Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking

Cigarette smokers inhale a broad range of carcinogens derived from tobacco and its pyrolysis products, including free radicals, which induce oxidative stress and subsequent lipid peroxidation (LPO). Miscoding carcinogen-DNA adducts are formed by cigarette smoke constituents and are thought to initiate lung carcinogenesis. The presence of various types of DNA damage was therefore analyzed in tumor adjacent uninvolved lung tissues of 13 smoking and 11 non-smoking operated lung cancer patients. O(4)-ethylthymidine (O(4)etT), 1,N(6)-ethenodeoxyadenosine ( epsilon dA) and 3,N(4)-ethenodeoxycytidine ( epsilon dC) were determined by immuno-enriched (32)P-postlabeling. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were measured as diagonal radioactive zones after nuclease P1 enriched (32)P-postlabeling. Mean O(4)etT and PAH-DNA adduct levels were higher in lung DNA of smokers than of non-smokers (O(4)etT/10(8) thymidine: 3.8 versus 1.6, P < 0.01; PAH-DNA adducts/10(8) nucleotides: 11.2 versus 2.2, P < 0.01). Pulmonary etheno-DNA adduct levels did not differ between smokers and non-smokers, but large inter-individual variations were observed (80- and 250-fold differences for epsilon dA and epsilon dC, respectively). As all smokers (except one) refrained from smoking at least for 1 week before surgery, our results demonstrate the persistence of O(4)etT and PAH-DNA adducts in human lung. A positive correlation obtained between O(4)etT and PAH-DNA adducts (R = 0.65, P < 0.01) suggests that both adducts are formed from cigarette smoke as the main exposure source. We conclude that in addition to the DNA adducts derived from PAH and tobacco-specific nitrosamines, miscoding O(4)etT lesions are formed by cigarette smoke that contribute to the increased genomic instability and increased lung cancer risk in smokers.     

Modulation of DNA and protein adducts in smokers by genetic polymorphisms in GSTM1,GSTT1, NAT1 and NAT2

The formation of DNA and protein adducts by environmental pollutants is modulated by host polymorphisms in genes that encode metabolizing enzymes. In our study on 67 smokers, aromatic-DNA adduct levels were examined by nuclease P1 enriched 32P-postlabelling in mononuclear blood cells (MNC) and 4-aminobiphenyl-haemoglobin adducts (4-ABP-Hb) by gas chromatography-mass spectroscopy. Genetic polymorphisms in glutathione S-transferase M1 (GSTM1), T1 (GSTT1) and N-acetyl-transferase 1 (NAT1) and 2 (NAT2) were assessed by polymerase chain reaction-based methods. DNA adduct levels, adjusted for the amount of cigarettes smoked per day, were higher in GSTM1(-/-) individuals (1.30 +/- 0.57 adducts per 108 nucleotides) than in GSTM1(+) subjects (1.03 +/- 0.56, P = 0.05), higher in NAT1 slow acetylators (1.58 +/- 0.54) than in NAT1 fast acetylators (1.11 +/- 0.58, P = 0.05) and were also found to be associated with the NAT2 acetylator status (1.29 +/- 0.64 and 1.03 +/- 0.46, respectively, for slow and fast acetylators, P = 0.06). An effect of GSTT1 was only found in combination with the NAT2 genotype; individuals with the GSTT1(-/-) and NAT2-slow genotype contained higher adduct levels (1.80 +/- 0.68) compared to GSTT1(+)/NAT2 fast individuals (0.96 +/- 0.36). Highest DNA adduct levels were observed in slow acetylators for both NAT1 and NAT2 also lacking the GSTM1 gene (2.03 +/- 0.17), and lowest in GSTM1(+) subjects with the fast acetylator genotype for both NAT1 and NAT2 (0.91 +/- 0.45, P = 0.01). No overall effects of genotypes were observed on 4-ABP-Hb levels. However, in subjects smoking less than 25 cigarettes per day, 4-ABP-Hb levels were higher in NAT2 slow acetylators (0.23 +/- 0.10 ng/g Hb) compared to fast acetylators (0.15 +/- 0.07, P = 0.03). These results provide further evidence for the combined effects of genetic polymorphisms in GSTM1, GSTT1, NAT1 and NAT2 on DNA and protein adduct formation in smoking individuals and indicate that, due to the complex carcinogen exposure, simultaneous assessment of multiple genotypes may identify individuals at higher cancer risk.     

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.     

Myeloperoxidase (MPO) -463G->A reduces MPO activity and DNA adduct levels in bronchoalveolar lavages of smokers.

The myeloperoxidase (MPO) -463G-->A genetic polymorphism is associated with a reduced risk for lung cancer, but the underlying mechanism is not yet elucidated. Therefore, the impact of this polymorphism on MPO activity and lipophilic DNA adducts was studied in respectively bronchoalveolar lavage (BAL) fluid and cells, from 106 smoking Caucasian lung patients. MPO activity was determined spectrophotometrically, aromatic DNA adducts by (32)P-postlabeling and MPO genotypes by RFLP analysis. Frequencies of MPO -463AA (13%), MPO -463AG (36%), and MPO -463GG (51%) were in line with earlier observations. MPO activity/neutrophil was lower in MPO -463AA (median 0.04 pU/cell) than in MPO -463AG (median 0.07 pU/cell) and MPO -463GG (median 0.14 pU/cell; P = 0.059) individuals. DNA adducts in BAL cells were measured in 11 MPO -463AA subjects and equal numbers of MPO -463AG and MPO -463GG subjects matched for smoking, age, gender, and clinical diagnosis. DNA adduct levels in MPO -463AA individuals (median 0.62 adducts/10(8) nucleotides) were lower than in MPO -463AG (median 1.51 adducts/10(8) nucleotides) and MPO -463GG (median 3.26 adducts/10(8) nucleotides; P = 0.003) subjects. Overall, no significant correlation was observed between amount of inhaled tar/day and DNA adduct levels. However, correlations improved considerably on grouping according to the MPO genotype; MPO -463AA subjects were the least responsive (R(2) = 0.73, slope = 0.4, P = 0.01) followed by MPO -463AG subjects (R(2) = 0.70, slope = 1.3, P = 0.01) and MPO -463GG patients (R(2) = 0.67, slope = 2.8, P = 0.02). These data demonstrate that MPO -463AA/AG genotypes are associated with (a) reduced MPO activity in BAL fluid and (b) reduced smoking-related DNA adduct levels in BAL cells in a gene-dose manner. These data provide a plausible biological explanation for the reduced risk for lung cancer as observed in MPO -463AA/AG compared with MPO -463GG subjects.     

Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non- smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.      

Slow wave sleep and a state resembling absence epilepsy induced in the rat by gamma-hydroxybutyrate.

The effect of gamma-hydroxybutyrate (GHB) in relatively low doses (12.5--200 mg/kg) on sleep stages, electrocorticogram (ECoG) patterns and behavior was investigated in the rat. 50-100 mg/kg GHB induced slow wave sleep but, in contrast to the cat, not paradoxical sleep. 200 mg/kg induced a hypersynchronous, bilaterally symmetrical ECoG pattern, which was different in amplitude and frequency distribution from normally occurring high amplitude patterns. When the hypersynchrony occurred in bursts, the rats displayed a sudden arrest of motor behavior. Convulsions were not induced. The results, together with the finding of others that GHB is a natural constituent of mammalian brain and our previous observation that the GHB-induced hypersynchrony can be antagonized by anti-absence (anti-petit mal) drugs are discussed in view of the possibility that GHB might play a role in the etiology of absence epilepsy in man.     

Older men manifest multifold synchrony disruption of reproductive neurohormone outflow

Under a working clinical hypothesis that aging putatively disrupts neuroendocrine control mechanisms, here we test a specific corollary notion that transitions in sleep stage, oscillations in nocturnal penile tumescence (NPT; a neurogenically organized signal), and the rates of instantaneous secretion of LH and/or testosterone are jointly synchronous in healthy young, but not older, men. To this end, we evaluated 10 young (aged 21-31 yr) and 8 older (aged 65-74 yr) men by intensive overnight multisite monitoring, viz. simultaneous electro-encephalogram and NPT recordings (every 30 s) and remote blood sampling (every 2.5 min) to quantitate LH and testosterone release. Waveform-independent deconvolution and cross-correlation analyses of these neurohormone outflow measures revealed that healthy young men sustain four salient physiological linkages overnight: 1) a strong inverse (confirmatory) relationship between sleep stage and NPT activity, such that deeper sleep is accompanied by suppression of NPT; 2) consistent coupling between NPT and testosterone secretion, wherein heightened NPT activity respectively precedes and follows increased testosterone secretion by 12.5-32.5 and 50-60 min; 3) evident synchrony between sleep stage and testosterone secretion, in which testosterone secretion increases over a 30-min window (-2.5 to 25 min) while sleep deepens; and 4) a close temporal linkage between instantaneous LH release and NPT oscillations, whereby LH secretion increases 55-62.5 min before and again 5-30 min after NPT declines. In contrast, older men manifested global loss of expected young adult synchrony; namely, 1) abolition of the inverse relationship between sleep stage and NPT, 2) decorrelation of NPT oscillations and testosterone secretion, 3) decoupling of testosterone release and deep sleep, and 4) abrogation of the linkage between LH secretion and penile detumescence. In summary, high intensity overnight monitoring of multiple reproductive neuroendocrine outflow measures simultaneously in young men delineates prominent neurophysiological coupling among sleep transitions and NPT activity, LH and testosterone secretion or NPT oscillations, and testosterone secretion and deepening sleep stage. In contrast, healthy older men exhibit near-universal disruption of physiological young adult synchronicity. Thus, we conclude that male reproductive aging is marked by erosion of coordinate regulation among sleep transitions, central nervous system-directed NPT activity, and hypothalamically driven episodic GnRH/LH (and thereby Leydig cell testosterone) secretion. Whether analogous multifold uncoupling of neurohormone signals emerges in the course of reproductive aging in women or in nonhuman species is not yet known.     

The Role of Genetic Variants in the Association between Dietary Acrylamide and Advanced Prostate Cancer in the Netherlands Cohort Study on Diet and Cancer

To investigate the association between dietary acrylamide and advanced prostate cancer, we examined acrylamide-gene interactions for advanced prostate cancer risk by using data from the Netherlands Cohort Study.

Participants (n = 58,279 men) completed a baseline food frequency questionnaire (FFQ), from which daily acrylamide intake was calculated. At baseline, 2,411 men were randomly selected from the full cohort for case-cohort analysis. Fifty eight selected single nucleotide polymorphisms (SNPs) and two gene deletions in genes in acrylamide metabolism, DNA repair, sex steroid systems, and oxidative stress were analyzed. After 20.3 years of follow-up, 1,608 male subcohort members and 948 advanced prostate cancer cases were available for Cox analysis.

Three SNPs showed a main association with advanced prostate cancer risk after multiple testing correction: catalase (CAT) rs511895, prostaglandin-endoperoxide synthase 2 (PTGS2) rs5275, and xeroderma pigmentosum group C (XPC) rs2228001. With respect to acrylamide-gene interactions, only rs1800566 in NAD(P)H quinone dehydrogenase 1 (NQO1) and rs2301241 in thioredoxin (TXN) showed a nominally statistically significant multiplicative interaction with acrylamide intake for advanced prostate cancer risk. After multiple testing corrections, none were statistically significant.

In conclusion, no clear evidence was found for interaction between acrylamide intake and selected genetic variants for advanced prostate cancer risk.     

Myeloperoxidase--463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients.

The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -463G-->A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts/10(8) nt for MPO-463AA/AG and -463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the anti-BPDE-DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The (32)P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE-DNA. The mean values of the BPDE-DNA adduct spots were 3.8 +/- 2.4 per 10(8) nt for MPO-463AA/AG (n = 3) and 18.4 +/- 11.0 per 10(8) nt for MPO-463GG (n = 7), respectively (P = 0.03). One individual with the homozygous mutant genotype (-463AA) even had a 13-fold lower adduct level (1.4 per 10(8) nt) as compared to MPO-463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.