Current status of healthy human skin models: can histone deacetylase inhibitors potentially improve the present replacement models?

Histone deacetylase inhibitors (HDACi), a relatively new group of epigenetic agents, are being investigated as powerful chemotherapeutics because of their antiproliferative and prodifferentiation effects both in vitro and in vivo, in various tumor cell lines. Only little is known with respect to their effects on normal cells. Yet, to understand tissue pathology and evaluate potential effects of new chemical entities in tissue homeostasis, insight into the physiology of healthy tissue is necessary. Therefore, this review addresses the effects of HDACi on healthy human primary skin cell cultures and three-dimensional epidermal models. In general, HDACi exert an effect on both the epidermal morphology and differentiation process of human skin. The latter is manifested through cell cycle arrest, disorganization of the basal layer, thinning of the stratum spinosum and thickening of the stratum corneum, reorganization of the cytoskeleton and increased formation of cornified envelopes. This overview shows that, although only a limited number of reports exist, these molecules might be an interesting tool for the development and study of new human skin models.

Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies.

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Inhibition of NF-kappaB activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytes.

OBJECTIVE: Benzoylaminoalkanohydroxamic acids, including 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), are structural analogues of Trichostatin A, a naturally occurring histone deacetylase inhibitor (HDACi). 4-Me(2)N-BAVAH has been shown to induce histone hyperacetylation and to inhibit proliferation in Friend erythroleukaemia cells in vitro. However, the molecular mechanisms have remained unidentified. MATERIALS AND METHODS: In this study, we evaluated the effects of 4-Me(2)N-BAVAH on proliferation in non-malignant cells, namely epidermal growth factor-stimulated primary rat hepatocytes. RESULTS AND CONCLUSION: We have found that 4-Me(2)N-BAVAH inhibits HDAC activity at non-cytotoxic concentrations and prevents cells from responding to the mitogenic stimuli of epidermal growth factor. This results in an early G(1) cell cycle arrest that is independent of p21 activity, but instead can be attributed to inhibition of cyclin D1 transcription through a mechanism involving inhibition of nuclear factor-kappaB activation. In addition, 4-Me(2)N-BAVAH delays the onset of spontaneous apoptosis in primary rat hepatocyte cultures as evidenced by down-regulation of the pro-apoptotic proteins Bid and Bax, and inhibition of caspase-3 activation.

Coatings in food contact materials: Potential source of genotoxic contaminants?

Up till now, no harmonized EU regulation exists on chemicals used in coatings for food contact materials (FCM). Therefore, these substances need to comply with the general provisions of EU Regulation 1935/2004 and, if present, with national legislation. Different 'inventory lists' of compounds that might be present in coatings are available, but for hundreds of these substances, the potential human health impact of their use in FCM coatings has not (recently) been evaluated. Since detailed evaluation of all compounds is not feasible, a pragmatic approach was developed to identify substances with a potential concern for human health. First, an inventory was assembled containing all substances potentially used in coatings. Afterwards, the genotoxic potential of the non-evaluated substances was predicted in silico using two structure-activity relationship (SAR) software programs. For substances yielding structural alerts in both models, genotoxicity data were collected from previous European evaluations in a non-FCM context and from the European CHemicals Agency (ECHA) website. In total, 53 substances were identified as genotoxic in both in silico models, of which ten were considered to be of high concern. For most of the substances, additional toxicological information is needed.

L. fermentum CECT 5716 prevents stress-induced intestinal barrier dysfunction in newborn rats.

BACKGROUND: Intestinal epithelial barrier (IEB) dysfunction plays a critical role in various intestinal disorders affecting infants and children, including the development of food allergies and colitis. Recent studies highlighted the role of probiotics in regulating IEB functions and behavior in adults, but their effects in the newborn remain largely unknown. We therefore characterized in rat pups, the impact of Lactobacillus fermentum CECT 5716 (L. fermentum) on stress-induced IEB dysfunction, systemic immune response and exploratory behavior. METHODS: Newborn rats received daily by gavage either L. fermentum or water. Intestinal permeability to fluorescein sulfonic acid (FSA) and horseradish peroxidase (HRP) was measured following maternal separation (MS) and water avoidance stress (WAS). Immunohistochemical, transcriptomic, and Western blot analysis of zonula occludens-1 (ZO-1) distribution and expression were performed. Anxiety-like and exploratory behavior was assessed using the elevated plus maze test. Cytokine secretion of activated splenocytes was also evaluated. KEY RESULTS: L. fermentum prevented MS and WAS-induced IEB dysfunction in vivo. L. fermentum reduced permeability to both FSA and HRP in the small intestine but not in the colon. L. fermentum increased expression of ZO-1 and prevented WAS-induced ZO-1 disorganization in ileal epithelial cells. L. fermentum also significantly reduced stress-induced increase in plasma corticosteronemia. In activated splenocytes, L. fermentum enhanced IFNγ secretion while it prevented IL-4 secretion. Finally, L. fermentum increased exploratory behavior. CONCLUSIONS & INFERENCES: These results suggest that L. fermentum could provide a novel tool for the prevention and/or treatment of gastrointestinal disorders associated with altered IEB functions in the newborn.

Triiodothyronine downregulates the periportal expression of alpha class glutathione S-transferase in rat liver.

Most drug-metabolizing phase I and phase II enzymes, including the glutathione S-transferases (GST), exhibit a zonated expression in the liver, with lower expression in the upstream, periportal region. To elucidate the involvement of pituitary-dependent hormones in this zonation, the effect of hypophysectomy and 3,3',5-triiodo-L-thyronine (T3) on the distribution of GST was studied in rats. Hypophysectomy increased total GST activity both in the periportal and perivenous liver region. Subsequent T3 treatment counteracted this effect in the perivenous zone. However, analysis for either mu class M1/M2-specific (1,2-dichloro-4-nitrobenzene) or alpha class A1/A2-specific (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) GST activity revealed that T3 treatment did not significantly affect the perivenous activity of these GST classes. In contrast, T3 was found to significantly counteract the increase of alpha class GST activity caused by hypophysectomy in the periportal zone. To establish whether this effect was T3-specific, hepatocytes were isolated from either the periportal and perivenous zone by digitonin/collagenase perfusion and cultured either as pyruvate-supplemented monolayer or as co-culture with rat liver epithelial cells. Only in the latter it was found that T3 suppressed the A1/A2-specific GST activity and alpha class proteins predominantly in periportal cells. The data demonstrate that T3 is an important factor responsible for the low expression of alpha GST in the periportal region. T3 may be involved in the periportal downregulation of other phase I and II enzymes as well.

Hormonal regulation of glutathione S-transferase expression in co-cultured adult rat hepatocytes.

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.

Hydroxypropyl-beta-cyclodextrin as delivery system for thyroid hormones, regulating glutathione S-transferase expression in rat hepatocyte co-cultures.

Thyroid hormones play a role in the regulation of glutathione S-transferase (GST) expression. Here, co-cultures of rat hepatocytes with bile duct epithelial cells have been used to study the direct effects of both triiodothyronine (T3) and thyroxine (T4) on GST activities and proteins. Because T3 and T4 are poorly water soluble and organic solvents used to dissolve them often interfere with biotransformation pathways, an alternative delivery system namely hydroxypropyl-beta-cyclodextrin (HPBC) has been applied. Appropriate control cultures contained either 0.02 or 0.10% (w/v) HPBC, the concentrations necessary to supply T3 and T4 (10(-9) to 10(-5) M) to the cells, respectively. No effect of the vehicle HPBC on the different GST isoenzyme activities and proteins could be observed. On the contrary, after 10 days of co-culture, T3 and T4 decreased GST protein concentrations as well as GST activities measured by 1-chloro-2,4-dinitrobenzene (broad spectrum), 1,2-dichloro-4-nitrobenzene (Mu class M1/M2-specific) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Alpha class A1/2-specific) in a concentration-dependent manner. The Alpha class subunits A1/2 and A3, and the Mu class subunit M2 were mostly affected. No effect was observed on the Pi class enzyme. These findings indicate that a combination of co-cultured hepatocytes with an HPBC-based delivery system for hydrophobic compounds represents a powerful in vitro tool in drug development.

Influence of culture system and medium enrichment on sulfotransferase and sulfatase expression in male rat hepatocyte cultures.

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.

Effect of ethanol on the expression of hepatic glutathione S-transferase: an in vivo/in vitro study.

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 liver. 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.

Effect of pyruvate on glutathione s-transferase expression in primary cultures of rat hepatocytes.

Recently, primary rat hepatocyte cultures supplemented with 30 mm pyruvate and various hormones have been proposed as suitable long-term in vitro models for xenobiotic metabolism studies. In this study, the effect of 30 mm pyruvate on the cytosolic phase II biotransformation enzyme glutathione S-transferase (GST, EC 2.5.1.18) has been investigated. Adult male rat hepatocytes were brought into primary culture in serum free Williams' medium E with and without supplementation of 30 mm pyruvate. Total, Mu and Alpha class GST activities as well as GST subunit patterns were determined on the day of the isolation and as a function of culture time. Cell morphology and albumin secretion were also examined. After 7 days of culture, the morphology of pyruvate-treated cultures was still intact and bile canaliculi were clearly visible, whereas control cells without pyruvate had deteriorated and lost their normal morphology. The albumin secretion rate was higher in pyruvate-supplemented than in non-supplemented cultures. Total, Mu and Alpha class GST activities were well maintained in the presence of pyruvate; conversely, without pyruvate, all GST isoenzyme activities were significantly decreased. These findings were confirmed at the protein level. However, when hepatocytes received medium containing pyruvate for 7 days, the Pi class subunit 7, normally not present in adult liver cells, was expressed at a much higher level than was the case in untreated hepatocyte cultures. This study clearly shows that GST activities and GST proteins, as well as cell morphology and albumin secretion, are better maintained in primary monolayer cultures of adult rat hepatocytes supplemented with 30 mm pyruvate than in control cultures. However, it was also found that pyruvate is a very strong inducer of GST Pi. Consequently, the percentage GST subunit pattern of cultured rat hepatocytes is significantly affected by pyruvate supplementation, which may have consequences for in vitro xenobiotic metabolism studies.

Effect of oxygen concentration on the expression of glutathione S-transferase activity in periportal and perivenous rat hepatocyte cultures.

Cultures of perivenous (PV) and periportal (PP) hepatocytes could provide suitable in vitro models for studying the zone-specific hepatotoxic potential of xenobiotics. However, it is not known whether cultured PP and PV hepatocytes keep their phenotypes when the microcirculation of the liver changes. This question has been studied by culturing rat hepatocytes at 13 and 4% (v/v) O(2), respectively, mimicking the acinar oxygen gradient. PP and PV adult rat hepatocytes were isolated by digitonin-collagenase in situ perfusion and cultured on plastic Falcon and gas-permeable Petriperm dishes in Williams' E medium and kept at 13 and 4% (v/v) O(2), respectively. Cultures at 20% (v/v) O(2) on plastic dishes served as a control. Two types of cultures were studied, namely conventional cultures either unsupplemented or supplemented with 30 mM pyruvate. The activities of glutamine synthetase (GS) and glutathione S-transferase (GST) were measured in freshly isolated PP and PV hepatocytes and all cultures. The heterogeneous expression of GS (PV>PP), observed in freshly isolated hepatocytes, was kept for at least 4 days in culture. Total, Mu and Alpha class GST activities were predominantly expressed in PV freshly isolated cells. However, no beneficial effect could be observed in culture by exposing the cells to their specific in vivo oxygen concentration. The best maintenance of GST PV predominance in culture was observed in Petriperm dishes at 20% (v/v) O(2), as well in pyruvate-supplemented as unsupplemented cultures. PV GST predominance was thus kept in particular when the highest oxygen concentration was used and made available to the cells through the gas-permeable membranes. The results on GS PV predominance support these findings.

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures.

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

[The role of intercellular communication via "gap junctions" in disease]. / De rol van intercellulaire communicatie via 'gap junctions' bij ziekte.

Gap junctional intercellular communication plays an important role in the maintenance of cellular homeostasis. The flow of chemical messengers through gap junctions, gap junctional intercellular communication, is essential in processes such as electrical coupling, embryonic development and adaptive tissue response. Gap junctions are formed by connexin proteins. Mutational alterations in the connexin genes are associated with the occurrence of multiple diseases, such as peripheral neuropathy, cardiovascular disease, dermatological disease, hereditary deafness and cataract. Consequently, modulation of gap junctional intercellular communication is a potential pharmacological target. Future research, based, for example, on the recent developments in genetics, may clarify gap junction physiology. This will in turn provide promising perspectives for the development of targeted drugs.

Drug-induced liver injury: mechanisms, types and biomarkers.

Drug-induced liver injury is a ubiquitous issue in clinical settings and pharmaceutical industry. Hepatotoxicity elicited by drugs may be intrinsic or idiosyncratic, both which are driven by different molecular mechanisms. Recently, a unifying mechanistic model of drug-induced liver injury has been introduced. According to this model, drug-induced hepatotoxicity relies on 3 consecutive steps, namely an initial cellular insult that leads to the occurrence of mitochondrial permeability transition, which in turn ultimately burgeons into the onset of cell death. Clinically, drug-induced liver injury can be manifested in a number of acute and chronic conditions, including hepatitis, cholestasis, steatosis and fibrosis. These pathologies can be diagnosed and monitored by addressing well-established physical, clinical chemistry and histopathological biomarkers. In the last few years, several novel read-outs of drug-induced liver injury have been proposed, involving genetic, epigenetic, transcriptomic, proteomic and metabolomic parameters. These new concepts and recent developments in the field of drug-induced liver injury are revised in the current paper.

A liver-derived rat epithelial cell line from biliary origin acquires hepatic functions upon sequential exposure to hepatogenic growth factors and cytokines.

Withdrawal of promising drug candidates is often due to the detection of liver toxicity. In particular the parenchymal liver cells or hepatocytes are targeted since they are the major sites of drug transport and of metabolite formation and thus also the place where not only detoxification, but also activation of new chemical (NCE) and biological (NBE) entities may occur. Therefore, primary hepatocyte- based cultures are currently the preferred in vitro model to screen for liver toxicity. However, within a few days, they undergo dedifferentiation with loss of liver-specific functionality, including xenobiotic biotransformation capacity, making them only suitable for short-term applications. A plausible alternative to primary hepatocyte cultures that can be maintained for longer periods of time could be the use of liver-derived epithelial cell lines and their optimized derivatives. Therefore, in the present study, we evaluated the stability and the hepatic differentiation potential of a neonatal liver-derived rat epithelial cell line from biliary origin (rLEC). Undifferentiated rLEC stably express the hepatic progenitor markers CEBPA, FOXA2, GJA1, ONECUT1, KRT18 and KRT19 for at least 15 consecutive passages after cryopreservation. Upon sequential exposure to hepatogenic growth factors and cytokines, rLEC generate functional hepatic progeny, expressing mature hepatic markers including Alb, Ahr, Car, C/ebpα, Cx32, Foxa2, Hnf1α, Hnf1ß and Onecut1. Furthermore, an active polarization is observed for the hepatic drug transporters Oatp4 and Ntcp. rLEC-derived hepatic cells also acquire the ability to store glycogen, express genes encoding for key hepatic enzymes as shown by Affymetrix microarray data, and display stable CYP1A1/2- and CYP2B1/2-dependent activities for several weeks at levels comparable to those observed in cultured primary rat hepatocytes. The acquisition of such a stable and active biotransformation capacity is key for the applicability of liver-based in vitro models for long-term toxicity testing.