Abstract: Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17β-glucuronide (E(2)17βG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17βG and MPP+. While the affinity of E(2)17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), Vmax and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17βG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, Vmax or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.
Abstract: HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.
Abstract: Solar radiation is one of the most common threats to the skin, with exposure eliciting a specific protective cellular response. To decrypt the underlying mechanism, we used whole genome microarrays (Agilent 44K) to study epidermis gene expression in vivo in skin exposed to simulated solar radiation (SSR). We procured epidermis samples from healthy Caucasian patients, with phototypes II or III, and used two different SSR doses (2 and 4 J/cm(2)), the lower of which corresponded to the minimal erythemal dose. Analyses were carried out five hours after irradiation to identify early gene expression events in the photoprotective response. About 1.5% of genes from the human genome showed significant changes in gene expression. The annotations of these affected genes were assessed. They indicated a strengthening of the inflammation process and up-regulation of the JAK-STAT pathway and other pathways. Parallel to the p53 pathway, the p38 stress-responsive pathway was affected, supporting and mediating p53 function. We used an ex vivo assay with a specific inhibitor of p38 (SB203580) to investigate genes the expression of which was associated with active p38 kinase. We identified new direct p38 target genes and further characterized the role of p38. Our findings provide further insight into the physiological response to UV, including cell-cell interactions and cross-talk effects.
Abstract: Practically all human hepatocyte cell lines are deficient in major cytochrome P450 (CYP)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the recently developed HepaRG cell line to determine the spectrum of most important CYP enzyme activities involved in xenobiotic metabolism (CYP1A1/2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) and the effect of the prototypical CYP-inducer phenobarbital and a panel of known CYP-selective inhibitors on these activities. Comparison of these activities was carried out with two human primary hepatocyte populations. We show that excluding CYP2A6 and CYP2E1, HepaRG cells express high functional levels of most of the major xenobiotic metabolising CYPs. These activities were found to be selectively inhibited and induced by prototypical CYP-selective inhibitors and inducer at comparable levels to primary hepatocytes. In conclusion, HepaRG cells may be a promising cell line for various applications, which currently employ hepatic subcellular preparations or cultured primary hepatocytes.
Abstract: Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification.Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa)channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.
Abstract: Merkel cells (MCs) are involved in mechanoreception, but several lines of evidence suggest that they may also participate in skin disorders through the release of neuropeptides and hormones. In addition, MC hyperplasias have been reported in inflammatory skin diseases. However, neither proliferation nor reactions to the epidermal environment have been demonstrated. We established a culture model enriched in swine MCs to analyze their proliferative capability and to discover MC survival factors and modulators of MC neuroendocrine properties. In culture, MCs reacted to bFGF by extending outgrowths. Conversely, neurotrophins failed to induce cell spreading, suggesting that they do not act as a growth factor for MCs. For the first time, we provide evidence of proliferation in culture through Ki-67 immunoreactivity. We also found that MCs reacted to histamine or activation of the proton gated/osmoreceptor TRPV4 by releasing vasoactive intestinal peptide (VIP). Since VIP is involved in many pathophysiological processes, its release suggests a putative regulatory role for MCs in skin disorders. Moreover, in contrast to mechanotransduction, neuropeptide exocytosis was Ca(2+)-independent, as inhibition of Ca(2+) channels or culture in the absence of Ca(2+) failed to decrease the amount of VIP released. We conclude that neuropeptide release and neurotransmitter exocytosis may be two distinct pathways that are differentially regulated.
Abstract: Cytochrome P450 (P450) induction may have considerable implications for drug therapy. Therefore, understanding the induction potential of a new chemical entity at an early stage in discovery is crucial to reduce the risk of failure in the clinic and help the identification of noninducing chemical structures. Availability of human viable tissue often limits evaluation of induction potential in human hepatocytes. A solution is to increase the time period during which the hepatocytes remain viable. In this study we have investigated the induction of several P450 isozymes in long-term cultured hepatocytes compared with short-term cultured hepatocytes from the same individuals. Short- and long-term cultured primary hepatocytes isolated from each individual were cultured in a 96-well format and treated for 24 h with a range of prototypical P450 inducers and Merck Research Laboratories compounds. CYP3A4, 1A1, 1A2, 2B6, and 2C9 mRNA levels were measured using quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan) from the same cultured hepatocyte wells. CYP3A4, 1A1, 1A2, 2B6, and 2C9 were shown to be inducible in long-term cultured hepatocytes. The -fold induction varied between donors, and between short- and long-term cultured hepatocytes from the same donor. However, this variability can be controlled by normalizing data from each hepatocyte preparation to a positive control. The use of long-term cultured hepatocytes on 96-well plates has proven to be sensitive, robust, and convenient for assessing P450 induction potential of new compound entities during the drug discovery process.
Abstract: Synopsis Octadecenedioic acid is known as a skin whitening agent but its activity is not mediated via a direct inhibition of tyrosinase. Based on the secondary properties of this molecule, such as its anti-inflammatory and anti-ageing effects, we postulated that octadecenedioic acid interacted with the peroxisome proliferator-activated receptor (PPAR) as this nuclear receptor also mediates these effects. Using reporter gene technology, we were indeed able to demonstrate binding of octadecenedioic acid to all three PPAR subtypes, in particular PPARgamma with an EC(50)-value of approx. 1 x 10(-6) m. Binding to PPARgamma of octadecenedioic acid or rosiglitazone, a known pharmaceutical PPARgamma agonist, led to reduced melanogenesis. Subsequently also tyrosinase mRNA (as measured by real-time polymerase chain reaction) and tyrosinase levels (as measured by Western blot) were reduced, suggesting the existence of a complete novel mechanism of skin whitening agents: binding to PPARgamma results in reduced tyrosinase mRNA expression which in turn results in less tyrosinase being formed. This in turn leads to reduced melanogenesis both in vitro and in vivo Because octadecenedioic acid binds not only to PPARgamma but also to PPARalpha and PPARdelta, other efficacies mediated via these receptors may also be expected.
Abstract: Immature dendritic cells (DCs) derived from CD34+ progenitor cells or peripheral monocytes, are used as in vitro sensitization models in many chemical allergen treatment studies. During the sensitization, DCs follow maturation process and gain the capacity to migrate to lymph nodes where they stimulate T cells. Chemokine receptor allows DCs to migrate along chemotactic gradients. In this work, we used immature DCs from peripheral monocytes to evaluate the influence of allergens on chemokine receptor and surface-marker expression. We tested the sensitizers dinitrochlorobenzene, Bandrowski's base, and coumarin, as well as the tolerogen dichloronitrobenzene, the irritant sodium dodecyl sulfate and the solvent dimethyl sulfoxide. All skin sensitizers up-regulated the co-stimulatory molecule CD86 and increased the CD83+ cell population. No expression of the chemokine receptors CCR2, CCR3, CCR6, or CXCR5 was observed on DCs exposed to the tested chemicals. The strong allergen dinitrochlorobenzene slightly increased CCR7 expression on DCs but down-regulated CCR1 surface expression. CCR1 down-regulation was not mediated by a classical maturation pathway, as it was unaffected by the corticosteroid dexamethasone.
Abstract: Drug metabolism in humans is essentially performed by three cytochrome P450 (P450) families (1 to 3), including 23 isoforms. The expression of these P450s is highly variable, and the rate and nature of the metabolites produced depend on the nature and the concentration of individual isoforms. P450 expression pattern is therefore a necessary tool to evaluate the effects of a given drug on P450 expression, its potential toxicity, and eventual interference with other drugs administered concomitantly. This pattern provides a general outline of the induction/repression effects of drugs leading to further mechanistic studies. A real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to evaluate the overall P450 expression pattern and measure human CYP1 to CYP3 mRNAs involved in drug metabolism. Our RT-PCR-based P450 mRNA assay enables us to quantify P450s rapidly with high specificity, a single annealing temperature, and low amounts of biological sample. All 23 single assays were validated by assessing the effects (induction or repression) of known inducers (ethanol, 3-methylcholanthrene, rifampicin, dexamethasone, phenobarbital) on P450 expression in human primary hepatocytes. Since this method may be used to determine human P450 expression in any human tissue or cell culture, it is a valuable tool for reliable prediction of drug safety, drug toxicity, and drug-drug interference.
Abstract: Langerhans cells (LCs) are dendritic cells localized in epidermis and mucosal tissues, where they are responsible for triggering the immune response. To study LCs in the oral epithelium, organotypic cultures were prepared using gingival explants. Immunochemical techniques using anti-CD1a, anti-HLA-DR, and anti-Langerin antibodies were used to detect and quantify LCs at various times. Observations were made by light and confocal microscopy. Quantification studies showed that there is a statistically significant drop in LC numbers in the epithelial tissue after 96 h of incubation. Gingival organotypic cultures thus are a good model for studying the migration of LCs and their involvement in contact hypersensitivity and periodontal diseases. The model offers potential utility as a tool for the study of periodontal tissue in the presence of different stimuli and for conducting immunotoxicologic experiments.
Abstract: Hepatocyte-based therapy has been proposed as an alternative to organ transplantation in the treatment of liver disorders. In the clinical context, a major issue is the constant supply of quality assurance-controlled hepatocytes, thereby requiring their cold storage in good conditions. We have analyzed the protective effects of alginate entrapment of rat hepatocytes after either 24 or 48 h of hypothermic storage or cryopreservation on the cell viability, cell yield, both mitochondrial and other cytoplasmic functional activities, and apoptosis. Decrease in viability, as evaluated by the MTT inclusion test, was 4% and 13% (24 h at 4 degrees C), 15% and 33% (48 h at 4 degrees C), and 9% and 19% (liquid nitrogen) for entrapped and free suspended hepatocytes, respectively. Viable cell yields were 86 +/- 8% and 51 +/- 6% for cryopreserved entrapped and free suspended hepatocytes, respectively. The mitochondrial (MTS assay), 7-ethoxyresorufin O-deethylase (EROD), and glutathione-S-transferase (GST) activities were better preserved in entrapped than in free suspended hepatocytes. Both hypothermic storage and cryopreservation were found to induce early caspase-3-like activities, being always much lower in entrapped hepatocytes, particularly after cryopreservation (98.4 +/- 42.4 vs. 6.4 +/- 4.0 fluorescence arbitrary units/hours/microg protein). Thus, cold-induced apoptosis in hepatocytes can be significantly reduced following their entrapment within alginate gel beads and this is associated with an improvement of both their viability and function.
Abstract: Most of the slimming products already developed for cosmetic applications did not result from strategies that integrate whole lipolysis-regulating mechanisms. We thus focused our attention on a more complete integration of these mechanisms and we developed slimming liposomes (SLC) containing two micro-circulation activators, i.e., esculoside and Centella asiatica extracts, one phosphodiesterase inhibitor, i.e., caffeine, and one fatty acid-beta oxidation activator, i.e., L-carnitine. The validity of our approach was assessed through (a) in vitro tests demonstrating that SLC induced a dramatic increase in the cyclic adenosine monophosphate (cAMP) content in human adipocytes, with a subsequent rise in the nonesterified fatty acids (NEFA) content of human adipocyte incubation medium, and (b) in vivo studies showing that SLC could provide an actual potent slimming effect on human volunteers. Moreover, we give here, through binding experiments, the unambiguous demonstration that SLC is able to antagonize the alpha(2)-adrenergic receptor that is known to reduce intracellular AMPc content and, subsequently, to down-regulate lipolysis. This alpha(2)-adrenergic antagonism has never been reported for any component of SLC, and this work is the first demonstration of the alpha(2)-adrenergic antagonism of such a combination of active liposome compounds.
Abstract: A prevalidation study on in vitro tests for acute skin irritation was conducted during 1999 and 2000. The overall objective of validation in this area, of which this prevalidation study is an initial stage, is to identify tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union (EU) risk phrases ("R38"; no classification) and the harmonised OECD criteria ("Irritant"; no label). This prevalidation study specifically addressed aspects of: protocol refinement (phase I), protocol transfer (phase II), and protocol performance (phase III), in accordance with the prevalidation scheme defined by the European Centre for the Validation of Alternative Methods (ECVAM). The tests evaluated were: EpiDerm (phases I, II and III), EPISKIN (phases I, II and III), PREDISKIN (phases I and II, and additional protocol refinement), the non-perfused pig ear method (phases I and II, and additional protocol refinement), and the mouse skin integrity function test (SIFT; phases I and II). Modified, standardised test protocols and well-defined prediction models were available for each of the tests at the end of phase I. The results of phase I (intralaboratory reproducibility) were sufficiently promising for all of the tests to progress to phase II. Protocol transfer between the Lead Laboratory and Laboratory 2 was undertaken for all five tests during phase II, and additional refinements were made to the test protocols. For EpiDerm, EPISKIN and the SIFT, the intralaboratory and interlaboratory reproducibilities were acceptable; however, better standardisation of certain aspects of the test protocols was needed prior to commencing phase III. Neither PREDISKIN nor the pig ear test performed sufficiently well in phase II to progress to phase III. The PREDISKIN protocol was overly sensitive, resulting in the prediction of all the NI chemicals as I. The variability in the pig ear test results was too great, indicating that the test would show limited predictive ability. In additional studies (a repeat of phase I), further modification of the PREDISKIN protocol and a change in the prediction model considerably improved the ability of the test to distinguish I from NI chemicals. However, attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. In phase III an initial assessment of the reproducibility and predictive ability, in three independent laboratories per test, was undertaken for the EpiDerm and EPISKIN tests (the SIFT was a late inclusion in the prevalidation study, and is being evaluated in a separate phase III study). A set of 20 coded chemicals (10 I, 10 NI) were tested with the final, refined, test protocols. The intralaboratory reproducibility was acceptable for both EpiDerm and EPISKIN. The interlaboratory reproducibility was considered to be acceptable for EPISKIN; however, for EpiDerm, analysis of variance (ANOVA) indicated that there was a statistically significant laboratory effect on the overall variability, suggesting that the interlaboratory transferability of the test needs to be improved. The EpiDerm test had an overall accuracy of 58%, with an over-prediction rate of 37% and an under-prediction rate of 47%. The EPISKIN test had an overall accuracy of 58%, showing an under-prediction rate of 23% and an over-prediction rate of 60%. It is concluded that, as yet, none of the tests evaluated in this prevalidation study are ready for inclusion in a formal validation study on in vitro tests for acute skin irritation. Overall protocol performance of the SIFT is currently being evaluated in a phase III study. Further studies are also in progress to improve the test protocols and prediction models for EpiDerm and EPISKIN.
Abstract: In the present study we evaluated viability and detoxifying enzyme capacity of cryopreserved hepatocytes from various species, including man, immobilized in calcium alginate gels. Ethoxyresorufin O-deethylase, phenacetin deethylase, pentoxyresorufin O-dealkylase, tolbutamide hydroxylase, S-mephenytoin hydroxylase, dextromethorphan demethylase, and nifedipine oxidation corresponding to the major cytochromes P450 (CYP) involved in xenobiotic metabolism as well as whole glutathione S-transferase (GST) activity were measured using specific substrates and after exposure or not to prototypical inducers. After deep-freeze storage, viability of immobilized hepatocytes was only slightly reduced and most CYP-related monooxygenase activities were well preserved, being expressed at levels close to those measured in unfrozen hepatocyte monolayers. By contrast, total GST activity was decreased by around 50%. However, as did CYP1A- and 3A-related enzymes, rat GST remained capable of responding to prototypical inducers. The fold increases were comparable in unfrozen and frozen immobilized hepatocytes and in unfrozen hepatocyte monolayers. The duration of storage, even when exceeding one year, did not affect viability and functions. In conclusion, after cryopreservation, alginate-entrapped hepatocytes remain highly viable and capable of expressing most detoxifying enzymes at levels close to those expressed in corresponding unfrozen hepatocyte monolayers and of responding to prototypical inducers.
Abstract: Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.
Abstract: Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.
Abstract: 1. The metabolism of Meloxicam (ME) and the cytochrome(s) P450 (CYPs) involved were analysed by using primary human hepatocytes, human liver microsomes and microsomes from recombinant human B-lymphoblastoid cell lines. 2. While human hepatocytes were capable of converting ME to a 5-hydroxymethyl metabolite (M7) and then to a 5-carboxyderivative (M5), human liver microsomes formed mostly only the 5-hydroxymethylderivative. The kinetics of the formation of M7 by human liver microsomes were biphasic with Km = 13.6 +/- 9.5 and 381 +/- 55.2 microM respectively. The corresponding Vmax were 33.7 +/- 24.2 and 143 +/- 83.9 pmol/min/mg protein respectively. 3. CYP2C9 and, to a much lesser extent, CYP3A4 were found to convert ME to M7. The involvement of 2C9 was demonstrated by inhibition of tolbutamide hydroxylase activity in the presence of ME, inhibition of ME metabolism by sulphaphenazole, correlation between ME metabolism and tolbutamide hydroxylase activity and active metabolism of ME by recombinant 2C9. The involvement of 3A4 was shown by inhibition of ME metabolism by ketoconazole, correlation between ME metabolism and nifedipine oxidase activity and metabolism of ME by recombinant 3A4. Kinetics of the formation of M7 by the individual enzymes resulted in a Km = 9.6 microM and Vmax = 8.4 pmol/min/mg protein for 2C9 and a Km = 475 microM and Vmax = 23 pmol/min/mg protein for 3A4.
Abstract: Rat hepatocytes immobilized in calcium-alginate beads were tested for their ability to survive and function in vitro by comparison with hepatocyte monolayer cultures before and after cryopreservation. The freeze-thaw protocol previously designed for suspended hepatocytes was used; the freezing medium was the Leibovitz medium containing 10% fetal calf serum and 16% dimethylsulfoxide. The functions investigated included protein, lipid, and urea synthesis, albumin secretion, glycogen content, and various phase I and phase II enzyme activities. Cell viability was not altered by immobilization and the freeze-thaw procedure. Most functions tested were expressed at similar levels in unfrozen immobilized hepatocytes and corresponding hepatocyte monolayers. Only protein neosynthesis and some phase II drug metabolizing enzyme activities were decreased. The freeze-thaw process had no detrimental effect, whatever the function investigated. In addition, cryopreserved immobilized hepatocytes remained responsive to inducers: ethoxyresorufin O-deethylase activity was increased 11-12 times by 3-methylcholanthrene treatment in both fresh and cryopreserved alginate-entrapped hepatocytes. These results clearly show that hepatocytes immobilized in calcium-alginate beads remain functional even after cryopreservation indicating that they represent a promising approach for xenobiotic metabolism and toxicity studies.
Abstract: Oxadiazon [5-ter-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)- 1,3,4-oxadiazol-2(3H)-one] was administered orally at 20-500 mg/ kg body wt/day to male Sprague-Dawley CD rats for 14 days, at 20-200 mg/kg body wt/day to male CD1 [CR1/CD-1(1GR)BR] mice for 28 days, and at 500 mg/kg body wt/day to male beagle dogs for 28 days. Although liver enlargement was observed in the three species, morphological studies indicated that peroxisome proliferation only occurred in rats and mice. Parallel biochemical investigations showed that there was a dose-dependent increase in the peroxisomal cyanide-insensitive palmitoyl CoA oxidase and acetyl carnitine transferase activities in treated rats and mice. Acetyl carnitine activity appeared to correlate well with the number and volume of peroxisomes as determined histologically. The increases in enzyme activities at 200 mg/kg body wt/day oxadiazon were comparable in rats and mice indicating that both rodent species were equally sensitive to oxadiazon-induced peroxisome proliferation. When added in vitro to cultured rat hepatocytes at concentrations ranging from 2.5 to 10 x 10(-5) M, oxadiazon induced a dose-dependent increase in the activities of palmitoyl CoA oxidase and acetyl carnitine transferase. The ratio obtained by comparing oxadiazon and clofibric acid on acetyl carnitine transferase activity at 5 x 10(-5) M in the present in vitro study on rat hepatocytes are equivalent to those that can be calculated from the results on this enzyme activity obtained in vivo in the rat with 500 mg/ kg body wt/day oxadiazon (this study) and clofibric acid (literature values), indicating that the rat hepatocyte cultures gave satisfactory results regarding peroxisome proliferation. Neither oxadiazon nor clofibric acid modified the activities of palmitoyl CoA oxidase and acetyl carnitine transferase in cultured human hepatocytes. The results presented here demonstrate clearly that oxadiazon induces peroxisome proliferation in rodents in vivo and in vitro, as determined both biochemically and morphologically, whereas dogs in vivo and human hepatocytes in vitro were refractory to peroxisome proliferation. This observation extends to the herbicide oxadiazon, which is structurally unrelated to other known peroxisome proliferators, the generally observed marked species difference in sensitivity to peroxisome proliferation, and has important implications in the human safety evaluation of this herbicide.
Abstract: The aim of this study was to analyse viability, growth, differentiation and drug metabolic capacity of cultured human keratinocytes obtained from post-mortem skin. Epidermal cells were prepared from 1-day post-mortem paired sun-exposed (outer) and sun-protected (inner) sites of the upper arm, of donors aged 47-80 years. The percentage of viable cells obtained from post-mortem skin was only slightly lower than that usually obtained for keratinocytes isolated from fresh skin, and no alterations of epidermal markers were noted. Keratinocytes isolated post-mortem from non-exposed skin had a higher viability (78 versus 73%), and a more active proliferation, while their attachment rate, keratin composition, lipid synthesis capacity and transglutaminase activity levels were similar to those of epidermal cells obtained from the sun-exposed skin. Keratinocytes isolated from post-mortem skin expressed various phase I and II activities at levels similar to those obtained with keratinocytes isolated from fresh skin while drug metabolizing enzyme activities were consistently higher in sun-exposed compared to sun-protected cells. The results support the conclusion that skin collected post-mortem can represent an alternative source of viable and functional epidermal cells, and that the functional changes that occur in adult keratinocytes habitually exposed to the sun, affect much more strongly the drug metabolism capacity than the expression of differentiation markers.
Abstract: We have studied the cytotoxity to human keratinocytes of three main classes of drugs known to induce cutaneous adverse reactions, namely beta-lactam antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs) and sulfonamides, using the neutral red uptake as an endpoint. IC(50) values were determined for 21 drugs, after 20 hr of exposure and compared with those obtained with rat hepatocytes. NSAIDs were found to be more cytotoxic than beta-lactum antibiotics to human keratinocytes. Large variations in IC(50) values were obtained between molecules of a same class, as well as between keratinocyte cultures from different donors, especially for beta-lactum antibiotics. All NSAIDs and beta-lactam antibiotics tested were more cytotoxic to rat hepatocytes (1.6- to 27-fold). Both cell types were only slightly sensitive to sulfonamides, if at all.
Abstract: In this study we analysed the expression and induction of several drug metabolizing enzymes involved in either phase I or phase II reactions, in adult human keratinocytes cultured in submerged conditions. We also evaluated the influence of confluence, subcultivation and cryopreservation on the expression of these enzymes. Besides ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities, which have been shown previously to be maintained in such cultures, three additional enzyme activities were measured (i.e. phenacetin deethylase, a phase I enzyme, and procainamide N-acetyltransferase and paracetamol sulfotransferase, two phase II enzymes). Post-confluent keratinocytes showed decreased activities in comparison with preconfluent cells and the different enzymes tested revealed different patterns. After confluence, some activities, such as those of procainamide N-acetyltrans-ferase, phenacetin deethylase and paracetamol sulfotransferase, showed only a slight decrease, whereas EROD and GST activities were decreased by 65 and 50%, respectively. No major differences were observed between keratinocytes in primary culture and those in second subculture. After freezing, xenobiotic metabolizing enzyme activities were only slightly reduced, if at all. Induction of EROD and GST enzymes was also analysed. Maximum EROD activity was obtained with 1 muM 3-methylcholanthrene (3-MC) and 20 muM benzanthracene (BA), in both pre-confluent and post-confluent cultures. At their optimal concentration 3-MC was a stronger inducer than BA. GST activity was slightly induced by the different compounds tested only in pre-confluent keratinocytes. In conclusion, the presence of a variety of drug metabolizing enzymes in adult human keratinocytes cultured in submerged conditions suggests that this model is suitable for investigating epidermal biotransformation of drugs and other chemicals and for determining the potential cutaneous toxicity of metabolites.
Abstract: To determine whether compounds are able to reach the neural microenvironment, a blood-brain barrier (BBB) co-culture model has been recently developed with bovine brain capillary endothelial cells and newborn rat astrocytes. In this study, the permeability of confluent endothelial cells to various compounds and the functional activity of P-glycoprotein (P-gp), an ATP-dependent pump known to efflux drugs from multidrug-resistant tumoral cells, was assessed. The permeability of the lipophilic compounds imipramine and sulpiride differed in relation to their structure. A good correlation was observed with in vivo brain extraction levels. P-gp activity was estimated by measuring the uptake of [(3)H]vinblastine by the endothelial cells, with or without verapamil, which is known to reverse drug resistance. Intracellular accumulation of the vinca alkaloid was strongly increased after addition of verapamil, suggesting that P-gp is active in these cells. These results provide further support for the use of the co-culture model of bovine brain endothelial cells and rat astrocytes to screen new centrally active drugs.
Abstract: A cell kit named PREDISAFE based on the use of confluent rabbit fibroblastic cells has been designed to predict eye irritancy of cosmetic raw materials and formulations. The kit can be stored for a few days and/or shipped at room temperature. Cytotoxicity was estimated after 1 min or 15 min contact with test compounds using the neutral red release assay. For the 84 products tested, IC50 values gave intervals similar to classes defined from the Draize test, i.e., mild, moderate, severe and extreme irritancy.
Abstract: Alterations in extracellular matrix occur in many chronic liver diseases leading to the formation of hepatic fibrosis. We have studied the effects of the putative hepatoselective fibrosuppressive compound HOE 077, a proinhibitor of prolyl 4-hydroxylase, on normal adult human and rat hepatocytes in primary culture. In human hepatocyte cultures, the cytotoxicity of HOE 077 was assessed after a 20-h treatment at concentrations ranging from 0.125 to 2 mg/ml of medium. No significant change was found in cell morphology, neutral red uptake, red oil staining, lactate dehydrogenase release, tetrazolium salt reduction, ethoxyresorufin O-deethylase activity and protein synthesis; however, HOE 077 slightly decreased DNA synthesis at 2 mg/ml. In rat hepatocyte cultures, the cytotoxicity of the compound was assessed by testing the same parameters after a daily exposure of cultures for 2 days or 4 days, at concentrations ranging from 0.25 to 4.5 mg/ml of medium. Whatever the concentration, the compound had no obvious morphological effect. However, hepatocytes were less spread at the concentration of 4.5 mg/ml. HOE 077 at 2 mg/ml slightly decreased neutral red uptake but was without obvious effect on protein synthesis after 2 days. By contrast, on day 4, protein synthesis was markedly reduced in hepatocyte cultures exposed to HOE 077 at 4.5 mg/ml. Hydroxyproline content determination in media from 4-day-old hepatocyte cultures incubated with HOE 077 at 0.5 to 4.5 mg/ml, showed a dose-dependent decrease in the hydroxyproline/proline ratio in acetic acid soluble material. By indirect immunoperoxidase, intracellular collagen IV was found to be inhibited in hepatocyte cultures after 4 days of exposure to 4.5 mg/ml HOE 077.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: To evaluate the potential use of cryopreserved hepatocytes for toxicological studies, rat, dog and human hepatocytes were frozen in Leibovitz medium containing 20% foetal calf serum and various concentrations of dimethylsulphoxide and stored in liquid nitrogen. 50% or more of the hepatocytes that attached and survived immediately after isolation still possessed these properties after freezing/thawing. Thawed hepatocytes from the three species showed only a slight reduction in their ability to metabolize phenacetin or to conjugate paracetamol with glucuronic acid. Sensitivity to the toxic effects of erythromycin was not affected by the MTT and neutral red assays. Similar observations were made with rat hepatocytes for five other toxic agents-chloramphenicol, chlorpromazine, acrylamide, chloroquine sulphate and p-chloromercuribenzoic acid. These results suggest that, after cryopreservation, isolated hepatocytes represent a suitable model for drug metabolism and toxicity screening studies.
Abstract: We explored the effect of transforming growth factor beta (TGF-beta), a cytokine that appears to play a central role in inflammatory events, on albumin expression by normal adult human hepatocytes and hepatoma cells. Addition of TGF-beta to primary human hepatocyte cultures resulted in a dramatic decrease in albumin accumulation and synthesis. This effect was dose-dependent, took place after a 48h incubation period and was maintained over 96h. TGF-beta-decreased albumin protein levels were associated with reduced albumin mRNA content. Actin mRNA levels were concomittantly increased. Comparable qualitative effects of TGF-beta were observed on human hepatoma HepG2 cells.
Abstract: Recent studies have clearly demonstrated that the hepatocyte requires a complex and well defined environment to survive and maintain differentiated functions in vitro. Soluble factors as well as cell-matrix and cell-cell interactions have been found to affect markedly hepatocyte functions. Thus co-culturing hepatocytes with another rat liver cell type results in a prolonged expression of liver functions including phase I and phase II drug-metabolizing enzymes. Addition of corticosteroids to the co-culture medium is a prerequisite, and accumulation of insoluble matrix components is observed within a few days primarily between the two cell types. Hepatocyte cultures have been widely used for pharmacology and toxicology studies during recent years, but most studies deal with short-term investigations. Although specific functions are not completely stabilized the use of long-term hepatocyte cultures represents a promising tool to investigate enzyme induction and inhibition, and drug chronic toxicity.
Abstract: Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, beta-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 +/- 30 x 10(6) viable hepatocytes vs. 520 +/- 40 x 10(6) viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin-preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin-preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Various parameters, including the nature and proportion of the constituents of the cryoprotective medium, the cooling rate, and the composition of the thawing medium, were evaluated for the cryopreservation of adult rat hepatocytes. The highest percentage of cells able to survive in culture was obtained by freezing in L15 medium containing 16% dimethyl sulfoxide, at a rate of 3 degrees C/min, and by adding 0.8 M glucose to the thawing medium. More than 50% of hepatocytes capable of attachment just after cell isolation kept this property after freezing and survived in primary culture. Dead cells could be eliminated before seeding by centrifugation on a Percoll layer. In culture, frozen cells exhibited a morphology similar to that of unfrozen cells and after 24 hr their protein secretion rate was reduced by only about 40%.
