Blue light is phototoxic for B16F10 murine melanoma and bovine endothelial cell lines by direct cytocidal effect.

UNLABELLED: The large number of studies devoted to the effect of ultraviolet light on biological systems, contrasts with the lack of experimental data concerning the direct effects of visible light. It has been shown that blue light inhibited the growth of B16F10 melanoma cell lines and reduced the percentage of S phase cells. Yet these effects are poorly understood. MATERIALS AND METHODS: Two cell lines and irradiation with blue light were used. Cell mortality and a possible mechanism of action were investigated. RESULTS: Exposure of B16F10 melanoma and bovine endothelial cells to blue light (wavelength 450 nm, 10 J/cm(2) from a Waldman lamp) induced a rapid and large reduction in viability followed by the death of virtually all the irradiated cells within 24 h. These results led us to expose a patient with haemorrhagic cutaneous melanoma metastasis to blue light. Irradiation led to an immediate arrest of haemorrhage, an inhibition of tumour growth and extensive tumour necrosis 24h after irradiation. CONCLUSION: Exposure to blue light may offer new approaches to the treatment of superficial skin carcinomas in humans.

Damage and functional recovery of the Atlantic cod (Gadus morhua) inner ear hair cells following local injection of gentamicin.

This study addresses the ultrastructural and functional damage and subsequent recovery of the inner ear in the Atlantic cod following intrasaccular gentamicin injection. Inner ear damage was assessed using SEM and measurements of AEP following 250-Hz pure-tone stimuli. Data from gentamicin-treated fish were compared with control (no injection) and sham (injection of saline) fish. Control fish had normal response thresholds associated with well-developed hair cell bundles in their macula sacculi. Sham fish had higher response thresholds compared with control fish during the first week post-intervention, but response thresholds were subsequently normal. Treated fish displayed significant inner ear damage associated with an increased average AEP threshold on the third day following treatment. Thereafter, inner ear tissue displayed signs of progressive regeneration until it was comparable to controls from the 14th day. Response thresholds were similar to those of control fish from the 17th day following treatment. These observations suggest that the macula sacculi of Atlantic cod can regenerate towards a near-complete functional and ultrastructural recovery within 17-21 days following ototoxic gentamicin treatment.

Impact of chronic cadmium exposure at environmental dose on escape behaviour in sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae).

The effect of chronic exposure to a low concentration (0.5 microg l(-1)) of cadmium ions was investigated on escape behaviour of sea bass, Dicentrarchus labrax, using video analysis. Observations were also performed on the microanatomy of lateral system neuromasts. When fish were exposed for 4h per day over 8 days to the cadmium ions, most of both types of neuromasts observed remained intact. However, some of them presented damaged sensory maculae. Whereas before cadmium exposure, fish responded positively to nearly all the lateral system stimulations, after exposure they decreased by about 10% their positive responses to stimulations. From the 15th day after the beginning of cadmium exposure, neuromasts presented progressively less damage, cadmium accumulation in gills and scales decreased significantly and fish escape behaviour had recovered. This study presents a new concept in ecotoxicology: using behavioural change to reveal the effects of pollution levels, scarcely detectable by currently used techniques (physiological responses).

Expression of 5-lipoxygenase (5-LOX) in T lymphocytes.

5-Lipoxygenase (5-LOX) is the key enzyme responsible for the synthesis of the biologically active leukotrienes. Its presence has been reported in cells of the myeloid lineage and B lymphocytes but has not been formally defined in T lymphocytes. In this study, we provide evidence for 5-LOX expression on both transcriptional and translational levels in highly purified peripheral blood T cells as well as in human T lymphoblastoid cell lines (MOLT4 and Jurkat). Messenger RNA (mRNA) of 5-LOX was amplified by conventional reverse transcription-polymerase chain reaction (RT-PCR; MOLT4 and Jurkat cells) and by in situ RT-PCR (T lymphocytes). 5-LOX protein expression was confirmed by Western blot and immunofluorescence studies. 5-LOX was present primarily in the cytoplasm with some nuclear localization and was translocated to the nuclear periphery after culture in a mitosis-supporting medium. Fluorescence-activated cell sorter analysis of different T-lymphocyte populations, including CD4, CD8, CD45RO, CD45RA, T helper type 2, and T-cell receptor-alphabeta and -gammadelta expressing cells, did not identify a differential distribution of the enzyme. Purified peripheral blood T lymphocytes were incapable of synthesizing leukotrienes in the absence of exogenous arachidonic acid. Jurkat cells produced leukotriene C(4) and a small amount of leukotriene B(4) in response to CD3-CD28 cross-linking. This synthesis was abolished by two inhibitors of leukotriene synthesis, MK-886 and AA-861. The presence of 5-LOX in T lymphocytes but the absence of endogenous lipoxygenase metabolite production compared to Jurkat cells may constitute a fundamental difference between resting peripheral lymphocytes and leukaemic cells.

Impact of acute cadmium exposure on the trunk lateral line neuromasts and consequences on the "C-start" response behaviour of the sea bass (Dicentrarchus labrax L.; Teleostei, Moronidae).

Behavioural responses of sea bass Dicentrarchus labrax were investigated after exposure to cadmium ions in laboratory-controlled conditions. The aim of this study was to discover whether environmental exposure to cadmium ions inactivates fish lateral line system neuromasts, and to determine the behavioural consequences of such a sensory blockage. For this, fish escape behaviour in response to an artificial water jet was recorded using a 25-frames s(-1) analog video camera before and after cadmium exposure. Experimental set up was tested with fish whose lateral line system was artificially inactivated by antibiotics (gentamicin and streptomycin). Histological analyses with scanning electron microscopy showed antibiotic treatment destroyed lateral line system neuromasts. In addition, these fish did not respond to stimulations provoked by the water jet after antibiotic treatment. Fish escape behaviour was then recorded before and after cadmium exposure at two different concentrations. When fish were exposed to the first concentration of cadmium tested (0.5 microg l(-1), which represents the maximal cadmium concentration encountered in contaminated estuaries), no alteration in neuromast tissue was observed. In addition, before cadmium exposure, fish responded positively in 98.41 +/- 4.95% of lateral line system stimulations (escape behaviour in response to the water jet). After cadmium exposure, no behavioural modification could be detected: the fish responded positively in 95.16 +/- 9.79% of stimulations (chi(2) = 2.464, p = 0.116). In contrast, the high cadmium concentration used (5 microg l(-1), which represents 10 times the concentration occurring in highly polluted estuarine areas) involved severe neuromast tissue damage. Just after such cadmium exposure, fish showed only 41.67 +/- 35.36% of positive responses to their lateral line system stimulations, while they responded positively in 95.93 +/- 9.10% of stimulations under control conditions (chi(2) = 24.562, p < 0.0001). Their lateral line system neuromasts seemed to regenerate about 1 month after cadmium exposure. Associated with this regeneration, from the 21st day after cadmium exposure, their escape behaviour had recovered and was not significantly different from that recorded under control conditions (86.74 +/- 20.82%, chi(2) = 2.876, p = 0.090). This study shows that although 5 microg l(-1) cadmium is able to damage lateral line system neuromasts and causes fish behavioural alterations, fish exposed to 0.5 microg l(-1) cadmium displayed neither tissue neuromast nor behavioural modification.

A quantitative RT-PCR method to determine topoisomerase I mRNA levels in human tissue samples.

DNA topoisomerase I (Topo I) is involved in DNA replication, transcription, recombination and repair. Clinical interest has focused on Topo I as it is the molecular target of camptothecin (CPT), used in first and second lines of treatment for different cancer types. Furthermore, it is well demonstrated that the patients who best responded to CPT-based chemotherapy were generally those with the greatest tumoral Topo I expression and/or activity. We developed a sensitive, simple and reproducible method to measure Topo I mRNA expression in human cancer samples. Experiments were performed in two steps. First, we checked the accuracy of the reverse transcription-polymerase chain reaction (RT-PCR) method by testing intra- and interassay reproducibility of Topo I and G6PDH gene amplification in different cell types. We observed that crossing-points (Cps) were different, depending on the cell type, dilution or cDNA concentration, but that the intra- and interassay Cp standard deviation (SD) never exceeded 0.77% and 1.39% for Topo I amplification, or 1.63% and 2.9% for G6PDH amplification, respectively. Secondly, we used our method to measure Topo I mRNA levels in primary tumor samples obtained from 27 patients with advanced colorectal cancer and 10 patients with pharyngeal/laryngeal cancer. The accuracy of G6PDH as a housekeeping gene was tested by analyzing its correlation with the mRNA level of a second housekeeping gene, porphobilinogen deaminase (PBG-D) in the tumoral samples. We found that the normalized Topo I/G6PDH mRNA ratios were significantly correlated with that of Topo I/PBGD in colorectal tumors (r(2)=0.47, p=0.02) but not in pharyngeal/laryngeal tumors (r(2)=0.35, p=0.3). Neither ratio showed any significant association with clinicopathological parameters, such as gender, age, tumor size, or grade and lymph node status. We believe that RT-PCR is a reliable and highly reproducible technique. However, the choice of the reference gene is an important point and must be defined based on the samples studied.

Overexpression of human GPX1 modifies Bax to Bcl-2 apoptotic ratio in human endothelial cells.

As they scavenge reactive oxygen species, antioxidants were studied for their ability to interfere with apoptotic processes. However, their mechanisms of action remain unclear. In this study, we measured the expression of two Bcl-2 family members, Bax and Bcl-2, in a human endothelial like cell-line overexpressing the organic hydroperoxide-scavenging enzyme glutathione peroxidase (GPX1), in the absence of any apoptotic/oxidant stimulus. ECV304 were stably transfected with the GPX1 cDNA and used for quantification of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic) mRNA and protein levels, by quantitative RT-PCR and Western-blot. We found that, compared to control cells, cells from a clone showing a 13.2 fold increase in GPX1 activity had unchanged mRNA or protein Bcl-2 levels but expressed 42.6% and 46.1% less Bax mRNA and Bax protein respectively. Subsequently to Bax decrease, the Bax/Bcl-2 ratio, reflecting the apoptotic state of the cells, was also lower in cells overexpressing GPX1. Noticeably, the mRNA and the protein level of the cell-cycle protein p53, known to activate Bax expression, was unchanged. Our study showed that overexpressing an antioxidant gene such as GPX1 in endothelial cells is able to change the basal mRNA and protein Bax levels without affecting those of p53 and Bcl-2. This phenomenon could be useful to antiatherogenic therapies which use antioxidants with the aim of protecting the vascular wall against oxidative stress injury.

Quantitative aspects of the spatial distribution and morphological characteristics of the sea bass (Dicentrarchus labrax L.; Teleostei, Serranidae) trunk lateral line neuromasts.

The results presented herein report quantitative data relative to the distribution and morphological characteristics of both types of neuromasts encountered on the trunk lateral line of the sea bass (Dicentrarchus labrax, L.). These data were obtained from scanning electron micrographs. They indicate that, as expected, each modified scale of the sea bass possessed a single canal neuromast with long axis oriented parallel to the fish's long axis. In contrast to several fish species, two thirds of superficial neuromasts observed herein were oriented perpendicular to the fish's long axis. However, whatever the main orientation of superficial neuromasts, two thirds of their hair bundles were oriented parallel to the long axis of the animal with approximately half of them in the direction of the head. Similar ratios were observed for canal neuromasts whatever the area of the maculae: central or peripheral. For both types of neuromasts it was not possible to clearly distinguish a paired organization of hair bundles with opposing polarities. Superficial neuromasts on each trunk canal scale were located on either the dorsal or ventral side of the canal and appeared to be distributed along the trunk lateral line with a higher probability to be encountered closer to the operculum. The frequency of presence and the average number of superficial neuromasts per scale increased with fish size. We observed a size gradient for canal neuromasts between the operculum and caudal peduncle. This gradation was correlated with a reduction of the width of the central area of the canal segment. Canal neuromasts were always localized in the larger portions of the canal segments. Taken together, these results point out some specific features associated with the sea bass trunk lateral line. With the previous report, they establish the first full description of the trunk lateral line of sea bass and will be useful for upcoming experiments regarding the function of the two types of neuromasts.

PHGPx overexpression induces an increase in COX-2 activity in colon carcinoma cells.

BACKGROUND: Lipid peroxidation is a constant problem that eukaryotic cells have to face. Glutathione peroxidases (GPx) are among the most effective systems that protect cells from hydroperoxide toxicity. The objective of this study was to evaluate the relationship between GPx and cyclooxygenase 2 (COX-2), implicated in cancer pathogenesis, particularly in colon cancer cells. MATERIALS AND METHODS: Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4), which metabolizes peroxidized phospholipids, was cloned in an expression plasmid, transfected in HT29 cl.19A colon carcinoma cells and the effects of PHGPx overexpression were measured on arachidonic acid metabolism by COX-2. Metabolites were studied by HPLC and EIA; COX-2 mRNA levels were analysed rising semi-quantitative PCR. RESULTS: Prostaglandins (PGE2, PGF2alpha, 6 keto-PGF1alpha) and thromboxane (TXB2) production were increased. COX-2 mRNA levels increased in PHGPx overexpressing cells. CONCLUSION: Surprisingly, our data suggest that PHGPx overexpression noticeably increases COX-2 metabolism.

Spatial distribution and morphological characteristics of the trunk lateral line neuromasts of the sea bass (Dicentrarchus labrax, L.; Teleostei, Serranidae).

The morphology and spatial distribution of the different types of neuromasts encountered on the trunk lateral line of the sea bass (Dicentrarchus labrax) were examined using scanning electron microscopy. The sea bass trunk lateral line exhibits a complete straight pattern. In their basic features, the two types of neuromasts present, canal and superficial, resemble what has been described in other fishes. They are similar in their general cellular organization but differ in sizes, and shapes, as well as in the densities and lengths of their hair bundles. However, the sea bass trunk lateral line distinguishes itself in several ways. For instance, the pores of the canal segments are partially obstructed due to the overlap of scales throughout the trunk. Moreover, based on the density and length of the hair bundles, two distinct areas, central and peripheral, could be distinguished within the maculae of canal neuromasts. Their cupulae are also peculiar as they possess two wing-like extensions and that their central core appears to be organized in layers instead of columns. In addition, the superficial neuromasts, up to 6 per scale, are either round or elliptical and seem to be distributed serendipitously. Finally, within the maculae of both types of neuromasts, a significant number of hair bundles do not follow the two-directional polarity pattern usually described. Although some hypotheses are proposed, the influence of these characteristics in terms of signal encoding and fish behavior is yet to be understood.