Impact of mono-culture vs. Co-culture of keratinocytes and monocytes on cytokine responses induced by important skin sensitizers.

Sensitization to a contact allergen brings with it a lifelong risk to develop allergic contact dermatitis. Inflammation is an important part of the skin sensitizing mechanism, and understanding how different haptens stimulate the immune system, as well as the role played by different cell types present in skin, may be helpful for developing optimized in vitro models for risk assessment of new chemicals or mixtures. The aim of this study was to compare the cytokine profile following exposure of cells representing keratinocytes (HaCaT), monocytes (THP-1) and a co-culture of these cells to three clinically important skin sensitizers: cobalt (II) chloride (CoCl2), methylisothiazolinone (MI) and p-phenylenediamine (PPD). Secretion of ten pro-inflammatory cytokines was measured using multiplexing. The results showed that the cytokine response differed substantially between the three cell assays. CoCl2 caused an increase of IL-8 in HaCaT cells, while the induction of also IL-13 and IL-1ß was observed in THP-1 cells and co-cultures. MI induced six cytokines in HaCaT cells but only IL-1ß in the THP-1 cells and four cytokines in the co-culture. Interestingly, the IL-1ß response was massive in the co-culture. PPD caused release of IL-1ß in all three models as well as IL-8 in the co-culture. Control experiments with two non-sensitizers and irritants (lactic acid and sodium dodecyl sulfate) showed no effect on IL-8 or IL-1ß in the co-culture. Taken together, results from this exploratory analysis show unique cytokine profiles dependent on the type of hapten and cell model. Importantly, all three haptens triggered secretion of IL-1ß and IL-8 in a co-culture of HaCaT cells and THP-1 cells, representing the most robust test system.

Dry Generation of CeO2 Nanoparticles and Deposition onto a Co-Culture of A549 and THP-1 Cells in Air-Liquid Interface-Dosimetry Considerations and Comparison to Submerged Exposure.

Relevant in vitro assays that can simulate exposure to nanoparticles (NPs) via inhalation are urgently needed. Presently, the most common method employed is to expose lung cells under submerged conditions, but the cellular responses to NPs under such conditions might differ from those observed at the more physiological air-liquid interface (ALI). The aim of this study was to investigate the cytotoxic and inflammatory potential of CeO2 NPs (NM-212) in a co-culture of A549 lung epithelial cells and differentiated THP-1 cells in both ALI and submerged conditions. Cellular dose was examined quantitatively using inductively coupled plasma mass spectrometry (ICP-MS). The role of serum and LPS-priming for IL-1ß release was further tested in THP-1 cells in submerged exposure. An aerosol of CeO2 NPs was generated by using the PreciseInhale® system, and NPs were deposited on the co-culture using XposeALI®. No or minor cytotoxicity and no increased release of inflammatory cytokines (IL-1ß, IL-6, TNFα, MCP-1) were observed after exposure of the co-culture in ALI (max 5 µg/cm2) or submerged (max 22 µg/cm2) conditions. In contrast, CeO2 NPs cause clear IL-1ß release in monocultures of macrophage-like THP-1, independent of the presence of serum and LPS-priming. This study demonstrates a useful approach for comparing effects at various in-vitro conditions.

A systems toxicology approach to compare the heavy metal mixtures (Pb, As, MeHg) impact in neurodegenerative diseases.

Conventional toxicological risk assessment methods mainly working on single chemicals that fail to adequately address the simultaneous exposure and their potential toxicity in humans. We herein investigated the toxic heavy metals lead (Pb), arsenic (As), and methylmercury (MeHg) and their binary mixtures role in neurodegenerative diseases. To characterize the toxicity of metal mixtures at the molecular level, we established a non-animal omics-based organ relevant cell model system. The obtained experimental data was refined by using the statistical and downstream functional analysis. The protein expression information substantiates the previous findings of single metal (Pb, As, and MeHg) induced alterations to mitochondrial dysfunction, oxidative stress, mRNA splicing, and ubiquitin system dysfunction relation to neurodegenerative diseases. The functional downstream analysis of single and binary mixtures protein data is presented in a comparative manner. The heavy metals mixtures' outcome showed significant differences in the protein expression compared to single metals that indicate metal mixtures exposure is more hazardous than single metal exposure. These results suggest that more comprehensive strategies are needed to improve the mixtures risk assessment in the future.

Emerging pathways to neurodegeneration: Dissecting the critical molecular mechanisms in Alzheimer's disease, Parkinson's disease.

Neurodegenerative diseases are usually sporadic in nature and commonly influenced by a wide range of genetic, life style and environmental factors. A unifying feature of Alzheimer's disease (AD) and Parkinson's disease (PD) is the abnormal accumulation and processing of mutant or damaged intra and extracellular proteins; this leads to neuronal vulnerability and dysfunction in the brain. Through a detailed review of ubiquitin proteasome, mRNA splicing, mitochondrial dysfunction, and oxidative stress pathway interrelation on neurodegeneration can improve the understanding of the disease mechanism. The identified pathways common to AD and PD nominate promising new targets for further studies, and as well as biomarkers. These insights suggested would likely provide major stimuli for developing unified treatment approaches to combat neurodegeneration. More broadly, pathways can serve as vehicles for integrating findings from diverse studies of neurodegeneration. The evidence examined in this review provides a brief overview of the current literature on significant pathways in promoting in AD, PD. Additionally, these insights suggest that biomarkers and treatment strategies may require simultaneous targeting of multiple components.

Differential protein expression of hippocampal cells associated with heavy metals (Pb, As, and MeHg) neurotoxicity: Deepening into the molecular mechanism of neurodegenerative diseases.

Chronic exposure to heavy metals such as Pb, As, and MeHg can be associated with an increased risk of developing neurodegenerative diseases. Our in vitro bioassays results showed the potency of heavy metals in the order of Pb < As < MeHg on hippocampal cells. The main objective of this study was combining in vitro label free proteomics and systems biology approach for elucidating patterns of biological response, discovering underlying mechanisms of Pb, As, and MeHg toxicity in hippocampal cells. The omics data was refined by using different filters and normalization and multilevel analysis tools were employed to explore the data visualization. The functional and pathway visualization was performed by using Gene ontology and PathVisio tools. Using these all integrated approaches, we identified significant proteins across treatments within the mitochondrial dysfunction, oxidative stress, ubiquitin proteome dysfunction, and mRNA splicing related to neurodegenerative diseases. The systems biology analysis revealed significant alterations in proteins implicated in Parkinson's disease (PD) and Alzheimer's disease (AD). The current proteomics analysis of three metals support the insight into the proteins involved in neurodegeneration and the altered proteins can be useful for metal-specific biomarkers of exposure and its adverse effects. SIGNIFICANCE: The proteomics techniques have been claimed to be more sensitive than the conventional toxicological assays, facilitating the measurement of responses to heavy metals (Pb, As, and MeHg) exposure before obvious harm has occurred demonstrating their predictive value. Also, proteomics allows for the comparison of responses between Pb, As, and MeHg metals, permitting the evaluation of potency differences hippocampal cells of the brain. Hereby, the molecular information provided by pathway and gene functional analysis can be used to develop a more thorough understanding of each metal mechanism at the protein level for different neurological adverse outcomes (e.g. Parkinson's disease, Alzheimer's diseases). Efforts are put into developing proteomics based toxicity testing methods using in vitro models for improving human risk assessment. Some of the key proteins identified can also potentially be used as biomarkers in epidemiologic studies. These heavy metal response patterns shed new light on the mechanisms of mRNA splicing, ubiquitin pathway role in neurodegeneration, and can be useful for the development of molecular biomarkers of heavy metals exposure.

Comparative In Vitro Toxicity Evaluation of Heavy Metals (Lead, Cadmium, Arsenic, and Methylmercury) on HT-22 Hippocampal Cell Line.

Heavy metals are considered some of the most toxic environmental pollutants. Exposure to heavy metals including lead (Pb), cadmium (Cd), arsenic (As), and methyl mercury (MeHg) has long been known to cause damage to human health. Many recent studies have supported the hippocampus as the major target for these four metals for inflicting cognitive dysfunction. In the present study, we proposed hippocampal relevant in vitro toxicity of Pb, Cd, As, and MeHg in HT-22 cell line. This study reports, initially, cytotoxic effects in acute, subchronic, chronic exposures. We further investigated the mechanistic potency of DNA damage and apoptosis damage with the observed cytotoxicity. The genotoxicity and apoptosis were measured by using the comet assay, annexin-V FTIC / propidium iodide (PI) assay, respectively. The results of cytotoxicity assay clearly demonstrated significant concentration and time-dependent effects on HT-22 cell line. The genotoxic and apoptosis effects also concentration-dependent fashion with respect to their potency in the range of IC10-IC30, maximal level of damage observed in MeHg. In conclusion, the obtained result suggests concentration and potency-dependent response; the maximal level of toxicity was observed in MeHg. These novel findings support that Pb, Cd, As, and MeHg induce cytotoxic, genotoxic, and apoptotic effects on HT-22 cells in potency-dependent manner; MeHg> As> Cd> Pb. Therefore, the toxicity of Pb, Cd, As, and MeHg could be useful for knowing the common underlying molecular mechanism, and also for estimating the mixture impacts on HT-22 cell line.

An in vitro cytotoxic approach to assess the toxicity of heavy metals and their binary mixtures on hippocampal HT-22 cell line.

Humans are exposed to a cocktail of heavy metal toxicants in the environment. Though heavy metals are deleterious, there is a paucity of information on the toxicity of mixtures. In this study, four common neurotoxicity heavy metals lead (Pb) cadmium (Cd), arsenic (As), and methylmercury (MeHg) were exposed individually and as mixtures to HT-22 cell line for 8days. The study established that low dose exposures induced toxicity to the HT-22 cell line during 8days. The results indicates potency dependent response, the toxicity of single metals on the HT-22 cells; MeHg > As > Cd > Pb. The cytotoxicity data of single metals were used to determine the mixtures interaction profile by using the dose additivity and effect additivity method. Metal mixtures showed higher toxicities compared to individual metals. Synergistic, antagonistic or additive effects of the toxicity were observed in different mixtures in low dose exposure. The interactive responses of mixtures depend on the co-exposure metal and their respective concentration. We concluded that the combined effects should be considered in the risk assessment of heavy metal co-exposure and potency. In future, comprehensive mechanistic based investigations needed for understanding the real interactive mixtures effects at molecular level.

Heavy metals (Pb, Cd, As and MeHg) as risk factors for cognitive dysfunction: A general review of metal mixture mechanism in brain.

Human exposure to toxic heavy metals is a global challenge. Concurrent exposure of heavy metals, such as lead (Pb), cadmium (Cd), arsenic (As) and methylmercury (MeHg) are particularly important due to their long lasting effects on the brain. The exact toxicological mechanisms invoked by exposure to mixtures of the metals Pb, Cd, As and MeHg are still unclear, however they share many common pathways for causing cognitive dysfunction. The combination of metals may produce additive/synergetic effects due to their common binding affinity with NMDA receptor (Pb, As, MeHg), Na+ - K+ ATP-ase pump (Cd, MeHg), biological Ca+2 (Pb, Cd, MeHg), Glu neurotransmitter (Pb, MeHg), which can lead to imbalance between the pro-oxidant elements (ROS) and the antioxidants (reducing elements). In this process, ROS dominates the antioxidants factors such as GPx, GS, GSH, MT-III, Catalase, SOD, BDNF, and CERB, and finally leads to cognitive dysfunction. The present review illustrates an account of the current knowledge about the individual metal induced cognitive dysfunction mechanisms and analyse common Mode of Actions (MOAs) of quaternary metal mixture (Pb, Cd, As, MeHg). This review aims to help advancement in mixture toxicology and development of next generation predictive model (such as PBPK/PD) combining both kinetic and dynamic interactions of metals.