[The Course of E-Cigarette Use in Adolescence: A Cohort Study over 18 Months]. / Der Verlauf des E-Zigarettenkonsums im Jugendalter: Eine Kohortenstudie über 18 Monate.

AIM: Description of adolescent e-cigarette use over time. METHOD: In 2017 and 2019, 261 adolescents from North Rhine-Westphalia who had used e-cigarettes at least once a month (mean age: 14.9 years; 33.5 % female) took part in a questionnaire study. RESULTS: In 2017, 84 adolescents (32.2 %) reported exclusive e-cigarette use (single users), 177 adolescents were classified as dual users (67.8 %) because they consumed a tobacco product (conventional cigarette and/or hookah) in addition to e-cigarettes. During the observation period of 18 months, 83 adolescents (31.8 %) quit nicotine products altogether. Dual users quit nicotine less often than single users (N = 39 or 22.0 % vs. N = 44 or 52.4 %, p < 0.001). Seven single users (8.3 %) did not change their behavior, 11 began to use tobacco exclusively (13.1 %), another 22 (26.2 %) started dual use. Seventy-eight dual users (44.1 %) did not change their behavior, 57 (32.1 %) switched to tobacco use only, 3 dual users (1.7 %) stopped tobacco use, but continued to use e-cigarettes. Taken together, at the end of the study, 10 (5.6 %) of the remaining 178 adolescents consumed only e-cigarettes, while 168 (94.4 %) smoked tobacco or were dual-users. CONCLUSIONS: More than two thirds of all young e-cigarette users and more than three quarters of dual users also used nicotine products 18 months later. The remaining consumers showed a less frequent stay or switch to single use, instead a more frequent use of tobacco or dual use.

The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

Inflammatory cell recruitment after experimental thromboembolic stroke in rats.

Inflammatory mechanisms were recently identified as contributors to delayed neuronal damage after ischemic stroke. However, therapeutic strategies are still lacking, probably related to the outstanding standardization on inflammatory cell recruitment emerging from predominantly artificial stroke models, and the uncertainty on functional properties of distinct subpopulations. Using a rodent model of stroke that closely reflects human embolic ischemia, this study was focused on the local recruitment of immunoreactive cells as well as their functional and regional characterization. Wistar rats underwent thromboembolic middle cerebral artery occlusion, followed by intravenous injection of the blood-brain barrier permeability marker fluorescein-conjugated albumin at 24h. One hour later, brain tissue was subjected to multi-parameter flow cytometry and Pappenheim staining to characterize cells invaded into the ischemia-affected hemisphere, compared to the contralateral side. Immunofluorescence labeling was applied to explore the distribution patterns of recruited cells and their spatial relationships with the vasculature. One day after ischemia onset, a 6.12-fold increase of neutrophils and a 5.43-fold increase of monocytes/macrophages was found in affected hemispheres, while these cells exhibited enhanced major histocompatibility complex class II expression and allocation with vessels exhibiting impaired blood-brain barrier integrity. Microglia remained numerically unaltered in ischemic hemispheres, but shifted to an activated phenotype indicated by CD45/CD86 expression and morphological changes toward an ameboid appearance in the bordering zone. Ischemia caused an increase of lymphoid cells in close vicinity to the affected vasculature, while further analyses allowed separation into natural killer cells, natural killer T cells, T cells (added by an unconventional CD11b(+)/CD3(+) population) and two subpopulations of B cells. Taken together, our study provides novel data on the local inflammatory response to experimental thromboembolic stroke. As concomitantly present neutrophils, monocytes/macrophages and lymphoid cells in the early stage after ischemia induction correspond to changes seen in human stroke, future stroke research should preferably use animal models with relevance for clinical translation.

[Universal newborn hearing screening : Definition of uniform parameters by the Association of German Hearing Screening Centers as a requirement for nationwide evaluation with valid results]. / Universelles Neugeborenen-Hörscreening : Definition einheitlicher Parameter durch den Verband Deutscher Hörscreening-Zentralen (VDHZ) als Voraussetzung für eine flächendeckende Evaluation mit validen Ergebnissen.

BACKGROUND: Since 2009, all newborns in Germany have been entitled to universal neonatal hearing screening (UNHS). UNHS with tracking of test results leads to earlier detection of hearing disorders. The Association of German Hearing Screening Centers (Verband Deutscher Hörscreening-Zentralen, VDHZ) was founded to promote nationwide tracking, validity and quality control of UNHS results. OBJECTIVES: A comparable data structure in the different screening centers, with uniform definitions of primary parameters is essential for the nationwide evaluation of UNHS results. To address the question of whether a data structure with comparable definitions already exists or still has to be created, the existing structures and primary parameter definitions in the hearing screening centers should be investigated and compared. METHODS: A survey was conducted in all hearing screening centers to assess how data on the primary UNHS parameters defined in pediatric guidelines was gathered. In the case of discrepancies, uniform definitions were created. Finally, the practicability of these definitions was evaluated. RESULTS: Due to differing definitions of primary parameters, some of the data were not comparable between the individual centers. Therefore, uniform definitions were created in a consensus process. In the centers, the screening method, the two-step first screening and the result of the first screening now correspond to these uniform definitions. Other parameters, e.g. the total number of newborns, still vary widely, rendering the comparison of screening rates almost impossible. CONCLUSION: Valid evaluation of UNHS not only requires nationwide establishment of hearing screening centers, but also unified data structures and parameter definitions.

DNA adducts in skin biopsies and 1-hydroxypyrene in urine of psoriasis patients and healthy volunteers following treatment with coal tar.

Coal tar ointments (CTO) are frequently used in the treatment of psoriasis and eczema, but CTO contain carcinogenic polycyclic aromatic hydrocarbons (PAH). PAH are absorbed and metabolized in the skin. In psoriasis, the skin barrier is altered and therefore, absorption and metabolism of PAH may differ from healthy skin. In this study, levels of urinary 1-hydroxypyrene and PAH-DNA adducts in the skin were studied in psoriatic patients and healthy volunteers. Three punch biopsies were taken from the lower back of 10 male volunteers and from a psoriatic plaque in 10 male patients. A surface of 6.25 cm(2) was treated with CTO. After 96 h CTO was removed and another three skin biopsies were collected from the treated area. DNA was isolated from skin biopsies and urine was collected during and after the exposure period. After 24h, a twofold lower 1-hydroxypyrene urinary excretion was observed in patients compared to healthy volunteers and after 48 h, this difference reached statistical significance (p<0.05). Over 96 h the median level of the sum of PAH-DNA adducts, analyzed by (32)P-post-labeling, increased from 3.5 before CTO administration to 21.1 adducts per 10(8) nucleotides in volunteers, and from 1.0 to 3.6 adducts per 10(8) nucleotides in patients. At 96 h, PAH-DNA levels were higher in healthy volunteers than in patients (p<0.05). Biomarkers for uptake, bioavailability and bioactivation of PAH were lower in patients compared to volunteers. These data suggest a lower risk of carcinogenic effects of CTO in psoriatic skin compared to healthy skin.

Effect-based and chemical analysis of polycyclic aromatic hydrocarbons in smoked meat: a practical food-monitoring approach.

Polycyclic aromatic hydrocarbons (PAHs), which are generated by heat treatment and smoke curing of meat, pose a risk to human health. At present, the determination of these unwanted contaminants requires costly, time-consuming chemical analysis of smoked meat. An alternative is effect-directed high-throughput bioassays, which could also be used as a pre-screening method. The authors recently adapted the in vitro chemical-activated luciferase gene expression (CALUX) assay as a rapid, sensitive, and inexpensive screening technique for compounds such as dioxins, polychlorinated biphenyls, and PAHs. The aim of the present study was to apply a practical approach under realistic conditions. Custom-made meat samples produced under defined conditions with different PAH levels were analysed using this bioassay and gas chromatography-mass spectrometry (GC/MS) to determine the influence of different smoking conditions (temperature and duration) on PAH levels. It was found that cold smoking for up to 6 h did not result in strong PAH contamination, whereas hot (65 degrees C) and longer smoking times caused a considerable increase in both the bioassay response and the levels of 31 individually determined PAHs. The response in the effect-based bioassay was in good agreement with the values of chemical analysis. The bioassay made it possible to determine accurately the degree of contamination. The results show that this assay is suitable for high-throughput screening for unknown levels of toxicologically relevant PAHs in meat samples and is sensitive enough to differentiate between different PAH levels generated under various smoking conditions. Effect-based screening techniques, therefore, provide a new instrument for official food monitoring.

Multilayer perceptron tumour diagnosis based on chromatography analysis of urinary nucleosides.

Nucleosides in human urine are of interest as a biochemical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. A reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode-array detection was used to quantitatively analyze urinary normal and modified nucleosides. 55 persons with malignant tumors of various types, 13 persons with benign tumors and 41 healthy controls were investigated within a clinical intervention study. Artificial neural networks (ANN) have been used as a practical pattern recognition tool to distinguish cancer patients from healthy persons. Using a multilayer perceptron (MPL), a specificity of 85%, and a sensitivity of 97% in differentiation between tumor patients and healthy persons was achieved. The differentiation between benign and malignant tumors had a sensitivity of 60% and a specificity of 84%. These results verify the usefulness of ANN and the RP-HPLC method for tumor recognition in agreement with existing studies.

Modified nucleosides: an accurate tumour marker for clinical diagnosis of cancer, early detection and therapy control.

Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies. To test their usefulness as tumour markers and to compare them with the conventional tumour markers, fractionated urine samples were analysed using chromatography. The excretion patterns of nucleosides of 68 cancer patients with malignant and benign tumours and 41 healthy controls have been studied. Significant elevations in the total sum and the concentrations of at least three (or four) of indicator nucleosides cytidine, pseudouridine, 2-pyridone-5-carboxamide-N1-ribofuranoside, N2,N2-dimethylguanine, 1-methylguanosine, 2-methylguanosine and 1-methyladenosine indicate a tumour with a sensitivity of 54% (77%) and a specificity of 86% (98%). Using an artificial neural network analysis, a sensitivity of 97% and a specificity of 85% were achieved in differentiating between tumour and control volunteers. The comparison with carcinoembryonic antigen, cancer antigen 15-3 und tissue polypeptide antigen indicates that urinary nucleosides may be useful tumour markers. This study suggests that the simultaneous determination of modified nucleosides and creatinine in urine samples of patients with cancer leads to an advantage to current methods and is a useful method to detect cancer early and to control the success of therapy.

The Influence of long-chain polyunsaturated fatty acids on total lipid fatty acid composition of a canine mastocytoma cell line.

Cutaneous mast cells are considered as key immune effectors in the pathogenesis of canine atopic dermatitis (CAD). These cells release immediate-phase and late-phase mediators of inflammation. Dietary fatty acids are incorporated in cellular membranes and seem to influence mediator production and release. A dietary intervention with n6- and n3-fatty acids is thought to alleviate clinical symptoms in atopic dogs. The purpose of this study was to examine the effects of n6- and n3-fatty acids on the fatty acid composition of canine mastocytoma cells (C2) as a possible model for CAD. The C2 was cultured in a basic medium called Dulbecco's modified Eagle's medium (DEH) or with additional 14 mum linoleate (C18:2n6, DEH-LA), gamma-linolenate (C18:3n6, DEH-GLA), arachidonate (C20:4n6, DEH-AA), alpha-linolenate (C18:3n3, DEH-LnA), eicosapentaenoate (C20:5n3, DEH-EPA) or docosahexaenoate (C22:6n3, DEH-DHA). Cell growth was examined for 11 days in all media. Cell growth increased from days 1 to 8 and decreased thereafter in all media conditions. The fatty acids supplied did not influence cell growth. The cells were harvested after 8 days for fatty acid analysis. The fatty acid composition was determined by gas chromatography after extraction and trans-esterification of the lipids. The added fatty acids increased the concentration of these fatty acids in C2 differently (LA 4.9-fold, GLA 6.9-fold, AA 6-fold, LNA 9.3-fold, EPA 6.5-fold and DHA 8.4-fold). Furthermore, elongated and Delta6-desaturated products of the corresponding fatty acids were significantly elevated. However, Delta5-desaturated products were not measurable. These results let us assume that C2 has no measurable activity of the Delta5-desaturase. In case the low activity of Delta5-desaturase is one of the mechanisms underlying the pathogenesis of CAD, C2 seems to be an adequate model for investigations in CAD.

Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted PAH mixture in human Caco-2 cells.

It was shown recently that in epithelial Caco-2 cells the food contaminant benzo[a]pyrene (B[a]P) is metabolized and B[a]P-sulfate metabolites were transported out of the cells. The aim of this study was to investigate whether B[a]P and other polycyclic aromatic hydrocarbons (PAH) such as chrysene, phenanthrene, benzo[k]fluoranthene (B[k]F), dibenzo[a,l]pyrene (DB[a,l]P), and pyrene alone or in a mixture in a ratio as they occur in tobacco smoke have effects on gene expression of intestinal cytochrome P450 enzymes (CYP), Phase II enzymes and ATP-binding cassette (ABC)-transport proteins in the human Caco-2 cells. B[a]P induced its own metabolism. Treatment of the Caco-2 cells with B[a]P, chrysene, B[k]F, or DB[a,l]P induced mRNA expression of CYP1A1 and CYP1B1 specifically as measured by RT-PCR. In contrast, the mRNA expression of the microsomal epoxide hydrolase (mEH) was not affected by PAH. The gene expression of the Phase II enzymes UDP-glucuronosyltransferase 1A6 (UGT1A6) and UGT1A7 was also induced by these PAH but treatment with them had no effect on gene expression of sulfotransferases (SULT) at all. Of the ABC-transport proteins, MDR1 mRNA expression was induced by treatment with carcinogenic PAH, whereas MRP2 mRNA expression was not changed. The mixture of PAH also induced CYP1A1, CYP1B1, UGT1A6, and UGT1A7 mRNA expression. We conclude that B[a]P, chrysene, B[k]F, and DB[a,l]P have specific effects on intestinal CYP1A1, CYP1B1, UGT1A6, and UDP1A7 mRNA expression but no effects on the expression of SULT.

Prevalence of varicose veins and venous anatomy in patients without truncal saphenous reflux.

OBJECTIVES: To determine the prevalence and distribution of primary venous reflux in the lower limbs in patients without truncal saphenous reflux. DESIGN: Prospective cohort study. PATIENTS AND METHODS: One thousand and seven hundred and twelve patients with suspected venous disease were examined by duplex ultrasonography. Seven hundred and thirty-five patients had primary varicose veins with competent saphenous trunks. Limbs with truncal saphenous reflux, deep vein reflux or obstruction, previous injection sclerotherapy or vein surgery, arterial disease and inflammation of non-venous origin were excluded from further consideration. The CEAP classification system was used for clinical staging. Systematic duplex ultrasound examination was undertaken to assess the distribution of incompetent saphenous tributaries. RESULTS: The prevalence of primary reflux with competent saphenous trunks was 43%. Reflux of GSV calf tributaries was the most common. The majority of the limbs (96%) belonged to chronic venous disease classes C1 and C2 of the CEAP classification. CONCLUSIONS: Superficial venous reflux causing varicose veins in the presence competent saphenous trunks is very prevalent in this series in contrast to other studies, presumably reflecting differing patient populations. Our data clearly show that varicose veins may occur in any vein and do not depend on truncal saphenous incompetence. Careful duplex ultrasound evaluation allows the pattern of venous reflux to be established in this group of patient ensuring appropriate management of varices.

Consequences of eicosapentaenoic acid (n-3) and arachidonic acid (n-6) supplementation on mast cell mediators.

Mast cells are important players in the pathogenesis of atopic diseases. These cells release immediate-phase and late-phase mediators of inflammation. Fatty acids are incorporated in cellular membranes and therefore seem to influence mediator production and release. A study was conducted to assess the effects of eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) on mast cell mediators in a canine mastocytoma cell line (C2). Cells were cultured in a basic medium (Dulbecco's modified Eagle's medium/HAM's F12 1 : 1, DEH), DEH supplemented with 14.0 microm EPA (DEH-EPA) or 14 microm AA (DEH-AA). The DEH-AA cultured cells had increased spontaneous and mastoparan-stimulated PGE2 production and histamine release. Furthermore, the tryptase activity was increased. The DEH-EPA cultured cells rendered elevated levels of PGE2 and histamine release compared with DEH only after stimulation. These levels were significantly lower in comparison to DEH-AA. The increased PGE2 production of C2 cultured in DEH-AA is the consequence of the AA enrichment, because AA is the precursor of PGE2. However, the different effects by AA and EPA on mast cell mediators possibly reflect the higher susceptibility of long chain polyunsaturated fatty acids (PUFA) to undergo lipid peroxidation, because it is known that altered cellular redox state influences mediator production and release.

Effects of essential fatty acids on mediators of mast cells in culture.

The objective of this study was to investigate the effects of alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) on the fatty acid composition and the activity and release of mast cell mediators in the canine mastocytoma cell line C2. Cells were cultured in Dulbecco's modified Eagle's medium mixed with 50% Ham's F12 (containing linoleic acid 0.14 micro M). The basic medium (DEH) was supplemented with 0.14 micro M alpha-linolenic acid. 14.0 micro M alpha-linolenic acid (DEH-n-3) or 14.0 micro M linoleic acid (DEH-n-6) was added. Eight days after culturing of C2 in DEH-n-3 we measured elevated levels of n-3 fatty acids up to 22:3. The tryptase activity and the stimulated PGE2 production and histamine release were reduced. In contrast, after culturing of C2 in DEH-n-6 we determined elevated levels of n-6 fatty acids up to 20:3, increased tryptase activity and stimulated histamine release. Thus 18:3n-3 has anti-inflammatory effects in cultured canine mastocytoma cells.

Effect of lung resection and sham surgery on the frequency of infection in alloxan-diabetic rats.

The present study was carried out in order to determine the effect of lung resection on the frequency of infections in alloxan-diabetic rats. Adult female Wistar rats were injected with alloxan (40 mg/kg, iv) to induce diabetes mellitus (group D; N = 45) or with vehicle (1.0 ml/kg, iv) to be used as controls (group C; N = 45). Thirty-six days after receiving alloxan both groups were randomly divided into three subgroups: no operation (NO; N = 15), sham operation (SO; N = 15), and left pneumonectomy (PE; N = 15). The rats were sacrificed 36 days after surgery and their lungs were examined microscopically and macroscopically. The occurrence of thoracic wall infection, thoracic wall abscess, lung abscess and pleural empyema was similar in groups D and C. In contrast, the overall infection rate was higher (P<0.05) in the diabetic rats (SO-D and PE-D subgroups, but not in the NO-D subgroup). Considering that the overall infection rate was similar in the SO-D and PE-D subgroups, we suggest that surgery but not pneumonectomy was related to the higher prevalence of infection in diabetic rats.

Effect of lung resection and sham surgery on the frequency of infection in alloxan-diabetic rats

The present study was carried out in order to determine the effect of lung resection on the frequency of infections in alloxan-diabetic rats. Adult female Wistar rats were injected with alloxan (40 mg/kg, iv) to induce diabetes mellitus (group D; N = 45) or with vehicle (1.0 ml/kg, iv) to be used as controls (group C; N = 45). Thirty-six days after receiving alloxan both groups were randomly divided into three subgroups: no operation (NO; N = 15), sham operation (SO; N = 15), and left pneumonectomy (PE; N = 15). The rats were sacrificed 36 days after surgery and their lungs were examined microscopically and macroscopically. The occurrence of thoracic wall infection, thoracic wall abscess, lung abscess and pleural empyema was similar in groups D and C. In contrast, the overall infection rate was higher (P<0.05) in the diabetic rats (SO-D and PE-D subgroups, but not in the NO-D subgroup). Considering that the overall infection rate was similar in the SO-D and PE-D subgroups, we suggest that surgery but not pneumonectomy was related to the higher prevalence of infection in diabetic rats

Effects of the (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo.

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.

Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol.

Most drug metabolizing cytochrome P450s (P450) are predominantly expressed in the liver. In contrast, human CYP1B1 is an extrahepatic P450 which is overexpressed in many tumours and has been strongly implicated in the activation of carcinogens. Rare allelic variants of the CYP1B1 gene which encode an inactive protein have been identified. However, four polymorphisms which most likely do not abolish functionality have been described. In this report, we have characterized the functional consequences of these. A CYP1B1 cDNA, identical to a cDNA published previously, served as a template to introduce allelic changes either separately or in combination. The resulting effects on CYP1B1 activity were determined in membranes isolated from Escherichia coli which coexpressed CYP1B1 together with P450 reductase. None of the allelic changes affected the CYP1B1 expression level. The allelic changes Arg48 to Gly, Ala19 to Ser and Asn453 to Ser had little influence on the Vmax and the Km of the CYP1B1 mediated 2- and 4-hydroxylation of estradiol. In contrast, the Km of these metabolic pathways was increased at least three-fold by the allelic change Va432 to Leu or by simultaneously changing Val432 to Leu and Asn453 to Ser. However, these alterations had little effect on the kinetic parameters of other CYP1B1 mediated reactions such as the epoxidation of (-)-trans-(7R,8R)-benzo[a]pyrene 7,8-dihydrodiol as determined by (r-7,t-8,t-9,c-10)-benzo[a]pyrene tetraol formation, or such as the O-dealkylation of ethoxyresorufin and the 1'-hydroxylation of bufuralol. Molecular modelling suggests that amino acid residue 432 of CYP1B1 may be involved in the interaction between CYP1B1 and P450 reductase. Since 4-hydroxyestradiol has been implicated in hormonal carcinogenesis and CYP1B1 is expressed in target tissues, the data presented demonstrate that polymorphisms in CYP1B1 have the potential to affect disease susceptibility.

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots.

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.

Detection of carcinogenic aromatic amines in the urine of non-smokers.

Smoking is thought to be one of the most important anthropogenic risk factors involved in the development of urinary bladder cancer in humans. Tobacco smoke contains a complex mixture of chemicals including potent carcinogens such as aromatic amines. In the present study the amounts of several freebase aromatic amines including the potent carcinogens 2-aminonaphthalene and 4-aminobiphenyl have been analyzed in the urine of 48 German urban living smokers and non-smokers. The results indicate that (i) both groups excrete the identical set of four aromatic amines; (ii) smokers excrete approximately twice the total amount of these amines, but similar amounts of 2-aminonaphthalene and 4-aminobiphenyl are found in non-smokers; and (iii) the excreted aromatic amines are decomposed in the urine within a few hours thus, explaining why aromatic amines are difficult to detect in this matrix. Their decomposition could be prevented by adding small amounts of p-toluidine to the freshly collected urine. Unlike smokers the origin of aromatic amines detected in the urine of non-smokers is at present unknown. Based on the cotinine levels found in the urine of non-smokers environmental tobacco smoke can be excluded as a major source of aromatic amines. In addition, neither diesel exhaust-related nitroarenes nor the corresponding amino-derivatives, to which they may be metabolically converted, were found. The detected urinary levels of aromatic amines arising from sources other than tobacco smoke or diesel exhaust may play a role in the bladder cancer etiology of non-smokers.