Background Bronchoalveolar lavage (BAL) fluid prostaglandin D2 (PGD2) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD2, which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G protein–coupled receptors, including chemoattractant receptor–homologous molecule expressed on TH2 lymphocytes (CRTH2) and PGD2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. Objective We sought to compare the expression and activation of PGD2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to TH2 pathway elements. Methods Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD2 pathway markers were then compared with asthma severity, level of control, and markers of TH2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). Results Confirming previous results, BAL fluid PGD2 levels were highest in patients with severe asthma (overall P = .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P = .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P = .001 and P = .0001, respectively). Asthma exacerbations, poor asthma control, and TH2 inflammatory markers were associated with higher PGD2, HPGDS, and CRTH2 levels. Conclusion The current study identifies coordinated upregulation of the PGD2 pathway in patients with severe, poorly controlled, TH2-high asthma despite corticosteroid use.

Benzo [a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6-and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8-and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-transdihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B [a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner providing an explanation for the lag-phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzop-dioxin, (TCDD) eliminated this lag-phase, but did not alter the levels of the individual metabolites observed, suggesting that both B[a]P and TCDD induction ultimately yield the same B[a]P-metabolic profile. The one exception was B[a]P-3,6-dione which was formed without a lag-phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH-activation yield their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [13C315N1]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [13C315N1]-pantothenate after three passages of the murine cells in culture. Charcoal−dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N(2)-ethylGua and the O(6)-ethylGua adducts by the DNA polymerases alpha and eta and extension from 3'-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N(2)-ethylGua adduct by DNA polymerase alpha is approximately 10(4)-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N(2)-ethylGua:Cyt 3'-terminus by DNA polymerase alpha is approximately 10(3)-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N(2)-ethylGua lesion by the DNA polymerase eta is about 370-fold more efficient than by the DNA polymerase alpha, and extension from the N(2)-ethylGua:Cyt 3'-terminus by the DNA polymerase eta is about 3-fold more efficient than by the DNA polymerase alpha. Furthermore, the DNA polymerase eta preferably inserts the correct nucleotide Cyt opposite the N(2)-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O(6)-ethylGua adduct by the DNA polymerases alpha and eta. In reactions containing both DNA polymerases alpha and eta, synthesis past the N(2)-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N(2)-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase alpha and lesion bypass catalyzed by DNA polymerase eta.

There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (+/-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-anti-trans-B[a]PDE-N2-2'-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (+/-)-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3',5'-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage.

Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis, sample extraction from complex biological matrices, and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by LC-MS. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

Table 1 are shown in red (213, 239, 244, 246, 251, 256, 273, 275, 276, 279, 281). The PAH o-quinone derived recessive mutations are shown in green (142, 144, 147, 152, 161, 167, 170, 173, 180, 181, 182, 196, 196, 198, 204, 213, 216, 220, 234, 236, 238, 242, 243, 245, 266, 267, 283, 285, 298, 301, 306, 316, 158, 165, 171, 180, 199, 203, 253, 224, 235, 255, 260, 275, 281, 325). (Bottom) The 8 unique BPDE derived dominant mutations are shown in red (156, 158, 176, 178, 196, 213, 279, 283). Note that the dominant mutations cluster in DNA contact regions. Structures were plotted using the online software of the IARC TP53 database (3).