DNA-damage, cell-cycle arrest and apoptosis induced in BEAS-2B cells by 2-hydroxyethyl methacrylate (HEMA)

The methacrylate monomer 2-hydroxyethyl methacrylate (HEMA) is commonly used in resin-based dental restorative materials. These materials are cured in situ and HEMA and other monomers have been identified in ambient air during dental surgery. In vitro studies have demonstrated a toxic potential of methacrylates, and concerns have been raised regarding possible health effects due to inhalation. In this study we have investigated the mechanisms of HEMA-induced toxicity in the human lung epithelial cell line BEAS-2B. Depletion of cellular glutathione (GSH) and an increased level of reactive oxygen species (ROS) were seen after 2h of exposure, but the levels were restored to control levels after 12h. After 24h, inhibited cell proliferation and apoptotic cell death were found. The results of the Comet assay and the observed phosphorylation of DNA-damage-associated signalling proteins including Chk2, H2AX, and p53 suggest that the toxicity of HEMA is mediated by DNA damage. Further, the antioxidant trolox did not counteract the HEMA-induced cell-cycle arrest, which indicates that the DNA damage is of non-oxidative origin.     

Adverse Effects of Epiphytic Crustose Lichens upon Stem Photosynthesis and Chlorophyll of Populus tremula L.

Dry cork layer (phellem) in stems of Populus tremula transmitted 35–55 percent of incident irradiation, depending upon moisture content. A cover of crustose Lecanora lichens reduced transmission through phellem to 10 percent or less of incident irradiation. The bark contains photosynthetically active cells. Apparent quantum yield for photosynthetic O₂-evolution was 0.017 in bark covered with dry Lecanora compared with 0.070 in naked bark. The capacity for gross photosynthesis in high light (1090 μmol photons m^?2 s^?1) was reduced by 50 percent in Lecanora-covered bark. Lecanora did not reduce the ratio between variable and maximal chlorophyll a fluorescence (F_v/F_m). Chlorophyll content per unit area was similar in leaves and naked bark of Populus tremula. The chlorophyll content in the bark decreased with increasing chlorophyll content in Lecanora. Chlorophyll a/b ratio was 2.5 in the bark compared with 4.0 in leaves and in Lecanora, and the ratio decreased down the stems. The a/b ratio was 2.3 in Lecanora covered bark compared with 2.6 in naked bark. The changes in bark photosynthesis below a Lecanora crust were probably due to acclimation of bark photosynthesis to shade, since the lichen acids in the measured lichens neither suppressed photosynthetic O₂-evolution nor changed the F_v/F_m in bark disks.     

Possible functional roles of cortical depsides and medullary depsidones in the foliose lichen Hypogymnia physodes

Secondary compounds were quantified in 75 thalli of the foliose lichen Hypogymnia physodes collected in habitats along a natural shade-sun gradient from dark spruce forests to sun-exposed sea cliffs. The irradiance in all the 75 lichen sites was estimated from hemispherical photographs. The content of lichen compounds per thallus area correlated positively with irradiance level (r²=0.73), and the mean concentration increased from 6.7% in the spruce forest to 14.4% on sea cliffs. The medullary depsidones, physodic, physodalic and protocetraric acids, constituted >95% of the total pool of extractable secondary compounds, the cortical depsides, atranorin and chloratranorin, represented <5%. Both cortical compounds correlated well with direct and with diffuse radiation, whereas the three medullary compounds correlated better with diffuse than with direct radiation. Mentioned trends are consistent with a solar radiation screening hypothesis of both groups of these colourless compounds occuring as tiny crystals outside fungal hyphae. However, the UV-B protective hypothesis of the compounds was further tested in a lab experiment. Unnaturally high UV-B doses were required to significantly reduce the PSII efficiency (F_V/F_M) of symbiotic algae. Removal of the major pool of secondary compounds with acetone did not increase photobiont susceptibility to UV-B. Therefore, the main functional role of the UV-B absorbing secondary compounds in H. physodes is hardly UV-B screening. Other roles such as PAR-screening and defence against herbivores and pathogenic organisms are discussed.     

High-light damage in air-dry thalli of the old forest lichen Lobaria pulmonaria-interactions of irradiance, exposure duration and high temperature

High-light damage in air-dry thalli of Lobaria pulmonaria were measured in the laboratory as reductions in maximal PSII efficiency (Fv/Fm) after a 48 h recovery in a hydrated state at low light to account for permanent damage. Thalli treated with the lowest light dose (90 mol photons m^-2) recovered normal Fv/Fm-values with increasing irradiances (400-700 nm) up to 1000 mol photons m^-2 s^-1. Doubling this dose lowered the threshold level for damage from 1000 to 320 mol photons m^-2 s^-1, and reduced Fv/Fm at 1000 mol photons m^-2 s^-1 by more than 50%. A second doubling of the dose to 360 mol photons m^-2 caused damage at 200 mol photons m^-2 s^-1, and a nearly complete cessation of PSII efficiency occurred at 1000 mol photons m^-2 s^-1. No reciprocity of irradiance and duration of illumination for PSII function was found. The measured time-dependent decrease in Fv/Fm was remarkably similar for the naturally coupled, but artificially separated, light and temperature factors. Therefore, the damage of high light on desiccated L. pulmonaria seemed to be an additive effect of high irradiance and high temperatures. Air-dry thalli were highly heat susceptible, being affected already at temperatures around 40C. Logging operations in forests are likely to raise the solar radiation at remaining lichen sites to destructive levels.Keywords: Lichens, high-light damage, heat stress, poikilohydric organisms, reciprocity.      

Seasonal Variation of Photoinhibition of Photosynthesis in Bark from Populus Tremula L.

In the bark of Populus tremula L. photochemical efficiency of photosystem 2 (PS2) determined as Fv/Fm decreased during winter. The strongest reduction was found after cold periods. The degree of reduction depended on irradiance since the lowest levels of Fv/Fm were found on the sun-exposed side of the stem and below thin phellem. Therefore, photoinhibition was partly responsible for the reduction in Fv/Fm. The photochemical efficiency of PS2 recovered in late April about a month before the trees got leaves. In the laboratory, Fv/Fm recovered within about a week under low irradiance at 20 °C. Rapid recovery of photochemical efficiency of PS2 in the bark may be important to reduce respiratory loss of CO2 from the stem before the trees get leaves.     

Ontogeny of neuronal nitric oxide synthase, NOS I, in the developing porcine kidney

To determine if the developing kidney differs from the adult in the expression of the neuronal nitric oxide synthase, NOS I, these experiments measured mRNA gene expression by RNase protection assay and protein content by Western blot of NOS I in piglets at ages newborn and 3, 7, 10, 14, and 21 days and adult pigs. Whole kidney NOS I mRNA was greatest at birth and decreased progressively during renal maturation to adult levels. NOS I protein content paralleled this developmental pattern. Cortical NOS I protein was equivalent in newborn and 14-day-old piglets and was greater at both ages than the adult. Medullary NOS I protein was relatively greater than cortical in both immature ages and decreased from a peak at birth to adult levels. We conclude the following. 1) During postnatal maturation, renal NOS I mRNA and protein content show a pattern that is developmentally regulated. 2) This developmental pattern of NOS I after birth may, in part, contribute to the enhanced functional role of NO during renal maturation.     

Expression of endothelial nitric oxide synthase in the postnatal developing porcine kidney

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.     

Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells

The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.     

Growth and ecophysiological acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light climates

This study aims to assess biomass and area growth of 600 thalli of the old forest lichen, Lobaria pulmonaria, transplanted to three successional boreal forest stands with (1) natural rainfall regime, (2) additional moistening during dry days, and (3) additional moistening with added nutrients. Mean biomass growth during 100 days varied from 8.3% in the dark young spruce forest to 23.1% in the clear-cut area, with the old forest in between (16.0%). Additional moistening did not enhance lichen growth, probably because the transplantation period was wet. Nutrient additions slightly increased area growth compared to artificial water additions only. Growth was determined by a combination of external (forest stand, site factors) and internal factors (chlorophyll content, biomass per area). Transplants acclimated to high light by increasing thickness and chlorophyll a/b-ratio. Some visible bleaching and a strong positive correlation between chlorophyll content per area and lichen growth in clear-cuts suggest some high light-induced chlorophyll degradation. We believe that biomass growth and natural occurrence of L. pulmonaria is controlled by a delicate balance between light availability and desiccation risk, and that the species is confined to old forests due to a physiological trade-off between growth potential and fatal desiccation damage, both of which increase with increasing light. The discrepancy between potential and realized ecological niches is probably caused by a long-term risk to be killed in open habitats by high light during long periods with no rain.