Biological evaluation of subgingivally placed direct resin composite materials.

Placement of composite resin restorations in deep subgingival cavities can damage surrounding soft tissues. In addition, commonly used resin-based composites (RBCs) might interfere with wound healing and periodontal health. To clarify cellular interactions with RBCs, we used an MTT assay to investigate adhesion of primary human gingival fibroblasts and human osteoblasts (hFOB 1.19) on five RBC materials with and without surface modifications (alumina blasting with 50- or 110-µm Al2O3). In addition, high-performance liquid chromatography (HPLC) was used to determine release of resin monomers from RBCs after 1 h, 1 day, and 7 days. As compared with tissue culture plastics (the control), cellular adhesion was significantly lower (P < 0.001) for human gingival fibroblasts and osteoblasts. Only minor, nonsignificant differences between individual RBCs were identified. HPLC analyses identified the release of three bifunctional methacrylates bisphenol A glycerolate dimethacrylate, triethylene glycol dimethacrylate, and diurethane dimethacrylate from RBCs and showed that monomer release increased between 1 h and 1 day but remained low. The present findings suggest that surface adhesion in the subgingival area is limited for the tested RBCs. Although residual monomer release was low for all tested RBCs, it might be sufficient to adversely affect cell adhesion.

Physiologically based pharmacokinetic modeling of POPs in Greenlanders.

Human exposure to persistent organic pollutants (POPs) and the potential health impact in the Arctic far from the emission sources have been highlighted in numerous studies. As a supplement to human POP biomonitoring studies, a physiologically based pharmacokinetic (PBPK) model was set up to estimate the fate of POPs in Greenlandic Inuit's liver, blood, muscle and adipose tissue following long-term exposure to traditional Greenlandic diet. The PBPK model described metabolism, excretion and POP accumulation on the basis of their physicochemical properties and metabolic rates in the organisms. Basic correlations between chemically analyzed blood POP concentrations and calculated daily POP intake from food questionnaire of 118 middle age (18-35years) Greenlandic Inuits from four cities in West Greenland (Qaanaaq: n=40; Qeqertarsuaq: n=36; Nuuk: n=20; Narsaq: n=22) taken during 2003 to 2006 were analyzed. The dietary items included were polar bear, caribou, musk oxen, several marine species such as whales, seals, bird and fish as well as imported food. The contaminant concentrations of the dietary items as well as their chemical properties, uptake, biotransformation and excretion allowed us to estimate the POP concentration in liver, blood, muscle and adipose tissue following long-term exposure to the traditional Greenlandic diet using the PBPK model. Significant correlations were found between chemically analyzed POP blood concentrations and calculated daily intake of POPs for Qeqertarsuaq, Nuuk and Narsaq Inuit but not for the northernmost settlement Qaanaaq, probably because the highest blood POP level was found in this district which might mask the interview-based POP calculations. Despite the large variation in circulating blood POP concentrations, the PBPK model predicted blood concentrations of a factor 2-3 within the actual measured values. Moreover, the PBPK model showed that estimated blood POP concentration increased significantly after consumption of meals. For individuals who had a high internal burden of POPs accumulated over years, the estimated blood levels were less influenced by recent meal intake. The model results also indicated that of the POPs accumulated in the body the concentrations were highest for CB-153 (oxychlordane: 0.6%; DDE and CB-99: 2.9%; HCB: 4.4%; CB-153: 34.5%). Furthermore, the model also estimated a significant internal body POP burden even several years after the mentioned dietetic shift and that contaminant accumulation was 2-6 folds faster than the decay after a shift to a diet low in contaminants. Using the PBPK model approach, we seek to improve the knowledge on contaminant body burden in humans of the Arctic. However, it should be noted that calculations of daily POP intake may be subject to considerable uncertainty due to imprecise information from the dietary interview. Based on these results we suggest that PBPK modeling is implemented as a tool in future human health exposure and effect assessments in Greenland.

Endocrine-disrupting effects of compounds in Danish streams.

Effluents from municipal wastewater-treatment plants and scattered dwellings, as well as runoff from agricultural fields, are sources of endocrine-disrupting compounds (EDCs) in the aquatic environment. The present study investigated the correlation between the occurrence of EDCs in nine Danish streams using passive samplers (polar organic integrative samplers and silicone membranes) and determined their possible biological effects as assessed by mammal cell cultures and the mussel (Unio tumidus). The passive samplers and mussels were exposed simultaneously at the study sites. The extracts from the passive samplers were used to measure the concentrations of EDCs and the biological effects on the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR)-receptor transactivation. Male mussels were investigated for biomarkers of endocrine effects, such as the levels of vitellogenin-like proteins measured as alkali-labile phosphate (ALP). EDC concentrations, hormone-receptor transactivation (ER, AR, AhR), and level of ALP were greater downstream of wastewater-treatment plants compared with upstream sites and sites supposed to be relatively nonimpacted by wastewater. Furthermore, there was a significant positive correlation between in vitro AhR transactivation and frequency of ALP of male mussels. We conclude that wastewater effluent is an important source of endocrine-disrupting effects in the aquatic environment and that the combination of biological effect measurements and chemical analyses based on passive sampling is useful in the assessment of the ecological state of the aquatic environment.

The combined effect of persistent organic pollutants in the serum POP mixture in Greenlandic Inuit: xenoestrogenic, xenoandrogenic and dioxin-like transactivities.

Greenlandic Inuit have high body burden of persistent organic pollutants (POPs). We analyzed the combined effect of the actual lipophilic serum POP mixture on estrogen-, androgen- and aryl hydrocarbon-receptor functions as effect biomarkers, and the associations between the effect biomarkers and serum POPs, and lifestyle characteristics. The serum POPs were extracted from 232 Inuit from Ittoqqortoormiit, Narsaq and Qeqertarsuaq. The POP-related receptor transactivities correlated negatively to the POP levels and were associated to the lifestyle characteristics. The POP-related receptor transactivities can be used as effect biomarkers. The serum POPs have hormone disruptive potentials.

Effects of selected phytoestrogens and their mixtures on the function of the thyroid hormone and the aryl hydrocarbon receptor.

Phytoestrogens (PEs) are natural plant components, which can induce biologic responses in vertebrates by mimicking or blocking the actions of natural hormones or influencing the hormone production in the body. This study investigated the effect of different mixtures composed of food-relevant PEs on the thyroid hormone (TH) system assessing the proliferation of the 3,3',5-triiodi-L-thryonine (T3) dependent rat pituitary GH3 cells using the T-screen assay, and the effect on the aryl hydrocarbon receptor (AhR) transactivation using an AhR-luciferase reporter gene assay. Most tested PEs and their mixtures showed effect on both the TH and AhR system. Single isoflavonoid metabolites and their mixture and coumestrol induced GH3 cell growth and AhR transactivity dose-dependently. Isoflavonoid metabolites elicited an additive effect on the T3-dependent GH3 cell growth, and a synergistic effect on the AhR transactivity. In conclusion, nutrition-relevant PEs, alone and in mixture may possess endocrine-disrupting potential by interfering with TH and AhR functions, which need to be considered when assessing the effects on human health.

Effects of phthalates on the human corneal endothelial cell line B4G12.

Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2-ethylhexyl phthalate (DEHP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DnOP), and di-isononyl phthalate (DINP). Gene expression and secretion of inflammatory cytokines were evaluated after exposure to DBP. Decreased cell proliferation was observed for the phthalates DBP, BBP, and DEHP, and cell toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1ß (IL-1ß) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell line B4G12 may be a potential model for inflammatory eye irritancy testing of phthalates.

Perfluorinated compounds are related to breast cancer risk in Greenlandic Inuit: a case control study.

BACKGROUND: Breast cancer (BC) is the most common cancer for women in the western world. From very few cases an extraordinary increase in BC was observed in the Inuit population of Greenland and Canada although still lower than in western populations. Previous data suggest that exposure to persistent organic pollutants (POPs) might contribute to the risk of BC. Rat studies showed that perfluorinated compounds (PFCs) cause significantly increase in mammary fibroadenomas. This study aimed at evaluating the association between serum levels of POPs/PFCs in Greenlandic Inuit BC cases and their controls, and whether the combined POP related effect on nuclear hormone receptors affect BC risk. METHODS: Thirty-one BC cases and 115 controls were sampled during 2000-2003 from various Greenlandic districts. The serum levels of POPs, PFCs, some metals and the combined serum POP related effect on estrogen- (ER), androgen- (AR) and Ah-receptor (AhR) transactivity were determined. Independent student t-test was used to compare the differences and the odds ratios were estimated by unconditional logistic regression models. RESULTS: We observed for the very first time a significant association between serum PFC levels and the risk of BC. The BC cases also showed a significantly higher concentration of polychlorinated biphenyls at the highest quartile. Also for the combined serum POP induced agonistic AR transactivity significant association to BC risk was found, and cases elicited a higher frequency of samples with significant POP related hormone-like agonistic ER transactivity. The AhR toxic equivalent was lowest in cases. CONCLUSIONS: The level of serum POPs, particularly PFCs, might be risk factors in the development of BC in Inuit. Hormone disruption by the combined serum POP related xenoestrogenic and xenoandrogenic activities may contribute to the risk of developing breast cancer in Inuit. Further investigations are needed to document these study conclusions.

Endocrine potency of wastewater: contents of endocrine disrupting chemicals and effects measured by in vivo and in vitro assays.

Industrial and municipal effluents are important sources of endocrine disrupting compounds (EDCs) discharged into the aquatic environment. This study investigated the endocrine potency of wastewater and the cleaning efficiency of two typical urban Danish sewage treatment plants (STPs), using chemical analysis and a battery of bioassays. Influent samples, collected at the first STP grate, and effluent samples, collected after the sewage treatment, were extracted using solid phase extraction. Extracts were analyzed for the content of a range of industrial chemicals with endocrine disrupting properties: phthalate metabolites, parabens, industrial phenols, ultraviolet screens, and natural and synthetic steroid estrogens. The endocrine disrupting bioactivity and toxicity of the extracts were analyzed in cell culture assay for the potency to affect the function of the estrogen, androgen, aryl hydrocarbon, and thyroid receptors as well as the steroid hormone synthesis. The early-life stage (ELS) development was tested in a marine copepod. The concentrations of all analyzed chemicals were reduced in effluents compared with influents, and for some to below the detection limit. Influent as well as effluent samples from both STPs were found to interact with all four receptors and to interfere with the steroid hormone synthesis showing the presence of measured EDCs. Both influent samples and one of the effluent samples inhibited the development of the copepod Acartia tonsa. In conclusion, the presence of EDCs was reduced in the STPs but not eliminated, as verified by the applied bioassays that all responded to the extracts of effluent samples. Our data suggest that the wastewater treatment processes are not efficient enough to prevent contamination of environmental surface waters.

Xenohormone transactivities are inversely associated to serum POPs in Inuit.

BACKGROUND: The persistent organic pollutants (POPs) are highly lipophilic and resistant to biodegradation and found in e.g. seafood and marine mammals. Greenlandic Inuit have high intake of marine food and thus high POP burden that varies according to local conditions and dietary preference. We do for the very first time report the serum POP related non-steroidal xenohormone activity of Inuit across Greenland. The aims were 1) to determine the integrated xenohormone bioactivities as an exposure biomarker of the actual lipophilic serum POP mixture measuring the effect on estrogen (ER) and androgen receptor (AR) transactivity in citizens from different Greenlandic districts and 2) to evaluate associations to serum POP markers (14 PCBs and 10 pesticides) and lifestyle characteristics. METHODS: Serum samples from 121 men and 119 women from Nuuk, Sisimiut and Qaanaaq were extracted using SPE-HPLC fractionation to obtain the serum POP fraction free of endogenous hormones. The serum POP fraction was used for determination of xenohormone transactivity using ER and AR reporter gene assays. RESULTS: In overall, the xenohormone transactivities differed between districts as well as between the genders. Associations between the transactivities and age, n-3/n-6 and smoker years were observed. The xenoestrogenic and xenoandrogenic transactivities correlated negatively to the POPs for the combined female and male data, respectively. CONCLUSION: The non-steroidal xenohormone transactivities can be used as an integrated biomarker of POP exposure and lifestyle characteristics. The actual serum POP mixtures antagonized the age adjusted sex hormone receptor functions. Comparison of different study populations requires in addition to age inclusion of diet and lifestyle factors.

Plastic components affect the activation of the aryl hydrocarbon and the androgen receptor.

Phenols and plasticizers are widely used in the plastic industry, in food packaging and to impart softness and flexibility to normally rigid plastic medical devices and children's toys. The effects on the aryl hydrocarbon receptor (AhR) and the androgen receptor (AR) were assessed using luciferase reporter gene assays of the following compounds: bisphenol A (BPA), 4-n-nonylphenol (nNP), 4-tert-octylphenol (tOP), bis(2-ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DNOP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), 4-chloro-3-methylphenol (CMP), 2-phenylphenol (2-PP), 2,4-dichlorophenol (DCP), resorcinol and bis(2-ethylhexyl) adipate (DEHA). Furthermore, a mixture of selected compounds was tested at the no-observed-effect concentration (NOEC), the low-observed-effect concentration (LOEC) and the half-maximum-effect/inhibitory concentration (EC50/IC50) of the single chemicals. Both receptors were affected by BPA, nNP, BBP, CMP, DCP and resorcinol whereas DEHP, DIDP and DBP affected only the AhR and tOP and 2-PP antagonised the AR activity. The mixture was composed of 6 compounds, of which one compound weakly induced the AhR but all compounds antagonized the AR activation. Using the concentration addition principle additive effects were observed at the NOEC, LOEC and EC50/IC50 for both receptors. Our in vitro data suggest that the effect of a mixture depends on the concentration, character, potency and composition of the single mixture compounds and that also the combined effects of the compounds should be taken into consideration for risk assessment of human health.

Xenobiotic activity in serum and sperm chromatin integrity in European and inuit populations.

Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans.

Relation between serum xenobiotic-induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations.

Persistent organic pollutants (POPs) can interfere with hormone activities and are suspected as endocrine disrupters involved in disorders, e.g. reproductive disorders. We investigated the possible relation between the actual integrated serum xenoestrogenic, xenoandrogenic and aryl hydrocarbon receptor (AhR) activities, and the sperm DNA damage and sperm apoptotic markers of 262 adult males (54 Inuits from Greenland, 69 from Warsaw (Poland), 81 from Sweden, and 58 from Kharkiv (Ukraine)) exposed to different levels of POPs. Xenobiotic-induced receptor activities were determined by receptor-mediated luciferase reporter gene expression. Sperm DNA damage was measured using terminal deoxynucleotidyl transferase-driven dUTP nick labeling assay (TUNEL) and pro- (Fas) and anti-apoptotic (Bcl-xL) markers were determined by immune methods. Different features of xenobiotic-induced receptor activity in serum and sperm DNA fragmentation and apoptotic markers existed between the Inuits and the European Caucasians. Negative correlations between xenobiotic-induced receptor activities and DNA damage were found for Inuits having relatively lower xenoestrogenic, lower dioxin-like activity, and lower sperm DNA damage, but higher xenoandrogenic activity. In contrast, in the European groups, xenobiotic-induced receptor activities were found to be positively correlated with the DNA damage. Further research must elucidate whether altered receptor activities in concerted action with genetic and/or nutrient factors may have protecting effect on sperm DNA damage of the Inuit population.

Semen quality in relation to xenohormone and dioxin-like serum activity among Inuits and three European populations.

BACKGROUND: Semen quality in humans may be influenced by exposure to endocrine-disrupting compounds. OBJECTIVES: We analyzed associations between semen characteristics and serum xenoestrogen receptor (XER), xenoandrogen receptor (XAR), and aryl hydrocarbon receptor (AhR) transactivity. XER and XAR activity were measured in serum samples cleared for endogenous steroid hormones and AhR activity in raw lipophilic serum extracts free of proteins. RESULTS: All together, 319 men from Warsaw (Poland), Greenland, Kharkiv (Ukraine), and Sweden provided semen and blood samples. No strong and consistent associations between xenobiotic activity and semen quality measures were observed in the four populations. However, when the data were combined across populations sperm concentration increased 40% per unit increase in XER activity [95% confidence interval (CI), 1-79%] in the subgroup with XER activity below the reference level. Among subjects with XER activity above the reference level an increase of 14% (95% CI, 2-28%) was found. Furthermore, an increase of 10% motile sperm per unit increase in XER activity below reference level (95% CI, 0.2-20) was found. We are unable to exclude that the associations are chance findings. CONCLUSION: Alteration of XER, XAR, or AhR transactivity within the range found in serum from the general European and Inuit population seems not to markedly deteriorate sperm cell concentration, motility, or morphology in adult men.

Xenoandrogenic activity in serum differs across European and Inuit populations.

BACKGROUND: Animal and in vitro studies have indicated that human male reproductive disorders can arise as a result of disrupted androgen receptor (AR) signalling by persistent organic pollutants (POPs). Our aim in the present study was to compare serum xenoandrogenic activity between study groups with different POP exposures and to evaluate correlations to the POP proxy markers 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE). METHODS: We determined xenoandrogenic activity in the serum fraction containing the lipophilic POPs but free of endogenous hormones. Adult male serum (n = 261) from Greenland, Sweden, Warsaw (Poland), and Kharkiv (Ukraine) was analyzed. Xenoandrogenic activity was determined as the effect of serum extract alone (XAR) and in the presence of the synthetic AR agonist R1881 (XARcomp) on AR transactivated luciferase activity. RESULTS: The study groups differed significantly with respect to XARcomp activity, which was increased in the Inuits and decreased in the European study groups; we observed no difference for XAR activity. We found the highest level of the AR antagonist p,p'-DDE in Kharkiv, and accordingly, this study group showed the highest percent of serum samples with decreased XARcomp activities. Furthermore, the percentage of serum samples with decreased XARcomp activities followed the p,p'-DDE serum level for the European study groups. No correlations between serum XAR or XARcomp activities and the two POP markers were revealed. CONCLUSIONS: The differences in XARcomp serum activity between the study groups suggest differences in chemical exposure profiles, genetics, and/or lifestyle factors.

Ex vivo induction of cytokines by mould components in whole blood of atopic and non-atopic volunteers.

We investigated the time-course release of IL-1beta and IL-8 protein as well as the steady state mRNA level of their genes in human whole blood after stimulation with LPS, beta-1,3-D-glucan and mould extracts. We compared the response of 10 non-atopic and 10 atopic individuals. In parallel, cytokine protein release and the corresponding steady state mRNA level was determined by the standard ELISA and real-time on-line RT-PCR methods, respectively. Glucan induced the highest level of IL-1beta mRNA and protein release after 3 h. IL-8 was induced at 3 h after glucan, but not after LPS, induction, which indicates different induction pathways. At the time-points found to elicit the optimal cytokine response significantly higher basal cytokine mRNA levels and significantly lower LPS induced cytokine mRNA levels were observed in the non-atopic group. Generally, mould components induced cytokine mRNA steady state to lower levels in the atopics compared to the non-atopics. In contrast, no differences were found between the two groups in their capacity to induce cytokine protein release. These findings persisted after correction for the percentage of mononuclear cells. The data supported our hypothesis that "exhaustion" of the atopic immune system elicits lower basal cytokine mRNA levels and that analysis of cytokine gene expression has the potential to differentiate between non-atopic and atopic individuals.