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.

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.

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.

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.

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).

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.

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.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects the actin cytoskeleton and calcium level of Swiss 3T3 mouse fibroblasts.

Organization of the actin cytoskeleton, the cytosolic free-calcium concentrations and ATP levels were analyzed in 3T3 mouse fibroblasts treated with 0.75 or 1.5 mM MPTP. In the presence of the drug actin filaments were time- and dose-dependently disorganized, ATP level was unaffected and intracellular calcium increased within 5 s. The correlation between MPTP cytotoxicity and [Ca2+]i level emerging from these results, suggests that the primary effect of the molecule itself is on the plasma membrane's integrity for calcium ion regulation.

Prevalence of valvular regurgitation in structurally normal hearts: a colour-Doppler study.

AIM: To evaluate, by colour-Doppler echocardiography, the effects of ageing on the continence of the heart valves in patients with normal hearts. METHODS: We reviewed all the consecutive echocardiographic examinations performed in our laboratory from 1988 to 1994. From a total of 4592 records, 1654 (977 from females and 677 from males) were selected as normal-that is, not having excluded any kind of valvular, chamber or wall pathology of the heart. These records alone were considered in the study. RESULTS: One or more valve regurgitations were evident on 286 of the records (17.3%; 202 from females and 84 from males); regurgitation was always mild and was more frequent in females (20.7% compared with 12.4% of males; P < 0.001). Mitral regurgitation was the most frequent (49.3% of all regurgitant valves), followed by tricuspid, aortic and pulmonary. In age groups 0-17 years and > or = 60 years there was no sex difference in regurgitation. With ageing, there was an increasing trend in prevalence of regurgitation of the mitral, aortic and tricuspid valves in females (P < 0.001, P < 0.001 and P < 0.05, respectively), and of aortic and mitral valves in males (P < 0.001 for both). Multiple regurgitation (two or three valves) was not sex-dependent, but showed an increasing trend with ageing (from 13% to 27.5% of records showing regurgitation; positive trend, P < 0.005). CONCLUSIONS: Prevalence of mild valvular regurgitation is age-dependent, but is more common in women than in men aged 18-59 years; there is no sex-related difference in patients aged 60 years or more.

Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes.

Rat primary hepatocyte cultures have been used to study the effect of Benomyl alone or in combination with Pirimiphos-methyl. The results presented demonstrate that Benomyl alone is responsible for the microtubular disorganization in both a time- and dose-dependent manner, that the effect is reversible after the agent is removed, and that Benomyl is a potent glutathione-depleting agent. Pirimiphos-methyl, alone or combined with Benomyl had no effect on microtubule organization, but reinforced the decrease in glutathione.

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.

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.

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.

Molecular approaches to evaluate pollutants.

Many organisms are in use to test pollutants and their extensive variability clearly emerges from reviews since researchers in the world are involved in continuous effort to set up new assays and to improve those already in use. In the present paper we focus the attention on the mixed function oxidase system and the DNA adduct formation which are two biomarkers widely used and extensively studied in mammals and fish by different Authors. We compare their results with the ones we obtained in amphibians, which result to be a good model. Moreover we present some significative results obtained by the use of cultured cell lines to test the herbicide MCPA. The results obtained demonstrate that the amphibian Xenopus is a suitable indicator for induction of cytochrome P-450 by B[a]P as well as for production of DNA adducts. Cultured cells evidenced that cytoskeletal array and thiol proteins are molecular targets of the herbicide used, demonstrating that risk assessment can be properly analysed in in vitro systems.

Human-derived cell lines to study xenobiotic metabolism.

In vitro systems make for rapid identification of xenobiotic effects and can be used to study cellular and subcellular toxicity mechanisms. In this report the metabolic competence of two human-derived cell lines, a hepatic (Hep G2) and a pulmonary one (A549) was tested. In the two cell systems the capability to activate Benzo[a]Pyrene through the cytochrome P450 enzyme system and to form reactive metabolites was analysed. 3H-BaP and the scintillation counting analysis were used to show the differences of the metabolic activity in Hep G2 and A549. A similar time course of 3H-BaP uptake was observed in the cell systems. Nevertheless, in the two cell lines the distribution of radioactive metabolites seemed to reflect a specific tissue response to toxicity.

Cellular targets in response to dioxin exposure.

Dioxins are contaminants present in a variety of environmental media. The 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD) is biologically reactive at very low concentrations. This research is based on the use of innovative bioindicators able to evidence the exposure to TCDD by evaluating the biological effects on the embryonic development of the amphibious Xenopus laevis. TCDD exposure effects analyzed found that an Lc 50 value of 342 ng/l, TCDD concentrations up to 400 ng/l caused significative developmental delays. No significative teratogenic effects were found. X. laevis proved to be sensitive to the P-450 induction and alteration of glutathione levels at TCDD low doses (0.1-0.5 ng/l).

Cellular and Molecular Targets of Benzo[a]pyrene and Metal Toxicity in Xenopus laevis Embryos and in Hep G2 Cells.

This paper describes the use of two in vitro systems (stage 35 Xenopus laevis embryos and the human hepatoblastoma cell line, Hep G2) to study effects of some environmental contaminants (benzo[a]pyrene, copper and zinc), which are representative of chemicals with different cell targets and mechanisms of action. The ability to activate benzo[a[pyrene and to metabolise it with the cytochrome P4501A isozyme were demonstrated in both in vitro systems by assessing the formation of water-soluble and protein-bound benzo[a]pyrene metabolites and by immunochemical analysis. In X. laevis embryos, the formation of DNA adducts demonstrated the ability to produce benzo[a]pyrene reactive metabolites. Moreover, in Hep G2 cells, the cytoskeletal protein, tubulin, and the reduced form of glutathione proved to be the cellular targets of copper and zinc toxicity. In response to metal-induced stress in Hep G2 cells, there was a cytoplasmic reorganisation of heat shock protein, Hsp 70. In conclusion, the in vitro systems provide for a rapid evaluation of heterogeneous compounds such as benzo[a]pyrene and heavy metals that differ in toxic potency and mechanisms of action. They could also be used to study the mechanisms of toxic action and to identify specific cellular and molecular targets.

[Primary non-Hodgkin's lymphoma of the intestine associated with asymptomatic celiac disease in adults]. / Linfoma non-Hodgkin primitivo dell'intestino associato a malattia celiaca asintomatica dell'adulto.

Celiac disease (CD), a gluten-induced enteropathy, is characterized by typical intestinal involvement with classical clinic features in childhood and less frequent features in adult patients. Recognizing pauci- and asymptomatic patients is a critical point in the clinical management of CD because of the high mortality associated with the onset of complications. Among these, malignant diseases are the most severe, particularly squamous cell carcinoma and lymphoma, the latter accounting for 50% of all malignancies occurring in CD patients. The authors describe a 57 years old patient with CD and Enteropathy-Associated-T-Cell Lymphoma, who had no intestinal symptoms but only severe pruritus and hypereosinophilia.