Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS

We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ⁴-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10⁻¹⁰ mol L⁻¹ and cross-reactivity with most Δ⁴-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry [UPLC-ESI(+)-MS/MS]. In multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to 10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species, that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone, were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea.     

Comparison of vascular plant diversity and species composition of coppice and high beech forest in the Banat region,Romania

Abandonment of coppicing is one of the main reasons of diversity decline in European temperate forests. To reverse this trend, coppicing has been reintroduced in several forests, especially in areas of high conservation value. However, empirical information on the effects of coppicing on plant diversity and composition is still scarce. By comparing vegetation data from active coppices and beech-dominated high forests in the Banat region in Romania, we tested the hypothesis that coppices have a higher plant diversity and different plant species composition than high forests. Data were collected in 60 randomly placed phytosociological relevés and were analysed using linear models and multivariate methods. As expected, we found differences in understorey plant composition. Herb species with various environmental demands–sciophytes-(hemi)-heliophytes and grassland species–were more frequent in coppices whereas high forests had more vernal species and sciophytes. Coppices also had slightly greater plant diversity but did not differ in herb species richness. Our results demonstrate that coppicing has a small positive effect on plant diversity but a large effect on species composition. Coppice restoration may be especially beneficial for populations of thermophilous and non-forest species.     

Variation in canopy openness among main structural types of woody vegetation in a traditionally managed landscape

The decrease in canopy openness after the abandonment of traditional coppicing in the twentieth century has been previously identified as the main reason behind changes in species composition and diversity in lowland woodlands in Europe. However, little is known about the role of other traditional practices in shaping woody vegetation and canopy cover in the past. In a traditionally managed landscape of the Banat region, western Romania, where long-established activities, such as human-induced burning, coppicing and grazing of woody vegetation are still being practised, we studied woody vegetation structure and canopy openness in 70 plots. Using a set of structural traits, we classified woody vegetation into four groups: active coppices, scrubs, abandoned coppices and high forests. Surprisingly, the lowest canopy openness was found in active coppices, probably due to high canopy recovery rate and selection coppice system. Scrubs, in which grazing and browsing were the most common activities in the past, were the most open type of woody vegetation. Our results emphasize the role of other traditional management practices in addition to coppicing in keeping canopy openness relatively high and spatially heterogeneous.     

Patterns of functional diversity of two trophic groups after canopy thinning in an abandoned coppice

Coppice abandonment had negative consequences for the biodiversity of forest vegetation and several groups of invertebrates. Most coppicing restoration studies have focused only on a single trophic level despite the fact that ecosystems are characterized by interactions between trophic levels represented by various groups of organisms. To address the patterns of functional diversity in the perspective of coppicing restoration, we studied the short-term effects of conservation-motivated tree canopy thinning in an abandoned coppice with standards in Central Europe, a region where such attempts have been rare so far. The functional diversity of vascular plants and spiders, chosen as two model trophic groups within the forest ecosystem, was compared between thinned and control forest patches. To characterize functional patterns, we examined several functional traits. These traits were assigned to two contrasting categories: response traits reflecting a change of environment (for both vascular plants and spiders) and effect traits influencing the ecosystem properties (only for vascular plants). Functional diversity was analysed by CCA using two measures: community-weighted means (CWM) and Rao’s quadratic diversity (RaoQ). CCA models revealed that the canopy thinning had a positive effect on the diversity of the response traits of both trophic groups and negatively influenced the diversity of effect traits. In addition, we found distinct seasonal dynamics in functional diversity of the spider communities, which was probably linked to leaf phenology of deciduous trees. We conclude that canopy thinning affected functional diversity across trophic groups during the initial phase of coppicing restoration. With necessary precautions, careful canopy thinning can be effectively applied in the restoration of functional diversity in abandoned coppices.     

Heat shock proteins hsp32 and hsp70 as biomarkers of an early response?: In vitro induction of heat shock proteins after exposure of cell culture to carcinogenic compounds and their real mixtures

Heat shock proteins (hsp) are a highly conserved group of proteins that are synthesized as a response to different forms of stress (heat, toxic chemicals, diseases, non-physiological pH changes). Because of their high sensitivity to changes in the environment, these proteins were suggested as possible early biomarkers of exposure in ecotoxicological studies. The purpose of the present study was to check the suitability of hsp32 and hsp70 as biomarkers of in vitro exposure to environmentally relevant carcinogens: polycyclic aromatic hydrocarbons (PAHs), their nitro-derivates, aromatic amines, acrylonitrile (ACN) and the mixture of organic compounds adsorbed onto ambient airborne particles (extractable organic matter, EOM).The expression of hsp32 and hsp70 was studied in human diploid lung fibroblasts (HEL cells) and human monocytic leukaemia cells (THP-1 cells) incubated in vitro with different concentrations of dibenzo[a,l]pyrene (DB[a,l]P), 1-nitropyrene, (NP), 4-aminobiphenyl (ABP), ACN and EOM for different periods of time. The incubation of cells with DB[a,l]P, NP, ABP and EOM did not result in increased levels of hsp32 or hsp70, either in dose- or time-dependent manner. ACN induced the expression of hsp32 as well as hsp70 in HEL and THP-1 cells, which probably reflects its ability to induce oxidative stress. We conclude that hsp32 and hsp70 are not suitable biomarkers of an early exposure to PAHs, their nitro-derivates, aromatic amines or EOM under the conditions used.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.     

Combination of two pseudoaneurysms in a patient 6 years after ascending aorta and aortic valve replacement

BACKGROUND: We report successful treatment of a patient 6 years after ascending aorta and aortic valve replacement suffering from a combination of two pseudoaneurysms. The first of them originated from the coronary ostial suture line and the second pseudoaneurysm originated from the distal suture line. METHODS: We performed re-replantation of the left main trunk to the prosthesis and then we resected the pseudoaneurysm originating from the distal suture line and we replaced the entire aorta by a vascular graft. RESULTS: The postoperative convalescence was uneventful, the patient was discharged two weeks after the surgery. The patient is currently in a very good condition one year after the surgery. CONCLUSIONS: Coronary ostial pseudoaneurysm in patients after ascending aorta and aortic valve replacement with the use of "button technique" is a quite rare situation. The combination of two pseudoaneurysms originating from different suture lines in one patient seems to be unique in literature reviews.     

Global environmental change effects on plant community composition trajectories depend upon management legacies

The contemporary state of functional traits and species richness in plant communities depends on legacy effects of past disturbances. Whether temporal responses of community properties to current environmental changes are altered by such legacies is, however, unknown. We expect global environmental changes to interact with land‐use legacies given different community trajectories initiated by prior management, and subsequent responses to altered resources and conditions. We tested this expectation for species richness and functional traits using 1814 survey‐resurvey plot pairs of understorey communities from 40 European temperate forest datasets, syntheses of management transitions since the year 1800, and a trait database. We also examined how plant community indicators of resources and conditions changed in response to management legacies and environmental change. Community trajectories were clearly influenced by interactions between management legacies from over 200 years ago and environmental change. Importantly, higher rates of nitrogen deposition led to increased species richness and plant height in forests managed less intensively in 1800 (i.e., high forests), and to decreases in forests with a more intensive historical management in 1800 (i.e., coppiced forests). There was evidence that these declines in community variables in formerly coppiced forests were ameliorated by increased rates of temperature change between surveys. Responses were generally apparent regardless of sites’ contemporary management classifications, although sometimes the management transition itself, rather than historic or contemporary management types, better explained understorey responses. Main effects of environmental change were rare, although higher rates of precipitation change increased plant height, accompanied by increases in fertility indicator values. Analysis of indicator values suggested the importance of directly characterising resources and conditions to better understand legacy and environmental change effects. Accounting for legacies of past disturbance can reconcile contradictory literature results and appears crucial to anticipating future responses to global environmental change.