Assessment of in vitro human dermal absorption studies on pesticides to determine default values, opportunities for read-across and influence of dilution on absorption.

Dermal absorption is an integral part of non-dietary human safety risk assessments for agrochemicals. Typically, dermal absorption data for agrochemical active substances are generated from the undiluted formulation concentrate and its spray dilutions. European Food Safety Authority (EFSA) guidance, which combines highly conservative default values, very limited opportunities for read-across from existing data and other overly conservative conclusions, was the driver for this assessment. To investigate the reliability of the EFSA guidance, a homogeneous data-set of 190 GLP and OECD guideline compliant in vitro human skin studies, chosen to match the test method preferred by EU data requirements, was evaluated. These studies represented a wide range of active substances, formulation types, and concentrations. In alignment with EFSA guidance on human exposure assessment, a conservative estimate of absorption (95th percentile) was chosen to define defaults, which were also based on the EFSA worst-case assumption that all material in skin, excluding the first two tape strips, is absorbed. The analysis supports dermal absorption defaults of 6% for liquid concentrates, 2% for solid concentrates, and 30% for all spray dilutions, irrespective of the active substance concentration. Relatively high dermal absorption values for organic solvent-based formulations, compared to water-based or solid concentrates, support their use as worst-case surrogate data for read-across to other formulation types. The current review also shows that dermal absorption of sprays does not increase linearly with increasing dilution, and provides a novel, science-based option for extrapolation from existing data.

Selenoprotein P, rather than glutathione peroxidase, as a potential marker of septic shock and related syndromes.

BACKGROUND/AIMS: Oxidative stress is involved in sepsis-related endothelium dysfunction. Selenoprotein-P (Sel-P), the main plasma selenoprotein, may have high antioxidant potential, and binds to endothelium. We hypothesize that, in septic shock, and similar syndromes such as systemic inflammatory response syndrome (SIRS), Sel-P binds massively to endothelium, causing a drop in Sel-P plasma concentration. METHODS: Plasma Se, Sel-P and albumin concentrations, and glutathione peroxidase (GPx) activity were measured in patients with septic shock and SIRS with organ failure (S group, n = 7 and n = 3, respectively) admitted to the intensive care unit (ICU) and compared to non-SIRS patients (NS group, n = 11) and healthy volunteers (HV group, n = 7). RESULTS: On ICU admission, plasma Sel-P concentrations were 70% lower in the S group than in the other groups [15 (10-26) vs. 44 (29-71) and 50 (45-53) nmol/l] and were lower in nonsurviving septic-shock patients. GPx activity did not differ between groups. Sel-P was significantly lower before ICU death in the 3 deceased patients of the S group (septic shock) than in the 3 patients of the non-SIRS group. CONCLUSIONS: Early decrease in Sel-P plasma concentrations was specifically observed in septic shock and was similar in SIRS patients whereas GPx activity remained unchanged. Further studies are needed to determine whether Sel-P can be an early marker of septic shock linked to microvascular injury.

Identification of an element within the promoter of human selenoprotein P responsive to transforming growth factor-beta.

Selenoprotein P (SeP) is a plasma protein that contains up to 10 selenocysteine residues and accounts for about 50% of total selenium in human plasma. We have previously shown that SeP expression in the human liver cell line HepG2 is inhibited by transforming growth factor (TGF)-beta1 on a transcriptional level. Smad proteins are the transcriptional mediators of TGF-beta signalling and putative Smad-binding elements (SBE) comprising the core sequence CAGACA are present at two positions in the SeP promoter. The aim of our study was to investigate whether Smad molecules are involved in inhibition of SeP expression by TGF-beta1 and to locate the promoter region critical for this effect. As seen in electrophoretic-mobility-shift assays, TGF-beta1 treatment led to enhanced binding of nuclear proteins to a putative SBE from the SeP promoter. Overexpression of Smad 3 and 4, but not of Smad 2, resulted in a marked down-regulation of SeP mRNA expression. Similar effects were observed for luciferase expression under control of a human SeP-promoter construct. Deletion as well as point-mutation of putative SBEs led to a loss of promoter sensitivity towards TGF-beta1 treatment. Hence, we demonstrated an involvement of Smad 3 and 4 in transcriptional regulation of SeP by TGF-beta1 and we were able to identify the TGF-beta-responsive element in the SeP promoter.

Modulation of selenoprotein P expression by TGF-beta(1) is mediated by Smad proteins.

Selenoprotein P (SeP) is a selenium-rich plasma protein which accounts for more than 50% this study, the effect of TGF-beta(1) on the expression of SeP in the human liver cell line HepG2 was investigated. Western analysis revealed a dose-dependent reduction of SeP content in cell supernatant. RT-PCR analysis of SeP-mRNA expression demonstrated a marked inhibition and a reporter gene under control of the SeP promoter was negatively regulated by TGF-beta(1). Smad proteins are the transcriptional mediators of TGF-beta signaling. A putative Smad-binding element (SBE) is present in the SeP promoter. In electrophoretic-mobility-shift assays, TGF-beta(1) enhanced the binding of nuclear proteins to this SBE. Overexpression of Smad3 and 4 resulted in a downregulation of SeP-promoter activity whereas deletion of the SBE led to a loss of TGF-beta(1) responsiveness. We conclude that SeP expression is modulated by the binding of Smad3/4 complexes to a functional SBE in the SeP promoter.

Cell-specific regulation of human aryl hydrocarbon receptor expression by transforming growth factor-beta(1).

Previous studies showed that TGF-beta down-regulates aryl hydrocarbon (AhR) expression in human lung carcinoma cells A549. Here we analyzed the molecular mechanisms by which TGF-beta modulates AhR expression. A 5799-nucleotide 5'-flanking region of human AhR gene was isolated. Transient transfection studies of full-length (hAhRP) and deletion promoter constructs indicate the requirement of a cis-regulatory element encompassing -1980 to -1892 for full constitutive activity. Basal hAhRP activity occurs in a cell-specific manner; human hepatoma HepG2 cells possess a 10-fold higher activity compared with A549 cells. TGF-beta exerts cell-specific effects on hAhRP activity. Treatment of cells with 100 pM TGF-beta leads to a 50% inhibition in A549 and a 3-fold induction in HepG2 cells. Deletion mutagenesis identified a TGF-beta-responsive sequence containing a functional conserved Smad-binding element. Transient overexpression of Smad 2, 3, and 4 indicates that these signal transducers modulate hAhRP activity. The down-regulation of AhR by TGF-beta is modulated by 5'-TG-3'-interacting factor (TGIF). Transient overexpression of TGIF in MDA-MB231 and HepG2 cells led to inhibition of hAhRP activity and a similar decrease of AhR mRNA expression. Our findings indicate that Smad proteins are involved in the cell-specific regulation of AhR expression by TGF-beta.

Selenoprotein P: properties, functions, and regulation.

Selenoprotein P (SeP) is a plasma protein which contains 10 selenocysteine residues per polypeptide. It accounts for more than 50% of the selenium content in rat and human plasma but its function is still not completely understood. However, a function as an extracellular antioxidant seems most probable. A protective function of SeP in human plasma against the potent endotoxin peroxynitrite and phospholipid hydroperoxide reducing activity was demonstrated in vitro. An association of SeP with the vascular endothelium, a prime target of peroxynitrite toxicity, was shown in vivo. SeP of bovine serum acts as a survival-promoting factor in neuronal cell culture. Analysis of the human SeP promoter indicates a transcriptional regulation of SeP by inflammatory mediators.

Transforming growth factor-beta1 inhibits expression of selenoprotein P in cultured human liver cells.

The effect of cytokines on the expression of selenoprotein P (SeP) in the human liver cell line HepG2 was investigated. Treatment with interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha had no effect on SeP levels in culture media or on SeP mRNA expression. Conversely, Western analysis revealed a dose-dependent reduction of SeP content in culture medium after treatment with transforming growth factor (TGF)-beta1 with an 1C50 of 31 pM. Treatment with 100 pM TGF-51 for 48 h led to a decrease to 21 +/- 9% of controls. RT-PCR analysis of SeP mRNA expression demonstrated an inhibition of SeP transcription to 40+/-2% of control levels after 24 h. The expression of a luciferase reporter construct under control of the human SeP promoter was downregulated by TGF-beta1 treatment in a dose-dependent fashion indicating a transcriptional regulation of the SeP gene by TGF-beta1.

Protection by selenoprotein P in human plasma against peroxynitrite-mediated oxidation and nitration.

In order to study functions of selenoprotein P in human plasma, its level was lowered via two techniques, chromatography on Sepharose-bound heparin, or immunoprecipitation; Western blot analysis showed that both techniques were effective at substantially lowering selenoprotein P levels in plasma. When peroxynitrite was infused to maintain a 0.9 microM steady-state concentration, plasma made deficient in selenoprotein P diminished benzoate hydroxylation significantly less than control plasma. Similar differences were found for protein 3-nitrotyrosine formation, determined by Western blot analysis. Conversely, in a selenoprotein P-enriched plasma preparation obtained via heparin-Sepharose chromatography, protection against benzoate hydroxylation was above controls. Likewise, a supernatant from control plasma that had been exposed to anti-selenoprotein P antibodies was less efficient in preventing oxidation and nitration reactions of peroxynitrite than the supernatant from plasma exposed to a non-specific antibody (rabbit anti-sheep IgG). These data demonstrate a role of selenoprotein P in human plasma in the defense against peroxynitrite.

A novel method for the purification of selenoprotein P from human plasma.

Selenoprotein P was purified from human plasma using conventional chromatographic methods featuring metal-chelate-affinity chromatography as the final step. Two distinct isoforms with different selenium content were isolated and identified by N-terminal sequencing and immunoblot analysis. Their molecular mass is 61 and 51 kDa, respectively. Both isoforms could be detected in fresh plasma from five individuals. This rules out the possibility of the second isoform being an artifact which results from degradation of full-length selenoprotein P during purification.