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Target Organ Toxicity in Marine and
Freshwater Teleosts
 New Perspectives: Toxicology and the Environment

Target Organ Toxicity in

Marine and Freshwater
Teleosts
Volume 2—Systems
Edited by

Daniel Schlenk
Department of Environmental Sciences University of
California Riverside, California USA
William H.Benson
Gulf Ecology Division, National Health and
Environmental Effects Research Laboratory US
Environmental Protection Agency Gulf Breeze, Florida
USA

London and New York

 First published 2001
by Taylor & Francis
11 New Fetter Lane, London EC4P 4EE
Simultaneously published in the USA and Canada
by Taylor & Francis
29 West 35th Street, New York, NY 10001
Taylor & Francis is an imprint of the Taylor & Francis Group
This edition published in the Taylor & Francis e-Library, 2005.
“To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of
thousands of eBooks please go to www.eBookstore.tandf.co.uk.”
© 2001 Daniel Schlenk and William H.Benson
All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or
by any electronic, mechanical, or other means, now known or hereafter invented, including
photocopying and recording, or in any information storage or retrieval system, without permission
in writing from the publishers.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging in Publication Data

ISBN 0-203-36141-5 Master e-book ISBN

ISBN 0-203-37397-9 (Adobe eReader Format)

ISBN 0-415-24839-6 (Print Edition)
 Contents

List of contributors vii

Foreword viii
Copyright acknowledgments ix

1 General mechanisms of toxicity 1

DANIEL SCHLENK
Introduction 1
Disposition of xenobiotics 1
Chemical reactions of reactive intermediates/ultimate toxicants 7
Exceeding capacity of cellular defense mechanisms 10
Specific cellular targets 12
Targets involving repair and cellular regeneration 19
Summary 23
2 Toxic responses of the nervous system 27
RUSSELL L.CARR AND JANICE E.CHAMBERS
Introduction 27
Nervous system physiology 28
Chlorinated hydrocarbon insecticides 41
Pyrethroids 48
Anticholinesterase insecticides 54
Metals 63
Organics 66
Natural toxins 67
Conclusions 74
 v

3 Fish immunotoxicology: understanding mechanisms of action 97

CHARLES D.RICE
Introduction 97
Innate, non-specific, and first-line defenses 103
Antigen processing and presentation for specific responses 109
Fish as models in biomedical research 114
Historical approaches to immunotoxicology 115
Mechanisms of action of some immunotoxicants 116
Autoimmunity and hypersensitivity: a frontier in fish immunotoxicology 119
The neuroendocrine-immune connection 122
Summary 127
4 Neurobehavioral toxicity in fish 141
EDWARD E.LITTLE AND SANDRA K.BREWER
Introduction 141
Neural basis of behavioral toxicity 144
Toxicant impacts on higher behavioral function 162
Ecologic relevance of behavior as a predictor of contaminant effects in the natural 164
environment
Conclusions 166
5 Toxic responses of the reproductive system 177
LISA D.ARCAND-HOY AND WILLIAM H.BENSON
Introduction 177
Hypothalamus-pituitary-gonadal axis 178
Gonadal development 182
Oocyte growth and maturation 186
Modes of nutrition 188
Targets for chemical toxicity 189
Egg-level effects 191
Alteration in egg yolk nutrients 191
Assessment of reproductive effects 195
Assessment of reproductive function 195
vi

Reproductive toxicity guidelines 196

Conclusions 199

Index 205
 Contributors

Lisa D.Arcand-Hoy is at Blasland, Bouck & Lee, Inc., Syracuse, NY, USA
William H.Benson is at the Gulf Ecology Division, National Health and
Environmental Effects Research Laboratory, US Environmental Protection Agency, Gulf
Breeze, FL, USA
Sandra K.Brewer is at the Department of Biological Sciences, University of
Missouri, Columbia, MO, USA
Russell L.Carr is at the Center for Environmental Health Sciences, Mississippi State
University, MS, USA
Janice E.Chambers is at the Center for Environmental Health Sciences, Mississippi
State University, MS, USA
Edward E.Little is at the US Geologic Survey, Columbia Environmental Research
Center, Columbia, MO, USA
Charles D.Rice is at the Department of Environmental Toxicology, Clemson
University, Pendleton, SC, USA
Daniel Schlenk is at the Department of Environmental Sciences, University of
California, Riverside, CA, USA
 Foreword

The field of aquatic toxicology is increasing in importance because it is now commonly

accepted that humans cannot continue to discharge their wastes and byproducts into
streams, rivers, and oceans without having impacts on those ecosystems. As a result,
efforts are focusing on reducing wastestreams from both point and non-point sources, as
well as reducing the impacts of historic discharges through remediation and cleanup. This
has led to considerable contention around the question “How clean is clean enough?” To
answer this question aquatic toxicologists have conducted extensive research and written
many books, looking at the effects of toxicants both at comparatively simple levels of
biological organization (e.g. the biomarker approach) and less commonly at highly
complex levels of organization (population and community impacts).
The challenge has been to link these two approaches. It is comparatively easy to determine
the effects and mechanisms by which chemical contaminants affect cellular function,
largely through laboratory investigations. Field investigations can demonstrate causal
relationships between certain classes of contaminants and effects on individual organisms,
but it is extremely rare for effects of chemical contaminants to be specifically discernible at
the population or community level, especially in open marine and estuarine ecosystems.
Increasingly, we are realizing that fitness of individual organisms may need to be the level
of biological function which we need to protect in aquatic ecosystems, much as we
currently do for human health. This requires assessing organ function, and it is surprising
that there are not good reference texts for assessing organ system function in fish species
exposed to chemical contaminants. This second volume addresses that void and will be a
valuable reference to the practicing aquatic toxicologist.
Tracy K.Collier
Northwest Fisheries Science Center
 Copyright acknowledgments

The authors and publishers would like to thank the following for granting permission to
reproduce material in this work.
W.B.Saunders Co., Philadelphia, for permission to reproduce Figure 3.1, originally
published as Figure 3.1 in Tizard, I.R. 1996. Veterinary Immunology—An Introduction.
W.B.Saunders Co., Philadelphia, for permission to reproduce Figure 3.2, originally
published as Figure 11.1 in Stoskopf 1996. Fish Medicine.
W.H.Freeman and Co., New York, for permission to reproduce Figure 3.6, originally
published as Figure 10.8 in Kuby, J. 1994. Immunology, 2nd edn.
The Company of Biologists Ltd, Cambridge, UK, for permission to reproduce
Figure 5.2, originally published as Figure 1 in Satoh, N. and Egami, N. 1972. Sex
differentiation of germ cells in the teleost, Oryzias latipes, during normal embryonic
development. Journal of Embryology and Experimental Morphology 28(2):385–395.
x
 1
General mechanisms of toxicity
Daniel Schlenk

Introduction
The overall purpose of this book is to provide a reference describing specific toxicities in
various organs of teleost fish. It is important to note that any given agent may have
multiple targets, and that the concentration of the agent to which the fish is exposed can
also have dramatic effects on the target(s) for the xenobiotic in question. As many of the
mechanisms eliciting tissue damage at a particular target are shared, this introductory
chapter is intended to focus upon general cellular mechanisms of toxicity. Most of the
mechanisms that are to be discussed have been identified primarily in mammalian systems
(see Gregus and Klaassen, 1996) with limited studies performed in teleosts.
Consequently, the purpose of this chapter is to discuss potential mechanisms of cellular
toxicity that have been elucidated primarily from mammalian studies and to identify
information gaps or mechanisms that have yet to be explored in fish.

Disposition of xenobiotics
As demonstrated in Figure 1.1, xenobiotic disposition as well as interaction with a target
involves several processes, including absorption, distribution, biotransformation, and
elimination. Involvement of these processes depends not only upon the physiochemical
parameters of the xenobiotic but also upon the location and molecular constitution of the
target (i.e. receptor/protein, lipid, DNA, etc.). There are primarily three mechanisms by
which a toxicant gains access to a cellular target: direct interaction with the target;
interaction with the target following absorption; and interaction with the target following
absorption and biotransformation.

Direct interaction with target

Because fish are in direct contact with the aqueous environment, exposure occurs over the
entire body surface. Many of the mechanisms involved in direct target interactions involve
disruption of plasma membranes or direct inhibition or destruction of specific receptors
located in the plasma membrane. Putative targets may include: (1) the dermal surface of
the fish which is susceptible to non-receptor-mediated plasma membrane disruption by
2 DANIEL SCHLENK

Figure 1.1 Dispositional processes of xenobiotic fate.

caustic agents (i.e. acids/bases), leading to a variety of lesions such as loss of sensory
tubricles or ulcerations of the epidermis and/or erosion of fins; (2) extracellular receptors
involved in osmoregulation and/or gas exchange found primarily in the gill (i.e. Na+, K+-
ATPases, H+ transporters, Cl• channels)—numerous agents, including organic
surfactants or metals, may have significant interactions with these targets; (3) extracellular
receptors involved in nutrient uptake or nitrogenous waste excretion (ammonia) in the gut
or gill respectively; (4) epithelial tissue of gut or eye which may undergo general
ulceration following interaction with caustic agents and/or metal oxides (gut).

Interaction with target following absorption

Absorption of xenobiotics may occur either passively or actively. Passive mechanisms of
absorption are controlled by a number of factors, including size, lipophilicity and/or
polarity of the compound as it moves down a concentration gradient, and the thickness of
the plasma membrane. Active processes of xenobiotic absorption usually involve the
substitution, or ‘hijacking’ by a structurally similar xenobiotic, with an endogenous ligand
for a specific receptor/transporter. Such receptor-mediated processes may or may not
require energy.
Intracellular damage may occur to the tissue which directly absorbs the xenobiotic
following passage through the plasma membrane. Such intracellular damage is common in
the gill. Alternatively, the compound may be transported or distributed to other organs,
where it may cause more significant damage. For example, the absorption of a
cholinesterase-inhibiting pesticide may not cause significant damage at the site of
absorption (i.e. gill) but will affect the central nervous system following transport to the
appropriate target(s) (neuron or neuromuscular junction). Transport and distribution of a
particular agent to a target is typically implemented by the circulatory system of the
organism and is influenced by several factors:
 GENERAL MECHANISMS OF TOXICITY 3

Binding and subsequent sequestration of the agent at the site of absorption

One of the best examples of this phenomenon is the binding of organic and inorganic
agents by melanin in the skin of fish (Van Leeuwen et al., 1986). The binding to melanin
efficiently sequesters the compound in the skin, preventing its distribution to other
organs.

Binding of the agent in the circulatory system by plasma proteins (i.e. albumin
and lipoproteins)
Several studies with therapeutic agents used in fish culture have indicated that plasma
protein binding significantly alters bioavailability and distribution in certain species such as
catfish and trout (Plakas et al., 1992; Jarboe et al., 1993). Metals are also primarily bound
to blood proteins (Roesijadi and Robinson, 1994). The specific form of plasma protein
may also be critical as some tissues have albumin-specific receptors and may preferentially
accumulate albumin-bound xenobiotics (Heath, 1995).

Specialized barriers
As in mammals, blood-brain barriers have been identified in several species of fish through
dye uptake studies and histologically (Castejon, 1983; Lane, 1991). In addition, evidence
for a blood-testes barrier has also been observed. Consequently, distribution to these
particular tissues would be diminished unless the xenobiotic is highly lipophilic or can
mimic endogenous ligands taken up by active transport mechanisms within those
particular systems.

Storage sites
In contrast to intracellular sequestration at the site of absorption, organic lipophilic agents
may be transported to specific storage sites that are abundant in lipid, such as fat bodies.
Alternatively, certain metals have the propensity to accumulate in bone. Although lipid
content in fish has been shown to have considerable impact on the accumulation of organic
agents, the role of bone metabolism has not been extensively examined.

Blood flow to and from potential target systems

Because most gut-absorbed material is transported via the portal venous system to the liver,
this tissue is generally considered to be the primary target for gut-absorbed chemicals.
Chemicals that are absorbed through the gill or skin would have a different primary
circulatory route from dietary chemicals. Blood transport from the gill would suggest that
the brain or, possibly, the kidney (depending on the species) may receive a more
significant blood flow and thus may be more susceptible targets.
4 DANIEL SCHLENK

Elimination away from the target

As mentioned above, cells possess numerous membrane transporters that are not only
important for cellular absorption but also involved in cellular excretion of absorbed
compounds. P-Glycoprotein homologs, also known as the multixenobiotic resistance
mechanism (MXR), are often found in cells undergoing neoplastic transformation (Juliano
and Ling, 1976). The MXR non-specifically exports a host of organic and inorganic
toxicants out of cells following absorption. The MXR has been observed in tumors and in
damaged tissues from several fish species (Kleinow et al., 1996; Kohler et al., 1998;
Cooper et al., 1999). Additional mechanisms to enhance elimination from the animal
through the urine or feces involve various biotransformation processes discussed below.

Interaction with target following biotransformation

Bioactivation versus detoxification

Most organic xenobiotics are absorbed in fish and other organisms as relatively non-polar
lipophilic chemicals. In an attempt to enhance the elimination of chemicals of this nature,
the cell utilizes a series of enzymes to modify radically the structures to polar, water-
soluble compounds. The enzymatic process of altering organic chemical structure
(biotransformation) consists of two phases. Phase I biotransformation reactions typically
involve the insertion of a polar atom (i.e. oxygen) or fragmentation of the molecule,
leading to access of polar atoms (i.e. hydrolysis). Such radical alterations of structure
often lead to the formation of reactive intermediates, necessitating a second phase of
biotransformation which conjugates the intermediates to endogenous macromolecules
which are in large cellular supply (i.e. glucose, water, peptides). If certain phase II
processes are compromised or overwhelmed, the reactive intermediates may adversely
affect proteins, lipids or nucleic acids which serve critical cellular functions, resulting in
toxicity. Although uncommon, reactive intermediates may also be formed through phase
II pathways. For example, in mammals, glutathione conjugates of halogenated aliphatic
hydrocarbons may eventually be hydrolyzed to thiols or methyl thioether metabolites
which may rearrange to electrophilic episulfonium species or become oxygenated by
phase I monooxygenases to electrophilic sulfoxides respectively. Certain glucuronides
may even become hydrolyzed by bacteria after reaching the gut, allowing the aglycone to
be reabsorbed; a process known as enterohepatic circulation. Although phase I
bioactivation has been observed in fish, bioactivation through phase II processes has yet to
observed.

Ultimate toxicants and reactive intermediates

Biotransformation of non-polar lipophilic compounds to polar hydrophilic products
typically involves radical structural modification which may result in the formation of
reactive intermediates or ultimate toxicants. Although there are some exceptions, most
 GENERAL MECHANISMS OF TOXICITY 5

reactive intermediates exist as either electron-deficient molecules (electrophiles) or

unstable molecular fragments (free radicals).

ELECTROPHILES
Electron-deficient molecules often result from phase I biotransformation reactions
(Table 1.1). Oxidation reactions catalyzed by cytochrome P450 or other enzymes can lead
to the formation of numerous electrophilic metabolites including, but not limited to,
epoxides, ketones, arene oxides, quinones, quinoneimines, or acyl halides. Heterolytic
bond cleavage can also lead to the formation of positively charged electrophiles. For
example, cleavage of the sulfoester conjugate of aromatic amines leads to the formation of
the highly reactive aryl nitrenium ion. The oxidation of AsO43• to AsO32• /(As3+) and
elemental mercury to Hg2+ are examples of inorganic electrophiles. Electron-rich
moieties found in amino acids and nucleic acids such as thiols and amines are generally
thought to be the molecular targets for these intermediates.

FREE RADICALS
Containing one or more unpaired electrons in its outer orbital, free radicals are typically
formed by accepting or losing single electrons during, respectively, reduction or oxidation
reactions. Toxicants capable of accepting single electrons from reduction reactions can
elicit toxicity by forming free radicals. The reduced toxicant typically transfers the extra
electron to molecular oxygen, leading to the formation of a superoxide anion radical and
the oxidized toxicant which can receive an additional electron. This process of the
acceptance and donation of electrons and regeneration of the parent compound is known
as ‘redox cycling’ (Figure 1.2) and can lead to the formation of a large number of
superoxide anion molecules from a single molecule of toxicant.
Single electron reduction reactions may also lead to homolytic bond fission in certain
xenobiotics, especially halogenated aliphatic hydrocarbons such as carbon tetrachloride.
Such reactions lead to a dehalogenation of carbon tetrachloride to the trichloromethyl
radical (Figure 1.3). Interaction of this reactive intermediate with molecular oxygen leads
to the formation of another highly reactive metabolite known as the trichloromethylperoxy
radical, which may abstract protons from lipids causing lipid peroxidation (see below).
Additional one-electron processes that lead to the formation of free radicals are described
by the Haber-Weiss and Fenton reactions (Figure 1.4). Transition metals such as iron and
copper act as electron donors to various peroxides (which may be the dismutated
products of superoxide anion), leading to the formation of the most reactive reduced
oxygen species, the hydroxyl radical.
In addition to electron-accepting xenobiotics, nucleophilic toxicants (chemicals
containing electron-donating atoms—aromatic amines, hydrazines, phenols, thiols) may
form superoxide anion radicals through peroxidase-catalyzed transfer of one electron from
the xenobiotic to molecular oxygen. Aromatic compounds having two ortho or para
phenolic groups may undergo two sequential one-electron oxidations, ultimately leading
to the formation of quinones. Depending upon the physicochemical properties of the
 6 DANIEL SCHLENK

Figure 1.2 Potential pathways for oxidative stress (see Di Giulio et al., 1995).
 GENERAL MECHANISMS OF TOXICITY 7

Figure 1.3 One-electron reduction of halogenated methanes.

Figure 1.4 Haber-Weiss and Fenton reactions.

specific toxicant, quinones not only may act as redox cyclers generating superoxide but
also are classic electrophiles (see above) and undergo attack by nucleophilic molecules
found in cellular proteins and nucleic acids.

Chemical reactions of reactive intermediates/ ultimate

toxicants

General cellular targets

Electron-deficient molecules formed through cellular biotransformation processes are
prone to attack by nucleophilic molecules within any given cell, leading to the potential
formation of toxicant-biomolecule adducts. Consequently, biomolecules having large
electron densities, such as thiols and basic amine moieties, would be susceptible targets of
electrophiles. As free radicals may donate or abstract electrons from targets, molecules
containing atoms that are inclined toward electron acceptance or loss would be prime
targets for free radical attack. Many biomolecules possess these general features, and it is
likely that more than one type of reaction may occur with the cellular target. Discussion of
general cellular targets will be limited to protein, lipid and nucleic acids.

Protein
As proteins contain numerous atoms that are excellent reducing agents (i.e. thiols),
oxidation of protein thiols to disulfides or sulfenic acids is a common outcome following
8 DANIEL SCHLENK

interactions with free radicals. Hydrogen abstraction of thiols by neutral free radicals may
also form thiyl radicals, eventually leading to similar disulfides and sulfenic acid products.
Hydroxyl radicals may also remove protons from methylene groups of amino acids,
leading to the formation of carbonyls which tend to bind amines of proteins or DNA
covalently, thus causing cross-linking. In addition, thiols are also excellent nucleophiles
and provide potential sites for covalent binding of electrophilic toxicants. Non-covalent
binding through hydrogen and ionic bonds may also occur with proteins having
appropriate tertiary structure (i.e. helices) such as membrane receptors or enzymes.
Covalent or non-covalent binding could significantly inhibit the catalytic activity of an
enzyme or signal transduction pathway initiated by a receptor. For example, the
mimicking of calcium by lead or 17β;-estradiol by nonylphenols can have significant
functional effects for their receptors or enzymes. Many enzymes and receptors require
cofactors such as metals or organic moieties such as porphyrins or folic acid. If cofactors
are modified (i.e. electron transfer to iron in hemoglobin) then protein function may be
severely altered (i.e. methemoglobinemia); also, if the affected protein is critical for cell
maintenance, it is likely that cell viability may be ultimately compromised.

Lipid
Possibly the most common reaction that occurs between toxicants and lipids is that of
lipid peroxidation. The primary targets are unsaturated lipids found throughout cellular
membranes. Bis-vinylic methylene protons are highly susceptible to abstraction by free
radicals (Figure 1.5). Abstraction of either of these protons by free radicals such as
hydroxyl radical leads to the formation of conjugated dienes, which may undergo Fenton
chemistry and form lipid peroxyl radicals, and lipid hydroperoxides. These intermediates
not only serve as endogenous toxicants but also result in significantly altered membrane
integrity and function. As most cellular organelles as well as the plasma membrane is
largely lipid in constitution, altered integrity could result in loss of osmotic gradients,
leading to cell death if unrepaired.

Nucleic acids
Like thiol-containing proteins, nucleic acids are electron-rich molecules that can
covalently bind electrophilic toxicants. In contrast to thiols, which tend to bind covalently
soft electrophiles (i.e. quinones), purine-associated oxygens and nitrogens are more likely
to attack hard electrophiles (i.e. alkyl carbonium ions). Covalent binding of DNA by
various electrophiles disrupts replicative and transcriptional activities of polymerases,
and, if unrepaired, may lead to genomic mutation. Because DNA exists as a double helix
through significant hydrogen bonding, non-covalent intercalation by planar toxicants may
also lead to mutation. DNA is also susceptible to attack by free radicals as hydroxyl
radicals tend to bind the C-8 position of guanine and abstraction of protons on the pentose
sugar of DNA by free radicals may lead to hydrolytic strand breaks. Alteration of the
genome through any of these mechanisms may lead to modified protein function which
Figure 1.5 Initiation of lipid peroxidation.
GENERAL MECHANISMS OF TOXICITY 9
10 DANIEL SCHLENK

could result in either cell death or carcinogenesis, depending upon the specific function of
the affected protein or gene product (see below).

Exceeding capacity of cellular defense mechanisms

General types of cell defense

Many cellular toxicants elicit their adverse effects through formation of electrophilic
reactive intermediates or free radical molecular fragments. Generally, cellular defense
mechanisms (i.e. glutathione, antioxidants) are chemically similar to molecular targets
(i.e. electron-rich molecules), but are in larger cellular concentrations and have greater
binding capacities. As stated above, other mechanisms of defense rely on structural
biotransformations (i.e. oxidation) to enhance polarity and cellular elimination. Since such
defense mechanisms rely heavily upon oxidative processes, it is imperative for the cell to
have adequate reducing equivalents (generally in the form of NADH or NADPH) to allow
oxidative biotransformation reactions to occur. Regeneration of defending molecules as
well as other vital cellular functions are also intimately related to the redox potential of
the cell and the ability of the cell to produce reducing equivalents. Cellular toxicity
generally occurs when either the scavenging capacity of defense mechanisms are exceeded
or the regenerative capacity of the cell to produce reducing equivalents is overwhelmed.

Defense of nucleophiles
To prevent the donation of electrons from nucleophilic toxicants such as hydroxylated,
thiol, or amine-containing chemicals, phase II conjugation reactions (i.e. glucuronidation,
sulfation, acetylation; see Table 1.1) are primarily responsible for detoxification. For
example, many amines may be acetylated through N-acetyl transferases to metabolites
that do not donate electrons. If such phase II processes are compromised, chemicals
containing these functional groups may undergo single electron reductions or oxidations
through respective reductases or peroxidases, leading to the formation of free radicals.
Some nucleophiles (amines, thiols, phosphines, selenides) are also detoxified by oxidation
reactions catalyzed by flavin-containing monooxygenases to corresponding N, S, P, or Se
oxides.

Defense of electrophiles
As stated above, many reactive intermediates from phase I processes are electrophilic and
are typically conjugated with nucleophilic phase II molecules. For example, mercury and
cellular concentrations of glutathione are very large and conjugate many reactive
intermediates such as epoxides or acyl halides. Glutathione, in addition to the polypeptide
metallothionein, also binds electrophilic metals such as cadmium. Epoxide hydrolases also
convert epoxides to the less reactive metabolites dihydrodiols. Reduction of hydroquiones
by DT diaphorase may afford protection from covalent binding and prevent redox
 GENERAL MECHANISMS OF TOXICITY 11

Table 1.1 General pathways of xenobiotic biotransformation and their major subcellular locations.

cycling. Covalent binding to cellular proteins by α, β;-unsaturated aldehydes can be

diminished through reduction by aldehyde dehydrogenase as well as by adduction to
glutathione. Glutathione is a unique defense mechanism because not only does it have the
capacity to bind many electrophiles non-enzymatically but also it may be enzymatically
transferred to electrophilic substrates through cytosolic and microsomal glutathione
transferase enzymes.

Defense of free radicals

Neutral free radicals such as lipid peroxyl or xenobiotic radicals are primarily detoxified
by non-enzymatic reduction by glutathione, α-tocopherol (vitamin E) or ascorbic acid
(vitamin C). Each of these systems can be regenerated through transfer of reducing
equivalents ultimately from NADPH via specific reductases (glutathione reductase and
glutaredoxin respectively) (Figure 1.6). Hydroxyl radicals, the most reactive endogenous
radical (see Figure 1.4) cannot be scavenged by any of the antioxidant systems described
above. Consequently, the cell’s primary strategy is to prevent the formation of hydroxyl
radical. Typically, superoxide is dismutated by cytosolic or mitochondrial superoxide
12 DANIEL SCHLENK

dismutase to hydrogen peroxide which undergoes catalase or glutathione peroxidase-

catalyzed peroxidation to water. Fenton reagents such as iron and copper are sequestered
by specific metal-binding proteins that limit their participation in peroxide
transformation. Metallothioneins have been shown not only to sequester potentially
reactive metals but also to scavenge free radicals because of the large content of thiols.
However, oxidation of thiols may also release metals such as copper, causing elevated
oxidative damage. The overall mechanism of protection by metallothioneins from free
radical damage is still unclear.

DNA repair
Although it may be argued that repair of DNA may not be classified as a defense
mechanism preventing DNA damage, DNA repair processes do, in fact, defend against the
transmission of damaged DNA to the next generation of cells, or, in other words, prevent
the occurrence of mutation. There are primarily four types of DNA repair pathways that
reverse DNA damage. The first of these is base-excision repair, in which apurinic
endonucleases, DNA glycosylases, polymerases and ligases remove and replace minor
lesions resulting from small molecule alkylation at specific bases. Second, if a bulky
adduct is present (such as an aflatoxin adduct or pyrimidine dimer), several nucleotides
surrounding the lesion may be removed by nucleotide excision, which utilizes an
adenosine triphosphate (ATP)-dependent nuclease, and replaced by subsequent DNA
polymerase and ligase activities. Third, direct repair mechanisms such as DNA photolyase
and O6-methylguanine-DNA methyltransferase specifically repair ultraviolet (UV)-
induced pyrimidine dimers and methylguanine adducts respectively. The last type of DNA
repair, recombinational repair, is reserved for massive structural alterations that are not
corrected prior to DNA replication by any of the other three types of repair processes.
Essentially, damaged DNA (i.e. double-strand breaks or massive single-stranded lesions)
is replaced by a segment from a ‘sister’ or homologous strand of DNA that ‘crosses over’
to the damaged strand, leading to the formation of a complete single or double strand of
DNA.

Specific cellular targets

Cellular regulation
Regulation of cell viability is ultimately controlled at the level of gene expression. Gene
expression can be self-regulating especially in multitissue organisms which require
extracellular signaling between tissues and intracellular signal transduction pathways for
appropriate signals to reach specified genes. Alteration in any of these pathways may
dramatically affect processes that control viability and/or proliferation of any given cell
type. As discussed above, modifications of gene expression may begin by a direct mutation
of a gene which encodes a specific product that is critical for cell survival. Alternatively,
the mutation may occur in a regulatory region of the gene, leading to enhanced or
Figure 1.6 Activation and detoxification of lipid peroxidation reactions (see Gregus and Klaasen,
GENERAL MECHANISMS OF TOXICITY 13

1996).
14 DANIEL SCHLENK

Table 1.2 Toxicants acting on ligand-activated transcription factors.

repressed expression of the gene product. A third possible scenario is disruption or

enhancement of specific transcription factors that are necessary for gene expression. Many
transcription factors also serve as intracellular receptors for various endogenous
substances such as hormones and vitamins (Table 1.2). Consequently, if a xenobiotic
competes for the binding site of the endogenous substance, the transcription factor may
antagonize or potentiate gene expression depending on the affinity of the xenobiotic for
the receptor and the specific activity or abundance of the transcription factor. Such is the
case with xenoestrogens and their activation or inactivation of the estrogen receptor,
 GENERAL MECHANISMS OF TOXICITY 15

which eventually serves as a transcription factor for estrogen-responsive genes (i.e.

hepatic expression of vitellogenin). The acute and chronic effects of such interactions on
reproduction and development can be significant depending on the dose of the xenobiotic
and the stage of development when exposure occurs. Transcription factors may never
interact directly with the xenobiotic, but may be activated through signal transduction
pathways originating upstream from ligand-receptor interactions which may occur in
another tissue. For example, extracellular signaling molecules such as macrophage-
derived cytokines and growth factors eventually trigger the activation of parenchymal
transcription factors, leading to gene expression. If any of the signal-transducing pathways
become altered (i.e. phosphorylation, calcium metabolism), activation of transcription
factors may be modified. In mammals, activation or increased expression of the
transducing protein protein kinase C (PKC) by a xenobiotic (i.e. tetradecanoylphorbol
acetate or lead) can cause stimulation of the transcription factor activation protein 1
(AP-1), which increases mitogenesis. Toxicants inducing oxidative stress activate other
transcription factors such as nuclear factor kappa beta (NF-KB), which may alter cellular
regulation.
Another potential mechanism affecting gene expression may involve disruption of an
extracellular signal or hormone which initiates the cascade to gene expression. As
mentioned above, preventing homeostatic function of circulating hormones either through
antagonism at any given receptor or through inhibition of hormone synthesis may have
serious effects on reproduction and development (i.e. alteration of sex steroids).
Likewise, impeding the normal degradation and elimination of signaling hormones also
impacts susceptible cellular systems through elevated gene expression.

Specialized cell functions

Tissues that are actively undergoing electrochemical modifications for specific functions
[i.e. central nervous system (CNS), cardiovascular, somatic muscle] are extremely
vulnerable to toxicant interference with ligand receptor and subsequent signal
transduction pathways. Indeed, many pesticides mediate their toxic effects through
disruption of one or more of the processes in these tissues. According to Gregus and
Klaassen (1996), adverse effects in these systems primarily are relegated to modifications
of (1) concentrations of neurotransmitters, (2) receptor function, (3) intracellular signal
transduction, or (4) the signal-terminating processes (Figure 1.7).

Alteration in neurotransmitter concentration

Xenobiotics may alter concentrations of ligands in electrochemically sensitive cells by
disrupting the ligand’s synthesis, storage, release or removal from the vicinity of the
receptor. An example of the last mechanism would involve the organophosphate-induced
inhibition of various acetylcholinesterases at the neuromuscular junction, brain or blood in
fish leading to accumulation of acetylcholine, resulting in cholinergic overload and
associated toxicities such as loss of opercular movement.
 16 DANIEL SCHLENK

Figure 1.7 Examples of signal transduction pathways (see Gregus and Klaasen, 1996).
 GENERAL MECHANISMS OF TOXICITY 17

Toxicant-neurotransmitter receptor interactions

There are several means by which xenobiotics may interact directly with neurotransmitter
receptors. Chemicals may act as direct or partial agonists, mimicking the endogenous
ligand and activating the receptor. Alternatively, the compound may antagonistically bind
to the receptor, thus failing to stimulate activity. If chemicals do not bind the active site of
the receptor but stimulate activity, they are classified as activators. In contrast, inhibitors
block receptor activation without binding directly to the active site of the receptor.

Toxicant-signal transducer interactions

Another mechanism of disrupting electrochemically active cells is that of interrupting
intracellular signal transduction pathways. Red tide-produced saxitoxins are excellent
examples as they block voltage-gated sodium channels. Opening of these channels is
triggered by excitatory signals generated by ligand-gated cation channels at the
neuromuscular junction. By blocking the voltage-gated channels, saxitoxin prevents
transduction of the original excitatory signal, resulting in paralysis of muscular tissue. In
contrast, DDT [1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane] causes an activation of
voltage-gated channels, leading to overstimulation and muscular tremors and/or
convulsions depending on the dose.

Toxicant-signal termination interactions

Cellular signals in electrochemically active cells are driven by cation flux into the cell.
Consequently, termination of the signal is mediated by cation efflux via transporters or
specific cationic channels. Disruption of cationic transporters may not be unique to
neurons or myocytes as a mechanism of toxicity. The Na+, K+-ATPase transporter in gills
is critical to osmoregulation in fish such that disruption of this transporter can have
dramatic effects on gill function.

General cellular function

There are primarily two major pathways that contribute to cell death in most tissues:
impairment of cellular energy and sustained increase in calcium. The former is brought
about by agents that disrupt ATP synthesis, whereas the latter may be effected through
several molecular pathways, including impairment of ATP synthesis.

Disruption of ATP synthesis

ATP is required for the maintenance of multiple cell functions, including cell division,
protein, lipid, carbohydrate and nucleic acid synthesis, muscle contraction,
polymerization of the cytoskeleton, regulation of cell volume, plasma membrane integrity,
and transport of essential nutrients and ions (i.e. Ca2+). The synthesis of ATP is
accomplished through oxidative phosphorylation occurring in the mitochondria. As
18 DANIEL SCHLENK

hydrogen is oxidized to water in the mitochondria, an electrochemical gradient along the

mitochondrial membrane drives ATP synthase, eventually phosphorylating adenosine
diphosphate (ADP) to ATP.
There are primarily four areas where agents may interrupt the synthesis of ATP (Gregus
and Klaassen, 1996). Agents may diminish the delivery of reducing equivalents to the
electron transport chain through disruption or inhibition of basic biochemical pathways
that produce reducing equivalents (i.e. citric acid cycle, glycolysis, gluconeogenesis, or
fatty acid oxidation). Reducing equivalents are critical in regenerating oxidized proteins
or lipids during cellular repair. Second, xenobiotics may inhibit electron transport either
by disrupting electron transport complexes (i.e. rotenone, diphenyl ether herbicides) or
by acting as electron acceptors (carbon tetrachloride). Third, agents leading to hypoxia or
reduction in oxygen transport to the electron transport chain (i.e. methemoglobinemia) will
adversely affect ATP synthesis. Lastly, ATP synthesis may be impaired by agents that
directly inhibit ADP phosphorylation either through a general inhibitor of protein
synthesis or through specific inhibition of ATP synthase (DDT, chlordecone), the ADP/
ATP antiporter (DDT), the phosphate transporter (p-benzoquinone), or through an
uncoupling of the mitochondrial membrane potential from ATP synthase
(pentachlorophenol, chlordecone) (Gregus and Klaassen, 1996).
When oxidative phosphorylation is diminished, ADP and its degradation products
accumulate in the cell leading to reductions in pH and increased phosphoric acid, which is
thought to bind excess calcium and accumulate because of dysfunctional ATP-driven Ca2+
exporters. The loss of ATP-dependent ion pumps eventually leads to an accumulation of
sodium, resulting in cellular swelling or blebbing. If no repair occurs, the intracellular pH
will begin to rise, stimulating phospholipase activity, rupturing blebs and forming free
fatty acids which cannot be reacylated (ATP dependent). In addition, cellular
concentrations of calcium will rise, leading to several other interrelated pathologic
events.

Prolonged excess of intracellular calcium

In certain cell types, influx of calcium is a regular occurrence. However, its sustained
increase and accumulation can significantly damage cells and lead to cell death. Cellular
membranes are impermeable to calcium and either actively transport it out of the cell or
sequester it in the endoplasmic reticulum and mitochondria (Figure 1.7). Thus, toxicants
either cause elevated calcium by increasing influx or decreasing efflux from the
cytoplasm. Mechanisms may vary from inhibiting specific organelle or plasma transporters
(including ATP synthesis which drives the transporters) to general membrane disruption,
allowing calcium to flow down its concentration gradient into the cytoplasm. There are
three significantly harmful consequences of sustained intracellular calcium concentrations:
depletion of energy reserves, dysfunction of microfilaments, and activation of hydrolytic
enzymes.
Cellular energy reserves may be depleted by inhibiting the synthesis of ATP (see
above). The resulting increase of intramitochondrial calcium activates mitochondrial
dehydrogenases which, through augmented electron transport chain activity, may lead to
 GENERAL MECHANISMS OF TOXICITY 19

the increased formation of partially reduced oxygen species (i.e. superoxide anion
radical). As mentioned above, increased formation of free radicals may lead to damage of
the inner mitochondrial membrane, further disrupting oxidative phosphorylation. Lastly,
as calcium export is ATP dependent, increases of intracellular calcium may deplete ATP
through consumption by specific calcium transporters. ATP depletion and increases of
intracellular calcium concentrations are processes that are significantly linked and may
potentiate cell damage through a cyclical interrelationship.
Sustained increases in intracellular calcium may also lead to dissociation of actin
microfilaments from membrane-anchoring proteins, causing blebbing and eventually
leading to membrane rupture. Cytoskeletal proteins may also be direct targets of
xenobiotics. However, this has only been observed in mammalian systems.
In addition to exacerbating energy depletion and its cytoskeletal effects, calcium
activates the endonuclease topoisomerase II, several proteases (i.e. calpains) and
phospholipases, leading to degradation of DNA, proteins and lipids respectively.
Hydrolytic enyzmes may also be released into the cytosol from general membrane
disruption of lysosomes, which also cause influx of calcium through destruction or
destabilization of plasma or mitochondrial or endoplasmic reticular membranes, thus
creating other cyclical reactions.

Targets involving repair and cellular regeneration

Apoptosis and cell proliferation

As mentioned throughout this chapter, cellular damage may not be life-threatening
because of numerous defense mechanisms that can limit or rapidly repair xenobiotic-
induced damage. However, when defenses are overwhelmed or compromised, the cell
may pass ‘the point of no return’ and either undergo apoptosis, which is a scheduled
mechanism of cell death, or necrosis, which tends to be an unorganized rupturing of the
damaged cell that leads to significant input by inflammation. The general characteristics of
each pathway are described in Table 1.3.
Generally, apoptosis is an intra- and extracellular signaled pathway which contributes
to cellular repair and is a means for tissue remodeling, which is critical in fetal
development (Gregus and Klaassen, 1996). Apoptosis allows DNA-damaged cells to be
placed in appropriate cell cycle phases for DNA repair to occur. For example, following
DNA damage, cells are cycled from the resting G0 to the G1 cell cycle phase by expression
of various gene products, one of which in mammals appears to be c-myc (Martin et al.,
1994). Further progression to a proliferative phase is blocked by p53 expression, allowing
DNA repair to occur. If repair does not occur, the cell is diverted to the apoptotic
pathway. Apoptosis may be blocked by expression of bcl-2 protein, which binds the
transcriptional activator (bax) that initiates apoptosis. If the apoptotic signaling pathway is
overwhelmed through massive cellular damage, cells typically undergo necrosis.
An additional repair mechanism is that of cellular proliferation. When cells experience
damage, adjacent undamaged cells in the G0 phase also rapidly (within 36 h) progress to
20 DANIEL SCHLENK

Table 1.3 Features of necrosis versus apoptosis.

the M (mitotic) phase and undergo proliferation. This is enhanced by the increased
expression of immediate-early growth response genes such as c-myc, c-fos and c-jun.
Expression of these transcription factors in addition to cytokines and other small
molecules which are thought to be released by damaged cells allows DNA synthesis to
gain top priority and override specialized cellular activities in adjacent cells. However,
many of these signaling molecules stimulate macrophage and endothelial cells to release
additional secondary signaling factors that not only aid in cellular proliferation but also
carry out a host of various inflammatory reactions, including microcirculatory alterations
and leukocyte recruitment which may engulf or destroy cells that could not be repaired.
An example of a macrophage-derived proliferative signal in mammals is hepatocyte
growth factor (HGF), which acts as a mitogen in many different tissues by stimulating
parenchymal regeneration and proliferation of endothelial cells. Unlike HGF, which has
yet to be observed in fish, the macrophage-derived transforming growth factor beta
(TGF-β;) has been observed in fish and stimulates non-parenchymal cells to synthesize
extracellular matrix which integrates with newly proliferated cells. Regenerating cells also
contribute to their own proliferation through the release of TGF-α. The function of TGF-
α is the proliferation of parenchymal cells and macrophages. Termination of proliferation
occurs as TGF-β; accumulates and eventually overrides the mitogenic activity of TGF-α.
If apoptosis and cellular proliferation are overwhelmed by massive cell injury,
macrophages and endothelial cells will release numerous other agents that can alter
microcirculation (i.e. NO), oxidatively degrade cells (i.e. reduced oxygen radicals) and
recruit peroxidative granulocytic lymphocytes (i.e. cytokines) which may potentially lead
to inflammation and/or tissue necrosis. The efficiency of cellular repair is an important
determinant of the dose-response relationship for toxicants that cause tissue necrosis. This
is not only because the dose ensures sufficient concentration of the ultimate toxicant at the
target site but also because that concentration of toxicant causes enough damage to
overwhelm repair, resulting in progression of the injury (Gregus and Klaassen, 1996).
 GENERAL MECHANISMS OF TOXICITY 21

Consequences of overwhelming tissue repair

Fibrosis
As discussed above, acute tissue damage that overwhelms apoptosis and cellular
proliferation typically leads to tissue necrosis. In an attempt to halt the spread of necrosis,
inflammatory signals from non-parenchymal tissues continue to be released (i.e. TGF-β;),
resulting in protracted production of extracellular matrix that eventually compromises
function through the formation of scar tissue. This ‘non-functional’ tissue causes the
production of a diffusional barrier which may lead to malnourishment of cells, obliteration
of blood vessels and parenchymal cells, and reduction in the flexibility of critical tissues such
as the heart and gills.

Carcinogenesis
An alternative pathway for a cell that has undergone toxicant insult is that of
carcinogenesis. Chemical carcinogenesis is a complex multistage process that can result
from the failure of three basic repair mechanisms: DNA repair, leading to mutation and
initiation; apoptosis, leading to uncontrolled cellular proliferation and promotion; and/or
termination of cellular proliferation, leading to propagation or progression of the neoplastic
tissue.
As mentioned earlier, DNA may be damaged through chemical adduction, oxidative
addition, and breakage of strands. Each of these lesions can be repaired through several
mechanisms (see above). However, if repair is compromised, a mutation may occur, with
the lesion transmitted to the mitotic daughter cells. Adverse effects may still not be
realized because of the large excess of non-protein-encoding DNA (99 percent of total
genome) that presumably acts as a buffer protecting against potential alterations of critical
regulatory proteins. If the lesion does occur in a coding or regulatory region, an
additional protective mechanism lies in the degeneracy of the genetic code. Thus, even
though mutations occur in the genome of an organism, they may still be ‘silent’ and not lead
to an adverse effect. However, if the mutation leads to an alteration of the amino acid
sequence which is important in the activation or function of the protein, two potential
initiation pathways may occur. Most mutations typically reduce or eliminate protein
function. If the protein is critical for cell viability then the mutation results in apoptotic cell
death. Unfortunately, elimination of specific proteins involved in suppression of cell
division (i.e. p53) may lead to uncontrolled proliferation of the cell. In mammalian
systems, p53 acts as a transcription factor and induces the transcription of growth arrest
and apoptotic genes through interaction with TATA-binding protein(s), inhibiting the
transcription of many genes that do not have p53 regulatory elements. Tumor-suppressor
genes have been isolated from fish (Van Beneden and Ostrander, 1994), but their
functional homology with mammalian systems has yet to be verified. For example, Kraus
et al. (1997) has identified the gene encoding for p53 in Japanese medaka, but sequencing
of DNA obtained from tumor-bearing fish exposed to the carcinogen N-methyl-N′-nitro-
N-nitrosoguanidine (MNNG) revealed no mutations within characteristic mutational
22 DANIEL SCHLENK

hotspots. In addition, studies with N-diethylnitrosoamine-treated Rivulus ocellatus

marmoratus indicated an increase of p53 protein in neoplasms rather than repression
(Goodwin and Grizzle, 1997). Clearly, more studies in fish are needed to determine
whether inhibition of tumor suppression is mechanistically similar to that observed in
mammals.
An alternative pathway following mutation is the activation of one or more transducing
proteins which may lead to uncontrolled cellular proliferation. The genes encoding such
proteins are referred to as oncogenes because of their association with carcinogenesis.
These gene products are generally growth factors, growth factor receptors, intracellular
signal-transducing proteins/kinases, and nuclear transcription factors (see above). When
these mutated cells undergo division, their descendants also have the propensity for
proliferation, leading to further mutations that potentiate proliferation which, in turn,
eventually override the apoptotic repair process. Although several oncogenic proteins
have been observed in fish, as indicated above, few have been observed to correlate with
tumors or cellular proliferation (for a review, see Van Beneden and Ostrander, 1994).
The most consistent relationship in fish has been between the expression of ras in N-
diethylmtrosamine-treated rainbow trout and Rivulus ocellatus marmoratus (Goodwin and
Grizzle, 1997). Ras proteins are transduction proteins with GTPase activity that are
normally triggered by various growth factor receptors and that initiate a kinase cascade
which eventually leads to mitogenic activity. Specific mutations in the ras gene allow the
expressed protein to remain in an activated state. The accumulation of genetic damage in
the form of mutant oncogenes (which encode activated proteins) and mutant tumor-
suppressor genes (which encode inactivated proteins) is the main driving force in the
transformation of normal cells with controlled proliferative activity to malignant cells
with uncontrolled proliferative activity in mammals (Figure 1.8). Whether this is the case
in fish remains to be verified.
As stated above, even after mutational damage occurs, initiated cells may be removed
by apoptosis. If tumor-suppressor protein expression is reduced, then the cell does not
undergo DNA repair or apoptotic death, but rather promotion and clonal expansion.
Thus, inhibition of apoptosis is detrimental because it facilitates both mutations and clonal
expansion of preneoplastic cells (Gregusand Klaassen, 1996). In addition to
underexpression of tumor-suppressor proteins, overexpression of apoptotic inactivating
proteins (i.e. bcl-2) have been shown to be associated with several mammalian cancers
(McDonnell et al., 1993). Consequently, enhancement of cellular proliferation through
the activation of oncogenes or through xenobiotic activation of factors that prevent
termination of cellular proliferation can result in non-genotoxic cancers. Thus, not all
carcinogens directly damage DNA. It must be emphasized, however, that a non-genotoxic
or epigenetic xenobiotic may actually cause cancer through a genotoxic mechanism by
enhancing the potential for mutation from endogenous sources such as oxygen radicals
resulting form respiratory bursts during chronic inflammation. Mitogenic activity can
significantly enhance the possibility of mutation by reducing DNA repair time by
shortening the G1 phase which also tends to reduce the time for DNA methylation
(transcriptionally active genes are typically hypomethylated). An additional effect of
cellular proliferation that may lead to invasiveness (or be a result of expansion) is the loss
 GENERAL MECHANISMS OF TOXICITY 23

Figure 1.8 Simplified scheme of neoplasia pathogenesis.

in cell-cell communication by disruption of intercellular adhesion gaps. Although several

promoting agents have been studied in fish (primarily rainbow trout), few epigenetic
carcinogens have actually been identified (McDonnell et al., 1993).

Summary
An understanding of the basic mechanisms of fundamental processes is considered essential
in traditional mammalian toxicology. Although this objective is viewed as highly desirable
in environmental toxicology and chemistry, often it is not considered essential in
24 DANIEL SCHLENK

addressing immediate environmental concerns. Thus, compared with the wealth of

information regarding cellular mechanisms of toxicity in mammalian systems, there have
been relatively few mechanistic studies in fish. Although understanding the mechanistic basis
of toxicant action is critical in assessing the risk of any given chemical to the environment,
there are numerous gaps in our knowledge of how many toxicants elicit cellular and
subsequent organelle damage. Fish share many common biochemical and physiologic
pathways with mammals, but, obviously, are quite distinct from mammals with regard to
life histories (i.e. reproductive strategies) and continual residence in aqueous
environments. Thus, it is the goal of this text to provide a current comparative review of
the unique interactions of various toxicants and their target organ systems in teleosts. It is
hoped that this framework will aid risk assessors in determining appropriate hazard
identifications for ecotoxicologic studies as well as biomedical researchers searching for
unique model systems for bettering human health.

References

Castejon, O.J. 1983. Light, scanning and transmission electron microscopy study of fish cerebellar
capillaries. Scanning Electron Microscopy 1:151–160.
Cooper, P.S., Vogelbein, W.K. and Van Veld, P.A. 1999. Altered expression of the xenobiotic
transporter P-glycoprotein in liver and liver tumors of mummichog (Fundulus heteroclitus) from
a creosote-contaminated environment. Biomarkers 4:48–58.
Di Giulio, R.T., Benson, W.H., Sanders, B.M. and Van Veld, P.A. 1995. Biochemical
mechanisms: metabolism, adaptation, and toxicity. In Fundamentals of Aquatic Toxicology: Effects,
Environmental Fate and Risk Assessment. Rand, G.M. (ed.), pp. 523–561. Taylor and Francis,
Washington, DC.
Goodwin, A.E. and Grizzle, J.M. 1997. Oncogene expression in hepatic and biliary neoplasms of
the fish Rivulus ocellatus marmoratus: Correlation with histologic changes. Carcinogenesis 15:1993–
2002.
Gregus, Z. and Klaassen, C.D. (1996). Mechanisms of Toxicity. In Casarett and Doull’s Toxicology:
The Basic Study of Poisons. Klaassen, C.D. (ed.), pp. 35–74. McGraw Hill, New York.
Heath, A.G. 1995. Water Pollution and Fish Physiology. Lewis Publishers, Boca Raton.
Jarboe, H., Toth, B.R., Shoemaker, K.E., Greenlees, K.J. and Kleinow, K.M. 1993.
Pharmacokinetics, bioavailability, plasma protein binding and disposition of nalidixic acid in
rainbow trout (Oncorhynchus mykiss). Xenobiotica 23:961–972.
Juliano, R.L. and Ling, V. 1976. A surface glycoprotein modulating drug permeability in Chinese
hamster ovary cell mutants. Biochimica et Biophysica Acta 455:152–162.
Kleinow, K., Venugopalan, C.S., Smith, A.A., Wiles, J.E. and Holmes, E. 1996. P-glycoprotein
distribution and inducibility in the catfish intestine. The Toxicologist 30: 35.
Kohler, A., Lauritzen, B., Bahns, S. and Van Noorden, C.J.F. 1998. Clonal adaptation of liver
tumor cells in flatfish to environmental contamination by multidrug resistance and metabolic
changes (G6PDH, CYP450, GST). Marine Environmental Research 46:191–195.
Krause, M.K., Rhodes, L.D. and Van Beneden, R.J. 1997. Cloning of the p53 tumor suppressor
gene from the Japanese medaka (Oryzias latipes) and evaluation of mutational hotspots in
MNNG-exposed fish. Gene 189:101–106.
Lane, N. 1991. Morphology of glial blood brain barriers. Annals of the New York Academy of Sciences
633:348–362.
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McDonnell, T.J., Marin, M.C. and Hsu, B. 1993. The bcl-2 oncogene: Apoptosis and neoplasia.
Radiation Research 136:307–312.
Martin, S.J., Green, D.R. and Cotter, T.G. 1994. Dicing with death: Dissecting the components of
the apoptosis machinery. Trends in Biochemical Science 19:26–30.
Plakas, S.M., Stehly, G.R. and Khoo, L. 1992. Pharmacokinetics and excretion of phenol red in the
channel catfish. Xenobiotica 22:551–557.
Roesijadi, G. and Robinson, W.E. (1994). Metal regulation in aquatic animals: mechanisms of
uptake, accumulation and release. In Aquatic Toxicology: Molecular, Biochemical and Cellular
Perspectives. Malins, D.C. and Ostrander, G.K. (eds), pp. 387–420. Lewis Publishers, Boca
Raton.
Van Beneden, R.J. and Ostrander, G.K. (1994). Expression of oncogenes and tumor suppressor
genes in teleost fishes. In Aquatic Toxicology: Molecular, Biochemical and Cellular Perspectives.
Malins, D.C. and Ostrander, G.K. (eds), pp. 295–325. Lewis Publishers, Boca Raton.
Van Leeuwen, C.J., Van Hameren, P., Bogers, M. and Griffioen, P.S. 1986. Uptake, distribution
and retention of zineb and ziram in rainbow trout (Salmo gairdneri). Toxicology 42:33–46.
26
 2
Toxic responses of the nervous system
Russell L.Carr and Janice E.Chambers

Introduction
Following exposure to any potentially toxic chemical, modification of behavior in an
organism frequently suggests that the nervous system is the toxicologic target of that
chemical. Although this is quite true in situations concerning mammalian species, the
same cannot be assumed for aquatic species. As the environments inhabited by the two
groups are distinctly different, the interaction of each group with its environment is also
distinctly different. Introduction of a contaminant into an aquatic system can produce
behavioral responses in the inhabitants that are quite different from those behavioral
responses observed in mammals exposed to a similar chemical. However, while
modification of behavior in fish may not be indicative of the nervous system as a target, it
is suggestive of neurotoxic activity.
In this chapter, the reported chemical-induced modifications of behavior in teleosts has
been used to identify chemicals which may have a neurologic target (Smith, 1984; Heath,
1995). The goal of this chapter is to report on the mechanistic interactions of toxicants
with neurologic systems present in teleosts. The majority of mechanistic work of
neurotoxicants has been directed towards mammalian toxicity, despite the fact that the
biochemical targets of many neurotoxic chemicals have been elucidated using non-
mammalian systems. Therefore, in searching for the target of toxicants in teleost systems,
one must use what is known concerning the effects of a chemical on targets in other
systems as a possible target in teleosts. Here, a brief overview of neuron physiology will
be provided and the similarity of these systems with teleost systems will be discussed.
Historical references in which the biochemical targets of certain neurotoxic chemicals
were first described and reports on whether or not teleosts and mammals share that target
will also be provided.
As stated earlier, the chemicals discussed in this chapter were selected based on their
reported disruption of behavior in fish. The organochlorine, pyrethroid, and
anticholinesterase insecticides disrupt behavior but are also known neurotoxicants and
have been responsible for many fish kills. Disruption of behavior in fish has been reported
for several metals (mercury, lead, copper, cadmium, and zinc) which have neurologic
targets. Other metals (i.e. chromium) disrupt behavior, but the literature suggests that
the effects observed may not be mediated by affecting a neurologic mechanism. Several
28 TOXIC RESPONSES OF THE NERVOUS SYSTEM

organic chemicals (solvents and polychlorinated biphenyls) have been reported to disrupt
behavior. Additionally, several natural toxins which have been demonstrated to be toxic
to fish (conotoxins), have been implicated in fish kills (cyanotoxins, saxitoxin,
neosaxitoxin, brevetoxin, and Pfiesteria piscicida), or have the potential for toxicity in fish
(kainic acid, domoic acid, and ciguatoxins) have been discussed.

Nervous system physiology

The teleost brain is largely similar to that of higher vertebrates. Many structures are the
same, but some regions differ in size among the vertebrate classes. For example, the
cerebellum and the optic and olfactory lobes are frequently quite large in teleosts,
whereas the cerebrum is small and does not possess a cerebral cortex (Heath, 1995).
Thus, many of the functions of the cerebrum in mammals are performed by other regions
of the brain in teleosts. There can also be variability among teleost species. For example,
the size of the optic and olfactory lobes is predicated on the method of feeding. Teleosts
that primarily utilize sight for foraging have larger optic lobes whereas teleosts that
primarily utilize olfaction for foraging have larger olfactory lobes. However, the
biochemical processes within the nervous system in teleosts are very similar to those in
mammals or other fish (i.e. elasmobranchs). In fact, much of the research which initially
characterized the biochemical systems responsible for nervous system function in
mammals and other higher vertebrates was performed using fish as an experimental
model (e.g. Torpedo). However, most research on nervous system function in fish is
oriented toward how that system is utilized to respond to natural changes in the environment
rather than how it is impacted by a contaminant.

Nerve function
The neuron is the fundamental unit of the nervous system. The typical neuron consists of
four regions, each of which carries out specialized functions of the cell. These include: (1)
the dendrites, which are multiple projections extending from the cell body that receive
signals from sense organs or other neurons, convert those signals to electrical impulses,
and transmit them toward the cell body; (2) the cell body or soma, which contains the
nucleus and the organelles for energy production and macromolecule synthesis; (3) the
axon, which is a single fiber that transmits impulses, termed action potentials, away from
the cell body; and (4) the axon (nerve) terminals, which are small branches of the axon
that form a junction with another neuron (synapse) or with an effector, such as a muscle
fiber (neuromuscular junction) (Figure 2.1). The signal passively spreads from the
dendrites through the cell body to the axon hillock (the junction of the cell body and the
axon). If the signal received by the axon hillock is sufficiently strong to change the
membrane potential from its resting state to a certain level called the threshold potential,
an action potential or larger electrical signal is generated at the axon hillock and
conducted down the axon to the axon terminal synapse.Once the action potential reaches
the junction, the impulse is transmitted to the post-junctional cell. This transmission may
be through either an electrical junction or a chemical junction. In an electrical junction,
 RUSSELL L.CARR AND JANICE E.CHAMBERS 29

Figure 2.1 Structure of a typical neuron.

the impulse passes directly from the presynaptic cell to the post-synaptic cell through gap
junctions. In a chemical junction, which is the most common type, the impulse stimulates
the release of neurotransmitters stored in synaptic vesicles at the axon terminal into the
space between the two cells, the synaptic cleft. The neurotransmitter release is stimulated
by increases in calcium (Ca2+), which enters the cell through voltage-gated Ca2+ channels.
Once released, the neurotransmitter crosses the cleft and binds to receptors on the post-
synaptic membrane, thereby transmitting the message to the effector cell. The chemical
junction may be excitatory or inhibitory. For example, if the junction is an excitatory
junction between two neurons (synapse), the neurotransmitter released would tend to
induce an action potential in the effector cell. If the synapse is an inhibitory junction
between two neurons (synapse), the neurotransmitter released would inhibit the
induction of an action potential in the effector cell. The type of neurotransmitter released
determines whether it is an excitatory or inhibitory junction.
The transmission of the action potential down the axon involves the use of
electrochemical gradients. When the neuron is resting, a normal gradient is maintained in
which the concentration of sodium (Na+) is greater on the outside of the cell while the
concentration of potassium (K+) is greater inside the cell. The cell membrane itself is
slightly negative and has a resting membrane potential of • 60 mV. In the axon
membrane, there are Na+ and K+ ionophores (channels) which are voltage dependent.
When the action potential is generated at the axon hillock, the electrical charge causes the
closed Na+ channels (closed but sensitive to a change in membrane potential) in that
region to open, allowing Na+ influx (Figure 2.2). This influx of Na+ depolarizes the
membrane in that area (i.e. increases the inside membrane potential toward the positive).
This change in the membrane potential in that area then stimulates the opening of the Na+
channels in the adjacent area, depolarizing that area, which then continues to stimulate the
Na+ channels to open in adjacent regions of the membrane. Thus, the action potential is
propagated down the axon. After a slight delay, the opened Na+ channels close and
become inactivated (i.e. no longer sensitive to a change in membrane potential). The K+
channels open, allowing K+ to flow outward. The outflux of K+ repolarizes the
membrane (i.e. decreases the membrane potential). The K+ channels close andthe Na+
channels return to the original closed state (i.e. sensitive to changes in membrane
potential). The enzyme Na+, K+-ATPase functions to re-establish the proper gradient
required for another action potential by pumping Na+ out and K+ in.
30 TOXIC RESPONSES OF THE NERVOUS SYSTEM

Figure 2.2 Simplistic representation of the process of sodium (Na+) channel and potassium (K+)
channel opening and closing during an action potential.

Sodium channels
Using pharmacologic agents, five distinct binding regions have been identified on the
mammalian Na+ channel (Catterall, 1988) (Figure 2.3). Some work has been carried out
 RUSSELL L.CARR AND JANICE E.CHAMBERS 31

Figure 2.3 Theoretical diagram of the structure of a sodium channel and the hypothesized binding
sites for ligands. The actual shape and structure of the sodium channel are not known. Binding sites:
(1) tetrodotoxin (TTX), saxitoxin (STX), and mu-O-conotoxins; (2) aconitine (ACN), veratridine
(VTD), grayanotoxin, and batrachotoxin (BTX); (3) North African scorpion venom α-polypeptide
toxin (α-ScV) and sea anemone nematocyst venom (ATX); (4) American scorpion venom β-
polypeptide toxin (β-ScV); and (5) ciguatoxins and brevetoxins.

using these same agents to characterize the fish brain Na+ channel. Although some binding
sites on the Na+ channel in fish are similar to those of mammals, some appear to be
different.
Site 1 binds sodium channel blockers tetrodotoxin (TTX), saxitoxin (STX) (see
Figure 2.11) and the mu-conotoxins which block Na+ permeability. Site 1 in fish seems
similar to that in mammals. TTX effectively blocks Na+ permeability in rainbow trout
(Oncorhynchus mykiss) synaptoneurosomes as it does in mammals (Stuart et al., 1987; Rubin
and Soderlund, 1992). However, although it appears that the binding characteristics of
[3H]-STX are similar in rainbow trout brain synaptoneurosomes (Rubin and Soderlund,
1993) and in garfish nerve membrane (Henderson et al., 1973), the binding characteristics
are slightly different from those in rat brain (Catterall et al., 1979).
Site 2 binds the lipophilic activators aconitine (ACN), veratridine (VTD), grayanotoxin,
and batrachotoxin (BTX), which cause persistent spontaneous opening of the Na+
channel. Site 2 in fish seems similar to that in mammals, but is not as responsive to
activation. ACN, VTD, and BTX will activate the opening of Na+ channels and allow Na+
uptake in both rainbow trout and mouse synaptoneurosomes (Stuart et al., 1987; Rubin
and Soderlund, 1992). Also, the ranking of potencies (BTX > VTD > ACN) in rainbow
trout is identical to that in mouse synaptosomes (Rubin and Soderlund, 1992). However,
the magnitude of Na+ uptake in response to these activators is much lower in rainbow
trout brain than in mice (Rubin and Soderlund, 1992) or rats (Eells et al., 1993).
Site 3 binds the α-polypeptide toxins isolated from the venom of North African
scorpions (α-ScV) (e.g. Leiurus quinquestriatus) and sea anemone nematocysts (ATX) (e.g.
32 TOXIC RESPONSES OF THE NERVOUS SYSTEM

Anthopleura xanthogrammica andAnemonia sulcata) which interfere with the Na+ channel
closing such that, once activated, the Na+ channel will remain open longer. Site 3 is
similar in some aspects but not in others. α-ScV and ATX enhance the activation of Na+
uptake by ACN, VTD, and BTX in mouse and rat preparations, but rainbow trout brain
preparations are less sensitive to this enhancement (Rubin and Soderlund, 1992; Eells et
al., 1993). While ACN-activated Na+ uptake is inhibited by the pyrethroid insecticide
deltamethrin in the mouse (Rubin and Soderlund, 1992) and by permethrin,
deltamethrin, and cypermethrin in the rat (Eells et al., 1993), these pyrethroids increase
ACN-activated Na+ uptake in rainbow trout.
Site 4 binds the β-polypeptide toxins isolated from the venom of American scorpions
(β-ScV) (i.e. Centruroides noxius and Tityus serrulatus) which also prolong the time that the
Na+ channel is open once activated. Little pharmacologic work has been done comparing
site 4 in fish and mammals. However, an antibody which effectively inhibits the binding of
Tityus serrulatus gamma scorpion toxin to site 4 in rats also antagonized the binding of the
toxin to electric eel (Electrophorus electricus) Na+ channels (Barhanin et al., 1985).
Site 5 binds the ciguatoxins produced by benthic dinoflagellates (Gambierdiscus toxicus)
and the brevetoxins produced by red tide dinoflagellates (Ptychodiscus brevis) which cause
persistent repetitive spontaneous opening of the Na+ channel. Site 5 is similar in mammals
and fish in some aspects but not in others. Although both ciguatoxins and brevetoxins bind
to the Na+ channel in fish (Capra et al., 1987; Stuart and Baden, 1988), in vitro the
brevetoxins are more potent activators of the Na+ channel in fish brain than the
ciguatoxins, whereas the opposite is true for mouse brain Na+ channels (Lewis, 1992).
Overall, a toxicant which binds to the Na+ channels in mammals should theoretically
bind to Na+ channels in fish. However, the species differences in the composition of the
channel may increase or decrease the efficacy of a toxicant in fish compared with
mammals.

Cholinergic system
Acetylcholine (ACh) is the neurotransmitter associated with the cholinergic system of all
developed organisms. ACh acts on two types of receptors, nicotinic and muscarinic
(Table 2.1). The nicotinic receptors were originally distinguished by the action of the
agonist nicotine and the muscarinic receptors by the action of the agonist muscarine, a
mushroom alkaloid. Subtypes of both nicotinic and muscarinic receptors have been
identified. Nicotinic receptors are associated with ligand-dependent ion channels (usually
Ca2+ and Na+ channels) and can be found in the neuromuscular junction of skeletal
muscles in the peripheral nervous system (PNS) and in the autonomic ganglia, adrenal
medulla, and other areas of the central nervous system (CNS). The nicotinic receptor has
been well characterized in mammals and consists of five subunits. In the neuromuscular
junction, the receptor consists of two α subunits, one β subunit, one γ subunit, and one δ
subunit. In the CNS, there are eight α subtypes and four β subtypes of nicotinic receptor,
but these receptors do not contain γ or δ subunits. Muscarinic receptors can be found in
the heart, smooth muscle, secretory glands, autonomic ganglia, and other areas of the
CNS. Muscarinic receptors are associated with guanine nucleotide-binding proteins (G
 RUSSELL L.CARR AND JANICE E.CHAMBERS 33

proteins) which are activated by the binding of the neurotransmitter to the receptor and,
in turn, activate (Gs) or inhibit (Gi) second messenger systems to produce the appropriate
cellular response. The type of second messenger system associated with the receptor
depends on the subtype of the receptor. For example, activation of mammalian M1, M3,
and M5 muscarinic receptor subtypes causes stimulation of phospholipase C to hydrolyze
phosphatidylinositol 4, 5-bisphosphate to the second messengers inositol 1, 4, 5-
trisphosphate (which increases intracellular Ca2+, thus activating other Ca2+-dependent
processes) and diacylglycerol (which, along with Ca2+, activates protein kinase C).
Activation of mammalian M2 and M4 muscarinic receptor subtypes causes inhibition of
adenyl cyclase, activation of K+ channels, and suppression of the activity of the voltage-
gated Ca2+ channels. The muscarinic subtypes are found in many different tissues (see
Ashkenazi and Peralta, 1994) with several subtypes present in the same tissue.
Teleosts possess nicotinic receptors which appear to be similar to mammalian nicotinic
receptors. The neuromuscular nicotinic receptor in electric eel has been purified and
sequenced and found to be highly homologous to that of mammals (Numa, 1986) and to
other non-teleosts (e.g. Torpedo californica) (Conti-Tronconi et al., 1984). Functionally,
the alpha-conotoxins from the marine snails of the genus Conus are effective antagonists
for nicotinic receptors in fish and other species (Hopkins et al., 1995), causing paralysis of
the skeletal muscles in all species. The nicotinic antagonist α-bungarotoxin will bind as
effectively to nicotinic receptors in teleosts as it does in other species (Deplano, 1988).
Additionally, the differential sensitivities of fish nicotinic receptors to nicotinic agonists
(e.g. nicotine) and antagonists (e.g. tubocurarine) suggest that different subtypes of
nicotinic receptors with differing functions are present in teleost nervous tissue (Deplano,
1988).
Little work has been carried out in identifying the presence of different subtypes of
muscarinic receptors or on the pharmacologic characterization of these subtypes in fish.
However, some muscarinic subtypes have been reported in teleosts. Using specific M1 and
M2 antagonists to block carbachol-stimulated contractions, the M2 subtype has been
suggested to be present in the smooth muscle of rainbow trout but not the M1 subtype
(Burka et al., 1989). The M3 subtype has been suggested to mediate the cholinergically
induced aggregation of pigment in the melanophores of glass crystal catfish (Kryptopterus
bicirrhis) and mailed catfish (Corydoras paleatus) (Hayashi and Fujii, 1994). With respect to
muscarinic receptors in the brain, few binding studies have been carried out, with
descriptive studies available only for goldfish (Carassius auratus) (Francis and Schechter,
1980), carp (Cyprinus carpio) (Szabó et al., 1989), and brook trout (Salvelinus fontinalis)
(Jones and King, 1995). In the last two, the reported numbers of the M1 subtype were
significantly greater in teleost brain than the M2 subtype, similar to mammals.
Once ACh has stimulated a cholinergic receptor, it is rapidly degraded by the enzyme
acetylcholinesterase (AChE). AChE is closely associated with the target receptors on the
post-synaptic membrane and functions to hydrolyze ACh to choline and acetate, thus
eliminating any further stimulation. In teleost brain, the number of molecular forms of
AChE can vary from one to five, and these mainly have a substrate preference for ACh
compared with other choline analogues such as propyl-, butyryl-, and methylcholine
(Kozlovskaya et al., 1993). The active site of AChE contains a serine hydroxyl group to
34 TOXIC RESPONSES OF THE NERVOUS SYSTEM

which ACh binds during hydrolysis. However, there are species differences in the
configuration of the active site of AChE. The active site of rainbow trout brain AChE is
smaller than that of rat brain AChE and has a weaker nucleophilic strength (Kemp and
Wallace, 1990). However, the active site of channel catfish (Ictalurus punctatus) brain
AChE appears to have a strong nucleophilic site similar to that of rat (Carr and Chambers,
1996). It is the characteristic of the binding site that influences its affinity for various
inhibitors of the enzyme (e.g. for different organophosphates and carbamates) and that can
lead to species differences in toxicity among teleosts for the same compound. Thus,
species differences in the characteristics of the enzyme AChE in teleosts could play a role
in the varying susceptibility to toxicants which bind to this enzyme. A discussion of
anticholinesterase insecticides follows in the section on acetylcholinesterase insecticides.

GABAergic system
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter which is widespread
in the vertebrate CNS (DeLory and Olsen, 1994). The inhibitory neurons which utilize
GABA as a neurotransmitter form a synapse on the cell body of other neurons. The
depolarization of the presynaptic inhibitory neurons stimulates the release of GABA into
the synaptic cleft which diffuses across the cleft to the target receptors on the post-
synaptic surface. In the brain, there are two major subtypes of GABA target receptors,
GABAA and GABAB (Table 2.1).
The GABAA receptor is directly coupled to a chloride (Cl• ) ionophore or channel.
When GABA binds to the GABAA receptor, the permeability of the post-synaptic
membrane to Cl• increases and Cl• flows into the post-synaptic cell through the Cl•
channel. This influx of Cl• drives the membrane potential toward the equilibrium
potential of Cl• , which is more negative than the resting potential. This hyperpolarization
of the post-synaptic membrane by Cl• makes the neuron less sensitive to the
depolarization resulting from the opening of voltage-gated Na+ channels. This reduces the
probability that an action potential can be initiated by the action of excitatory
neurotransmitters. The permeability of the membrane to Cl• induced by GABA is long
lasting (seconds) compared with the rapid depolarizing effects of Na+ (milliseconds), so
GABA will induce a slow, inhibitory post-synaptic response.
The GABAA receptor-ionophore complex is believed to be made up of five similar
subunits (Burt and Kamatchi, 1991). Five binding domains have been identified
pharmacologically: (1) the GABA binding site to which both agonists, such as muscimol
from the mushroom Amanita muscaria, and antagonists, such as the convulsant bicuculline,
can bind; (2) the benzodiazepine site to which benzodiazepines such as the drugs diazepam
(Valium) and chlordiazepoxide (Librium) bind as well as several other non-
benzodiazepines, including β-carbolines, cyclopyrrolones, and imidazopyridines; (3) the
barbiturate site to which barbiturates such as phenobarbital and pentobarbital bind; (4) the
neuroactive steroid site to which steroids bind; and (5) the Cl• binding site to which
channel blockers such as the plant alkaloid metabolite picrotoxin or the compound t-
butylbicyclophosphorothioate (TBPS) can bind. Binding of the benzodiazepines,
barbiturates, and neuroactive steroids to the GABA site potentiate GABA action, resulting
 Table 2.1 Nervous system receptors and their associated neurotransmitters, agonists, and antagonists.

GABA, γ-aminobutyric acid; NMDA, N-methyl-D-aspartate; D-AP5, D-amino-5-phosphonopentanoiacid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole

propionic acid; NBQX, 6-nitro-7-sulfamobenzo[f]quinoxaline-2, 3-dione; GAMS, γ-D-glutamylaminomethylsulfonate; L-AP4, 1, 2-amino-4-
RUSSELL L.CARR AND JANICE E.CHAMBERS 35

phosphonopropionic acid; ACPD, aminocyclopentyldicarboxylic acid; L-AP3, L-2-amino-3-phosphonopropionic acid.

36 TOXIC RESPONSES OF THE NERVOUS SYSTEM

in depression of the CNS. Binding of a channel blocker to the Cl• site results in a state of
uncontrolled excitation.
It has been suggested that the GABAA receptor has been conserved over vertebrate
evolution (Cole et al., 1984; Eshleman and Murray, 1990). However, such sweeping
statements about the receptor as a single unit cannot be made. Although some of the
binding sites on the GABAA receptor in fish brain are similar to those in other species,
there are insufficient data on the physiologic effects of pharmacologic agents in fish to
compare them with other species.
The subunits of the GABA binding site in goldfish demonstrate about 75 percent
homology with those of rodent and bird (Bahn et al., 1996). Two binding sites for γ-
aminobutyric acid have been demonstrated in channel catfish brain (Mathis and Tunnicliff,
1984), and similar dual binding systems for γ-aminobutyric acid have been demonstrated
in mammals (Olsen et al., 1981; Tunnicliff and Smith, 1981). The potencies of several
inhibitors of [3H]-GABA binding were similar in the channel catfish to that reported or
rats (Mathis and Tunnicliff, 1984). The receptor density, affinity constants, and specific
activity for [3H]-muscimol binding in codfish (Gadus morhua morhua) brain are similar to
those in mammalian brain (Deng et al., 1991). Additionally, the effect of different free
fatty acids on the binding of [3H]-muscimol was similar among seventeen fish species and
various mammals and amphibians (Witt and Nielsen, 1994). However, there may be
differences in the functional aspects of the site. The stimulation of 36C1• uptake into brain
membrane vesicles by the agonists GABA and muscimol was significantly lower in
freshwater eel (Anguilla anguilla) brain than in rat brain (Corda et al., 1989). Additionally,
the antagonist bicuculline inhibited 36C1• uptake into brain membrane vesicles in
freshwater eel brain to a greater extent than in rat brain.
The binding site for benzodiazepines has been identified in fish brain as well. This site is
suggested to be highly conserved, but only with higher bony fish and tetrapods. For
example, it has been suggested that cyclostomes and elasmobranchs do not possess
benzodiazepine binding sites (Fernholm et al., 1979), whereas teleosts, mammals, and
birds possess these sites that have similar affinities (Friedl et al., 1988; Deng et al., 1991)
but are composed of different subunits (Hebebrand et al., 1987). Additionally, there seems
to be an age-dependent decrease in benzodiazepine binding sites in both rats and
freshwater and saltwater teleosts (Corda et al., 1991; Giannaccini et al., 1997). At least
three benzodiazepine binding sites have been postulated to exist in the brain of marine
flatfish plaice (Pleuronectes platessa) and codfish (Nielsen et al., 1978) but only one has been
identified in the brain of saltwater mullet (Mullus surmeltus) (Giannaccini et al., 1997). In
the saltwater mullet, the benzodiazepine recognition site was similar to that in mammalian
brain, but was not identical pharmacologically (Giannaccini et al., 1997). Wilkinson et al.
(1983) reported only one benzodiazepine binding site in rainbow trout brain but
Eshleman and Murray (1989) utilized several different ligands and found two sites in
rainbow trout brain which were similar pharmacologically in some aspects to mouse, calf,
and human brain but which were different in others. Thus, multiple benzodiazepine
binding sites may exist in both mammalian and fish brain but they are not
pharmacologically identical. Additionally, differences in the benzodiazepine binding site
exist between different species of fish. Witt and Nielsen (1994) demonstrated differences
 RUSSELL L.CARR AND JANICE E.CHAMBERS 37

in the brain benzodiazepine binding site of several fish species by comparing the effect of
different free fatty acids on the binding of [3H]-diazepam to brain membranes. However,
although different, it appears that the benzodiazepine binding sites in fish serve similar
physiologic functions to those of mammalian systems (Rehnberg et al., 1989).
Review of the literature found little information about the barbiturate site in fish.
However, pentobarbital acted as an agonist and stimulated the influx of 36C1• into trout
brain synaptoneurosomes (Eshleman and Murray, 1991). This is consistent with the
normal response of pentobarbital binding to the GABAA receptor. Likewise, review of the
literature found little information about the neuroactive steroid site in fish. However, the
GABAA receptor has been demonstrated to be involved in the release of gonadotropin
from goldfish pituitary (Kah et al., 1992; Trudeau et al., 1993); thus a possible feedback
binding site may exist for sex-related steroids.
The Cl• binding site (picrotoxm/TBPS site) has been identified in several species of
fish. The binding characteristics of [35S]-TBPS in the brain of channel catfish (Carr et al.,
1998), Sacramento blackfish (Orthodon microlepidotus) (Cole et al., 1984) and rainbow trout
(Eshleman and Murray, 1990) are similar to reported values for human, cow, rat, and
chicken brain. Thus, based on binding studies, it can be hypothesized that the picrotoxin/
TBPS binding site in fish and mammals may be similar in many aspects. However, the
functional interaction of this site with the other binding sites on the GABAA receptor
differs between mammals and fish. For example, modulation of [35S]-TBPS binding to the
GABAA receptors by the GABA site ligands (GABA, muscimol, and bicuculline) was
different in freshwater eel and rat brain (Corda et al., 1989). Whereas GABA and
muscimol completely inhibited [35S]-TBPS binding in rat brain, less inhibition was
observed in freshwater eel brain. Bicuculline significantly increased [35S]-TBPS binding in
freshwater eel brain but did not in rat brain. Additionally, there may be differences in the
picrotoxin/TBPS site between fish species. The number of picrotoxin/TBPS binding sites
in freshwater eel brain was similar to those reported for the species of fish listed above but
the affinity of these sites was twofold lower (Corda et al., 1989), whereas the affinity of
the picrotoxin/TBPS binding sites in mosquitofish (Gambusia affinis) was similar to those
reported for the species of fish listed above but the number of binding sites was lower
(Bonner and Yarbrough, 1987).
Less is known about the GABAB receptor. The GABAB receptor is thought to be
indirectly associated with a potassium (K+) ionophore or channel (Bowery, 1989). When
GABA binds to the post-synaptic GABAB receptor, cyclic adenosine 3′, 5′-cyclic
monophosphate (cAMP) production is decreased via intracellular mechanisms mediated
by G proteins. This may result in the dephosphorylation of the K+ channel, which opens
the channel and increases the outward movement of K+. The outward movement of K+ is
normally associated with repolarization of the membrane of a neuron after it has been
depolarized during an action potential. If K+ is flowing outward during a stimulus, the
membrane potential will remain at resting potential or even be slightly hyperpolarized. As
with the GABAA receptor, this resistance to the effect of the depolarization resulting from
the opening of voltage-gated Na+ channels reduces the probability that an action potential
can be initiated by the binding of an excitatory neurotransmitter. It has also been
proposed that GABAB can inhibit presynaptic release of the neurotransmitter by causing a
38 TOXIC RESPONSES OF THE NERVOUS SYSTEM

decrease in Ca2+ influx, which is necessary for release, but most work suggests that the
main response of the GABAB receptor is K+ flux (Gähwiler and Brown, 1985). The
GABAB receptor was initially distinguished from the GABAA receptor by its insensitivity
to several GABAA agonists, THIP [4, 5, 6, 7-tetrahydroisoxazolo(5, 4-c)pyridin-3-ol],
APS (3-aminopropane sulfonic acid), and isoguvacine, and to the GABAA antagonist
bicuclline. Also, GABAB can be stimulated by baclofen and blocked by 2-hydroxysaclofen
or phaclofen, whereas GABAA is not. A large amount of work investigating the physiology
of the GABAB receptor has been carried out using the spinal cord of the cyclostome
lamprey eel as a study animal (Leonard and Wickelgren, 1986; Alford and Grillner, 1991;
Christenson and Grillner, 1991; Dubuc et al., 1993; Matsushima et al., 1993; Tegnér et
al., 1993). In teleosts, however, little work has been done on GABAB receptors. The
GABAB agonist baclofen has been used as a pharmacologic tool to eliminate the presence
of GABAB receptors while investigating GABAergic functions in rainbow trout pineal
gland (Meissl and Ekstrom, 1991), catfish retina (Dong et al., 1994; Takahashi et al.,
1995), carp retina (Han et al., 1997), and goldfish retina (Ishida and Cohen, 1988;
Heidelberger and Matthews, 1991; Kah et al., 1992; Trudeau et al., 1993; Matthews et
al., 1994). Although the inhibition of Ca2+ currents by baclofen suggested the presence of
GABAB receptors in the retinal ganglion cells of goldfish using patch clamp methodology,
this inhibition of Ca2+ currents was not sensitive to the GABAB receptor antagonists 2-
hydroxysaclofen or phaclofen (Bindokas and Ishida, 1991). As the GABAB receptor is
found in the cyclostome spinal cord, it could be present in the teleost spinal cord as well.
More recently, a third type of GABA receptor, GABAc, has been identified in
vertebrate retina and to some extent in other parts of the CNS (Lukasiewicz, 1996). As
with GABAA receptors, GABAC receptors are closely associated with a Cl• channel.
However, using fish retina as a source of GABAC and GABAA receptors, these two
receptors have been shown to be pharmacologically, molecularly, and functionally
distinct. The retinal GABAc receptor has been shown to be insensitive to the GABAA agonist
bicuculline in white perch (Roccus americand) (Qian et al., 1997), carp (Han et al., 1997),
hybrid bass (Morone chrysops×Morone saxitilis) (Qian and Dowling, 1995), goldfish
(Matthews et al., 1994), and catfish (Dong et al., 1994; Takahashi et al., 1994, 1995;
Dong and Werblin, 1996). Likewise, the GABAc receptor differs from the GABAB
receptor by its insensitivity to the GABAB agonist baclofen in catfish retina (Dong et al.,
1994; Takahashi et al., 1995). A binding site for the Cl• channel blocker picrotoxin has
also been demonstrated on the GABAC receptor in catfish retina (Takahashi et al., 1994,
1995; Dong and Werblin, 1996). It appears that these receptors play an important role in
the transmission and processing of visual information. Thus, if a toxicant alters
functioning of the GABAc receptor, it could affect the teleost in many ways (e.g.
detection of prey, detection of predators, and detection of changes in photoperiod
necessary for controlling circadian rhythm).

Glutamate system
Glutamate and aspartate mediate the majority of the excitatory synaptic transmission in
neurons. These amino acid transmitters act on ionotropic and metabotropic receptors
 RUSSELL L.CARR AND JANICE E.CHAMBERS 39

(Table 2.1). The ionotropic receptors include the N-methyl-D-aspartate (NMDA), α-

amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), and kainate receptors.
The metabotropic receptors include the aminocyclopentyl dicarboxylic acid (ACPD) and
L-2-amino-4-phosphono-propionic acid (L-AP4) receptors.
The three ionotropic receptors have been identified in fish neurons (Henley and
Oswald, 1988; Maler and Monaghan, 1991; Barnes and Henley, 1994; Peng et al., 1995).
In mammals, the activation of each of the ionotropic receptors opens an ion channel,
allowing the influx of Ca2+ and/or Na+, which depolarizes the neuronal membrane. The
NMDA receptor is activated by the simultaneous binding of glycine and either glutamate
or aspartate, whereas the AMPA and kainate receptors are activated only by glutamate
(Dingledine and McBain, 1994). There is a great deal of similarity between these
receptors in fish and other species. The cDNA sequence coding for a portion of the
NMDA receptor in the electric fish Apteronotus leptorhynchus demonstrated 80 percent
homology with DNA coding for the rat, human, and duck receptor (Bottai et al., 1997).
The cDNA sequence coding for the AMPA receptor in the brain of the tilapia Oreochromis
mossambicus demonstrated 85 percent homology with the DNA coding for the rat brain
receptor (Kung et al., 1996; Chang et al., 1998). Additionally, antibodies against specific
domains of the AMPA receptor recognized similar proteins in rat, human, frog, chick, and
goldfish brain samples (Bahr et al., 1996). A purified kainate receptor polypeptide from
goldfish brain containing the kainate binding site exhibited a similar size and an amino acid
sequence homology of 40–60 percent compared with that of frog, chick, and pigeon brain
(Ziegra et al., 1992). However, the size of the kainate binding site in goldfish brain is very
different from that in mammalian brain (Murphy et al., 1993). Additionally, a kainate
binding site, which is thought to be a metabotropic receptor rather than an ionotropic
receptor, has been identified in goldfish brain but not in mammals (Willard et al., 1991;
Barnes et al., 1993; Wo and Oswald, 1996).
The ionotropic receptors display a similar mechanism (mediate the influx of Ca2+ and/
or Na+) in fish to that in other species. Specific agonists for all three ionotropic receptors
have been shown to increase Ca2+ influx into channel catfish retinal horizontal cells (Linn
and Christensen, 1992). Additionally, the stimulated Ca2+ influx by each agonist was
blocked by its specific receptor antagonist. This is similar to that observed in mammalian
systems.
In mammals, the activation of metabotropic ACPD receptors by glutamate activates
phospholipase C to convert phosphoinositides to inositol 1, 4, 5-trisphosphate (IP3),
which in turn stimulates the release of intracellular Ca2+ (Dingledine and McBain, 1994).
In mammals, the activation of L-AP4 receptors by glutamate activates phosphodiesterase
to hydrolyze a cyclic nucleotide (i.e. cAMP/cGMP to AMP/GMP) which causes the
ionotropic Ca2+ ion channels to remain open, thus hyperpolarizing the neuron and leading
to reduced synaptic transmission (Dingledine and McBain, 1994). Metabotropic glutamate
receptors have been identified in goldfish olfactory epithelium (Cao et al., 1998), and
channel catfish olfactory neurons (Medler et al., 1998). The ACPD receptor-mediated
stimulation of IP3 formation has been demonstrated in the olfactory rosettes of Atlantic
salmon (Pang et al., 1994), in salmon brain (Kubokawa et al., 1996), and in carp retinal
cells (Janssen-Bienhold et al., 1994). However, little is known about the physiologic role
Other documents randomly have
different content
 By the late Lieut.-Colonel JOSEPH ANDERSON, C.B., K.H.
With Photogravure Portrait. Demy 8vo. 10s. 6d. net.
The late Lieut.-Colonel Joseph Anderson was born in 1790, and from
the age of fifteen, when he received a commission as Ensign in the
78th Regiment, to within a few years of his death in 1877, his career
was almost continuously as adventurous as it was distinguished. In
1806 he saw active service for the first time, when he took part in
the expedition to Calabria; in the following year he served in the
Egyptian Campaign of that date; and during the Peninsular War he
fought at the battles of Maida, Busaco, Fuentes d'Onoro, was
wounded at Talavera, and accompanied Wellington on the retreat to
the lines of Torres Vedras. A few years later Captain Anderson, now
a Captain in the York Chasseurs, was sent with his regiment to
Barbadoes, and was present at the capture of Guadeloupe in 1815.
He was appointed Colonel Commandant of the Penal Settlement at
Norfolk Island in 1834, where his humane endeavours to reform the
prevailing penal system, and his efforts to quell mutinous convicts,
met with marked success. Nine years later Colonel Anderson went to
India to take part in the Mahratta Campaign, and at the Battle of
Punniar (where he commanded a Brigade) was severely wounded
when charging the enemy's guns. After retiring from the Service,
Colonel Anderson settled down in Australia, and it was at his home
near Melbourne that these memories were compiled, during the later
years of a strenuous and active life, for the edification of his family.
They are written in a simple, unaffected style, which renders them
peculiarly readable, and form a most instructive record of the
manners and customs, of the mode of warfare, and the military and
social life of a past age, and a bygone generation.

MEMORIES OF A SOLDIER'S LIFE.

 By Major-General Sir H. M. BENGOUGH, K.C.B.
With Portrait. Demy 8vo. 8s. 6d. net.
Major-General Sir H. M. Bengough joined the army in 1855, and
retired in 1898, after more than forty years of distinguished service
in all quarters of the Empire. His first experience of active warfare
dates from the Crimea; later on he took the field in the Zulu War and
the Burma Expedition of 1885. In days of peace he held various high
commands in India, South Africa, and Jamaica, and finally
commanded a brigade of infantry at Aldershot. In this volume of
personal recollections the author narrates the many varied incidents
and experiences of a long military career and vividly describes the
campaigns in which he took part. He also gives an interesting
account of his adventures in the realm of sport—pig-sticking, tiger-
shooting, and pursuing other forms of game in India and elsewhere;
subjects upon which a long experience enables him to write with
expert knowledge. It will be strange indeed if so interesting an
autobiographical volume from the pen of a deservedly popular
soldier and sportsman fails to appeal to a wide public.

ZACHARY STOYANOFF.
Pages from the Autobiography of a Bulgarian
Insurgent.
Translated by M. POTTER.
One Volume. Demy 8vo. 10s. 6d. net.
In this volume Zachary Stoyanoff gives us the narrative of his
personal experiences during the Bulgarian outbreaks of 1875 and
1876. Almost by accident he became an "apostle" of rebellion, and
was sent out forthwith to range the country, stirring up the villagers
and forming local committees. It is an amazing story. With
unsurpassable candour he portrays for us the leaders, their
enthusiasm, their incredible shortsightedness, and the pitiful
inadequacy of their preparations. The bubble burst, and after a
miserable attempt at flight, Stoyanoff was taken prisoner and sent to
Philippopolis for trial. There is no attempt at heroics. With the same
Boswellian simplicity he reveals his fears, his cringing, his mendacity,
and incidentally gives us a graphic picture, not wholly black, of the
conquering Turk. The narrative ends abruptly while he is still in peril
of his life. One is glad to know that, somehow, he escaped. A very
human document, and a remarkable contrast to the startling
exhibition of efficiency given to the world by the Bulgarians in their
latest struggle with the Turks.

SPLENDID FAILURES.
By HARRY GRAHAM,
Author of "A Group of Scottish Women," "The Mother of
Parliaments," etc.
With Portraits. Demy 8vo. 10s. 6d. net.
It is perhaps unlikely that any two individuals will agree as to the
proper definition of the term "A Splendid Failure"—a phrase of which
the origin would appear to be obscure. It may, however, be roughly
stated that the "Splendid Failures" of the past divide themselves
naturally into three classes: those whom their contemporaries
invested with a fictitious or exaggerated splendour which posterity is
quite unable to comprehend or appreciate; those whom the modern
world regards with admiration—but who signally failed in impressing
the men of their own generation; and those who, gifted with genius
and inspired with lofty ideals, never justified the world's high opinion
of their talents or fulfilled the promise of their early days. In this
volume of biographical essays, the author of "A Group of Scottish
Women" and other popular works has dealt with a selection of
"splendid failures" of whose personal history the public knows but
little, though well acquainted with their names. Wolfe Tone, "the first
of the Fenians"; Benjamin Haydon, the "Cockney Raphael"; Toussaint
L'Ouverture, the "Napoleon of San Domingo"; William Betty, the
"Infant Roscius"; and "Champagne" Townshend, the politician of
Pitt's day, may be included under this category. The reader cannot
fail to be interested in that account which the author gives of the ill-
fated Archduke Maximilian's attempt to found a Mexican monarchy;
in his careful review of the work and character of Hartley Coleridge;
and in his biographical study of George Smythe, that friend of
Disraeli whom the statesman-novelist took as his model for the hero
of "Coningsby." This book, which should appeal strongly to all
readers of literary essays, is illustrated with eight excellent portraits.

THE CORINTHIAN YACHTSMAN'S HANDBOOK.

By FRANCIS B. COOKE.
With 20 Folding Plates of Designs for Yachts, and numerous
black and white Illustrations. Demy 8vo. 10s. 6d. net.
This new handbook covers the sport of yachting in all its branches.
The writer, who has had many years' experience of cruising and
racing in yachts and boats of all types, has treated the subject in a
thoroughly practical manner. The book is divided into six parts.
In Part I., which deals with the selection of a yacht, the various
types and rigs suitable for Corinthian yachting are discussed. The
designing and building of new craft are also dealt with at some
length, and designs and descriptions of a number of up-to-date
small cruisers are given.
In Part II. some hints are given as to where to station the yacht. All
available headquarters within easy reach of London are described,
and the advantages and disadvantages of each pointed out.
Part III. is devoted to the equipment of yachts, and contains a
wealth of information as to the internal arrangement, rigging, and
fittings of small cruisers.
Part IV. treats of the maintenance of small cruising vessels, with
notes on the cost of upkeep, fitting out and laying up. Other matters
dealt with in this section are the preservation of sails and gear, and
insurance.
Part V., on seamanship, covers the handling of fore-and-aft vessels
under all conditions of weather, and upon every point of sailing.
Part VI. covers the racing side of the sport in a comprehensive
manner. An exhaustive exposition of the International Sailing Rules is
followed by hints on racing tactics. The appendix contains, inter alia,
an illustrated description of the British Buoyage System.
Mr. Cooke's well-known handbooks have come to be regarded by
yachtsmen as standard works, and a new and more ambitious work
from his pen can hardly fail to interest them.

THE FALL OF PROTECTION.

By BERNARD HOLLAND, C.B.,
 Author of "Imperium et Libertas."
One Volume. Demy 8vo. 12s. 6d. net.
This volume is a political-historical study of the great change which
took place in British commercial and financial policy mainly between
the years 1840 and 1850. The writer examines the state of things in
these respects which existed before this revolution, and describes
the previous protective system, navigation system, and colonial
system. He then narrates the process by which those systems were
overthrown, devoting special attention to the character, career, and
changes in opinion of Sir Robert Peel, and to the attitude and action
of the Tory, Whig, and Radical parties, and of their leading men,
especially Mr. Disraeli, Lord John Russell, and Mr. Cobden. He
analyses with care the arguments used on all sides in these
controversies, especially with regard to the Repeal of the Corn Laws,
and he shows the extent to which questions of imperial preference
and the relations between the United Kingdom and the Colonies
entered into the issues. One chapter is devoted to the Bank Act of
1844, and to the consideration of its causes and results. The author
concludes by tracing very briefly the chain of events which connect
the period in question with our own day, in respect of commercial
and fiscal policy, and expresses his own views as to existing
tendencies and future developments.
Mr. Bernard Holland is known as the author of the Life of the Duke of
Devonshire, and of "Imperium et Libertas." In a sense the present
volume is a continuation of the latter book, or rather is an attempt to
deal more expansively and in detail with certain history and
questions connected with the same theme, for the full treatment of
which there was insufficient space in that book. Mr. Holland having
acted for a number of years as Private Secretary to two successive
Secretaries of State for the Colonies, has been brought into close
touch in a practical way with colonial questions. This book, it is
hoped, will be of some service both to students of economic history
and to politicians in active life.
 PAINTING IN THE FAR EAST.
By LAURENCE BINYON.
A New Edition, thoroughly Revised, with many new and
additional Illustrations. Crown 4to. 21s. net.
Since the first edition of this book was published in 1907, much has
happened, and a quantity of new material has been brought to light.
Interest in the subject has been immensely widened and
strengthened. The museums of Europe and America are vying with
each other to procure fine specimens of Chinese and Japanese art.
The opening this autumn of a new museum at Cologne, exclusively
devoted to the arts of Eastern Asia, is a symptom of the times.
Collections, public and private, both European and American, have
been greatly enriched; and the exhibition in 1910 at Shepherd's
Bush, of treasured masterpieces lent from Japanese collections, has
provided a standard for the student.
Six years ago, again, scarcely any of the voluminous literature of art
existing in Chinese and Japanese had been translated. On this side,
too, an added store of information has been made accessible,
though still in great part scattered in the pages of learned
periodicals. Above all, the marvellous discoveries made of recent
years in China and Chinese Turkestan have substituted a mass of
authentic material for groping conjectures in the study of the art of
the early periods.
In preparing a new edition of this book and bringing it up to date,
Mr. Binyon has therefore been able to utilize a variety of new sources
of information. The estimates given of the art of some of the most
famous of the older masters have been reconsidered. The sections
dealing with the early art have been in great measure rewritten; and
the book has been revised throughout. In the matter of illustrations
it has been possible to draw on a wider range and make a fuller and
more representative selection.

PAINTING IN EAST AND WEST.

By ROBERT DOUGLAS NORTON,
Author of "The Choice."
Crown 8vo. 5s. net.
The art of painting, which in the days of Gothic church-building
contributed so much both to the education and the pleasure of the
community at large, has admittedly come to appeal to ever-
narrowing circles, until to-day it cannot be said to play any part in
popular life at all. This book seeks to discover the causes of its
decline in influence. A brief review of the chief contemporary
movements in painting gives point to a suggestion made by more
than one thoughtful critic that the chief need of Western painting is
spirituality. Since this is a quality which those competent to judge
are at one in attributing to Eastern art, the author, in a chapter on
Far Eastern Painting, sets forth the ideals underlying the great
painting of China and Japan, and contrasts these ideals with those
which have inspired painters and public in the West. This leads to an
inquiry into the uses of imagination and suggestion in art, and to an
attempt to find a broad enough definition for "spirituality" not to
exclude many widely divergent achievements of Western painting.
Finally, the possibility of training the sense of beauty is discussed in
the light of successful instances.
Incidentally the book touches on many questions which, though of
interest to picture-lovers, often remain unasked; such, for instance,
as what we look for in a picture; how far subject is important; why it
may happen that the interest of one picture, which pleases at first,
soon wanes, while that of another grows steadily stronger; the value
of technique, of different media of expression, of mere resemblance,
etc.
Without going into the technicalities of æsthetics, the author aims at
investigating certain first principles which are overlooked at times by
possessors of even the widest knowledge of individual schools.

SHAKESPEARE'S STORIES.
By CONSTANCE MAUD and MARY MAUD.

As You Like It—The Tempest—King Lear—Twelfth Night—The

Merchant of Venice—A Midsummer Night's Dream—Macbeth—
Hamlet—Romeo and Juliet.

With Illustrations from the famous Boydell prints. Crown

8vo.
5s. net.
Miss Constance Maud is the author of "Wagner's Heroes" and
"Wagner's Heroines," two books on similar lines to these tales which
have had a great vogue among young people of all ages. In the
present volume she tells the charming stories of nine of the most
famous of Shakespeare's Tragedies and Comedies in prose of
delightful and unstudied simplicity. On occasion the actual text has
been used for familiar passages and phrases. These great world-
tales, regarded merely as tales, with the elemental motives and
passions displayed in them, appeal strongly to the imagination, and
when narrated by a competent pen there cannot be finer or more
absorbing reading. In addition to this, he must be a dull reader in
whom they do not awaken a desire to make a closer acquaintance
with the plays themselves.
The book forms a companion volume to Sir A. T. Quiller-Couch's
well-known "Historical Tales from Shakespeare."

THE MUSE IN MOTLEY.

By HARRY GRAHAM.
Author of "Ruthless Rhymes for Heartless Homes," etc., etc.
With 24 Illustrations by Lewis Baumer.
Fcap. 4vo. 3s. 6d. net.
All lovers of humorous verse will welcome a fresh volume of lyrics by
the author of "Deportmental Ditties," "Canned Classics," and other
deservedly popular products of the Minor Muse. Readers of Captain
Graham's new collection of light verse will agree with the Daily
Chronicle in describing its author as "a godsend, a treasure trove, a
messenger from Olympus; a man who really does see the ludicrous
side of life, a man who is a genuine humorist." Once again the
author of these amusing poems attempts to "shoot Folly as she
flies," and genially satirizes the foibles of the age in a fashion that
will certainly add to his reputation as a humorist; and his work is
rendered still more delightful by the drawings of Mr. Lewis Baumer,
the well-known Punch artist, with which it is lavishly illustrated. "It is
a great and good thing," as the Pall Mall Gazette remarked with
reference to another of Captain Graham's books, "to have a man
among us who is witty all the time and lets himself go. We ought to
be duly thankful. And we are!"
 HANNIBAL ONCE MORE.
By DOUGLAS W. FRESHFIELD, M.A.,
Vice-President of the Royal Geographical Society; Treasurer of the
Hellenic and Roman Societies; formerly President of the Alpine
Club.
8vo. 5s. net.
In this little volume Mr. Freshfield has put into final shape the results
of his study of the famous and still-debated question: "By which
Pass did Hannibal cross the Alps?" The literature which has grown up
round this intricate subject is surprisingly extensive, and various
solutions have been propounded and upheld, with remarkable
warmth and tenacity, by a host of scholars, historians, geographers,
military men, and mountaineers. Mr. Freshfield has a solution of his
own, which, however, he puts forward in no dogmatic spirit, but in
such a fashion that his book is practically a lucid review of the whole
matter in each of its many aspects. To an extensive acquaintance
with ancient and modern geographical literature he unites a wide
and varied experience as an alpine climber and a traveller, and a
minute topographical knowledge of the regions under discussion;
and these qualifications—in which many of his predecessors in the
same field of inquiry have been conspicuously lacking—enable him
to throw much new light on a perennially fascinating problem.

THE PASTORAL TEACHING OF ST. PAUL.

 By the Rev. Canon H. L. GOUDGE,
Principal of the Theological College, Ely; Author of "The Mind of
St. Paul," etc.
Crown 8vo. Cloth. 2s. 6d. net.
These lectures were delivered at the end of May, 1913, at the
Palace, Gloucester, to the clergy of the diocese, and are now
published in response to the request of those who heard them. They
do not constitute a detailed commentary on the Pastoral Epistles,
though a good deal of detailed exegesis necessarily finds a place in
them. The writer's aim has been to collect and arrange St. Paul's
teaching as to the work of the Christian pastor, and to point out its
applicability to modern conditions and modern difficulties. The writer
has often found, through his experience in conducting Retreats, that
the Pastoral Teaching of St. Paul is of the greatest value to the clergy
to-day, but that this teaching is often obscured by the unsystematic
character of St. Paul's writing and by the passing controversies with
which he has to deal. In these lectures the First Epistle to Timothy is
used as the basis, but continually illustrated by passages from the
other Pastoral Epistles, and from St. Paul's earlier writings. The first
lecture deals with the pastor's aim, the second with the pastor's
character, the third with the pastor's work, and the fourth with the
adaptation of his message to men and to women, to old and to
young, to rich and to poor. The ground already covered by the
writer's earlier book, "The Mind of St. Paul," has been carefully
avoided, but it is hoped that the one book may throw light upon the
other. An index of texts has been added for those who may wish to
use this second book, as far as that is possible, as a commentary.

NEW NOVELS
 SOMETHING AFAR.
By MAXWELL GRAY,
Author of "The Silence of Dean Maitland," "The Great Refusal,"
etc.

Crown 8vo. Cloth. 6s.

The scene of Maxwell Gray's new story is laid in London and in Italy,
where the gradual unfolding of an elaborate but absorbing plot holds
the reader's attention until the very last page of the book. This is a
tale of heroism, of self-sacrifice, of romance, full of incident and
adventure, illumined by those tender and imaginative touches, that
vivid portrayal of character, which the public has learnt to expect
from the author of "The Silence of Dean Maitland." From these
pages we may learn that there is "something afar from the sphere of
our sorrow," the highest aspiration of the lover, the artist, the poet
and the saint, which, beautiful beyond all that man's heart can
divine, is yet within the reach of every one of us.

THE GENTLE LOVER.

A Comedy of Middle Age.
By FORREST REID,
Author of "The Bracknells," "Following Darkness," etc.
Crown 8vo. 6s.
This extremely interesting story, of which the title gives a most apt
description, is written in a lighter vein than the author's previous
work. It is a love story, and while the tale itself is enthralling, it
depends in great measure for its charm on the attractiveness of the
characters who figure in the drama and who are all very pleasant
company. The book is essentially human, the note is never forced,
yet the interest goes on increasing right up to the end. It is actual
life with its comedy and tragedy so closely intermingled that it is not
always easy to distinguish one from the other. The scene is laid
abroad, partly in Bruges, and partly in Italy, but the characters are,
with one or two exceptions, natives of that part of Ireland with
which the author is most familiar, and they lose none of their
individuality by being transplanted to those beautiful old-world cities
where we follow their varied fortunes. Mr. Reid's previous novels
have already secured for his work the warm appreciation of some of
the best judges of literary values, and the present novel may be
confidently stated to exhibit his undoubted power as a writer of
fiction in an advanced and progressive stage.

NEW SCIENTIFIC WORKS

INDUSTRIAL POISONING
From Fumes, Gases, and Poisons of
Manufacturing Processes.
By Dr. J. RAMBOUSEK,
 Professor of Factory Hygiene, and Chief State Health Officer,
Prague
Translated and Edited by Dr. T. M. LEGGE,
H.M. Medical Inspector of Factories.
Fully Illustrated. Demy 8vo. 12s. 6d. net.

MALINGERING
And Feigned Sickness.
By Sir JOHN COLLIE, M.D., J.P.,

Medical Examiner, London County Council; Chief Medical Officer,

Metropolitan Water Board; Consulting Medical Examiner to the
Shipping Federation; Medical Examiner to the Sun Insurance Office,
Central Insurance Company, London, Liverpool, and Globe Insurance
Company, and other Accident Offices; late Home Office Med. Ref.
Workmen's Compensation Act.

Assisted by ARTHUR H. SPICER, M.B., B.S. (Lond.), D.P.H.

Illustrated, xii + 340 pp. Demy 8vo. 10s. 6d. net.
In this work Sir John Collie, whose wide experience has eminently
fitted him for the task, has given an interesting and lucid description
of the methods and peculiarities of the malingerer. He describes fully
and in detail the methods of examination for the detection of
malingering and the diseases usually simulated, and discusses the
attitude required by the medical attendant towards unduly prolonged
illness.
 OLD AGE:
Its Care and Treatment in Health and Disease.
By ROBERT SAUNDBY, M.D., F.R.C.P., L.L.D., J.P.,

Member General Medical Council: Ex-President British Medical

Association; Professor of Medicine, University of Birmingham;
Physician to the Birmingham General Hospital.

320 pp. 7s. 6d. net.

No English writer having recently dealt with this subject, it has been
felt that there is room for a book which should bring together the
various contributions made to it in modern times, including the
results of the author's extensive experience during forty years of
medical practice. The author discusses the principles of health, by
due attention to which healthy old age may be attained. The
diseases to which the aged are especially liable are fully described,
their causes are clearly indicated, and the author shows in a
practical way by what means they may be avoided and how they
may be appropriately treated. Special attention is given to such
important subjects as diet, exercise, etc. Suggestive dietary tables
are given, both for use in health and in particular diseases, while the
chapters devoted to methods of exercise most suitable in advanced
age will also prove of value.

LONDON: EDWARD ARNOLD, 41 & 43 MADDOX STREET, W.

 Footnotes:

[1] Napoleon III.

[2] Lord Lyons to Lord J. Russell, July 9.
[3] Lord Lyons to the Duke of Newcastle, Oct. 29.
[4] Lord Lyons to Mr. Griffith, Nov. 10.
[5] Lord Clarendon, upon the death of Lord Palmerston, became
Foreign Secretary in place of Lord Russell.
[6] British minister at Bucharest.
[7] Lord Lyons to Mr. Stuart.
[8] In consequence of the change of Government, Lord Stanley
(subsequently Earl of Derby) had now become Foreign Secretary.
[9] It used to be said that it took a Franco-German war to secure
the correct spelling of this name. It is certainly a curious fact that
another Foreign Secretary also used to spell it incorrectly.
[10] Alaska.
[11] The vanity which was responsible for Prince Gortschakoff's
love of conferences is frequently referred to in Busch's 'Bismarck.'
[12] Subsequently Lord Ampthill.
[13] Prussian Ambassador in London.
[14] Now Wilhelmshafen.
[15] British Ambassador at Berlin.
[16] Lord Lyons to Lord Clarendon, Feb. 18, 1870.
[17] As Minister for Foreign Affairs.
[18] French Ambassador at Berlin.
[19] 'The Life of Lord Granville.'
 [20] Bavarian Minister.
[21] Prussian Under Secretary of State for Foreign Affairs.
[22] Foreign Minister.
[23] Representative at Tours of the French Foreign Office.
[24] 'Memoirs of Sir Robert Morier.'
[25] Minister at Madrid; subsequently Ambassador at
Constantinople.
[26] Col. the Honble. Percy Fielding.
[27] Now Sir Frank Lascelles, G.C.B.
[28] Now Lord de Saumarez.

Transcriber's notes:
P.ix. 'inpressions' changed to 'impressions'.
P.27. 'proferred' changed to 'proffered'.
P.58. 'on or' changed to 'or on'.
P.120. 'inclned' changed to 'inclined'.
P.192. 'Russia' changed to 'Prussia'.
P.256. 'ne' changed to 'me'.
Various punctuation fixed.
*** END OF THE PROJECT GUTENBERG EBOOK LORD LYONS: A
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