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Even though the number of studies aiming to improve comprehension of ADHD pathology has increased in recent years, there still is an urgent need for more effective studies, particularly in understanding adult ADHD, both at preclinical and clinical levels, due to the increasing evidence that adult ADHD is highly distinct and a different entity from childhood ADHD. This review paper outlines the symptoms, diagnostics, and neurobiological mechanisms of ADHD, with emphasis on how adult ADHD could be different from childhood-onset. Data show a difference in the environmental, genetic, epigenetic, and brain structural changes, when combined, could greatly impact the behavioral presentations and the severity of ADHD in adults. Furthermore, a crucial aspect in the quest to fully understand this disorder could be through longitudinal analysis. In this way, we will determine if and how the pathology and pharmacology of ADHD change with age. This goal could revolutionize our understanding of the disorder and address the weaknesses in the current clinical classification systems, improving the characterization and validity of ADHD diagnosis, specifically those in adults.

ABSTRACTPrenatal exposure to endocrine disrupting chemicals can interfere with development, and has been associated with social-cognitive functioning and adverse health outcomes later in life. Exposure-associated changes of DNA methylation (DNAm) patterns have been suggested as a possible mediator of this relationship. This study investigated whether prenatal low-dose exposure to polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) is associated with altered DNAm patterns across the genome in a Western urban-industrial population. In 142 mother-infant pairs from the Duisburg Birth Cohort Study, PCBs and PCDD/Fs levels were quantified from maternal blood during late pregnancy and associated with DNAm levels in cord blood using the Illumina EPIC beadchip. The epigenome-wide association studies (EWAS) identified 32 significantly differentially methylated positions (DMPs) and eight differentially methylated regions (DMRs) associated with six congeners of PCB and PCDD in females or males (FDRs < 0.05). DMPs and DMRs mapped to genes involved in neurodevelopment, gene regulation, and immune functioning. Weighted gene correlation network analysis (WGCNA) showed 31 co-methylated modules (FDRs < 0.05) associated with one congener of PCDF levels in females. Results of both analytical strategies indicate that prenatal exposure to PCBs and PCDD/Fs is associated with altered DNAm of genes involved in neurodevelopment, gene expression and immune functioning. DNAm and gene expression levels of several of these genes were previously associated with EDC exposure in rodent models. Follow-up studies will clarify whether these epigenetic changes might contribute to the origin for adverse mental and health outcomes.

Acetaldehyd kommt natürlicherweise in zahlreichen Lebensmitteln vor und wird aufgrund seines fruchtigen Aromas auch als Aromastoff eingesetzt. Die Internationale Agentur für Krebsforschung (IARC) stufte Acetaldehyd als möglicherweise krebserregend sowie in Verbindung mit der oralen Aufnahme über alkoholhaltige Getränke als humanes Kanzerogen ein. Vor diesem Hintergrund stellt sich die Frage, ob die Verwendung von Acetaldehyd als Aromastoff weiterhin vertretbar ist. Die Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) hat die aktuelle Datenlage zur Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff geprüft und hierzu eine Stellungnahme verabschiedet. Demnach kann die Frage, ob Acetaldehyd nach oraler Exposition in vivo gentoxisch und mutagen wirkt, derzeit nicht abschließend beantwortet werden. Weiterhin ist auch unklar, welchen Beitrag die Verwendung von Acetaldehyd als Aromastoff zur Gesamtexposition des Verbrauchers gegenüber Acetaldehyd leistet. Eine wissenschaftliche Bewertung des gesundheitlichen Risikos der Verwendung von Acetaldehyd als Aromastoff ist daher weiterhin nicht möglich. Die SKLM weist darauf hin, dass aufgrund des gentoxischen Gefährdungspotenzials sowie zahlreicher Datenlücken, die für eine vollständige Risikobewertung geschlossen werden müssen, Zweifel an der Sicherheit von Acetaldehyd als Aromastoff bestehen. Nach Ansicht der SKLM sollte der gezielte Zusatz von Acetaldehyd als Aromastoff aus Gründen des vorsorgenden Verbraucherschutzes neu beurteilt werden.

Hintergrund: Harnblasenkrebs kann durch die Freisetzung krebserzeugender aromatischer Amine aus Azofarbstoffen verursacht werden (BK-Report 1/2019). Zielstellung: Ziel der vorliegenden Untersuchung war es, aus nach BK 1301 angezeigten Fällen Informationen über die stoffliche Natur entsprechender früher verwendeter Azofarbstoffe zu gewinnen. Material und Methoden: Es wurden 14 Fälle einer angezeigten Berufskrankheit BK 1301, die gegen unterschiedliche Rissprüfsprays bei Tätigkeiten in der Metallbranche exponiert waren, untersucht. Eine ausführliche Berufsanamnese wurde erhoben und, falls möglich, der genetische Status der N-Acetyltransferase 2 (NAT2) bestimmt. Ergebnisse: Die untersuchten Personen waren erstmals im Zeitraum zwischen 1957 und 1986 gegen Rissprüfsprays exponiert. Das Alter bei der ersten Exposition lag zwischen 14 und 33 Jahren. Das Alter bei der Erstdiagnose des Harnblasenkrebses variierte zwischen 35 und 64 Jahren. Die Latenzzeiten lagen zwischen 17 und 45 Jahren. Die längste berichtete Expositionsdauer betrug 29 Jahre. Sechs von 10 genotypisierten Fällen waren langsame NAT2-Acetylierer. Das meist rote Rissprüfmittel wurde auf den zu prüfenden Metallkörper gesprüht. Danach wurde die Metallfläche mit einem Lappen gereinigt. In Poren oder Risse eingedrungener Farbstoff verbleibt. Die Rissprüfer waren sowohl durch Hautkontakt als auch durch Inhalation gegen die Farbbestandteile exponiert. In Großbritannien wurden jedoch bei einer Fallserie von 8 Harnblasenkrebspatienten auch Rissprüfmittel mit Farbstoffen auf der Basis von Naphthalimid oder Naphthalazin angegeben. Schlussfolgerung: Harnblasenkrebspatienten mit einer beruflichen Tätigkeit in der Metallbranche müssen explizit nach der Anwendung von Rissprüfsprays gefragt werden.

•Classification of hepatotoxicity based on cytotoxicity assays and RNAseq measurements.•Summarizing high-dimensional gene expression data into interpretable measurements.•Comparison of classification accuracy for different classification methods.•Superior performance of tree-based classification methods over linear methods.•Synergistic effect of combining cytotoxicity data and gene expression.

Infiltration of T and B/plasma cells has been linked to NSCLC prognosis, but this has not been thoroughly investigated in relation to the expression of programmed death ligand 1 (PD-L1). Here, we determine the association of lymphocytes and PD-L1 with overall survival (OS) in two retrospective cohorts of operated NSCLC patients who were not treated with checkpoint inhibitors targeting the programmed death 1/PD-L1 axis. Moreover, we evaluate how PD-L1 positivity and clinicopathologic factors affect the prognostic association of lymphocytes.

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Numerous protein biomarkers have been analyzed to improve prognostication in non-small cell lung cancer, but have not yet demonstrated sufficient value to be introduced into clinical practice. Here, we aimed to develop and validate a prognostic model for surgically resected non-small cell lung cancer. A biomarker panel was selected based on (1) prognostic association in published literature, (2) prognostic association in gene expression data sets, (3) availability of reliable antibodies, and (4) representation of diverse biological processes. The five selected proteins (MKI67, EZH2, SLC2A1, CADM1, and NKX2-1 alias TTF1) were analyzed by immunohistochemistry on tissue microarrays including tissue from 326 non-small cell lung cancer patients. One score was obtained for each tumor and each protein. The scores were combined, with or without the inclusion of clinical parameters, and the best prognostic model was defined according to the corresponding concordance index (C-index). The best-performing model was subsequently validated in an independent cohort consisting of tissue from 345 non-small cell lung cancer patients. The model based only on protein expression did not perform better compared to clinicopathological parameters, whereas combining protein expression with clinicopathological data resulted in a slightly better prognostic performance (C-index: all non-small cell lung cancer 0.63 vs 0.64; adenocarcinoma: 0.66 vs 0.70, squamous cell carcinoma: 0.57 vs 0.56). However, this modest effect did not translate into a significantly improved accuracy of survival prediction. The combination of a prognostic biomarker panel with clinicopathological parameters did not improve survival prediction in non-small cell lung cancer, questioning the potential of immunohistochemistry-based assessment of protein biomarkers for prognostication in clinical practice.

Numerous protein biomarkers have been analyzed to improve prognostication in non-small cell lung cancer, but have not yet demonstrated sufficient value to be introduced into clinical practice. Here, we aimed to develop and validate a prognostic model for surgically resected non-small cell lung cancer. A biomarker panel was selected based on (1) prognostic association in published literature, (2) prognostic association in gene expression data sets, (3) availability of reliable antibodies, and (4) representation of diverse biological processes. The five selected proteins (MKI67, EZH2, SLC2A1, CADM1, and NKX2-1 alias TTF1) were analyzed by immunohistochemistry on tissue microarrays including tissue from 326 non-small cell lung cancer patients. One score was obtained for each tumor and each protein. The scores were combined, with or without the inclusion of clinical parameters, and the best prognostic model was defined according to the corresponding concordance index (C-index). The best-performing model was subsequently validated in an independent cohort consisting of tissue from 345 non-small cell lung cancer patients. The model based only on protein expression did not perform better compared to clinicopathological parameters, whereas combining protein expression with clinicopathological data resulted in a slightly better prognostic performance (C-index: all non-small cell lung cancer 0.63 vs0.64; adenocarcinoma: 0.66 vs0.70, squamous cell carcinoma: 0.57 vs0.56). However, this modest effect did not translate into a significantly improved accuracy of survival prediction. The combination of a prognostic biomarker panel with clinicopathological parameters did not improve survival prediction in non-small cell lung cancer, questioning the potential of immunohistochemistry-based assessment of protein biomarkers for prognostication in clinical practice.

Stable reconstitution of vascular endothelial beds upon transplantation of progenitor cells represents an important challenge due to the paucity and generally limited integration/expansion potential of most identified vascular related cell subsets. We previously showed that mouse fetal liver (FL) hemato/vascular cells from day 12 of gestation (E12), expressing the Stem Cell Leukaemia (SCL) gene enhancer transgene (SCL‐PLAP+cells), had robust endothelial engraftment potential when transferred to the blood stream of newborns or adult conditioned recipients, compared to the scarce vascular contribution of adult bone marrow cells. However, the specific SCL‐PLAP+hematopoietic or endothelial cell subset responsible for the long‐term reconstituting endothelial cell (LTR‐EC) activity and its confinement to FL developmental stages remained unknown. Using a busulfan‐treated newborn transplantation model, we show that LTR‐EC activity is restricted to the SCL‐PLAP+VE‐cadherin+CD45−cell population, devoid of hematopoietic reconstitution activity and largely composed by Lyve1+endothelial‐committed cells. SCL‐PLAP+Ve‐cadherin+CD45−cells contributed to the liver sinusoidal endothelium and also to the heart, kidney and lung microvasculature. LTR‐EC activity was detected at different stages of FL development, yet marginal activity was identified in the adult liver, revealing unknown functional differences between fetal and adult liver endothelial/endothelial progenitors. Importantly, the observations that expanding donor‐derived vascular grafts colocalize with proliferating hepatocyte‐like cells and participate in the systemic circulation, support their functional integration into young livers. These findings offer new insights into the engraftment, phonotypical, and developmental characterization of a novel endothelial/endothelial progenitor cell subtype with multiorgan LTR‐EC activity, potentially instrumental for the treatment/genetic correction of vascular diseases. StemCells2017;35:507–521 (A): SCL‐PLAP+VE‐cad+CD45−, Lyve1+/‐(P+V+C−) fetal liver derived cells generate long term vascular endothelial clusters in different organs when intravenously transplanted into busulfan conditioned newborn recipient mice. P+V+C−cells contributed to endothelial cells (arrows) and also to rare blood cells (asterisks). Scale bars 50 μm. (B): As early as 3 weeks post‐transplantation, transferred fetal liver progenitor cells already form PLAP+vascular clusters (vc) in the host liver. The functionality of the vascular grafts is indicated by the presence of circulating cells in the vessels lumen (i, asterisk), implying their participation in the systemic circulation. Also the higher frequency of BrdU+hepatocytes‐like cells detected in expanding vascular cluster (in vc) areas (ii) compared with tissue areas outside the vascular clusters (out vc) (iv), suggests that donor‐host cellularity cross‐talk activity involved in proliferation control occurs during organ growth. At later stages (12 weeks), when the organ growth slows down and vascular graft expansion stabilizes, almost no BrdU+cells are observed within the vascular cluster areas (iiiand iv) indicating the absence of continuing proliferation in the grafted region. Scale bars 60 μm.

Exposure to serious or traumatic events early in life can lead to persistent alterations in physiological stress response systems, including enhanced cross talk between the neuroendocrine and immune system. These programming effects may be mechanistically involved in mediating the effects of adverse childhood experience on disease risk in adulthood. We investigated hormonal and genome-wide mRNA expression responses in monocytes to acute stress exposure, in a sample of healthy adults (n=30) with a history of early childhood adversity, and a control group (n=30) without trauma experience. The early adversity group showed altered hypothalamus-pituitary-adrenal axis responses to stress, evidenced by lower ACTH and cortisol responses. Analyses of gene expression patterns showed that stress-responsive transcripts were enriched for genes involved in cytokine activity, cytokine–cytokine receptor interaction, chemokine activity, and G-protein coupled receptor binding. Differences between groups in stress-induced regulation of gene transcription were observed for genes involved in steroid binding, hormone activity, and G-protein coupled receptor binding. Transcription factor binding motif analysis showed an increased activity of pro-inflammatory upstream signaling in the early adversity group. We also identified transcripts that were differentially correlated with stress-induced cortisol increases between the groups, enriched for genes involved in cytokine–cytokine receptor interaction and glutamate receptor signaling. We suggest that childhood adversity leads to persistent alterations in transcriptional control of stress-responsive pathways, which—when chronically or repeatedly activated—might predispose individuals to stress-related psychopathology.

•Improving precision in concentration-response estimation for cytotoxicity assays with varying exposure durations.•Predicting cytotoxicity at exposure durations and concentrations not conducted in the experiment.•New, mechanistically motivated two-dimensional model for exposure duration-concentration-response cytotoxicity data.•R-package td2pLL for easy application of proposed two-dimensional model and modeling pipeline.

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P< 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2and NFIL3in the ER+/HER2- subgroup, CLOCKand NPAS2in the ER-/HER2- subtype, and ARNTL2in HER2+ breast cancer. In the multivariate Cox model, only PER3(HR = 0.66; P = 0.016) and RORC(HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.

Endometrial carcinoma differential 3 (EDI3) was the first member of the glycerophosphodiesterase (GDE) protein family shown to be associated with cancer. Our initial work demonstrated that endometrial and ovarian cancer patients with primary tumors overexpressing EDI3 had a higher risk of developing metastasis and decreased survival. Further analysis indicated that EDI3 cleaves glycerophosphocholine to choline and glycerol-3-phosphate, increases the levels of active PKC, and enhances the migratory activity of tumor cells. Despite these initial findings, EDI3 remained mainly uncharacterized. Therefore, to obtain an overview of processes in which EDI3 may be involved, gene array analysis was performed using MCF-7 breast cancer cells after EDI3 knockdown compared with a non-targeting control siRNA. Several biological motifs were altered, including an enrichment of genes involved in integrin-mediated signaling. More specifically, silencing of EDI3 in MCF-7 and OVCAR-3 cells was associated with reduced expression of the key receptor subunit integrin β1, leading to decreased cell attachment and spreading accompanied by delayed formation of cell protrusions. To confirm these results, we stably overexpressed EDI3 in MCF-7 cells which led to elevated integrin β1 expression associated with enhanced cell attachment and spreading - two processes critical for metastasis. In conclusion, our data provide further insight into the role of EDI3 during cancer progression.

Despite the progress made in developmental toxicology, there is a great need for in vitro tests that identify developmental toxicants in relation to human oral doses and blood concentrations. In the present study, we established the hiPSC-based UKK2 in vitro test and analyzed genome-wide expression profiles of 23 known teratogens and 16 non-teratogens. Compounds were analyzed at the maximal plasma concentration (Cmax) and at 20-fold Cmaxfor a 24 h incubation period in three independent experiments. Based on the 1000 probe sets with the highest variance and including information on cytotoxicity, penalized logistic regression with leave-one-out cross-validation was used to classify the compounds as test-positive or test-negative, reaching an area under the curve (AUC), accuracy, sensitivity, and specificity of 0.96, 0.92, 0.96, and 0.88, respectively. Omitting the cytotoxicity information reduced the test performance to an AUC of 0.94, an accuracy of 0.79, and a sensitivity of 0.74. A second method, which used the number of significantly deregulated probe sets to classify the compounds, resulted in a specificity of 1; however, the AUC (0.90), accuracy (0.90), and sensitivity (0.83) were inferior compared to those of the logistic regression-based procedure. Finally, no increased performance was achieved when the high test concentrations (20-fold Cmax) were used, in comparison to testing within the realistic clinical range (1-fold Cmax). In conclusion, although further optimization is required, for example, by including additional readouts and cell systems that model different developmental processes, the UKK2-test in its present form can support the early discovery-phase detection of human developmental toxicants.

Recently, EDI3 was identified as a key factor for choline metabolism that controls tumor cell migration and is associated with metastasis in endometrial carcinomas. EDI3 cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P). Choline is then further metabolized to phosphatidylcholine (PtdC), the major lipid in membranes and a key player in membrane-mediated cell signaling. The second product, G3P, is a precursor molecule for several lipids with central roles in signaling, for example lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). LPA activates intracellular signaling pathways by binding to specific LPA receptors, including membrane-bound G protein-coupled receptors and the intracellular nuclear receptor, PPARγ. Conversely, PA and DAG mediate signaling by acting as lipid anchors that bind and activate several signaling proteins. For example, binding of GTPases and PKC to PA and DAG, respectively, increases the activation of signaling networks, mediating processes such as migration, adhesion, proliferation or anti-apoptosis—all relevant for tumor development. We present a concept by which EDI3 either directly generates signaling molecules or provides “membrane anchors” for downstream signaling factors. As a result, EDI3 links choline metabolism to signaling activities resulting in a more malignant phenotype.

Genotyping N-acetyltransferase 2 (NAT2) is of high relevance for individualized dosing of antituberculosis drugs and bladder cancer epidemiology. In this study we compared a recently published tagging single nucleotide polymorphism (SNP) (rs1495741) to the conventional 7-SNP genotype (G191A, C282T, T341C, C481T, G590A, A803G and G857A haplotype pairs) and systematically analysed if novel SNP combinations outperform the latter. For this purpose, we studied 3177 individuals by PCR and phenotyped 344 individuals by the caffeine test. Although the tagSNP and the 7-SNP genotype showed a high degree of correlation (R=0.933, P<0.0001) the 7-SNP genotype nevertheless outperformed the tagging SNP with respect to specificity (1.0 vs. 0.9444, P=0.0065). Considering all possible SNP combinations in a receiver operating characteristic analysis we identified a 2-SNP genotype (C282T, T341C) that outperformed the tagging SNP and was equivalent to the 7-SNP genotype. The 2-SNP genotype predicted the correct phenotype with a sensitivity of 0.8643 and a specificity of 1.0. In addition, it predicted the 7-SNP genotype with sensitivity and specificity of 0.9993 and 0.9880, respectively. The prediction of the NAT2 genotype by the 2-SNP genotype performed similar in populations of Caucasian, Venezuelan and Pakistani background. A 2-SNP genotype predicts NAT2 phenotypes with similar sensitivity and specificity as the conventional 7-SNP genotype. This procedure represents a facilitation in individualized dosing of NAT2 substrates without losing sensitivity or specificity.

Recently, a genome-wide single nucleotide polymorphism association study has identified a sequence variant 30 kb upstream of the c-Mycgene (allele T of rs9642880) that confers susceptibility to bladder cancer. However, the role of exposure to bladder carcinogens has not been considered. This prompted us to analyse the relevance of this polymorphism in 515 bladder cancer cases and 893 controls where the quality and quantity of occupational exposure to bladder carcinogens has been documented. When we analysed a hospital-based case–control series not selected for occupational exposure, rs9642880T was influential, in contrast to GSTM1 00. However, in a case–control series of patients that have been occupationally exposed to aromatic amines and polycyclic aromatic hydrocarbons, rs9642880T was not influential but GSTM1 00was significantly associated with bladder cancer risk. Therefore, the degree to which rs9642880T and GSTM1 00confer susceptibility to urinary bladder cancer seems to depend on the extent of exposure to urinary bladder carcinogens.

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

Remarkable interindividual differences in 3,4-methylenedioxymethamphetamine (‘Ecstasy’)-mediated toxicity have been reported in humans. Therefore, we tested whether CYP2D6 or its variant alleles as well as CYP3A4 influence the susceptibility to 3,4-methylenedioxymethamphetamine.

Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, α-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte does not yet exist there is a good chance that this aim may be achieved in the future.

There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. In addition to mechanical devices supporting liver function, such as bioreactors, the transplantation of hepatocyte-like cells derived from (adult) stem cells offer great perspectives. We have generated hepatocyte-like (NeoHep) cells from terminally differentiated peripheral blood monocytes and, in this study, have evaluated these cells as a possible tool for autologous cell therapy.

Co-exposure to cadmium, cobalt, lead and other heavy metals occurs in many occupational settings, such as pigment and batteries production, galvanization and recycling of electric tools. However, little is known about interactions between several heavy metals. In the present study we determined DNA single strand break (DNA-SSB) induction and repair capacity for 8-oxoguanine in mononuclear blood cells of 78 individuals co-exposed to cadmium (range of concentrations in air: 0.05–138.00 μg/m3), cobalt (range: 0–10 μg/m3) and lead (range: 0–125 μg/m3). Exposure to heavy metals was determined in air, blood and urine. Non-parametric correlation analysis showed a correlation between cadmium concentrations in air with DNA-SSB (P = 0.001, R = 0.371). Surprisingly, cobalt air concentrations correlated even better (P < 0.001, R = 0.401), whereas lead did not correlate with DNA-SSB. Logistic regression analysis including 11 possible parameters of influence resulted in a model showing that cobalt in air, cadmium in air, cadmium in blood and lead in blood influence the level of DNA-SSB. The positive result with cobalt was surprising, since exposure levels were much lower compared with the TRK-value of 100 μg/m3. To examine, whether the positive result with cobalt is stable, we applied several logistic regression models with two blocks, where all factors except cobalt were considered preferentially. All strategies resulted in the model described above. Logistic regression analysis considering also all possible interactions between the relevant parameters of influence finally resulted in the following model: Odds ratio = 1.286Co in air × 1.040Cd in air × 3.111Cd in blood × 0.861Pb in air × 1.023Co in air × Pb in air. This model correctly predicts an increased level of DNA-SSB in 91% of the subjects in our study. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium, cobalt and lead. For instance increasing lead air concentrations from 1.6 to 50 μg/m3 in the presence of constant exposures to cobalt and cadmium (8 μg/m3 and 3.8 μg/m3) leads to an almost 5-fold increase in the odds ratio, although lead alone does not increase DNA-SSB. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or cobalt. Indeed, repair of 8-oxoguanine decreased with increasing exposures and inversely correlated with the level of DNA-SSB (P = 0.001, R = −0.427). Protein expression patterns of individuals exposed to cobalt concentrations of ∼10 μg/m3 were compared with those of unexposed individuals using two-dimensional gel electrophoresis. Qualitative and apparent quantitative alterations in protein expression were selective and certainly occurred in <0.1% of all proteins. In conclusion, the hazard due to cobalt exposure – that has been classified only as IIB by the IARC – seems to be underestimated, especially when individuals are co-exposed to cadmium or lead. Co-exposure may cause genotoxic effects, even if the concentrations of individual heavy metals do not exceed TRK-values.

Co-exposure to cadmium, cobalt, lead and other heavy metals occurs in many occupational settings, such as pigment and batteries production, galvanization and recycling of electric tools. However, little is known about interactions between several heavy metals. In the present study we determined DNA single strand break (DNA-SSB) induction and repair capacity for 8-oxoguanine in mononuclear blood cells of 78 individuals co-exposed to cadmium (range of concentrations in air: 0.05-138.00 micro g/m(3)), cobalt (range: 0-10 micro g/m(3)) and lead (range: 0-125 micro g/m(3)). Exposure to heavy metals was determined in air, blood and urine. Non-parametric correlation analysis showed a correlation between cadmium concentrations in air with DNA-SSB (P = 0.001, R = 0.371). Surprisingly, cobalt air concentrations correlated even better (P < 0.001, R = 0.401), whereas lead did not correlate with DNA-SSB. Logistic regression analysis including 11 possible parameters of influence resulted in a model showing that cobalt in air, cadmium in air, cadmium in blood and lead in blood influence the level of DNA-SSB. The positive result with cobalt was surprising, since exposure levels were much lower compared with the TRK-value of 100 micro g/m(3). To examine, whether the positive result with cobalt is stable, we applied several logistic regression models with two blocks, where all factors except cobalt were considered preferentially. All strategies resulted in the model described above. Logistic regression analysis considering also all possible interactions between the relevant parameters of influence finally resulted in the following model: Odds ratio = 1.286(Co in air) x 1.040(Cd in air) x 3.111(Cd in blood) x 0.861(Pb in air) x 1.023(Co in air x Pb in air). This model correctly predicts an increased level of DNA-SSB in 91% of the subjects in our study. One conclusion from this model is the existence of more than multiplicative effects for co-exposures of cadmium, cobalt and lead. For instance increasing lead air concentrations from 1.6 to 50 micro g/m(3) in the presence of constant exposures to cobalt and cadmium (8 micro g/m(3) and 3.8 micro g/m(3)) leads to an almost 5-fold increase in the odds ratio, although lead alone does not increase DNA-SSB. The mechanism behind these interactions might be repair inhibition of oxidative DNA damage, since a decrease in repair capacity will increase susceptibility to reactive oxygen species generated by cadmium or cobalt. Indeed, repair of 8-oxoguanine decreased with increasing exposures and inversely correlated with the level of DNA-SSB (P = 0.001, R = -0.427). Protein expression patterns of individuals exposed to cobalt concentrations of approximately 10 micro g/m(3) were compared with those of unexposed individuals using two-dimensional gel electrophoresis. Qualitative and apparent quantitative alterations in protein expression were selective and certainly occurred in <0.1% of all proteins. In conclusion, the hazard due to cobalt exposure - that has been classified only as IIB by the IARC - seems to be underestimated, especially when individuals are co-exposed to cadmium or lead. Co-exposure may cause genotoxic effects, even if the concentrations of individual heavy metals do not exceed TRK-values.

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) encodes a dual specific protein and phospholipid phosphatase that affects cell proliferation, apoptosis and migration. In our study, we examined protein expression of PTEN in renal carcinogenesis. PTEN protein levels were examined in 42 clear cell renal cell carcinomas (ccRCC) and oncocytomas as well as in the corresponding normal renal tissue of the same patients using Western blot analysis. Cellular localization was analyzed by immunohistochemistry. PTEN was highly expressed in all investigated normal renal tissue specimens. Immunohistochemical analysis showed an almost exclusive staining of proximal tubulus epithelial cells known to be precursor cells of ccRCC. Within the proximal tubulus cells, PTEN exhibited a membrane predominant immunostaining pattern. In ccRCCs PTEN expression was markedly reduced to an average of less than 10% compared with normal tissue as evidenced by Western blot analysis (p &lt; 0.001). The degree of reduction was similar in highly differentiated (G1) carcinomas and in less differentiated (G2–G4) carcinomas. These observations were reproduced by immunohistochemical studies, which revealed a loss of the characteristic membrane predominant immunostaining pattern in ccRCC. In contrast to the PTEN positive proximal tubulus epithelial cells, the distal tubulus epithelial cells, which are precursor cells of the benign oncocytomas, exhibited only a very weak PTEN expression. Compared with the distal tubulus epithelial cells, no downregulation of PTEN was seen in oncocytomas. We conclude that PTEN expression and PTEN membrane localization are lost during early renal cell carcinogenesis and may therefore be a valuable RCC tumor marker. © 2002 Wiley-Liss, Inc.

Mutagenic and co‐mutagenic effects of static, pulsed bipolar gradient, and high‐frequency magnetic fields, as well as combinations of them, were examined using the Ames test. The Ames test using Salmonella typhimuriumbacteria, wild‐type strain RTA, preincubation assay, without metabolic activation, was performed. All combinations of magnetic fields were tested with and without co‐exposure to N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and benzo[a]pyrene‐4,5‐oxide, ethylene oxide, carboplatin, or cisplatin. As expected, chemical mutagens caused a clear‐cut increase of the revertants in the Ames test. However, neither the static fields nor a combination of a static magnetic field with the time‐varying bipolar gradient field or a pulsed high‐frequency magnetic field caused an alteration in the number of revertants in the Ames test. No co‐mutagenic effect of any magnetic field combination was observed. In conclusion, magnetic fields used during clinical magnetic resonance imaging (MRI) were neither mutagenic nor co‐mutagenic. J. Magn. Reson. Imaging 2001;14:779–788. © 2001 Wiley‐Liss, Inc.

Mutagenic and co-mutagenic effects of static, pulsed bipolar gradient, and high-frequency magnetic fields, as well as combinations of them, were examined using the Ames test. The Ames test using Salmonella typhimurium bacteria, wild-type strain RTA, preincubation assay, without metabolic activation, was performed. All combinations of magnetic fields were tested with and without co-exposure to N-methyl-N'-nitro-N-nitrosoguanidine and benzo[a]pyrene-4,5-oxide, ethylene oxide, carboplatin, or cisplatin. As expected, chemical mutagens caused a clear-cut increase of the revertants in the Ames test. However, neither the static fields nor a combination of a static magnetic field with the time-varying bipolar gradient field or a pulsed high-frequency magnetic field caused an alteration in the number of revertants in the Ames test. No co-mutagenic effect of any magnetic field combination was observed. In conclusion, magnetic fields used during clinical magnetic resonance imaging (MRI) were neither mutagenic nor co-mutagenic. J. Magn. Reson. Imaging 2001;14:779–788. © 2001 Wiley-Liss, Inc.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1–/– null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1–/– mice aged 9–12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1–/– and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1–/– mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1–/– fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1–/– fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release—a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

The DNA-repair protein O⁶-methylguanine-DNA methyltransferase (alkyltransferase; MGMT) is a major determinant of resistance of cells to various alkylating cytostatic drugs. Its expression in tissues is highly variable, indicating complex regulatory mechanisms involved. Transfection-mediated expression of wild-type p53 has been shown to negatively regulate basal promoter activity of MGMT in vitro. To elucidate whether p53 is involved in regulation of MGMT in tumor tissue, we examined MGMT expression and the p53 status of 140 primary ovarian carcinomas and analyzed the data as to the correlation between MGMT and p53, as well as the survival response of the patients after chemotherapy. We show that MGMT expression is highly variable in ovarian carcinomas, ranging from zero level up to 2500 fmol/mg protein. MGMT activity was significantly lower in tumors with wild-type p53 (p53wt) than in tumors with mutant p53 (p53mt) (p = 0.045). As expected, the percentage of tumors with p53mt increased with increasing histologic grade of the tumors. Thus, p53mt was observed in 4, 45 and 64% of grades 1, 2 and 3 tumors, respectively (p = 0.001). Increase in p53mt was accompanied by an increase in MGMT activity, which was, on average, 460 ± 66, 624 ± 63 and 662 ± 60 fmol/mg protein in grades 1, 2 and 3 tumors, respectively (p = 0.047). In addition, MGMT activity as well as p53mt were associated with the FIGO stage of the tumors. Mean MGMT activity was 472 ± 48 fmol/mg for patients with FIGO stages I and II, as compared with 675 ± 50 fmol/mg for patients with FIGO stages III and IV, (p = 0.0179). The percentage of p53mt was 27% and 54% in ovarian tumors with FIGO stages I/II and FIGO stages III/IV, respectively (p = 0.004). Thus, progression of ovarian tumors was clearly associated with increase of both MGMT activity and the percentage of p53mt. In tumors expressing low MGMT (&lt;100 fmol/mg), p53mt was very rarely found. No significant association was observed between MGMT level in ovarian carcinomas and the survival of patients treated with cyclophosphamide and carboplatin. On the other hand, a clear correlation was found between histological type, grading, residual tumor mass and p53wt expression and duration of the patient's survival. The finding that p53wt expression was associated with low MGMT level in primary ovarian cancer supports the view that down-regulation of basal MGMT promoter activity by p53wt is also relevant in tumor cells in vivo. Int. J. Cancer (Pred. Oncol.) 84:388–395, 1999. © 1999 Wiley-Liss, Inc.

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and posttranslationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129for CYP2E1 function, Ser129was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDNAs were transfected into Chinese hamster lung fibroblast V79 cells. Despite the mutation in the PKA phosphorylation motif, all strains produced catalytically active CYP2E1. However, there was a marked change in the substrate preference: The Gly129-containing strains hydroxylatedp-nitrophenol (PNP) to a markedly higher extent than the wild-type cDNA-containing cells, while they demethylatedN-nitrosodimethylamine (NDMA) to a markedly lower extent than the wild-type cells. All the strains activated NDMA to mutagenic products. Treatment with the membrane-permeating cAMP derivative db-cAMP reduced markedly both the PNP hydroxylase and the NDMA demethylase activities as well as the mutation frequency induced by NDMA in the Ser129-containing strain. This decrease in activity was not accompanied by a decrease in CYP2E1 content. In addition, the catalytic activities of CYP2E1 were decreased in microsomes from rat hepatocytes treated with db-cAMP. Also in this case, the decrease in activities was not accompanied by a decrease in enzyme protein. These findings argue that involvement of Ser129and its phosphorylation is not in determining CYP2E1 protein level, but rather in controlling its catalytic activity. In contrast, in the strains containing Ala129or Gly129, treatment with db-cAMP caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129or Gly129substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129is not involved in CYP2E1 degradation but dictates requirements for its specific activities.

In 28 smoking and 37 non-smoking male metal workers from 7 small to medium sized plants the genotoxic effect caused by the occupational exposure measured as DNA single strand breaks in the peripheral mononuclear blood cells was investigated. Metal workers using synthetic cutting fluids are possibly exposed to the carcinogenic N-nitrosodiethanolamine (NDELA). In this investigation NDELA was detected in the air of the working halls in a concentration up to 1000 ng/m3and in the cutting fluids in a concentration up to 135 mg/1 (mean values per plant).

Infiltration of plasma cells is associated with better prognosis in breast, lung and colon cancer. Immunoglobulin κ chain (IGKC) is now available as a single, robust immune marker predicting metastasis-free survival and response to chemotherapy. This will facilitate a deeper understanding of the role of the humoral immune system in cancer development.

Nervenzellen vernetzen sich miteinander — es sei denn, Neurotoxine stören sie dabei. Eine neue In‐vitro‐Methode ermittelt, wie schnell Zellfortsätze auf einer Analysenplattform wachsen, und misst so die Neurotoxizität von Chemikalien.