Toxicology in Vitro 12 (1997) 77±100
Liver Slices in In Vitro Pharmacotoxicology with Special Reference to the Use of Human Liver Tissue P. OLINGA*, D. K. F. MEIJER, M. J. H. SLOOFF1 and G. M. M. GROOTHUIS $Groningen Institute for Drug Studies, Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, Ant. Deusinglaan 1, 9713 AV Groningen and 1Division of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University Hospital, Hanzeplein 1, 9713 EZ Groningen, The Netherlands (Accepted 25 June 1997) SummaryÐIn the early years of research in in vitro pharmacotoxicology liver slices have been used. After a decline in the application of slices in favour of the use of isolated hepatocytes and the isolated perfused liver preparation, the development of the Krumdieck slicer in the 1980s led to a `comeback' of the technique. This review will focus on the use of human liver, with special reference to the comparison of slices with isolated hepatocytes in in vitro pharmacotoxicology. In addition, an overview on the predictive value of these in vitro systems for drug disposition and toxicity in vivo will be given. Preservation techniques for liver slices and hepatocytes will also be discussed. These techniques ensure an ecient utilization of the scarce human material. For long-term storage of liver slices and hepatocytes, cryopreservation seems most promising. However, cryopreservation is still in its infancy, and reports mainly deal with drug metabolism studies after cryopreservation. Drug toxicity, metabolism and transport data determined in slices and isolated hepatocytes, from both human and animal liver showed good correlation with the corresponding parameters measured in vivo. Therefore, the results obtained in such studies may give rise to more in-depth research on the mechanisms of pharmactoxicology in the human liver. # 1998 Elsevier Science Ltd Abbreviations: CDNB = 1-chloro-2,4-dinitrobenzene; DOC = dynamic organ culture system; EC = energy charge; 7-EC = 7-ethoxycoumarin; GSH = glutathione; 7-HC = 7-hydroxycoumarin; LDH = lactate dehydrogenase; LU = lucigenin; RB = rhodamine B; UW = University of Wisconsin organ preservation solution.
1. Introduction The liver is an important organ for the uptake, biliary excretion and metabolism of xenobiotics. As a consequence, many xenobiotics, including drugs, are converted to inactive metabolites but may also exert toxicity in the liver due to bioactivation reactions. These liver functions reside predominantly in hepatocytes, which represent 65% of the total liver cell population and more than 90% of the total liver mass. The current knowledge on mechanisms of transport, metabolism and toxicity of xenobiotics in the liver is mainly derived from animal experiments. The possibilities for studying liver function and drug disposition in detail in man in vivo are restricted due to obvious ethical considerations. Therefore, to explore these liver functions in man, in vitro human liver preparations (Table 1) are indispensable and are used increasingly. Results *Author for correspondence.
from such in vitro experiments might result in a better understanding of the functions of the human liver. Such knowledge will not only be valuable for drug development and toxicity studies, but also for transplantation research. In the latter area, in vitro research may contribute to a better understanding of preservation and reperfusion damage, and can also be employed for the development of viability tests for donor livers. In drug development research, animal experiments are extensively used. However, because of large interspecies dierences, especially in metabolic and transport functions, extrapolation of the results to man is not always adequate or is even impossible (Ruelius, 1987). Therefore, comparison of the functions of in vitro liver preparations of human and animal origin may contribute to a more rational choice of adequate animal models for toxicity and metabolism studies. In the future, the use of human in vitro preparations in drug research can lead to
0887-2333/98 $19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(97)00097-0
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P. Olinga et al. Table 1. Human liver preparations Cloned carriers Isolated, puri®ed enzymes Plasma membranes vesicles Subcellular fractions Isolated cells/cell cultures Tissue slices Isolated perfused organ Liver in vivo
Increasing structural organization
reduction and replacement of animal use in drug development research. Essential for the relevance of in vitro systems is the assessment of to what extent the in vitro results can be extrapolated to the process under study in vivo. Consequently, the usefulness of an in vitro preparation depends for a great part on the ability to predict the fate of drugs in vivo. This review on the use of liver slices in pharmacotoxicology will focus on the use of human liver, with special reference to the comparison with isolated hepatocytes. In addition, an overview on the predictive value of these in vitro systems for drug disposition and toxicity in vivo will be given. Pivotal to the use of human in vitro preparations is ecient utilization of the scarce human liver material that is available for research. Therefore, the preservation techniques reported for liver slices and hepatocytes will also be discussed.
2. Human liver tissue In most countries, human liver tissue can only be used on the basis of strict ethical guidelines including approval by a local medical ethical committee. Such strict ethical guidelines are of paramount importance. Although it is obvious that the guidelines may be dierent for each source of human liver, international uniformity of guidelines would facilitate ecient and adequate evaluation of research proposals. Various sources of human liver material have been employed. Autopsy livers Human liver tissue obtained at autopsy has been used to study the metabolism of xenobiotics (Kapitulnik et al., 1977; Moore and Gould, 1984; von Bahr et al., 1980). However, such livers show a marked decrease in hepatic xenobiotic metabolizing activity compared with fresh liver (Powis et al., 1988). Surgical residual material Another source of human liver tissue is healthy parts from partial hepatectomy specimens of the liver (Ballet et al., 1984; Morel et al., 1990; Olinga et al., 1997e; Vons et al., 1990) of patients with primary or secondary liver tumours. The amount of tissue that can be used for research depends on the localization and size of the tumour. Liver resection involves clamping of the aerent and eerent blood
vessels during resection, and therefore warm ischaemia of the resection specimen is inevitable. Liver biopsies The amount of liver material taken as a needle biopsy for diagnostic purposes is often small (20± 100 mg). Nevertheless, small pieces of leftover material can still be used for in vitro studies as described by Sandker (1993) and Olinga et al. (1997c). Obviously, the quality of the biopsy depends on the status of the liver from which the tissue was taken (diseased, donor liver, etc.). When the biopsy is immersed in cold University of Wisconsin organ preservation solution (UW) immediately after it is taken from the liver, warm ischaemia is avoided and functional hepatocytes can be isolated (Sandker, 1993). In addition, drug metabolism, measured in these biopsies, appears to re¯ect this function of the liver in vivo (Olinga et al., 1997c). Furthermore, wedge biopsies weighing about 1±2 g can be obtained from patients undergoing for instance, cholescytectomy surgery, after informed consent has been given by the patients. Although the amount of tissue obtained is limited, sucient hepatocytes can be isolated and used for functional studies (GoÂmez-LechoÂn et al., 1990; Kim et al., 1995). Human foetal liver Liver tissue is also dissected from human foetal tissue (Wiebkin et al., 1985). However, in vitro preparations isolated from this kind of tissue exhibit typical foetal characteristics that can be quite dierent from those of adult livers with respect to drug disposition data (Guguen-Guillouzo et al., 1984). Diseased livers In vitro tissue preparations of diseased livers removed from patients with terminal liver cirrhosis during transplantation have also been used for metabolic studies, although the predictive value of such studies for the normal liver remains to be established. However, these liver specimens may be useful in order to gain more insight into the particular disease (Sandker, 1993). Such data may contribute to a better understanding of the pathophysiology and can therefore be of value for the development of therapeutic strategies. Organ donors Human liver material can be obtained from livers procured from organ donors. Such material, however, should only be used within a strict ethical framework which may vary from country to country. In our institution, the medical ethical committee has approved such protocols when the research has a transplantation purpose and consent is given by the family of the donor to use the organs for transplantation purposes. Liver tissue remaining after size reduction or split liver transplantation, which is regarded as surgical residue, is obviously very valuable for research
Human liver slices in in vitro pharmacotoxicology
(Dorko et al., 1994; Groothuis et al., 1995; Olinga et al., 1997e; Sandker et al., 1994a,b). In addition, donor livers that are discarded for transplantation on the basis of medical grounds have been used to prepare in vitro preparations (Groothuis et al., 1995). Liver tissue is also obtained from kidney donors (Iqbal et al., 1991; Moshage et al., 1988), in cases where the liver is not considered for transplantation, or when no appropriate recipient can be found. Generally, owing to the organ procurement procedures, the transport to the transplantation centre and the size-reduction procedure, these liver samples can only be used for research after considerable cold ischaemia time. In general, one should realize that the entire procedure for the procurement of the liver tissue from dierent sources may largely in¯uence the functions to be measured. This is not only due to the surgical procedure but also to the necessity to store the tissue until use. This issue will be addressed in Section 4. 3. History The basic concept of in vitro research was re¯ected in the statements of physiologist Claude Bernard, in 1856 ``... physiological occurrence must, as far as possible, be isolated outside the organism by means of experimental procedures. This isolation can then allow us to see and understand better the deepest associations of the phenomena, so that their vital role may be followed later in the organism.'' (Bernard, 1957). In vitro research began with organ culture of embryonic organ rudiments (Trowell, 1959). The slice technique, using slices of tumour and liver tissue, was performed as early as 1923 by Otto Warburg (1923) and in the following years by H. A. Krebs (1933), who investigated the metabolism of amino acids in liver slices of cats, dogs and rats. Liver slices were prepared manually with limited reproducibility and viability (Stadie and Riggs, 1944). After a decline in the application of slices in favour of the use of isolated hepatocytes as well as the isolated perfused liver preparation, the development of the Krumdieck slicer in the eighties led to a `comeback' of the technique enabling the reproducible production of thin and viable slices (Krumdieck et al., 1980). This technology induced a renaissance of the slice technology. In 1936, Potter and Elvehjem used liver homogenates for the study of enzymatic reactions in biological material (Potter and Elvehjem, 1936). The use of homogenates evolved ®nally into the use of microsomes for metabolism experiments, while plasma membrane vesicles where employed to study transport phenomena (Boyer and Meier, 1990). Recently, sophisticated systems have been developed for the cloning and expression of speci®c human liver transport carriers (Hagenbuch and Meier, 1994; Kullak-Ublick et al., 1995) and cytochrome P-450 isoenzymes (Waterman et al., 1995),
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and these methods are now extensively used to shed more light on mechanisms of transport and metabolism of drugs in man. Since in 1969 a high-yield isolation procedure of rat hepatocytes was described (Berry and Friend, 1969), hepatocytes became the model of choice for transport and metabolism studies in vitro (Skett, 1994). With this procedure, isolated hepatocytes from many species were prepared. This includes hepatocytes from rat, mouse, chicken, dog, ®sh, hamster, pig, cow, sheep and monkey liver [for an extensive review see Berry et al., 1991]. Before 1976, only small numbers of human hepatocytes could be isolated, due to the use of non-perfusion techniques (Guillouzo et al., 1972). Bojar et al. (1976) were the ®rst to use a perfusion technique on human livers which greatly enhanced the yield of human hepatocytes. In principle the procedure that is now commonly used is based on the one described by Seglen (1976) for rat hepatocytes. Either a biopsy wedge with intact capsula on three sides (Groothuis et al., 1995; Reese and Byard, 1981), or single lobes of liver (Dorko et al., 1994) and even entire human livers (Moshage et al., 1988) are used. One or more cannulas are inserted into (branches of) the portal vein(s) (Dorko et al., 1994; Groothuis et al., 1995). In general, the yield of human hepatocytes [5± 20 106 hepatocytes/g liver (Groothuis et al., 1995; Ryan et al., 1993; Sandker, 1993; SchroÈder et al., 1994; Takahashi et al., 1993)] is low compared with rat hepatocytes (60±70 106 hepatocytes/g liver, average yield in our lab). The ®rst studies with isolated human hepatocytes concentrated on the characterization of these hepatocytes, as well as on the improvement of the isolation procedure, and the possibilities of culturing these cells (Ballet et al., 1984; Houssin et al., 1983; Hsu et al., 1985; Miyazaki et al., 1981; Ryan et al., 1993; Strom et al., 1982; Trevisan et al., 1982). Thereafter, studies were performed to investigate the metabolism of drugs (Blaauboer et al., 1985; Guillouzo et al., 1985; Tee et al., 1985; Wiebkin et al., 1985), in which emphasis was often placed on the activity and concentration of cytochrome P-450 isoforms. Nowadays, hepatocytes are more generally used in metabolic studies of speci®c compounds in order to unravel potential species dierences (Bader et al., 1994; Ballet et al., 1986; Berthou et al., 1989; Blaauboer et al., 1985; Blom et al., 1988; Chenery et al., 1987; Donato et al., 1993; Emmison and Agius, 1988; Green et al., 1986; Guillouzo et al., 1985; Le Bigot et al., 1987; Richard et al., 1991; Seddon et al., 1989; Tee et al., 1985; Valles et al., 1993; Wiebkin et al., 1985) and are also increasingly used in a variety of other research ®elds including pharmacology and toxicology. The isolated perfused liver preparation, which is extensively used to study rat liver functions (Meijer et al., 1981), has not been employed for this purpose in the case of human liver. This is partly due to the large size of the organ, which requires an excessively large volume of perfusion medium but
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mainly related to the limited availability of human livers. 4. Preparation of liver slices As described in the forementioned historical sketch of in vitro liver preparations, liver slices were initially made manually using razor blades or mechanical instruments like the Stadie Riggs tissue slicer (Stadie and Riggs, 1944). The reproducibility of the thickness of the slices at that time was largely dependent on the skills of the operator. Of note, the minimal slice thickness that could be produced was about 0.5 mm. This dimension appeared to limit the penetration of nutrients and oxygen into the inner cell layers: central necrosis in the slice occurred during incubation (Smith et al., 1986).
The introduction of the Krumdieck slicer enabled a more optimal and reproducible preparation of much thinner liver slices. With this technique the thickness of the slices is adjustable to a value as low as 100 mm. The particular slicing procedure is performed in a buer that ensures minimal trauma of the tissue. In addition, the Krumdieck slicer provides a rapid and automatic production of slices with reproducible thickness (Fig. 1). Recently, the so-called `Brendel slicer' was introduced, which exhibits the same advantages as the Krumdieck slicer but oers the advantage of a more constant oxygenation and temperature control of the recirculating buer. However, with this technique the slices have to be prepared manually (Parrish et al., 1995). To prepare liver slices with the Krumdieck slicer, ®rst cylindrical cores of tissue are made from the
Fig. 1. Schematic representation of the slicing technique.
Human liver slices in in vitro pharmacotoxicology
liver specimens. These tissue cores are preferably prepared by advancing a sharp rotating metal tube in the liver tissue using a drilling press, ensuring the preparation of real cylindrical cores. If a biopsy punch is used to prepare the cores, more skill is demanded from the operator to obtain properly formed cylindrical cores. The cores are subsequently placed in the slicer, and the slicing procedure is performed by advancing the core over an oscillating knife in a controlled environment. Cold (48C) Krebs±Henseleit buer (pH 7.4 and saturated with 95% O2 and 5% CO2) supplemented with 25 mM glucose, is commonly used in preparing the slices (de Kanter and Koster, 1995; Ekins et al., 1995; Olinga et al., 1993; Smith et al., 1986), but also Williams' medium E (Leeman et al., 1995), Sacks preservation medium (Fisher et al., 1991c) and V-7 preservation buer (Fisher et al., 1995a,b) are used. The optimal thickness for liver slices to retain their viability during culture is approximately 200±250 mm. In thicker slices the inner cell layers suer of lack of oxygen and substrates and in thinner slices the ratio of damaged cells in the outer cell layers to the living cell mass becomes unfavourable (Bach et al., 1996; Brendel et al., 1987; Fisher et al., 1995a,b; Parrish et al., 1995; Smith et al., 1989). For cryopreservation, however, slightly thicker slices appear to give better results (de Kanter and Koster, 1995). 5. Incubation and culture The use of primary suspensions of hepatocytes for metabolic and transport studies is restricted to a few hours (Tee et al., 1985). Culturing of the hepatocytes allows experiments to last for a longer period of time (up to approx. 5±7 days). The hepatocytes form monolayers and develop bile canalicular-like spaces in between the cells (Ballet et al., 1984). However, speci®c liver functions such as albumin secretion, transport activity and cytochrome P-450 activity decrease considerably during incubation (Kwekkeboom et al., 1989; Paine, 1991). Already after 24 hr of culturing, drug metabolism activity is decreased about 50%. This is very likely because of dedierentiation on the level of gene transcription (Padgham et al., 1993). In recent years much eort has been put into improving the culture conditions of hepatocytes by adding extracellular matrix components or co-culturing with other cell types to maintain their dierentiation status (Dich et al., 1988; Dunn et al., 1992; Guillouzo et al., 1993; Jeerson et al., 1984; Koebe et al., 1994; Miyazaki et al., 1985; Paine, 1991; Rogiers, 1993; Saad et al., 1993). Although survival and functioning of these cells was greatly improved, complete maintenance of dierentiated isoenzymes patterns has not yet been achieved. Initially, liver slices were incubated in static organ cultures (Trowell, 1959). Hart et al. (1983) cultured rat liver slices for 24 hr spread out on wet ®lter paper, ¯oating on top of the incubation med-
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ium. Several slice-containing vessels were placed in a box with saturated 95% O2 and 5% CO2 at 378C. However, the slices employed were rather thick (approx. 0.3 mm) and only the upper cell layers in the slice contained viable cells (0.2 mm). Simultaneously, with the introduction of the Krumdieck slicer (Krumdieck et al., 1980), a new incubation technique for slices, the dynamic organ culture system (DOC), was introduced by Smith et al. (1986). The characteristic of this system is the intermittent exposure of the slice to incubation medium and to the gas phase. The DOC is in fact a modi®ed version of the Trowell incubation system (Trowell, 1959). Meanwhile, many incubation systems have been developed, mostly based either on DOC or on culturing the slices in multiwell incubation systems (de Kanter and Koster, 1995; Dogterom, 1993; Fisher et al., 1991c and 1995b; Goethals et al., 1990; Leeman et al., 1995; Olinga et al., 1993 and 1997a), and these have been used in pharmacological and toxicological research (Parrish et al., 1995). The most remarkable phenomenon emerging from these studies is the observation that the liver slices can be cultured up to 72 hr with maintenance of biotransformation activities (Fisher et al. 1995b). Only a few studies have been published in which the various incubation systems were evaluated. Smith et al. (1986) showed that slices in the dynamic organ culture maintain their viability, as measured by ATP and potassium concentration, for up to 20 hr. Connors et al. (1990) used a 24-well incubation system, in which the medium was stirred by a magnetic stirrer. In this incubation system rat liver slices were cultured for 8 hr and human liver slices for 9 hr while maintaining high potassium concentration in the slice. Recently, Connors et al. (1996) reported that the 24-well incubation system and the DOC gave similar metabolite patterns after 24 hr of incubation with a somatostatin analogue. Vickers et al. (1992) also used the 24-well incubation system for 24 hr, but no viability parameters were described. Dogterom and Rothuizen (1993) showed that in a 12-well culture plate that is put on a gyratory shaker, rat liver slices maintain their viability up to 11 hr as determined by potassium concentration and ATP content. However, they found an impairment of the rat liver slices in a 24-well incubation system on the gyratory shaker after 11 hr. This was explained by the insucient agitation of the medium in the 24-well incubation system. Leeman et al. (1995) described a modi®cation of the dynamic organ culture: a Netwell insert (200 mm polyester mesh carrier) placed in the wells of a sixwell culture plate on a rocker platform. In this system, as in the DOC, the slices are intermittently exposed to the gas phase, (in this system being 40% O2/5% CO2) and to medium. This incubation system maintained MTT reduction in the slices up to 72 hr (Leeman et al., 1995). Simple incubation of slices in 25 ml Erlenmeyer in a shaking water-bath was reported by de Kanter and Koster, (1995).
Fig. 2. Schematic representation of ®ve liver slice incubation systems, divided into two groups: incubation systems where the slices are continuously submerged in the culture medium; dynamic organ culture related incubation systems, where the slices are intermittently exposed to the medium and the air.
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Human liver slices in in vitro pharmacotoxicology
On the basis of these ®ndings, we made a thorough comparison of ®ve incubation systems (Fig. 2) in a collaborative study of four laboratories (Olinga et al., 1997a). The ®ve systems that were evaluated included: the shaken ¯ask [an Erlenmeyer in a shaking water-bath (de Kanter and Koster, 1995)], the stirred well [24-well culture plate equipped with stainless-steel grids and magnetic stirrers (Connors et al., 1990; Olinga et al., 1993)], the rocker platform [a DOC system using six-well culture plates with Netwell inserts, rocked on a platform (Leeman et al., 1995)], the roller system [DOC rolled on an insert in a glass vial (Smith et al., 1986)] and the six-well shaker (six-well culture plates in a shaking water-bath). In the rocker platform, 40%O2/5%CO2 was used whereas in the other four systems 95%O2/5%CO2 was used to oxygenated the tissue. The liver slices were incubated in these incubation systems for 0.5, 1.5 and 24.5 hr, and subsequently subjected to viability and metabolic function tests. The viability of the incubated liver slices was evaluated by potassium content, MTT assay, energy charge, histomorphology and lactate dehydrogenase (LDH) leakage. Their metabolic functions were studied by determination of the metabolism of lidocaine, testosterone and antipyrine. For up to 1.5 hr of incubation, all ®ve incubation systems gave similar results with respect to viability and metabolic function of the slices. However, after 24 hr, the shaken ¯ask, the rocker platform and the six-well shaker incubation systems appeared to be superior to the stirred well and the roller incubator. Recently, Brendel and co-workers compared two incubation systemsÐthe DOC (Smith et al., 1986) and the multiwell plate culture (Dogterom, 1993) (the plates were put on a gyratory shaker), with respect to their ability to maintain the functionality of rat liver slices over 72 hr of culturing. The slices were evaluated with respect to ATP concentration, potassium retention, MTT reduction and protein synthesis, as well as to alanine aminotransferase and LDH leakage. The metabolic function was investigated by oxidative O-deethylation of 7-ethoxycoumarin (7-EC) (Fisher et al., 1995b). It was concluded that the dynamic organ culture was superior to the multiwell plate culture (Fisher et al., 1995b). Also, for human liver slices, various incubation systems have been used, such as the 24 well plates with magnetic stirrers (Olinga et al., 1993 and 1995; Vickers et al., 1992), the six-well plates in shaker (P. Olinga, I. H. Hof, M. Smit, M. H. de Jager, E. Nijenhuis, M. J. H. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997), the DOC roller system (Fisher et al., 1993; Lake et al., 1996a), but no direct comparison has yet been made. Human liver slices can be cultured for 72 hr in DOC, maintaining the ability to response to a speci®c inducer of cytochrome P-450 (Aroclor 1254) (Lake et al., 1996a). In addition, they showed that human liver slices do not respond to methylclofena-
83
pate. This was as anticipated from in vivo studies with this class of compound (Lake et al., 1996a). In addition to the incubation system, the oxygen and the nutrient concentration of the medium are also important for the viability of liver slices (Brendel et al., 1987; Smith et al., 1989). Fisher et al. (1995a) compared dierent media to investigate the in¯uence on viability parameters. It appeared that an enriched medium containing bicarbonate maintained the slice viability better than a simpler medium like Krebs±HEPES buer (Fisher et al., 1995a). They showed that K+ retention, protein synthesis and LDH leakage was maintained in rat liver slices for 5 days in a Waymouth's/bicarbonate medium in the DOC (Fisher et al., 1995a). Oxygenation of the hepatocytes, especially those in the centre of the slice, has been a major concern. In this respect it is important to realize that too high oxygen concentrations may be toxic, due to the tissue damage by oxygen radicals, whereas too low oxygen concentrations may cause ischaemia. Both 95% air/5% CO2 and 95%O2/5%CO2 are commonly used. In long-term culture for up to 5 days (Fisher et al., 1995a), the DOC system or DOC-related incubation systems are recommended, because of the intermittent exposure of the slice to culture medium and to air. This feature was claimed to be important for optimal gas exchange. In our experiments (Olinga et al., 1997a), however, slices that were continuously submerged in medium performed equally well or even better than in the DOC system or DOC-related system (six-well culture plate on the rocker platform (Leeman et al., 1995)). It can be calculated that liver slices consume only 0.3±1% of the dissolved oxygen per minute. This implies that, provided the medium is continuously oxygenated, the availability of oxygen is unlikely to be a limiting factor. This also explains why the lower oxygen percentage of 40% used in the rocker platform system did not seem to in¯uence the functionality or viability of the rat liver slices. Therefore, the intermittent exposure of the slice to medium and gas phase does not seem to be an important factor for slice viability. Agitation of the medium seems more important to ensure ecient oxygen delivery and to maintain liver slice viability. Another unresolved issue is the usefulness of preincubation of the slices. We showed that at least 1.5 hr of incubation are necessary to restore K+ and ATP levels. It has been suggested that a change of medium after preincubation is useful to remove cell debris from cells damaged during slicing. This is of special importance if leakage of cell components, such as LDH and alanine transaminase (ALT), is used as a marker of cell damage in toxicity studies. However, no conclusive evidence on the necessity and time of preincubation has yet been published. In conclusion, for short-term incubation the choice of the incubation system is not critical for slice viability and may be determined by other features, such as the volume of the medium, the possi-
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bility for rapid sampling and the costs. For studies where a rapid sampling of the slices is necessary, for instance in studies on drug uptake, incubation in the shaken six-well system is recommended. In the 24-well system the agitation of the medium appeared insucient, whereas in DOC systems drug uptake rate may be in¯uenced by the limited supply of substrate from the medium during the exposure to the gas phase. For longer-term incubations, the choice of the incubation system and medium seems to be more critical, and more basic studies on the slice technology have to be performed to ensure optimal long-term culture of liver slices. Among others, incubation experiments should be performed with dierent oxygen concentrations with concomitant measurement of oxygen consumption, to establish the optimal oxygen concentration. Also the agitation of the medium should be studied in more detail. Care should be taken to extrapolate the results obtained with rat liver slices to human liver slices, because considerable species dierences with respect to the in¯uence of incubation systems on slice viability have been reported (Fisher et al., 1995a). Finally, a standardization of incubation system and culture media would allow a better comparison of the results obtained from dierent laboratories. 6. Viability tests for slices For pharmacological and toxicological studies it is of the utmost importance to assess not only the viability but also the functionality of the liver slices. This is essential both for endpoint determination of toxic cell damage, and for assessing the quality of the tissue during incubation. Several viability tests have been developed for liver slices, in line with those for isolated hepatocytes: K+ retention, ATP content, energy charge, enzyme [LDH, ALT, aspartate transaminase (AST)] leakage, protein synthesis and MTT reduction (Dogterom, 1993; Fisher et al., 1995a,b; Leeman et al., 1995; Olinga et al., 1997a; Parrish et al., 1995). Speci®c liver function tests include urea synthesis, gluconeogenesis, biotransformation of test substrates (such as testosterone, 7ethoxycoumarin) and glutathione (GSH) concentration (de Kanter and Koster, 1995; Miller et al., 1993; Valentovic et al., 1995; de Kanter et al., 1997). Potassium retention is generally used to assess the viability of liver slices (Fisher et al., 1995b; Parrish et al., 1995). However, in our studies on the comparison of incubation systems and on cold storage of slices, the potassium concentration in the slice was retained while metabolic capacity was already clearly decreased (Olinga et al., 1997a). This illustrates that in drug metabolism studies the metabolic rate of a standard drug should be included as vaibility test. The determination of the energy charge (EC) is of limited value to assess slice viability, because changes in ATP, ADP and AMP have to be quite
large before a signi®cant variation of EC will be observed. Fisher et al. (1995b) proposed the following rank of sensitivity for tests aimed at the detection of cellular viability: ATP content > K+ retention > protein synthesis > enzyme leakage > MTT reduction. Because these dierent viability tests all re¯ect dierent aspects of cell viability, the choice of the viability test depends on the aim of the study. For toxicity studies where biotransformation is an important bioactivation or detoxi®cation step, metabolic function tests should be included to judge the validity of the method, whereas viability tests are needed to assess toxic eects. Both positive and negative controls should be included in such studies. Especially when human liver is used, the metabolic characterization with respect to the activity of the various drug metabolizing (iso)enzymes of that particular liver is necessary because of the large interindividual variability in this respect (Olinga et al., 1997e). Such metabolic characterization could be performed by including standard incubations with reference substrates (such as testosterone, lidocaine, 7-hydroxycoumarin and 1-chloro-2,4-dinitrobenzene) in each experiment. The viability and function tests described above are used to evaluate the hepatocytes within the slice. Up to now, tests to measure the viability of the non-parenchymal cells have not been published. The presence of the latter cell types is one of the conceptual advantages of slices compared with isolated hepatocytes. As the interaction of these nonparenchymal cells with the hepatocytes may play an important role in liver damage, the development of tests for the sinusoidal cell types deserves more attention. For example, the uptake of succinylated human serum albumin (Suc-HSA), which is speci®cally endocytozed by endothelial cells (Jansen et al., 1993) or of hyaluronic acid (Sutto et al., 1994), can be used to assess the functionality of endothelial cells in the slice (Olinga et al., 1997a). Other modi®ed proteins that are speci®cally taken up by Kuper cells, such as mannosylated HSA, may be used to assess Kuper cell functionality (Jansen et al., 1991). 7. Preservation Human liver tissue for research purposes is scarce, but when available, the amount may exceed the experimental capacity. Therefore, it is of importance to be able not only to preserve the tissue itself but also to preserve the cells and slices prepared. To ensure ecient use of the scarce human material and to limit the use of animals in drug screening tests, preservation methods for isolated cells and slices were developed. Cold storage We and others have shown that rat hepatocytes can be stored for 24 hours in cold UW, while maintaining transport capacity, morphology, viability
Human liver slices in in vitro pharmacotoxicology
and ATP content (Hammond and Fry, 1993; Marsh et al., 1991 and 1993; Sandker et al., 1990 and 1992; Sorrentino et al., 1991; Vreugdenhil et al., 1993). In a study on the in¯uence of cold preservation on metabolic function, we preserved rat hepatocytes in UW for 48 hours with good maintenance of phase 1 and phase 2 metabolic rate (Olinga et al., 1997h). In some studies, a slight reduction in viability and cell functions after 48 hr of storage in UW was seen (Poullain et al., 1992; Sorrentino et al., 1991). UW is also used for the storage of human hepatocytes. Sandker et al. (1990) in our lab showed that hepatocytes can be stored for up to 18 hours while maintaining drug transport capacity. In addition, cells isolated from both human transplantation livers and from partial hepatectomy specimens from patients undergoing resection of the liver can be stored for 18 hr with maintenance of drug metabolism and transport functions (P. Olinga, M. T. Merema, I. H. Hof, M. H. de Jager, K. P. de Jong, M. J. H. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997). It can be concluded that the use of preservation solutions as UW is valuable for short-term storage of human hepatocytes and can contribute to a more ecient use of these scarce hepatocytes. However, care should be taken if data on storage techniques for rat hepatocytes are extrapolated to human hepatocytes: human hepatocytes appear to be more resistant to anoxia and subsequent reoxygenation than rat hepatocytes (Caraceni et al., 1994). There are only a few known studies regarding cold preservation of liver slices. Fisher et al. (1993) prepared human liver slices and subsequently preserved the liver slices in UW, Sacks and Eurocollins solution. After 24 hr of storage in UW, the intracellular potassium concentration was 75% of freshly prepared slices and the protein synthesis was 58% of initial values. 24 hr of storage in Sacks or Eurocollins maintained the viability tests of the liver slices at only 45% of fresh values. Recently we showed that the viability and the metabolic capacity of slices derived from donor livers is well maintained during 18 hr of storage in UW. However, in slices prepared from partial hepatectomy specimens, the metabolic capacity is decreased after 18 hr of storage. In such tissue, warm ischaemia occurs during surgery, and this may lead to deterioration of hepatocytes in this tissue (P. Olinga, I. H. Hof, M. Smit, M. H. de Jager, E. Nijenhuis, M. J. H. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997; P. Olinga, M. T. Merema, I. H. Hof, M. H. de Jager, K. P. de Jong, M. J. H. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997). Until now most of the tests on liver preservation have been focused on the eect of preservation on the hepatocytes. Yet, it seems worthwhile to study slice preservation also with respect to the functions of the non-parenchymal cells. Recently, a paper was presented in which rat liver slices were used to
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study the in¯uence of Kuper cells on protein synthesis following cold storage and reperfusion. The large decrease of protein synthesis found in liver slices compared with isolated hepatocytes was suggested to be due to the presence of Kuper cells in liver slices. This may be related to release of cytokines and other mediators from the macrophages (Lindell et al., 1994). These results indicate that liver slices can be used to shed more light on the interactions between non-parenchymal cells and hepatocytes after cold storage or reperfusion. Cryopreservation Through cryopreservation, prolonged storage periods of human liver in vitro preparations would be possible. Proper cryopreservation could therefore facilitate a more ecient utilization of the tissue and would permit the use of liver preparations at any desired time. It would also enable a comparison of liver specimens of dierent species sampled in widely dierent periods. In the development of drugs, cryopreserved in vitro preparations from various species, including man, could be applied to select the best animal species as a model for man in toxicological studies. Such a rational approach could prevent toxicity studies in inadequate animal models and also result in safer ®rst administration of drugs to humans. In addition, cryopreserved human liver tissue can be used for the development of viability tests for donor livers. Finally, cryopreserved in vitro preparations of human liver could be used `on demand' for extracorporeal liver support. Dierent cryoprotectants [dimethyl sulfoxide (DMSO) and 1,2-propanediol] and freezing methods (controlled freezing, slow freezing and vitri®cation) have been reported (Chesne et al., 1993; Coundouris et al., 1990; de Kanter and Koster, 1995; Fisher et al., 1991c; Wishnies et al., 1991; Zaleski et al., 1993). Thawing of the cryopreserved in vitro preparations is performed by incubating the frozen preparation at 378C in the chosen medium. After thawing, the cryoprotectant is washed out by an excess of incubation medium used in the particular experiment (de Kanter and Koster, 1995; Fisher et al., 1991c). Important for the maintenance of the viability in such preparations after cryopreservation is the prevention of ice-crystal formation. If liver slices or cells are exposed to temperatures below 08C, ice-crystal formation can occur. However, also after thawing of the preparation there may be ice formation in the tissue, whereby additional damage of the cells may occur (Ren et al., 1994). Vitri®cation (i.e. very rapid up to 10008C/min cooling) inhibits such ice-crystal formation and through this procedure the intracellular and extracellular solutions appear as amorphous glass (MacFarlaine, 1987). Storage in liquid nitrogen seems to be essential because during storage at ÿ808C ice-crystal formation appears to take place (de Kanter and Koster, 1995). Hepatocytes have been cryopreserved with variable success. For isolated hepatocytes, 10% DMSO
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is used as the cryoprotectant in most protocols. After thawing of cryopreserved rat and human hepatocytes the recovery of viable cells is generally low, and only a small percentage of the cells can be cultivated successfully (Chesne et al., 1993; Coundouris et al., 1990 and 1993; Powis et al., 1987; Rijntjes et al., 1986; Utesch et al., 1992; Zaleski et al., 1993). In addition, the metabolic capacity of the remaining hepatocytes is diminished to variable extent, in particular with respect to phase II metabolism (Adams et al., 1995; Coundouris et al., 1993; Diener et al., 1993 and 1995; Diener and Oesch, 1995; Powis et al., 1987; Utesch et al., 1992). Thus, the art of cryopreservation of isolated cells is still in its infancy. The cryopreservation of liver slices is often more successful: the slices retain viability and phase I and phase II drug metabolism after thawing. The optimal cooling rate seems to be higher for slices than for cells, possibly due to the collagen present in the liver slices (de Kanter and Koster, 1995). This idea is supported by the observation that a higher optimal cooling rate is observed for the cryopreservation of cultured hepatocytes embedded in collagen, as compared with cells in suspension (Hubel et al., 1991). Fast-freezing of liver slices seems to be a very promising technique: the metabolic capacity can be preserved to 80±100% of fresh values after cryopreservation of monkey and rat liver slices (de Kanter and Koster, 1995). The cryopreservant used in this method is 12% DMSO in Williams' medium E and the slices are rapidly frozen by quick immersion in liquid nitrogen (Fig. 3). The same method has been
used successfully in our lab with human liver slices (de Kanter et al., 1997). Functional integrity after thawing compared with that of freshly prepared liver slices was determined by various parameters. Functionality in the cryopreserved human liver slices was maintained at 662 8% (ALT activity retained in the slices), 78 27% (rate of urea production), 88 214% (testosterone hydroxylation), 84 27% (N-deethylation of lidocaine) and 88 210% (O-deethylation of 7-EC) of values from freshly prepared human liver slices. These results indicate successful cryopreservation of slices prepared from human livers, with preservation of functional integrity close to values obtained with fresh liver material. This method does not require the use of sophisticated freezing equipment and is applicable in any standard biochemical laboratory. A proper preservation of viability and phase I and II metabolism [as measured by 7-EC and 7hydroxycoumarin (7-HC) conversion], after vitri®cation of human liver slices was also reported by Wishnies et al. (1991). These authors used 1,1-propanediol as a cryoprotectant. However, the success of their method was quite variable. Ekins (1996b) used the same method with rat liver slices, but also with variable success. Both phase I and phase II metabolic rate of 7-EC and 7-HC conversion were decreased by 50%. The `cryocassette' described in this report seems to contribute to optimal storage of the slices in liquid nitrogen. In addition, cryopreservation of human liver slices was reported using 10% DMSO, a cooling rate of 18C/min and storage in liquid nitrogen (Fisher et al., 1991c and 1993).
Fig. 3. Schematic representation of the fast-freezing cryopreservation procedure of liver slices as described by de Kanter and Koster, 1995.
Human liver slices in in vitro pharmacotoxicology
After thawing and incubation, the human liver slices maintained 80% of the potassium concentration, protein synthesis and glucose production compared with the values obtained with fresh liver slices, while the rate of urea synthesis was 112% of control (Fisher et al., 1991c and 1993). However, drug biotransformation experiments were not performed in these studies. An interesting approach to improve the cryopreservation success may be the use of antifreeze proteins to protect the slice from damage of ice-crystal formation (Ekins et al., 1996c). Cryopreservation could facilitate more ecient utilization of the in vitro preparations and permits their use at any desired time. Cryopreservation studies on liver slices or hepatocytes reported until now mainly deal with drug metabolism studies, but are far from complete. In addition, more research is necessary to obtain information on the maintenance of drug toxicity and membrane transport capacity after cryopreservation.
8. Use of in vitro liver preparations to study metabolism of xenobiotics in man As stated before, considerable species dierences exist in biotransformation of drugs (Chenery et al., 1987; Le Bigot et al., 1987; Le Bot et al., 1988), making extrapolation from animals to man hazardous. The metabolism of a plethora of compounds by speci®c cytochrome P-450 isoenzymes as observed in man in vivo have been reviewed extensively (Nedelcheva and Gut, 1994; Spatzenegger and Jaeger, 1995). Human in vitro preparations may be used to identify human-speci®c metabolites, to characterize unclassi®ed metabolic pathways in man, and can also be employed for the detection of drug±drug interactions in man. Comparison of the data obtained using in vitro preparations of human liver with those from animals provides important data for the rational choice of the most suitable animal species for toxicological and metabolism studies. In current practice in drug development, metabolism and toxicity studies in one rodent and one non-rodent species are obligatory. The choice of the species is primarily a standard one of rat and dog. It is only after these studies have been completed and human studies begun that the choice of animal species can be evaluated. If at that time large dierences in drug metabolism pro®les or toxic responses are found between the standard animal and man, animal studies have to be repeated in other species. For this reason, experiments with liver slices from a range of species before actual toxicity studies are started could strongly support a rational choice for the species to be used. Because in this manner the choice of the animal model is better funded, this can lead to a marked reduction in the use of experimental animals in the framework of drug development.
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Numerous studies have been performed with animal hepatocytes, mainly from rat, to study drug metabolism (Rogiers and Vercruysse, 1993). In fact, a major part of our knowledge on drug metabolism is based on studies with isolated rat hepatocytes. In rat hepatocytes, the prediction of the metabolic clearance in vivo appeared very satisfying (Houston, 1994). Since the introduction of the isolation technique for human hepatocytes, many papers have also been published on metabolism of drugs in human hepatocytes compared with hepatocytes from other species. For most compounds, the metabolism in human liver cells was lower compared with that in animals (Chenery et al., 1987; Le Bigot et al., 1987; Martelli et al., 1986; Olinga et al., 1997e; Powis et al., 1989). Not only were quantitative dierences observed between rat and man, but also qualitative dierences. In isolated rat hepatocytes, 7-EC was metabolized to the same extent as 7-HC sulfate and 7-HC glucuronide. Only minor amounts of 7-HC were found. However, in human liver in vitro preparations 7-EC was predominantly metabolized to 7HC and 7-HC glucuronide. Only in a few human livers was sulfate conjugation observed in vitro (Olinga et al., 1997e; P. Olinga, M. T. Merema, I. H. Hof, M. H. de Jager, K. P. de Jong, M. H. J. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997). In general, human hepatocytes very well predict the in vivo metabolic pattern observed of various drugs, for example caeine (Berthou et al., 1989), minaprine (Lacarelle et al., 1991), paracetamol (Tee et al., 1985), diazepam (Chenery et al., 1987), pindolol and ¯uperlapine (Guillouzo et al., 1988). In addition, stereoselectivity in drug metabolism and drug±drug interactions could be assessed in human hepatocytes (Le Corre et al., 1991; Pichard et al., 1990). Human-speci®c metabolites of Org GB 94 and Org 3770 were detected in vivo and in vitro, and were not found in any of the previously studied animals (Sandker et al., 1994a). However, for Org 3770 an O-glucuronide was the main phase II metabolite in vitro, whereas the Nglucuronide was the main conjugate found in vivo. This was also found in studies with pimobendan (Pahernik et al., 1995). These results indicated that N-glucuronidation of these substrates may be mainly performed extrahepatically (Sandker et al., 1994a). Bosentan, an antagonist of endothelin (ET-1), which is almost exclusively eliminated through hepatic metabolism and biliary excretion, was used to study the in vitro clearance and to predict in vivo kinetics from these in vitro data in dierent species (Ubeaud et al., 1995). The plasma clearance in man was successfully predicted based on the similarity of the in vitro metabolism in microsomes and hepatocytes from man and dog and the in vivo data in dog. By comparing a large number of literature data on isolated hepatocytes and in vitro studies,
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Houston et al. (1994) showed that isolated rat hepatocytes can be used successfully to predict metabolic clearance in the rat in vivo also quantitatively. Dierences in metabolic pro®les that are sometimes observed can be caused by dierences in drug concentrations and the duration of exposure (Monteith et al., 1988). Liver slices are now increasingly used to investigate species dierences in drug metabolism. Toxicological aspects are the main points of interest here. A great number of reports have been published on the metabolism of drugs in liver slices, among others: halothane (Ghantous et al., 1990a), cyclosporin A (Vickers et al., 1992), tolbutamide (Dogterom and Rothuizen, 1993), lidocaine (Olinga et al., 1995), 7-EC (Olinga et al., 1997e), testosterone (de Kanter and Koster, 1995) and 1-chloro-2,4dinitrobenzene (CDNB) (Ekins et al., 1995), and this is reviewed extensively by Ekins (1996a). Taxol metabolism was shown to be comparable in human liver slices and microsomes (Harris et al., 1994), the main metabolite in vitro was also found in vivo. Also other novel drugs have been tested such as the HIV-I transcriptase inhibitor L-629,229. The same major metabolite was found in vivo as in vitro in rat liver slices (Balani et al., 1995). Theophylline was studied in rat and human liver slices by Salyers et al. (1994), similar metabolites were found in vitro and in vivo. Vickers et al. (1992) found a sevenfold faster metabolism of cyclosporin A in human liver microsomes compared with human liver slices. Cross-species comparisons with respect to drug metabolism were recently reviewed by Vickers et al. (1995). The authors concluded that the slice system represents a versatile in vitro model with applications to biotransformation, pharmacological and toxicological studies. For instance, Stearns et al. (1992) used human, monkey and rat liver slices to produce metabolites of the non-peptide angiotensin II receptor antagonist DuP 753. In vivo observed species dierences in metabolic pattern of this compound were also detected in slices (Stearns et al., 1992). Only a few studies are known that compare the disposition kinetics of compounds in rat and human liver slices with those in vivo. Vickers et al. (1993) studied the clearance of an ergot derivative in liver slices, in microsomes and in vivo. This compound was predominantly eliminated by the liver, only minor amounts were found in the urine. These authors found that the intrinsic clearance calculated from the in vitro data was lower than the plasma clearance found in vivo. The values calculated from the liver microsomes were closer to the in vivo plasma clearance than the data obtained with the liver slices. However, liver slices from the dierent species correctly predicted species dierences in in vivo plasma clearance (Vickers et al., 1993). Similar results were found by Worboys et al. (1995) for 7EC and tolbutamide kinetics: in vivo disposition was well predicted by the liver slices. In studies on the kinetics of drug metabolism diazepam, pheny-
toin, caeine and ondansetron in rat liver slices it was found that the intrinsic clearance was underestimated and the Km for a particular pathway in slices exceeded the corresponding value in isolated hepatocytes (Worboys et al., 1996a,b). It was proposed that the rate of metabolism is not uniform throughout the slice due to the relatively slow transport within the tissue. These results imply that liver slices in principle can be used to assess metabolic pathways of drugs and also to indicate potential species dierences in drug metabolism, but are less suitable to predict intrinsic clearance in vivo. Comparison of liver slices and hepatocytes for drug metabolism studies To compare the metabolic rates obtained in liver slices and hepatocytes, the amount of metabolite formed in the liver slice can be expressed either per mg liver wet weight, per mg liver protein or per million hepatocytes. For the latter, the number of hepatocytes that are present in the liver must be assessed. Values, obtained from various publications, show similar results: the number of rat hepatocytes found per gram liver are 114 106/g (Seglen, 1976), 120 106/g (Moldeus et al., 1978), 125 106/g (Le Cam et al., 1976); for human hepatocytes 100 106/g was used (Berthou et al., 1989).1 g of fresh liver is approximately equivalent to 180 mg protein (Bock et al., 1976). These ®gures were used in dierent reports to express metabolic rate per number of hepatocytes in isolated cell suspensions and liver slices. It is more appropriate to express the metabolic rate per hepatocyte number than per mg protein: other cell types in the slice contribute to the protein content of the slice, while metabolism of drugs is predominantly performed in hepatocytes. The ®rst systematic comparison of drug metabolism in liver slices and hepatocytes was performed in 1975 (Gerayesh-Nejad et al., 1975). Biphenyl, thiabendazole, benzo[a]pyrene and ethylmorphine oxidase activities were investigated. Biphenyl oxidase activity was lower in rat liver slices than in rat hepatocytes; however, the activities of the other three oxydases were higher in slices. This was speculated to be due to the involvement of non-parenchymal cells in the metabolism of the particular drugs. In another study (Powis et al., 1989), biphenyl metabolism was also investigated in human, rat and dog liver slices and compared with hepatocytes. Metabolic rate in dog and human liver slices was higher compared with the respective hepatocytes, which in this case could be explained by the low viability of the hepatocytes that were isolated by a non-perfusion technique. The metabolic rate of biphenyls in rat hepatocytes, isolated by the standard perfusion technique, was higher compared with the rat slices (Powis et al., 1989). Berthou et al. (1989) investigated the metabolism of caeine in liver slices, hepatocytes and microsomes. However, in this study slices were used that were prepared
Human liver slices in in vitro pharmacotoxicology
after thawing of frozen human liver while metabolism of caeine was only investigated in the presence of an exogenously added NADPH-generating system. In addition, these authors used dierent concentrations of caeine for the dierent in vitro preparations, which makes a fair comparison dicult. If these results were expressed per million cells and normalized to a concentration of 1 mM caeine, human liver slices and hepatocytes and microsomes all exhibited the same metabolic rate. This implies that the hepatocytes used in this study had sucient co-factors to perform the metabolism of caeine even compared with microsomes and liver slices incubated in the presence of an NADPH-generating system with optimal co-substrate concentrations. Ekins et al. (1995) compared the metabolism of 7-EC, testosterone and CDNB in rat hepatocytes and liver slices and found a two to 29-fold lower metabolic rate in slices compared with hepatocytes. In line with these ®ndings, we observed that metabolism of lidocaine to monoethylglycinexylidide (MEGX) in liver slices was three-fourfold lower compared with that in isolated hepatocytes (P. Olinga, unpublished data). In contrast to rat preparations, a quantitatively comparable rate of metabolism of 7-EC, testosterone and lidocaine (Fig. 4) was found in hepatocytes and liver slices prepared from human liver (Olinga et al., 1995 and 1997e) and monkey liver (P. Olinga, M. T. Merema, I. H. Hof, M. H. de Jager, K. P. de Jong, M. H. J. Sloo, D. K. F. Meijer and G. M. M. Groothuis, unpublished data, 1997). These ®ndings could be explained assuming that the uptake of compounds into the rat liver slices was impaired compared with human and monkey slices. A possible explanation for this dierence may be the fact that human and monkey livers were perfused with UW to ¯ush out blood and to properly preserve the livers before the slicing procedure starts. In contrast, rat livers were not ¯ushed and were directly used to prepare slices. Blood that is remaining in the non-perfused rat liver may clot in the sinusoids, and may thereby in¯uence the penetration of com-
Fig. 4. Biotransformation of lidocaine to MEGX in human liver slices and human hepatocytes (left axis) and human liver microsomes (right axis) in ®ve dierent human livers. Interindividual dierences are equally expressed in microsomes, slices and isolated hepatocytes.
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pounds into the slice. In addition, it is known from transplantation research that during cold storage of livers, interstitial oedema may occur (Martelli et al., 1985). This may result in wider sinusoids, which may lead to better penetration of the compound. More experiments are necessary to elucidate these factors. To evaluate the involvement of the various cell layers in the functioning of the slice, visualization of the number of hepatocytes involved in the metabolism by using substrates that are converted into ¯uorescent products could be helpful. Our ®rst results indicate that after a lag time which seems to be dependent on the lipophilicity of the substrate, they do reach all cells in the slices (see Study of drug transport). The idea may arise that using human hepatocytes, the metabolic clearance is underestimated because of the relatively low viability of the human cells, caused by the greater diculty in isolation of viable human cells as compared with rat cells. However, this is not supported by our data, which shows that isolated human hepatocytes very well re¯ect in vivo drug transport functions (Olinga et al., 1997f; Sandker et al., 1994b). In conclusion, both isolated hepatocytes and slices are useful for studying drug metabolism qualitatively with respect to metabolite patterns, stereoselectivity, species dierences and drug±drug interactions. For quantitative prediction of in vivo clearance, rat slices, in contrast to rat cells, appear to underestimate the in vivo value, whereas human slices appear to operate comparable to isolated cells. In general, in vitro preparations of the human liver show a slower metabolic rate than rat liver in vitro preparations, which is in line with in vivo data. The results obtained so far with human and animal slices again emphasize the dierences in drug disposition existing between species. 9. Study of drug transport The mechanisms of uptake and excretion of drugs by the liver is widely studied using isolated hepatocytes and isolated perfused livers of rodents. The subject was extensively treated by Oude Elferink et al. (1995), summarized in a comprehensive special issue of the Journal of Hepatology in 1996 (Tiribelli et al., 1996) and reviewed in several papers from our group (Meijer, 1989; Meijer and Ziegler, 1993). In general, the rate and mechanism of drug uptake in isolated rat hepatocytes are very well in line with that found in vivo. Only a few studies are known that investigate transport of drugs in human hepatocytes, and even fewer have been performed with liver slices. Kwekkeboom et al. (1989) showed accumulation of the anionic bile acid taurocholic acid in cultured human hepatocytes, demonstrating that the capacity of accumulation clearly decreased with increasing culture time. Azer and Stacey (1993a,b) observed that uptake and accumulation of taurocholic acid and cholic acid was inhibited by CsA, whereas the
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uptake and accumulation of glycocholic acid was not aected by CsA. This indicates that increases in serum bile acids concentration in CsA-treated patients can, at least partly, be explained by interference with the hepatocellular uptake and accumulation of certain bile acids (Azer and Stacey, 1993a,b). As in rat liver, in human hepatocytes taurocholic acid uptake was shown to be ATP and partly Na+dependent (Sandker et al., 1994b). In addition, the rate of uptake appeared to be age dependent (P. Olinga, M. T. Merema, G. W. Sandker, M. J. H. Sloo, D. K. F. Meijer and G. M. M. Groothius, unpublished data, 1997). Apart from bile acids, the uptake of two other classes of compounds, namely the uncharged cardiac glycosides (ouabain and digoxin), and the cationic muscle relaxants (vecuronium and rocuronium), was studied. These three classes of drugs were commonly supposed to be transported by separate carriers in the sinusoidal domain of the liver plasma membrane of the rat (Meijer, 1989; Oude Elferink et al., 1995; Steen et al., 1992). For these substrates the observed uptake rates were higher in the isolated rat cells than in the human cells, but they appeared to properly re¯ect the relative hepatic uptake rates found in the intact organism (Sandker et al., 1994b). We extended this study to investigate mechanisms of drug uptake of cationic drugs and cardiac glycosides (Olinga et al., 1997f). Temperature-dependent uptake indicated carrier mediated transport. Substrate speci®city studies and inhibition studies seem to indicate that the dierentiation in type I and type II cation uptake systems, as proposed for rat hepatocytes (Steen et al., 1992) is not necessarily valid in man. Yet the cloned human organic anion transporting polypeptide (OATP) that also seems to accommodate certain organic cations clearly dierentiates between relatively small (type I) and more bulky (type II) cationic drugs (Bossuyt et al., 1996). Unexpectedly, the uptake of digoxin in human hepatocytes was not inhibited by quinidine or quinine. This is in contrast to experiments in rat hepatocytes, where a marked inhibition of digoxin uptake by quinine and virtually no inhibition by the diastereomer quinidine was demonstrated. These results may indicate that the well known pharmacokinetic interaction between cardiac glycosides and quinine-like compounds, as is found in patients, may not be related to the uptake process in the liver. More experiments have to be performed to elucidate the mechanism of this interaction. Zhou et al. (1994), found a signi®cant correlation between uptake rates of various vinca alkaloids in human hepatocytes and the in vivo plasma clearance of the drugs. De Jong et al. (1993) showed that there is a high degree of similarity between the inhibition patterns in the uptake and subsequent conversion of thyroid hormones in human and rat hepatocytes, although the absolute rates of uptake and conversion were found to be lower in human hepatocytes.
These dierences in drug transport rate and mechanism in rat and man again emphasize that extrapolation of pharmacokinetic data from rat to man is hazardous. Moreover, in general the uptake of drugs in human hepatocytes is slower than in rat hepatocytes. In vitro±in vivo scaling calculations (Olinga et al., 1997f; Sandker et al., 1994b) showed that these dierences in uptake rate between rat and human cells really re¯ect interspecies dierences rather than being due to dierences in viability. Most of the current knowledge on drug transport carriers is derived from experiments in rats. Therefore, more drug transport studies have to be performed in human liver preparations to further study the relevance of rat experiments for the situation in man. These results also indicate that human hepatocytes are an appropriate model to study interspecies dierences and the mechanisms of hepatic transport in man. Mechanisms of drug uptake in liver slices were studied in vitro as early as 1963 by Schanker and Solomon (1963). The results obtained in these experiments are still valuable. They show that the in¯uence of temperature, anoxia, metabolic inhibitors and substrate inhibition can be studied successfully in this preparation. However, as previously mentioned, in that time the preparation of reproducible precision-cut slices was not feasible. Therefore, the slice incubation technique was virtually abandoned in transport studies after the introduction of the successful isolation of rat hepatocytes. To investigate the possibilities and limitations of the use of precision-cut liver slices prepared by the mechanical slicer, in drug transport studies, dierent aspects of the mechanism of the uptake of several classes of drugs in human and rat liver slices were investigated in our laboratory. Four model compounds were investigated that enter hepatocytes via entirely dierent membrane transport mechanisms: the ¯uorescent dyes rhodamine B (RB) and lucigenin (LU), the cardiac glycoside digoxin and the neo-glycoprotein lactosylated albumin. Receptor-mediated endocytosis into endothelial cells was studied with succinylated and aconylated albumin. The rate of penetration into the rat liver slice was studied with the lipophilic cationic compound RB and hydrophilic organic cation LU. RB, which enters hepatocytes by passive diusion, was homogeneously distributed throughout the rat liver slice (250 mm thickness) within 5 min (Plate 1), indicating that for very lipophilic components the penetration rate into the slice and the diusion rate into all of the cells are rapid. In contrast, after incubation with LU that is taken up by hepatocytes through adsorptive endocytosis, the inner cell layers contained ¯uorescence only after 15 min. Digoxin uptake into the rat slices showed a temperature-dependent component, compatible with involvement of carrier-mediated uptake mechanisms. Quinine markedly inhibited the uptake of digoxin, in contrast to its diastereomer quinidine,
Plate 1. Distribution of 25 mM Rhodamine B in cross-section of a rat liver slice (2250 mm) after 5 min of incubation (¯uorescence microscopy, bar = 100 mm).
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which only slightly inhibited the digoxin uptake in rat liver slices. This stereoselective inhibition is in line with results obtained in isolated rat hepatocytes and isolated perfused rat livers (Fig. 5). The temperature-dependent uptake and immunohistochemical localization of modi®ed proteins, Lactose27-Human Serum Albumin (Lact27-HSA), Succinylated-Human Serum Albumin (Suc-HSA) and Aconylated Human Serum Albumin (AcoHSA), in rat liver slices showed that large molecules can enter the slice and are probably taken up by receptor mediated endocytosis. Thompson et al. (1993) clearly showed that liver slices are capable of excreting bile acid derivatives into the incubation medium. In conclusion, liver slices appear to be a valuable tool for assessing mechanisms of drug uptake of very dierent compounds. If the uptake rates of drugs in liver slices are to be compared with parameters obtained in vivo, some diculties may arise as already indicated in the previous section. The uptake rate of compounds into the slice may not only re¯ect the uptake rate of the cells in the slice involved, but can also be in¯uenced by the rate of penetration of the substrates (i.e. diusion through sinusoidal spaces) into the slice. From our results with the lipophilic agent RB it is clear that the penetration process into the slice takes at least 5 minutes. Therefore, for substrates that are taken up in hepatocytes relatively quickly, penetration into the slice may limit the uptake rate of the drug in the slice. Although human hepatocytes seem to be valuable for the study on mechanisms of drug transport, the isolation procedures involve collagenase digestion for the disruption of cell to cell contacts. Clearly, the digestion may also damage plasma membranes
Fig. 5. The uptake of 25 nM digoxin (q) in the presence of 50 mM quinidine (*) or 50 mM quinine (w) in rat liver slices. Points are the mean of three separate experiments2SEM; *P < 0.05.
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and transport systems therein. In addition, the normal polarity of the hepatocytes is lost after isolation. In contrast, liver slices retain the cellular architecture of the liver while no digestion with collagenase is needed. This makes a systematic comparison of transport data from isolated hepatocytes and slices an attractive scienti®c item. Evidently more research is necessary to elucidate the potentials and limitations of the liver slice model in this ®eld.
10. Toxicological studies Many eorts have been made to develop in vitro models to investigate hepatotoxicity, because the liver is the most commonly aected target organ in preclinical studies. Hepatotoxicity is primarily investigated in hepatocytes (George et al., 1996). However, using isolated hepatocytes, only acute eects can be studied reliably. Subchronic and chronic studies are hampered by the decrease in dierentiation status of cultured hepatocytes. Ulrich et al. (1995) showed that human hepatocytes are useful in toxicity studies to examine the relevance of animal data for the human situation. Since the introduction of the precision-cut liver slices they have been used to determine hepatotoxicity (Azri et al., 1990b; Smith et al., 1989). As mentioned earlier, the liver slices retain the intact liver structure, thus the cellular heterogeneity of the liver is preserved, making slices an ideal tool for investigating celland zone-speci®c cytotoxicity. Incubation of slices with carbon tetrachloride, one of the zone-speci®c toxicants, resulted in leakage of glucose-6-phosphate dehydrogenase and b-glucuronidase mainly from centrilobular hepatocytes. However, less leakage of the enzymes (e.g. LDH) from the periportal hepatocytes occured, indicating site-speci®c hepatotoxicity (Azri et al., 1990a and 1992). Michaud et al. (1996) showed that hepatotoxic interaction of carbon tetrachloride and trichloroethylene found in vivo can be observed in liver slices. Incubations with two toxic agents, CHBr3 and CHCl3 revealed synergetic toxicity in the liver slice model (Azri-Meehan et al., 1994). Also other volatile compounds, for instance anaesthetics, were studied using slices in the DOC system. Sevo¯urane and des¯urane showed only minimal toxicity, as was expected from in vivo data (Ghantous et al., 1991 and 1992b). However, incubations of guinea pig liver slices with halothane, which has been shown to exert hepatoxicity in vivo (Ghantous et al., 1990c), resulted in clear toxicity in the slices (Ghantous et al., 1990a,b,c and 1992a,c). A comparison of dierent in vitro rat liver preparations with respect to rank order of toxicity of bromobenzene and ®ve of its ortho-substituted derivatives showed that liver slices were more representative for the in vivo toxicity than hepatocytes (Fisher et al., 1991b). The toxicity of dichloroaniline and its isomers were studied with liver slices
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and was comparable with in vivo data (Valentovic et al., 1995). Dichlorobenzenes, which are widely dispersed within the environment, have been investigated with human liver slices to rank the toxicity of the isomers. The rank order of hepatotoxicity in human tissue found by Fisher et al. (1991a) was 1,3DCB>1,2-DCB>1,4-DCB. With liver slices they also showed rat strain dierences in the rank order of toxicity of these dichlorobenzenes (Fisher et al., 1990). Species dierences in hepatotoxicity, found in vivo, were also observed in studies in liver slices with acetaminophen, cocaine and furosemide (Connors et al., 1990; Miller et al., 1993; Wormser et al., 1990). Liver slices were also used to evaluate species dierences in xenobiotic-induced genotoxicity (Beamand et al., 1995; Lake et al., 1996a,b) and appeared to be a useful alternative for the primary hepatocyte cultures, which are currently quite popular in this ®eld of research. Only few studies take advantage of a special feature of the liver slice model: the presence of nonparenchymal cells. Endotoxin-induced cytokine secretion was investigated in mouse and human liver slices, and this study elegantly demonstrated that cytokine-responses can be measured in liver slices (Luster et al., 1994). The same group investigated cadmium-induced hepatotoxicity and subsequent cytokine secretion from non-parenchymal cells in the liver slice (Kayama et al., 1995). Ishiyama et al. (1995) showed that Kuper cell function in slices was involved in the hepatotoxicity of diethyldithiocarbamate. In conclusion, although the amount of data is still limited, the data show clearly that liver slices are a very useful in vitro preparation to study drug toxicity in animals and man. 11. Use of liver slices or hepatocytes: advantages and disadvantages Isolated hepatocytes have proved to be a valuable tool in drug disposition and toxicity research. The data described in this review indicate clearly that the use of human liver slices may also be developed as a valuable method. Moreover, the use of isolated hepatocytes and liver slices from human liver is of vital importance for studying drug disposition and toxicity in man. The isolation of hepatocytes involves collagenase digestion and causes disruption of cell to cell contacts. The digestion is a necessary step for ecient cell isolation but it may also damage plasma membranes and transport systems therein. An important drawback is that the normal polarity of the hepatocytes is lost after isolation. Liver slices, however, retain the normal cellular architecture of the liver. Furthermore, the liver slice contains all the dierent cell types present in the liver while the lobular (or acinar) organization of the liver is maintained. This aspect may be very important for toxicological research: non-parenchymal cells such as Kuper cells may largely in¯uence the function and viability
of the hepatocytes. Moreover, the preparation of the slices seems less operator and species dependent while it lacks the potentially membrane damaging eect of the digestion procedure for hepatocytes. To date, the issue of viability and functionality of hepatocytes in the slices, especially in the inner cell layers, is insuciently elucidated and requires additional research. Both hepatocytes and liver slices have the ability for an integrated phase I and phase II metabolism of xenobiotics. This in contrast to microsomes in which the metabolic reactions of drugs are restricted to phase I metabolism and glucuronidation, and only if sucient co-factors are added. In studies on the metabolism of drugs, slices may become superior to isolated hepatocytes. Hepatocytes in suspension are only viable for a few hours. In culture dedierentiation of the cells occurs already within 24 hr which can only partly be overcome by co-culturing hepatocytes with epithelial cells (Guillouzo et al., 1993), which is a technically complicated system. We (Olinga et al., 1997a) and others (Connors et al., 1996) have shown that liver slices do not dierentiate with respect to drug metabolism in 24 hours. Fisher et al. (1995b) even showed that liver slices can be incubated for up to 3 days without losing metabolic capacity, suggesting that longterm culture of liver slices seems possible without loss of phase I and II metabolism. However, others (MuÈller et al., 1996) reported loss of drug metabolism of liver slices after 48 hours of culture. It has been insuciently investigated as to what extent dedierentiation also occurs in liver slices in longterm culture. Another advantage of the use of liver slices is based on the ®nding that cryopreservation permits the ecient use of liver slices for metabolism studies at any desired time. For the investigations on the mechanisms of the uptake of drugs, isolated cells and slices seem to be equally valuable. However, rate-limited penetration into the slice of the substrates under study may in¯uence some of the quantitative aspects of uptake characteristics (e.g. Vmax and Km; Worboys et al., 1996a,b). Other advantages to the use of slices include the relatively easy preparation, and the more ecient use of the available tissue. Preparation of liver slices does not impose restrictions on the size or con®guration of the tissue specimen, and in principle slices can be prepared even if only small amounts of (human) tissue are available. Conclusion Drug toxicity, metabolism and transport data determined in in vitro preparations such as slices and isolated hepatocytes, from both human and animal liver, showed a good correlation with the corresponding parameters measured in vivo. Therefore, the results obtained in such studies may give an onset to more in-depth research on the mechanism of drug transport, metabolism and
Human liver slices in in vitro pharmacotoxicology
toxicity in the human liver. In addition, the knowledge of species dierences in drug metabolism, as detected in isolated hepatocytes and liver slices, can be applied to select the best animal species as a model for man in toxicological studies. A more rational choice on this point may lead to less super¯uous toxicity studies in inadequate animal models and may also result in a safer ®rst administration to humans. Finally, it may in general lead to a reduction of animal use in drug development research.
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