Keratin 17 as a Marker of UVB-Induced Stress in Human Epidermis and Modulation by Vitis vinifera Extract.

Human epidermis responds to ultraviolet (UV)B-induced damage by tolerating it, restoring it, or undergoing programmed cell death when the damage is massive. Recently, compounds rich in polyphenols, such as Vitis vinifera L. leaf extract (VVLe), have attracted a lot of interest for skin protection. We investigated the effect of VVLe pre-treatment (1 h) in a 2D model of HaCaT cells and in 3D organotypic cultures of normal human skin exposed to a single UVB dose to study the immediate specific events 1 h and the response orchestrated in the epidermal layer 24 h after irradiation, respectively. In both models, transmission electron microscopy analysis was carried out. The expression of the inducible keratin K17, the activation of both pSTAT3 and Nuclear Factor (NF)-κB signalling pathways, and the epidermal distribution of Toll-Like Receptor (TLR) 4 were assessed by immunofluorescence in the 2D and 3D model. In 3D organotypic cultures, thanks to the preservation of a multi-layered structure, the epidermal distribution of the differentiation biomarkers K10 and K14 as well as of K16 was analysed by immunofluorescence, while the release of interleukin (IL)-8 was evaluated by ELISA. In skin bioptic fragments, cytotoxicity and genotoxicity were investigated by LDH assay and Alkaline Comet assay, respectively, and then compared to cell proliferation. The epidermal distribution of the histone γ-H2AX, indicating the fragmented DNA, was analysed by immunofluorescence. In both experimental models, VVLe tuned UVB-induced K17 expression to a different extent in HaCaT cells and in the skin. In HaCaT cells, pSTAT3 activation was induced by UVB and reverted by VVLe pre-treatment. TLR4 expression was triggered by UVB in both models, but VVLe pre-treatment abolished this event only in HaCaT cells. NF-κB immunostaining increased both in the nucleus and in the cytoplasm only in HaCaT cells after UVB irradiation. In all irradiated skin samples, VVLe pre-treatment was not able to revert the inhibition of epidermal proliferation, K16 expression, and IL-8 secretion. The effectiveness of VVLe in contrasting the irradiation-induced genotoxicity still remains unclear. In conclusion, our study clearly shows that K17 is a robust marker induced in keratinocytes upon UVB stimulation and that this event can be reverted by a pre-treatment with VVLe. On the whole, these observations represent a novelty in the scenario of the complex relationships between the effects exerted by UVB rays on human skin and significantly improve the knowledge regarding the modulation of the early epidermal response induced by a single exposure to UVB in the presence of VVLe.

Mutant p53 reactivator SLMP53-2 hinders ultraviolet B radiation-induced skin carcinogenesis.

The growing incidence of skin cancer (SC) has prompted the search for additional preventive strategies to counteract this global health concern. Mutant p53 (mutp53), particularly with ultraviolet radiation (UVR) signature, has emerged as a promising target for SC prevention based on its key role in skin carcinogenesis. Herein, the preventive activity of our previously disclosed mutp53 reactivator SLMP53-2 against UVR-induced SC was investigated. The pre-treatment of keratinocyte HaCaT cells with SLMP53-2, before UVB exposure, depleted mutp53 protein levels with restoration of wild-type-like p53 DNA-binding ability and subsequent transcriptional activity. SLMP53-2 increased cell survival by promoting G1-phase cell cycle arrest, while reducing UVB-induced apoptosis through inhibition of c-Jun N-terminal kinase (JNK) activity. SLMP53-2 also protected cells from reactive oxygen species and oxidative damage induced by UVB. Moreover, it enhanced DNA repair through upregulation of nucleotide excision repair pathway and depletion of UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPD) levels. SLMP53-2 further suppressed UVB-induced inflammation by inhibiting the nuclear translocation and DNA-binding ability of NF-κB, and promoted the expression of key players involved in keratinocytes differentiation. Consistently, the topical application of SLMP53-2 in mice skin, prior to UVB irradiation, reduced cell death and DNA damage. It also decreased the expression of inflammatory-related proteins and promoted cell differentiation, in UVB-exposed mice skin. Notably, SLMP53-2 did not show signs of skin toxicity for cumulative topical use. Overall, these results support a promising protective activity of SLMP53-2 against UVB-induced SC.

Conservation of mechanisms regulating emotional-like responses on spontaneous nicotine withdrawal in zebrafish and mammals.

BACKGROUND: Nicotine withdrawal syndrome is a major clinical problem. Animal models with sufficient predictive validity to support translation of pre-clinical findings to clinical research are lacking. AIMS: We evaluated the behavioural and neurochemical alterations in zebrafish induced by short- and long-term nicotine withdrawal. METHODS: Zebrafish were exposed to 1 mg/L nicotine for 2 weeks. Dependence was determined using behavioural analysis following mecamylamine-induced withdrawal, and brain nicotinic receptor binding studies. Separate groups of nicotine-exposed and control fish were assessed for anxiety-like behaviours, anhedonia and memory deficits following 2-60 days spontaneous withdrawal. Gene expression analysis using whole brain samples from nicotine-treated and control fish was performed at 7 and 60 days after the last drug exposure. Tyrosine hydroxylase (TH) immunoreactivity in pretectum was also analysed. RESULTS: Mecamylamine-precipitated withdrawal nicotine-exposed fish showed increased anxiety-like behaviour as evidenced by increased freezing and decreased exploration. 3H-Epibatidine labeled heteromeric nicotinic acethylcholine receptors (nAChR) significantly increased after 2 weeks of nicotine exposure while 125I-αBungarotoxin labeled homomeric nAChR remained unchanged. Spontaneous nicotine withdrawal elicited anxiety-like behaviour (increased bottom dwelling), reduced motivation in terms of no preference for the enriched side in a place preference test starting from Day 7 after withdrawal and a progressive decrease of memory attention (lowering discrimination index). Behavioural differences were associated with brain gene expression changes: nicotine withdrawn animals showed decreased expression of chrna 4 and chrna7 after 60 days, and of htr2a from 7 to 60 days.The expression of c-Fos was significantly increased at 7 days. Finally, Tyrosine hydroxylase (TH) immunoreactivity increased in dorsal parvocellular pretectal nucleus, but not in periventricular nucleus of posterior tuberculum nor in optic tectum, at 60 days after withdrawal. CONCLUSIONS: Our findings show that nicotine withdrawal induced anxiety-like behaviour, cognitive alterations, gene expression changes and increase in pretectal TH expression, similar to those observed in humans and rodent models.

Effects of Vitis vinifera L. leaves extract on UV radiation damage in human keratinocytes (HaCaT).

Vitis vinifera L. water extract from red grapevine leaves contains high levels of polyphenols in quantities similar to those found in red grape and grape seeds. Phenolic compounds are the largest group of natural antioxidants with also an anti-inflammatory activity, widely demonstrated both in vitro and in vivo. Interestingly, their antioxidant effect relies not only on the direct radical scavenging activity but also on their ability in modulating cellular signalling transduction pathways. UV radiation exerts multiple effects on skin cells inducing apoptosis, senescence and carcinogenesis. The aim of this study was to investigate the antioxidant and the DNA protective potentials of Vitis vinifera L. water extract against UV-A and UV-B radiation in HaCaT cells, a human keratinocytes cell line. Comet and É£H2AX assays were used to assess DNA damage in UV irradiated cells pre-treated or not with the extract (100 µg/mL). For UV-B, DNA damage resulted significantly increased at 40 mJ/cm2 dose determining cell cycle arrest and apoptosis. For UV-A, DNA damage was significant at 10 J/cm2 while cell cycle arrest and apoptosis were evident only at 25 J/cm2. The extract (1h of pre-treatment) highlights the antioxidant and scavenger activity on the UV-A, while the maintenance of the apoptosis with both UV-A and UV-B must be interpreted as an anti-mutagenic effect.

Vitis vinifera L. Leaf Extract Inhibits In Vitro Mediators of Inflammation and Oxidative Stress Involved in Inflammatory-Based Skin Diseases.

Psoriasis is a chronic cutaneous condition characterized by the release of pro-inflammatory mediators and oxidative stress. The reduction of these factors is currently the most effective strategy to inhibit the symptoms of pathology. Antioxidants from natural sources are increasingly used to improve skin conditions. Dried red leaves from grapevine (Vitis vinifera L., cv Teinturiers) showed anti-inflammatory and anti-bacterial activities, but their possible effects on keratinocytes have not been previously investigated. In this study we tested the ability of a water extract from grapevine leaves (VVWE) to inhibit inflammatory conditions in human keratinocytes (HaCaT cells), challenged with proinflammatory (tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS)) or prooxidant (ultraviolet B radiation (UVB) or H2O2) mediators. VVWE inhibited interleukin-8 (IL-8) secretion induced by proinflammatory stimuli, acting on the IL-8 promoter activity, but the effect was lower when prooxidant mediators were used. The effect was partly explained by the reduction of nuclear factor-κB (NF-κB)-driven transcription and nuclear translocation. Furthermore, vascular endothelial growth factor (VEGF) secretion, a regulator of angiogenesis, was inhibited by VVWE, but not matrix metalloproteinase-9 (MMP-9), a protease involved in matrix remodeling. VVWE, assayed on Franz diffusion cell system, showed a marked reduction of High Performance Liquid Chromatography (HPLC)-identified compounds. Pure molecules individually failed to reduce TNF-α-induced IL-8 release, suggesting synergistic effects or the presence of other bioactive compounds still unknown.

Effect of plant extracts on the genotoxicity of 1'-hydroxy alkenylbenzenes.

Food-borne alkenylbenzenes are potential risks for human health because they are known to induce liver tumors in rodent bioassays at high dose levels. This carcinogenicity is ascribed to the conversion of their 1'-hydroxymetabolites to the ultimate DNA reactive and carcinogenic 1'-sulfoxymetabolites. The aim of this study was to investigate the in vitro genotoxicity of some botanical extracts used as Plant Food Supplements (PFS) and to compare it with the individual substances, estragole, safrole and their 1'-hydroxy-derivative activity. The genotoxicity of the PFSs was evaluated in HepG2 cell line by comet and micronucleus assays. Unlike the 1'-hydroxy derivatives, PFS extracts and parent alkenylbenzenes did not show genotoxicity at any of the tested concentrations. The sulfotransferase inhibitor pentachlorophenol (PCP) reduced the 1'-hydroxy compound-induced response in the comet and micronucleus assays, thus confirming that the formation of sulfoxy-metabolites is essential for inducing genotoxic effects. When the cells were treated with hydroxylated alkenylbenzenes in the presence of PFSs, a reduction in genotoxic activity of synthetic compounds was observed.

DNA damage in human skin fibroblasts from patients with dermatitis herpetiformis.

Dermatitis herpetiformis (DH) and celiac disease (CD) are considered to be autoimmune diseases that share a specific trigger (gluten) and a common genetic background (HLA-DQ2/DQ8). However, the pathogenesis of DH is not yet fully understood and no data are available regarding a possible role of fibroblasts in this disease. The aim of this study was to assess baseline DNA damage in fibroblasts in DH-diagnosed patients vs. fibroblasts of controls without DH or CD. Primary fibroblast cultures were derived from dermal biopsies from DH patients and controls (without DH or CD). In vitro genotoxic damage was investigated using the comet assay and ɣH2AX test after different treatments (with 33mer peptide and digested gliadin [DG]) in order to investigate a correlation between oxidative stress (evaluated by reactive oxygen species formation) and glutathione content. Our results demonstrate a difference in baseline DNA damage between cutaneous fibroblasts of controls and DH patients, moreover, DNA damage significantly increased after exposure to gluten (DG and 33mer peptide) in fibroblasts from DH patients. DNA damage in fibroblasts from patients under dapsone treatment was similar to that of the control group. Our data indicate that oxidative stress and DNA damage may be characteristics of fibroblasts from DH patients who are not treated with dapsone, particularly after exposure to gliadin peptides.

Ultrafine particles (UFPs) from domestic wood stoves: genotoxicity in human lung carcinoma A549 cells.

In this paper, results on the potential toxicity of ultrafine particles (UFPs d<100nm) emitted by the combustion of logwood and pellet (hardwood and softwood) are reported. The data were collected during the TOBICUP (TOxicity of BIomass COmbustion generated Ultrafine Particles) project, carried out by a team composed of interdisciplinary research groups. The genotoxic evaluation was performed on A549 cells (human lung carcinomacells) using UFPs whose chemical composition was assessed by a suite of analytical techniques. Comet assay and γ-H2AX evaluation show a significant DNA damage after 24h treatment. The interpretation of the results is based on the correlation among toxicological results, chemical-physical properties of UFPs, and the type and efficiency conditions in residential pellet or logwood stoves.

Effects of UV Rays and Thymol/Thymus vulgaris L. Extract in an ex vivo Human Skin Model: Morphological and Genotoxicological Assessment.

Ultraviolet (UV) radiation is the major environmental factor affecting functions of the skin. Compounds rich in polyphenols, such as Thymus vulgaris leaf extract and thymol, have been proposed for the prevention of UV-induced skin damage. We compared the acute effects induced by UVA and UVB rays on epidermal morphology and proliferation, cytotoxicity, and genotoxicity. Normal human skin explants were obtained from young healthy women (n = 7) after informed consent and cultured at the air-liquid interface overnight. After 24 h, the samples were divided in 2 groups: the former exposed to UVA (16 or 24 J/cm2) and the latter irradiated with UVB (0.24 or 0.72 J/cm2). One hour after the end of irradiation, supernatants were collected for evaluation of the lactate dehydrogenase activity. Twenty-four hours after UVB exposure, biopsies were processed for light and transmission electron microscopy analysis, proliferation, cytotoxicity, and genotoxicity. UVB and UVA rays induced early inhibition of cell proliferation and DNA damage compared to controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA ones. For this reason, we evaluated the effect of either T. vulgaris L. extract (1.82 µg/ml) or thymol (1 µg/ml) on all samples treated for 1 h before UVB irradiation. While Thymus had a protective action for all of the endpoints evaluated, the action of the extract was less pronounced on epidermal proliferation and morphological features. The results presented in this study could be the basis for investigating the mechanism of thymol and T. vulgaris L. extract against the damage induced by UV radiation.

Thymol and Thymus Vulgaris L. activity against UVA- and UVB-induced damage in NCTC 2544 cell line.

Many authors focused on the research of natural compounds in order to protect skin from indirect (UVA) and direct (UVB) ultraviolet radiation side effects. The aim of this study to evaluate the protective effect of a dry extract from T. vulgaris L. and of its major synthetic compound thymol (about 60%), against oxidative and genotoxic UVA- and UVB damage. Experiments were reproduced in a low differentiated keratinocytes cell line (NCTC 2544) Cells were pretreated for 1h, in serum-free medium, with thymol (1µg/mL) or T. vulgaris L. (1.82µg/mL) then exposed to different UVA (8-24J/cm(2)) or UVB doses (0.016-0.72J/cm(2)). Immediately after the UV exposure the intracellular redox status was evaluated by ROS quantification and by LPO. Genotoxic aspects were evaluated 24h after the end of irradiations using the alkaline comet assay, the micronucleus formation assay and the immunostaining of phosphorylated H2AX histone protein (detected 1h after the end of UV exposure). Thymol and T. vulgaris L. extract inhibited ROS generation in UVA and UVB-irradiated cells. On the contrary, MDA formation was reduced only in UVA treated cells. Both agents decreased the DNA damage evaluated by the alkaline comet assay, but not in the micronucleus and H2AX tests probably because of the severity of damage (double strands) detected.

Protective effect of Vaccinium myrtillus extract against UVA- and UVB-induced damage in a human keratinocyte cell line (HaCaT cells).

Recently, the field of skin protection have shown a considerable interest in the use of botanicals. Vaccinium myrtillus contains several polyphenols and anthocyanins with multiple pharmacological properties. The purpose of our study was to examine whether a water-soluble V. myrtillus extract (dry matter 12.4%; total polyphenols 339.3mg/100 g fw; total anthocyanins 297.4 mg/100 g fw) was able to reduce UVA- and UVB-induced damage using a human keratinocyte cell line (HaCaT). HaCaT cells were pretreated for 1h with extract in a serum-free medium and then irradiated with UVA (8-40 J/cm(2)) and UVB (0.008-0.72 J/cm(2)) rays. All experiments were performed 24h after the end of irradiation, except for oxidative stress tests. The extract was able to reduce the UVB-induced cytotoxicity and genotoxicity (studied by comet and micronucleous assays) at lower doses. V. myrtillus extract reduced lipid peroxidation UVB-induced, but had no effect against the ROS UVB-produced. With UVA-induced damage V. myrtillus reduced genotoxicity as well as the unbalance of redox intracellular status. Moreover our extract reduced the UVA-induced apoptosis, but had no effect against the UVB one. V. myrtillus extract showed its free radical scavenging properties reducing oxidative stress and apoptotic markers, especially in UVA-irradiated cells.

Genotoxic effects of polychlorinated biphenyls (PCB 153, 138, 101, 118) in a fish cell line (RTG-2).

Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. The primary aim of this study was to investigate the genotoxic effects of some PCBs (PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) and 138 (2,2',3,4,4',5'-hexachloro-biphenyl), both non-dioxin-like compounds, and the pentachlorobiphenyls PCB118 (2,3',4,4',5-) and 101 (2,2',4',5,5'-), the former an ortho-substituted, low-affinity dioxin-like compound and the latter a non-coplanar congener classified as non-dioxin-like) in fish cells (RTG-2). These congeners are mostly present in surface waters and in edible aquatic organisms and the loss of DNA integrity in vitro serves as a sensitive biomarker of cytogenetic alterations and is considered as an initial step for the identification of genotoxic effects. The alkaline comet assay and the micronucleus test show clear genotoxic damage after short and longer exposure (2 and 24h) to maximum soluble, non-cytotoxic doses, evident sooner with PCBs 101 and 118. Oxidative stress situations involving ROS release, reduction in total GSH, lipid peroxidation and alteration to superoxide dismutase, seen after exposure with all the congeners, though with different kinetics, seem the most likely explanation for the genotoxic damage. This appears to be confirmed by the modified comet assay (pH 10) for detection of oxidized bases using endonuclease III. The increased generation of intracellular ROS might explain the apoptosis seen after treatment with the single PCBs and evaluated on the basis of the rise in 3-7 caspase activity. Therefore both the non-coplanar, non-dioxin-like PCBs (153, 138, 101) and the low-affinity dioxin-like compound PCB118 cause evident genotoxic damage, probably as a consequence of oxidative stress.

Protective effect of erdosteine metabolite I against hydrogen peroxide-induced oxidative DNA-damage in lung epithelial cells.

It has been shown that the mucolytic agent erdosteine (N-carboxymethylthio-acetyl-homocysteine thiolactone, CAS 84611-23-4) has anti-inflammatory and anti-oxidant properties, and an active metabolite I (MET I) containing pharmacologically active sulphydryl group has been found to have a free radical scavenging activity. The aim of this study was to assess the ability of erdosteine metabolite I to protect A549 human lung adenocarcinoma cell against hydrogen peroxide (H2O2)-mediated oxidative stress and oxidative DNA damage. When A549 cells were pre-treated with the active metabolite I (2.5-5-10 microg/ml) for 10-30 min and then exposed to H2O2 (1-4 mM) for two additional hours at 37 degrees C, 5% at CO2, the intracellular peroxide production, reflected by dichlorofluorescein (DCF) fluorescence, decreased in a concentration-dependent manner. Furthermore, using a comet assay as an indicator for oxidative DNA damage, it was found that the metabolite I prevented damage to cells exposed to shortterm H2O2 treatment. The data suggest that this compound is effective in preventing H2O2-induced oxidative stress and DNA damage in A549 cells. The underlying mechanisms involve the scavenging of intracellular reactive oxygen species (ROS).

Different effects of PCB101, PCB118, PCB138 and PCB153 alone or mixed in MCF-7 breast cancer cells.

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that can be a potential health hazard. In the present study we analyzed the potential estrogenic effect in MCF-7 cells of four biologically relevant PCB congeners, alone or in mixtures, present in dairy products, vegetable oil and fish: PCB101, PCB118, PCB138 and PCB153. The mixture of four PCB was tested at seven different concentrations. We investigated the ability of these PCBs, alone or mixed, to induce cell proliferation, and the level of estrogen-regulated protein pS2, in human MCF-7 breast cancer cells. PCB153 (35 microM) stimulated cell proliferation from 48 h up to day 6, PCB118 (40 microM) only at 48 h, but PCB101 (45 microM) and PCB138 (15 microM) applied to the cells for 6 days had no effect. In contrast, the various concentrations of mixtures significantly reduced cell proliferation at different times. No change in pS2 levels was seen after treatment with the PCBs alone or mixed. In exploring the mechanism of these events, we found that PCB153 induced mitogen-activated protein kinase (MAPK) ERK1/2 at 4, 8 and 12 h, while the antiproliferative effect seemed to be related to an apoptotic action beginning at 12 h and ending at 48 h. These findings indicate that these PCBs alone or mixed have no estrogenic effect in MCF-7 cells, although PCB153 induce an ERK1/2-mediated mitogenic effect. On the contrary the mixture of PCBs induces an antiproliferative effect, ascribable to an apoptotic action.

Toxicity evaluation of surface water treated with different disinfectants in HepG2 cells.

It is well known that water disinfection through chlorination causes the formation of a mixture of disinfection by-products (DBPs), many of which are genotoxic and carcinogenic. To demonstrate the formation of such compounds, a pilot water plant supplied with water from Lake Trasimeno was set up at the waterworks of Castiglione del Lago (PG, Italy). The disinfectants, continuously added to pre-filtered lake water flowing into three different basins, were sodium hypochlorite, chlorine dioxide and peracetic acid, an alternative disinfectant used until now for disinfecting waste waters, but not yet studied for a possible use in drinking water treatment. The aim of this study was to evaluate the formation during the disinfection processes of some toxic compounds that could explain the genotoxic effects of drinking waters. Differently treated waters were concentrated by solid-phase adsorption on silica C(18) columns and toxicity was assessed in a line of human hepatoma cells (HepG2), a metabolically competent cellular line very useful for human risk evaluation. The seasonal variability of the physical-chemical water characteristics (AOX, UV 254 nm, potential formation of THM, pH and temperature) made indispensable experimentation with water samples taken during the various seasons. Autumn waters cause greater toxicity compared to those of other seasons, in particular dilution of the concentrate at 0.5l equivalent of disinfected waters with chlorine dioxide and peracetic acid causes a 55% reduction in cellular vitality while the cellular vitality is over 80% with the all other water concentrates. Moreover it is very interesting underline that non-cytotoxic quantities of the autumnal water concentrates cause, after 2h treatment, a decrease in GSH and a statistically significant increase in oxygen radicals, while after prolonged treatment (24h) cause a GSH increase, without variations in the oxygen radical content. This phenomenon could be interpreted as the cellular adaptation response to an initial oxidative stress.

Estrogenic effect of procymidone through activation of MAPK in MCF-7 breast carcinoma cell line.

Procymidone modifies sexual differentiation in vitro and induces estrogenic activity in primary cultured rainbow trout hepatocytes, as shown by an increase in the contents of vitellogenin and heat shock proteins. Since this dicarboximide fungicide is found in human tissues, it was considered of interest to investigate its ability to induce endocrine damage in the MCF-7 human cell line. The mechanism of this estrogenic action was also evaluated. Procymidone 100 microM stimulated cell growth from day 3 up to day 12 and raised the level of pS2 on day 3. Although procymidone does not bind the estrogen receptor (ER), the antiestrogen ICI 182780 inhibited its effect on cell growth and pS2 content, suggesting that the ER is involved indirectly in these effects. In exploring the mechanism of ER indirect activation we found that the antibody against c-Neu receptor (9G6) did not modify procymidone's effects on cell growth and pS2 expression. Thus, procymidone does not bind the c-Neu membrane receptor, excluding this indirect ER activation pathway. We also found that procymidone induced mitogen-activated protein kinase (MAPK) at 15 and 30 min, and that PD 98059, a MAPK (Erk1/2) inhibitor, prevented procymidone's effects on cell growth and pS2, indicating that MAPK activation is responsible for procymidone ER activation. The production of reactive oxygen species (ROS) with these times and elimination of the phenomenon by alpha-tocopherol (alpha-T), a ROS scavenger, is proof that oxygen free-radical production is at the basis of the MAPK activation by procymidone.

Study of potential toxic effects on rainbow trout hepatocytes of surface water treated with chlorine or alternative disinfectants.

This study evaluates the effects of disinfection on the formation of toxic compounds in lake water treated with sodium hypochlorite, chlorine dioxide and peracetic acid (a disinfectant not previously used in drinking water processes). Chlorine reacts with the natural organic matter or contaminants in surface waters and produces a complex mixture of disinfection by-products (DBPs), some of which have been shown to be carcinogenic, mutagenic and/or teratogenic in animal studies. To define the potential toxicity on aquatic animals, disinfected drinking waters obtained from a pilot plant fed with water coming from Lake Trasimeno (Perugia) were collected, adsorbed by using silica C18 cartridges, and then eluted in sequence with ethylacetate, dichloromethane and methanol. The eluates were concentrated under vacuum and resuspended in dimethylsulfoxide (DMSO). Primary cultures of rainbow trout hepatocytes were exposed to three amounts of water concentrate (equivalent to 0.5, 0.25 and 0.125 l of disinfected water per ml of culture medium) for 24 h at 20 degrees C, after which their viability, glutathione content (GSH), free radical production (ROS) and cytochrome P4501A3 expression were determined. The disinfected water samples were collected during different seasons in order to evaluate a possible correlation between seasonal water variations and formation of toxic compounds. None of the water concentrates showed any cytotoxic effect or variations in GSH content, but significant increases in ROS production were detected in the autumn water concentrates from the treatments with sodium hypochlorite and chlorine dioxide. Cytochrome P4501A3 expression was not modified in the hepatocytes exposed to the water concentrates irrespective to the sampling season.

Genotoxicity and cytotoxicity assessment in lake drinking water produced in a treatment plant.

Chemical analyses and short-term mutagenicity bioassays have revealed the presence of genotoxic disinfection by-products in drinking water. In this study, the influence of the different steps of surface water treatment on drinking water mutagen content was evaluated. Four different samples were collected at a full-scale treatment plant: raw lake water (A), water after pre-disinfection with chlorine dioxide and coagulation (B), water after pre-disinfection, coagulation and granular activated carbon filtration (C) and tap water after post-disinfection with chlorine dioxide just before its distribution (D). Water samples, concentrated by solid phase adsorption on silica C18 columns, were tested in human leukocytes and HepG2 hepatoma cells using the comet assay and in HepG2 cells in the micronuclei test. A significant increase in DNA migration was observed in both cell types after 1 h treatment with filtered and tap water, and, to a lesser extent, chlorine dioxide pre-disinfected water. Similar findings were observed for the induction of "ghost" cells. Overloading of the carbon filter, with a consequent peak release, might explain the high genotoxicity found in water samples C and D. Cell toxicity and DNA damage increases were also detected in metabolically competent HepG2 cells treated with a lower concentration of tap water extract for a longer exposure time (24 h). None of the water extracts significantly increased micronuclei frequencies. Our monitoring approach appears to be able to detect contamination related to the different treatment stages before drinking water consumption and the results suggest the importance of improving the technologies for drinking water treatment to prevent human exposure to potential genotoxic compounds.

Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action.

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.

Early oxidative damage in primary cultured trout hepatocytes: a time course study.

The aim of this study was to evaluate the influence of the two-step hepatocyte isolation procedure on primary cultured trout (Oncorhynchus mykiss) hepatocytes over time. We characterised the possible changes of a variety of some cellular parameters within the first 24-48 h after seeding. We followed the time dependent changes of these parameters during subsequent culture times in order to see if the cells maintained a differentiated status. Scanning electron microscopy revealed bleb formation and 20% cell damage in freshly isolated hepatocytes. During subsequent culture times the bleb dimension appear to be reduced. Heat shock proteins 70 and 50 (HSP70, HSP50) were induced by hepatocyte isolation. During the first 4 h of culture, the hepatocytes showed a variation in mitochondrial activity, an increase in free radical species (ROS), and a decrease in both glutathione (GSH) content and catalase (CAT) activity; the generation of free radicals led to an increase in the formation of 8-hydroxydeoxyguanosine (8-OHdG) in the DNA. The cells showed detectable ethoxyresorufin-O-deethylase activity after 4 h of culture, which had rapidly increased by the 24th hour. After 24 h, mitochondrial and CAT activity, free radical production, and the content of GSH and 8-OHdG returned to their original levels. P450 activity was retained for at least 48 h after seeding. Our data show that trout hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells metabolically recover from this stress after a few hours: they are capable of repairing their damaged surfaces, recovering their antioxidant defences and retaining their ability to repair DNA. Our results also confirm that trout hepatocytes in a primary culture maintain their in vivo-like metabolic activities for 3-8 days.