Stroke-derived neutrophils demonstrate higher formation potential and impaired resolution of CD66b + driven neutrophil extracellular traps.

BACKGROUND: Recent evidence suggests a merging role of immunothrombosis in the formation of arterial thrombosis. Our study aims to investigate its relevance in stroke patients. METHODS: We compared the peripheral immunological profile of stroke patients vs. healthy controls. Serum samples were functionally analyzed for their formation and clearance of Neutrophil-Extracellular-Traps. The composition of retrieved thrombi has been immunologically analyzed. RESULTS: Peripheral blood of stroke patients showed significantly elevated levels of DNAse-I (p < 0.001), LDG (p = 0.003), CD4 (p = 0.005) as well as the pro-inflammatory cytokines IL-17 (p < 0.001), INF-γ (p < 0.001) and IL-22 (p < 0.001) compared to controls, reflecting a TH1/TH17 response. Increased counts of DNAse-I in sera (p = 0.045) and Neutrophil-Extracellular-Traps in thrombi (p = 0.032) have been observed in patients with onset time of symptoms longer than 4,5 h. Lower values of CD66b in thrombi were independently associated with greater improvement of NIHSS after mechanical thrombectomy (p = 0.045). Stroke-derived neutrophils show higher potential for Neutrophil-Extracellular-Traps formation after stimulation and worse resolution under DNAse-I treatment compared to neutrophils derived from healthy individuals. CONCLUSIONS: Our data provide new insight in the role of activated neutrophils and Neutrophil-Extracellular-Traps in ischemic stroke. Future larger studies are warranted to further investigate the role of immunothrombosis in the cascades of stroke. TRIAL REGISTRATION: DRKS, DRKS00013278, Registered 15 November 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013278.

The Carcinogenic Properties of Overlooked yet Prevalent Polycyclic Aromatic Hydrocarbons in Human Lung Epithelial Cells.

The WHO classified air pollution as a human lung carcinogen and polycyclic aromatic hydrocarbons (PAHs) are components of both indoor (e.g., tobacco smoke and cookstoves) and outdoor (e.g., wildfires and industrial and vehicle emissions) air pollution, thus a human health concern. However, few studies have evaluated the adverse effects of low molecular weight (LMW) PAHs, the most abundant PAHs in the environment. We hypothesized that LMW PAHs combined with the carcinogenic PAH benzo[a]pyrene (B[a]P) act as co-carcinogens in human lung epithelial cell lines (BEAS-2B and A549). Therefore, in this paper, we evaluate several endpoints, such as micronuclei, gap junctional intercellular communication (GJIC) activity, cell cycle analysis, anti-BPDE-DNA adduct formation, and cytotoxicity after mixed exposures of LMW PAHs with B[a]P. The individual PAH doses used for each endpoint did not elicit cytotoxicity nor cell death and were relevant to human exposures. The addition of a binary mixture of LMW PAHs (fluoranthene and 1-methylanthracene) to B[a]P treated cells resulted in significant increases in micronuclei formation, dysregulation of GJIC, and changes in cell cycle as compared to cells treated with either B[a]P or the binary mixture alone. In addition, anti-BPDE-DNA adducts were significantly increased in human lung cells treated with B[a]P combined with the binary mixture of LMW PAHs as compared to cells treated with B[a]P alone, further supporting the increased co-carcinogenic potential by LMW PAHs. Collectively, these novel studies using LMW PAHs provide evidence of adverse pulmonary effects that should warrant further investigation.

In-Vitro Identification and In-Vivo Confirmation of DNA Methylation Biomarkers for Urothelial Cancer.

We identified DNA methylation targets specific for urothelial cancer (UC) by genome-wide methylation difference analysis of human urothelial (RT4, J82, 5637), prostate (LNCAP, DU-145, PC3) and renal (RCC-KP, CAKI-2, CAL-54) cancer cell lines with their respective primary epithelial cells. A large overlap of differentially methylated targets between all organs was observed and 40 Cytosine-phosphate-Guanine motifs (CpGs) were only specific for UC cells. Of those sites, two also showed high methylation differences (≥47%) in vivo when we further compared our data to those previously obtained in our array-based analyses of urine samples in 12 UC patients and 12 controls. Using mass spectrometry, we finally assessed seven CpG sites in this "bladder-specific" region of interest in urine samples of patients with urothelial (n = 293), prostate (n = 75) and renal (n = 23) cancer, and 143 controls. DNA methylation was significantly increased in UC compared to non-UC individuals. The differences were more pronounced for males rather than females. Male UC cases could be distinguished from non-UC individuals with >30% sensitivity at 95% specificity (Area under the curve (AUC) 0.85). In summary, methylation sites highly specific in UC cell lines were also specific in urine samples of UC patients showing that in-vitro data can be successfully used to identify biomarker candidates of in-vivo relevance.

Environmentally prevalent polycyclic aromatic hydrocarbons can elicit co-carcinogenic properties in an in vitro murine lung epithelial cell model.

Low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAH) are the most abundant PAHs environmentally, occupationally, and are in cigarette smoke; however, little is known about their carcinogenic potential. We hypothesized that LMW PAHs act as co-carcinogens in the presence of a known carcinogen (benzo[a]pyrene (B[a]P)) in a mouse non-tumorigenic type II cell line (C10 cells). Gap junctions are commonly suppressed and inflammation induced during tumor promotion, while DNA-adduct formation is observed during the initiation stage of cancer. We used these endpoints together as markers of carcinogenicity in these lung adenocarcinoma progenitor cells. LMW PAHs (1-methylanthracene and fluoranthene, 1-10 µM total in a 1:1 ratio) were used based on previous studies as well as B[a]P (0-3 µM) as the classic carcinogen; non-cytotoxic doses were used. B[a]P-induced inhibition of gap junctional intercellular communication (GJIC) was observed at low doses and further reduced in the presence of the LMW PAH mixture (P < 0.05), supporting a role for GJIC suppression in cancer development. Benzo[a]pyrene diol-epoxide (BPDE)-DNA adduct levels were significantly induced in B[a]P-treated C10 cells and additionally increased with the LMW PAH mixture (P < 0.05). Significant increases in cyclooxygenase (Cox-2) were observed in response to the B[a]P/LMW PAH mixture combinations. DNA adduct formation coincided with the inhibition of GJIC and increase in Cox-2 mRNA expression. Significant cytochrome p4501b1 increases and connexin 43 decreases in gene expression were also observed. These studies suggest that LMW PAHs in combination with B[a]P can elicit increased carcinogenic potential. Future studies will further address the mechanisms of co-carcinogenesis driving these responses.

Effects of benzo[a]pyrene, aromatic amines, and a combination of both on CYP1A1 activities in RT-4 human bladder papilloma cells.

The interaction of arylamines and polycyclic aromatic hydrocarbons (PAH) is of particular interest in the etiology of bladder cancer. The aim of this study was to (1) examine the metabolic capacity of RT-4 human bladder papilloma cells and (2) investigate the influence of aromatic amines on the induction of cytochrome P-450 1A1 (CYP1A1) activity and their effects on benzo[a]pyrene (BaP)-induced CYP1A1 activities. Cells were incubated for 24 h with different concentrations of BaP, 1- or 2-naphthylamine (NA), 2-, 3-, or 4-aminobiphenyl (ABP), and binary mixtures consisting of 1 µM BaP and different concentrations of each arylamine. Changes in CYP1A1 activities were measured at concentrations with no or only low cytotoxicity and accompanied by specific protein detection. Several phase I and II enzymes relevant to metabolism of PAH and arylamines were present in RT-4 cells. Concentration-dependent elevation in CYP1A1 activities accompanied by increasing protein levels was found after treating cells with BaP and 1- or 2-NA. The majority of synergistic effects in binary mixtures were less than additive. In contrast, concentration-dependent inhibition was observed for 2-, 3-, and 4-ABP and in both the presence and absence of BaP. Our results suggest that RT-4 cells represent a reliable model cell line to study arylamine- and PAH-induced effects in vitro and that BaP-induced CYP1A1 activities are modulated by aromatic amines. In general, the direction of the effect depends upon the aromatic amine, rather than being unidirectional for aromatic amines.

Polymorphisms in circadian genes, night work and breast cancer: results from the GENICA study.

OBJECTIVES: The role of genetic variants and environmental factors in breast cancer etiology has been intensively studied in the last decades. Gene-environment interactions are now increasingly being investigated to gain more insights into the development of breast cancer, specific subtypes, and therapeutics. Recently, night shift work that involves circadian disruption has gained rising interest as a potential non-genetic breast cancer risk factor. Here, we analyzed genetic polymorphisms in genes of cellular clocks, melatonin biosynthesis and signaling and their association with breast cancer as well as gene-gene and gene-night work interactions in a German case-control study on breast cancer. METHODS: GENICA is a population-based case-control study on breast cancer conducted in the Greater Region of Bonn. Associations between seven polymorphisms in circadian genes (CLOCK, NPAS2, ARTNL, PER2 and CRY2), genes of melatonin biosynthesis and signaling (AANAT and MTNR1B) and breast cancer were analyzed with conditional logistic regression models, adjusted for potential confounders for 1022 cases and 1014 controls. Detailed shift-work information was documented for 857 breast cancer cases and 892 controls. Gene-gene and gene-shiftwork interactions were analyzed using model-based multifactor dimensionality reduction (mbMDR). RESULTS: For combined heterozygotes and rare homozygotes a slightly elevated breast cancer risk was found for rs8150 in gene AANAT (OR 1.17; 95% CI 1.01-1.36), and a reduced risk for rs3816358 in gene ARNTL (OR 0.82; 95% CI 0.69-0.97) in the complete study population. In the subgroup of shift workers, rare homozygotes for rs10462028 in the CLOCK gene had an elevated risk of breast cancer (OR for AA vs. GG: 3.53; 95% CI 1.09-11.42). Shift work and CLOCK gene interactions were observed in the two-way interaction analysis. In addition, gene-shiftwork interactions were detected for MTNR1B with NPAS2 and ARNTL. CONCLUSIONS: In conclusion, the results of our population-based case-control study support a putative role of the CLOCK gene in the development of breast cancer in shift workers. In addition, higher order interaction analyses suggest a potential relevance of MTNR1B with the key transcriptional factor NPAS2 with ARNTL. Hence, in the context of circadian disruption, multivariable models should be preferred that consider a wide range of polymorphisms, e.g. that may influence chronotype or light sensitivity. The investigation of these interactions in larger studies is needed.

Miniaturization of cytotoxicity tests for concentration range-finding studies prior to conducting the pH 6.7 Syrian hamster embryo cell-transformation assay.

The Syrian hamster embryo (SHE) cell-transformation assay (SHE assay) is a promising alternative method to animal testing for the identification of potential carcinogens in vitro. Prior to conducting the SHE assay the appropriate concentration range for each test chemical must be established, with a maximum concentration causing approximately 50% cytotoxicity. Concentration range-finding is done in separate experiments, which are similar to the final SHE assay but with less replicates and more concentrations. Here we present an alternative for the cytotoxicity testing by miniaturization of the test procedure by use of 24-well plates and surpluses from feeder-cell preparations as target cells. In addition, we integrated the photometry-based neutral red (NR) assay. For validation of the assay, incubations with dimethyl sulf-oxide, p-phenylenediamine-2HCl, aniline, o-toluidine-HCl, 2,4-diaminotoluene, and 2-naphthylamine were carried out in the miniaturized approach and compared with the standard procedure in terms of calculating the relative plating efficiencies (RPEs). To directly compare both methods, concentrations that produced 50% cytotoxicity (IC50) were calculated. Excellent associations were observed between the number of colonies and NR uptake. For all test substances a concentration-dependent, concomitant decrease of NR uptake in the miniaturized approach and RPEs in the standard test was observed after a 7-day incubation. The results from both test setups showed a comparable order of magnitude and the IC50 values differed by a factor <2 (1.4-1.9), depending on the substance in question. Overall, the miniaturized approach should be considered an improved alternative for cytotoxicity testing in the SHE assay, as it saves valuable SHE cells and speeds-up the time, to obtain test results more rapidly.

Effects of cigarette smoke condensate on primary urothelial cells in vitro.

Cigarette smoking is a risk factor for bladder cancer. Since urothelial cells express phase I and II enzymes these cells are able to metabolize precarcinogens into DNA reactive intermediates. Cigarette smoke is a complex mixture containing at least 80 known carcinogens. In this context especially aromatic amines and polycyclic aromatic hydrocarbons are discussed as being responsible for bladder-carcinogenicity. Cell cultures of primary porcine urinary bladder epithelial cells (PUBEC) have been useful models for studies on bladder-specific effects. These cells are metabolically competent and found to be a valuable tool for examining effects of cigarette smoke constituents. In the present study PUBEC were utilized to investigate the effects of the complex mixture cigarette smoke condensate total particulate matter (CSC TPM) with emphasis on induction of cytochrome P-450 1A1 (CYP1A1) and genotoxic effects. CYP1A1 induction was investigated by Western blot and flow cytometry. The most pronounced effects were found after 24 h of incubation with 1-10 µg/ml CSC TPM. Maximal induction was observed at 5 µg/ml by flow cytometry and at 10 µg/ml by Western blot analysis. Genotoxic effects were investigated by means of alkaline single-cell gel electrophoresis ("comet assay") with and without the use of the DNA repair enzyme formamidopyrimidine-DNA glycosylase (Fpg) and the micronucleus (MN) test. A numerical concentration-dependent increase in Fpg-sensitive sites indicating oxidative DNA damage and a quantitative rise in MN formation were noted. The CSC utilized in this study contained low amounts of benzo[a]pyrene, 4-aminobiphenyl, and 2-naphthylamine. With regard to the observed CYP1A1 induction, these substances cannot explain the CYP1A1 inducing effect of CSC TPM. It is possible that other compounds within CSC TPM contribute to CYP1A1 induction in our cellular model.

Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1.

Cultured primary porcine urinary bladder epithelial cells (PUBEC) represent an adequate and easy to handle in vitro system for studies of urothelial toxicity. PUBEC maintain in vivo-like metabolic activities and physiological functions. They express inducible cytochrome P4501A isoenzymes, which are of particular relevance, since they contribute to activation of bladder carcinogens. A possible drawback of PUBEC is their isolation from common domestic pigs that do not represent an inbred strain. In order to further establish PUBEC as a standard in vitro toxicity test system we analysed possible interindividual differences in CYP1A1 inducibility. Interestingly, we observed by flow cytometry that PUBEC obtained from individual pigs consist of two distinct subpopulations with inducible and non-inducible cells. A strong, concentration-dependent CYP1A1 induction was observed in the responsive subpopulation when incubated with benzo[a]pyrene (B[a]P) in a concentration range between 1 and 10 muM. In contrast, no CYP1A1 induction was obtained in the non-responsive subpopulation up to the highest tested concentrations of 100 muM. The fraction of responsive cells showed large interindividual differences ranging from 10 to 65% of the total cell number. For practical purposes it might be reasonable to analyse pools of PUBEC from five pigs which substantially reduce batch to batch variability. In conclusion, we have identified two functionally distinct subpopulations of urinary bladder epithelial cells. It will be interesting to study whether the CYP1A inducible subtype is more susceptible to bladder carcinogens.

Synergism of aromatic amines and benzo[a]pyrene in induction of Ah receptor-dependent genes.

Aromatic amines have been shown to cause bladder cancer. However, epithelial cells of the urinary bladder, cells of origin of bladder cancer, may be exposed to numerous substances besides aromatic amines. In the present study, we analysed possible interactions between the aromatic amines 4-aminobiphenyl (4-ABP) as well as 2-naphthylamine (2-NA) and the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). For this purpose we incubated primary porcine urinary bladder epithelial cells (PUBEC) with concentrations of 1 to 50 microM 4-ABP with and without co-exposure to B[a]P. As expected B[a]P increased mRNA expression of cytochrome P450 1A1 (CYP1A1), whereas 4-ABP had no effect. However, when co-exposed 4-ABP enhanced the induction of CYP1A1 by B[a]P. This result was confirmed by Western blot analysis of CYP1A1 protein expression. A similar effect as for CYP1A1 was also observed for cyclooxygenase-2 (COX-2) and UDP-glucuronosyltransferase 1 (UGT1). Next, we studied co-exposures of 2-NA and B[a]P. Similar as for 4-ABP also 2-NA enhanced B[a]P-mediated induction of CYP1A1. Our results demonstrate that some aromatic amines may enhance the influence of B[a]P on Ah receptor-dependent genes.

Analysis of CYP1A1 induction in single cells of urothelial cell populations by flow cytometry.

As carcinogenesis is a process starting at the single-cell level it is desirable to study carcinogen-mediated effects in individual cells. A primary step in chemically induced carcinogenesis is the formation of reactive DNA-binding metabolites by cytochromes P450 (CYP). We applied indirect immunofluorescence to stain CYP1A1 in urothelial cells for quantification by flow cytometry. Our studies were carried out with metabolically competent primary porcine urinary bladder epithelial cells (PUBECs) and the human urothelial cell line 5637 for which we have previously demonstrated CYP1A1 mRNA induction by the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) applying real-time RT-PCR. Flow cytometric analysis revealed that for PUBEC and 5637 cells the fraction of CYP1A1-induced cells increased with B[a]P concentration. Furthermore, in 5637 cells this effect was time-dependent, being more pronounced after 48 h than after 24 h. However, CYP1A1 induction could not be detected in all analyzed PUBEC and 5637 cells after treatment with up to 50 muM B[a]P. The reason for this remains unknown at the moment. Overall, B[a]P-treated cells could be divided into fractions of clearly CYP1A1-induced and clearly uninduced cells. Another fraction of "unclear" CYP1A1-induced cells and one of unclassifiable cells remained, as quantification of CYP1A1 induction by flow cytometry was hampered by B[a]P-related fluorescence. This is ascribed to phenolic B[a]P metabolites formed by CYP1A1 and which are known to fluoresce at wavelengths above 500 nm, whereas B[a]P does not. Overall, the method permits the detection of CYP1A1 protein level in large numbers of individual cells, thereby providing an adequate basis for statistical analyses. Flow cytometric detection of CYP1A1 induction in individual cells allows further insight into the metabolic competence of single cells and therefore could be a valuable tool for toxicological studies.

Evaluation of time dependence and interindividual differences in benzo[a]pyrene-mediated CYP1A1 induction and genotoxicity in porcine urinary bladder cell cultures.

Exposure to tobacco smoke is an established cause of cancer in humans and cigarette smoking is a risk factor for urinary bladder cancer development. Aromatic amines are believed responsible for the bladder-specific carcinogenic effect, but polycyclic aromatic hydrocarbons (PAHs) are also of potential relevance. Urothelial cells contain a number of xenobiotic-metabolizing enzymes, which enable them to convert pro-carcinogens into reactive intermediates. In a preceding study, it was demonstrated using cultured porcine urinary bladder epithelial cells (PUBEC) that CYP1A1 mRNA is induced in a potent manner by treatment with benzo[a]pyrene (BaP). In the present study, the time dependence of these effects was evaluated and whether PUBEC cultures derived from individual donors respond differently to BaP treatment was determined. CYP1A1 induction was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR), and genotoxic effects were studied using the Comet assay. Incubation of PUBEC with BaP increased CYP1A1 expression and induction of DNA strand breaks in a time-dependent manner. Interindividual differences were found between PUBEC cultures derived from several donor animals with respect to the response to BaP, such that the extent of CYP1A1 induction and magnitude of DNA damage was interrelated. Hence, individual differences in metabolic capacities and responsiveness to xenobiotics of urothelial cells from individual donors may be factors in susceptibility to genotoxic effects induced by PAHs.

Genotoxicity and potential carcinogenicity of 2,4,6-TNT trinitrotoluene: structural and toxicological considerations.

Environmental contamination with 2,4,6-TNT (trinitrotoluene) represents a worldwide problem. Concern for carcinogenicity can be derived from chemically related compounds, especially the dinitrotoluenes. In the metabolism of TNT, the reductive routes are preponderant. The main urinary metabolites of TNT are 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene. In humans exposed to TNT, the formation of hemoglobin adducts of the amino-dinitrotoluenes is in general concordance with the ratio of urinary excretion. The variations in quantities of excreted metabolites among the different occupational cohorts studied are likely explained by the different routes of exposure to TNT, including dermal uptake. Most studies show that urinary excretion of the amino-dinitrotoluenes (4-amino-dinitrotoluene plus 2-amino-dinitrotoluene) in a range of 1 to 10 mg L(-1) (5-50 microM) are not uncommon--for instance in persons employed with the disposal of military waste. Trinitotoluene is mutagenic in Salmonella typhimurium strains TA98 and TA100, with and without exogenous metabolic activation. Mutagenic activity has been found in urine from workers who were occupationally exposed to TNT. An unpublished 2-year study was reported in 1984 by the IIT Research Institute, Chicago, IL. Fischer 344 rats were fed diets containing 0.4, 2.0, 10, or 50 mg/kg TNT per day. In the urinary bladder, hyperplasia (12 of 47 animals p < .01) and carcinoma (11 of 47 animals, p < .05) were observed at significant levels in high-dose (50 mg kg(-1)) females and in one or two females, respectively, at 10 mg kg(-1). Taking all the available evidence together, the appropriate precautions should be taken.

The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.