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University of Groningen Human and rat organ slices de Kanter, Ruben IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2002 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): de Kanter, R. (2002). Human and rat organ slices: a tool to study drug metabolism and toxicity Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 15-06-2017 1 Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues Ruben de Kanter Mario Monshouwer Dirk Meijer Geny Groothuis Groningen University Institute for Drug Exploration, Department of Pharmacokinetics and Drug Delivery, Groningen, Netherlands (RdK, DM, GG) Pharmacia, Global Drug Metabolism , Nerviano, Italy (MM) adapted from Current Drug Metabolism 3, 39-59 (2002) Chapter one Abstract Metabolism of xenobiotica is often seen as an exclusive function of the liver, but some current findings support the notion that the lungs, kidneys and intestine may contribute considerably. After the establishment of the use of liver slices as a useful in vitro model to study metabolism and toxicity of xenobiotica, the same concept is currently being used for slices from lung, kidney and intestine. It is the aim of this review to discuss the use of organ slices in biotransformation research. The basic idea behind the use of tissue slices in biomedical research is the assumption that the cells under study will function optimally in vitro if they are cultivated in an environment that is most alike to their natural in vivo embedding, which is the case in tissue slices. Advantages in the use of organ slices are the relatively easy preparation as well as the potential standardization of both the preparation and use. Moreover, a direct interspecies comparison can be made between liver, lungs, kidneys and intestines, for example with respect to their metabolic capacity and their sensitivity for toxicants. Of major importance is that organ slices can be made with a similar procedure from organs/tissues originating from different species, including man. This latter aspect is useful in drug development in general but also for a better insight in the metabolic fate of compounds in man. Importantly the use of slices may largely contribute to a reduction in the use of experimental animals. § 1. Introduction Despite the fact that the liver is rightfully considered to be the most important organ involved in drug metabolism, some recent findings illustrate the importance of extra-hepatic drug metabolism for overall drug disposition in humans. For example, the use of St John’s Wort extracts (hypericum perforatum), an herbal product that is frequently used as antidepressant was reported to substantially decrease blood/plasma concentrations and efficacy of many drugs, including the immunosuppressant cyclosporine [275], the HIV protease inhibitor indinavir [250], the cardiac glycoside digoxin [43] as well as oral contraceptives [85]. As an herbal remedy, St. John’s Wort has not been subjected to the rigorous clinical testing of modern drug candidates. After the recognition that St. John’s Wort interacts with drugs that are metabolized by cytochrome P450 isoform (CYP) 3A4, it was suggested that St. John’s Wort might induce CYP3A4 expression. This was confirmed in vivo [192] and in vitro [208] and it appeared that St John’s Wort preparations induced not only hepatic CYP3A4 but also both intestinal CYP3A4 and human MDR1 P-glycoprotein (P-gp) [78]. Another example of the importance of the expression of intestinal drug metabolizing enzymes is the finding that grapefruit juice selectively downregulates 6 Introduction CYP3A4 expression in the intestinal wall. Clinically relevant interactions with grapefruit juice have been described for drugs that have a narrow therapeutic range like cyclosporin, and also for most dihydropyridines, terfenadine, saquinavir, midazolam, triazolam and verapamil [3,13]. These drugs can reach higher (toxic) concentrations in the blood as a result of the lowered CYP3A4 activity in the intestines due to grapefruit juice components. These examples illustrate the need for methods to study not only hepatic but also extrahepatic transport and metabolism. However, the relative contribution of individual organs in vivo is rarely investigated, because of technical reasons, but also because these studies are time consuming, expensive and, finally yet importantly, require a large number of experimental animals. In addition, animal data can often not be simply extrapolated to the human situation and animal models appear often inadequate for the screening of many compounds (figure 1). Thus, to prevent unexpected side effects, interactions and toxicity findings in man, there is a need for appropriate in vitro techniques, that reflect the in vivo situation in both animals and man, are technically simple and efficient in use. We consider the use of slices as a good option because tissue architecture is retained for liver as well as lung, kidney and intestine and because slices can be made both from experimental animal and from human organs. In this review we will discuss the use of organ slices in the study of xenobiotic metabolism and toxicity. The principle of the use of slices is to make multiple animal in vivo man in vivo animal in vitro man in vitro Figure 1. The relationship between in vitro and in vivo experiments in animals and in man. Results of the combined in vitro and in vivo experiments of man and animal species can be used for extrapolation (pharmacokinetic scaling) from data obtained with human organ slices, to the in vivo situation in humans. 7 Chapter one reproducible tissue samples, in such a way that in vitro experiments can be carried out on viable cells in the slice without disrupting the natural embedding of the cells. To compensate for the absence of blood flow that normally provides oxygen, nutrients and removes waste products, slices are prepared in a thickness (200-250 µm for liver, kidney, intestine and 500 µm for lung slices) that is thin enough to allow diffusion of substrates from the surrounding medium towards the even innermost cell layers of the slice. On the other hand, slices should be made thick enough (> 150 µm) to maintain the normal organ architecture and keep the proportion of the damaged cells (on the cutting edges) small. The latter aspect is also addressed by using a reasonable diameter: 6-10 mm [96]. Because the use of slices or the liver was reviewed earlier [171,227,322], here we mainly focus on lung, kidney and gut slices. Practical aspects, such as the preparation (§ 4) and incubation conditions (§ 4.1), cold- and cryopreservation (§ 5 and § 6), and their viability during incubation are addressed (§ 7), as well as the use of slices in studies on toxicity and xenobiotic metabolism (including clearance and enzyme induction and inhibition) (see § 8-10). Because of the similarity of preparation, and also because of their similar physical characteristics, slices are especially useful to compare different organs (e.g. liver and kidney) within one species, as well as between species (e.g. rat and man). The use of human tissue is given special attention with respect to ethical considerations and tissue sources (§ 2.2). As every in vitro system, it is important to recognize the inherent disadvantages and limitations of the use of slices. In addition, in this case one should carefully interpret the relevance of the in vitro results for the in vivo situation (§ 11). § 2. Why in vitro research? There are many reasons to use in vitro approaches instead of performing in vivo experiments in drug metabolism and toxicity studies. In vitro systems are ideal when large numbers of substances have to be compared. This is relevant for the screening of novel compounds to select future drugs, that is if a specific endpoint can be defined. In vitro experimentation provides an environment where the various conditions variables can be carefully manipulated and controlled. Ideally, in vitro methods are easy to set up, often yield results quickly and are inexpensive as well as simple to run. In addition, in vitro systems can be a very efficient way to reduce the amount of experimental animals. In the pre-clinical setting, in vitro testing is the only option to produce human data. This is especially relevant if major differences exist between laboratory animals and man. Pharmacokinetic (PK) differences between species may be the result of functional differences or differential expression of transporter proteins, which determine cellular influx and efflux rates of drugs into or from cells. PK differences can also be due to the unequal presence or distribution of drug-metabolizing enzymes, or differences in the intrinsic catalytic activity within a particular fam8 Introduction ily of enzymes. For example, in man, CYP3A4 is the most abundant cytochrome P450 isoenzyme, whereas this isoenzyme is not present in the rat at all. Moreover, rodent CYP2A enzymes have steroid 7a- and 15a-hydroxylation activities while human CYP2A enzymes do not [124]. Species differences in metabolism are widely recognized, however, only a few studies compared metabolism of a single substrate in whole cell systems from different species, as rat, dog and man [22,84]. This type of study is most common using microsomal preparations [123]. Certain species differences appear to be almost specific for a particular compound under study, and despite of extensive research, systematic structure-activity relationships are difficult to asses. In general, extrapolation of results obtained in animal experiments to the human condition is hazardous. As most of the abovementioned processes cannot be studied in detail in man in vivo, studies using human tissue in vitro have been elaborated. The data obtained with these in vitro studies have to be extrapolated to the in vivo situation. To enlarge the predictive quality of such extrapolations, the in vivo-in vitro relation obtained in animal experiments should be determined in parallel or beforehand. Apart from species comparisons, the purpose of many in vitro metabolism studies is to establish the metabolic stability of drugs [79], expressed as the metabolic half-life [138]. Another goal is to study drug-drug interactions on the level of metabolism [146,326]. Such interactions may be the result of inhibition of certain metabolizing enzymes of the parent compound, or of induction of enzymes, resulting in decreased concentrations of the parent drug. § 2.1. An industrial perspective During the process of drug development, pharmaceutical companies are mandated by regulatory agencies to investigate and to describe the metabolism and toxicity of a given drug candidate. Traditionally, assessment of drug metabolism and xenobiotic toxicity has depended largely on information gained in vivo, using animal models such as the rat, dog, and monkey, and extrapolation of this data to man. Unfortunately, there have been several instances where either the route or rate of drug metabolism varies substantially from experimental animals to man, as can also be the case for the susceptibility of man to xenobiotic induced toxicity. As a consequence of this, in the process of introducing new drugs into the clinic, scientists within pharmaceutical companies would prefer to have as much as possible preliminary human information early on in the process of drug development and safety assessment. In recent years, there is an extensive use of human model systems for studying the in vitro metabolism and in vitro toxicity of drug candidates. With respect to metabolism, studies have focused mainly on the liver, since this is considered to be the major site of metabolism, and have included primary cultures of hepatocytes, precision-cut liver slices, subcellular fraction and heterologously expressed drug-metabolizing enzymes. It should be mentioned that all these in 9 Chapter one vitro approaches have their own advantages and limitations and depending on the purpose of the study the most appropriate method should be selected. Within pharmaceutical companies it is the balance between cost, speed and accuracy, which determines the selection of the most appropriate in vitro assay. To which of the three previous mentioned aspects the highest priority is given, should really depend on the question to be addressed. Looking at drug metabolism, the information obtained by using in vitro metabolic studies to select candidate drugs before making them into full development, can be summarized in five points (table 1). Table 1. Information obtained by using in vitro metabolic studies 1. extent, rate and routes of metabolism 2. comparative metabolic profiles to choose the non rodent toxicological species 3. isolation, production and identification of major metabolites 4. predictions of whole body clearance and estimates of first-pass metabolism 5. identification of metabolic enzymes to predict polymorphism, induction or inhibition It is not possible to obtain all the information described in table 1, with one single in vitro approach. As this review is focusing on tissue slices we would like to highlight a few examples where we believe slices are an useful in vitro tool to obtain the required information, also keeping in mind the cost, speed and accuracy triangle. Currently, slices can be especially helpful to obtain information about the items described under 1, 2 and 4 in table 1. With the recent improvements of the cryopreservation technologies of liver slices (§ 6), the preparation of extra-hepatic tissue slices (§ 4) and the higher throughput incubation possibilities using multiwell plates (§ 4.1), the applications of tissues slices has increased substantially (§ 8–10). In particular, the possibility of using cryopreserved human liver slices with both phase I and II metabolic enzyme capacities comparable to freshly prepared liver slices is of interest. This allows one not only to use preciously human livers in a more efficient way, but also to obtain human metabolism information at any time we need. The same is true for other species such as monkey and dog, which provides an in vitro tool to investigate a complete (both phase I and II) metabolite profiles across different species. Another useful application of tissue slices is the possibility to study extra-hepatic metabolism. In those situations where there are indications for a significant role of extra-hepatic metabolism, precision cut tissues slices of organs with drug metabolizing enzyme capacity is probably one of the easiest in vitro tools cur10 Introduction rently available. The ease of preparation (which is species independent) and scaling to in vivo (based on total organ weight) make the tissues slice technique a valuable in vitro tool for relatively short time incubations. The last interesting aspect of tissue slices that we would like to address at this point is the possibility of combining information on toxicity with metabolism data. Although pharmaceutical companies realize increasingly that toxicity and metabolism are two areas, which are closely related, very often the in vitro experiments performed to obtain toxicity or metabolism data are carried out separately. The fact that tissue slices can be seen as small ‘intact’ organs allow us to look at toxicity parameters, as described in § 7, after exposure to potential drug candidates. Performing these experiments, using different species and/or organs, in combination with metabolite profiles of the drug could help us to identify possible species specific toxicity due to metabolism already at an early stage of drug development. § 2.2. The use of human tissue Human tissue for in vitro research can be obtained from various sources: parts of donor tissue that is donated for transplantation, but for technical reasons cannot be used for transplantation purposes. This may concern surgical waste tissue that is acquired during the donor- or transplantation operation procedure, or donor tissue that appears not suitable for transplantation (discarded donor tissue). Furthermore tissue can be obtained as surgical waste from patients from whom tissue is removed for clinical reasons, and tissue can be obtained merely for scientific purposes. As stated above, the most important argument to use human tissue for biomedical research is the risky extrapolation of kinetic data from animal to man due to interspecies differences. Secondly, not only for human but also for animal experiments ethical considerations are at stake. For studies with human tissue, the privacy, safety and potential discomfort of the patient, including the discomfort produced by seeking informed consent should be carefully considered. In addition, the safety of the laboratory staff with respect to protection against possible infectious material should be guaranteed. With respect to animal experiments also the discomfort, the species and the number of animals needed should be carefully considered. In the third place, the quality of the tissue is often easier to control in animal experiments than is the case for human tissue. In general, one can assume that human donor tissue is preserved optimally, but in contrast, surgical waste tissue often is obtained under sub-optimal conditions. Moreover, the time between harvesting of the tissue and the start of the experiment is largely determined by the logistic and surgical procedures, and may sometimes result in decreased viability of the tissue [228]. Lastly, the amount of human tissue available for research purposes is constrained by legal, ethical, cultural and practical considerations. If proper planned and approved by the ethical committee, animals can be ordered at one’s 11 Chapter one wish and at times that are convenient for the investigators. Fresh human tissue is not available on command, is scarce and available with irregular intervals on unusual working h. For this reason appropriate cryopreservation methods (§ 6) will increase the efficiency of the use of the scarce tissue. In addition, the creation of tissue banks can provide an effective infrastructure whereby ethical aspects are assured while researchers can be supplied with various types of human tissues [6]. Such tissue banking highly facilitates the availability of human tissue and increases the transparency of the followed protocols. Human tissue banks require a professional setup, comparable to the United Kingdom Human Tissue Bank (www.ukhtb.org). This non-profit organization can organize the collection, processing, characterization, storage and distribution of the tissue for biomedical and pharmaco-toxicological research to ethically approved institutions [5]. It is considered ethically permissible, and in most countries compulsory, to obtain informed consent for the use of human material for research purposes from the involved patients/tissue donors or their family or relatives. It is considered ethically acceptable to make use of human tissue for fundamental and applied research if the use contributes directly or indirectly to optimization of medical treatment. In general, there are strong arguments against the commercial acquisition and supply of human tissue and tissue banks should therefore function on a not-for-profit basis. In vitro test systems with human tissue can be important both to prevent unnecessary or inappropriate animal experimentation and to achieve better and safer conditions for phase I and phase II studies. One has to keep in mind that the use of human tissue can reduce, but cannot always replace the use of experimental animals. The development of the in vitro technology is usually performed with animal tissue, as human tissue is scarce and should be used only if technical problems are solved to a reasonable extent. Nevertheless, one should be aware of species differences in the applicability of techniques as well. Moreover, studies with human material will increase our fundamental knowledge on the processes of drug metabolism and toxicity in man. An additional advantage of studies with human tissue is that a more appropriate choice of the animal species to be made for toxicological studies becomes possible, thereby reducing the number of animals used for drug development. § 2.3. In vitro methods: microsomes and isolated cells Several in vitro systems are available for studying the metabolism or toxicity of compounds, ranging from purified (recombinant) enzymes or microsomes to isolated cells, tissue slices or perfused organs. Among them, microsomal fractions of cell homogenates have been most extensively used to study drug metabolism because of the easy preparation. Microsomes can be prepared from different organs and species, be stored at –80°C and used when needed [346]. However, for organ and species comparisons and for quantitative measurements in general, some problems can arise with the use of microsomes. In the first 12 Introduction place, a very variable preparation yield is found between different laboratories, ranging from 33 mg [236] and 52.5 mg [147] microsomal protein per g tissue for human liver, or ranging from 6.1 mg/g [215] to 60 mg/g for rat liver [40]. In addition, the recovery of microsomes from other organs is relatively low, 3.8, 5.3 and 3.9 mg/g for respectively human lung, kidney and gut [236]. In addition, the concentration of cytochrome P450, as a marker protein of microsomes, is much lower in extra-hepatic microsomes than in liver microsomes. There are strong indications that these differences do not always represent the actual levels of smooth endoplasmic recticulum or concentration of cytochrome P450 but are at least partly due to the different recovery of microsomes or cytochrome P450 in the 100,000g pellet [248]. Reasons for this include the differences in the experimental procedure for preparation of microsomes [212], degradation of cytochrome P450 and/or contamination with bacteria, mucus (in the case of intestine and lung) or erythrocytes [48]. In addition, the intrinsic catalytic activity of microsomes largely depends on the experimental procedure used to prepare and incubate microsomes. This was recently shown in a detailed examination of the three primary variables in the preparation of human liver microsomes [212]. All these aspects complicate the use of microsomes for inter-organ comparisons and can be misleading when comparing the microsomal concentrations of extra-hepatic organs with liver microsomal values. Although microsomes are used extensively and can be of great value, one should realize that the metabolic conversions found are limited to those that are catalyzed by enzymes present in the endoplasmic recticulum membrane. Moreover, the in vitro reaction rate is not only determined by the sometimes non-physiological concentrations of cofactors that are added, but also that for some reactions (i.e. glucuronidation) the addition of detergents is necessary but is noxious for others (i.e. CYP mediated reactions). Metabolic conversions catalyzed by enzymes that are present in the cytosol can be studied using cytosolic preparations or a so-called S9 mix (a combination of microsomes and cytosol). Yet, the question which co-factors should be added remains a problem. A more complete in vitro model for drug metabolism, where in principle all the enzymes and co-factors are present, is the use of isolated cells. Isolated cells have also been used extensively to study biotransformation and toxicity. Especially the use of hepatocytes is quite established. However experimentation is limited to short term experiments due to a marked cytochrome P450 loss that occurs during culturing [238]. To overcome such dedifferentiation processes, more complex culture models have been developed to reproduce the interactions between different cell types present in the liver or between cells and the matrix, as co-culture [23] and collagen sandwich culture [77]. The use of isolated cells for metabolism and toxicity studies from lung, kidney or intestine is very limited, in contrast to hepatocytes. These organs contain many cell types (see § 3.1.1–§ 3.1.3) and it is often difficult to separate the different cell 13 Chapter one types and to keep cells viable and differentiated in culture. In many cases, the isolation of a particular cell type is a tricky process, requiring special skills, equipment or enzymes. Isolation techniques for the major drug-metabolizing cell types have been described. Yet, there is no apparent consensus on the most optimal preparation method for the isolation of type II pneumocytes [325], renal fragments and epithelial and glomerulal cells [9] or for intestinal epithelial cells [55]. A major drawback of the use of isolated cells is that the polarity of the cells is lost [121] and that quite aggressive enzymes have to be used for isolation that can damage the plasma membrane and largely affect its function. For instance it has been described that by collagenase digestion used to isolate hepatocytes, the amount of asialoglycoprotein receptor present on the membrane of the hepatocyte is decreased [351] and endogenous NO production is greatly induced [183]. § 2.4. In vitro methods: Slices The use of tissue slices is certainly not new. Liver slices have in fact been used since1923 (for an overview, see [171,227]-and references therein), and kidney slices were first used in 1948 (for a overview, see [24] and references therein). These slices were mainly used to study intermediate metabolism (liver) or transport processes (kidney). The use of liver slices as an in vitro tool to study metabolism and toxicity has become more widespread after the introduction of the Krumdieck tissue slicer (Alabama Research and Development Corp. Munford, AL, USA) and the Brendel-Vitron slicer (Vitron Inc., Tucson, AZ, USA). With these new slicers, the concept of precision-cut slices was introduced, which means that tissue slices can be cut with a consistent thickness, around 0.3 mm, and with minimal cutting-induced damage [290]. After the introduction of precision-cut liver slices [162], soon similar methods for lung [294] and kidney have been developed [273]. Also heart [243], brain [175], spleen [148], testis [337], and other tissues have been subjected to the slice technique in order to obtain precision-cut slices. The latter organs are unlikely to contribute much to the overall drug metabolism in the body and will therefore not be reviewed here. Of note, organ specific metabolism in these tissues does sometimes take place, which can result in organ specific toxicity. Precision-cut slices have some typical advantages compared with other slices prepared in other ways, e.g. using hand-held razor blades, McIIwain or StadieRiggs slicers. Briefly, thickness of precision-cut slices is far more constant and the amount of damaged cells is greatly reduced. Non-precision-cut slices are generally thicker slices (0.3-1 mm) and due to lack of O2 and substrates the inner cell layers show signs of necrosis very rapidly. In addition, it was found for lung slices, that in thicker slices (1 mm compared with 0.5 mm) higher rates of metabolism were found, due to the lower percentage of cells that were damaged as a result of the slicing procedure [172,219]. Very thick (2 mm) rat and human liver slices, cut by a self-made slicer were claimed to be more viable than preci14 Introduction sion-cut slices [285]. However, in this study penetration into the inner layers of the thick slices was studied only for small molecules, while incubations were performed at 22°C, apparently to slow down overall metabolism. In contrast, for precision-cut liver slices the contrary phenomenon was found; thinner instead of thicker slices are found to have the highest rate of metabolism on a per protein basis, because of the more optimal diffusion to the inner cell layers [71,255,340]. There are several obvious advantages to the use of precision-cut slices. In contrast to microsomes, all enzymes are situated in their physiological environment and the cofactors are present in more physiological concentrations. In contrast to the use of isolated cells, no proteolytic enzymes are necessary for preparation and normal polarity of the cells is preserved in the slices. Further, the cell-cell contacts remain intact and all cell types that are present in vivo are also present in the slices. This is important because the presence of matrix and other cell types can strongly influence both phase I and phase II metabolism [287] - and references therein]. Additionally, the procedure (see also § 4) to cut slices is quite simple and is in principle similar for different species. In addition, the procedure to prepare slices from different organs is standardized (§ 4). Importantly, slices can be made from quite small (pieces of) organs (less than 1 g), so that for example surgical waste material can be used to prepare human organ slices, as is routinely done in our lab. Finally, successful cryopreservation methods have been published allowing successful metabolism studies after thawing (§ 6). § 3. Extrahepatic metabolism Although the liver plays a major role in xenobiotic metabolism, other organs can contribute significantly to the biotransformation of a compound in the body. The relative role of different organs in the body with respect to xenobiotic metabolism depends on how much of the xenobiotic is presented to the organ as well as the amount and activity of transporting and metabolizing enzymes present. The route of administration and organ blood flow primarily determines the first aspect while the activity of the transporters and the phase I and phase II enzymes determines the latter aspect. Currently, informative reviews are available about the different (super) families of transporter proteins, for example, for the liver [136], lung [12], kidney [74] and intestine [302]. With respect to drug metabolizing enzymes, the reader is referred to reviews on phase I enzymes as P450s [122], acetyltransferases [331], epoxide hydrolases [106], carbonyl reductases [103], aldehyde dehydrogenases [317], quinone oxidoreductases [181,271], methyltransferases [234], peroxidases [218], xanthine oxidoreductase [259] and phase II conjugation enzymes as UDP-glucuronosyltransferases [268], glutathione-transferases [190], amino acid conjugation [37] and sulfotransferases [235]. In general, these xenobiotic metabolism reactions are regarded as detoxification mechanisms. However, depending on the properties of the compound involved, 15 Chapter one organs liver lungs tissue cores small intestine kidneys colon agarose filled agarose filled lung kidney intestine slice slice slice compound(s) of interest INCUBATION 37°C, 0-24 hr liver colon control slice slice 6 wells plate Figure 2. Schematic overview of the use of slices prepared from cylindrical cores of liver, lung (agarose filled), kidney and intestines (agarose filled), all incubated in 3.2 ml of medium, and placed together in single 6-well plate containing compound(s) of interest, including a control incubation without slice material. bioactivation is also possible, resulting in a (more) toxic products. The concentrations of the individual members of xenobiotic metabolizing enzymes vary considerable between organs. The liver has the highest overall cytochrome P450 content, followed by the intestine, whereas kidneys and lungs have relatively low amounts of overall cytochrome P450. However, up to 40-50 different isoenzymes of cytochrome P450 are present per species [214]. The organ specific expression of the isoenzymes involved in metabolism of a given compound, determines the role of different organs in the body - and not the overall tissue content of cytochrome P450. In spite of extensive research, it is not yet possible to predict which isoenzymes of cytochrome P450 are involved in the metabolism of a given compound. Neither is it possible to precisely predict which metabolites are formed. CYP3A4 is the most abundant isoenzyme in man, and 60% of prescribed drugs are substrates for CYP3A4 [198]. The human liver and duodenum have the highest content of CYP3A4: about 350 and 160 pmol/mg microsomal protein respectively [69]. Due to the lower mass of the small intestine compared with liver, and the low amount of microsomal protein recovered during preparation, the duodenum is believed to have 30 times less CYP3A4 content than the liver [69]. However, the total recovery of intestinal microsomes used for these quantifications, was very low [236], and consequently the CYP3A4 content in the intestines might be underestimated. Further, the relative importance of the small intestine in the metabolism of a compound is more prominent for orally taken drugs that first have to pass the intestinal drug-metabolizing enzymes. 16 Introduction This even applies, if at high doses, active back transport (intestinal secretion) and / or, the metabolizing enzymes in the gut wall, become saturated. In addition, efflux transporter proteins, as the well-studied P-gp in the intestines can work synergistically with drug metabolizing enzymes [302]. Drug molecules that are substrates for P-gp and that escape metabolic conversion can be directly excreted back into the lumen via P-gp and are then ready to be taken up again and face another chance of metabolism by intestinal enzymes. Some isoenzymes exhibit an organ specific localization, for example the isoenzymes CYP4B1 and CYP4B11 are only expressed in human lung and kidney, respectively. When a given compound is metabolized by CYP4B1, the lungs can be very important in total drug clearance. In addition, possible interactions with other CYP4B1 substrates, and toxicity due to toxic metabolites formed by CYP4B1 can affect the lungs specifically. In the rabbit, for example, the lungselective toxicity of 4-ipomeanol is the consequence of relatively large amounts of CYP2B1 and CYP4B1 in nonciliated bronchiolar epithelial cells (Clara cells) of the terminal airways [288]. Another example is the formation of reactive intermediates from several pneumotoxic chemicals by members of the CYP2F gene subfamily, which are selectively expressed in the lung [165,329]. In this respect, the CYP1A family deserves additional attention because many pro-carcinogens are known to be converted by CYP1A1 and CYP1A2 to carcinogens, and CYP1A1 is mainly located in extra-hepatic tissues, mostly in lung [286] and intestine [239]. The same considerations can be made for the other types of xenobiotic families of metabolizing enzymes, e.g. for the human UDP-glucuronosyltransferases, from which isoenzymes UGT1A7, UGT1A8 and UGT1A10 were found to be exclusively expressed in the human gastrointestinal tract [44,297,308], although the liver has the highest overall UDP-glucuronosyltransferase (UGT) content. This is significant because UGT1A10 has one of the widest range of substrate specificities of any of the UGTs [308]. Thus, the drug biotransformation activity of an organ in the body depends, among other factors, on the drug-metabolizing enzymes present. However, the contribution of an organ towards the total metabolic body clearance can be largely dependent on the size of the organ and other factors such as blood flow. Due to both its high drug-metabolizing enzyme content and its size, the liver is considered to be the main organ involved in biotransformation, but the contribution of the intestines, lungs and kidneys can be considerable. Most other organs contain drug-metabolizing enzymes but their contribution is limited when compared with liver, lung, kidney and intestine, due to their limited size. For instance, the nasal epithelium contains high concentrations of drug metabolizing enzymes, such as cytochrome P450 [182]. The CYPs in the olfactory mucosa are considered to be important for maintaining acuity in the sense of smell, but not to the fate of drugs in the body, due to the limited size of the tissue. In addition, the size of the organs of the reproductive system limits their 17 Chapter one importance, although they contain several drug metabolizing enzymes, which are important for the synthesis of endogenous hormones. On the other hand, the skin is a very large organ that is not considered as a major biotransformation organ as a result of its low content of metabolizing enzymes. Most of the present enzymes are expressed in keratinocytes [17]. Of note, in particular cases, for instance when transdermal, or nasal administration is applied, nose and skin may play a more important role in the metabolism of drugs. In addition, tissuespecific CYPs have been described both in nasal epithelium [191] and in keratinocytes [154]. § 3.1. Organ specific toxicity Up to 50% of the known or suspected carcinogens require metabolic activation, and many of those carcinogens exhibit marked tissue specificity [119]. The occurrence of cancer (or toxicity in general) in a certain organ is dependent on specific metabolic activation processes, but also on potential inactivation pathways that are present. Because of its major role in drug metabolism, the liver is a prominent site for toxic injury for agents that are metabolically activated. The liver can also be the source of reactive metabolites that can damage extrahepatic organs after transport to the systemic circulation, or to the biliary system and gastrointestinal tract. Extrahepatic tissues generally are much more heterogeneous whereas in the liver drug metabolizing enzymes are mainly expressed in the major liver cell type: the hepatocytes. Therefore, in spite of a relatively low cytochrome P450 content per gram extra-hepatic tissue, the specific localization of drug metabolizing enzymes and membrane transporters in these cell populations can result in high local concentrations of drugs and their active metabolites and thus in tissue selective toxic injury. § 3.1.1. Metabolism and toxicity in the lung The cells and tissues of the respiratory system can be exposed to xenobiotica both via inhalation and via the systemic circulation (the lungs receive 100% of the cardiac output). The epithelium of the respiratory tract consists of more than twenty types of cells. However, the most important cells for xenobiotic metabolism and toxicity are the alveolar and Clara cells. In addition, smooth muscle cells are important in bronchial hyper-reactivity, while fibroblasts play a prominent role in the development of lung fibrosis, and both cell types can be activated by xenobiotica. The alveolar and Clara cells possess a variety of xenobiotic metabolizing enzymes [31]. Clara cells show the highest rates of cytochrome P450 mediated metabolism of any pulmonary cell type. The pattern of expression of CYPs in the lung is increasingly characterized. Briefly, human lung express CYP1A1, CYP2A6, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7 [8,158,286] and additionally CYP2B6/7, CYP2F1 and CYP4B1 were detected on the mRNA level [262]. CYP1A1 is known to be induced by smoking and is involved in the activation of the pro-carcinogen benzo(a)pyrene and many 18 Introduction other chemicals from tobacco smoke. In man, CYP4B1 is exclusively expressed in the lungs [118], and this gave rise to the hypothesis that a unique endogenous substrate for CYP4B1 is present in the lung [213]. Apart from P450s, also prostaglandin synthases are known to be involved in carcinogen activation in the lung, as is the case in the kidney [288]. An example of toxicity due to specific accumulation in the lung is paraquat, an herbicide widely used in agriculture [120]. Much research has also been done on mutagenic and carcinogenic compounds from tobacco smoke [120]. Other specific lung toxicants as 4-ipomeanol (natural product of potatoes fungus), antitumor drug bleomycin and industrial products as naphthalene and dichloroethylene, have been widely studied and reviewed [120]. § 3.1.2. Metabolism and toxicity in the kidney The primary functions of the kidneys are the elimination of waste products and the regulation of the whole body fluid and salt homeostasis. As a selective filter, the kidney receives about 25% of the cardiac output while the kidneys represents only 1% of total bodyweight. This results in an extensive exposure of the kidney cells to blood borne compounds. During the process of concentration of the tubular fluid, the cells of the nephron can be exposed from the luminal side to potentially high concentrations of toxicants. Compounds that are substrate for one of the organic solute transport systems may accumulate in the kidney cells and damage the proximal tubular cells. Several human CYPs have been identified in human kidney, among them are CYP1A1, CYP2D6 [118], and most abundant, CYP3A4 [157]. The human kidney was found to express CYP4B11 and CYP4F2 exclusively [52,166]. Except for cytochrome P450 activity, the kidney contains a range of other xenobiotic metabolizing enzymes, such as prostaglandin H synthase, glutathione transferase and epoxide hydroxylase. The distribution of these enzymes varies markedly within different cells in the kidney and also differs between cortex and medulla [237]. There are some well-studied examples of drug induced kidney toxicity. b-lactam antibiotics as cephaloridine are nephrotoxic because they accumulate in proximal tubular cells, and express intrinsic reactivity of their b-lactam ring or are activated by cytochrome P450 [309]. In addition, acetaminophen (and related aminophenol) can be activated by cytochrome P450 in the kidney. The latter process accounts for 0.6-1.2% incidence of clinical renal failure [76]. The uptake of mercapturic acids and glutathione conjugates in the kidney can result in the well-studied toxicity mediated by renal cysteine conjugate b-lyase. After uptake in the kidney cells, mercapturic acids of hydrocarbons, especially alkenes, can be deacetylated to cysteine conjugates. After cleaving off most of the conjugated cysteine residu, a reactive (toxic) thiol molecule is formed [4]. Although glucuronidation generally results in less biologically active and more polar metabolites, that can be rapidly excreted, this is not true for the reactive acyl glucuronides. Renal toxicity has been shown to occur as a result from this 19 Chapter one class of glucuronidated metabolites of nonsteroidal anti-inflammatory drugs (NSAIDs) as benoxaprofen, indoprofen, alclofenac, ticrynafen and ibufenac [180]. Another group of compounds that are toxic for the kidney, are heavy metals, such as lead, cadmium, mercury and arsenic, which easily react with nucleophilic sites such as the thiol group of proteins, peptides and amino acids. The resulting cysteine conjugates are accumulated primarily in the kidneys, resulting in renal damage [350]. § 3.1.3. Metabolism and toxicity in the intestines The intestinal tract, especially the mucosal lining, is exposed continuously to a large variety of chemicals because this organ acts as a major portal for entry into the body of orally taken substances. The intestinal tract possesses many of the major phase I and phase II biotransformation enzymes that are found in the liver. In general, the metabolizing potential closely parallels the absorptive capacity since both phenomena increase from stomach to duodenum and than decrease towards the colon [69]. On a cellular level, the metabolizing potential and the absorptive capacity increase during maturation of the mucosal crypt cells during their migration and differentiation into columnar epithelium at the villous tip [48]. Human intestine expresses CYP1A1, CYP2C8, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, of which CYP3A4 is most abundant [118]. An example of specific expression of a drug metabolizing enzymes in the intestine is flavin-containing monooxygenase-1 (FMO1) that is expressed at high levels in human inestine and kidney, but not in liver where mainly FMO3 is expressed [130]. Intestinal glucuronosyltransferases and sulfotransferases can reach concentrations between 10% and 300% of the hepatic values, making the intestine a major biotransformation organ for many chemicals that pass the mucosal cells during absorption as well as via the blood stream [280]. Also, compounds can undergo enterohepatic circulation: after elimination from the liver into the bile, reabsorption from the gastrointestinal tract can take place, and the compound or its metabolites can be subject to intestinal and hepatic biotransformation again. In addition, the intestinal microflora can activate compounds by de-conjugation. Intestinal toxicity was mainly studied in relation to the metabolic activation of carcinogens, especially in the colon, due to the great incidence of tumors diagnosed in the large intestine. Another field of interest lies in the side effects of NSAIDs, which can cause inflammation of the small intestine or the colon. This has been reported for 65% of patients receiving long-term treatment with NSAIDs for osteoarthritis or rheumatoid arthritis [27]. § 4. Practical aspects: preparation of precision-cut slices The preparation of tissue slices is in principle similar for all organs. Tissues are removed from anesthetized experimental animals or, in the case of human mate20 Introduction rial, tissue is used which is regarded as surgical waste. After removal, tissues are placed in ice-cold buffer, as soon as possible and are subsequently transferred to the laboratory for slicing. For short periods, up to 1h, physiological buffers as Krebs-Henseleit buffer (based on the extracellular environment) are often used for storage. For longer cold-ischemia periods, cold-preservation buffers are used which are based on intracellular ion concentrations combined with colloids. The rationale for this is that in the cold, cell metabolism is slowed down and cells are not able to maintain normal ion gradients across their cellular membrane while colloids can reduce cell swelling due to osmotic water influx. At body temperature, it is obvious that an ionic environment close to the plasma composition should be created. Therefore, slices should not be incubated in preservation solutions at body temperature. The most frequently used cold-preservation solution is the University of Wisconsin solution (Belzers UW, DuPont Pharmaceuticals, Waukegan, IL, USA), which has been proven useful to preserve liver and kidney slices [36,98,202,230]. From solid or agarose-filled organs, cylindrical tissue cores are made by pushing a rotating thin-wall sharpened tubing through the tissue. Cores of 3, 5, 8 and 10 mm diameter are commercially available (Alabama R&D. Munford, AL, USA). For this purpose, a common drill motor with variable speed can be used, rotating at approximately 300 rpm. Solid organs can be used directly for tissue coring and subsequent slicing. However lung tissue have to be filled with melted agarose, to obtain a more solid material, and to keep the alveoli open. Low melting agarose (1.5%) solution in water is used at 37°C, which gels when the tissue is put on ice. To keep osmotic pressure normal, 0.9% NaCl is added to the agarose solution. In addition, intestines can be filled with agarose (3%) solution and then embedded in agarose in a core-shaped container to obtain solid cores that than can be sliced directly (without coring). Also small organs, e.g. from mice, may be embedded in agarose. For this purpose a commercially available tissue-embedding unit may be used (Alabama R&D. Munford, AL, USA). The obtained tissue cores are subsequently sliced using a Krumdieck or Vitron Tissue slicer. Both slicers are considered to produce ‘precision-cut slices’. No differences were found between the rat liver slices prepared in the two apparatus with respect to viability and rate of metabolism [254]. These slicers use a moving, disposable knife (oscillitating or rotating) that cuts the cores (that are in fact moved over the knife) into slices. By adjusting the distance between the knife and the tissue core, the thickness of the slices is set. The optimal thickness of liver, kidney and intestine slices is 200-250 µm [245]. For lung slices, 500 µm is optimal [245] because diffusion distances in lung slices are shorter [105]. The diameter of the slices is determined by the diameter of the cores. These slicers are filled with ice-cold physiological solution as Krebs-Henseleit buffer or commercially available culture media. For experiments, the slices are incubated 21 Chapter one Table 2. Incubation systems incubation system vol-ume mixing type (freq. /min) availability Dynamic Organ Culture (DOC) 1.6 ml 3 bubble culture 10 ml bubbles submersion self-made [274] stirred 24 well 1.4 ml stirred submersion self-made [228] 12 wells 1.5-1.7 ml 90* submersion [71,127] 6 wells 3.2 ml 90* submersion commercial / self-made shaken flask 5.0 ml 110* submersion commercial [59,62] netwell 3.3 ml 10 alternately commercial air/submersi (Alabama on R&D. alternately air commercial / submersion (Vitron, ref. [290] Tucson, AZ, USA) [64,306] [170] Munford, AL, USA) * back-and-forward shaking directly at body-temperature (usually 37°C). They can also be cold-stored (04°C) in physiological buffer or preservation solution in order to collect all the prepared slices before starting the incubations. § 4.1. Practical aspects: incubation a. Incubation systems Several incubation systems for slices have been used. After the introduction of the Krumdieck slicer, a ‘Dynamic Organ Culture’ (DOC) [290] was developed, in which slices are incubated and exposed to medium and to the atmosphere alternately. This is considered to allow optimal gas exchange. Other incubations systems are used as well, such as multiwell system (6, 12 or 24 wells), Erlenmeyer flasks, and netwell inserts [224]. In all incubation systems, O2 and CO2 supply should be sufficient to compensate for the amount of oxygen used and to equilibrate the carbondioxide produced by the slices. The latter is important in order to keep the pH constant in the case of CO2/HCO3- buffered media. In a primary comparison using rat liver slices, the DOC was found superior to the multiwell system, which is in fact a submersion system [101]. However, a 22 Introduction disadvantage of the DOC incubator is the possible mechanical damage of the slice due to the contact with the mesh. This is avoided in submersion systems. In a multiwell system the gas exchange occurs through the surface area of the medium in the well. In 6 wells plates, this surface is about 36 mm2, which apparently is enough for optimal gas exchange. Indeed, a comparison or rat liver slices prepared by 4 laboratories, including that of the authors, five incubation systems were tested with regard to viability and metabolic activity of the incubated slices. This study showed that the DOC is less optimal compared with the slice-submersion systems in 6 wells system and Erlenmeyers [224]. Three more recent papers compared the multiwell (6 and 12 wells) plate culture to the DOC, using rat [127,306] and trout [279] liver slices, and concluded that the multiwell plate culture system for culturing slices is superior to the DOC. Further, our own experience is that use of the DOC is very laborious and expensive and therefore unsuitable for larger experiments (more than 20 slices). Which incubation system is to be preferred depends further on the volume of medium needed per slice (smaller volumes should be used if the sensitivity of the analytical method is limited. If volatile compounds are used, closed (capped) systems are required. A brief summary of frequently used incubation systems is given in table 2. b. Mixing In all incubation systems shaking or rotating is used to keep a homogenous incubation solution (easily tested by adding a drip of Trypan Blue), and also to facilitate gas exchange with the used atmosphere. Higher shaking speed is found to be beneficial because of the better oxygen exchange with the atmosphere [219]. On the other hand, the shaking speed should be limited to prevent physical damage and deterioration of the slices. Therefore a compromise should be sought between optical gas exchange and mechanical damage. For optimal results, multiwell plates and Erlenmeyer should be shaken back-and–forward, and not gyratory (unpublished observations from our own laboratory). c. Gas supply Most commercially available culture media used for slice incubation are bicarbonate buffered and an atmosphere of 5% CO2 is therefore used. CO2 is either Table 3. Antibiotics Organ slices Antibiotic / antifungic liver gentamicin 50 µg/ml lung gentamicin 50 µg/ml kidney penicillin G 100 U/ml and streptomycin 100 µg/ml intestine gentamicin 50 µg/ml and amphotericin B 2.5 µg/ml 23 Chapter one mixed with air (21% O2) or pure oxygen (95% O2). Oxygen can reach the cells in the slice by diffusion only. Due to consumption of O2, in the outer cell layers, the actual oxygen tension in the inner cell layers might be substantially lower than that of the atmosphere used in the system. To compensate for this, many researchers use a high oxygen tension: 40%, 70% or even 95% O2. This higher O2 tension was indeed found beneficial for rat liver slices when compared with air [306] (unpublished observations from our own laboratory). However, in a study on rat lung slices, the use of 70% oxygen appeared to produce toxic reactions, and the authors concluded that lung slices incubated under air have better metabolic properties [206]. This can also be due to the fact that lung slices contain less cells (because most volume is taken by the gelled agarose) that consume oxygen so that oxygen gradients will be smaller in lung slices than in liver slices. This would imply that liver as well as kidney and intestine slices are preferably incubated under higher oxygen atmosphere, and that lung slices require lower O2 tension. On the other hand, a previous study showed that 95% O2 was to be preferred over to air, in the case of lung slices [219]. This might be related to use of a different incubation system but anyway, more systematic research is needed to address this issue satisfactorily. d. Medium The choice of a suitable medium is dependent on the purpose of the experiment. Many commercially available media have been used for the incubation of slices. They exhibit several characteristics necessary to maintain optimal viability. Their components must represent total osmotic strength as in vivo and must also provide a rich source of carbohydrates (usually glucose), amino acids, minerals and vitamins. Antibiotics and antifungicals are usually added (table 3). Hormones, such as insulin, glucagon, testosterone, dexamethasone, triodothyronine and estradiol are occasionally added [318]. Fetal calf serum is sometimes supplemented up to 5% (v/v). In our group, if necessary, we use serum of the same experimental animal, where the tissues is taken from, to reduce experimental animal use. In addition, in this manner serum does not need to be frozen, eliminating possible loss or break down of components. To keep pH on a physiological 7.4 value, a suitable buffer should be included. This is useful to compensate for acidification by lactate production. Mostly, buffers are based on bicarbonate because of its similarity with blood buffering and because Hepes buffer was found less optimal for slices [96]. For easy pH monitoring, phenol red is included. Of note, phenol red can interfere with glucuronidation [75] and can be taken up in kidney slices and induce enzymes [9], which may interfere with the particular experimental outcome. Williams’ medium E, which contains a rich amino acid mixture is often used and was found very suitable for liver slices [96], although is was found that Williams’ medium E can also increase basal cell proliferation in liver slices during 48 h of culture [58]. For slices from lung, kidney and intestine it remains to be investigated which culturing medium is most opti24 Introduction mal. For long-term incubation, slices are usually transferred to fresh prewarmed oxygenated medium every 24 h. § 4.2. Practical aspects: normalization of slice activity For proper interpretation, up-scaling, or comparisons to other in vitro models, metabolic activities and other characteristics of slices have to be normalized for instance by wet or dry weight, protein or DNA content. When using agar-filled (lung or intestine) slices, wet weight cannot be used since agar represents an important portion of the slice weight. In addition, for liver and kidney slices, wet weight is only a rough measure because adhering water easily cause errors (e.g. 5 µl adhering fluid adds 5 mg to the usual slice weigh of 10-15mg). Dry weight is influenced by the technique of drying and the final weight is usually very low (25 mg), and is therefore inaccurate. A more accurate normalization parameter is total protein or DNA content. Protein content of liver slices can significantly decrease during long-term incubation [315]. This is due to damaged cells that detach from the slices and thereby are lost for protein determination when the slices are transferred to fresh medium. However, the activity of those slices still can be expressed per (lower) protein content because this value reflects the number of cells actually present. For protein and DNA normalization slices have to be homogenized and washing of the slices is necessary if serum containing media are used. Care should be taken for the influence of DNAses. § 5. Cold preservation Preservation by lowering the temperature just above the freezing point is meant to slow down metabolic processes in the cells. Cold preservation for 16 – 48 h is described to have only a minor effect on the viability and function of liver [209,230], lung [36] and kidney slices [97,98] using UW or comparable cold storage solutions. These cold preservation procedures for slices are based on those for whole organ preservation. Interestingly, slices have proven to be a valuable tool to improve hypothermic storage technology for transplantation purposes, as has been shown using slices from the liver [209,230], as well as from lung [36,93] and kidney [299]. It is preferable to store slices on melting ice, or even just below 0°C, above preservation at 4°C in the refrigerator [86]. § 6. Cryopreservation By freezing to temperatures under –130°C, biological activity is stopped completely and biological material stored at those temperatures can be stored infinitely [199]. This is especially useful for slices prepared from organs from man, monkeys and dogs of which availability is limited. In addition, if the amount of tissue exceeds the actual experimental capacity, storage by deep freezing is helpful. Cryopreservation therefore facilitates a broader and more efficient utiliza25 Chapter one tion of such tissues and allows their use at any desired time. For cryopreservation of cells, normally a slow freezing rate of 1-10°C/min with 10-20% (v/v) cryoprotectant (most often dimethylsulphoxide) is applied. Slow freezing dehydrates the cells and thus avoids intracellular ice formation [199]. This approach has been used to cryopreserve liver slices [98,99,186]. Surprisingly for slices, a fast freezing rate of 250°C / min gave better results [62,186]. An important advantage of fast freezing is its practical simplicity: vials with slices are put straight into liquid nitrogen, instead of the need of a computer-controlled cooling device. To date, most cryopreservation approaches for liver slices use a high freezing rate [56,59,62,65,66,113-115,186,196,291]. Alternatively, vitrification (formation of an amorphous state) has been used [81,84,335]. However, this requires high cryoprotectant concentrations (35-50% v/v) in combination with very fast freezing. Vitrification is a promising technique although it remains to be investigated if vitrification results in better viability of the slices after thawing than fast freezing. Using the fast-freezing technique, it was shown that cryopreserved liver slices were viable for 3-24 h after thawing and were found suitable for xenobiotic metabolism studies [56,59,62,65,66,186,187,196,291,316] and also for in vitro induction of CYP enzymes [113-115]. For kidney slices only a few studies were performed with regard to cryopreservation [97,98], while for lung and intestinal slices no reports are available. Obviously, when cryopreservation is feasible, as now appears to be the case for liver slices, it has the potential to greatly facilitate the use of slices, especially from larger mammals as man. Therefore, more research is needed to develop methods for proper cryopreservation of lung, kidney and intestine slices. § 7. Viability parameters and endpoints When using intact cell preparations, biological viability is a crucial factor when considering the usefulness of the system, even when the method used is established and routinely applied in the laboratory. There are many ways to check viability, varying from simple and fast procedures to thorough and time-consuming methods. The most frequently applied approaches for slices are mentioned here (see for references table 4 and 5): • ATP content. Being the central energy-storing molecule, ATP content is a sensitive and practically easy to measure parameter of viability. When a luciferase bioluminescence assay is used, ATP levels as low as 10-15 moles can be quantified. Care should be taken to inhibit the present ATPases completely, to avoid ATP loss. In most cases perchloric acid or ethanol precipitation is used, in combination with snap-freezing by immersing into liquid nitrogen. • Histopatholgy. From the slices, cryostat or paraffin / plastic embedded sections can be easily made perpendicular to the surface of the slice. Morphology has proven to be a very sensitive tool to estimate viability, although it is not easy to 26 Introduction quantify the results. Obviously, a whole range of staining and immunohistochemical techniques can be applied. • Protein synthesis. By using 14C-leucine, the radioactivity incorporated into percipitable protein can be used as a measure of protein synthesis. Clearly this requires many complex processes to be active in the slice and therefore is a sensitive measure of viability. Protein synthesis inhibitors such as ricin and cycloheximde can be applied as controls. • Potassium. The amount of intracellular potassium is often applied as a sensitive measure of viability, and found to be positively correlated with ATP level. Due to the limited sensitivity of most potassium assays, this parameter is not applicable for small amounts of tissues, as is the case for lung slices. To avoid contamination with extracellular potassium, it is necessary to wash the slices in a potassium free solution. • Enzyme leakage. Enzyme assays, as used in clinical chemistry, are also applied for slices. Sometimes enzyme activities in the incubation medium are given. However, interpretation is more feasible if the amount of leaked enzyme activity is expressed as the percentage of total enzyme content in the slice. Lactate dehydrogenase (LDH) and alkaline phosphatase (AP) leakage is often applied in the case of liver, lung and kidney slices. Alanine and aspartate aminotransferase (ALT, AST) data are used for liver slices and gamma-glutamyl transpeptidase (gGT) for kidney slices, because of their relatively high content in these organs. Directly after slicing, a high enzyme leakage is found (10-20%), because of the loss of enzymes from the damaged cells on the cutting edges. • Glutathione. Oxidized (GSSG) and reduced (GSH) glutathione levels or the ratio of GSH/GSSG is used occasionally to monitor oxidative stress. • Lipid peroxidation (LPO). As a specific parameter of the action of toxic radicals, lipid peroxidation can be determined by measuring thiobarbituric reactive substances such as malondialdehyde. • Tetraethylammonium (TEA) / p-aminohippuric acid (PAH) uptake. Accumulation of the 14C-labeled organic cation TEA and the anion PAH in kidney slices, expressed as slice to medium ratio (S/M) is applied both as a viability and as a functional parameter. A control of carrier mediated PAH uptake is its inhibition by probenecid, and TEA uptake can be inhibited by mepiperphenidol. • MTT tetrazolium assay. MTT can be converted by succinate dehydrogenase to strongly absorbing formazan products that precipitates in the cells. After extraction the absorbance is measured as a measure of viability. The MTT assay is frequently used in cell cytotoxicity tests but does not always correlate with other viability tests, and appears to have a relatively low sensitivity for monitoring changes in viability [321]. The reason for this is that MTT is not only converted by mitochondrial succinate dehydrogenase, but for the greatest part by extramitochondrial NADH- and NADPH-dependent reactions [26]. Most of these viability tests are also used as endpoints in toxicity testing. Yet, to study specific toxic responses, many other endpoints are used such as DNA dam27 Chapter one age and cytokine release. Further, the accumulation of 14C-labeld putrescine in lung slices was applied as a specific viability parameter in relation to the study on uptake of other substrates of the diamine uptake system, e.g. paraquat and cystamine. The action of reactive metabolites can be studied by using labeled compounds of which the appearance of radioactivity in the protein precipitate is a measure of the formation of protein adducts. Several of these viability endpoints are used in combination to assess the quality of the slices during incubation. For liver, lung and kidney slices, incubation up to 24 h can be performed without a significant loss of viability. Extra attention should be given to the viability of intestine slices, because this organ is known to be very susceptible towards hypothermic and ischemic damage in vitro. From isolated segments of rat small intestine it is recommended that incubations should be limited to only 5 min. Histological examination of the tissue indeed showed significant loss of structural integrity after 10 to 20 min at 37°C [233]. Therefore, routine monitoring of the integrity of intestinal preparations in vitro is desirable and for this purpose histological assessment is an appropriate technique [252]. The many studies on this aspect do not entirely agree with regard to the relative impact of these parameters. Yet, it is generally agreed that ATP and K+ content, protein synthesis, and morphology are more sensitive than enzyme leakage or MTT tests [101]. The values found for ATP and potassium can be related to in vivo values and can give a more absolute insight in the viability than parameters such as enzyme leakage where no direct quantitative comparison with in vivo values are possible. To validate whether slices are a good model to study certain cell functions, it is advisable to also incorporate functional tests that are related to the subject under investigation. For instance, metabolism of reference substrates can be used to assess metabolic capacity. § 8. Applications of lung slices in metabolism and toxicology The preparation of precision-cut lung slices using the agarose instilling technique (§ 4) has been developed by the same group that pioneered with liver slices [294,295] and is since then used by many others. The toxicity and metabolism studies performed using precision-cut lung slices are summarized in table 4. A very interesting application of precision-cut lung slices is their use in pharmacodynamic research. Following exposure of lung slices to bronchoconstrictors, the effect on the diameter of the smaller airways was measured in lung slices, using microscopic techniques [128,193-195]. The use of precision-cut lung slices in these studies made it possible to investigate the effects of drugs on very small airways (50 µm diameter) and measurements on airway diameter were found more reproducible in precision-cut than razor blade cut lung slices [195]. 28 Introduction Table 4. Applications of lung slices species rat rat toxicant 3-methylindole, 1nitro-naphthalene, paraquat acrolein end-point rat acrolein and nitrofurantoin acrolein O2 utilization, GSH, protein synthesis GSH, ATP, phospholipid synthesis LDH release ATP, TNFa, IL-1b, nucleosomes histopathology stress response parameters rat diesel engine exhaust rat sodium arsenite rat rat prostaglandin 7-ethoxy-coumarin, coumarin tobacco specific nitrosamines metabolism rat, human cyclosporines metabolism rat, mouse, human 7-ethoxy-coumarin, agaritine metabolism rat, human lidocaine, testosterone, 7ethoxycoumarin, 7hydroxy-coumarin metabolism hamster ref. [257] acrolein is a component of cigarette smoke [294] protein synthesis protein synthesis man remarks histopathology, metabolism, histopathology metabolism [102] effects compared with L2 cell line [207] [169] localization of transcription factors by confocal microscopy compared with liver slices compared with liver and kidney slices compared with liver slices and scaled to whole organs compared with liver slices,also P450 levels and gGT activities compared with liver and kidney slices [333] [188] [256] [264] [319] [258] [64] 29 Chapter one There are also many examples of toxicity studies using non-precision-cut lung slices, which were either made with the McIlwain tissue chopper or hand-held razor blades. Lungs used for these studied were usually not filled with agarose, except if histopathology was at state. The use of non precision-cut lung slices was reviewed earlier [105] and therefore only some additional examples are given. GSSG levels, 14C-glucose metabolism and protein synthesis have been used to study cobalt toxicity in hamster lung slices [173]. Toxicity of sulphur mustard (mustard gas) in rat lung slices was studied by the MTT assay [334], histopathology [278] and by measuring GSH content [164]. Protein synthesis was measured in rabbit lung slices under influence of b-receptor drugs, betamethasone and cigarette smoke as well as one of its components, acrolein [129]. NADP and NADPH were measured under influence of paraquat and analogs in rat slices [1,300]. Paraquat, which is a well-studied toxic herbicide has been studied widely using lung slices [1,53,126,134,174,257,277]. The cytostatic agents bleomycin and cyclophosphamide cause acute injury to the lungs and their effects and metabolism have been studied using rat and mouse lung slices [141-143]. Many metabolism studies with lung slices deal with ethanol metabolism in the lung, e.g. [25]. Metabolism of resorufines was studied, under influence of CYP inducers b-naphthoflavone and metylcholanthrene in hamster and human lung slices [133]. A two-fold induction was found of CYP1A1 although viability was low (shown by LDH leakage, glucose metabolism, putrescine uptake and protein synthesis). The latter may have been due to the McIlwain chopper slicing technique used, and the effect of the (toxic) inducers [133]. However, it is promising that CYP induction in lung slices is possible and more research is needed to establish (CYP) enzyme induction in precision-cut lung slices, as was shown using precision-cut liver slices (see [185]-and references therein). § 9. Applications of kidney slices in metabolism and toxicology Soon after the introduction of precision-cut liver slices, a similar method for kidney tissue has been developed by the same group [273]. A difference with precision-cut liver or lung slices is that kidney slices can be prepared perpendicular to their cortical-papillary axis so that positional slices are produced. However, most studies use slices prepared from kidney cortex. The use of free-hand cut slices or slices prepared with the Stadie-Riggs or McIlwain slicers has been widespread used and is reviewed before [24,203,289]. The applications of precisioncut kidney slices are summarized in table 5. § 10. Applications of intestinal slices in metabolism and toxicology Until now, slices from the intestine are not systematically investigated. 30 Introduction Table 5. Applications of kidney slices species rabbit, human rat, rabbit rabbit human, dog toxicant HgCl2, K2Cr2O7, hypoxia halogenated hydrocarbons quarter-nary amines cis-platin rat rabbit mycotoxin HgCl2 rat benzo(a)pyrene rabbit arsenic salts rat, pig atractyl-oside rat 1-benzyl quinolinium end-point K+, histopathology remarks K+, ATP, O2 consumption LDH, AP, gGT, LPO, histopathology, covalent binding K+, ATP, O2 consumption TEA, PAH, histopathology K+, ATP, GSH/GGSG, protein synthesis, uptake of glucose, TEA, PAH, histopathology spingoid levels K+, proton-induced X-ray emission, Western, PCR and EMSA on stress response parameters ATP, K+, EMSA, histopathology, PCR, Northern on stress response parameters K+, Western, PCR and EMSA on stress response parameters ATP, GSH/GSSG, PAH, LPO, gluconeogenesis, AP, LDH ATP, K+, O2 consumption, histopathology, TEA, PAH [54,296,338,33 b-lyase activity, covalent binding, 9] effects of antioxidants [283] organs human, rat, dog cyclo-sporines MTT, GST and nuclear protein-leakage, CYP3A levels metabolism human, rat, dog tropisetron tissue cores hamster rat, compound(s) human liver metabolism lungs kidneys agarose filled tobacco spemetabolism cific nitrosamines lung slice lidocaine, metabolism of interest testosterone, 7–ethoxyliver coumarin, slice 7–hydroxy6 wells plate coumarin kidney slice ref. [100,272,273] [100,307] [217] effect of chelators [155,311] also CYP induction; compared with liver slices [244] nanomolair conc. [246] [222,223] fos/jun, CYP in[283] duction; compared with liver slices comparison with [318,321,324] liver slices, intestine slices, and jejunum microsomes comparison with [323] all liver slices,s mjejucolon intestine num microsomes and in vivo metabolism agarose filled compared with [264] liver and lung slices intestine slice compared with [64] liver and lungI N C U B A T I O N 37°C, 0-24 hr slices colon control slice 31 Chapter one Metabolism of cyclosporin A and its derivative SDZ IMM 125 has been studied in human colonic mucosal slices where it was shown that the gastrointestinal tract contributes to the first-pass effect of the drugs [318,324]. Metabolic conversion of a cooked-food carcinogen was studied in rat colon slices [189] and phase-II activity has been shown to take place, but P450 activity was not found. However, in this study viability was checked only marginally by MTT conversion, which is not a very sensitive parameter (§ 7). § 11. Concluding remarks and future perspectives A promising approach to reduce the use of experimental animals, as well as to reduce the costs of drug development and also to get more insight of the action and fate of novel compounds in man, is the use of in vitro systems. Although powerful, in vitro systems will never replace in vivo studies completely because several complex interactions are missing. Therefore, in the interpretation of in vitro results one should always be aware of the limitations of the model used. The use of slices from liver, lung and kidney as an in vitro tool has been proven successful for biotransformation and toxicity studies as is shown by many reports from both university and industrial researchers. In contrast to liver, lung and kidney slices, of which the viability has been studied extensively and proven to be sufficient at least for 24 h of incubation, viability of intestine slices is hardly studied. Therefore, some basic work needs to be done on the testing of intestinal slices before their usefulness can be established. Because of the significant contribution of the intestine to the overall metabolism of many compounds, the introduction of intestinal slices as an in vitro technique might be a promising additive to the existing slice techniques and needs to be further developed. Some intrinsic properties of slices, no matter how well they are prepared and how excellent their viability is, impose limitations on their relevance. One obvious limitation is that the separation between the basolateral (blood) side and the apical side of the tissue is lost, because both sides are in contact with medium: constant mixing produces in fact a closed system of slice incubation. The consequence of this lack of polarity in slices is that uptake and export processes can take place from (or towards) both sides simultaneously, a condition that may differ from the condition normally present in the body. Vectorial transport across the cells contained in the slice can in principle occur but the process of excretion towards bile or urine cannot be directly studied quantitatively. This implies that metabolic clearance can be estimated, but not for instance, the net effect of secretion and reabsorption. Yet, microscopic techniques, especially confocal laser scanning microscopy, can be used to study uptake and possible secretion processes qualitatively [225]. Normally, proximal tubular cells in the kidney are very active in reabsorbing water and salts from the glomerular filtrate. This process probably is retained in kidney slices, but there is a lack of supply of filtrate resulting in reduced 32 Introduction intratubular pressure. As a result, kidney slices were reported to contain only a few open tubuli [272]. The process can be reversed by inhibiting the salt and water uptake with mercury [272]. A functional implication of the closed tubules in kidney slices is a higher accumulation of PAH at steady state, compared with renal fragments, which are short segments of tubuli, obtained by collagenase digestion [104]. Studies are lacking to investigate the diffusion of (larger) compounds into the innermost layers of lung, kidney and intestine slices. In liver slices of both rat and man it was shown that rhodamine B, lucigenin [227] and even large proteins, such as albumin [225], diffuse readily inside liver slices. On the other hand, the probe CPPCQ did not readily diffuse towards the inner cell layers of rat liver slices, because it was metabolized to a fluorescent product in the outer cell layers of the slice before it could reach the inner cell layers [82]. Thus, one should control whether diffusion rate might be rate limiting in the uptake processes under study. Diffusion may also be the limiting step for metabolizing processes, particularly for high clearance compounds [38,139]. High clearance in the outer cell layers limits accessibility of substrate to the inner cell layers of the slice, which results in a reduced number of cells involved in uptake and metabolism, and hence a reduced clearance is found when normalized for hepatocellularity [341,342]. Also, when the rate of metabolism of other in vitro techniques, such as primary hepatocyte cultures or hepatic microsomal fractions, are compared with in vivo values, often an underestimation is found, e.g. [7,321]. A reasonable explanation for this might be that extra-hepatic metabolism takes place, which is not accounted for in the liver based in vitro models used. A proper quantitative comparison of in vitro and in vivo metabolic clearance should therefore include not only liver data but also data from lung, kidney and intestine. Precision-cut organ slices are mostly used for short-term incubations, because drug metabolizing enzyme activities often diminish in time and de-differentiation of cell function may take place. Apart from efforts to develop methods that retain cellular functions for longer incubation periods, there is a continuing search towards a better characterization of long-term processes in vitro. Examples under study are enzyme induction, inflammation, and delayed toxic responses. Molecular biology techniques can be applied to detect these processes in an early stadium. PCR techniques haven proven to be of great value for studying the induction of genes, encoding for transcription factors, drug metabolizing enzymes, transporter proteins and cytokines. The detection of cytokines or ‘stress’ or ‘repair’ markers is also increasingly used to define long-term processes in vitro. Such processes require cell-cell interactions of various cell types, which makes the use of slices attractive. One example is the up-regulation of the mRNA of the inducible nitric oxide synthase (iNOS) gene, in response of lipopolysaccaride (LPS). This inflammation process can be detected in rat liver slices already after 3 h of exposure to LPS [229]. In addition, more chronic 33 Chapter one inflammation processes as induction of fibrosis induced by CdCl2 was studied in lung slices [176]. Taken together, the development of the use of extra-hepatic tissue slices is a very promising and powerful in vitro technique. In combination with the more established use of liver slices, now for the first time it becomes feasible to compare the activity of different organs in vitro by using slices. This opens possibilities to predict total metabolic body clearance from in vitro studies. In addition, the interaction between different organs with respect to metabolism and toxicity may be studied by combined incubation of slices from different organs. For instance, a toxic effect of a compound activated by the liver, but manifested in the kidney can be studied by simultaneously incubating liver and kidney slices together. The advantages of the use of slices (species independent, easy, cells embedded in matrix) can be fully exploited not only in slices of the liver but also in the more heterogeneous organs as the lung, kidney and intestinal tract. For a further understanding of the usefulness and limitations of the use of extra-hepatic tissue slices, more research is necessary, especially for the intestine, to optimize viability and to study characteristics of the slice preparations. Finally, the development and application of the slice technique in drug development will contribute to the 3R’s (replacement, reduction and refinement) defined by Russel and Burch and is endorsed by the European Center for the Validation of Alternative Methods (ECVAM) [11]. Replacement of the use of animal experiments is achieved when human tissues are used instead of animal tissues. Reduction is achieved because several drugs can be tested simultaneously in slices from one animal. Moreover, a more adequate selection of the species used in toxicological studies can be made based on slice experiments, avoiding experiments in non-relevant species. And a considerable refinement of animal experiments will be reached with in vitro toxicity testing thus avoiding administration of toxic compounds in vivo, thereby reducing the discomfort of the animals. 34