Molecular and Cellular Toxicology E-Book

Lesley Stanley

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 902

Veröffentlichungsjahr: 2014

Beschreibung

Toxicology is the study of the adverse effects of chemical, physical, or biological agents on people, animals, and the environment. Toxicologists are trained to investigate, interpret, and communicate the nature of those effects. 

Over the last ten years the subject of toxicology has changed dramatically, moving from a discipline which was once firmly wedded to traditional methods to one which is keen to embrace the innovative techniques emerging from the developing fields of cell culture and molecular biology. There is an acute need for this to be reflected in a paradigm shift which takes advantage of the opportunities offered by modern developments in the life sciences, including new in vitro and in silico approaches, alternative whole organism (non-mammalian) models and the exploitation of ‘omics methods, high throughput screening (HTS) techniques and molecular imaging technologies.

This concise, accessible introduction to the field includes the very latest concepts and methodologies. It provides MSc, PhD and final year undergraduate students in pharmacy, biomedical and life sciences, as well as individuals starting out in the cosmetics, consumer products, pharmaceutical and testing industries, with everything they need to know to get to grips with the fast moving field of toxicology and the current approaches used in the risk assessment of drugs and chemicals.

Leseprobe

Table of Contents

Molecular and Cellular Toxicology

An Introduction

Dr Lesley A. Stanley

Consultant in Investigative Toxicology, UK

This edition first published 2014 © 2014 by John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data

Stanley, Lesley A., author.

Molecular and cellular toxicology : an introduction / Dr. Lesley A. Stanley.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-1-119-95207-7 (cloth)— ISBN 978-1-119-95206-0 (paper)

I. Title.

[DNLM: 1. Toxicity Tests— methods. 2. Molecular Biology. 3. Xenobiotics— toxicity. QV 602]

RA1199.4.A38

615.9′07— dc23

2013048370

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Dedication

Dedicated to the memory of Elizabeth Stanley and Margaret Orr, two wonderful mothers

Foreword

by Dr Marilyn J. Aardema

Marilyn Aardema Consulting, LLC

Technical advances and bold initiatives like the National Academy of Science's Toxicology in the 21st Century, along with socio-political pressures such as the 3Rs (replace, reduce and refine the use of animals in experiments) have led to remarkable changes in the field of toxicology over the past several years. Toxicology is undergoing a major shift towards assessing and understanding damage at the cellular and tissue level. This comprehensive, well-written book focusses on the timely topic of current advances in the field of molecular and cellular toxicology. The book starts with a review of how cells and tissues respond to damage and the consequences of damage that overwhelm normal cellular protective mechanisms. With this background, new technologies for evaluating cellular and tissue damage along with investigating the toxicological outcomes are described. This includes the use of ‘omics technologies (transcriptomics (changes in RNA), proteomics (changes in proteins)), metabolomics (changes in products of metabolism; Chapter 3), the use of 3D tissue models to obtain a more biologically relevant assessment of toxicity (Chapters 4, 8) and the use of in silico approaches for predicting toxicological effects (Chapter 6). The final Chapter 10 provides a glimpse forward at emerging technologies that are sure to impact the field of molecular and cellular toxicology further in the years ahead. These and the other chapters in the book not only provide essential reading on recent technology developments, but also provide up-to-date information on the drivers behind these advances, and the global efforts towards validation and incorporation of new approaches into the toxicology paradigm.

This book will be invaluable to all those interested in the latest advances in toxicology including postgraduate/graduate life science students interested in toxicology as well as individuals starting out in the field of cosmetics, consumer products, pharmaceutical and testing industries who need knowledge of current approaches in toxicology. I commend the editors and author, Dr Lesley Stanley, on this valuable contribution to the field of Toxicology.

Preface

Acknowledgements

My Editor, Nicky McGirr, has been a source of inspiration throughout the course of this project. I am immensely grateful to her for her excellent advice, and particularly for her unflagging enthusiasm which kept me going through all the difficult bits.

I would like to thank a number of people who were kind enough to let me use some of their material in this book. Dr Elaine Johnstone (Department of Oncology, University of Oxford) gave me permission to use material from one of her lectures on pharmacogenetics and genome wide association studies in Chapter 3; part of the text upon which Chapters 4 and 6 are based was provided by Dr Paul Brantom (Brantom Risk Assessment Ltd) with permission from the cosmetics industry association Colipa; and Dr Gill Clare (Independent Consultant on Genetic Toxicology) provided invaluable material and advice for Chapter 8. In addition, many people (some of whom do not even know me) generously allowed me to use their illustrations; they are too numerous to list individually, but would like to record my thanks to all of them.

I am also grateful to Mrs Roberta Logan and Drs Eian Massey, Robin Whelpton and Gary Hutchison, all of whom provided constructive comments on various versions of the manuscript during its preparation.

This project could not have been completed without the encouragement of many of my friends. In particular, I would like to thank Sarah Young for letting me work in her house while the builders were in mine and Julie McDowell for being my gym buddy.

Finally, it is my duty and pleasure to thank my husband, Nigel Orr, for his unfailing support, his infinite tolerance and for never being without a secret supply of chocolate.

Abbreviations

3Rs

replacement, refinement and reduction (of the use of animals in research)

4-ABP

4-aminobiphenyl

AAF

acetylaminofluorene

ADME

Absorption, Distribution, Metabolism and Excretion

AFB1

aflatoxin B1

AhR

arylhydrocarbon receptor

ALT

alanine aminotransferase

ASO

allele-specific oligonucleotide

ASPCR

allele-specific polymerase chain reaction

AST

aspartate aminotransferase

ATP

adenosine triphosphate

AUC

area under the plasma concentration–time curve

BAC

bacterial artificial chromosome

BBB

blood-brain barrier

BMD

benchmark dose

bp

base pair

BrdU

bromodeoxyuridine

CAR

constitutive androstane receptor

cdk

cyclin-dependent kinase

cDNA

copy DNA

CEBS

Chemical Effects on Biological Systems

ChIP

chromatin immunoprecipitation

CHO

Chinese hamster ovary

CIN

cervical intraepithelial neoplasia

CITCO

6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde

CL

INT

intrinsic clearance

C

MAX

maximum (plasma) concentration

CNS

central nervous system

COMET

Consortium for Metabonomic Toxicology

CPMP

European Committee for Proprietary Medical Products

CYP

cytochrome P450

DDI

drug–drug interaction

DEHP

diethylhexylphthalate

DEN

diethylnitrosamine

DILI

drug-induced liver injury

DMBA

7,12-dimethylbenz(a)anthrancene

DMN

dimethylnitrosamine

DMSO

dimethyl sulphoxide

EC

50

concentration giving 50% of maximal effect

ECHA

European Chemical Agency

ECVAM

European Centre for the Validation of Alternative Methods

EFSA

European Food Safety Authority

EMA

European Medicines Agency

ENU

ethylnitrosourea

EPA

US Environmental Protection Agency

ESC

embryonic stem cell

EST

embryonic stem cell test

EU

European Union

FABP

fatty acid binding protein

FDA

US Food and Drug Administration

floxed

flanked with loxP sites

GC

gas chromatography

GFP

green fluorescent protein

γGT

gamma glutamyl transpeptidase

GI

gastrointestinal

GLP

Good Laboratory Practice

GSH

glutathione

GST

glutathione

S

-transferase

GTP

guanosine triphosphate

GWAS

genome-wide association study

HCC

hepatocellular carcinoma

hERG

human ether-a-go-go related gene

HO-1

haem oxygenase 1

HPRT

hypoxanthine phosphoribosyltransferase

HRN

TM

Hepatic Reductase Null

TM

HTS

high throughput screening

i.p.

intraperitoneal

i.v.

intravenous

IC

50

concentration giving 50% inhibition

ICH

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use

ILSI

International Life Sciences Institute

iPSC

induced pluripotent stem cell

IVIVE

in vitro–in vivo

extrapolation

kb

kilobase

kD

kilo Dalton

KEGG

Kyoto Encyclopedia of Genes and Genomes

K

M

Michaelis constant

K

OW

octanol-water partition coefficient

LC

liquid chromatography

LC-MS/MS

liquid chromatography-tandem mass spectrometry

LD

50

dose giving 50% lethality

LDH

lactate dehydrogenase

LOAEL

lowest observed adverse effect level

LPS

lipopolysaccharide

MALDI

matrix-assisted laser desorption ionisation

MAPK

mitogen-activated protein kinase

MDCK

Madin-Darby canine kidney

MDR

multidrug resistance protein

MHLW

Japanese Ministry of Health, Labour and Welfare

MHRA

Medicines and Healthcare Products Regulatory Agency

MIAME

Minimum Information About a Microarray Experiment

MIAPE

Minimum Information About a Proteomics Experiment

MNU

methylnitrosourea

MOE

margin of exposure

mRNA

messenger RNA

MRP

multi-drug resistance-associated protein

MS

mass spectrometry

MTD

maximum tolerated dose

NADPH

nicotinamide adenine dinucleotide phosphate

NAT

N

-acetyltransferase

NHS

UK National Health Service

NIEHS

US National Institute of Environmental Health Sciences

NIH

US National Institutes of Health

NMR

nuclear magnetic resonance

NOAEL

no observed adverse effect level

NTP

US National Toxicology Programme

OECD

Organisation for Economic Co-operation and Development

p.o.

perioral

PAH

polycyclic aromatic hydrocarbon

PAMPA

Passive Artificial Membrane Permeability Assay

P

app

apparent permeability

PB

phenobarbital

PBBK

physiologically based biokinetic

PBPK

physiologically based pharmacokinetic

PBTK

physiologically based toxicokinetic

PCN

pregnenlonone 16α-carbonitrile

PCR

polymerase chain reaction

PhIP

2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine

pK

a

acid dissociation constant

PPARα

peroxisome proliferater activated receptor α

PPD

p

-phenylene diamine

PXR

pregnane X-receptor

QA

quality assurance

QC

quality control

QSAR

quantitative structure–activity relationship

QSPR

quantitative structure–permeability relationship

RFLP

restriction fragment length polymorphism

RHE

reconstructed human epidermis

RIVM

Netherlands National Institute for Public Health and the Environment

RSMN

reconstructed skin micronucleaus

RT-PCR

reverse transcriptase polymerase chain reaction

SDS

sodium dodecyl sulphate

SELDI

surface-enhanced laser desorption/ionization

SHE

Syrian hamster embryo

SNP

single nucleotide polymorphism

SOD

superoxide dismutase

SULT

sulphotransferase

SXR

steroid X receptor

t

½

half life

TCDD

2,3,7,8-tetrachlorodibenzo-

p

-dioxin

TCPOBOP

1,4-bis[2-(3,5-dichloropyridyloxy)]benzene

TGP

Toxicogenomics Project in Japan

T

M

melting temperature

TNF

tumour necrosis factor

TOF

time-of-flight

TPA

12-

O

-tetradecanoyl phorbol 13-acetate

TTC

threshold of toxicological concern

UDS

unscheduled DNA synthesis

UGT

UDP-glucuronyl transferase

ULN

upper limit of normal

UV

ultraviolet

V

max

maximum velocity

About the Companion Website

This book is accompanied by a companion website:

www.wiley.com/go/stanley/molecularcellulartoxicology

The website includes:

Powerpoints of all figures from the book for downloading

PDFs of all tables from the book for downloading

Chapter 1Background to Molecular and Cellular Toxicology

1.1 What do we mean by molecular and cellular toxicology?

The Society of Toxicology1 defines Toxicology as ‘the study of the adverse effects of chemical, physical or biological agents on people, animals and the environment’ and toxicologists as ‘scientists trained to investigate, interpret and communicate the nature of those effects’. In the disciplines of molecular and cellular toxicology, toxicologists make use of the many new techniques which are becoming available in the molecular life sciences to understand the underlying mechanisms by which these agents damage cells, tissues and entire organisms. The main aims of toxicity testing, whether during pre-clinical drug development, in the course of safety assessment of cosmetic ingredients and consumer products or while evaluating the potential consequences of exposure to industrial and environmental chemicals, are to construct a toxicological profile of the chemical and to identify a threshold dose (if any).

The topic of this book is how molecular and cellular techniques can be used to study the toxicity of exogenous chemicals, referred to in the trade as xenobiotics. The primary target organs for xenobiotic toxicity are usually those which are exposed to xenobiotics and their metabolites because of the roles they play as portals of entry, sites of metabolism and/or organs of excretion. The molecular and cellular consequences of exposure are summarised in Figure 1.1.

Figure 1.1Consequences of exposure to a toxic insult (source: Dr Cliff Elcombe, CXR Biosciences Ltd. Reproduced with permission of Dr Cliff Elcombe)

Despite the many scientific advances made in the life sciences over the last couple of decades, which include spectacular advances in the fields of molecular biology, biotechnology and bioinformatics, the basic concepts of regulatory toxicology have hardly changed over the same period. For example, although the classical LD50 (dose giving 50% lethality) test for oral toxicity and the Draize tests for eye or skin irritancy are widely considered to cause unacceptable suffering to laboratory animals, they are still widely used and the development of non-animal alternatives has been slow, to say the least. However, the implementation of both the 7th Amendment to the European Union (EU) Cosmetic Ingredient Directive and the Registration, Evaluation, Authorisation and restriction of Chemicals (REACH) regulations during the early 2000s has provided a strong stimulus for further developments.

There is an acute need for this to be reflected in a paradigm shift in the field of toxicology to take advantage of the new opportunities offered by modern developments in the life sciences, including new in vitro models, alternative whole organism (non-mammalian) models and the exploitation of ‘omics methods, high throughput screening (HTS) technologies and molecular imaging technologies.2

1.2 Tissues and their maintenance

Tissues are made up of cells of various types plus the extracellular space which surrounds them. The extracellular space is filled with extracellular matrix, the proportion and structure of which depends on the tissue type. Epithelia, for example, consist mainly of sheets of epithelial cells with very little extracellular matrix whereas connective tissue contains few cells and a lot of extracellular matrix. The proteins of the extracellular matrix are linked to cytoskeletal proteins through the plasma membrane and are able to influence cell development, migration, proliferation, shape and function.

All tissues have certain basic requirements including mechanical strength, access to nutrients and removal of waste, connection to the nervous system, removal of debris and protection against infection. Specialised (differentiated) cells provide these and other functions.

During the process of embryonic development, the fertilised ovum proliferates and the resulting daughter cells differentiate to form three germ layers:

The endoderm gives rise to the epithelia of the gut and its associated organs (lung, liver and pancreas).

The ectoderm gives rise to the outer surface epithelia (epidermis, buccal epithelium and outer cervical epithelium) and neuroectodermal tissues.

The mesoderm gives rise to the embryonic mesenchyme and thence to the connective tissue and supporting tissues including bone, cartilage, muscle, vascular tissue and haematopoietic system.

The products of this process are the various differentiated tissues of the body. Even when removed from their normal environment, differentiated cells retain their specialised characteristics; for example, glandular cells still secrete mucin, fibroblasts still make extracellular matrix and macrophages still carry out phagocytosis. Differentiated cells can still respond to the environment and some cell types can adapt quite dramatically: for example, fibroblasts can convert into cartilage cells, liver cells can express different enzymes and mammary cells can switch milk proteins on or off. Some cells, however, are terminally differentiated, having become so specialised that they have lost the ability to divide.

1.2.1 Stem cells

Terminally differentiated tissues are maintained by stem cells, precursors which are not themselves differentiated but are committed to produce a particular type of terminally differentiated cell. A stem cell can be defined as ‘a cell which can proliferate either symmetrically or asymmetrically in response to an appropriate external signal’;3 in other words, under one set of circumstances it will divide to produce two stem cells and under other circumstances it will divide to generate one stem cell and one progenitor cell which can give rise to a differentiated cell lineage. The signals to which stem cells can respond include growth factors, levels of oxygen and antioxidants and growth substrates (e.g. feeder layers, extracellular matrix).

Stem cells can divide without limit and on division the daughter cells have a choice either to remain as a stem cell or embark on terminal differentiation. The final differentiated state of the majority of stem cells is pre-determined (e.g. muscle satellite cell, spermatogonium), although some stem cells are pluripotent (can differentiate into many cell types). Organ-specific stem cells have two defining properties, the ability to self-renew and the potential to differentiate into organ-specific cell types. The various types of stem cells have different potencies (i.e. abilities to generate different classes of progeny):

A totipotent stem cell

can generate an entire new organism. The definitive totipotent stem cell is the fertilised egg; following implantation, the totipotent fertilised egg becomes committed to form an embryonic pluripotent stem cell.

A pluripotent stem cell

can give rise to any other type of cell but not to an entire new organism. Pluripotent cells give rise to committed progenitor cells which can only mature into one type of cell (i.e. each one is unipotent) and this maturation process involves differentiation, which is controlled by growth factors and the surrounding environment.

Multi-potent stem cells

can produce a limited number of cell types and are committed to become part of a particular organ. They give rise to lineages of progenitor cells.

Progenitor cells

are committed to a particular lineage (e.g. the haematopoietic system) and give rise to terminally differentiated cells, which do not divide further.

1.3 Tissue damage

Living tissues are constantly exposed to environmental changes to which they respond with modifications of metabolism and growth.

Primary

(

direct

)

injury

involves an interaction between the chemicals and the components of the cell. Toxic cell injury requires high concentrations of toxic compounds and, in some cases, metabolic activation. It may involve membrane damage (e.g. lipid peroxidation induced by carbon tetrachloride in the liver).

Secondary (indirect) injury

involves changes in the cellular environment (e.g. oxygen tension, nutrient supply, hormone levels).

1.3.1 Consequences of tissue injury

The primary responses following tissue damage due to an injury are cell death and acute inflammation. The pathological stimuli responsible may be endogenous (e.g. hormones, autoimmunity, anoxia) or exogenous (e.g. radiation, drugs/chemicals, infections, mechanical trauma, heat or nutritional imbalances). The pathological changes observed following a toxic insult give an indication of the vulnerability of certain organ systems, and their nature and severity may give an insight into the toxicity of the compound. However, pathological changes, as revealed by microscopy, do not necessarily provide information about the sub-cellular and molecular processes involved.

The long-term consequences of injury depend on the ability of the tissue to regenerate and on whether the damaging agent persists. They include regeneration, healing by repair and chronic inflammation. The final outcome may be restoration (complete healing with full functionality) or fibrosis/scar formation.4

Following an episode of tissue damage, the following may occur:

Full regeneration: an optimal response, but only occurs in the liver in higher organisms.

Removal/repair of necrotic tissue leading to restitution or fibrosis (scar formation).

Alterations to necrotic tissue (e.g. calcification).

Acute and chronic inflammation

Acute inflammation is the commonest early response to tissue damage and destruction. The classical clinical indications of acute inflammation are rubor (redness), calor (heat), dolor (pain), tumor (swelling) and loss of function. If the injury is not too severe and the damaging agent has been removed, this will rapidly subside and the tissue will start to heal itself, either by restoration or by scar formation. Restoration occurs when there is minimal damage to the tissue architecture and comprises restoration of the normal structure and function of the tissue without forming a scar. It requires the supporting stroma to be intact and the damaged cells must be able to regenerate as in, for example, liver regeneration following acute liver damage. Regeneration depends on the ability of cells to divide, which means that it usually involves stem cells such as those in the gastrointestinal (GI) tract, urinary tract, skin, lymphoid tissue and the haemopoietic system. Cells such as hepatocytes which can come out of quiescence are also able to regenerate, but terminally differentiated cells such as cardiac myocytes and neurons cannot regenerate.

If, however, the damaging agent persists, the tissue will become chronically inflamed. In chronic inflammation, the processes of necrosis, organisation and repair all occur simultaneously. Chronic inflammation occurs in situations such as long-term alcohol abuse, where the ability of the liver to restore itself is overwhelmed by continual exposure to alcohol and progresses through chronic inflammation to fibrosis, cirrhosis and ultimately liver failure. The macrophage, which arises as a result of monocyte differentiation in response to interferon γ, is the main effector cell in chronic inflammation. Activated macrophages are also called epithelioid cells and can fuse to form multi-nucleate histiocyte giant cells, which have both phagocytic and secretory roles in chronic inflammation.

Restoration

Restoration occurs when there has been minimal damage to the tissue architecture and cells can re-grow. The end result of this process is restoration of normal tissue structure and function without scarring. In order for this to occur the acute inflammatory response must be terminated appropriately. The support stroma must remain intact and the damaged cells must be able to regenerate. This process is most clearly seen in response to acute liver damage (e.g. two-thirds partial hepatectomy), when the full mass and function of the liver is restored within a few days. The ability of a tissue to regenerate depends on the ability of its cells to divide, so this process is dependent upon the presence of stem cells (e.g. in the GI tract, urinary tract, skin, lymphoid tissue and haematopoietic system) or cells which can come out of quiescence (e.g. liver, kidney). Terminally differentiated cells (e.g. cardiac myocytes, neurones) cannot regenerate, so tissues made up of these cell types are particularly vulnerable to injury.

Scar formation

If a tissue is too severely damaged for restoration to be possible, healing can occur by means of organisation and repair, leading to the formation of a scar. Macrophages phagocytose dead tissue and inflammatory exudate and existing capillaries bud into the damaged area leading to the formation of vascular granulation tissue (organisation). Proliferation of fibroblasts within this tissue causes it to develop into fibrovascular granulation tissue, which gradually fills with collagen secreted by the fibroblasts to form a collagenous scar (repair). This process can be impaired by inadequate nutrition, ischaemia , infection, disease (e.g. diabetes) and the presence of foreign material.

1.3.2 Reversible changes in cells and tissues

Each tissue is an intricate mixture of different cell types and this organisation is maintained even though individual cells are constantly dying and being replaced. An appropriate balance between cell growth and cell death is therefore essential for the maintenance of homeostasis. An excess of cell growth over cell death leads to disorders of cell accumulation (e.g. cancer) and insufficient cell growth combined with excessive cell death leads to disorders of cell loss (toxicity/atrophy). Changes in cellular growth patterns may involve changes in the size of cells or in their number. Alterations in the differentiation state of cells (dedifferentiation or metaplasia) may also occur.

Xenobiotics may induce the following reversible changes in the pattern of cellular growth:

Hypertrophy

is an increase in the size of the individual cells within a tissue. It may be a physiological adaptation (e.g. hypertrophy of skeletal and cardiac muscle in athletes), an adaptive response to stress (e.g. hepatocyte hypertrophy due to enzyme induction and proliferation of the smooth endoplasmic reticulum) or a pathological effect (e.g. heart muscle in hypertension). The opposite of hypertrophy (cell shrinkage) is called

atrophy

.

Hyperplasia

is an increase in cell number leading to an increase in the volume of an organ. By definition, it can only occur in cell types which have retained the ability to divide, and is therefore not seen in terminally differentiated tissues (brain, skeletal muscle). Hyperplasia may be a physiological process (e.g. in the lactating breast) or a repair process (e.g. wound healing). It can also have pathological consequences because it is necessary for fixing (i.e. making irreversible) DNA damage, which increases the risk of neoplasia. Hyperplasia can occur in response to toxic stimuli in epithelial cells (e.g. renal tubule, pulmonary alveolar epithelium, intestinal epithelium and epidermis), blood cells, thyroid cells and bone tissue. The liver, despite having a very low rate of cell proliferation under normal circumstances, can come out of quiescence and respond with a spectacular proliferative response following chemical or physical damage. The opposite of hyperplasia (reduced cell proliferation) is called

hypoplasia

.

Metaplasia

refers to the reversible replacement of one type of adult cell by a simpler mature cell type due to abnormal differentiation of a stem cell. This is commonly an adaptation to stress (e.g. as a result of chronic inflammation). A classical example of metaplasia is the replacement of the columnar epithelium of respiratory tract (the bronchial lining) with squamous epithelium in smokers (squamous metaplasia). This is a reversible event and therefore not classified as part of the neoplastic process, but it can lead to dysplasia, which is irreversible.

1.3.3 Irreversible changes in cells and tissues

When a tissue is exposed to a sub-lethal dose of a toxin it may undergo reversible adaptive changes in order to cope with the insult. Such changes may be accompanied by morphological alterations, but these will regress if the insult is removed. However, if adaptive changes are insufficient to overcome the insult, the cell will progress to irreversible damage. The likelihood of this happening depends upon the cell type and its metabolic state at the time of injury; for example, ischaemia will cause irreversible damage after a few minutes in neurons, after 1–20 min in cardiac myocytes and after 1–2 h in epithelial cells of the renal proximal tubule. Severe or chronic toxic insults can therefore lead to irreversible changes in cell growth and differentiation.

Dysplasia

is an abnormal change in the arrangement and size of cells in a tissue. It can sometimes be reversed, but is generally considered to represent a point of commitment to the carcinogenic process.

Neoplasia

literally means new growth: a group of cells which are growing in an uncontrolled manner.

Anaplasia

refers to the regression of the physical characteristics of a cell towards a more primitive or undifferentiated type and is a common feature of malignant tumours.

1.4 Tissue responses to injury

The main targets for damage within the cell are the cell membrane, mitochondria, cytoskeleton and DNA. The biochemical mechanisms involved include ATP loss, release of calcium into the cytoplasm, reactive oxygen metabolites, structural damage to membranes and cytoskeleton and DNA damage which can be lethal or lead to mutations.

1.4.1 Oxidative stress

Oxidative stress underlies a vast number of human diseases as well as mechanisms of toxicity of drugs and chemicals; in addition, exposure to environmental chemicals can cause a variety of human diseases by mechanisms which involve oxidative stress.

Oxidative stress has been implicated in the toxicity of a plethora of drugs and environmental chemicals. One of the key intracellular molecules involved in this process is the tripeptide glutathione (γ-glutamyl-cysteinyl-glycine (GSH) (Figure 1.2). Glutathione is the most abundant low-molecular-weight thiol found within cells; by cycling between its reduced state (GSH) and its oxidised state (glutathione disulfide) it helps to maintain the appropriate redox status within the cell. Many inducers of oxidative stress exert their toxic effects by causing GSH depletion as a consequence of generation of reactive oxygen species, which arise from a variety of sources, being either endogenously generated or produced by environmental agents such as xenobiotics, UV irradiation and infectious organisms.5 In the event that a cell produces more reactive oxygen species than can be detoxified the result is DNA damage, lipid peroxidation and cell death. The consequence of this is an acute or a chronic disease.

Figure 1.2Structure of glutathione

The three major types of reactive oxygen species are as follows:

Superoxide anion radical (O

2

−

)

, which is present constitutively in cells because of leakage from the mitochondrial respiratory chain.

Hydrogen peroxide (H

2

O

2

)

, resulting from the dismutation of O

2

−.

or directly from the action of oxidase enzymes.

Hydroxyl radical (

⋅

OH)

, a highly reactive species that can modify purine and pyrimidine bases and cause DNA strand breaks resulting in DNA damage.

Reactive oxygen species are natural by-products of cellular metabolism, and oxidative stress is tightly regulated by the balance between their production and removal. Stress response pathways have evolved to protect cells against oxidative stress and environmental challenge, as well as to repair damage. Indeed, all organisms have enzymes which can scavenge superoxide and H2O2 (Imlay, 2008). Oxidative stress is a very complex problem because of the number of different pathways which exist in order to protect against different oxidants. In mammalian cells, for example, the enzymes involved include superoxide dismutases (SODs), catalase, glutathione S-transferases (GSTs), glutathione peroxidase, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase, haem oxygenase-1 (HO-1), dual-specificity phosphatases, thioredoxin and peroxiredoxins.

The main intracellular site where reactive oxygen species are generated is the mitochondrion, and mitochondrial energy metabolism is quantitatively the most important source of reactive oxygen species in the majority of eukaryotic cell types (Kowaltowski et al., 2009; Khansari et al., 2009; Murphy, 2009; Kagan et al., 2009). The production of reactive oxygen species in mitochondria is a normal consequence of respiration, but the different species generated during this process can differ markedly in their reactivity and lifetime; for example, hydroxyl radical reacts almost instantaneously with adjacent molecules whereas semiquinones may be stable for days, weeks or months (Pryor et al., 2006). In fact, mitochondria can produce reactive oxygen species even under conditions of hypoxia and this may have implications for mitochondrial redox signalling (Murphy, 2009).

The primary reactive oxygen species generated by mitochondria is superoxide, which is produced by one-electron reduction of O2 and metabolised by SOD in the inter-membrane space. The resulting H2O2 is relatively unreactive; however, if it is not metabolised, it may go on to form hydroxyl radical via a Fenton reaction (Kowaltowski et al., 2009; Pryor et al., 2006). If there is a lack of balance between reactive oxygen species generation and antioxidant defence mechanisms, reactive oxygen species can leak from the mitochondrion causing damage to cellular targets, including cell membrane fatty acids (forming lipid peroxides), cellular proteins (damaged proteins may accumulate up to toxic levels causing cell death) and DNA (causing DNA strand breaks and deletions). The consequent damage has a number of sequelae including modulation of survival signalling molecules, triggering of cell death pathways and production of proinflammatory cytokines and chemokines (Khansari et al., 2009; Roberts et al., 2009; Pan et al., 2009).

Oxidative stress and chronic inflammation are characteristic of a wide variety of human diseases (Brenneisen et al., 2005), but it is often difficult to determine whether oxidative stress is a primary cause of cell death or a physiological consequence of the induction of cell death pathways. This has led to the identification of a ‘growing need for simple, convenient, and reliable markers for the assessment both in vitro and in vivo of the metabolic/oxidative distress and of its modulation…[by]…pharmaceutical products’ (D'Alessandro et al., 2011).

Oxidative stress also plays a role in cellular senescence and cancer. Unbalanced regulation of the production of reactive oxygen species appears to initiate cellular senescence programmes via multi-faceted mechanisms including the direct induction of mutations (Pan et al., 2009), and the altered metabolic state of cancer cells (associated with aerobic glycolysis) makes them particularly susceptible to reactive oxygen species damage linked to the accumulation of mutations (D'Alessandro et al., 2011). Indeed, the interaction between reactive oxygen species and cellular senescence has been suggested as a target for cancer therapy (Pan et al., 2009).

The transcription factor Nrf2 plays a key role in cellular responses to oxidative stress.6 Under normal conditions, Nrf2 is located in the cytoplasm and is bound to the accessory protein Keap1, which targets Nrf2 for proteasome-mediated degradation. Under conditions of oxidative stress, however, Keap1 loses its ability to bind Nrf2, which is then able to translocate to the nucleus and bind to the antioxidant response elements in the 5′ regulatory regions of target genes.

Example: Oxidative stress and cardiovascular disease

Oxidative stress is known to play a role in the aetiology of cardiovascular diseases (atherosclerosis, coronary artery disease and myocardial infarction) as well as stroke, dementia, Parkinson's disease and cancer. Excessive oxidative stress and chronic inflammation are characteristic features of cardiovascular disease, which features increased production of reactive oxygen species, compromised antioxidant defences (e.g. GSH depletion) and increased circulating levels of proinflammatory cytokines (Lee et al., 2011). Oxidative stress may be involved in the pathogenesis of cardiovascular disease, and the Nrf2 pathway has been implicated in this process, especially in the sedentary elderly. Proposed strategies for prevention range from lifestyle changes including dietary supplementation (increased consumption of broccoli, curcumin) and increased exercise to pharmaceutical interventions such as prophylactic treatment with the ubiquitin-proteasome inhibitor MG132, which may protect cardiomyocytes against oxidative stress via Nrf2-mediated up-regulation of antioxidant genes.

1.4.2 Necrosis and apoptosis

The two main mechanisms by which cells can die are necrosis and apoptosis (also known as programmed cell death).7 If repair mechanisms and changes in gene expression are insufficient to allow the cell to cope with a toxic insult, the consequence is cell death by one of these two mechanisms. Many of the key players in apoptosis have now been identified and it turns out that many of the genes which control cell growth are also involved in apoptosis, allowing the processes of cell division and death to be tightly coregulated. This is crucial for the maintenance of homeostasis; indeed, evasion of apoptosis signals is a hallmark of human cancer, and the genes involved in regulation of the cell cycle and apoptosis represent potential molecular targets for the control of human diseases where inappropriate apoptosis is prominent (e.g. cancer and degenerative disorders).

The occurrence of necrosis is not determined by factors that are intrinsic to the cell but by changes in the cellular environment. In contrast, apoptosis is a physiological process and plays a role in the maintenance of tissue homeostasis. Apoptosis is an active process leading to cell death via an ordered sequence of events. Its morphologic appearance is very different from that of necrosis (Figure 1.3). Necrosis involves the swelling and rupture of the injured cells, whereas apoptosis involves a specific series of events that leads to the dismantling of the internal contents of the cell. Apoptosis is a normal part of development, being involved in processes including

Figure 1.3Structural changes of cells in necrosis and apoptosis (Source: Goodlett and Horn ({); figure 1. Reproduced with permission of Charles Goodlett)

resorption of the tail during metamorphosis of a tadpole;

removal of the webbing between the digits of hands and feet during mammalian embryonic development.

Necrosis is an unregulated mechanism involving dilation of the endoplasmic reticulum, dissolution of lysosomes and ribosomes, mitochondrial swelling and an increase in cell volume. Necrosis does not, however, involve gross changes in chromatin structure. In contrast, during apoptosis, the cytoplasmic organelles are well preserved and there is actual shrinkage of the cell and nucleus. The biochemical changes which occur during apoptosis include a moderate increase in intracellular [Ca2+] and total shutdown of protein and RNA synthesis. Following condensation of the nuclear chromatin, activation of Ca2+/Mg2+ endonuclease produces distinctive chromatin fragments which may be viewed as a ladder on an agarose gel (Hooker et al., 2012).

Apoptosis is characterised by distinctive morphological changes including decreased cell volume, increased cell density, compaction of cytoplasmic organelles (except for the mitochondria, which remain morphologically normal) and dilation of the endoplasmic reticulum. Nucleolar disintegration, budding and separation of nucleus and cytoplasm into multiple small membrane-bound apoptotic bodies occurs, followed by progressive degeneration of residual nuclear and cytoplasmic structures and condensation of chromatin into crescent-shaped caps at the cell periphery.

A key process in apoptosis is the activation of a series of highly conserved cysteine proteases called caspases which act as common effector molecules in various forms of cell death. Caspases are produced as inactive precursors called procaspases which can be activated either by oligomerisation (initiator caspases e.g. caspases 8 and 9) or by proteolytic cleavage to create active enzymes in a proteolytic cascade (effector caspases e.g. caspase 3). Once they are activated, caspases cleave other proteins within cells resulting in efficient and precise killing of the cell in which they are activated.

Apoptosis can be initiated via two different pathways, the extrinsic and intrinsic pathways, each of which involves the activation of specific caspases:8

The extrinsic pathway

is triggered by the binding of so-called death signals such as tumour necrosis factor (TNF), Fas ligand or TNF-related apoptosis-inducing ligand to the corresponding receptors (TNFR, Fas receptor, DR4/5). This triggers the recruitment of adaptor proteins and activation of the initiator caspases 8 and 9. Interestingly, caspase 8 mutations have been detected in some cancers and can act as dominant negative mutations, blocking apoptotic cell death and having a profound impact on the cancer cell's ability to undergo apoptosis (Fulda, 2009).

The intrinsic pathway

is mediated by mitochondrial damage, which leads to cytochrome c release. Cytochrome c interacts with an adaptor molecule (Apaf-1) and procaspase 9 to form a complex called an

apoptasome

leading to the activation of caspase 9. Inhibitor of Apoptosis Proteins (IAPs) inhibit caspases and procaspases and are themselves controlled by other mitochondrial proteins (Smac/DIABLO and Omi/HtrA2). This process is regulated by various factors including members of the Bcl-2 family, Bax and Bak.

Both pathways lead to the activation of so-called executioner caspases (caspase 3 and caspase 7) by caspases 8 and 9.

Apoptosis appears to be the major pathway of cell death triggered by DNA damage. It is thought to eliminate genetically damaged cells and, therefore, counteracts carcinogenesis. The genes involved in regulating this process represent potential molecular targets for the control of human diseases which feature inappropriate apoptosis (e.g. cancer and degenerative disorders).

Role of calcium in cell death

Calcium signals are responsible for the regulation of many vital cell functions; cellular Ca2+ overload or perturbation of intracellular Ca2+ compartmentalisation can cause cytotoxicity. The point of no return is thought to be when the sarcolemma can no longer bind Ca2+ and the mitochondria start to take up the excess calcium (Rasola and Bernardi, 2011). Cell death can be brought about by a loss of Ca2+ homeostatic control, but can also be triggered by more subtle changes in Ca2+ distribution within intracellular compartments.9

Normal intracellular calcium levels range between 10−7 and 10−6 M, approximately four orders of magnitude lower than in the extracellular fluid, thanks to the activity of plasma membrane Ca2+ ATPases and the Na+/Ca2+ exchanger which remove Ca2+ from the cell. This concentration gradient allows rapid influx of Ca2+ into the cell if the plasma membrane channels open, for example in response to a toxic insult. An unphysiological increase in cytosolic calcium concentration (to >10−5 M) can either cause necrosis or contribute to apoptosis by activating physiological calcium-dependent processes and cellular responses that are not normally affected by calcium. These include changes in cellular shape, blebbing (a bleb is an irregular bulge in the plasma membrane of a cell caused by localised decoupling of the cytoskeleton from the plasma membrane), changes in ionic conduction, excessive contraction/transmitted release, enzyme activation (proteases, nucleases, phospholipases, transglutaminases) and inhibition (adenylate cyclase). This is associated with a cycle of decreased ATP levels due to activation of the Ca2+ pump, subsequently causing reduced functioning of the pump itself due to insufficiency of ATP.

1.4.3 Neoplasia

The term neoplasia refers to the development of a collection of cells which grow in an uncontrolled manner, usually resulting in the formation of a tumour. Neoplasia may be benign or malignant and its causes include foreign chemicals (xenobiotics), hormones, viruses, inherited genes, radiation and physical agents (e.g. inert implanted plastic or metal film).

The cellular changes observed during neoplasia include hypertrophy, hyperplasia and alterations in differentiation which may be associated with changes in the histological appearance of cells. It is important to note that the terms cancer and tumour are not synonymous: the word tumour applies to any readily defined mass of tissue distinct from normal tissue but not necessarily made up of abnormal cells; in other words, the term tumour just means a lump. The definition of a benign tumour is that it is restricted to its site of origin; a malignant tumour is one which is metastatic.

Cancer is defined as a heritably altered, relatively autonomous growth of tissue which occurs when a cell or group of cells begins to multiply more rapidly than normal leading to the development of a malignant tumour: it is, by definition, a disease of aberrant cells and is a consequence of both uncontrolled cell division and the loss of normal patterns of differentiation leading to the autonomous growth of these abnormal cells. A true cancer is a malignant neoplasm made up of morphologically transformed, autonomously replicating (malignant) cells. The characteristics of morphologically transformed cells include uncontrolled cell division, loss of contact inhibition, defective cell cycle control, lack of balance between cell division and cell death, breakdowns in cell–cell communication and dedifferentiation.

Metastasis occurs when individual cells or small groups of cells break away from the primary tumour and migrate to other sites within the body, where they begin to grow into secondary tumours (metastases). Malignant tumours tend to be locally invasive, fast-growing and anaplastic whereas benign tumours are usually encapsulated and are generally slow growing.

1.4.4 The initiation–promotion paradigm

Carcinogenesis is defined as the process by which normal cells are transformed into cancer cells.10 A carcinogen, therefore, is any substance that causes cancer. The link between cancer and exposure to specific exogenous substances was first identified in the 18th century, when in 1761 John Hill noticed an increase in nasal cancer associated with long-term use of snuff and in 1775 Percival Pott observed that chimney sweeps often suffered from scrotal cancer. The role of industrial exposure to aromatic amines in the induction of bladder cancer was demonstrated in 1895, when Rehn reported an increase in bladder cell tumours in workers in the dye and rubber industries.11

Some cancers arise as a result of genetic susceptibility or alterations in homeostasis (e.g. hormonal changes) while others are caused by exposure to carcinogenic substances. Experimentally, a compound is considered to be carcinogenic if its administration to laboratory animals induces a statistically significant increase in the incidence of one or more histological types of neoplasia compared with animals in the control group, which are not exposed to the substance.

According to the European Chemicals Agency (ECHA)12:

Chemicals are defined as carcinogenic if they induce tumors, increase tumor incidence and/or malignancy, or shorten the time to tumor occurrence. Benign tumors that are considered to have the potential to progress to malignant tumors are generally considered along with malignant tumors. Chemicals can induce cancer by any route of exposure (e.g. when inhaled, ingested, applied to the skin, or injected), but carcinogenic potential and potency may depend on the conditions of exposure (e.g., route, level, pattern, and duration of exposure).

The concept of cancer as a multi-step process was first proposed by Armitage and Doll in 1954 and extended by Moolgavkar and Knudsen in 1981 (Armitage and Doll, 1954, Moolgavkar and Knudson, 1981). The idea that the process of carcinogenesis could be divided into discrete stages arose when it was observed that tumours could be induced by painting mouse skin with polycyclic aromatic hydrocarbons (PAHs) then with croton oil, but not if these substances were applied in the reverse order. This is now known as the initiation–promotion paradigm (Figure 1.4) and reflects the fact that the carcinogenic process involves both genetic damage and changes in the growth of cells and tissues. Cell replication is necessary for fixing DNA damage (i.e. making it irreversible), therefore increasing the risk of neoplasia, and hyperplasia is central to the promotion phase during which tumour growth occurs.

Figure 1.4 The initiation–promotion–progression paradigm

Initiation

A cancer originates from an initiated cell which multiplies clonally, escapes apoptosis and accumulates a collection of genetic and/or epigenetic alterations which allow it to escape from normal control mechanisms. Mutagenesis, the process by which mutations occur, is a key aspect of carcinogenesis. A mutation is a change in the sequence of bases in a DNA molecule, and any insult which causes a mutation is known as a mutagen. Carcinogenesis almost always involves mutagenesis, and many carcinogens are also mutagens, but it is a complex process involving defects in many different biological control mechanisms so mutagenesis is not, in and of itself, sufficient to generate cancer.

Initiation involves the acquisition of irreversible genetic alterations as a consequence of mutation of one or more key genes. This may occur spontaneously or as a result of DNA damage caused by chemical or physical damage (e.g. due to ionising radiation). Initiation gives rise to a single initiated cell which is phenotypically indistinguishable from the surrounding normal cells but is predisposed to give rise to a neoplastic lesion. It is a rapid and irreversible process and the characteristics of initiation are transmitted to daughter cells when the initiated cell divides. The ability of chemicals which induce DNA damage (genotoxicity) to cause initiation and ultimately tumour development has led to an intense focus on the identification and regulation of genotoxic chemicals.

Promotion

Promotion involves an increase in the number of initiated cells to produce a benign tumour. Increases in cell number can result from either increased cell proliferation or reduced cell death, so increased cell division and evasion of apoptosis are both considered to be critical in the promotion process. Promotion is believed not to require further DNA damage.

The importance of cell proliferation in the promotion process is twofold: (i) prior to DNA replication, DNA damage can be repaired, but proliferating cells have less time to repair DNA damage, and DNA replication during cell division can fix this damage, making it permanent and irreversible. This can be particularly important if the DNA damage occurs in a stem cell which can give rise to multiple progeny, and pluripotent stem cells are now believed to be the key target cells in carcinogenesis. Indeed, cells which are fully differentiated or committed to differentiation are unlikely to give rise to tumours as they are already programmed to die. (ii) Cell proliferation converts an individual initiated cell to a detectable lesion (a preneoplastic focus and subsequently a benign tumour).

By definition, the promotion process involves hyperplasia which may be augmentative (proliferation over and above that which is required for tissue maintenance) or regenerative (proliferation which is necessary for tissue repair) (Figure 1.5). Tumour promoters contribute towards the fixation of DNA damage in the form of mutations, enhance epigenetic processes which alter gene expression and cause changes in cellular growth control. Some promoters are tissue specific whereas others can act on more than one tissue type.

Figure 1.5 Augmentative and regenerative hyperplasia

Long-term treatment with certain promoting agents can induce neoplasia, apparently without the need for initiation, the process known as non-genotoxic carcinogenesis. Some investigators believe that this indicates a genotoxic effect which has been missed by conventional genotoxicity assays, but it is equally possible that non-genotoxic carcinogenesis occurs as the result of promotion of pre-existing, spontaneously initiated cells.

The cell cycle

During the cell cycle, a cell must replicate its DNA and duplicate its contents, then divide in two. In the case of unicellular organisms each cell division cycle makes a new organism whereas in the case of multi-cellular organisms many rounds of cell division are required to make a new organism. Cell division is also needed in the adult body in order to replace cells which have been lost as a consequence of apoptosis or necrosis.

Eukaryotic cells divide and grow at different rates. The eukaryotic cell cycle can be resolved into four phases (Figure 1.6):

Figure 1.6 The cell cycle and cell division. (a) The phases of the cell cycle (b) DNA content of cells at different stages of the cell cycle (source: Dr Jerry Styles. Reproduced with permission of Dr Jerry Styles)

G1 phase (growth of the cell)

S-phase (DNA replication)

G2 phase (growth and error checking)

M phase (cell divides)

Cells can leave the cell cycle during and go into quiescence (G0 phase); indeed, some cell types (e.g. hepatocytes) spend most of their time in G0. During M phase the cell divides (cytokinesis) and its DNA is distributed evenly between the daughter cells.

The cell cycle must be controlled to allow time for synthesis of new proteins, replication of DNA and checking for DNA damage. Checkpoints are required in order to ensure that each process is complete before the next one starts. If this does not happen, delays and interruptions occur; this is what happens in cancer. At a cell cycle checkpoint the cell cycle pauses until the appropriate feedback signals have been received. The key checkpoints in the eukaryotic cell cycle are

start (in G1, just before the beginning of S-phase), when the cell becomes committed to the cell cycle

entry to M phase (at the end of G2)

exit from M phase

Cell cycle checkpoints are tightly controlled and monitored by proteins which include the cyclins and cyclin-dependent kinases (CDKs). The mitotic cyclins bind to CDK molecules during G2, permitting entry to mitosis, while the G1 cyclins bind to CDK molecules during G1 and are required for entry into S-phase. The CDKs act by phosphorylating specific proteins on serine or threonine residues.

As a consequence of the importance of cell cycle control, increases in cell replication are indicative of tumour promotion. Changes in cell replication may be detected by a variety of methods including:

Incorporation of

3

H- or

14

C-labelled thymidine into cellular DNA (detected by autoradiography and scintillation counting, respectively)

Incorporation of bromodeoxyuridine (BrdU) into cellular DNA

Up-regulation of proliferating cell nuclear antigen, an endogenous protein whose expression increases during S-phase

These techniques can, in theory, be applied to any tissue but it is easier to detect increased cell proliferation over background in tissues with a low background rate of replication, such as the liver.

Under normal circumstances adjacent cells communicate with each other to ensure that proliferation is properly controlled. Cell–cell contact is a key aspect of this process. When non-transformed adherent cells are grown in culture they become arrested in G0 when they are touching each other on all sides, thus forming a confluent monolayer. This process, which is known as contact inhibition, is controlled by cyclin-dependent kinases and mediated via cell membrane proteins such as N-cadherin.

Cell–cell adhesion in epithelial cells is mediated by integrins, transmembrane proteoglycans and calcium-dependent cell–cell adhesion molecules called cadherins. In particular, the calcium-dependent homophilic interactions of E-cadherin, which induce contact inhibition, maintain the epithelial cell phenotype and prevent migration. E-cadherin communicates with the cellular interior by catenin-mediated interactions with the actin cytoskeleton. One of the functions of E-cadherin is to sequester β-catenin, reducing the levels observed in the cytoplasm. Cytoplasmic levels of β-catenin are also regulated by proteolysis. Accumulation of β-catenin occurs physiologically during embryonic development and pathologically during tumorigenesis. In contrast, α-catenin, which associates with desmosomal cadherins as well as E-cadherin, is believed to have an inhibitory effect on processes associated with tumour development.

Disruption of cell–cell communication facilitates the dedifferentiation of cells to a more mesenchymal phenotype. This is a normal process during embryonic development but occurs pathologically as benign tumours progress towards a more invasive/metastatic phenotype. Loss of expression of E-cadherin and β-catenin is a key aspect of this process, which is known as the epithelial–mesenchymal transition and is associated with the induction of proliferative, mesenchymal and invasive genes leading towards a more malignant phenotype.

Preneoplastic lesions

A preneoplastic lesion is a recognisable group of cells which has undergone the early stages of the carcinogenic process but is not yet fully committed to forming a tumour and can, under certain circumstances, regress to form apparently normal tissue. For example, a well-characterised series of phenotypic changes occurs during the early stages of rodent liver carcinogenesis in vivo. The methods used to detect these changes often involve the use of so-called initiation-promotion protocols (Pitot, 2007),13 in which animals are treated with a single dose of a potent genotoxic compound such as 2-acetylaminofluorene (2-AAF) (initiation) followed by regular treatment with a compound such as phenobarbital (PB) which induces cell proliferation (promotion). In some cases two-thirds partial hepatectomy is used to stimulate cell proliferation because the rodent liver can survive removal of two-thirds of the liver. Following this surgery it undergoes a period of rapid growth as a result of which the full weight of the liver is restored in less than a week.

Other early markers of tumour development include preneoplastic enzyme changes (e.g. up-regulation of γ-glutamyl transpeptidase (γGT) and glutathione-S-transferases (GSTs) in rodent liver), development of morphologically altered preneoplastic foci, early changes in ploidy and nuclearity, oncogene activation, activation of growth factors and altered cell–cell communication. All these can be seen to be logically related to the acquisition of cancerous properties by the cells: up-regulation of detoxifying enzymes may confer resistance to the cytotoxic effects of the carcinogen, morphological changes are diagnostic of the transformed phenotype, oncogene activation and growth factor up-regulation enhance proliferation and loss of cell–cell communication allows the transformed cell to ignore growth retarding signals from surrounding cells.

Examples of types of preneoplastic lesions which can arise in animals during chemical carcinogenesis include altered hepatic foci, prepapillomas of the skin and aberrant crypt foci in the colon. Among the best-characterised preneoplastic lesions of toxicological relevance are hepatic preneoplastic foci (Figure 1.7), which develop in rodent liver in response to treatment with hepatic carcinogens.14 They are easy to identify, being characterised by an altered pattern of expression of various markers which can be detected by means of immunohistochemistry. The most commonly used markers are GST-P and gamma glutamyl transpeptidase (γGT); others include the diubiquitin-like molecule FAT10, α2-macroglobulin, fatty acid synthase and α-fetoprotein. One of the most useful of these markers is FAT10, which is also overexpressed in 70–90% of human hepatocellular carcinomas (HCCs). Single FAT10-positive cells appear early in the carcinogenic process, possibly as a consequence of epigenetic alterations. They have a growth advantage compared with normal hepatocytes, and are thought to represent a subpopulation of initiated cells which are resistant to cytotoxicity in the presence of a strong growth stimulus (French, 2010).

Figure 1.7 Photomicrograph of a GST-P positive preneoplastic nodule. This example is taken from a male Wister rat subjected to an initiation-promotion regime as follows: injected intraperitoneally with a single dose of diethylnitrosamine (DEN) dissolved in water (100 mg kg body weight) on day 1, then dosed with 2-acetylaminofluorene (2-AAF) (20 mg kg body weight p.o.) in dimethyl sulfoxide/carboxymethylcellulose on days 7, 8 and 9, and 70% partial hepatectomy on day 10. Transverse histological sections (6 µm thick) were stained using a rabbit primary antibody against the placental form of glutathione S-transferase (GST-P, 1:250). The GST-P positive hepatocytes are stained brown while normal hepatocytes are blue. (Bar = 100 µm, haematoxylin counterstain). (source: Gonzalez de Mejia et al. ({); figure 6. Reproduced with permission of Elsevier)

Studies on preneoplastic stem cells suggest that, at least in rats, susceptibility to the early stages of hepatocarcinogenesis is a consequence of the activation of several low penetrance genes and a single predominant susceptibility gene (French, 2010). Epigenetic phenomena also play a role; for example, hypomethylation is observed in the hepatocytes of susceptible F344 rats.

Preneoplastic lesions in humans

If clearly defined and easy to identify, preneoplastic lesions can be of great value both to the toxicologist and to the oncologist. Because they occur early in the carcinogenic process, the toxicologist can use them as an early indicator of potential future tumour development, while the oncologist can use them for early diagnosis and treatment. For example, dysplastic foci (<1 mm diameter) and nodules (1 mm—1 cm diameter) have been identified as preneoplastic lesions in human liver. These may have either a large cell or small cell morphology; large cell dysplasia is not a precursor for HCC whereas small cell dysplasia is considered to be a preneoplastic lesion (French, 2010).

Identification of preneoplastic lesions can also play a key role in cancer screening. Cancer causes approximately 840 000 deaths annually in the EU and over 1 300 000 new cases are reported each year. In many cases, by the time a tumour causes symptoms, it is too advanced to treat, but if tumours can be detected when they are very small, or better still, people who have not yet developed a tumour but have a high risk of doing so can be identified; the chances of successful treatment are much improved. As well as saving lives, a well-organised screening programme can save the health services an immense amount of money and time. In order to establish a screening programme, two key requirements must be met: There must be a test or procedure which will detect the cancer before symptoms develop (preferably at the preneoplastic stage) and there must be evidence that treatment at this earlier stage of the disease will result in an improved outcome. The most successful screening programme to date is the one for cervical cancer.

Example: Early diagnosis and treatment: the cervical smear test

The cervical cancer screening programme depends upon the identification of preneoplastic cells in otherwise healthy women. In this programme, precancerous changes in the cervix are detected by looking for abnormal cells which arise in its lining.

Precancerous change in the cervix is called cervical intraepithelial neoplasia (CIN). In this condition, abnormal cells with large, oddly shaped nuclei are seen in the lining of the cervix. The process starts with CIN I and progresses through CIN II to CIN III. The majority of these changes will eventually revert to normal, but in a few cases they precede the development of a tumour.

When a woman goes for a smear test, cells are scraped from the cervix using a spatula, spread on a slide, stained using a method developed by Papanicolau in 1942 (the Pap test), and examined to identify any which are a strange colour or have an enlarged or bizarrely shaped nucleus. If a mild abnormality (dyskaryosis) is detected, the woman will be asked to return in 3–6 months. If the changes persist, she will be referred for colposcopy. In the colposcopy procedure a gynaecologist examines the cervix directly and removes any abnormal tissue. This procedure cures the problem at the same time as confirming the diagnosis.

The problem of cervical cancer screening is that the current screening programme depends upon the detection of dyskaryotic cells by trained cytology screeners. The process is labour-intensive, expensive, subjective and prone to a significant incidence of errors. Any method giving a simple yes/no answer would be a significant improvement, especially if it lent itself to automatic sample processing and analysis.

Could CYP1B1 be the answer? Immunohistochemical analysis of the expression of a protein called CYP1B1 in human tumours and corresponding histologically normal tissues (Murray et al., 1997), suggested that this cytochrome P450 (CYP) isozyme might have a future as a tumour marker since CYP1B1 expression was detected in 122/127 tumours and 0/130 normal tissue samples.

The question that arose from this was: At what stage of tumour development is CYP1B1 expression initiated? Cervical cancer was selected for further study for the reasons outlined earlier, and the results of this study indicated that expression of CYP1B1 was detectable in cervical lesions and individual exfoliated cervical epithelial cells from a subject with CIN. The location of the staining corresponded with the area occupied by abnormal cells. In contrast with the difficulty of picking out abnormal cells in a Pap smear, individual CYP1B1 positive cells could readily be detected in smear samples from patients with CIN (Figure 1.8). Thus, CYP1B1 appeared to be a good marker for precancerous changes in the cervix. The potential advantage of this test is that it gives an unequivocal result: normal cells are blue whereas abnormal cells are brown. This test would be much easier to interpret than the Pap test, and would lend itself to automation, making screening cheaper and less labour-intensive.

Figure 1.8