PCR program. During the amplification cycle, fluorescence was measured at the end of each elongation step.

Fluorescence was measured continuously during the slow heating step of the melting curve cycle. The PCR products were analyzed once by conventional agarose gel electrophoresis to verify that a single band of the correct size was amplified. Each RNA sample was also tested in the Q-PCR without a reverse transcription step to exclude PCR signals being obtained from genomic DNA instead of mRNA. Primer pairs were deduced from the literature or obtained from the Primer Bank (41). The sequences of the primers used for Q-PCR were as follows: Mt-1: 5'-TCT CGG AAT GGA CCC CAA CTG-3' and 5'-TTT ACA CGT GGT GGC AGC GC-3' (26); FIII: 5'-AAC CCA CCA ACT ATA CCT ACA CT-3' and 5'-GTC TGT GAG GTC GCA CTC G-3'; Areg: 5'-TCA TGG CGA ATG CAG ATA CA-3' and 5'-GCT ACT ACT GCA ATC TTG GA-3' (14); HO-1: 5'-AAG CCG AGA ATG CTG AGT TCA-3' and 5'-GCC GTG TAG ATA TGG TAC AAG GA-3'; Txnrd-1: 5'-CGG AGG AAC GTG TGT GAA TGT-3' and 5'-TCA GAG CTT GTC CGA GCA AA-3' (13); Hprt-1: 5'-TCA GTC AAC GGG GGA CAT AAA-3' and 5'-GGG GCT GTA CTG CTT AAC CAG-3'. A BLAST search with the primer sequences confirmed the specificity. For each mouse, the individual level of initial target cDNA was expressed as the difference in threshold cycle (Ct) between the cDNA of interest and Hprt1 cDNA. {Delta}Ct was calculated by subtracting the mean difference in Ct of the exposed mice from the mean difference in Ct of the untreated mice. The gene expression ratio was calculated as 2–{Delta}Ct.

Statistical Analysis

All data of biological effect parameters were log-transformed and subjected to a two-way ANOVA. Two-way ANOVA analyses were used to assess differences due to ozone exposure, genotype, and their interaction. The Bonferroni method was used for post hoc analyses to reveal differences between genotypes and/or ozone exposure of specific groups. Since the cell differentials were expressed as percentages (no normal distribution), the nonparametric Wilcoxon rank test was used. ANOVA analyses show that the fact that the exposure was conducted on two separate days does not affect the results found for the terms "ozone" and "genotype" in the analyses, except for the ozone term for the cell differential parameters. Repair-deficient Csb–/– mice were considered to be more sensitive to ozone than their repair-proficient counterparts, Csb+/– mice, when a significance was found in the interaction term between the ozone exposure and the genotype (exposure x genotype). Such a significance would imply that the repair-deficient Csb–/– mouse reacts differently to the ozone exposure than the repair-proficient Csb+/–mouse.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Body and Organ Weights

The body, heart, and lung weights, as well as the lung-to-body weight ratios, were unaffected by the ozone exposure of both genotypes (Table 1). However, body weight data for repair-deficient Csb–/– mice are lower than those of repair-proficient Csb+/– mice, although not significantly.


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  		Table 1. Body and organ weights and results of BALF analyses

 
BALF Analysis

A two-way ANOVA performed on the complete data set revealed significant ozone effects for the following parameters: PMN percentages, ALP, LDH, MIP-2, TNF-{alpha}, and total glutathione. Significant effects due to the genotype for TNF-{alpha}, total glutathione, and LDH were also found.

Further post hoc Bonferroni and Wilcoxon rank tests revealed significantly increased PMNs in BALF of 0.1% to 15% after ozone exposure (Table 1) for repair-proficient Csb+/– mice. The amount of ALP was approximately five times greater, which was significant. Ozone exposure increased the LDH or total glutathione concentrations in BALF 4 h after exposure. However, ozone exposure of the repair-proficient Csb+/– mice increase the MIP-2 (not significant) and TNF-{alpha} (significant, Table 1).

Analysis of BALF showed significant increases of ALP and PMNs in response to ozone in the repair-deficient Csb–/– mice, which were similar to those of the repair-proficient variant (Table 1). The total glutathione concentrations in the sham-exposed Csb–/– mice were significantly greater than those in the sham-exposed Csb+/– mice (Table 1). The total glutathione concentration was not significantly affected by exposure to ozone. Ozone exposure of the Csb–/– mice resulted in significant increases of the cytokines MIP-2 and TNF-{alpha} in BALF (Table 1). In addition, there was a significant difference in the concentration of TNF-{alpha} found in the ozone-exposed Csb–/– mice and that of the Csb+/– mice. However, no statistical significance could be calculated for the interaction term (exposure x genotype) of this parameter.

Lung Pathology

Clear pathological changes in the lung were observed in repair-proficient Csb+/– mice after ozone exposure (Table 2). All pathological lesions were scored semiquantitatively as minimal. A minimal centriacinar increase of alveolar macrophages was observed. In addition, two animals showed a minimal perivascular and peribronchiolar infiltrate of PMNs that corresponded with observations of the BALF. One animal had many macrophages in the bronchial lumen. Four animals showed a focal minimal hypertrophy of the bronchiolar epithelium. As expected, the unexposed groups did not show any histological abnormalities.


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  		Table 2. Pathological changes observed in the lungs of repair-proficient Csb+/– and repair-deficient Csb–/– mice exposed to ozone

 
Similar to those in the repair-proficient lung, clear pathological changes in the lung of the repair-deficient Csb–/– mice due to ozone were observed after ozone exposure (Table 2). There was a minimal centriacinar increase of alveolar macrophages. In addition, three animals had minimal perivascular influxes of PMNs. Each of two animals had a minimal influx of macrophages in the bronchial lumen. The unexposed control group did not show any histological abnormalities.

Gene Expression: Profiling of Ozone-Responsive Genes

We used arrays containing ~22,000 oligonucleotides to profile the changes in gene expression after ozone exposure. Principal component analyses (PCAs) of the genes affected by exposure to ozone resulted in two distinct groups, a sham-exposed group and an ozone-exposed group (Fig. 1). A one-way ANOVA on all four different Csb groups resulted in 269 significant genes (P < 0.001 and FR > 1.5). With a two-way ANOVA, no significant effect of the genotype was found, whereas 332 significant genes were affected upon exposure to ozone (P < 0.001 and FR > 1.5). For the interaction "exposure x genotype" five significant genes were found, namely, BH3 interacting domain death agonist, membrane-spanning 4-domains, subfamily A; member 6D, T-cell receptor beta chain; membrane-spanning 4-domains, subfamily A; and member 6B, lecithin-retinol acyltransferase. A 22,000-oligonucleotide array is expected to give 22 false positives for a significance of less than 0.001.


Figure 1
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  		Fig. 1. Principal component analysis of expression data for the 332 genes affected by exposure to ozone (P < 0.001 and fold ratio > 1.5). The numbers shown are animal identification numbers. Axes are in arbitrary units.

 
After ozone exposure of the repair-proficient Csb+/– mice, 275 genes appeared to be affected in the lung tissue (p < 0.001 and FR > 1.5). Of these genes, 110 were induced and 165 were suppressed upon exposure. The differentially expressed genes were assigned to functional classes (see Supplemental Table 1S, available with the online version of this article). It is clear that the classes "oxidative stress," "signal transduction," "lipid metabolism," "coagulation," and "cytoskeleton" are dominated by the upregulated expression of genes. The classes "polysaccharide," "calcium binding," and "transduction" are dominated by the downregulated expression of genes. Table 3 shows the genes affected by oxidative stress after ozone exposure (FR > 1.5).


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  		Table 3. Genes affected by ozone exposure in the lungs of repair-proficient Csb+/– and repair-deficient Csb–/– mice involved in oxidative stress

 
In addition, an EASE with DAVID (12, 17) was used to find out which set of genes, on the basis of their functions, had the most modified expression after exposure. No categories were found for genes with upregulated expression. The EASE found categories like "defense response," "antigen presentation/exogenous antigen via major histocompatibility complex (MHC) class II," and "MHC class II receptor activity" (data not shown).

As already stated, 275 affected genes in the lungs of the repair-proficient Csb+/– mice were found. We found 156 affected genes in lung tissue of the repair-deficient Csb–/– mice (P < 0.001 and FR > 1.5). Of these genes, 89 were induced and 67 were suppressed upon exposure. The FRs of the ozone-affected genes of the repair-deficient Csb–/– mice are somewhat lower than those of the repair-proficient Csb+/– mice, in particular the genes with downregulated expression (Fig. 2). In 77% of the cases (256 of the 332 significantly affected genes), the FRs of the affected genes of the repair-deficient Csb–/– mice are lower than those of the repair-proficient Csb+/–mice.


Figure 2
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  		Fig. 2. Gene expression ratio due to ozone exposure of repair-deficient Csb–/– mice vs. repair-proficient Csb+/– mice. Genes with fold ratios (FRs) greater than 1.5 in both genotypes are shown. FR is the ratio of the maximum to the minimum of gene expression under ozone and control conditions, and it is negative if the gene expression of control is greater than the gene expression after ozone exposure.

 
Quantitative Gene Expression

We used Q-PCR with a selection of genes in lung tissue and liver tissue to verify the findings from the total gene expression profiling. Liver was taken as an indirect target tissue. Three oxidative stress genes, Mt-1, Txnrd-1, and HO-1; a cytokine of the epidermal growth factor family, Areg; and the cardiovascular related gene FIII were selected for Q-PCR. The data were normalized to the expression of the household gene Hprt1 (9, 39). Table 4 shows the gene expression ratios after ozone exposure. All the genes in the lung tissue showed significant changes in expression that correlated closely with the changes calculated in the microarray analysis. As expected, Q-PCR analysis of the same genes in liver tissue shows no significant increase of expression due to ozone exposure, except for Mt-1 and FIII.


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  		Table 4. Gene expression ratio due to exposure to ozone in lung and liver tissue as determined by quantitative real-time polymerase chain reactions

 
Similar to the ratios found by microarray analyses, the gene expression ratio found with Q-PCR in the lungs of repair-deficient Csb–/– mice was lower than that of the repair-proficient Csb+/– mice (Table 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
In this study, a clear ozone effect was shown by significant increases in PMN percentages and ALP in the BALF, which indicates an inflammatory response and damage of type II epithelial cells, respectively. The increased neutrophil chemoattractant MIP-2 represents a typical cytokine response, the most likely sources of which (after ozone exposure) are macrophages, PMNs, and the epithelial cells (19). Clear pathological changes (such as the disappearance of the protruding apical parts of the Clara cells) are commensurate with the findings that ozone causes lung inflammation and damage characterized by the release of cytokines and chemokines (such as MIP-2 and TNF-{alpha}), as well as by an influx of PMNs in both repair-deficient Csb–/– and repair-proficient Csb+/– mice. Our data are consistent with those of previous studies in which C57BL/6J mice were exposed to ozone (5, 34). In addition, we have identified a clear and significant ozone exposure-related effect on the expression of 332 genes in this study, which is confirmed by PCA (Fig. 1). Most of the genes affected by ozone are repressed (60%), which correlated with the findings of Gohil et al. (15). Similarly to their findings, our data show induction of the serum amyloid A3 gene, which suggests that inflammatory cytokines activate NF-{kappa}B and CCAAT/enhancer binding protein-mediated pathways (15). Further, ozone increased the mRNA concentrations of many oxidative-stress-related genes in the glutathione system, HO-1, and Mt-1, as has been previously described in in vivo and in vitro gene expression studies (15, 40). The suppression of immune response-related genes indicated by the EASE is in line with previous findings suggesting that ozone-exposed mice may also have suppressed immune responses (7).

Except for the strong response to ozone of both types of mice, repair-deficient Csb–/– mice do not respond significantly differently than repair-proficient Csb+/– mice, both at the protein level and the gene expression level (according to calculations using oligonucleotide arrays, in which there is a lack of significance in the interaction term "exposure x genotype"). The gene expression analysis shows five significant genes for this term, but we do not consider them relevant because of the number of expected false positives (22). However, Q-PCR data do show significance for the interaction term "exposure x genotype" for the gene expression of the individual genes Mt-1, HO-1, and FIII in the lung. More detailed analysis of the data reveals further subtle discrepancies between the genotypes.

First, the data for gene expression after ozone exposure point toward the finding that Csb–/– mice respond to ozone exposure to a lesser extent than the repair-proficient Csb+/– mice. We found fewer genes in Csb–/– mice that respond to ozone exposure than in Csb+/– mice: 156 and 275 genes, respectively. The FR of the gene expression due to ozone exposure is lower for the repair-deficient Csb–/– mice in 77% of the cases than for the repair-proficient Csb+/–mice. We confirmed this with Q-PCR for a selected set of genes, and the overall results suggest that repair-deficient Csb–/– mice have a lower response, both in the number of responding genes and in the FR of the gene expression.

Second, there was a genotype-dependent increase of TNF-{alpha} in the mice 4 h after ozone inhalation. This clearly significant increase in TNF-{alpha} concentrations in the ozone-exposed, repair-deficient Csb–/– mice with respect to the repair-proficient Csb+/– mice is not confirmed by an increase of TNF-{alpha} at the gene expression level. One possible explanation is that the increase in gene expression took place earlier, but it is more likely that TNF-{alpha} is mainly released from storage granules, and the repair-deficient Csb–/– mice responded more than the repair-proficient Csb+/– mice. Whether this relative increase of TNF-{alpha} for the repair-deficient Csb–/– mice results in a difference in specific leukocyte subpopulations at the site of injury is not clear from this study.

Another interesting observation is the increase in gene expression of MT-1 in the liver due to ozone exposure in the lung as shown by Q-PCR, which is analogous to the findings of Last et al. (25). They hypothesize that exposure of mice to ozone might activate various transcriptionally regulated pathways of gene expression in the lungs, which in turn could cause systemic effects via cross-talk with other tissues by release of cytokines or other signaling molecules (25). Cytokines such as TNF-{alpha} or IFN-{gamma} produced by inflammatory or epithelial cells in the lung in response to the injury likely play a role in this by activating the NF-{kappa}B pathway (24, 33). In this respect, the greater TNF-{alpha} concentrations in BALF after ozone exposure and, analogously, the greater expression of the MT-1 gene in the liver in Csb–/– mice than the respective values for Csb+/– mice is remarkable, and it is compatible with the hypothesized cross-talk.

Although the trend was not significant, whole body weights of the repair-deficient Csb–/– mice were slightly less than those of repair-proficient Csb+/– mice. This is consistent with human data: Cockayne syndrome patients have a growth failure, both in weight and length (29). However, we believe that this difference in weight and the accompanying differences in metabolic rate and breathing frequency are unlikely to influence the amount of ozone inhaled, considering the study exposure time (8 h) and concentration (0.8 ppm).

This initial in vivo study in which Csb mice were exposed to ozone began with the premise that repair-deficient Csb–/– mice would be more sensitive to this oxidative stressor than the repair-proficient control mice. However, although significant increases of TNF-{alpha} were found in repair-deficient Csb–/– mice after ozone exposure, and their responses in gene expression (both in number and FR of genes) were weaker, no significance was included in the interaction term "exposure x genotype" for either protein or gene-expression parameters. Nonetheless, it might well be that the early time of necropsy [chosen because this study was designed to determine gene expression profiles shortly after ozone exposure (22)] masked differences between the repair-deficient Csb–/– and repair-proficient Csb+/– mice. The Csb gene functions in the transcription-coupled repair in both nucleotide excision repair and base excision repair (NER and BER), and in doing so, it is involved in removing transcription-blocking lesions such as those induced by oxidative stress. In the present necropsy conditions, when transcription-blocking lesions do not dominate, no major, significant difference between the knockout and repair-proficient mice would be expected. However, our results showing that there is somewhat less transcription in general in Csb–/– mice are compatible with the idea that, owing to enhanced DNA damage, especially in actively transcribed genes, transcription is hampered somewhat in Csb–/– mice since lesions persist. Whether such effects are more prevalent at later times remains to be studied.

In conclusion, this study has shown clearly significant biological responses to ozone for both repair-deficient Csb–/– and repair-proficient Csb+/– mice, as determined by biochemical, pathological, and gene expression analyses. In the present experimental conditions, there was significantly more TNF-{alpha} in repair-deficient Csb–/– mice than in repair-proficient Csb+/– mice after ozone exposure. In addition, we observed a clear trend of fewer differentially expressed genes with a lower FR in repair-deficient Csb–/– mice than in repair-proficient Csb+/– mice. Moreover, repair-deficient Csb–/– mice do not, or do not significantly, respond more sensitively to ozone than repair-proficient Csb+/– mice, at least under the conditions we used here. Therefore, we conclude that the repair-deficient Csb–/– mice were not more sensitive to ozone exposure than the repair-proficient controls and that the TCR pathway is not crucial to ozone-induced lung injury.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Gijsbertus T. J. van der Horst (Erasmus Medical Center, Rotterdam) for providing us with the Csb mouse model, and Jan Bos, Ron F. Vlug, Ruud W. M. van Kinderen, Liset J. J. de la Fonteyne, Yvonne C. Wallbrink, Eugène H. J. M. Jansen, Piet K. Beekhof, Rija H. A. van Loenen, G. van Leuveren, F. K. Gielis-Proper, and F. de Vlugt van den Koedijk for experimental assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. M. Kooter, TNO, Postbus 342, 7300 AH Apeldoorn, the Netherlands (e-mail: ingeborg.kooter{at}tno.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 

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