Asbjørn Magne Nilsen

Closely related colon cancer cell lines display different sensitivity to polyunsaturated fatty acids, accumulate different lipid classes and downregulate sterol regulatory element‐binding protein 1

N‐6 polyunsaturated fatty acids (PUFAs) may be associated with increased risk of colon cancer, whereas n‐3 PUFAs may have a protective effect. We examined the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid and arachidonic acid on the colon carcinoma cell lines SW480 derived from a primary tumour, and SW620 derived from a metastasis of the same tumour. DHA had the strongest growth‐inhibitory effect on both cell lines. SW620 was relatively more growth‐inhibited than SW480, but SW620 also had the highest growth rate in the absence of PUFAs. Flow cytometry revealed an increase in the fraction of cells in the G2/M phase of the cell cycle, particularly for SW620 cells. Growth inhibition was apparently not caused by increased lipid peroxidation, reduced glutathione or low activity of glutathione peroxidase. Transmission electron microscopy revealed formation of cytoplasmic lipid droplets after DHA treatment. In SW620 cells an eightfold increase in total cholesteryl esters and a 190‐fold increase in DHA‐containing cholesteryl esters were observed after DHA treatment. In contrast, SW480 cells accumulated DHA‐enriched triglycerides. Arachidonic acid accumulated in a similar manner, whereas the nontoxic oleic acid was mainly incorporated in triglycerides in both cell lines. Interestingly, nuclear sterol regulatory element‐binding protein 1 (nSREBP1), recently associated with cell growth regulation, was downregulated after DHA treatment in both cell lines. Our results demonstrate cell‐specific mechanisms for the processing and storage of cytotoxic PUFAs in closely related cell lines, and suggest downregulation of nSREBP1 as a possible contributor to the growth inhibitory effect of DHA.

The adjuvant activity of diesel exhaust particles and carbon black on systemic IgE production to ovalbumin in mice after intranasal instillation

The adjuvant activity of diesel exhaust particles (DEP) on systemic IgE production to ovalbumin (OA) was studied in mice after intranasal administration. The main purpose was to elucidate which part of the particles was responsible for the effect, the carbon core and/or the adsorbed organic substances. Female Balb/cA mice were immunized with OA either alone or in combination with DEP or carbon black particles (CB), the latter used as a surrogate for the non-extractable carbon core of DEP. Controls were given DEP, CB or buffer alone. The animals were immunized four times. 1 and 2 weeks after the last immunization anti OA IgE antibody in serum was analysed by enzyme linked immunosorbent assay (ELISA). An increased response to the antigen was observed in animals receiving OA together with DEP or CB, compared with animals receiving OA alone. The increased response was seen as both increased number of responding animals and increased serum anti OA IgE antibody. For OA + DEP 37% of the animals showed a serum anti OA IgE response, whereas 22% of the OA + CB animals and 10% of the OA animals responded. In conclusion, this work shows that not only DEP, but also CB have an adjuvant activity for specific IgE production after intranasal instillation. However, the activity of DEP may be more pronounced than that of CB. The results imply that both the organic matter adsorbed to DEP and the non-extractable carbon core are responsible for the observed adjuvant effect.

The mycotoxins citrinin and gliotoxin differentially affect production of the pro-inflammatory cytokines tumour necrosis factor-alpha and interleukin-6, and the anti-inflammatory cytokine interleukin-10.

Background Microbial growth is considered one of the major causes of indoor air problems. Moulds have been associated with asthma, allergy and a wide range of diffuse indoor air‐related symptoms. However, mechanisms of the adverse health effects are not well understood.

Intraphagosomal pH in Alveolar Macrophages after Phagocytosis In Vivo and In Vitro of Fluorescein-Labeled Yeast Particles

The pH in phagolysosomes of rabbit alveolar macrophages was studied using yeast particles labeled with fluorescein isothiocyanate (FITC). The yeast particles were added to the macrophages in vitro a few hours or 1 day after they had been lavaged from the lung and in vivo 3 h or 1 day before the lungs were lavaged. Intracellular pH was estimated from the ratio between the fluorescence intensity at wavelength 519 nm with excitation at wavelengths of 495 and 450 nm. In both the in vitro and in vivo experiments pH decreased significantly during the first hours after lavage, but after a few hours reached almost the day-2 levels, i.e., 4.9-5.4. The decrease in pH was related to time after lavage and not to time after phagocytosis of the particles. It is suggested that intracellular measurements of pH in alveolar macrophages should be combined with determinations of lung clearance of metal particles.

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

Reduction of IL-12 p40 Production in Activated Monocytes after Exposure to Diesel Exhaust Particles

Background: A reduction of IL-12 production by lung macrophages may partly explain the presumed adjuvant effect of diesel exhaust particles (DEP) in allergy and asthma. IL-12 stimulates T helper type 1 (Th1) lymphocytes, which inhibit Th2 cells via Th1-specific cytokines. The aim of this study was to investigate the influence of DEP on the production of IL-12 p40 in lipopolysaccharide (LPS)-activated monocytes. Methods: The human monocytic cell line Mono-Mac-6 was stimulated with LPS (200 ng/ml) and grown with DEP (0–200 µg/ml) for 0, 6 or 24 h. IL-12 p40 and the pro-inflammatory cytokine TNF were analysed in the cell supernatants by ELISA and a cell assay, respectively. Results: Levels of IL-12 p40 correlated inversely with the DEP exposure concentrations, whereas TNF increased in parallel to the DEP concentrations. At a DEP concentration of 200 µg/ml, the amount of IL-12 p40 was 35% of that observed without DEP. The corresponding TNF value was 230% of the control. Reduced viability, binding of cytokines to DEP or endotoxin in the DEP samples cannot fully explain the changes in the concentrations of these two cytokines. Conclusion: DEP seem to inhibit the production of IL-12 p40 and stimulate that of TNF in activated monocytes. This may partly explain the presumed adjuvant effect of DEP in atopy; by altering the Th1/Th2 balance via down-regulation of IL-12, the Th2 response characteristic of allergy and asthma may be favoured.

Nitrogen dioxide: no influence on allergic sensitization in an intranasal mouse model with ovalbumin and diesel exhaust particles

The role of traffic-related air pollution in the development of allergic diseases is still unclear. We therefore investigated if NO2, an important constituent of traffic-related air pollution, promotes allergic sensitization to the allergen ovalbumin (OVA). We also examined if NO2 influenced the allergy adjuvant activity of diesel exhaust particles (DEP). For this purpose, mice were exposed intranasally to OVA with or without DEP present, immediately followed by exposure to NO2 (5 or 25 parts per million [ppm]) or room air for 4 h in whole body exposure chambers. Eighteen hours after the last of three exposures, the lungs of half of the animals were lavaged with saline and markers of lung damage and lung inflammation in the bronchoalveolar lavage fluid (BALF) were measured. Three weeks later, after intranasal booster immunizations with OVA, the levels of OVA-specific IgE and IgG2a antibodies in serum were determined. Both NO2 (25 ppm) and DEP gave lung damage, measured as increased total protein concentration in BALF, whereas only NO2 seemed to stimulate release of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast, only DEP significantly increased the number of neutrophils. Furthermore, DEP in combination with OVA stimulated the production of serum allergen-specific IgE antibodies. NO2, however, neither increased the production of allergen-specific IgE antibodies, nor influenced the IgE adjuvant activity of DEP. Thus, based on our findings, NO2 seems to be of less importance than combustion particles in the development of allergic diseases after exposure to traffic-related air pollution.

Beryllium sensitivity among workers at a Norwegian aluminum smelter

BACKGROUND Sensitivity to beryllium was investigated among workers at an aluminum smelter in Norway as a consequence of the findings in an occupational exposure survey. METHODS Three hundred and sixty-two employees and 31 reference persons were tested for sensitization to beryllium with the beryllium lymphocyte proliferation test (BeLPT) based on specifications by the US Department of Energy in 2001. The results are reported as abnormal, borderline, or normal. RESULTS One person (0.28%) from the aluminum smelter was found to have abnormal results in two separate blood samples and is sensitized to beryllium. Three other persons had one abnormal test that was not confirmed by a second test. One person in the reference group had one abnormal and one normal test result. No borderline samples were detected. None of the employees with one or more abnormal sample results had pot room asthma. The sensitized individual worked in a Soederberg line in 1972-1974. The beryllium concentration in the work atmosphere is estimated to have been similar as today (0.1-0.3 microg/m(3)), but work routines, etc. would cause higher total exposures. CONCLUSIONS Only one sensitized person of 362 is in line with what is found in other studies in the aluminum industry. The low number, compared with the beryllium handling industry, may be attributable to lower work atmosphere concentrations, beryllium speciation effects, or use of respiratory protection equipment. Pot room asthma does not appear to be associated with beryllium sensitization.

Iron oxide nanoparticles modulate lipopolysaccharide-induced inflammatory responses in primary human monocytes.

Co-stimulation of the immune system to more than one agent concomitantly is very common in real life, and considering the increasing use of engineered nanoparticles and nanomaterials, it is highly relevant to assess the ability of these materials to modulate key innate immune responses, which has not yet been studied in detail. We investigated the immunomodulatory effects of 10 nm and 30 nm iron oxide nanoparticles (IONPs) on primary human monocytes in the presence and absence of Toll-like receptor 4 agonist lipopolysaccharide (LPS). Prior to the cell studies, we characterized the physicochemical properties of the nanoparticles in cell culture medium and ensured that the nanoparticles were free from biological contamination. Cellular uptake of the IONPs in monocytes was assessed using transmission electron microscopy. Using enzyme-linked immunosorbent assay, we found that the IONPs per se did not induce the production of proinflammatory cytokines tumor necrosis factor-α, interleukin-6, and interleukin-1β. However, the IONPs had the ability to suppress LPS-induced nuclear factor kappa B activation and production of proinflammatory cytokines in primary human monocytes in an LPS and a particle dose-dependent manner. Using confocal microscopy and fluorescently labeled LPS, we showed that the effects correlated with impaired LPS internalization by monocytes in the presence of IONPs, which could be partly explained by LPS adsorption onto the nanoparticle surface. Additionally, the results from particle pretreatment experiments indicate that other cellular mechanisms might also play a role in the observed effects, which warrants further studies to elucidate the additional mechanisms underlying the capacity of IONPs to alter the reactivity of monocytes to LPS and to mount an appropriate cellular response.

Combined cell wall polysaccharide, mycotoxin and bacterial lipopolysaccharide exposure and inflammatory cytokine responses

Human exposure to environmental microbes occurs regularly. Microbial compounds may interact with each other to affect cellular responses. We hypothesized that interactions between microbial compounds could modulate inflammatory cytokine responses in vitro. We investigated monocyte production of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α) and the regulatory cytokine interleukin‐10 (IL‐10) after combined exposure to the fungal cell wall polysaccharide mannan and to the β‐glucan laminarin, the mycotoxin citrinin and bacterial lipopolysaccharide (LPS). Interactions between the cell wall microbial compounds were estimated statistically in a general linear mixed model. We found that LPS (100 ng/ml) and the used β‐glucan (up to 1000 μg/ml) significantly interacted with each other to reduce TNF‐α production. Mannan (up to 100 μg/ml) did not interact with the β‐glucan, but interacted with LPS. IL‐10 production was induced by LPS only. The mycotoxin citrinin did not induce cytokine production, but was toxic to the cells in a dose‐ and time‐dependent manner. However, non‐toxic doses of citrinin reduced LPS‐induced IL‐10 production while LPS‐induced TNF‐α production was not similarly reduced by citrinin. In conclusion, interactions between microbial compounds can modulate cellular inflammatory cytokine production and experimental investigations of one compound at a time could give misleading conclusions about these combined effects.