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Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.

The temperature dependent carrier transport characteristics of n-type gate-all-around nanowire field effect transistors (GAA NW-FET) on bulk silicon are experimentally compared to bulk fin field effect transistors (FinFET) over a wide range of temperatures (25&ndash, 125 &deg, C). A similar temperature dependence of threshold voltage (VTH) and subthreshold swing (SS) is observed for both devices. However, effective mobility (&mu, eff) shows significant differences of temperature dependence between GAA NW-FET and FinFET at a high gate effective field. At weak Ninv (= 5 &times, 1012 cm2/V∙s), both GAA NW-FET and FinFET are mainly limited by phonon scattering in &mu, eff. On the other hand, at strong Ninv (= 1.5 &times, 1013 cm2/V∙s), GAA NW-FET shows 10 times higher d&mu, eff/dT and 1.6 times smaller mobility degradation coefficient (&alpha, ) than FinFET. GAA NW-FET is less limited by surface roughness scattering, but FinFET is relatively more limited by surface roughness scattering in carrier transport.

Cholestasis can be defined as any situation of impaired bile secretion with concomitant accumulation of bile acids in the liver or in the systemic circulation. A variety of factors may evoke cholestasis, including genetic disorders, metabolic pathologies, infectious diseases, immunogenic stimuli, and drugs. Drug-induced cholestasis is a mechanistically complex process. At least three triggering factors of drug-induced cholestasis have been described, including effects on drug transporters, various hepatocellular changes, and altered bile canaliculi dynamics. These stimuli induce two cellular responses, each typified by a number of key events, namely a deteriorative response activated by bile acid accumulation and an adaptive response aimed at decreasing the uptake and increasing the export of bile acids into and from the liver, respectively. The mechanistic scenario of drug-induced cholestasis is described in this chapter.

An in-depth study of scaled nanowire Ge pFETs for digital and analog applications is proposed. Improved device characteristics are first obtained after gaining a good understanding of the HPA on device performance. Up to 45% higher ID,SAT is obtained at I OFF =3nA/fin when comparing to best Si GAA nFET and similar ID,SAT is found when benchmarking to mature 14/16nm pFinFET technology at −0.5 V DD . The temperature dependent study of I D,SAT highlights that the mechanism limiting the transport in Ge at short channel are neither purely diffusive nor fully ballistic.

Pistacia atlantica belongs to the Anacardiaceae family, and originated from the Atlas mountains of north Africa, which has been employed in traditional medicine for the treatment of various diseases. There are many limitations regarding their activities. The bioactive compounds present in the extract of P. atlantica leaves are not well defined. For these identifications HPLC fingerprint methodology can be used. In the present study, 28 extracts were obtained from male and female leaves of P. atlantica, collected monthly during 6 months, in two different growing regions. The total antioxidant capacity (TAC) values of P. atlantica extracts were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method and the potassium ferricyanide (PFC) reduction method. The fingerprints were pre-treated using several techniques, such as column centering; normalization followed by column centering, and standard normal variate (SNV) transformation followed by column centering; each after initial alignment of the chromatographic profiles with Correlation Optimized Warping (COW). The multivariate calibration methods, Partial Least Squares (PLS) and Orthogonal Projections to Latent Structures (O-PLS), were employed to model TAC. The regression coefficients of the best models were evaluated to indicate the peaks probably responsible for the antioxidant activity. The O-PLS model seems preferable because of its better predictive and describing abilities, and its good interpretability of the contribution of compound peaks to the TAC. Several peaks in the chromatograms were recognized as importantly contributing to the model due to their large regression coefficients. The structural elucidation of the relevant peaks was achieved by negative ionization LC–QTOF–MS. The phenolic compounds galloylquinic acid, quinic acid and gallic acid mainly seemed to be responsible for scavenging the DPPH radical, while glucogallin, trigalloylglucose, gallic acid and tetragalloylquinic acid were considered specifically important for the prediction of TAC determined by the PFC method.

In Europe, hydroquinone is a forbidden cosmetic ingredient. It is, however, still abundantly used because of its effective skin-whitening properties. The question arises as to whether the quantities of hydroquinone used become systemically available and may cause damage to human health. Dermal absorption studies can provide this information. In the EU, dermal absorption has to be assessed in vitro since the Cosmetic Regulation 1223/2009/EC forbids the use of animals. To obtain human-relevant data, a Franz diffusion cell protocol was validated using human skin. The results obtained were comparable to those from a multicentre validation study. The protocol was applied to hydroquinone and the dermal absorption ranged between 31 and 44%, which is within the range of published in vivo human values. This shows that a well-validated in vitro dermal absorption study using human skin provides relevant human data. The validated protocol was used to determine the dermal absorption of illegal skin-whitening cosmetics containing hydroquinone. All samples gave high dermal absorption values, rendering them all unsafe for human health. These results add to our knowledge of illegal cosmetics on the EU market, namely that they exhibit a negative toxicological profile and are likely to induce health problems.

This work presents Si/SiGe superlattice finFETs (FF) for 1.8V/2.5V I/O applications in vertically-stacked Gate-All-Around horizontal nanowire technology (hNW) technology. Superlattice FF have a higher ION than I/O hNW reference devices and can be more easily integrated into a GAA hNW technology than Si I/O FF. These novel I/O FET structures exhibit competitive analog performance and are superior as ESD protection devices.

An important group of suspected illegal cosmetics consists of skin bleaching products, which are usually applied to the skin of the face, hands and décolleté for local depigmentation of hyper pigmented regions or more importantly, for a generalized reduction of the skin tone. These cosmetic products are suspected to contain illegal active substances that may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. In that respect, illegal and restricted substances in cosmetics, known to have bleaching properties, are in particular hydroquinone, tretinoin and corticosteroids. From a legislative point of view, all cosmetic products containing a prohibited whitening agent are illegal and must be taken off the EU market. A newly developed screening method using ultra high performance liquid chromatography-time off flight-mass spectrometry allows routine analysis of suspected products. 163 suspected skin whitening cosmetics, collected by Belgian inspectors at high risk sites such as airports and so-called ethnic cosmetic shops, were analyzed and 59% were classified as illegal. The whitening agents mostly detected were clobetasol propionate and hydroquinone, which represent a serious health risk when repeatedly and abundantly applied to the skin.

Background/Aims: Silicone excipients are commonly used ingredients because of their emollient and skin-conditioning effects, and their ability to form uniform, water-resistant, yet permeable films. Based on comparisons with organic materials and conflicting knowledge from silicones used in scar treatment, the misconception still exists that silicone topical excipients are occlusive substances that may block the passive loss of water through the upper skin layers. Therefore, 3 types of common silicone excipients and 3 water-in-(oil-plus-silicone) or W/(O + Si) creams, containing 10% (w/w) of the respective silicones, were investigated as a function of time and compared to petrolatum. Methods: Transepidermal water loss (TEWL) and skin hydration measurements were carried out after a single topical application on forearm skin of 26 healthy young female volunteers. Results: Both petrolatum and silicones significantly decreased TEWL 15 min after application, but the measurements for the silicones were not significantly different from the untreated control values. The tested silicones did not moisturize the skin. Petrolatum formed an occlusive layer, creating an increase in skin hydration for more than 4 h. The results measured for the W/(O + Si) creams indicated that they moisturized the skin, without any effect on TEWL. Conclusion: A clear difference was shown between the skin occlusive properties of petrolatum and the water vapor permeability of the common silicone excipient materials.

Tissue homeostasis is driven by a myriad of extracellular, intracellular and intercellular signaling networks. Direct communication between neighboring cells is typically controlled by gap junctions (Decrock et al. 2009; Nielsen et al. 2012; Vinken et al. 2011). Gap junctions mediate the intercellular diffusion of small and hydrophilic substances, including cyclic adenosine monophosphate, adenosine triphosphate, inositol triphosphate, glutathione, glutamate, glucose and several ions (Alexander and Goldberg 2003) (Fig. 1a). This flux is denoted as gap junctional intercellular communication (GJIC) and is considered as a key mechanism in the maintenance of tissue functioning (Decrock et al. 2009; Nielsen et al. 2012; Vinken et al. 2011). Over the last decades, GJIC has been shown indispensable for the establishment of metabolic or electrical intercellular coupling in all vital organs, such as the brain (Eugenin et al. 2012), the heart (Kurtenbach et al. 2014) and the liver (Vinken et al. 2008). Gap junctions are formed by the docking of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. At present, 21 different connexins have been identified in humans, all which are expressed in a cell-specific way (Kar et al. 2012; Nielsen et al. 2012). They are named based upon their molecular weight. Thus, the most widespread connexin species has a molecular mass of 43 kDa and hence is called Cx43. Connexin proteins share a common molecular structure consisting of four transmembrane domains, two extracellular loops, one cytosolic loop, one cytosolic aminotail and one cytosolic carboxytail (Fig. 1b) (Decrock et al. 2009; Nielsen et al. 2012). Fig. 1 a Gap junctions consist of two connexin hemichannels of adjacent cells and mediate intercellular communication. Connexin hemichannels also provide a pathway for communication, namely between the cytosol and the extracellular environment. Similar messengers ... Although considered as merely structural precursors of gap junctions for a long time, an abundance of reports published in the last few years shows that connexin hemichannels as such can provide a pathway for cellular communication, albeit between the cytosol of individual cells and their extracellular environment and not between adjacent cells as is the case for GJIC. Nonetheless, the messengers that are conveyed through connexin hemichannels are very similar to those involved in GJIC, including adenosine triphosphate, nicotinamide dinucleotide, glutamate, glutathione, prostaglandin, sodium and calcium ions (Fig. 1a) (Chandrasekhar and Bera 2012; Decrock et al. 2009; Kar et al. 2012). Furthermore, connexin hemichannels are regulated by mechanisms that equally affect gap junctions, yet an identical factor can have opposing effects on the two channel types, such as shown for certain inflammatory triggers (De Vuyst et al. 2007; Retamal et al. 2007). In line with this notion, connexin hemichannels, unlike their full channel counterparts, display a low open probability. In fact, connexin hemichannels seem to be preferably activated by pathological stimuli, including ischemia–reperfusion insults and oxidative stress, and thereby drive processes like cell death and inflammation (Chandrasekhar and Bera 2012; Decrock et al. 2009; Kar et al. 2012). For this very reason, connexin hemichannels are sometimes considered as “pathological pores” (Decrock et al. 2009). This concept is clearly relevant to toxicologists, as several toxic substances open up connexin hemichannels. For example, ultrafine carbon black particles activate Cx43-based hemichannels in astrocytes, which may represent a novel mechanism of neurotoxicity (Wei et al. 2014). Similarly, cobalt–chromium nanoparticles can damage human fibroblasts by a mechanism involving transmission of adenosine triphosphate and signaling through connexin hemichannels (Bhabra et al. 2009). The connexin research field has been surrounded by a lot of controversy in the last few years. Specifically, the concept of (dys) functional connexin hemichannels has been debated heavily on several occasions (Saez and Leybaert 2014; Spray et al. 2006). A major reason for this impediment is the ubiquitous lack of tools and technologies to distinguish between the different channel types, in casu between gap junctions and connexin hemichannels (Bodendiek and Raman 2010; Iyyathurai et al. 2013). Classical strategies, such as the use of RNA interference-based technologies, genetically modified animals or even antibodies, are indeed not applicable, as they target connexins, which are the shared building stones of gap junctions and connexin hemichannels. Furthermore, most, if not all, of the routinely used gap junction inhibitors, including long-chain alcohol substances, anaesthetic substances, the glycyrrhetinic acid derivative carbenoxolone and the fenamate family of blockers, equally suppress connexin hemichannel activity (Bodendiek and Raman 2010). Great expectations now lie with peptides that reproduce sequences in the cytosolic loop regions of connexins, as they suppress connexin hemichannel activity without influencing GJIC (Iyyathurai et al. 2013). Among those, a very prominent one is Gap19, a peptide that inhibits Cx43-based hemichannels while leaving the corresponding gap junctions unaffected (Abudara et al. 2014; Wang et al. 2013). In fact, Gap19 was found to reduce experimentally induced cell death in a mouse model of cardiac ischemia–reperfusion (Wang et al. 2013). This area is still in its infancy, but upon further exploration, it can be expected that this will also open new perspectives for the field of toxicology. Indeed, such specific connexin hemichannel inhibitors are not only interesting experimental tools to shed light onto yet unraveled toxicological mechanisms of action, but they may also form the basis for the development of antidotes pertinent for clinical use.

During the last years, the EU market is flooded by illegal cosmetics via the Internet and a so-called "black market". Among these, skin-bleaching products represent an important group. They contain, according to the current European cosmetic legislation (Directive 76/768/EEC), a number of illegal active substances including hydroquinone, tretinoin and corticosteroids. These may provoke as well local as systemic toxic effects, being the reason for their banning from the EU market. To control this market there is a need for a fast screening method capable of detecting illegal ingredients in the wide variety of existing bleaching cosmetic formulations. In this paper the development and validation of an ultra high pressure liquid chromatographic (UHPLC) method is described. The proposed method makes use of a Waters Acquity BEH shield RP18 column with a gradient using 25 mM ammonium borate buffer (pH 10) and acetonitrile. This method is not only able to detect the major illegal (hydroquinone, tretinoin and six dermatologic active corticosteroids) and legal whitening agents, the latter having restrictions with respect to concentration and application (kojic acid, arbutin, nicotinamide and salicylic acid), but can also quantify these in a run time of 12 min. The method was successfully validated using the "total error" approach in accordance with the validation requirements of ISO-17025. During the validation a variety of cosmetic matrices including creams, lotions and soaps were taken into consideration.

Since the publication of REACH guidelines, the need for in vitro tools for toxicity testing has increased. We present here the development of a hepatotoxicity testing tool using human HepaRG cell cultures and metabolomics. HepaRG cells were exposed to either 4 mM acetaminophen (APAP) as reference toxicant for oxidative stress or 50 μM triphenyl phosphate (TPHP) as toxicant with unknown toxicity pathways (TPs). After 72 h exposure, cells were subjected to quenching and liquidliquid extraction which resulted in a polar and an apolar fraction. Analysis of fractions was performed by ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-QTOF-MS). Significantly up or down regulated metabolites were selected by univariate statistics prior to identification. In order to obtain robust and specific TP biomarkers, the experiment was also repeated using a different culture medium composition to assess which metabolites show consistent changes. Potential biomarkers belonging to different TPs were found for APAP and TPHP. For APAP, the biomarkers were related to a decrease in unsaturated phospholipids, and for TPHP to an accumulation of phosphoglycerolipids and increase of palmitoyl lysophosphatidylcholine. This first proof-of-concept opens new perspectives for the analysis of other (reference) toxicants with different TPs and it can be used to expand the in vitro tool for hepatotoxicity screening of various compounds.

Made available in DSpace on 2018-12-11T17:18:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-03-12 European Research Council Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5′-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease. Department of In Vitro Toxicology and Dermato-Cosmetology Vrije Universiteit Brussel Department of Pathology School of Veterinary Medicine and Animal Science University of São Paulo Department of Food and Experimental Nutrition Faculty of Pharmaceutical Sciences University of São Paulo Laboratory of Hepatogastroenterology Institut de Recherche Expérimentale et clinique Université Catholique de Louvain Department of Pathology Botucatu Medical School UNESP-São Paulo State University Department of Basic Medical Sciences Physiology Group Ghent University Department of Pathology Botucatu Medical School UNESP-São Paulo State University European Research Council: 335476

Homeostasis relies on the intimate interplay between extracellular, intracellular and intercellular signaling networks. Direct communication between adjacent cells is mediated by gap junctions [1-3]. They allow the intercellular diffusion of small (i.e. less than 1 kDa) and hydrophilic substances, including adenosine triphosphate (ATP), cyclic adenosine monophosphate, inositol triphosphate, glucose, glutathione, glutamate and several ions, like sodium, calcium and potassium (Fig. 1) [4]. This flux is called gap junctional intercellular communication (GJIC) and is considered as a basic mechanism in the maintenance of tissue functioning [1-3]. Over the last decades, GJIC has been shown indispensable for the establishment of metabolic or electrical intercellular coupling in all vital organs, such as the brain [5], the heart [6] and the liver [7], to name a few.

Recent advances in genomics, recombinant expression technologies and peptide synthesis have led to an increased development of protein and peptide therapeutics. Unfortunately this goes hand in hand with a growing market of counterfeit and illegal biopharmaceuticals, including substances that are still under pre-clinical and clinical development. These counterfeit and illegal protein and peptide substances could imply severe health threats as has been demonstrated by numerous case reports. The Belgian Federal Agency for Medicines and Health Products (FAMHP) and customs are striving, together with their global counterparts, to curtail the trafficking and distributions of these substances. At their request, suspected protein and peptide preparations are analysed in our Official Medicines Control Laboratory (OMCL). It stands to reason that a general screening method would be beneficiary in the battle against counterfeit and illegal peptide drugs. In this paper we present such general screening method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of counterfeit and illegal injectable peptide preparations, extended with a subsequent quantification method using ultra-high performance liquid chromatography with diode array detection (UHPLC-DAD). The screening method, taking only 30 min, is able to selectively detect 25 different peptides and incorporates the proposed minimum of five identification points (IP) as has been recommended for sports drug testing applications. The group of peptides represent substances which have already been detected in illegal and counterfeit products seized by different European countries as well as some biopharmaceutical peptides which have not been confiscated yet by the controlling agencies, but are already being used according to the many internet users forums. Additionally, we also show that when applying the same LC gradient, it is also possible to quantify these peptides without the need for derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the «total error» approach in accordance with the validation requirements of ISO-17025.

An adequate permeability barrier function of the mammalian epidermis is guaranteed by the characteristic architecture of the stratum corneum. This uppermost layer consists of a highly organized extracellular lipid compartment which is tightly joined to the corneocytes. The generation of the extracellular lipid compartment and the transformation of the keratinocytes into corneocytes are the main features of epidermal differentiation. However, equally important is the continuous renewal of the stratum corneum, which is insured by a careful balance between the replenishment of new keratinocytes from the proliferating basal layer, and the well-orchestrated loss of the most superficial cells after the so-called ‘epidermal programmed cell death’. In this overview, the complete life of keratinocytes is described, from the proliferative organization to the process of desquamation.

Study question: Is it possible to derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine? Summary answer: We developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. What is known already: The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. Study design, size, duration: Human cadaveric testicular tissue was exposed for 24 h or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulfate (SDS). Acellular samples were used for further scaffold characterization purposes. Participants/materials, setting, methods: The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. Retention of testicular tissue-specific characteristics was evaluated by mass spectrometry, immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. Main results and the role of chance: Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure and the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, using proteomic analysis, we revealed numerous additional ECM proteins in DTM, indicating its complex nature. The mass spectrometry data were deposited to the ProteomeXchange with identifier PXD001524. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay not showing an aberrant fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration. Limitations, reasons for caution: The functionality of human testicular cells in DTM needs to be investigated. Wider implications of the findings : Our results suggest that the insights into the molecular composition of the testicular ECM provide new clues for the unravelling of its important yet poorly understood role in regulating testicular function, and DTM-based bioscaffolds are promising components for the development of human in vitro spermatogenesis as a treatment for various types of male fertility disorders. Study funding/competing interest(s): This study is supported by a Ph.D. grant from the Agency for Innovation by Science and Technology (IWT) and research grants from the Flemish League Against Cancer-Public Utility Foundation (VLK), the Scientific Research Foundation Flanders (FWO), the Vrije Universiteit Brussel, the Swedish Research Council/ Finnish Academy of Science, Emil och Wera Cornells Stiftelse, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. J.B.S. was supported by the German Research Foundation (DFG-Grant No.: STU 506/3-1). E.G. and J.D.K. are postdoctoral fellows of the Scientific Research Foundation Flanders. The authors declare that no competing interests exist. Trial registration number: Not applicable

Background/aim: Transepidermal water loss (TEWL) is one of the most important biophysical parameters for evaluating the efficiency of the human skin water barrier. Different approaches exist to measure TEWL. The most commonly used methodology consists of the open chamber diffusion technique in which the water vapor pressure gradient is measured in g/h m2 according to Fick's law. A typical apparatus is the Tewameter®. Recently, a portable device – the VapoMeter – became available with a humidity sensor in a closed chamber. Methods: In the present work, the closed chamber VapoMeter is compared with the open chamber Tewameter® for its applicability to assess TEWL. A comparative study – including parallel in vivo measurements with both devices – was carried out on human forearm skin. Results: It could be concluded that both instruments are reliable tools. A good correlation between recordings (r=0.503–0.966) was found with a consistent feature of measuring higher TEWL values for the Tewameter® than for the VapoMeter. Probe pressure, probe temperature and relative humidity were revealed to be important parameters inducing significant differences in data outcome. Conclusions: From skin barrier damage experiments it became clear that the Tewameter® is able to detect significantly smaller differences than the VapoMeter. In addition, the closed chamber device is currently not sensitive enough to discriminate for the effects induced by diurnal rhythm and fluctuations as a function of time. On the other hand, the small and handy VapoMeter allows more flexibility in measuring protocols and in in-use performance.

Potential allergenic fragrances are part of the Cosmetic Regulation with labelling and concentration restrictions. This means that they have to be declared on the ingredients list, when their concentration exceeds the labelling limit of 10 ppm or 100 ppm for leave-on or rinse-off cosmetics, respectively. Labelling is important regarding consumer safety. In this way, sensitised people towards fragrances might select their products based on the ingredients list to prevent elicitation of an allergic reaction. It is therefore important to quantify potential allergenic ingredients in cosmetic products. An easy to perform liquid extraction was developed, combined with a new headspace GC-MS method. The latter was capable of analysing 24 volatile allergenic fragrances in complex cosmetic formulations, such as hydrophilic (O/W) and lipophilic (W/O) creams, lotions and gels. This method was successfully validated using the total error approach. The trueness deviations for all components were smaller than 8%, and the expectation tolerance limits did not exceed the acceptance limits of ??20% at the labelling limit. The current methodology was used to analyse 18 cosmetic samples that were already identified as being illegal on the EU market for containing forbidden skin whitening substances. Our results showed that these cosmetic products also contained undeclared fragrances above the limit value for labelling, which imposes an additional health risk for the consumer. ?? 2014 Elsevier B.V.

Cosmetic products containing illegal whitening agents are still found on the European market. They represent a considerable risk to public health, since they are often characterised by severe side effects when used chronically. The detection of such products at customs is not always simple, due to misleading packaging and the existence of products containing only legal components. Therefore there is a need for easy to use equipment and techniques to perform an initial screening of samples. The use of attenuated total reflectance-infrared (ATR-IR) spectroscopy, combined with chemometrics, was evaluated for that purpose. It was found that the combination of ATR-IR with the simple chemometric technique k-nearest neighbours gave good results. A model was obtained in which a minimum of illegal samples was categorised as legal. The correctly classified illegal samples could be attributed to the illegal components present. © 2014 Elsevier B.V.

Cytochrome P450 enzymes are a diverse group of catalytic enzymes in the liver that are mainly responsible for the biotransformation of organic substances. Cytochrome P450 activity as well as both its induction and inhibition are key factors in drug biotransformation and can be involved in deactivation, activation, detoxification, and toxification processes. Thus, the modulation of cytochrome P450 activity is an important parameter when evaluating the potential toxicity of chemical compounds using an in vitro system. The cytochrome P450 3A subfamily proteins are among the most important drug-metabolizing enzymes in human liver and are responsible for about half of all cytochrome P450- dependent drug oxidations. In vitro, these enzymes are active not only in primary human hepatocyte cultures, but also in differentiated human hepatoma HepaRG cells. The present protocol describes the culture of cryopreserved differentiated HepaRG cells and the evaluation of its cytochrome P450 activityupon exposure to a chemical compound using a commercially available luminogenic cytochrome P450 assay. This in vitro model can be used to monitor the induction and inhibition of cytochrome P450 3A following exposure to a particular test compound.

Glutathione S-transferases (GSTs) are subject to regulation by thyroid and sex hormones and by GH. We have used an in vitro experimental system comprising adult rat hepatocytes co-cultured with rat liver epithelial cells of primitive biliary origin, to distinguish between direct and indirect effects of various hormones on GSTs; to identify the GST subunits affected by individual hormones; and to investigate the level at which the hormones act. Tri-iodothyronine (T3), thyroxine (T4) and 17beta-oestradiol (OE2) reduced GST activities, whereas testosterone, dihydrotestosterone, and human growth hormone (hGH) had little effect on total GST activity. HPLC separation of the various GST subunits revealed that T3 and T4 reduced total GST content, in particular the abundance of subunits M1 and M2. The amount of the Pi-class subunit P1 was reduced by OE2. Treatment of the co-cultured cells with this hormone altered the GST subunit profile to one that is more similar to that observed in freshly isolated hepatocytes. Analysis of mRNAs demonstrated that some of the hormones act at a pre-translational level, whereas others act at a translational or post-translational level to regulate the expression of various GST subunits.

The effect of age and gender on the in vitro biotransformation of 2-methylpropene, an alkene metabolized to 2-methyl-1,2-epoxypropane, was studied. The epoxide concentration and the epoxide metabolizing enzymatic activities were investigated in male and female Brown Norway rats of different ages. Liver tissue of senescent rats was exposed to smaller 2-methyl-1,2-epoxypropane concentrations than that of young animals, although changes during ageing were rather modest. With advancing age a feminization of male glutathione S-transferase and cytosolic epoxide hydrolase activities was found, as well as a significant decline of the female microsomal epoxide hydrolase activity and an increase of the cytochrome P-450 content in the oldest female rats.

Ca(2+) is known as a universal messenger mediating a wide variety of cellular processes, including cell death. In fact, this ion has been proposed as the 'cell death master', not only at the intracellular but also at the intercellular level. The most direct form of intercellular spread of cell death is mediated by gap junction channels. These channels have been shown to propagate cell death as well as cell survival signals between the cytoplasm of neighbouring cells, reflecting the dual role of Ca(2+) signals, i.e. cell death versus survival. Its precursor, the unopposed hemichannel (half of a gap junction channel), has recently joined in as a toxic pore connecting the intracellular with the extracellular environment and allowing the passage of a range of substances. The biochemical nature of the so-called intercellular cell death molecule, transferred through gap junctions or released/taken up via hemichannels, remains elusive but several studies pinpoint Ca(2+) itself or its messenger inositol trisphosphate as the responsible masters in crime. Although direct evidence is still lacking, indirect data including Ca(2+) involvement in intercellular communication and cell death, and effects of intercellular communication on intracellular Ca(2+) homeostasis, support this hypothesis. In addition, hemichannels and their molecular building blocks, connexin or pannexin proteins, may exert their effects on Ca(2+)-dependent cell death at the intracellular level, independently from their channel functions. This review provides a cutting edge overview of the current knowledge and underscores the intimate connection between intercellular communication, Ca(2+) signalling and cell death.

Monolayer cultures of primary hepatocytes, isolated from freshly removed livers, represent widely used in vitro tools in the area of liver physiology and pathology, pharmacology and toxicology. However, a major shortcoming of these systems is that they cope with dedifferentiation, which is accompanied by spontaneous cell death. The goal of the present study was to elucidate the mechanisms that drive the process of self-generated cell demise in primary hepatocyte cultures. For this purpose, isolated rat hepatocytes were cultivated under conventional conditions, and the occurrence of apoptosis and necrosis was monitored during 4 days by performing a set of acknowledged cell death assays. These included examination of cell morphology by light microscopy, quantification of apoptotic and necrotic cell populations by Hoechst 33342 and propidium iodide in situ staining, assessment of apoptotic and necrotic activities by measuring caspase 3-like activity and extracellular leakage of lactate dehydrogenase, and studying the expression of apoptosis regulators through immunoblot analysis. In essence, two cell death peaks were observed, namely shortly after cell seeding and in the final stages of the cultivation period, both involving apoptotic and necrotic actions. The outcome of this study not only sheds new light onto the molecular processes that underlie spontaneous cell death in primary hepatocyte cultures, but also opens perspectives for the establishment of strategies to increase cell survival in these popular in vitro systems.

Long-term cultures of hepatocytes could represent a suitable in vitro model for botransformation studies of xenobiotics. At present however, no ideal culture system can be proposed since, in all existing models, phenotypic changes occur, affecting selectively some components of phase I and/or phase II xenobiotic metabolism. From the authors' own results and recent studies of several other investigators, carried out on rat hepatocytes, it becomes clear that four groups of factors may affect biotransformation capacity: soluble medium factors, extracellular matrix components, cell-cell interactions and factors affecting replication. For the maintenance of liver-specific functions, it seems of utmost importance that the tridimensional shape of the hepatocytes is kept. Usually, phase II enzymatic activity is better kept than that of phase I. The cytochrone P450 dependent monoxygenases, in particular, are easily lost. Interesting is the observation that co-cultures of rat hepatocytes with rat liver epithelial cells exhibit higher and much better preserved phase I and phase II biotransformation than monocultures. Clearly, further research is needed to improve this promising in vitro model.

A quantitative, non-destructive cellular autofluorescence based in vitro imaging assay has been developed and applied to study the cytotoxicity of Sodium Lauryl Sulfate (SLS) and HgCl2 on Balb/c 3T3 cells. A phenomenological double logistic model was proposed to quantify and relate the observed kinetic changes of fluorescence to the toxic potency of chemical compounds. This work forms the basis for cellular autofluorescence measurements in in vitro toxicity screening assays.

In order to investigate whether collagen gel sandwich and immobilization cultures of rat hepatocytes are suitable in vitro models for long-term pharmaco-toxicological studies, the expression of the key phase II biotransformation enzyme, glutathione S-transferase (GST, EC 2.5.1.18), has been studied in the presence or absence of l -proline (60μg/ml) in the culture medium. Additionally, hepatocytes morphology was followed and albumin secretion into the medium measured. As judged by inverse phase light microscopy and transmission electron microscopy, cells cultured in both organotypical models remained viable and well differentiated for at least 14 days. Albumin secretion increased 2.5-fold after 7 days of culture, in comparison with the values found after 2 days, and remained thereafter relatively constant. When l -proline was added to the medium of sandwich and immobilization gel cultures, steady-state secretion levels of 7.1 and 5.1 μg albumin/hr, respectively, were already obtained after 4 days of culture. Total, Mu, Alpha and Pi class GST activities were determined using a general substrate and isoenzyme specific substrates, respectively. After 7 days of culture, total GST activities were decreased as compared with the values obtained for freshly isolated cells. On the contrary, Mu class GST activities were kept at a constant level. Alpha class GSTs were maintained at a 50% activity level and GST 7-7 activity was shown to be slightly induced. l -proline prevented an initial decline in total and Mu class GST activities in both culture models. The GST subunit pattern, measured after affinity chromatography by reversed phase HPLC, reflected the GST activity results.

To examine the effects of ethanol (EtOH) on rat liver glutathione S -transferase (GST, E.C. 2.5.1.18), a key phase 2 biotransformation enzyme, an in vitro system consisting of rat hepatocytes co-cultured with rat epithelial cells derived from primitive biliary duct cells was used. Cells were either untreated or treated for 10 days with 0.1 or 0.4% EtOH. Cytosolic GST activities were assayed spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) as substrates. When cultures were exposed to 0.4% EtOH the total GST activity towards the substrate CDNB increased by 35%. However, the activity of GSTs towards DCNB did not increase significantly in the cells treated with this concentration of EtOH. Affinity chromatography followed by reversed phase HPLC was used to separate the various GST subunits. EtOH was found to affect the GST subunit pattern. Total GST proteins increased after exposure to 0.4% EtOH; this was largely due to a three-fold and 2.5-fold increase in the GST μ-class subunits 3 and 4 respectively. Since EtOH induces GST expression in liver parenchymal cells, particularly the μ-class GSTs, its addition as a solvent to cultured hepatocytes or its use in vivo may result in important changes in the metabolism and toxicity of the xenobiotics under study.

BACKGROUND: Carrier-added [(123)I]-2-iodo-D-phenylalanine (CA [(123)I]-2-I-D-Phe) was previously found to have a preferential retention in tumors with a high tumor background contrast in animal models. A previous human dosimetry study demonstrated a favorable biodistribution and radiation burden in human subjects. AIM: The aim of this study was to investigate the potential of CA [(131)I]-2-I-D-Phe as an agent for radionuclide therapy. METHODS: Sixty (60) nude athymic mice were inoculated subcutaneously with firefly luciferase-transduced R1M rhabdomyosarcoma cells. The mice in the therapy group were injected intravenously (i.v.) with 148 MBq [(131)I]-2-I-D-Phe (432 GBq/mmol) in kit solution. Controls were injected with kit solution without radioactivity, with physiological saline, or with 148 MBq [(131)I](-) in physiological saline. Tumor growth was quantified using bioluminescent imaging and caliper measurements. RESULTS: [(131)I]-2-I-D-Phe clearly reduced tumor growth in the treated mice compared with thecontrol groups. A tumor growth-rate reduction of at least 33% was found for mice receiving a therapeutic dose. There were no serious adverse side-effects of the therapy. CONCLUSIONS: In conclusion, i.v. injection of CA 148 MBq [(131)I]-2-I-D-Phe specifically reduces tumor growth in athymic nude mice without relevant side-effects on the animals' health.

Objective: Hydrocolloid patches are developed with 10%, 20% and 30% (w/w) glycerol as the main active ingredient. They are intended for post-treatment of the skin after peeling or laser therapy. These patches are expected to protect and hydrate the skin intensively when applied repetitively as single patches remaining on the skin for 7 consecutive days. Methodology: By making use of two experimental forearm models, the efficacy of glycerol patches on the skin and its dependency on the glycerol concentration applied, were compared with a blank reference patch and a commercialized protective patch. Testing versus a cosmetic barrier cream was also carried out. As a preliminary test, measurements were done after a single application on healthy forearm skin. Then a long-term application protocol was developed for skin, mechanically irritated by stripping with cyanoacrylate tape. Skin hydration and transepidermal water loss measurements were combined with skin erythema assessments. Results and conclusions: On healthy skin, a clear concentration dependent effect of glycerol-containing patches versus the reference patch was observed - with for the highest glycerol content - a 31% increase of skin hydration and an improvement of skin barrier properties with 15%. Moreover, the glycerol-containing patch accelerated skin barrier recovery after tape stripping. After 1 week of repetitive application, a significantly hydrating effect of the moisturizing patch versus the brand product was observed. The hydrating efficacy obtained was of the same order of magnitude as observed for the cosmetic barrier cream, the latter being applied twice daily. In conclusion, the 30% glycerol-containing moisturizing patch had a significant effect on hydration and barrier recovery after skin damage. The effects seen were maximal after 3 days of patch application.

Until now, no data are available concerning the biotransformation and toxicity of 2-methylpropene (or isobutene), a gaseous alkene widely used in industry (rubber, fuel additives, plastic polymers, adhesives, antioxidants). In this work, the biotransformation of 2-methylpropene (MP) has been studied, using total liver homogenates of mice, supplemented with a NADPH-generating system. In analogy to other olefins, 2-methylpropene is metabolized to its epoxide 2-methyl-1,2-epoxypropane (MEP), as proved by the identification by gas chromatography coupled with mass spectrometry. The epoxidation is cytochrome P-450 dependent, as shown by experiments in the absence of the NADPH-generating system and in the presence of various concentrations of metyrapone and SKF 525-A, two known inhibitors of the mono-oxygenases. A simple gas chromatographic headspace method has been developed for the quantitative determination of the epoxide formed. The formation of MEP is never linear in function of time and it reaches a maximum after 20 min. Thereafter is decreases continuously to undetectable levels. This observation can be explained by the immediate action of epoxide hydrolase and glutathione S-transferase, converting the epoxide to 2-methyl-1,2-propanediol and to the glutathione conjugate respectively. The involvement of both enzymes has been demonstrated by the addition of 3,3,3-trichloropropene oxide and indomethacin. These inhibitors of, respectively, epoxide hydrolase and glutathione S-transferase increase the epoxide formation in a significant way. The actual concentration of MEP is therefore not only dependent on its formation by cytochrome P-450 dependent mono-oxygenases, but also on its conversion by epoxide hydrolase and glutathione S-transferase, both very active in liver tissue.

In the present work, nasolabial skin condition and the influence of seasonal changes during autumn and winter were studied in 16 healthy female volunteers. Apart from visual scoring of erythema and skin scaliness, transepidermal water loss (TEWL), skin hydration, apparent skin pH, skin colour and skin desquamation were biophysically measured. The study results showed that nasolabial TEWL was significantly higher during wintertime than in autumn. Also skin colour measurements and squamometry scorings revealed higher values, indicating a more reddish and scaly nasolabial skin during winter compared to autumn. Results from tape stripping and skin surface lipid analysis by high-performance thin-layer chromatography demonstrated significant differences for triglycerides and cholesterol esters, indicating a functionally inferior hydrolipidic layer during the winter season.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay. Copyright © 2007 S. Karger AG.

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes.

Primary cultures of hepatocytes are useful tools for both short- and long-term pharmacotoxicological research. Under conventional conditions, isolated hepatocytes form a monolayer and survive for about 1 wk but lose some liver-specific functions, including xenobiotic biotransformation. In comparison with the conventional monolayer culture model, cocultures with rat liver epithelial cells (RLECs) have an extended life-span and better maintain their drug-metabolizing capacity, owing to the presence of cell-cell interactions. In this chapter, techniques for setting up conventional monolayer cultures and cocultures of hepatocytes with RLECs (including isolation, culture, and cryopreservation of RLECs) are described in detail. In addition, comments derived from our own experience are given for successfully culturing primary hepatocytes.

Isolated hepatocytes are a physiologically relevant in vitro model exhibiting intact subcellular organelles, xenobiotic transport, and integrated phase I and phase II biotransformation. They represent the "gold standard" for investigating xenobiotic biotransformation and metabolic bioactivation. When used in suspension, they provide an easy-to-handle and relatively cheap in vitro system that can be used for up to 4 h. The use of animal- and human-derived hepatocytes allows interspecies comparisons of metabolic properties. In contrast with microsomes, which are easily prepared from human liver tissue and can be stored in liquid nitrogen with minimal loss of functionality, cryopreservation of isolated human hepatocytes has been shown to be more difficult: after thawing losses of cell viability and biotransformation capacity occur. We provide general recommendations for the appropriate use of hepatocytes in suspension for pharmaco-toxicological studies. We also provide protocols for the cryopreservation of freshly isolated hepatocytes and their handling on thawing.

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.

Chronic exposure of the skin to sunlight causes damage to the underlying connective tissue with a loss of elasticity and firmness. Silicon (Si) was suggested to have an important function in the formation and maintenance of connective tissue. Choline-stabilized orthosilicic acid (''ch-OSA'') is a bioavailable form of silicon which was found to increase the hydroxyproline concentration in the dermis of animals. The effect of ch-OSA on skin, nails and hair was investigated in a randomized, double blind, placebo-controlled study. Fifty women with photodamaged facial skin were administered orally during 20 weeks, 10 mg Si/day in the form of ch-OSA pellets (n=25) or a placebo (n=25). Noninvasive methods were used to evaluate skin microrelief (fore- arm), hydration (forearm) and mechanical anisotropy (forehead). Volunteers evaluated on a virtual analog scale (VAS, ''none=0, severe=3'') brittleness of hair and nails. The serum Si concentration was significantly higher after a 20-week supplementation in subjects with ch-OSA compared to the placebo group. Skin roughness parameters increased in the placebo group (Rt:+8%; Rm: +11%; Rz: +6%) but decreased in the ch-OSA group (Rt: 16%; Rm: 19%; Rz: 8%). The change in roughness from baseline was significantly different between ch-OSA and placebo groups for Rt and Rm. The difference in longitudinal and lateral shear propa- gation time increased after 20 weeks in the placebo group but decreased in the ch-OSA group suggesting improvement in isotropy of the skin. VAS scores for nail and hair brittleness were significantly lower after 20 weeks in the ch-OSA group compared to baseline scores. Oral intake of ch-OSA during the 20 weeks re- sults in a significant positive effect on skin surface and skin mechanical properties, and on brittleness of hair and nails.

A method was optimalised for the quantitative determination of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid. The principle of the method was that bound desfuroylceftiofur is first released by dithioerythritol, a reducing agent, followed by the derivatization of the free sulfhydryl group with iodoacetamide. The stable derivative—desfuroylceftiofuracetamide—was then further purified using an Oasis HLB solid-phase extraction column. Chromatography was performed using a PLRP-S polymeric column (100 A, dp: 5 μm, 150 mm ×2.1 mm i.d.), with a mixture of 0.1% trifluoro acetic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. The flow-rate was 0.4 ml/min and the UV detector was set at a wavelength of 266 nm. The method was validated in plasma and synovial fluid (linearity, precision, trueness, LOQ, LOD, specificity, susceptibility to interferences). Calibration graphs were prepared over a concentration range of 0–20 μg/ml and good linearity was achieved (r≥0.99, g≤10%). A limit of quantification of 0.5 μg/ml was obtained for ceftiofur in both matrices. Limits of detection were 0.36 and 0.27 μg/ml for ceftiofur in plasma and synovial fluid, respectively. The results of the within-run and between-run precision and the trueness fell within the ranges specified. The main advantage of our method, compared to previously reported methods, was that the sample preparation procedure was less time consuming, resulting in a higher sample throughput (up to 40 samples a day). In addition, the analysis cost was reduced due to the consumption of a lower amount of solvents and reagents and of only one solid-phase extraction column per sample. The method was successfully applied during a pharmacokinetic study in horses after the administration of ceftiofur sodium via regional intravenous perfusion and systemic intravenous injection.

This paper focuses on the use of liver-derived in vitro systems for biotransformation studies during early drug development, as exemplified by the two molecules recently studied in our laboratory: Trichostatin A (TSA) and its structural analogue 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me2N-BAVAH). Phase I biotransformation of TSA, a histone deacetylase inhibitor with promising antifibrotic and antitumoural properties, was investigated in liver microsomal (rat and human) and in hepatocyte (rat) suspensions. Within 40 minutes, 50μM of TSA was completely metabolised by 2 × 106 hepatocytes/ml. Reduction of the hydroxamic acid function to its corresponding amide and N-demethylation were the two major phase I biotransformation pathways, while hydrolysis products of TSA were minor metabolites. Lower concentrations of TSA (5μM and 25μM) were N-demethylated faster. Liver microsomes, however, metabolised TSA incompletely with the formation of two major metabolites, N-mono- and N-didemethylated TSA. Unlike TSA, 4-Me2N-BAVAH (50μM) could still be detected after 3 hours of incubation with 2 × 106 rat hepatocytes/ml suspension. Hydrolysis and reduction of the hydroxamic acid function to its corresponding acid and amide, respectively, were shown to be the major phase I biotransformation pathways. Lower concentrations of 4-Me2N-BAVAH were hydrolysed more readily. 4-Me2N-BAVAH and its metabolites were less subjected to N-demethylation than TSA.

The synthesis of new amide type histone deacetylase inhibitors is described, having an (R)-methyl substituent and a diene or saturated structure of the chain linking the hydroxamic acid and dimethylaminobenzoyl groups. The saturated compound shows stronger HDAC inhibition than the unsaturated analogue. Molecular modeling suggests that the flexibility of the linker chain is important for an optimal orientation of the dimethylaminobenzoyl group in the enzyme.

Background It is generally acknowledged that well-formulated moisturizing skin care products can restore disturbed barrier function that can be assessed by transepidermal water loss (TEWL) measurements. When ceramides and/or other barrier lipids are incorporated, it is, however, not always clearly demonstrated which ingredients of the formulation exert the beneficial effects. Objectives In this study the effects of topically applied ceramide-containing mixtures on the barrier repair of sodium lauryl sulphate (SLS)- and acetone-induced skin damage have been studied in human volunteers. TEWL and stratum corneum hydration measurements were carried out. The emulsions applied contained either a mixture of two types of ceramides, CerIII and CerIIIB (emulsion 1) or a complete mixture of ceramides III, IIIB and VI together with phytosphingosine, cholesterol and the free fatty acid linoleic acid (emulsion 2). Results After SLS damage, it was observed that barrier recovery was significantly accelerated by topical application (14 days, 2 ×/d) of emulsion 2 compared with the results obtained with emulsion 1. Corneometrical results were not relevant due to the occurrence of scaly fissured skin, failing to provide a good skin/probe contact. Although no effect on TEWL could be observed, the improvement of skin hydration after acetone treatment and a single application of the emulsions, was significantly more positive for emulsion 2 than for emulsion 1. Conclusions The investigative methods used in this study show that ceramides combined with other skin lipids can improve barrier repair after damage.

After removing lipophilic material, the ground root bark of Quassia africana Baill. (Simaroubaceae) was extracted with ethanol 95 %. Partitioning between chloroform, ethyl acetate and water yielded three crude extracts. Pronounced activities were shown by the chloroform and ethyl acetate crude extracts against Herpes simplex, Semliki forest, Coxsackie and Vesicular stomatitis viruses. By repeated column chromatography and preparative thin layer chromatography on silica gel, two quassinoids, i. e., quassin and simalikalactone D were isolated. Structures of the pure compounds were established primarily using NMR spectroscopy. Mass spectral information confirmed the assigned structures. Simalikalactone D was responsible, at least in part, for the high antiviral activity observed for the chloroform crude extract. Quassin showed no activity. For quassinoids the ester group at C-15 and the epoxymethano bridge between C-8 and C-13 appeared to be important structural features in order to exhibit a pronounced antiviral activity.

Rice starch added to bath water was studied for its possible beneficial effects on impaired barrier function as evaluated by transepidermal water loss measurements. The forearm skin of healthy volunteers was irritated by sodium lauryl sulphate. Exposure to rice-starch-containing bath water--twice daily for 15 min--led to a 20% improvement on the healing capacity of damaged skin. The beneficial effect was also observed for a rice-starch-containing lipid-free bath formulation, and an oil-in-water bath lotion enriched with evening primrose oil. Skin barrier function in patients with atopic dermatitis also improved after the addition of starch powder to bath water. Rice starch in powder or formulated in a bath product can therefore be recommended as a skin repair bathing additive for barrier damaged skin, particularly in the case of atopic dermatitis patients.

The expression of sulfotransferase and steroid sulfatase was studied in rat liver using the most promising culture models of hepatocytes, including monolayer culture with a pyruvate (30 mM) enriched medium, co-culture with rat epithelial cells from primitive biliary origin and collagengel sandwich culture. In the latter, addition of dexamethasone (1 microM) to the medium was examined. Phenol sulfotransferase enzymes (SULT1) were studied by measuring activities towards 4-methylphenol and estradiol, hydroxysteroid sulfotransferase (SULT2A) activity was determined towards dehydroepiandrosterone (DHEA). Microsomal steroid sulfatase activity was measured towards estrone sulfate. Western blot analysis was carried out using polyclonal antibodies raised against rat phenol sulfotransferase SULT1A1 (ASTIV), estrogen sulfotransferase SULT1E1 (EST) and hydroxysteroid sulfotransferase (HST). SULT2A activity towards DHEA was maintained at a high level during the whole culture time. In the co-culture it even reached the level of freshly isolated cells. Addition of pyruvate had no positive effect on the activity measured in monolayer cultures. High SULT1A1 activity towards 4-methylphenol was found in the co-culture system. In the monolayer culture, the activity initially decreased with 35% but was then kept at a constant level, while in the sandwich culture low activities were measured. For dexamethasone, an inducing effect on the various SULT activities could not be detected. Independently of the culture model used, the SULT1E1 activity towards estradiol decreased to 20% and 5% of the initial activity after four and seven days of culture, respectively. Microsomal steroid sulfatase activity was best maintained in collagengel sandwich cultures. During the first four days in culture it retained 73% of the initial activity, afterwards it decreased to 40% of the activity found in freshly isolated hepatocytes, irrespective of the culture conditions. High expectations exist for collagengel sandwich cultures, however, in our study the results were rather disappointing. Monolayer is a suitable culture model for short-term purposes. For long-term in vitro biotransformation studies, co-culture is preferred but is rather complex.

Albumin secretion, expression of cytochrome P450 dependent mono-oxygenases (CYPs) and their inducibility by well-known inducers were evaluated during 1 week in collagen type I gel sandwich and immobilisation cultures of adult primary rat hepatocytes. Albumin secretion increased during culture time and, following an initial decrease, CYP biotransformation activities remained stable for at least 7 days. Better preservation results were observed in the collagen gel sandwich culture than in the immobilisation model. The inducibility of CYPs by beta-naphthoflavone (beta-NF), 3- methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (DEX) was studied in both collagen gel hepatocyte cultures. Exposure of the cells to either 5microM 3-MC or 25 microM beta-NF, added to the culture medium, resulted in strong increases of CYP1A1/2 activity in both culture models. Treatment with PB (3.2 mM) resulted in an increase in the CYP2B activity and a higher hydroxylation of testosterone in the 16alpha-position (CYP2B1/2 and CYP2C11), the 7alpha-position (CYP2A1/2), and the 6beta-position (CYP3A1). DEX (10 microM) markedly increased testosterone 6beta- and 7alpha-hydroxylation. Expression and induction experiments of CYP proteins exposed to these molecules confirmed the results of the CYP activity measurements. The patterns of CYP induction in collagen gel cultures of rat hepatocytes were similar to those observed in vivo. Consequently, collagen gel cultures and, more specifically, collagen gel sandwich cultures seem to be suitable as in vitro models for evaluating xenobiotics as potential inducers of CYP-enzymes.

Ethanol, a human toxicant and a solvent in pharmacological research, is known to interfere with biotransformation of xenobiotics. We compared the in vivo and in vitro long-term effects of ethanol exposure on the expression of glutathione S-transferases (GST, EC 2.5.1.18) in rat river. Long-term in vivo ethanol treatment to achieve blood ethanol levels ranging between 10-50 mM was by liquid diet feeding. For in vitro experiments, rat hepatocytes co cultured with rat liver epithelial cells were exposed to 17 and 68 mM ethanol for up to 10 days. Two weeks of liquid diet ethanol treatment increased total GST activity. Both Mu and Alpha classes and in particular the A1 and A2 subunits and the amount of their corresponding mRNAs were increased. Total GST activity was also increased in co cultures after exposure to 68 mM ethanol for 10 days. However, the Mu class subunits M1 and M2 and the corresponding mRNAs were increased, rather than the Alpha class subunits. Thus, long-term exposure to ethanol induces hepatic GST both in vivo and in, vitro, but different isoenzymes are affected. Consequently, extrapolation of in vitro data on GST expression and regulation to the in vivo situation must be judicious. During xenobiotic metabolism in cell culture, a shift in relative expression and induction of different GST forms may occur, resulting in either an under or overestimation of effects. (C) 2000 Elsevier Science Inc.