Carbon nanotubes have a deleterious effect on the nose: the first in vitro data.

BACKGROUND: The information currently available concerning carbon nanotubes toxicity is disturbing and conflicting. Moreover, little is known about their effect on the nasal cavities, which are the first target for nanoparticles. MATERIAL AND METHOD: We investigated the cytotoxicity (50 to 0.5 microg/mL) of double-walled carbon nanotube with two independent tests (MTT, Wst-1) on normal human nasal epithelial cells after 12-day exposure (control untreated nasal cells and A549). Nasal cell differentiation function, oxidative stress, the morphological features of cells in contact with DWCNTs and the localizations of the latter were also investigated. RESULTS: Exposure revealed a dose-dependent decrease in cell metabolic activity and cell growth. In nearly all conditions, normal human nasal epithelial cells were more sensitive than malignant ones. Even with both tests, the cytotoxic threshold dose could not be accurately determined because of dye adsorption by DWCNTs. Nasal cells showed stronger cytokeratin 7 and persistent UEA-I immunostaining. Cytokeratin 19 production was increased at 25 microg/mL and mucus production was stimulated from 0.5 microg/mL. A significant increase in Reactive Oxygen Species was observed from 25 microg/mL. The cell plasma membrane showed several holes and DWCNTs were present in the cytoplasm. CONCLUSION: DWCNTs seem to have a deleterious effect on nasal cells after 12-day exposure.

[Research ethics: French regulations and applications in radiation oncology]. / Éthique de la recherche : réglementations françaises et applications en oncologie radiothérapie.

French regulations about research ethics are based on the so-called Jardé law, which defines researches involving human beings. Researches involving human beings require the submission of research protocols to a committee for protection of persons with a precise list of documents to submit for a favourable opinion. This law describes different categories of researches and determines the ethical procedures to apply before setting up a research protocol. This issue of categorisation is central and must be taken into account by researchers from the beginning of the research process. Researches considered as not involving human beings also require a set of ethical precautions focused on patients' information and the collection of their non-opposition (due to the application of the General Data Protection Regulation adopted by the European Parliament). Thus, many regulations exist and they require a real work for researchers to meet these requirements in research ethics. This article aims to summarise French regulations. Selected examples are specifically taken into the field of radiation oncology research.

Circadian rhythms of renal hemodynamics in unanesthetized, unrestrained rats.

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (Cin) and p-amino-hippuric acid (CPAH) clearances; Cin represents glomerular filtration rate and CPAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, Cin exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 +/- 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in CPAH was similar with the minimum between 16:00 and 20:00 h (2.5 +/- 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.

Prevention of cyclosporin-induced vasoconstrictive effect in rat isolated glomeruli with pharmacological vasoactive agents.

This study was designed to demonstrate the ability of some physiological or pharmacological agents to diminish or even suppress the vasoconstrictive effects of cyclosporin (CsA) in rat isolated glomeruli. The area of glomeruli isolated from the superficial cortex of male Sprague-Dawley rats was assessed by an image analyser with a video camera. Each glomerulus was imaged before treatment and 5, 10, 15, 20 and 30 min after incubation with CsA alone or in association with another vasoactive agent. CsA alone induced a marked time- and dose-dependent decrease in glomerular area (-4.7% at 10 min, -10.3% at 20 min and -12.0% at 30 min); cremophore or control solute did not induce any decrease. CsA with 10(-6)m-verapamil brought about only a slight decrease (-1.5% at 10 min, -3.0% at 20 min and -4.8% at 30 min), as was the case with 10(-8)m-prostacyclin analogue (Iloprost) (-1% at 10 min, -1.8% at 20 min and -3.4% at 30 min); finally, 10(-6)m-thromboxane synthesis inhibitor (CGS 12970) brought about a reduction in area of -2% at 10 min, -3.6% at 20 min and -4.3% at 30 min. In conclusion, the marked vasoconstriction induced by CsA in rat isolated glomeruli can be partially prevented by various pharmacological vasoactive agents that act on different vascular targets of CsA.

Inhibition of cyclosporin-induced in vitro glomerular contraction by theophylline.

Cyclosporin A (CsA) induces in vivo severe nephrotoxicity characterized by a large decrease in renal haemodynamics. The aim of this study is to show the ability of theophylline, a xanthic derivative, to diminish the CsA-induced vasoconstrictive effects by using two in vitro rat glomerular models, for example isolated glomeruli and cultured mesangial cells. Isolated glomeruli are obtained from the superficial renal cortex of male Sprague-Dawley rats by a sieving method. Mesangial cells are cultured in RPMI 1640 medium with 15% foetal calf serum (FCS). The area of either isolated glomeruli or mesangial cells is assessed by an image analyser with a video camera. Each glomerulus or mesangial cell serves as its own control by photographing them before any drug incubation and after incubation for 10, 20 and 30 min either in control solution or control solution with CsA or CsA and theophylline. CsA (10(-6)m) induces an important time-dependent decrease in the glomerular area (about 13.4% after 30 min). When theophylline is added only a slight decrease is noticed (about 3.4% after 30 min). The same results are obtained with mesangial cells. In conclusion, a direct vasoconstrictive effect of CsA in isolated glomeruli and mesangial cells can be confirmed. In addition, this effect can be partially prevented by theophylline.

In vitro effects of cadmium on two different animal cell models.

The aim of this study was to assess comparatively the effects of cadmium on two different in vitro cell models, a cell line derived from proximal tubule renal cells (LLC-PK1) and haemocytes or blood cells of mussels (Mytilus galloprovincialis). Cells were seeded in 96-well microplates and exposed in vitro to different concentrations of cadmium (CdCl(2)) ranging from 10 to 2000 microM for haemocytes and from 1 to 100 microM for LLC-PK1 cells, added to the culture medium. After 24 h of exposure, different assays were performed on haemocytes: neutral red uptake, phagocytosis of neutral red-stained zymosan, XTT assay, activity of lysosomal acid phosphatase and demonstration of the actin cytoskeleton using TRITC-labeled phalloidin. Cell viability expressed as LC50 was 750 microM when using the neutral red assay and 400 microM with the XTT assay. The phagocytic ability and the activity of acid phosphatase increased significantly in cells treated with Cd in a non dose-dependent manner. Doses of Cd above 100 microM caused disruption of the actin cytoskeleton. In LLC-PK1 cells, cell viability expressed as LC50 was found to be around 40 microM when using the neutral red assay and 50-60 microM with MTT and LDH assays, respectively. These results show that mussel haemocytes are in general more resistant to Cd exposure than LLC-PK1 cells. Furthermore, Cd appears to stimulate phagocytic and lysosomal activities in haemocytes in vitro.

Influence of cadmium speciation for the evaluation of in vitro cadmium toxicity on LLC-PK(1) cells.

The main objective of the present work was to assess the potentiality of in vitro models to improve our understanding of cadmium-induced toxicity, especially on epithelial renal cells. Indeed cadmium, a potent toxic metal, poses a serious environmental threat and the mechanisms of its renal toxicity need to be clarified. Cytotoxicity studies presented here were performed in a tubular proximal original established porcine kidney cell line (LLC-PK(1)). We have compared cytotoxicity induced by different chemical cadmium forms in LLC-PK(1) cells as a function of media cell culture pH and protein content. Cadmium stock solutions were prepared either by dissolving cadmium chloride or cadmium sulphate with increasing protein concentrations in the media cell culture. Its pH was monitored during experiments. Cytotoxicity was measured by neutral red uptake after 24 h of exposure. Dose-dependent cytotoxicity curves, calculated with REGTOX, were systematically correlated with pH and protein content. Experiments in vitro revealed that cadmium was dose-dependently toxic for LLC-PK(1) for concentrations ranging from 10(-4) to 10(-6) M. We have noticed a lack of influence of the media cell culture pH on the cadmium cytotoxicity. REGTOX determines closely the EC(50) values but EC(50)CdCl(2)>EC(50)CdSO(4) and cadmium have been assayed with an inductively coupled atomic emission spectrometer (ICP/AES) directly in the media cell culture and the cellular pellet.

[Protective effect of verapamil and dopamine against cyclosporine-induced vasoconstriction in isolated glomeruli in rats]. / Effet protecteur du vérapamil et de la dopamine à l'égard de la vasoconstriction induite par la ciclosporine sur glomérules isolés de rat.

Cyclosporin A (CsA)-induced nephrotoxicity is characterized by dramatic changes in glomerular filtration rate and renal plasma flow, largely limiting the clinical use of this drug. The vasoconstrictive response of CsA could explain, in part, these hemodynamic alterations. The present study compares the area changes in rat-isolated glomeruli incubated with CsA alone or after pre-treatment with verapamil and dopamine. In verapamil-pretreated CsA-intoxicated glomeruli, size decrease was reduced (-1.5 percent at T10, -3.1 percent at T20 and -4.8 percent at T30), when compared with CsA alone (-4.7 percent at T10, -10.1 percent at T20 and -12 percent at T30). The results obtained with dopamine were similar. In conclusion, verapamil and dopamine can be regarded as fair protective agents against CsA-induced vasoconstriction in rat-isolated glomeruli.

[Effect of verapamil on cyclosporine-induced vasoconstriction in human or murine isolated glomerules]. / Influence du vérapamil sur la vasoconstriction induite par la ciclosporine sur glomérules isolés humains et murins.

Cyclosporin is a very potent immunosuppressant, but it often produces renal disturbances which limit its clinical use. Using an image analyzer which determines the areas of isolated glomerules, we were able to demonstrate that cyclosporin in various concentrations exerts a direct vasoconstrictive effect on human and murine glomerules. We also showed that verapamil has an almost total inhibitory effect on cyclosporin-induced vasoconstriction. These findings seem to be of interest in clinical practice to reduce the nephrotoxicity of cyclosporin.

Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies.

Cadmium represents a major environmental pollutant that may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC(50) cytotoxicity index was obtained with 3.55 micromol/L using neutral red assay for 24 h. After cadmium exposure (1 micromol/L, determined as nonlethal concentration), 0.38 microg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle alpha-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.

In vitro endothelial cell susceptibility to xenobiotics: comparison of three cell types.

In three different endothelial cell (EC) cultures (primary human umbilical cord vein, so-called HUVEC; and immortalized cell lines HBMEC and EA-hy-926), the effects of different xenobiotics were studied in order to standardize vascular EC models for in vitro pharmacotoxicological studies. Cell characteristics were first investigated by the production and the mRNA levels of known endothelial markers in the three EC culture models. EC secretory products, tissue plasminogen activator (tPA) and von Willebrand factor (vWF), were present in the supernatant of the immortalized cell lines. The mRNA levels of vWF, tPA, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and beta -integrin subunit, which are involved in the control of platelet function, coagulation, and fibrinolysis as well as in cell-matrix interactions, were investigated in all EC types. For at least three parameters, cultured cells provided marked characteristics of EC phenotype, in HUVEC and in immortalized cell lines, regardless of their origin from the macro- or microcirculation. Toxicity experiments were assessed after 24 h exposure to cadmium, cyclosporin A and cisplatin by MTT assay. These experiments show nonsignificant difference in susceptibility to cyclosporin A and cadmium on HUVEC, HBMEC, and EA-hy-926. However, HBMEC, seems to be highly susceptible to cisplatin compared to HUVEC, the latter being more sensitive than EA-hy-926. For experiments conducted with cyclosporin and cadmium, cell lines could constitute an alternative material for routine cytotoxicity studies.

Isolated glomeruli and cultured mesangial cells as in vitro models to study immunosuppressive agents.

Immunosuppressive agents, such as cyclosporin A (CsA), by their vasoconstrictive properties, induce in vivo in patients and rodents a dramatic fall in renal hemodynamics. The aim of this study is to review the ability of some physiological and/or pharmacological agents which are supposed to be involved in the renal physiopathology of CsA to prevent the contraction induced by CsA in two in vitro glomerular models. Isolated glomeruli are obtained by a sieving method from male Sprague-Dawley rat superficial cortex. Mesangial cells from these isolated glomeruli are cultured in RPM1 1640 medium with 20% FCS in 5% CO2 atmosphere. The area of isolated glomeruli and cultured mesangial cells is assessed by an image analyzer with a video camera. Each glomerulus and cell is its own control and is photographed before incubation with any drug (T0) and then during incubation at 5, 10, 20, and 30 min. Incubations are performed during 30 min with 10(-6) mol/L CsA either with a 10 min pretreatment with the vasoactive agent or without pretreatment. CsA alone induces a time- and dose-dependent decrease in glomerular structure area (-4.7% at 10 min, -10.3% at 20 min, and -12.0% at 30 min for isolated glomeruli); Cremophore excipient or control solute does not induce any significant decrease in surface area. CsA with 10(-6) mol/L verapamil pretreatment induces only a slight decrease: -1.5% at 10 min, -3.0% at 20 min, and -4.8% at 30 min. Calcium blockers nifedipine and felodipine produce similar results. Likewise, with 10(-8) mol/L prostacyclin analog (iloprost), only a slight area decrease in mesangial cells is noted: -1.3% at 5 min, -1.8% at 10 min, and -3.3% at 20 min; with 10(-6) mol/L TXA2 synthesis inhibitor (CGS 12970) the results are -2.0% at 10 min, -3.6% at 20 min, and -4.3% at 30 min. Finally, a similar protective effect can be noted with 10(-5) mol/L theophylline: -0.4; -1.5 and -1.9% at 10, 20, and 30 min. In conclusion, CsA-induced contraction in two in vitro glomerular models can be partially or even totally prevented by pretreatment with various pharmacological agents.

Image analyser as a tool for the study of in vitro glomerular vasoreactivity.

Many drugs used in clinics can dramatically reduce renal hemodynamics. For some years there have been developed in our laboratory two in vitro glomerular models, isolated glomeruli and mesangial cell cultures, to quantitate, by video image analyzer, the direct glomerular effect of vasoreactive agents. The present study shows the vasoconstrictive effects of angiotensin II and cyclosporin in both models and compares their glomerular vasoconstriction with or without vasodilating agents such as verapamil. This drug-induced glomerular vasoreactivity is time- and dose-dependent; moreover, it can be reversible after perfusion in control conditions. The interest of these in vitro glomerular models is validated by fair correlations between in vivo and in vitro data and between the responses of both. These models can be considered as tools for assessing glomerular vasoreactivity of nephrotoxic agents.

Protective effect of verapamil in cyclosporin-incubated human and murine isolated glomeruli.

Cyclosporin A (CsA)-induced nephrotoxicity is characterized by a decrease in the glomerular filtration rate (GFR) which is associated with a large increase in renal vascular resistance (RVR). Using a video image analyzer, we have demonstrated CsA-induced glomerular vasoconstriction in rat isolated glomeruli as assessed by a significant reduction of glomerular area. This vasoactive response explains in part the renal hemodynamic changes and the development of CsA-induced reversible decline in renal function. To confirm the direct vasoactive effect of CsA on glomeruli and to determine if calcium-blocking agents modified this response, we compared the changes in area of isolated rat and human glomeruli incubated either with CsA alone or with CsA plus verapamil. The area of the isolated glomeruli was quantitatively evaluated by a camera video image analyzer; each glomerulus served as its own control. Area kinetics were studied at 5 min intervals over 30 min. CsA-induced glomerular size reduction is dose dependent (-4.2% for 10(-10) M and -10.2% for 10(-6) M) and time dependent (-2.3% at 5 min, -4.7% at 10 min, and -12.1% at 30 min for 10(-6) M). With verapamil pretreatment, CsA-induced reduction in glomerular size was reduced (-0.6% and -3.6%, respectively, for 10(-6) M and 10(-7) M verapamil). Thus, verapamil can be considered as a protective agent against CsA-induced vasoconstriction in rat and human isolated glomeruli.

Potential use of isolated glomeruli and cultured mesangial cells as in vitro models to assess nephrotoxicity.

The purpose of this short review is to present the potential of using isolated glomeruli and cultured mesangial cells as two different in vitro models to assess the glomerular effect of molecules with nephrotoxic properties. The advantage of using isolated renal glomeruli is that they conserve the architecture of this anatomical region of the kidney; moreover, they are free of any vascular, nervous or humoral influences derived from other regions of the kidney. Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Mesangial cells have a variety of functions including synthesis and assembly of the mesangial matrix, endocytosis and processing of plasma macromolecules, and control of glomerular hemodynamics, mainly the ultrafiltration coefficient Kf, via mesangial cell contraction or release of vasoactive hormones. Most authors agree that mesangial cells play a major role in glomerular contraction, filtration surface area, and Kf regulation. One of the major effects of toxicants on glomerular structures is contraction. We can assess quantitatively the degree of toxicant-induced mesangial cell contraction or glomerular contraction by measuring the changes in planar cell surface area or apparent glomerular cross-sectional area after exposition to the toxicant. These in vitro models can also reveal glomerular effects of xenobiotics that are difficult or impossible to observe in vivo. In addition, these studies permit a fundamental examination of the mechanism of action of xenobiotics on glomerular cells, including the possibility that at least a part of their effects are mediated by local mediators released by glomerular cells. We review the effects and the mechanisms of action of several toxicants such as gentamicin, cyclosporin, cisplatin, and cadmium on isolated glomeruli or cultured mesangial cells. As such in vitro results confirm in vivo renal hemodynamic changes caused by toxicants, we conclude that these models are fruitful tools for the study of renal toxicity. These in vitro systems might also serve as a predictive tool in the evaluation of drugs inducing changes in glomerular filtration rate and as a way to propose protective agents against these dramatic hemodynamic effects.

Effect of cyclosporin and the prostacyclin analogue iloprost on human glomerular vasoreactivity.

Cyclosporin is known to cause a decrease in renal blood flow and glomerular filtration rate in both humans and animals. These acute modifications are reversible if the drug is withdrawn, and seem to be caused by a local hormonal imbalance between vasoconstricting and vasodilating substances. We studied human glomeruli incubated with either CsA alone, iloprost alone, or CsA + iloprost. Photomicrographs of glomeruli were taken at t0, t1, t2 and t5 min and glomerular areas were measured with a videoanalyser linked to a computer. Results show a significant vasoconstriction with CsA alone, and no significant modification of glomerular areas with iloprost or CsA + iloprost. We conclude that iloprost prevents CsA-induced vasoconstriction in human glomeruli in vitro.

In vitro models to study mechanisms involved in cyclosporine A-mediated glomerular contraction.

The immunosuppressive drug, cyclosporin A (CsA), which is successfully used to prevent rejection in organ transplantation, induces renal side-effects as shown by a decrease in glomerular filtration rate and ultrafiltration coefficient regulated by the tone of mesangial cells.The aim of the present study was to investigate the effect of CsA on isolated glomeruli and mesangial cells, which constitute appropriate in vitro models for renal vasoreactivity studies. The roles of different intracellular and extracellular mediators such as calcium, endothelin-1 (ET-1), prostaglandins (TXA(2 )and PGI(2)) and reactive oxygen intermediates (ROIs) were analysed. CsA caused a concentration- and time-dependent decrease in the planar cross-sectional areas of isolated glomeruli and mesangial cells as determined by image analysis. Intracytosolic free calcium concentration determined by fluorimetric analysis was significantly increased after 30 min CsA (10 microM) incubation. In the contraction experiment, the calcium antagonist verapamil inhibited the CsA response. ET-1, TXB(2) and keto-PGF(1alpha) were determined directly, however no changes were found statistically significantly different from respective controls. In contrast to these results, the ET-1 specific antibody was able to reduce CsA-mediated cell contraction. In the presence of a prostacyclin agonist iloprost, CsA-induced contraction was also modified. The role of ROIs using a 2'7'-dichlorofluorescein diacetate (DCFdAc) fluorimetric method was directly determined by observing, with 10 microM CsA, a significant production of hydrogen peroxide (H(2)O(2)), which was able alone to induce mesangial cell contraction. Coincubation with the antioxidants led to a significant inhibition of mesangial cell contraction. These results suggest that CsA caused an imbalance in the normal level of all investigated vasoconstrictive and vasodilator mediators, which shifted towards the advantage of vasoconstrictive action.

Effects of cadmium and uranium on some in vitro renal targets.

Metals are major pollutants not only in occupational settings but also in the general environment. Chronic exposure of workers has been related to severe damage, especially at the renal level. While toxic compounds such as metals are well known to severely impair tubular functions, it is clear that nephrotoxicants can act on various other renal targets, i.e., vascular and glomerular ones. In vitro models are available to assess these toxicities and can be used to better understand the different cell targets. This paper summarizes data obtained in our laboratory after exposure of isolated renal structures such as glomeruli, and cell cultures such as glomerular mesangial and tubular epithelial cells, to cadmium and uranium. Morphometric studies by image analysis of isolated glomeruli and mesangial cultured cells showed that cadmium and uranium induced a dose- and time-dependent glomerular contraction accompanied by disorganization of the cytoskeleton. Classical viability tests demonstrated various factors influencing the metal toxicity. The important roles of pH, extracellular protein concentrations and the nature of the anion accompanying the metal were demonstrated. These data obtained in in vitro models provide better understanding of the cytotoxicity after metal uptake and accumulation in glomerular and tubular cells. Moreover, the glomerular and tubular cytotoxicity they induce may be correlated with severe renal hemodynamic changes in vivo. Finally, we briefly present eventual improvements for in vitro renal models by the use of new cell models such as immortalized human cell lines or by the introduction of porous supports and perifusion devices.