To spread or not to spread? Assessing the suitability of sewage sludge and other biogenic wastes for agriculture reuse.

Sewage sludge (biosolids) management represents a worldwide issue. Due to its valuable properties, approximately one half of the EU production is recovered in agriculture. Nevertheless, growing attention is given to potential negative effects deriving from the presence of harmful pollutants. It is recognized that a (even very detailed) chemical characterization is not able to predict ecotoxicity of a mixture. However, this can be directly measured by bioassays. Actually, the choice of the most suitable tests is still under debate. This paper presents a multilevel characterization protocol of sewage sludge and other organic residues, based on bioassays and chemical-physical-microbiological analyses. The detailed description of the experimental procedure includes all the involved steps: the criteria for selecting the organic matrices to be tested and compared; the sample pre-treatment required before the analyses execution; the chemical, physical and microbiological characterisation; the bioassays, grouped in three classes (baseline toxicity; specific mode of action; reactive mode of action); data processing. The novelty of this paper lies in the integrated use of advanced tools, and is based on three pillars:•the direct ecosafety assessment of the matrices to be reused.•the adoption of innovative bioassays and analytical procedures.•the original criteria for data normalization and processing.

Removal of Antibiotics Using an Algae-Algae Consortium (Chlorella protothecoides and Chlorella vulgaris).

The intensive use of antibiotics (for human, veterinary, and agricultural purposes) has steadily increased over the last 30 years. Large amounts of antibiotic residues are released into aquatic systems, mostly due to inefficient wastewater treatment. Conventional wastewater treatments are not designed to remove emerging contaminants (such as antibiotics) from wastewater. Therefore, algae treatment (phycoremediation) has emerged as a promising choice for cost-effective, eco-friendly, and sustainable wastewater treatment. For this reason, we investigated the removal performance of a well-established algal consortia (Chlorella protothecoides and Chlorella vulgaris) used in passive wastewater treatment ponds (Mosselbay, South Africa). Five antibiotics (sulfamethoxazole, amoxicillin, trimethoprim, ofloxacin, and clarithromycin) were selected for their ubiquity and/or low removal efficiency in conventional wastewater treatment plants (WWTPs). For each antibiotic, two concentrations were used: one environmentally relevant (10 ppb) and another 10 times higher (100 ppb), tested in triplicate and collected at two-time points (7 and 10 days). The algae remained viable over the exposure period (which is similar to the retention time within maturation ponds) and exhibited the capacity to remove sulfamethoxazole (77.3% ± 3.0 and 46.5% ± 5.3) and ofloxacin (43.5% ± 18.9 and 55.1% ± 12.0) from samples spiked with 10 and 100 ppb, respectively. This study demonstrates the potential and innovation of algal remediation for contaminants in a developing country context, where minimal infrastructure is available.

Detection of a functional xenobiotic response element in a widely employed FoxO-responsive reporter construct.

FHRE-Luc is a promoter reporter construct that is widely used to assess the activity of FoxO (forkhead box, class O) transcription factors. We here demonstrate that this promoter construct responds to exposure of HepG2 human hepatoma cells to known agonists of the aryl hydrocarbon receptor (AhR), 3-methylcholanthrene, benzo(a)pyrene, and 6-formylindolo[3,2-b]carbazole. However, FHRE-Luc activation did not coincide with FoxO DNA binding or changes in Akt-induced FoxO phosphorylation after treatment with AhR agonists. Testing FHRE-Luc deletion constructs and using AhR-deficient cells, we found that FHRE-Luc activation by AhR agonists is due to a functional xenobiotic-response element (XRE) spanning the backbone/insert border of the reporter plasmid. In conclusion, care must be taken when using FHRE-Luc to assess FoxO activity in response to stimuli that potentially interfere with xenobiotic signaling.

Adaptation of topoisomerase I paralogs to nuclear and mitochondrial DNA.

Topoisomerase I is essential for DNA metabolism in nuclei and mitochondria. In yeast, a single topoisomerase I gene provides for both organelles. In vertebrates, topoisomerase I is divided into nuclear and mitochondrial paralogs (Top1 and Top1mt). To assess the meaning of this gene duplication, we targeted Top1 to mitochondria or Top1mt to nuclei. Overexpression in the fitting organelle served as control. Targeting of Top1 to mitochondria blocked transcription and depleted mitochondrial DNA. This was also seen with catalytically inactive Top1 mutants, but not with Top1mt overexpressed in mitochondria. Targeting of Top1mt to the nucleus revealed that it was much less able to interact with mitotic chromosomes than Top1 overexpressed in the nucleus. Similar experiments with Top1/Top1mt hybrids assigned these functional differences to structural divergences in the DNA-binding core domains. We propose that adaptation of this domain to different chromatin environments in nuclei and mitochondria has driven evolutional development and conservation of organelle-restricted topoisomerase I paralogs in vertebrates.

The proteasome is an integral part of solar ultraviolet a radiation-induced gene expression.

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

Functional consequences of mitochondrial DNA deletions in human skin fibroblasts: increased contractile strength in collagen lattices is due to oxidative stress-induced lysyl oxidase activity.

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor beta-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-alpha-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both beta-aminopropionitrile and N-tert-butyl-alpha-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.

Role of mitochondria in photoaging of human skin: the defective powerhouse model.

The exact pathogenesis of photoaging of the skin is not yet known. Earlier, a number of molecular pathways explaining one or more characteristics of photoaged skin have been described, but a unifying mechanistic concept is still missing. Here we propose the "Defective Powerhouse Model of Premature Skin Aging", which reconciles most of the earlier conducted research as one concept. In this model, the persistence of UV radiation-induced mtDNA deletions or the infrared radiation-induced disturbance of the electron flow of the mitochondrial electron transport chain leads to inadequate energy production in dermal fibroblasts. As a consequence of this defective powerhouse, retrograde mitochondrial signaling pathways are triggered that then they transduce functional and structural alterations in the skin. This model, which is supported by a growing number of recent studies, is of direct clinical importance in preventing and treating photoaging in human skin.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 44-49; doi:10.1038/jidsymp.2009.1.

Ultraviolet-A irradiation but not ultraviolet-B or infrared-A irradiation leads to a disturbed zinc homeostasis in cells.

Changes of the redox balance in cells alter the availability of intracellular free Zn(2+). Here, cells were exposed to ultraviolet (UV)-A, UV-B, or infrared (IR)-A light irradiation, and the intracellular free zinc pool was monitored. Under sublethal conditions only UV-A irradiation resulted in a transient cytoplasmic and nuclear increase of intracellular free Zn(2+). Likewise, tert-butyl hydroperoxide and singlet oxygen, but not H(2)O(2) or intracellular generation of O(2)(*-) by redox cyclers, mimicked the effects of UV-A irradiation, while disulfide stress by diamide only led to a transient cytoplasmic zinc release. These results show that only certain types of subtoxic cellular stress massively disturb the zinc homeostasis in cells.

The role of near infrared radiation in photoaging of the skin.

Infrared (IR) radiation is non-ionizing, electromagnetic radiation with wavelengths between 760 nm and 1 mm, which is further divided into IRA, IRB and IRC. IR accounts for more than half of the solar energy that reaches the human skin. While IRB and IRC do not penetrate deeply into the skin, more than 65% of IRA reaches the dermis. Human skin is increasingly exposed to IRA-radiation; most relevant sources are (i) natural solar radiation consisting of over 30% IRA, (ii) artificial IRA sources used for therapeutic or wellness purposes and (iii) artificial UV sources contaminated with IRA. As part of natural sunlight, IRA significantly contributes to extrinsic skin aging. This article reviews the cutaneous effects of IRA-radiation, the underlying molecular mechanisms and the available protective strategies.

Changes of MMP-1 and collagen type Ialpha1 by UVA, UVB and IRA are differentially regulated by Trx-1.

Exposure of human skin to solar radiation, which includes ultraviolet (UV) radiation (UVA and UVB) visible light and infrared radiation, induces skin aging. The effects of light have been attributed to irradiation-induced reactive oxygen species (ROS) formation, but the specific signaling pathways are not well understood. Detrimental effects of solar radiation are dermal diseases and photoaging. Exposure of cultured human dermal fibroblasts to UVA, UVB or IRA increased ROS formation in vitro. One important redox regulator is the oxidoreductase thioredoxin-1 (Trx). Trx is ubiquitously expressed and has anti-oxidative and anti-apoptotic properties. Besides its function to reduce H(2)O(2), Trx binds to and regulates transcription factors. The aim of this study was to investigate whether Trx influences the regulation of MMP-1 and collagen Ialpha1 by UVA, UVB and IRA. We irradiated human dermal fibroblasts with UVA, UVB and IRA. UVA, UVB and IRA upregulated MMP-1 expression. Trx inhibited UVA-induced MMP-1 upregulation in a NFkappaB dependent manner. UVA, UVB and IRA reduced collagen Ialpha1 expression. Incubation with Trx inhibited the effects of UVB and IRA on collagen Ialpha1 expression. In conclusion, MMP-1 and collagen Ialpha1, which play important roles in aging processes, seems to be regulated by different transcriptional mechanisms and Trx can only influence distinct signaling pathways induced by UVA, UVB and probably IRA. Thus, Trx may serve as an important contributor to an "anti-aging therapeutic cocktail".

Nuclear redox-signaling is essential for apoptosis inhibition in endothelial cells--important role for nuclear thioredoxin-1.

OBJECTIVE: The redox regulator thioredoxin-1 (Trx) is a potent antioxidative enzyme and exerts important cellular functions. Physiological concentrations of reactive oxygen species (ROS) and of nitric oxide (NO) act as second messengers. Previously, we demonstrated that ROS and NO reduced apoptosis in a Trx-dependent manner. The aim of this study was to determine the underlying mechanisms. METHODS AND RESULTS: First, we investigated the localization of Trx after H2O2 and NO. Both induced nuclear import of Trx, which required karyopherin-alpha. siRNA against karyopherin-alpha inhibited nuclear import of Trx. Analysis of the Trx amino acid sequence and subsequent immunoprecipitation studies revealed that Trx(K81/82E) is not imported into the nucleus under H2O2 treatment and Trx(K81/82/85E) was retained in the cytosol and induced cell death. Trx(K81/82E) abolished the antiapoptotic capacity of H2O2. Glutathione S-transferase P1 (GST-P1) was identified as one major target regulated by H2O2. siRNA against GST-P1 abolished the antiapoptotic effect of H2O2. Cysteine 69, but not cysteines 32 and 35, which are all required for the complete antiapoptotic function of Trx, is not imported into the nucleus. CONCLUSION: H2O2-induced nuclear import of Trx depends on karyopherin-alpha and NO. Trx-dependent induction of GST-P1 expression is required for apoptosis inhibition in endothelial cells.

Cellular response to infrared radiation involves retrograde mitochondrial signaling.

Infrared A radiation (IRA) is a major component of sunlight. Similar to ultraviolet (UV) B and UVA, IRA induces gene transcription. In contrast to the UV response very little is known about the IRA response. In the present study, IRA-induced expression of matrix metalloproteinase-1 (MMP-1) was found to be mediated by the formation of intracellular reactive oxygen species (ROS). Staining of IRA-irradiated cells with MitoSox revealed an increase in mitochondrial superoxide anion production and treatment of fibroblasts with the mitochondrial targeted antioxidant MitoQ completely abrogated the IRA, but not the UVB or UVA1, response. ROS relevant for IRA-induced signaling originated from the mt electron transport chain, because (i) chemical inhibition of the electron transport chain prevented IRA, but not UVB or UVA1, radiation-induced MMP-1 expression, (ii) rho0 fibroblasts specifically failed to increase MMP-1 expression in response to IRA, and (iii) peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) overexpressing fibroblasts with increased electron transport chain content were hypersensitive to IRA radiation-induced gene expression. Thus, IRA, in contrast to UV, elicits a retrograde signaling response in human skin.

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes.

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.

A highly versatile and easily configurable system for plant electrophysiology.

In this study we present a highly versatile and easily configurable system for measuring plant electrophysiological parameters and ionic flow rates, connected to a computer-controlled highly accurate positioning device. The modular software used allows easy customizable configurations for the measurement of electrophysiological parameters. Both the operational tests and the experiments already performed have been fully successful and rendered a low noise and highly stable signal. Assembly, programming and configuration examples are discussed. The system is a powerful technique that not only gives precise measuring of plant electrophysiological status, but also allows easy development of ad hoc configurations that are not constrained to plant studies. •We developed a highly modular system for electrophysiology measurements that can be used either in organs or cells and performs either steady or dynamic intra- and extracellular measurements that takes advantage of the easiness of visual object-oriented programming.•High precision accuracy in data acquisition under electrical noisy environments that allows it to run even in a laboratory close to electrical equipment that produce electrical noise.•The system makes an improvement of the currently used systems for monitoring and controlling high precision measurements and micromanipulation systems providing an open and customizable environment for multiple experimental needs.

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.

The presence of nitrite during UVA irradiation protects from apoptosis.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways.

Peroxynitrite, generated for example in inflammatory processes, is capable of nitrating and oxidizing biomolecules, implying a considerable impact on the integrity of cellular structures. Cells respond to stressful conditions by the activation of signaling pathways, including receptor tyrosine kinase-dependent pathways such as mitogen-activated protein kinases and the phosphoinositide-3-kinase/Akt pathway. Peroxynitrite affects signaling pathways by nitration as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephosphorylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphorylation/dephosphorylation balance. The flavanol (-)-epicatechin is a potent inhibitor of tyrosine nitration and may be employed as a tool to distinguish signaling effects due to tyrosine nitration from those that are due to oxidation reactions.

Infrared A radiation influences the skin fibroblast transcriptome: mechanisms and consequences.

Infrared A (IRA) radiation (760-1440 nm) is a major component of solar radiation and, similar to UVR, causes photoaging of human skin by increasing the expression of matrix metalloproteinase-1 in human skin fibroblasts. In this study, we assessed the IRA-induced transcriptome in primary human skin fibroblasts. Microarray analysis revealed 599 IRA-regulated transcripts. The IRA-induced transcriptome differed from changes known to be induced by UV. IRA-responsive genes include the categories extracellular matrix, calcium homeostasis, stress signaling, and apoptosis. Selected results were confirmed by real-time PCR experiments analyzing 13 genes representing these four categories. By means of chemical inhibitors of known signaling pathways, we showed that ERK1/2, the p38-, JNK-, PI3K/AKT-, STAT3-, and IL-6 as well as the calcium-mediated signaling pathways, are functionally involved in the IRA gene response and that a major part of it is triggered by mitochondrial and, to a lesser extent, non-mitochondrial production of reactive oxygen species. Our results identify IRA as an environmental factor with relevance for skin homeostasis and photoaging.

Reactive oxygen species as mediators of membrane-dependent signaling induced by ultrafine particles.

Cell-membrane-dependent proliferative signal transduction activated by ultrafine carbon particles in lung epithelial cells involves the specific induction of Akt and ERK1/2 phosphorylation. Particle-induced generation of reactive oxygen species (ROS) and oxidative stress are regarded as initial molecular mechanisms leading to the induction of diverse cellular responses. Therefore, we aimed to analyze the ROS dependence of the induced activation of the Akt/ERK1/2 signaling pathway upon exposure to ultrafine particulate matter (UPM). For this, ultrafine carbon black (ufCB) and ferric sulfate (FS) were used as a model representing the carbonaceous core and a nonparticulate Fenton-reactive transition metal salt often found in combustion-derived UPM. Cell-free as well as intracellular particle-induced ROS generation was assessed and related to the induced Akt and ERK1/2 phosphorylation by inhibiting oxidative stress with catalase, superoxide dismutase, and N-acetylcysteine. We show here that the activation of this signal transduction pathway was mainly due to intracellular, rather than extracellular, ROS production induced by both ufCB and FS. Further inhibitor studies on the role of cell membrane receptors pointed to the epidermal growth factor receptor as a common mediator for particle- as well as transition metal-induced signaling, whereas integrin-dependent Akt and ERK1/2 activation seems to be particle-specific.

Assessment of successful experiments and limitations of phytotechnologies: contaminant uptake, detoxification and sequestration, and consequences for food safety.

PURPOSE: The term "phytotechnologies" refers to the application of science and engineering to provide solutions involving plants, including phytoremediation options using plants and associated microbes to remediate environmental compartments contaminated by trace elements (TE) and organic xenobiotics (OX). An extended knowledge of the uptake, translocation, storage, and detoxification mechanisms in plants, of the interactions with microorganisms, and of the use of "omic" technologies (functional genomics, proteomics, and metabolomics), combined with genetic analysis and plant improvement, is essential to understand the fate of contaminants in plants and food, nonfood and technical crops. The integration of physicochemical and biological understanding allows the optimization of these properties of plants, making phytotechnologies more economically and socially attractive, decreasing the level and transfer of contaminants along the food chain and augmenting the content of essential minerals in food crops. This review will disseminate experience gained between 2004 and 2009 by three working groups of COST Action 859 on the uptake, detoxification, and sequestration of pollutants by plants and consequences for food safety. Gaps between scientific approaches and lack of understanding are examined to suggest further research and to clarify the current state-of-the-art for potential end-users of such green options. CONCLUSION AND PERSPECTIVES: Phytotechnologies potentially offer efficient and environmentally friendly solutions for cleanup of contaminated soil and water, improvement of food safety, carbon sequestration, and development of renewable energy sources, all of which contribute to sustainable land use management. Information has been gained at more realistic exposure levels mainly on Cd, Zn, Ni, As, polycyclic aromatic hydrocarbons, and herbicides with less on other contaminants. A main goal is a better understanding, at the physiological, biochemical, and molecular levels, of mechanisms and their regulation related to uptake-exclusion, apoplastic barriers, xylem loading, efflux-influx of contaminants, root-to-shoot transfer, concentration and chemical speciation in xylem/phloem, storage, detoxification, and stress tolerance for plants and associated microbes exposed to contaminants (TE and OX). All remain insufficiently understood especially in the case of multiple-element and mixed-mode pollution. Research must extend from model species to plants of economic importance and include interactions between plants and microorganisms. It remains a major challenge to create, develop, and scale up phytotechnologies to market level and to successfully deploy these to ameliorate the environment and human health.