Paul Skett

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Cryopreservation of hepatocytes: a review of current methods for banking

Cryopreservation, the freezing of hepatocytes in liquid nitrogen for storage, has been investigated for many years, as a method of long-term storage for hepatocytes. Unfortunately an agreed acceptable protocol has been elusive, in part due to the susceptibility of hepatocytes to the freeze thaw process involved. A method for long-term storage (months, possibly years) of human hepatocytes, in particular, is desirable for the development of a clinically applicable bioartificial liver, hepatocyte transplantation and for pharmacotoxicological research. The sources of human liver tissue from which hepatocytes can be derived are limited. Many groups have modified and improved the process of cryopreservation and many new techniques have been published, including the incorporation of such cryopreserved cells in clinically based studies. Further evaluation is still required to develop a universally acceptable protocol. This article reviews the difficulties involved in cryopreserving hepatocytes for banking and examines recent technical advances within this field.

Induction of rat hepatic glucocorticoid-inducible cytochrome P450 3A by metyrapone

The bipyridyl compound metyrapone is a potent inhibitor of cytochromes P450, a gene superfamily of haemoproteins involved in the metabolism of many xenobiotics as well as endogenous compounds such as steroid hormones. Administration of metyrapone to male rats induces the expression of the cytochrome P450 sub-family 3A (CYP3A). In order to determine whether metyrapone was causing the induction of CYP3A by blocking endogenous glucocorticoid metabolism, CYP3A levels were examined in rat hepatocytes cultured in serum-free medium supplemented with hydrocortisone 21-hemisuccinate plus or minus metyrapone. Western blotting indicated that metyrapone alone induces CYP3A and that hydrocortisone 21-hemisuccinate is ineffective. However, hydrocortisone 21-hemisuccinate enhanced the levels of CYP3A induced by metyrapone. In contrast, glucocorticoid-inducible tyrosine aminotransferase (TAT) activity was unaffected by metyrapone but metyrapone enhanced the levels induced by hydrocortisone 21-hemisuccinate. An examination of the metabolism of hydrocortisone by rat hepatocytes in vitro indicated that metyrapone perturbed the catabolism of hydrocortisone under conditions which give rise to an enhancement of hydrocortisone 21-hemisuccinate and hydrocortisone-dependent TAT induction. However, evidence is presented to suggest that such a perturbation of hydrocortisone metabolism could not account for the glucocorticoid potency amplifying property of metyrapone. Thus the induction of CYP3A and the enhancement of glucocorticoid-mediated TAT induction appears not to be associated with any perturbation in glucocorticoid metabolism but with some other as yet undefined mechanism(s).

Pathways of drug metabolism

The routes by which drugs may be metabolised or biotransformed are many and varied and include the chemical reactions of oxidation, reduction, hydrolysis, hydration, conjugation and condensation. It is important that these pathways are studied as the route of metabolism of a drug can determine whether it shows any pharmacological or toxicological activity. Drug metabolism is normally divided into two phases, phase I (or functionalisation reactions) and phase II (or conjugative reactions). The chemical reactions normally associated with phase I and phase II drug metabolism are given in Table 1.1.

Techniques and experiments illustrating drug metabolism

This chapter is designed to illustrate experimentally some of the concepts discussed in this book, and the experiments described herein are derived from undergraduate practicals that have been running in the authors’ laboratories for several years. The design of adequate experiments illustrating drug metabolism is not an easy task. For example, practical classes can suffer from time constraints in that the experiments usually have to be completed in one day. In addition, experimental design must also take account of practical constraints such as availability of reagents or analytical instrumentation. Accordingly, we have described a series of experiments that are flexible and can be tailored to suit either the size of a practical class, the time available or access to specific instrumentation.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco–toxicology ☆

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Induction and inhibition of drug metabolism

The study of drug metabolism in experimental animals in general and Man in particular is ideally studied under strictly controlled conditions, such that we only observe the influence of the normal physiological and biochemical processes that contribute to the metabolism of the drug in question. However, this ideal situation is rarely achieved, and the metabolism of drugs is substantially influenced by the deliberate or passive intake of many chemical substances that Man is increasingly being exposed to either in his environment, for medical reasons or as a result of his life style. These chemical substances are derived from a variety of sources and include pharmaceutical products, cosmetics, food additives and industrial chemicals. As summarized in Table 3.1, the magnitude of the various chemicals in use today, and hence the potential exposure to Man, is staggering.

Pharmacological and toxicological aspects of drug metabolism

In general, the intensity and duration of drug action is proportional to the concentration of the drug at the site of action and the length of time it remains there. Therefore any factor that effectively alters the drug concentration at the active site will result in a changed pharmacological response to the drug. As indicated in previous chapters, the processes of drug metabolism result in biotransformation of the drug to metabolites that are chemically different from the parent drug and would therefore be expected to have an altered affinity for the drug receptor. Thus the processes of drug metabolism change the structure of the drug and essentially result in the production of a different chemical that often is not recognised by the relevant receptor system, and hence results in little or no pharmacological response. In this case, drug metabolism results in pharmacological deactivation. In contrast to the above, many drugs are pharmacologically inert and absolutely require metabolism to express their pharmacological effect. Therefore in this case, the process of drug metabolism results in pharmacological activation.

Enzymology and molecular mechanisms of drug metabolism reactions

As described in chapter 1, drugs and xenobiotics are transformed by a variety of pathways in two distinct stages. The phase I (or functionalisation) reactions serve to introduce a suitable functional group into the drug molecule, thereby changing the drug in most cases to a more polar form and hence more readily excretable. In addition, the product of phase I drug metabolism may then act as the substrate for phase II metabolism, resulting in conjugation with endogenous substrates, increased water solubility and polarity, and drug elimination or excretion from the body. In a quantitative sense, the liver is the main organ responsible for phase I and phase II drug metabolism reactions, although this is by no means the only organ involved. Drug localisation, and hence probably metabolism, in a given tissue is dependent on many factors including the physico-chemical properties of the drug (pK a, lipid solubility and molecular weight), chemical composition of the organ and the presence of uptake mechanisms which allow the drug to be ‘trapped’. As drugs are often given several times per day in high doses for long periods, it is not surprising that the drug binding and metabolism sites become saturated in a given organ. This would then lead to drug diffusion to other sites in the body and may well explain extrahepatic drug metabolism and some bizarre side effects observed after prolonged drug treatment. Other organs where drug metabolism reactions have been observed include skin, gastrointestinal tract, gastrointestinal flora, lung, blood, brain, kidney and placenta, amongst others.

The Preparation and Culturing of Rat Hepatocytes

Isolated, cultured hepatocytes would seem at first sight to be an ideal model system for the study of hepatocarcinogenesis and the testing of chemicals that may be carcinogens as the liver is the major site of activation of many potential carcinogens and, thus can act as generator of the toxic metabolite as well as the target.The great potential of this model is, however, diluted by the diversity of approaches taken by various workers in the field to the preparation and culturing of hepatocytes.There is no accepted or recommended method for preparing the cells from intact liver (although many methods have been published) and little work on the optimisation of the medium and medium additions needed for culturing of hepatocytes.