Mechanistic studies on the adverse effects of manganese overexposure in differentiated LUHMES cells.

Manganese (Mn) is an essential trace element, but overexposure is associated with toxicity and neurological dysfunction. Accumulation of Mn can be observed in dopamine-rich regions of the brain in vivo and Mn-induced oxidative stress has been discussed extensively. Nevertheless, Mn-induced DNA damage, adverse effects of DNA repair, and possible resulting consequences for the neurite network are not yet characterized. For this, LUHMES cells were used, as they differentiate into dopaminergic-like neurons and form extensive neurite networks. Experiments were conducted to analyze Mn bioavailability and cytotoxicity of MnCl2, indicating a dose-dependent uptake and substantial cytotoxic effects. DNA damage, analyzed by means of 8-oxo-7,8-dihydro-2'-guanine (8oxodG) and single DNA strand break formation, showed significant dose- and time-dependent increase of DNA damage upon 48 h Mn exposure. Furthermore, the DNA damage response was increased which was assessed by analytical quantification of poly(ADP-ribosyl)ation (PARylation). Gene expression of the respective DNA repair genes was not significantly affected. Degradation of the neuronal network is significantly altered by 48 h Mn exposure. Altogether, this study contributes to the characterization of Mn-induced neurotoxicity, by analyzing the adverse effects of Mn on genome integrity in dopaminergic-like neurons and respective outcomes.

A fast and reliable method for monitoring genomic instability in the model organism Caenorhabditis elegans.

The identification of genotoxic agents and their potential for genotoxic alterations in an organism is crucial for risk assessment and approval procedures of the chemical and pharmaceutical industry. Classically, testing strategies for DNA or chromosomal damage focus on in vitro and in vivo (mainly rodent) investigations. In cell culture systems, the alkaline unwinding (AU) assay is one of the well-established methods for detecting the percentage of double-stranded DNA (dsDNA). By establishing a reliable lysis protocol, and further optimization of the AU assay for the model organism Caenorhabditis elegans (C. elegans), we provided a new tool for genotoxicity testing in the niche between in vitro and rodent experiments. The method is intended to complement existing testing strategies by a multicellular organism, which allows higher predictability of genotoxic potential compared to in vitro cell line or bacterial investigations, before utilizing in vivo (rodent) investigations. This also allows working within the 3R concept (reduction, refinement, and replacement of animal experiments), by reducing and possibly replacing animal testing. Validation with known genotoxic agents (bleomycin (BLM) and tert-butyl hydroperoxide (tBOOH)) proved the method to be meaningful, reproducible, and feasible for high-throughput genotoxicity testing, and especially preliminary screening.

A matter of concern - Trace element dyshomeostasis and genomic stability in neurons.

Neurons are post-mitotic cells in the brain and their integrity is of central importance to avoid neurodegeneration. Yet, the inability of self-replenishment of post-mitotic cells results in the need to withstand challenges from numerous stressors during life. Neurons are exposed to oxidative stress due to high oxygen consumption during metabolic activity in the brain. Accordingly, DNA damage can occur and accumulate, resulting in genome instability. In this context, imbalances in brain trace element homeostasis are a matter of concern, especially regarding iron, copper, manganese, zinc, and selenium. Although trace elements are essential for brain physiology, excess and deficient conditions are considered to impair neuronal maintenance. Besides increasing oxidative stress, DNA damage response and repair of oxidative DNA damage are affected by trace elements. Hence, a balanced trace element homeostasis is of particular importance to safeguard neuronal genome integrity and prevent neuronal loss. This review summarises the current state of knowledge on the impact of deficient, as well as excessive iron, copper, manganese, zinc, and selenium levels on neuronal genome stability.

Characterizing effects of excess copper levels in a human astrocytic cell line with focus on oxidative stress markers.

BACKGROUND: Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer's disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far. METHODS: In this study, excess copper levels affecting human astrocytic cell model and their involvement in the neurotoxic mode of action of copper, as well as, effects on the homeostasis of other trace elements (Mn, Fe, Ca and Mg) were investigated. RESULTS: Copper induced substantial cytotoxic effects in the human astrocytic cell line following 48 h incubation (EC30: 250 µM) and affected mitochondrial function, as observed via reduction of mitochondrial membrane potential and increased ROS production, likely originating from mitochondria. Moreover, cellular GSH metabolism was altered as well. Interestingly, not only cellular copper levels were affected, but also the homeostasis of other elements (Ca, Fe and Mn) were disrupted. CONCLUSION: One potential toxic mode of action of copper seems to be effects on the mitochondria along with induction of oxidative stress in the human astrocytic cell model. Moreover, excess copper levels seem to interact with the homeostasis of other essential elements such as Ca, Fe and Mn. Disrupted element homeostasis might also contribute to the induction of oxidative stress, likely involved in the onset and progression of neurodegenerative disorders. These insights in the toxic mechanisms will help to develop ideas and approaches for therapeutic strategies against copper-mediated diseases.

Cellular toxicological characterization of a thioxolated arsenic-containing hydrocarbon.

Arsenolipids, especially arsenic-containing hydrocarbons (AsHC), are an emerging class of seafood originating contaminants. Here we toxicologically characterize a recently identified oxo-AsHC 332 metabolite, thioxo-AsHC 348 in cultured human liver (HepG2) cells. Compared to results of previous studies of the parent compound oxo-AsHC 332, thioxo-AsHC 348 substantially affected cell viability in the same concentration range but exerted about 10-fold lower cellular bioavailability. Similar to oxo-AsHC 332, thioxo-AsHC 348 did not substantially induce oxidative stress nor DNA damage. Moreover, in contrast to oxo-AsHC 332 mitochondria seem not to be a primary subcellular toxicity target for thioxo-AsHC 348. This study indicates that thioxo-AsHC 348 is at least as toxic as its parent compound oxo-AsHC 332 but very likely acts via a different mode of toxic action, which still needs to be identified.

Subcellular Localization of Copper-Cellular Bioimaging with Focus on Neurological Disorders.

As an essential trace element, copper plays a pivotal role in physiological body functions. In fact, dysregulated copper homeostasis has been clearly linked to neurological disorders including Wilson and Alzheimer's disease. Such neurodegenerative diseases are associated with progressive loss of neurons and thus impaired brain functions. However, the underlying mechanisms are not fully understood. Characterization of the element species and their subcellular localization is of great importance to uncover cellular mechanisms. Recent research activities focus on the question of how copper contributes to the pathological findings. Cellular bioimaging of copper is an essential key to accomplish this objective. Besides information on the spatial distribution and chemical properties of copper, other essential trace elements can be localized in parallel. Highly sensitive and high spatial resolution techniques such as LA-ICP-MS, TEM-EDS, S-XRF and NanoSIMS are required for elemental mapping on subcellular level. This review summarizes state-of-the-art techniques in the field of bioimaging. Their strengths and limitations will be discussed with particular focus on potential applications for the elucidation of copper-related diseases. Based on such investigations, further information on cellular processes and mechanisms can be derived under physiological and pathological conditions. Bioimaging studies might enable the clarification of the role of copper in the context of neurodegenerative diseases and provide an important basis to develop therapeutic strategies for reduction or even prevention of copper-related disorders and their pathological consequences.

Assessing neurodevelopmental effects of arsenolipids in pre-differentiated human neurons.

SCOPE: In the general population exposure to arsenic occurs mainly via diet. Highest arsenic concentrations are found in seafood, where arsenic is present predominantly in its organic forms including arsenolipids. Since recent studies have provided evidence that arsenolipids could reach the brain of an organism and exert toxicity in fully differentiated human neurons, this work aims to assess the neurodevelopmental toxicity of arsenolipids. METHODS AND RESULTS: Neurodevelopmental effects of three arsenic-containing hydrocarbons (AsHC), two arsenic-containing fatty acids (AsFA), arsenite and dimethylarsinic acid (DMAV ) were characterized in pre-differentiated human neurons. AsHCs and arsenite caused substantial cytotoxicity in a similar, low concentration range, whereas AsFAs and DMAV were less toxic. AsHCs were highly accessible for cells and exerted pronounced neurodevelopmental effects, with neurite outgrowth and the mitochondrial membrane potential being sensitive endpoints; arsenite did not substantially decrease those two endpoints. In fully differentiated neurons, arsenite and AsHCs caused neurite toxicity. CONCLUSION: These results indicate for a neurodevelopmental potential of AsHCs. Taken into account the possibility that AsHCs might easily reach the developing brain when exposed during early life, neurotoxicity and neurodevelopmental toxicity cannot be excluded. Further studies are needed in order to progress the urgently needed risk assessment.

Toxicity of two classes of arsenolipids and their water-soluble metabolites in human differentiated neurons.

Arsenolipids are lipid-soluble organoarsenic compounds, mainly occurring in marine organisms, with arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) representing two major subgroups. Recently, toxicity studies of several arsenolipids showed a high cytotoxic potential of those arsenolipids in human liver and bladder cells. Furthermore, feeding studies with Drosophila melanogaster indicated an accumulation of arsenolipids in the fruit fly's brain. In this study, the neurotoxic potential of three AsHCs, two AsFAs and three metabolites (dimethylarsinic acid, thio/oxo-dimethylarsenopropanoic acid) was investigated in comparison to the toxic reference arsenite (iAsIII) in fully differentiated human brain cells (LUHMES cells). Thereby, in the case of AsHCs both the cell number and cell viability were reduced in a low micromolar concentration range comparable to iAsIII, while AsFAs and the applied metabolites were less toxic. Mechanistic studies revealed that AsHCs reduced the mitochondrial membrane potential, whereas neither iAsIII nor AsFAs had an impact. Furthermore, neurotoxic mechanisms were investigated by examining the neuronal network. Here, AsHCs massively disturbed the neuronal network and induced apoptotic effects, while iAsIII and AsFAs showed comparatively lesser effects. Taking into account the substantial in vitro neurotoxic potential of the AsHCs and the fact that they could transfer across the physiological barriers of the brain, a neurotoxic potential in vivo for the AsHCs cannot be excluded and needs to be urgently characterized.

Gender and Programming.

The current study examines U.S. prison programming availability and participation by gender on a national level. The authors build upon previous literature by using national-level data, something that has been done in very limited cases previously. The main concern of this study is gender and its effects on programming availability and participation. The U.S. corrections field has undergone major changes in regard to population trends, fiscal constraints, policies, and research over the last few decades without a large-scale examination of the effects of these changes on programming across the United States. In this study, multiple types of programming areas were examined and results indicated that often female prisons (i.e., prisons housing only females) were more likely to offer programs (e.g., mental health options) and women were more likely to participate in many programming options compared with male prisons and men, respectively. We discuss the possible reasons for this and implications for future research.

Evaluating long-term cellular effects of the arsenic species thio-DMA(V): qPCR-based gene expression as screening tool.

Thio-dimethylarsinic acid (thio-DMA(V)) is a human urinary metabolite of the class 1 human carcinogen inorganic arsenic as well as of arsenosugars. Thio-DMA(V) exerts strong cellular toxicity, whereas its toxic modes of action are not fully understood. For the first time, this study characterises the impact of a long-term (21days) in vitro incubation of thio-DMA(V) on the expression of selected genes related to cell death, stress response, epigenetics and DNA repair. The observed upregulation of DNMT1 might be a cellular compensation to counterregulate the in a very recent study observed massive global DNA hypomethylation after chronic thio-DMA(V) incubation. Moreover, our data suggest that chronic exposure towards subcytotoxic, pico- to nanomolar concentrations of thio-DMA(V) causes a stress response in human urothelial cells. The upregulation of genes encoding for proteins of DNA repair (Apex1, Lig1, XRCC1, DDB2, XPG, ATR) as well as damage response (GADD45A, GADD45G, Trp53) indicate a potential genotoxic risk emanating from thio-DMA(V) after long-term incubation.

Cytotoxicity and genotoxicity of nano - and microparticulate copper oxide: role of solubility and intracellular bioavailability.

BACKGROUND: Nano- or microscale copper oxide particles (CuO NP, CuO MP) are increasingly applied as catalysts or antimicrobial additives. This increases the risk of adverse health effects, since copper ions are cytotoxic under overload conditions. METHODS: The extra- and intracellular bioavailability of CuO NP and CuO MP were explored. In addition, different endpoints related to cytotoxicity as well as direct and indirect genotoxicity of the copper oxides and copper chloride (CuCl2) were compared. RESULTS: Comprehensively characterized CuO NP and CuO MP were analysed regarding their copper ion release in model fluids. In all media investigated, CuO NP released far more copper ions than CuO MP, with most pronounced dissolution in artificial lysosomal fluid. CuO NP and CuCl2 caused a pronounced and dose dependent decrease of colony forming ability (CFA) in A549 and HeLa S3 cells, whereas CuO MP exerted no cytotoxicity at concentrations up to 50 µg/mL. Cell death induced by CuO NP was at least in part due to apoptosis, as determined by subdiploid DNA as well as via translocation of the apoptosis inducing factor (AIF) into the cell nucleus. Similarly, only CuO NP induced significant amounts of DNA strand breaks in HeLa S3 cells, whereas all three compounds elevated the level of H2O2-induced DNA strand breaks. Finally, all copper compounds diminished the H2O2-induced poly(ADP-ribosyl)ation, catalysed predominantly by poly(ADP-ribose)polymerase-1 (PARP-1); here, again, CuO NP exerted the strongest effect. Copper derived from CuO NP, CuO MP and CuCl2 accumulated in the soluble cytoplasmic and nuclear fractions of A549 cells, yielding similar concentrations in the cytoplasm but highest concentrations in the nucleus in case of CuO NP. CONCLUSIONS: The results support the high cytotoxicity of CuO NP and CuCl2 and the missing cytotoxicity of CuO MP under the conditions applied. For these differences in cytotoxicity, extracellular copper ion levels due to dissolution of particles as well as differences in physicochemical properties of the particles like surface area may be of major relevance. Regarding direct and indirect genotoxicity, especially the high copper content in the cell nucleus derived after cell treatment with CuO NP appears to be decisive.

Can resveratrol in wine protect against the carcinogenicity of ethanol? A probabilistic dose-response assessment.

Resveratrol, which may occur in wine, was suggested to act as a chemopreventive agent against the carcinogenic effects of ethanol. The assumption was based on data from experimental animals, which have shown that resveratrol above certain thresholds may reduce the incidence of tumours in several of the alcohol-related cancer sites (colon, liver and female breast). Using a probabilistic Monte Carlo type methodology, we estimated daily intake based on chemical analysis of resveratrol (n = 672) and ethanol (n = 867). Benchmark dose (BMD)-response modelling was conducted for resveratrol based on eight animal experiments, whereas BMD data for ethanol were taken from the literature. The margin of exposure (MOE) was calculated for both substances as an indicator if the intake may reach effective dosages. For intake of one 100-mL glass of wine, the average MOE was found to be 4.1 for ethanol and 459,937 for resveratrol. In the best-case scenario for resveratrol (e.g., very high contents and assuming a low effective dosage), the minimum MOE would be 111, which means that 111 glasses of wine need to be consumed daily to reach the BMD. The MOE ratio between resveratrol and ethanol is 166,128 on average, meaning that per glass of wine, ethanol is more than 100,000 times more potent than resveratrol. As resveratrol intake may not optimally reach the effective dosage, our study excludes a preventive effect of this substance on alcohol-related cancer. Commercial information about cancer-preventive or -protective effects of resveratrol in wine is misleading and must be prohibited.