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Human and rat organ slices
de Kanter, Ruben
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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