D. M. F. A. Pachen

Activated neutrophils inhibit nucleotide excision repair in human pulmonary epithelial cells: role of myeloperoxidase

Neutrophils are thought to affect pulmonary carcinogenesis by promoting the metabolism of inhaled chemical carcinogens, causing enhanced formation of promutagenic DNA adducts. We hypothesized that neutrophils interfere with the removal of such DNA adducts by inhibiting nucleotide excision repair (NER) in target cells. Human alveolar epithelial cells (A549) were cocultured with activated neutrophils, and we observed a significant reduction of NER in the A549 cells, which was abrogated by addition of the myeloper‐oxidase (MPO) inhibitor 4‐aminobenzoic acid hydra‐zide. The inhibitory effect of neutrophils could be mimicked by the MPO product hypochlorous acid (HOCl), which caused an acute, dose‐dependent inhibition of NER in A549 cells. This was independent of cytotoxicity or ATP loss and persisted up to 24 h. These data were supported by showing that HOCl caused a delayed removal of DNA adducts in benzo[a]pyrene‐diolepoxide‐exposed A549 cells. The acute HOCl‐in‐duced inhibition of NER can only partly be explained by oxidative modification of repair proteins. To explain the more persistent effects of HOCl, we analyzed the expression of NER genes and found that HOCl significantly reduced XPC expression. In conclusion, these data indicate that neutrophils are potent inhibitors of nucleotide excision repair. This may provide a further biological explanation for the association between inflammation and lung cancer development.—Güngör N., Godschalk, R. W. L., Pachen, D. M., Van Schooten F. J., Knaapen A. M. Activated neutro‐phils inhibit nucleotide excision repair in human pulmonary epithelial cells: role of myeloperoxidase. FASEB J. 21, 2359–2367 (2007)

Bile Acid Concentrations, Cytotoxicity, and pH of Fecal Water from Patients with Colorectal Adenomas

In the multistage model of human colorectaltumorigenesis, both genetic and environmental factorsplay an important role. The identity of theenvironmental factors involved, however, still remainsto be elucidated. As fecal bile acids are proposed ascandidates, we compared the concentration of bile acidsin fecal water from patients at different risk ofdeveloping colorectal cancer. In addition, pH of fecal water as well as its cytotoxicity toHT-29 colonic cells was determined. The high-risk groupconsisted of individuals diagnosed with one or more(tubulo)villous colorectal adenomas larger than 1 cm in diameter and containing moderate or severedysplasia (N = 20). Subjects with colorectal adenomassmaller than 1 cm and showing only minor dysplasia wereassigned to the medium risk group (N = 19). The control group consisted of persons with normalfindings by colonoscopy (N = 25). The results show nosignificant differences in fecal water bile acidconcentrations between the three groups. However, 46% of the observed cytotoxicity is explained in aregression model that includes pH and the concentrationsof deoxycholic acid, cholic acid, and ursodeoxycholicacid. The pH of fecal water is found to be significantly lower in the high risk group as compared to thecontrols, suggesting that a relatively high fecal pH hasa protective effect on the development of colorectaladenomas. Although hyperproliferation as a result of cytotoxicity has been suggested tocontribute to tumor formation in the colon, thepH-dependent cytotoxicity of bile acids in fecal waterwas not found to be associated with adenoma formation inthe present study.

Induction of DNA adducts by several polychlorinated biphenyls.

It is known that lower‐chlorinated biphenyls are metabolically activated to electrophilic quinoid species capable of binding to DNA. Also, certain metabolites are capable of redox cycling, thereby increasing oxidative stress in biological systems. In the present study, we tested mono‐, di‐, tri‐, tetra‐, penta‐, hexa‐, and heptachlorinated biphenyls for their ability to bind with DNA and to induce oxidative DNA damage. We present additional evidence that several PCB congeners form DNA adducts after metabolic activation, which can be detected by the nuclease P1‐ or butanol‐enrichment procedures of the 32P‐postlabeling technique. Butanol and nuclease P1 enrichments showed different adduct recoveries, depending on the level of chlorination of the biphenyls. Application of the nuclease P1 enrichment showed that the incubation of 2‐chloro‐; 3,4‐dichloro‐; 2,4,4′‐trichloro‐; 3,4,5‐trichloro‐; and 2,2′,5,5′‐tetrachlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital, followed by oxidation with a peroxidase, produced five to eight different DNA adducts. For these lower‐chlorinated biphenyls, butanol enrichment generally showed a lower recovery. For some higher substituted congeners (3,3′,4,4′,5‐pentachloro‐, 2,2′,3,4,4′,5′‐hexachloro‐, 2,2′,4,4′,5,5′‐hexachloro‐, and 2,2′,3,4,4′,5,5′‐heptachlorobiphenyl), after butanol enrichment a single dominant spot was observed, which was absent in the nuclease P1 procedure. After incubation of calf thymus DNA with either higher‐ or lower‐chlorinated PCB congeners, we were not able to detect significantly increased levels of oxidative DNA damage above background levels, measured as 8‐oxo‐7,8‐dihydro‐2′deoxyguanosine. In view of the carcinogenicity of PCB mixtures in animals and the ability of PCB metabolites to bind covalently to DNA, rats were orally treated with a mixture of PCBs (Aroclor 1242). PCB‐DNA adduct levels were analyzed in PCB target organs: liver, thymus, glandular stomach, spleen, testes, seminal vesicles and prostate DNA. In vivo PCB‐DNA adducts could not be detected by either the butanol‐ or by the NP1‐enrichment procedure in rat target tissue DNA. Also, no differences in oxidative DNA damage could be observed between PCB‐treated rats and controls. These results indicate a lack of DNA reactivity of PCB mixtures in vivo. Environ. Mol. Mutagen. 36:79–86, 2000.

Volatile N-nitrosamines in gastric juice of patients with various conditions of the gastrointestinal tract determined by gas chromatography-mass spectrometry and related to intragastric pH and nitrate and nitrite levels

Gastric juice samples of 71 patients undergoing upper gastrointestinal endoscopy were collected as well as saliva samples from 40 of these patients. Age, sex, endoscopic diagnosis and medication were recorded. The gastric juice samples were analyzed for the presence and quantity of individual volatile N-nitrosamines, which were detected by gas chromatography-mass spectrometry, without prior derivatization. The samples were screened for eight nitrosamines, i.e. N-nitrosodimethylamine, N-nitrosoethylmethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-nitrosomorpholine. The pH of the fresh gastric juice as well as nitrate and nitrite levels of gastric juice and saliva were determined. The mean total level of volatile N-nitrosamines in gastric juice was found to be 4.84 nmol/l (range 0-17.7 nmol/l). The main N-nitrosamines found were N-nitrosodiethylamine (mean concentration 3.1 nmol/l), N-nitrosodimethylamine (mean concentration 0.90 nmol/l) and N-nitrosopyrrolidine (mean concentration 0.38 nmol/l). Significant correlations between mean intragastric pH values and mean N-nitrosodi-n-butylamine level (P = 0.005) and total volatile N-nitrosamine contents (P = 0.009) were observed.

Lower placental telomere length may be attributed to maternal residential traffic exposure; a twin study

BACKGROUND High variation in telomere length between individuals is already present before birth and is as wide among newborns as in adults. Environmental exposures likely have an impact on this observation, but remain largely unidentified. We hypothesize that placental telomere length in twins is associated with residential traffic exposure, an important environmental source of free radicals that might accelerate aging. Next, we intend to unravel the nature-nurture contribution to placental telomere length by estimating the heritability of placental telomere length. METHODS We measured the telomere length in placental tissues of 211 twins in the East Flanders Prospective Twin Survey. Maternal traffic exposure was determined using a geographic information system. Additionally, we estimated the relative importance of genetic and environmental sources of variance. RESULTS In this twin study, a variation in telomere length in the placental tissue was mainly determined by the common environment. Maternal residential proximity to a major road was associated with placental telomere length: a doubling in the distance to the nearest major road was associated with a 5.32% (95% CI: 1.90 to 8.86%; p=0.003) longer placental telomere length at birth. In addition, an interquartile increase (22%) in maternal residential surrounding greenness (5 km buffer) was associated with an increase of 3.62% (95% CI: 0.20 to 7.15%; p=0.04) in placental telomere length. CONCLUSIONS In conclusion, we showed that maternal residential proximity to traffic and lower residential surrounding greenness is associated with shorter placental telomere length at birth. This may explain a significant proportion of air pollution-related adverse health outcomes starting from early life, since shortened telomeres accelerate the progression of many diseases.

Exposure to genotoxic compounds alters in vitro cellular VOC excretion

Genotoxic carcinogens significantly damage cells and tissues by targeting macromolecules such as proteins and DNA, but their mechanisms of action and effects on human health are diverse. Consequently, determining the amount of exposure to a carcinogen and its cellular effects is essential, yet difficult. The aim of this manuscript was to investigate the potential of detecting alterations in volatile organic compounds (VOCs) profiles in the in vitro headspace of pulmonary cells after exposure to the genotoxic carcinogens cisplatin and benzo[a]pyrene using two different sampling set-ups. A prototype set-up was used for the cisplatin exposure, whereas a modified set-up was utilized for the benzo[a]pyrene exposure. Both carcinogens were added to the cell medium for 24 h. The headspace in the culture flask was sampled to measure the VOC content using gas chromatography-time-of-flight-mass spectrometry. Eight cisplatin-specific VOCs and six benzo[a]pyrene-specific VOCs were discriminatory between treated and non-treated cells. Since the in vivo biological effects of both genotoxic compounds are well-defined, the origin of the identified VOCs could potentially be traced back to common cellular processes including cell cycle pathways, DNA damage and repair. These results indicate that exposing lung cells to genotoxins alters headspace VOC profiles, suggesting that it might be possible to monitor VOC changes in vivo to study drug efficacy or exposure to different pollutants. In conclusion, this study emphasizes the innovative potential of in vitro VOCs experiments to determine their in vivo applicability and discover their endogenous origin.

Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status

A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity.