Cadmium elicits alterations in mitochondrial morphology and functionality in C3H10T1/2Cl8 mouse embryonic fibroblasts.

BACKGROUND: Cadmium is a widespread carcinogen. We previously showed that the administration of low CdCl2 doses for 24 h to healthy C3H10T1/2Cl8 mouse embryonic fibroblast cell line at the beginning of Cell Transformation Assay (CTA), up regulates genes involved in metal scavenging and antioxidant defense, like metallothioneines, glutathione S-transferases and heat shock proteins. Still, although most cells thrive normally in the following weeks, malignancy is triggered by CdCl2 and leads to the appearance of foci of transformed cells at the end of the CTA. In this work we aim at elucidating the early metabolic deregulation induced by cadmium, underlying healthy cell transformation into malignant cells. METHODS: Respiratory metabolism was investigated through Seahorse Agilent assays, while oxidative stress level was assessed through fluorescent probes; DNA damage was evaluated by Comet assay, and mitochondrial morphology was analyzed in confocal microscopy. RESULTS: Results show that the initial response to CdCl2 involves mitochondria rearrangement into a perinuclear network. However, SOD1 and SOD2 activities are inhibited, leading to increased superoxide anion level, which in turn causes DNA strand breaks. From the metabolic point of view, cells increase their glycolytic flux, while all extra NADH produced is still efficiently reoxidized by mitochondria. CONCLUSIONS: Our results confirm previously shown response against cadmium toxicity; new data about glycolytic increase and mitochondrial rearrangements suggest pathways leading to cell transformation. GENERAL SIGNIFICANCE: In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand early events in cadmium-induced carcinogenesis.

Neuronal specific and non-specific responses to cadmium possibly involved in neurodegeneration: A toxicogenomics study in a human neuronal cell model.

Epidemiological data have linked cadmium exposure to neurotoxicity and to neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease), and to increased risk of developing ALS. Even though the brain is not a primary target organ, this metal can bypass the blood brain barrier, thus exerting its toxic effects. The coordination chemistry of cadmium is of strong biological relevance, as it resembles to zinc(II) and calcium(II), two ions crucial for neuronal signaling. A toxicogenomics approach applied to a neuronal human model (SH-SY5Y cells) exposed to cadmium (10 and 20 µM) allowed the identification of early deregulated genes and altered processes, and the discrimination between neuronal-specific and unspecific responses as possible triggers of neurodegeneration. Cadmium confirmed its recognized carcinogenicity even on neuronal cells by activating the p53 signaling pathway and genes involved in tumor initiation and cancer cell proliferation, and by down-regulating genes coding for tumor suppressors and for DNA repair enzymes. Two cadmium-induced stress responses were observed: the activation of different members of the heat shock family, as a mechanism to restore protein folding in response to proteotoxicity, and the activation of metallothioneins (MTs), involved in zinc and copper homeostasis, protection against metal toxicity and oxidative damage. Perturbed function of essential metals is suggested by the mineral absorption pathway, with MTs, HMOX1, ZnT-1, and Ferritin genes highly up-regulated. Cadmium interferes also with Ca2+ regulation as S100A2 is one of the top up-regulated genes, coding for a highly specialized family of regulatory Ca2+-binding proteins. Other neuronal-related functions altered in SH-SY5Y cells by cadmium are microtubules dynamics, microtubules motor-based proteins and neuroprotection by down-regulation of NEK3, KIF15, and GREM2 genes, respectively.

Cadmium-transformed cells in the in vitro cell transformation assay reveal different proliferative behaviours and activated pathways.

The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1µM CdCl2 for 24h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.

In vitro-to-in vivo correlation of the skin penetration, liver clearance and hepatotoxicity of caffeine.

This work illustrates the use of Physiologically-Based Toxicokinetic (PBTK) modelling for the healthy Caucasian population in in vitro-to-in vivo correlation of kinetic measures of caffeine skin penetration and liver clearance (based on literature experiments), as well as dose metrics of caffeine-induced measured HepaRG toxicity. We applied a simple correlation factor to quantify the in vitro and in vivo differences in the amount of caffeine permeated through the skin and concentration-time profiles of caffeine in the liver. We developed a multi-scale computational approach by linking the PBTK model with a Virtual Cell-Based Assay to relate an external oral and dermal dose with the measured in vitro HepaRG cell viability. The results revealed higher in vivo skin permeation profiles than those determined in vitro using identical exposure conditions. Liver clearance of caffeine derived from in vitro metabolism rates was found to be much slower than the optimised in vivo clearance with respect to caffeine plasma concentrations. Finally, HepaRG cell viability was shown to remain almost unchanged for external caffeine doses of 5-400 mg for both oral and dermal absorption routes. We modelled single exposure to caffeine only.

Cadmium Impairs p53 Activity in HepG2 Cells.

Cadmium and cadmium compounds are contaminants of the environment, food, and drinking water and are important constituents of cigarette smoke. Cd exposure has also been associated with airborne particulate CdO and with Cd-containing quantum dots in medical therapy. Adverse cadmium effects reported in the literature have stimulated during recent years an ongoing discussion to better elucidate cadmium outcomes at cell and molecular level. The present work is designed to gain an insight into the mechanism of p53 impairment at gene and protein level to understand Cd-induced resistance to apoptosis. We used a hepatoma cell line (HepG2) derived from liver, known to be metal responsive. At genotoxic cadmium concentrations no cell cycle arrest was observed. The p53 at gene and protein level was not regulated. Fluorescence images showed that p53 was correctly translocated into the nucleus but that the p21(Cip1/WAF-1), a downstream protein of p53 network involved in cell cycle regulation, was not activated at the highest cadmium concentrations used. The miRNAs analysis revealed an upregulation of mir-372, an miRNA able to affect p21(Cip1/WAF-1) expression and promote cell cycle progression and proliferation. The role of metallothioneins and possible conformational changes of p53 are discussed.

The acute effects of daily nicotine intake on heart rate--a toxicokinetic and toxicodynamic modelling study.

Joint physiologically-based toxicokinetic and toxicodynamic (PBTK/TD) modelling was applied to simulate concentration-time profiles of nicotine, a well-known stimulant, in the human body following single and repeated dosing. Both kinetic and dynamic models were first calibrated by using in vivo literature data for the Caucasian population. The models were then used to estimate the blood and liver concentrations of nicotine in terms of the Area Under Curve (AUC) and the peak concentration (Cmax) for selected exposure scenarios based on inhalation (cigarette smoking), oral intake (nicotine lozenges) and dermal absorption (nicotine patches). The model simulations indicated that whereas frequent cigarette smoking gives rise to high AUC and Cmax in blood, the use of nicotine-rich dermal patches leads to high AUC and Cmax in the liver. Venous blood concentrations were used to estimate one of the most common acute effects, mean heart rate, both at rest and during exercise. These estimations showed that cigarette smoking causes a high peak heart rate, whereas dermal absorption causes a high mean heart rate over 48h. This study illustrates the potential of using PBTK/TD modelling in the safety assessment of nicotine-containing products.

Application of physiologically-based toxicokinetic modelling in oral-to-dermal extrapolation of threshold doses of cosmetic ingredients.

The application of physiologically based toxicokinetic (PBTK) modelling in route-to-route (RtR) extrapolation of three cosmetic ingredients: coumarin, hydroquinone and caffeine is shown in this study. In particular, the oral no-observed-adverse-effect-level (NOAEL) doses of these chemicals are extrapolated to their corresponding dermal values by comparing the internal concentrations resulting from oral and dermal exposure scenarios. The PBTK model structure has been constructed to give a good simulation performance of biochemical processes within the human body. The model parameters are calibrated based on oral and dermal experimental data for the Caucasian population available in the literature. Particular attention is given to modelling the absorption stage (skin and gastrointestinal tract) in the form of several sub-compartments. This gives better model prediction results when compared to those of a PBTK model with a simpler structure of the absorption barrier. In addition, the role of quantitative structure-property relationships (QSPRs) in predicting skin penetration is evaluated for the three substances with a view to incorporating QSPR-predicted penetration parameters in the PBTK model when experimental values are lacking. Finally, PBTK modelling is used, first to extrapolate oral NOAEL doses derived from rat studies to humans, and then to simulate internal systemic/liver concentrations - Area Under Curve (AUC) and peak concentration - resulting from specified dermal and oral exposure conditions. Based on these simulations, AUC-based dermal thresholds for the three case study compounds are derived and compared with the experimentally obtained oral threshold (NOAEL) values.

Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions.

Regulation of metallothioneins and ZnT-1 transporter expression in human hepatoma cells HepG2 exposed to zinc and cadmium.

Essential and non-essential metals can affect vital cellular processes, when over-accumulated within the cells. For this reason, cells have evolved multiple protein sensors, transporters, and other type of proteins to regulate and control free metal homeostasis. Among these, metallothioneins (MT) and ZnT-1 transporter play a key role in the regulation of free Zn concentrations. Herewith, MT expression in Zn (170microM) and Cd (0.1 and 10microM) exposed HepG2 cells is analyzed and compared. In addition, the modulation and localization of the membrane transporter ZnT-1 has been investigated. MT-I and MT-II were up-regulated in response to both Zn and Cd exposure and, as expected, Cd represented the most potent inducer. Namely, 0.1microM Cd was able to up-regulate MT-I, and -II in a way comparable to 170microM Zn. This is in agreement with MT general function of metal-chelating protein, acting with higher tolerance to essential metals than to non-essential ones. ZnT-1 protein, a plasma membrane specific Zn transporter, was up-regulated as well by both Zn and Cd, although in the same way. Immunofluorescence technique provided evidence that high levels of ZnT-1 measured by biochemical techniques, are related to an increased localization of the transporter at the plasma membrane.

Image analysis and automatic classification of transformed foci.

Carcinogenesis is a multi-step process involving genetic alterations and non-genotoxic mechanisms. The in vitro cell transformation assay allows the monitoring of the neoplastic phenotype by foci formation in suitable cells (e.g. C3H10T1/2 mouse embryo fibroblasts) showing aberrant morphology of massive build-up, polar and multi-layered densely stained cells. The classification of transformed foci in C3H cells relies on light microscopy scoring by a trained human expert based on standard rules. This procedure is time-consuming and prone, in some cases, to subjectivity, thereby leading to possible over- or under-estimation of the carcinogenic potential of tested compounds. Herewith we describe the in vitro neoplastic transformation induced by B[a]P and CdCl2, and the development of a foci classifier based on image analysis and statistical classification. The image analysis system, which relies on 'spectrum enhancement', is quantitative and extracts descriptors of foci texture and structure. The statistical classification method is based on the Random Forest algorithm. We obtained a classifier trained by using expert's supervision with a 20% classification error. The proposed method could serve as a basis to automate the in vitro cell transformation assay.

Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure.

Cadmium is a widely distributed industrial and environmental pollutant. Principle target organs are soft tissues such as the liver, where cadmium accumulates with a biological half-life of approximately 20-30 years causing a variety of toxic responses. In HepG2, CdCl(2) exposure for short periods (from 1 to 24h) induces differential expression of stress proteins, including MT and hsp70. However, less is known about the stress response during a prolonged exposure to this metal. MTT assay showed a low cytotoxicity of CdCl(2) (0.1, 0.5, 1, 2, 5, 10microM), over a period of 72h. Cadmium uptake by ICP-AES technique and the corresponding expression of stress proteins (MT, hsp70) during the same prolonged time were also analysed. Results show that Cd was continuously and increasingly accumulated, at the highest of the concentrations tested. Metallothionein expression was up-regulated with a saturation curve at 48 as well as 72h after CdCl(2) exposure. High levels of MT probably confer an acquired tolerance to the stress and protection against cell injury as demonstrated by low cytotoxicity values. On the contrary, the unchanged pattern of hsp70 expression suggests that this protective mechanism, unlike other members of the family, is less involved during CdCl(2) prolonged exposure.

Quantitative kinetics of damage and recovery of cytoskeletal structure by means of image analysis.

The cytoskeleton is a network of proteins which structurally and dynamically organise the cytoplasm of living cells. Microtubules are among its constituents. Morphological alterations of microtubules are related to functional impairment. Therefore cytoskeletal morphology is a valuable indicator of cell injury and functionality. This paper focuses on the comparison between normal and altered cytoskeletal microtubules by means of image analysis and classification with the aim of replacing visual assessment. Morphology has been quantified by the extraction of some descriptors yielded by spatial differentiation, fractal analysis and Fourier analysis followed by non-linear filtering. The principal component analysis of these descriptors has led to image recognition and has been applied to hepatocytes and fibroblasts exposed to some xenobiotics. In the case of hepatocytes, images have been ranked according to the severity of cytoskeletal damage, a dose-response relation has been derived from the regression of the first principal component and the percentage of structural recovery after exposure has been estimated.

Cytotoxicity and induction of protective mechanisms in HepG2 cells exposed to cadmium.

Cadmium is a widespread industrial pollutant. The primary route of exposure occurs via contaminated drinking water or food supplies, and tobacco. Its chronic introduction and ingestion lead to bio-magnification in target organs, as the liver. The aim of this paper is to determine Cd cytotoxic concentrations in the human hepatoma cell line HepG2. Further aims are the study of the activation and involvement of protection mechanisms against Cd hepatotoxicity. Cd was accumulated within the cells, as measured by ICP-AES. Metallothioneins (MT-1 and -2), a family of metal-binding proteins, were induced in a dose-dependent way after treatment with concentrations below the IC(50) value (mean value 22 microM). The over-expression of MT by Zn pre-treatment was able to defend against Cd cytotoxicity. Heat shock protein 70 kDa (hsp70) was induced at high non-cytotoxic concentrations (5, 10 microM) probably as a consequence of proteotoxicity, but its over-expression by a sub-lethal heat shock was not able to protect the cells from Cd cytotoxic concentrations (20, 50, 100 microM).

Different induction of metallothioneins and Hsp70 and presence of the membrane transporter ZnT-1 in HepG2 cells exposed to copper and zinc.

Eukaryotic cells respond to stressful environmental stimuli, such as toxic concentrations of heavy metals, by rapidly synthesising defence proteins: the metallothioneins (MT) and the heat shock protein 70 (Hsp70). In this study we have analysed how the human hepatoblastoma cell line HepG2 responds to exposure to excess copper (30 microg/ml) and zinc (50 microg/ml) for long exposure times (48 and 72 h). Accumulation of the two metals, as measured by ICP-AES, was time-dependent reaching a plateau after 72 h. HepG2 cells responded by dramatically increasing levels of MT during stress, mostly during zinc exposure. A time lag in Hsp70 induction was observed as the levels of this protein increased only after removal of the stress from culture medium (recovery) for 24 h, thus suggesting that the two defence mechanisms are not coordinated in a metal-induced stress response. Moreover in HepG2 cells, immunochemical and fluorescence techniques showed the presence and the localisation of the zinc membrane exporter ZnT-1 as a further mechanism of defence/homeostasis against zinc toxicity.

Overexpression of HSP70 is induced by ionizing radiation in C3H 10T1/2 cells and protects from DNA damage.

Various kinds of stress such as heat, UV, gamma-rays and chemicals that cause DNA damage induce heat shock proteins (Hsps), and in particular Hsp70. The Hsps cytoprotective function is not fully understood, although these proteins act as molecular chaperones or modulators of intracellular levels of reactive oxygen species (ROS). Recently, Hsps have been proposed to play a significant role in DNA repair after UV or gamma-ray irradiation. Ionizing radiation targets DNA molecules either via direct interaction or via production of free radicals and ROS. When exposed to gamma-rays C3H 10T1/2 cells are radiosensitive, therefore we decided to use them to investigate Hsp induction after ionizing radiation and their protective role against DNA damage. Here we demonstrate the induction of Hsps by gamma-rays, and investigate the kinetics of expression after irradiation at different doses. We also show that Hsp70 overexpression acts as a radioprotective mechanism towards the first event of DNA damage and increases long term viability. A preliminary investigation on the cell cycle does not evidence a significant protective action of inducible Hsp70 on it.

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells.

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of 137CS gamma-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation.

Copper and zinc uptake and hsp70 expression in HepG2 cells.

The aim of this work is to study the accumulation in HepG2 cells of two essential metals with toxic potency and to analyse the induction of the heat shock protein 70 kDa (hsp70) consequent to metal exposure. Cu and Zn were the metals considered and were analysed both as single compounds and in combination in order to evidence synergic effects of the mixture. The use of HepG2 cells provided an in vitro system that retains morphological and metabolic properties and the expression of specific genes typical of liver parenchymal cells. Moreover, the hepatic cells represent a suitable model for their susceptibility to metal toxicity since liver, gastrointestinal tract and renal tubular cells are involved in the uptake, transport, detoxification and secretion of these compounds. The uptake of Cu and Zn followed a time-dependent accumulation when they were used separate. The combination of the two metals produced a higher accumulation of Zn. The stress protein hsp70 was expressed before the metals accumulated within the cells, as shown by the measures obtained with the ICP-AES technique. Moreover, the accumulation of hsp70 by a sublethal shock provided a protective mechanism against metal cytotoxicity.

Patent foramen ovale and its embolic implications.

This study investigates the usefulness of the echocardiographic characteristics of patent foramen ovale (PFO) in the stratification of stroke recurrence risk in patients with acute ischemic cerebral disease. Shunting at rest and a highly mobile fossa ovalis membrane are more frequently detected in stroke patients with PFO as the only identifiable cause of embolism. For PFO patients with both rest patency and membrane mobility > 6.5 mm, the risk of stroke/transient ischemic attack recurrence was 7.6% (95% CI, 0-18.0) at 12 months and 12.5% (95% CI, 0-26.1) at 24 months (p = 0.05). The association of both rest patency and high membrane mobility seems to identify those stroke patients with PFO at higher risk for further brain embolism.