Jan Tore Samuelsen

Involvement of NADPH oxidase and iNOS in rodent pulmonary cytokine responses to urban air and mineral particles.

We have investigated the potential of two complex mineral particles (feldspar and mylonite), quartz (Min-U-Sil), and suspended particulate matter (SRM-1648) (SPM) from urban air to induce inflammatory cytokine responses in primary rat alveolar type 2 cells and alveolar macrophages, and the involvement of cellular formation of free radicals in these responses. All particle types induced an increased release of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-2 from type 2 cells. Diphenyleneiodonium chloride (DPI), a selective inhibitor of NADPH-oxidase, reduced the IL-6 and MIP-2 responses to quartz, SPM and mylonite. N-(3-[Aminomethyl] benzyl) acetamidine (1400W), a selective inhibitor of inducible nitric oxide synthase (iNOS), significantly reduced the Il-6 response to SPM and feldspar in the type 2 cells. The macrophages displayed significantly increased TNF-α and MIP-2 release upon exposure to quartz or SPM. Here, DPI significantly reduced the tumor necrosis factor (TNF)-α and MIP-2 responses to quartz, and the MIP-2 response to SPM. No significant effect of 1400 W was detected in the alveolar macrophages. The role of particle-induced cellular generation of free radicals in lung cytokine responses was further elucidated in mice that lacked either NADPH-oxidase or iNOS as well as in wild-type (wt) mice. All particles were able to elicit increased cytokine levels in the bronchoalveolar lavage (BAL) fluid of the mice, although the levels depended on particle type. The NADPH-oxidase knockout (KO) mice demonstrated a significantly lower IL-6 and MIP-2 responses to SPM compared to their respective wt mice. The iNOS KO mice displayed significantly reduced IL-6, TNF-α, and MIP-2 responses to SPM. The overall results indicate the involvement of cellular free-radical formation in the pulmonary cytokine responses to particles of varying composition.

Expression of Cyp2B1 in Freshly Isolated and Proliferating Cultures of Epithelial Rat Lung Cells

Bronchiolar Clara cells and alveolar type 2 cells of the lung are known to express relatively high levels of P450 enzymes compared to other pulmonary cells. Populations of enriched type 2 cells and Clara cells were isolated from rat lung by a procedure including lung perfusion, protease digestion, centrifugal elutriation, and differential attachment. Alveolar macrophages were removed by lavage. The purity of the type 2 cell-enriched population was approximately 90%, and the purity of the Clara cell-enriched population was 40-50%. Both type 2 cells and the cells of the Clara cell-enriched population proliferated in culture. CYP2B1 mRNA was expressed approximately to the same level in type 2 cells and the Clara cell-enriched population. The mRNA levels remained roughly constant for both cell types throughout the culture period, except for an early transient reduction. The apoenzyme level of CYP2B1 was 2-3 times higher in freshly isolated cells of the Clara cell-enriched population than in the type 2 cells. Both epithelial cell types showed decreased level of CYP2B1 apoenzyme in culture. The differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated cells and cultured cells suggest the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells. The characterization of specific functions of lung cells in culture, such as P450 gene expression, provides necessary information for the use of the cells in in vitro pulmonary toxicology.

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl2 blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca2+], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.

DNA damage and DNA damage responses in THP-1 Monocytes after exposure to spores of either Stachybotrys chartarum or Aspergillus versicolor or to T-2 toxin

We have characterized cell death in THP-1 cells after exposure to heat-treated spores from satratoxin G-producing Stachybotrys chartarum isolate IBT 9631, atranone-producing S. chartarum isolate IBT 9634, and sterigmatocystin-producing Aspergillus versicolor isolate IBT 3781, as well as the trichothecenes T-2 and satratoxin G. Spores induced cell death within 3-6 h, with Stachybotrys appearing most potent. IBT 9631 induced both apoptosis and necrosis, while IBT 9634 and IBT 3781 induced mostly necrosis. T-2 toxin and satratoxin G caused mainly apoptosis. Comet assay +/- formamidopyrimidine DNA glycosylase showed that only the spore exposures induced early (3h) oxidative DNA damage. Likewise, only the spores increased the formation of reactive oxygen species (ROS), suggesting that spores as particles may induce ROS formation and oxidative DNA damage. Increased Ataxia Telangiectasia Mutated (ATM) phosphorylation, indicating DNA damage, was observed after all exposures. The DNA damage response induced by IBT 9631 as well as satratoxin G was characterized by rapid (15 min) activation of p38 and H2AX. The p38 inhibitor SB 202190 reduced IBT 9631-induced H2AX activation. Both IBT 9631 and T-2 induced activation of Chk2 and H2AX after 3 h. The ATM inhibitor KU 55933, as well as transfection of cells with ATM siRNA, reduced this activation, suggesting a partial role for ATM as upstream activator for Chk2 and H2AX. In conclusion, activation of Chk2 and H2AX correlated with spore- and toxin-induced apoptosis. For IBT 9631 and satratoxin G, additional factors may be involved in triggering apoptosis, most notably p38 activation.

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis.

Abstract Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI2C2) and type II (RII2C2) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIα, RIIα, Cα, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes.

Cell toxicity of 2‐hydroxyethyl methacrylate (HEMA): the role of oxidative stress

2-Hydroxyethyl methacrylate (HEMA) is a methacrylate monomer used in polymer-based dental-restorative materials. In this study, the viability of human lung epithelial cells, BEAS-2B, was investigated after exposure to this monomer. Exposure to HEMA reduced the viability of the BEAS-2B cells as a result of increased apoptosis, interruption of the cell cycle, and decreased cell proliferation. Depletion of cellular glutathione and increased levels of reactive oxygen species (ROS) were seen after exposure of BEAS-2B cells to HEMA. The glutathione synthase inhibitor, L-buthioninesulfoximine (BSO), was used to study whether the reduced viability was caused by glutathione depletion and increased levels of ROS. Similarly to incubation with HEMA, incubation with BSO resulted in glutathione depletion and increased ROS levels, without increasing cell death or inhibiting cell growth. The results indicate that HEMA-induced cell damage is not caused exclusively by these mechanisms. Mechanisms other than glutathione depletion and ROS formation seem to be of importance for the toxic effect of HEMA on lung epithelial cells.

Filler particles used in dental biomaterials induce production and release of inflammatory mediators in vitro

Although dental composites are in extensive use today, little is known about the biological effects of the filler particles. As composite materials are gradually broken down in the aggressive environment of the oral cavity, the filler particles may leak and induce toxic effects on the surrounding tissue and cells. The aim of this study was to elucidate possible adverse biological effects of commonly used dental filler particles; bariumaluminiumsilica (BaAlSi) and bariumaluminiumfluorosilica (BaAlFSi) with mean size of 1 microm. BEAS-2B cells were used as a model system. Particle morphology, mean particle size in solution, and particle surface charge were determined by scanning electron microscopy and Malvern zetasizer technology, respectively. Enzyme-linked immunosorbent assay was used to detect secretion of cytokine and chemokine (IL-8 and IL-6) and quantitative PCR for detection of gene activity. Both types of particle increased the release of IL-6 and IL-8 in a dose-dependent manner. BaAlFSi particles induced a more marked IL-8 response compared to BaAlSi particles, whereas no significant difference was observed for the IL-6 response. Mechanistic studies using specific inhibitors and activators indicated that cyclic AMP-dependent protein kinase A is partly involved in the observed IL-8 response. In conclusion, we consider dental filler particles to have potential to induce adverse biological response in cell cultures.

The dental monomer hydroxyethyl methacrylate (HEMA) counteracts lipopolysaccharide-induced IL-1β release—Possible role of glutathione

Methacrylate monomers, like 2-hydroxyethyl methacrylate (HEMA), are common components of resin based dental materials. Leakage of unpolymerized monomers after placement and curing leads to human exposure. HEMA is known to inhibit lipopolysaccharide (LPS) induced cytokine release. In this study we explore a possible role of the antioxidant glutathione (GSH) in this effect. In the RAW 264.7 murine macrophage cell line, HEMA (<2mM) did not induce cell death, but reduced cellular GSH levels, increased cellular ROS and decreased the IL-1β release from LPS-stimulated cells. Moreover, the IL-1β mRNA levels were reduced after 3-6h exposure, suggesting transcriptional effects of HEMA. The GSH modulators butylsulfoximine (BSO; inhibitor of GSH synthesis) and 2-oxothiazolidine-4-carboxylate (OTC; Cysteine precursor) caused a decrease and increase in the LPS-induced IL-1β release, respectively, suggesting a role for GSH in negative regulation of LPS-induced IL-1β release. However, the magnitude and dynamics of the effects of HEMA and BSO on LPS-induced IL-1β release and GSH depletion differed considerably. Thus, GSH depletion alone could not explain the strong attenuation of LPS-induced IL-1β release caused by HEMA. Formation of HEMA-protein conjugates due to the thiol reactivity of HEMA emerges as a likely candidate for the molecular mechanism accounting for this effect.

Di-n-butyl phthalate modifies PMA-induced macrophage differentiation of THP-1 monocytes via PPARγ

The present study examined the effects of di-n-butyl phthalate (DBP) on phorbol myristate acetate (PMA)-induced macrophage differentiation of THP-1 monocytes, determined by morphological classification and flow cytometry. Focusing on the expression of the surface marker CD36, the potential role of peroxisome proliferator-activated receptor gamma (PPARγ) was examined using various PPARγ agonists and antagonists. As the PPARγ ligand-binding domain contains multiple ligand-binding sites (LBS), agonist and antagonists targeting the different sites were used. DBP accelerated PMA-induced morphological changes and increased expression of CD36, although to a lesser degree than the PPARγ agonists rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). A proteomics screening revealed that DBP enhanced the expression of PPARγ-regulated proteins. During combined exposures, DBP partly attenuated the effect of rosiglitazone, an agonist binding reversibly to PPARγs canonical LBS. In contrast, DBP increased expression of CD36 in combination with 15d-PGJ2 which binds irreversibly to the canonical LBS. Thus, DBP appears to interact with both the canonical and alternative LBS. Accordingly, the antagonist GW9662, which binds to the canonical LBS, only partly reduced the DBP-induced CD36 expression, while the dual-site antagonist SR16832 completely blocked the effects of DBP. Overall, the results show that DBP modifies PMA-induced differentiation of THP-1 cells through interaction with PPARγ.

Presence and leaching of bisphenol a (BPA) from dental materials

Abstract BPA has been reported to leach from some resin based dental restorative materials and materials used for orthodontic treatment. To confirm and update previous findings, especially in light of the new temporary lower threshold value for tolerable daily BPA intake, we have investigated the leaching of BPA from 4 composite filling materials, 3 sealants and 2 orthodontic bonding materials. The materials were either uncured and dissolved in methanol or cured. The cured materials were kept in deionized water for 24 hours or 2 weeks. Samples were subsequently analyzed by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS-MS). The composite filling material Tetric EvoFlow® and the fissure sealant DELTON® showed significantly higher levels of BPA leaching compared to control samples for all test conditions (uncured, 24 h leaching and 2 weeks leaching). There were no significant differences in amount of leached BPA for any of the tested materials after 24 hours compared to 2 weeks. These results show that BPA is still released from some dental materials despite the general concern about potential adverse effects of BPA. However, the amounts of BPA were relatively low and most likely represent a very small contribution to the total BPA exposure.