Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72 h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

Arsenic-containing hydrocarbons disrupt a model in vitro blood-cerebrospinal fluid barrier.

Lipid-soluble arsenicals, so-called arsenolipids, have gained a lot of attention in the last few years because of their presence in many seafoods and reports showing substantial cytotoxicity emanating from arsenic-containing hydrocarbons (AsHCs), a prominent subgroup of the arsenolipids. More recent in vivo and in vitro studies indicate that some arsenolipids might have adverse effects on brain health. In the present study, we focused on the effects of selected arsenolipids and three representative metabolites on the blood-cerebrospinal fluid barrier (B-CSF-B), a brain-regulating interface. For this purpose, we incubated an in vitro model of the B-CSF-B composed of porcine choroid plexus epithelial cells (PCPECs) with three AsHCs, two arsenic-containing fatty acids (AsFAs) and three representative arsenolipid metabolites (dimethylarsinic acid, thio/oxo-dimethylpropanoic acid) to examine their cytotoxic potential and impact on barrier integrity. The toxic arsenic species arsenite was also tested in this way and served as a reference substance. While AsFAs and the metabolites showed no cytotoxic effects in the conducted assays, AsHCs showed a strong cytotoxicity, being up to 1.5-fold more cytotoxic than arsenite. Analysis of the in vitro B-CSF-B integrity showed a concentration-dependent disruption of the barrier within 72 h. The correlation with the decreased plasma membrane surface area (measured as capacitance) indicates cytotoxic effects. These findings suggest exposure to elevated levels of certain arsenolipids may have detrimental consequences for the central nervous system.

Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

In vitro toxicological characterisation of arsenic-containing fatty acids and three of their metabolites.

Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMAV, DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at µM concentrations. DMAV causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMAV in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.

In vitro toxicological characterisation of three arsenic-containing hydrocarbons.

Arsenic-containing hydrocarbons are one group of fat-soluble organic arsenic compounds (arsenolipids) found in marine fish and other seafood. A risk assessment of arsenolipids is urgently needed, but has not been possible because of the total lack of toxicological data. In this study the cellular toxicity of three arsenic-containing hydrocarbons was investigated in cultured human bladder (UROtsa) and liver (HepG2) cells. Cytotoxicity of the arsenic-containing hydrocarbons was comparable to that of arsenite, which was applied as the toxic reference arsenical. A large cellular accumulation of arsenic, as measured by ICP-MS/MS, was observed after incubation of both cell lines with the arsenolipids. Moreover, the toxic mode of action shown by the three arsenic-containing hydrocarbons seemed to differ from that observed for arsenite. Evidence suggests that the high cytotoxic potential of the lipophilic arsenicals results from a decrease in the cellular energy level. This first in vitro based risk assessment cannot exclude a risk to human health related to the presence of arsenolipids in seafood, and indicates the urgent need for further toxicity studies in experimental animals to fully assess this possible risk.

The impact of domain-specific beliefs on decisions and causal judgments.

Extensive evidence suggests that people often rely on their causal beliefs in their decisions and causal judgments. To date, however, there is a dearth of research comparing the impact of causal beliefs in different domains. We conducted two experiments to map the influence of domain-specific causal beliefs on the evaluation of empirical evidence when making decisions and subsequent causal judgments. Participants made 120 decisions in a two-alternative forced-choice task, framed in either a medical or a financial domain. Before each decision, participants could actively search for information about the outcome ("occurrence of a disease" or "decrease in a company's share price") on the basis of four cues. To analyze the strength of causal beliefs, we set two cues to have a generative relation to the outcome and two to have a preventive relation to the outcome. To examine the influence of empirical evidence, we manipulated the predictive power (i.e., cue validities) of the cues. Both experiments included a validity switch, where the four selectable cues switched from high to low validity or vice versa. Participants had to make a causal judgment about each cue before and after the validity switch. In the medical domain, participants stuck to the causal information in causal judgments, even when evidence was contradictory, while decisions showed an effect of both empirical and causal information. In contrast, in the financial domain, participants mainly adapted their decisions and judgments to the cue validities. We conclude that the strength of causal beliefs (1) is shaped by the domain, and (2) has a differential influence on the degree to which empirical evidence is taken into account in causal judgments and decision making.

[Do media reports and public brochures facilitate informed decision making about cervical cancer prevention?]. / Ermöglichen Medienberichte und Broschüren informierte Entscheidungen zur Gebärmutterhalskrebsprävention?

With the introduction and recommendation of the new HPV (human papillomavirus) vaccination in 2007, cervical cancer prevention has evoked large public interest. Is the public able to make informed decisions on the basis of media reports and brochures? To answer this question, an analysis of media coverage of HPV vaccination (Gardasil®) and Pap (Papanicolaou) screening was conducted from 2007-2009, which investigated the minimum requirement of completeness (pros and cons), transparency (absolute numbers), and correctness (references concerning outcome, uncertainty, magnitude) of the information. As a bench mark, facts boxes with concise data on epidemiology, etiology, benefits, harms, and costs were compiled in advance. Although all vaccination reports and brochures covered the impact of prevention, only 41% provided concrete numbers on effectiveness (90/220) and 2% on absolute risk reductions for the cancer surrogate dysplasia (5/220), whereby none of the latter numbers was correct. The prevention potential was correctly presented once. Only 48% (105/220) mentioned pros and cons. With regard to screening, 20% (4/20) provided explicit data on test quality and one expressed these in absolute numbers, while 25% (5/20) reported the prevention potential; all given numbers were correct. Finally, 25% (5/20) mentioned the possibility of false positive results. Minimum requirements were fulfilled by 1/220 vaccination and 1/20 screening reports. At present, informed decision making based on media coverage is hardly possible.

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity.

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4(+) and CD8(+) HLA-A1-specific T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.

Reticular dysgenesis: HLA non-identical bone marrow transplants in a series of 10 patients.

Reticular dysgenesis is a very rare congenital immunodeficiency classified within the severe combined immunodeficiencies (SCID) and characterized by impairment of both lymphoid and myeloid cell development. We report our experience in 10 patients with RD, treated between 1979 and 1999 with HLA-haploidentical hematopoietic stem cell transplantation (HSCT). All children but one were symptomatic within the first days of their lives. Five patients required two HSCT. Five patients received conditioning therapy with busulfan (16 mg/kg) and cyclophosphamide. Three of them are alive and well with myeloid and T and B cell lymphoid reconstitution, whereas two patients died (one chronic graft-versus-host disease, one pneumonitis). Transplantation without or with other conditioning regimens in the other five cases led to absent or incomplete engraftment and none of these cases survived. These results demonstrate the mandatory need for intensive conditioning before haploidentical HSCT in RD to achieve full lymphoid and myeloid engraftment.

[The relationship between fovea capitis femoris and weight bearing area in the normal and dysplastic hip in adults: a radiologic study]. / Die radiologische Beziehung der Fovea capitis femoris zur azetabulären Belastungszone bei der normalen und dysplastischen Hüfte des Erwachsenen.

AIM: In residual hip dysplasia the fovea capitis femoris lays often more cranial than in the normal hip morphology, the ligamentum capitis femoris thereby articulating with the weight-bearing area of the acetabular cartilage. The aim of this study was to quantitate this aspect with regard to its potential negative effect for the degeneration of the dysplastic acetabulum. METHOD: The relation between the fovea capitis femoris and the weight-bearing area were studied using the a.p.-pelvis view in normal and dysplastic hips. The measurements were made by digital image analysis. RESULTS: The hypothesis that the "fovea alta" is characteristic for the dysplastic hip was confirmed. In dysplastic hips the typically wider fovea lays on average 30 degrees more cranial, touching the weight-bearing area over 11 degrees. The fovea in normal hips has on average a distance of 26 degrees to the acetabular roof. In dysplastic hips the steeper roof, the tendency for decentering of the femoral head as well as a higher CCD angle explain this phenomenon to a certain degree. In a theoretical model to correct the acetabular position over the femoral head alone one quarter of the foveae would still touch the weight-bearing area. Nevertheless, in our series after periacetabular osteotomy this was never the case due to better centering. CONCLUSION: lt is our hypothesis that a "fovea alta", which further reduces the already smaller loaded cartilage surface, is one part leading to the early degeneration of the dysplastic hip. Therefore, it should be considered in biomechanical models and in the planning of corrective procedures.

Transplacentally acquired maternal T lymphocytes in severe combined immunodeficiency: a study of 121 patients.

A study in 121 infants with severe combined immunodeficiency (SCID) was performed to determine the prevalence of an engraftment by transplacentally acquired maternal T cells and to explore clinical and immunological findings related to this abnormality. Each newly diagnosed patient with SCID presenting with circulating T cells was evaluated for chimerism by performing selective HLA typing of T cells and non-T cells. In patients with engraftment, maternal T cells were characterized phenotypically and functionally, and results were correlated with clinical findings in the patients. Maternal T cells were detected in the circulation in 48 patients; these cells ranged from fewer than 100/microL in 14 cases to more than 2000/microL in 4 cases (median, 415/microL). Clinical signs of graft-versus-host disease (GVHD) were absent in 29 patients. In the other cases, manifestations of GVHD were present, involving the skin and in 14 cases also the liver. Skin GVHD was mild in 8 patients. In these patients, as well as in patients with no signs of GVHD, maternal T cells were predominantly CD8(+) and, with one exception, failed to respond to mitogen stimulation. In 9 patients, manifestations of skin GVHD were prominent. T cells in these cases were predominantly CD4(+) and responded, with one exception, to mitogen stimulation. In 8 of the cases with prominent skin GVHD, the underlying SCID variant was characterized by the absence of B cells. In this study, further understanding is provided of a phenomenon that is responsible for the significant heterogeneity of clinical and immunological findings in SCID.

The effect of under and over refractive correction on visual performance and spectacle lens acceptance.

As a follow-on from a previous study by Miller, Kris and Griffiths (1997, Optom. Vis. Sci. 84, 521-526), we investigated the effect of small prescription errors on spatial visual performance and spectacle lens acceptability. We included both negative and positive errors and binocular and monocular errors. Data were collected on 15 young adult subjects. Clinical measures were taken of pupil size, ocular dominance, binocular visual acuity, negative and positive relative accommodation, distance and near heterophorias, and stereopsis. Visual performance was measured with the best correction and for +/-0.50 D spherical binocular and monocular errors. Subjects wore spectacles, each containing a different error in turn, for 2 days and compared them with a reference pair. Following the wearing period subjects assessed the performance of the spectacles by completing a short questionnaire. The only ocular tests for which these small prescription errors had significant effects were binocular visual acuity and negative relative accommodation. No significant relationship was observed between any of the clinical test results and overall lens acceptance. Despite this, the reference pair was generally perceived as better than the test pairs containing errors, and a considerable proportion of subjects reported pairs with errors as being unacceptable. In conclusion, the questionnaire findings support the need for both accurate monocular and binocular refractions. Subjects differed in their criteria for judging lens acceptability.

V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations.

Severe combined immunodeficiency (SCID) comprises a heterogeneous group of primary immunodeficiencies, a proportion of which are due to mutations in either of the 2 recombination activating genes (RAG)-1 and -2, which mediate the process of V(D)J recombination leading to the assembly of antigen receptor genes. It is reported here that the clinical and immunologic phenotypes of patients bearing mutations in RAGs are more diverse than previously thought and that this variability is related, in part, to the specific type of RAG mutation. By analyzing 44 such patients from 41 families, the following conclusions were reached: (1) null mutations on both alleles lead to the T-B-SCID phenotype; (2) patients manifesting classic Omenn syndrome (OS) have missense mutations on at least one allele and maintain partial V(D)J recombination activity, which accounts for the generation of residual, oligoclonal T-lymphocytes; (3) in a third group of patients, findings were only partially compatible with OS, and these patients, who also carried at least one missense mutation, may be considered to have atypical SCID/OS; (4) patients with engraftment of maternal T cells as a complication of a transplacental transfusion represented a fourth group, and these patients, who often presented with a clinical phenotype mimicking OS, may be observed regardless of the type of RAG gene mutation. Analysis of the RAG genes by direct sequencing is an effective way to provide accurate diagnosis of RAG-deficient as opposed to RAG-independent V(D)J recombination defects, a distinction that cannot be made based on clinical and immunologic phenotype alone.

Similar pattern of thymic-dependent T-cell reconstitution in infants with severe combined immunodeficiency after human leukocyte antigen (HLA)-identical and HLA-nonidentical stem cell transplantation.

Donor T cells after stem cell transplantation reconstitute by 2 different pathways: by expansion from grafted, mature T cells and by intrathymic maturation from progenitor cells. This study characterized thymic-dependent reconstitution of CD4(+) T cells following different transplant modalities in patients with severe combined immunodeficiency (SCID). Three groups of patients were studied: one group after transplantation from human leukocyte antigen (HLA)-identical siblings with unmanipulated grafts without conditioning, a second group after transplantation from HLA-nonidentical parents with T-cell-depleted grafts without preconditioning, and a third group with prior conditioning. Reconstitution of the T-cell compartment was monitored by determining the expression of CD45 isoforms by developing CD4(+) cells in the peripheral blood and in discriminating expanded (CD45RO(+)) and newly generated (CD45RA(+)) T cells. Concomitantly, changes in the size of the thymus were evaluated sequentially by ultrasonography. Reconstitution of CD4(+)CD45RA(+) cells was delayed in all patients for several months, including patients after HLA-identical transplantation, and was always paralleled by normalization of the size of the thymus. No engraftment of donor progenitor cells was observed, as studied in one patient transplanted without conditioning. CD4(+)CD45RO(+) cells were detected early after transplantation only in patients given unmanipulated grafts. The study showed that thymic-dependent T-cell maturation in these patients with SCID runs an autonomous course, independent of graft manipulation, of major HLA disparities, and of whether conditioning is used or not. In addition, thymic maturation may not require engraftment of donor-derived CD34(+) cells in the marrow. (Blood. 2000;96:4344-4349)

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas.

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n = 10; basal cell carcinomas: n = 16) on frozen sections using a highly sensitive streptavidin-peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1-3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.

Regulation of urokinase receptor transcription by Ras- and Rho-family GTPases.

How cell adhesion is coordinated with extracellular proteolysis is a key question in understanding cell migration. Potentially, the small GTP-binding proteins that affect actin organisation and signal transduction may also regulate the expression of genes associated with extracellular proteolysis. We investigated the ability of Ras, Rac-1, Cdc42Hs, and RhoA to regulate transcription from the1.55-kb promoter region of the human urokinase plasminogen activator receptor (uPAR) gene. Constitutively active V12 H-Ras and Rho-A stimulated uPAR transcription while Cdc42Hs and Rac-1 did not. The use of Ras effector-loop mutants indicated that signalling via multiple Ras-effectors is necessary for the maximum activation of transcription.