Underlying mechanisms of cytotoxicity in HepG2 hepatocarcinoma cells exposed to arsenic, cadmium and mercury individually and in combination.

BACKGROUND: Toxicity data regarding combinational exposure of humans to arsenic, cadmium and mercury is scarce. Although hepatotoxicity has been reported, limited information is available on their mechanistic underpinnings. The cytotoxic mechanisms of these metals were determined in HepG2 hepatocarcinoma cell lines after individual and combinational exposure. METHODS: HepG2 cells were exposed to heavy metals (sodium arsenite, cadmium chloride, and mercury chloride) individually or in combination for 24 h, after which cell density, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), reduced glutathione (GSH), adenosine triphosphate (ATP) and caspase-3/7 activity was assessed. RESULTS AND DISCUSSION: Cadmium (IC50 = 0.43 mg/L) and the combination (0.45 mg/L, arsenic reference) were most cytotoxic, followed by arsenic (6.71 mg/L) and mercury (28.23 mg/L). Depolarisation of the ΔΨm and reductions in ROS, GSH and ATP levels occurred. Arsenic, cadmium and the combination increased caspase-3/7 activity, while mercury reduced it. CONCLUSION: The combination produced a greater, albeit mechanistically similar, cytotoxicity compared to individual metals. Cytotoxicity was dependent on altered mitochondrial integrity, redox-status, and bioenergetics. Although the combination's cytotoxicity was associated with caspase-3/7 activity, this was not true for mercury. Heavy metal interactions should be assessed to elucidate molecular underpinnings of cytotoxicity.

A randomized, 5-arm dose finding study with a mite allergoid SCIT in allergic rhinoconjunctivitis patients.

BACKGROUND: The safety and tolerability of a mite allergoid subcutaneous allergen immunotherapy (SCIT) product was previously established. The aim of this study (EudraCT number: 2011-000393-61) was to find the optimally safe and effective allergoid dose by evaluating several dosages in patients with house dust mite (HDM)-induced allergic rhinoconjunctivitis (ARC) using a titrated nasal provocation test (TNPT). METHODS: In total, 290 adult ARC patients (148 females; 142 males) with established HDM allergy and with a positive TNPT were randomized to receive placebo or mite allergoid SCIT 6667, 20 000, 50 000 or 100 000 AUeq/ml for 12 months. Patients were updosed weekly, followed by monthly maintenance dosing. The primary study endpoint comprised the clinical response to TNPT after 12 months of treatment. Secondary endpoints included response to TNPT after 6 months, PNIF measurements, symptom and medication scores during the last 8 weeks of treatment, serum immunoglobulins and safety assessments. RESULTS: After 12 months, a dose-response was observed showing statistically significant improvements in the TNPT with SCIT concentrations of ≥20 000 AUeq/ml, while no significantly different outcomes were reached after 6 months. Specific serum IgG and IgG4 levels were dose dependently increased. In the highest dose group, more treatment-emergent adverse events were observed compared with the lower dose groups. CONCLUSION: In this mite allergoid SCIT dose finding study in HDM-induced ARC, concentrations of ≥20 000 AUeq/ml showed both immunological effects and clinical efficacy in the TNPT compared with placebo. The risk-benefit ratio favours 20 000 AUeq/ml and 50 000 AUeq/ml strengths for further clinical development.

The anti-tumour properties and biodistribution (as determined by the radiolabeled equivalent) of Au-compounds intended as potential chemotherapeutics.

The anti-tumour activity of the Au (I) phosphine complex [Au(dppe(2)]Cl was first discovered in the mid 1980s although promising results were obtained it did not pass clinical studies because of its toxicity to organs such as the liver and heart. The aim of this study was to determine whether the two novel gold compounds (MM5 and MM6), selected for this study, have higher selectivity for cancer cells with less toxicity towards normal cells than [Au(dppe)(2)]Cl, and also to determine whether they have improved bio distribution compared to [Au(dppe)(2)]Cl. The Au-compounds as potential chemotherapeutic drugs were evaluated by using radioactive tracers in the in vitro and in vivo studies. Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However; the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. In terms of the total uptake it was found that the compounds that were less lipophilic (MM5, MM6) were taken up less efficiently in cells than those that are more lipophilic. Therefore hydrophilic drugs are expected to have a limited biodistribution compared to lipophilic drugs. This might imply a more selective tumour uptake.

Efficacy of bactericidal/permeability-increasing protein in experimental otitis media with effusion in rats: a new therapy for mucosal infections.

Otitis media with effusion (OME) is characterized by the presence of fluid in the middle ear without signs or symptoms of acute infection and by persistent changes in the middle ear mucosa. These are mainly induced by gram-negative bacterial infection and dysfunction of the eustachian tube (ET). Gram-negative bacteria (GNB) contain lipopolysaccharide (LPS) in their outer membrane that is responsible for inflammatory reactions in the middle ear. In this study we investigated the therapeutic effect of a recombinant LPS-binding protein, bactericidal/permeability-increasing protein (rBPI21), on the repair of mucosal damage in rats with experimentally induced OME. OME was induced by obstruction of the eustachian tube in combination with LPS injection. Twelve weeks after OME induction, secretory cells in the tympanic orifice of the middle ear were increased from an average of 14 +/- 2 to 31 +/- 5, ciliated cells were decreased from 24 +/- 4 to 6 +/- 4, and the number of macrophages in the subepithelial layer increased from 13 +/- 4 to 27 +/- 3. A single dose of rBPI21 was administered directly into the middle ear cavity 2 weeks after the induction of OME. Histologic examination of the middle ear mucosa at 4 and 12 weeks after OME induction showed that mucosal changes were restored by rBPI21 treatment. These results demonstrate that the middle ear mucosa recovers from inflammatory changes associated with OME after treatment with rBPI21. This suggests that rBPI21 may be useful in the treatment of OME and of mucosal infections of the respiratory tract.

Inhibition of endotoxin effects on cultured human middle ear epithelium by bactericidal permeability-increasing protein.

HYPOTHESIS/BACKGROUND: Endotoxin can induce morphologic changes to middle ear epithelium, which can disturb the mucociliary clearance system (MCS) and lead to otitis media with effusion (OME). The bactericidal/permeability-increasing (BPI) protein is a major component of neutrophil granules and binds with high affinity to endotoxin. In this study, the capacity to inhibit the effects of endotoxin by rBPI21, a recombinant amino-terminal analog derived from BPI, was investigated on cultured human middle ear epithelium using light microscopy and scanning- and transmission electron microscopy. METHODS: Human middle ear epithelium was air-exposed cultured on a collagenous underlayer with different additions of endotoxin and rBPI21 to the culture medium. The tissue specimens were inspected after 4 weeks for the number of ciliated and secretory cells, thickness of the mucosal layer, and cell size. RESULTS: The morphologic changes induced by endotoxin were increased thickness of the mucosal layer and increased number of secretory cells. These changes were significantly diminished or even absent when endotoxin was added with rBPI21 to the culture medium. CONCLUSION: rBPI21 can inhibit morphologic changes in the middle ear epithelium due to endotoxin. Hence, the authors believe that rBPI21 can be a new therapeutic agent in the treatment of OME.

Bactericidal/permeability-increasing protein prevents mucosal damage in an experimental rat model of chronic otitis media with effusion.

In this study, the efficacy of bactericidal/permeability-increasing protein (BPI) was assessed in a rat model of chronic otitis media with effusion. BPI injection prevented disturbance of the mucociliary clearance system of the middle ear. Hence, it is postulated that BPI can be a new therapy for chronic otitis media with effusion.

Endotoxin and tumor necrosis factor-alpha in middle ear effusions in relation to upper airway infection.

OBJECTIVES/HYPOTHESIS: This study was performed to elucidate the role of endotoxin and tumor necrosis factor-alpha (TNF-alpha) in the middle ear effusions (MEEs) of children with otitis media with effusion (OME) in relation to the chronicity of the disease and the presence of upper respiratory tract infection (URTI). STUDY DESIGN: In a retrospective study 140 MEEs were collected from 101 children between 2 and 12 years of age, and evaluated for the cytokine TNF-alpha and the lipopolysaccharide endotoxin. The amounts were quantified and correlated with the type of MEE, OME duration, and the presence of URTI. METHODS: Endotoxin levels were measured using a limulus amebocyte lysate assay and TNF-alpha levels were measured with an enzyme-linked immunosorbent assay (ELISA). Means of the different variables were compared using the one-way ANOVA least significance difference test with P<.05. RESULTS: In MEEs classified as mucopurulent (22.8%) both endotoxin and TNF-alpha levels (11.9+/-3 ng/mg total protein and 61.1+/-21 pg/mg total protein, respectively) were significantly higher compared with serous- (23.6%) or mucoid- (53.6%) type effusions. Fifty-five percent of the children who were classified as having chronic OME also had significantly higher amounts of endotoxin and TNF-alpha. The majority of the children (61%) had no URTI, although children with URTI (36%) did also have significantly higher levels of endotoxin and TNF-alpha in their middle ears. CONCLUSIONS: These results indicate that there is a strong correlation between the endotoxin and the TNF-alpha concentration in the middle ear and the type of MEE, the presence of URTI, and the chronicity of the disease.

Effect of endotoxin on cultured human middle ear epithelium.

The effect of endotoxin was investigated on air-exposed cultured human middle ear epithelium. Concentrations of 0, 1 and 100 ng/ml endotoxin were used. Complete differentiation of the cells was not reached at 12 days. After 21 days, endotoxin had induced an increased proliferation of the epithelial layer. Furthermore, an increase in the number of secretory cells and in the amount and length of microvilli was observed at this time. There were no significant morphological differences between the high and the low endotoxin concentrations, which supports our hypothesis that endotoxin induces an all-or-nothing reaction. These findings are in agreement with our previous results on serially submerged cultured rat middle ear epithelium. From these results we conclude that endotoxin is an important factor in the disturbance of the morphology of the middle ear epithelium, which may lead to chronic otitis media with effusion. In addition, our tissue culture method proved to be a good model for further studies on human middle ear mucosa.

Structural changes in the rat middle ear mucosa due to endotoxin and eustachian tube obstruction.

The middle ears of 48 rats were used to examine the effects of endotoxin injection, eustachian tube obstruction or a combination of eustachian tube obstruction and endotoxin injection. Animals were killed after 1, 2, 4, or 12 weeks and the middle ears processed for light and scanning electron microscopy. Compared to the normal middle ear mucosa, the epithelial layer was more pseudostratified, cuboidal or cylindrical after endotoxin injection or obstruction of the eustachian tube. In the early phase, numerous ciliated cells occurred in areas originally almost devoid of these cells. At 3 months, degeneration of ciliated cells was observed. The combination of eustachian tube obstruction and endotoxin injection also induced a more pseudostratified, cuboidal or cylindrical epithelium with an increased number of goblet cells. However, an early decrease occurred in the number of ciliated cells in the tympanic orifice of the eustachian tube. Furthermore, inflammatory cells, mainly PMNs, macrophages and lymphocytes, invaded the subepithelial layer after eustachian tube obstruction and endotoxin injection. These structural changes resulted in an impairment of the mucociliary transport system for clearance of the middle ear cavity. For this reason we believe that both endotoxin and eustachian tube obstruction or dysfunction play an important role in inducing persistent mucosal changes in the middle ear cavity, thereby prolonging otitis media with effusion.