Target Organ Toxicity in Marine and Freshwater Teleosts

Organs - 1st Edition

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 Contents
List of contributors vii
Foreword viii
Copyright acknowledgments ix
1 Toxic responses of the gill
CHRIS M.WOOD 1
2 Target organ toxicity in the kidney
BODIL K.LARSEN AND EVERETT J.PERKINS JR 102
3 Toxic responses of the skin
JAMES M.MCKIM AND GREGORY J.LIEN 165
4 Toxic responses of the liver
DAVID E.HINTON, HELMUT SEGNER, AND THOMAS
BRAUNBECK 248
5 Response of the teleost gastrointestinal system to xenobiotics
KEVIN M.KLEINOW AND MARGARET O.JAMES 299
Index 395
 Contributors

Thomas Braunbeck is at the Department of Zoology I, University of Heidelberg,

Germany
David E.Hinton is at the Nicholas School of the Environment, Duke University,
Durham, NC, USA
Margaret O.James is at the Department of Medicinal Chemistry, University of Florida,
FL, USA
Kevin M.Kleinow is at the Department of Comparative Biomedical Sciences, School of
Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA
Bodil K.Larsen is at the Department of Pharmacology, University of Mississippi, MS,
USA
Gregory J.Lien is at the Mid-continent Ecology Division, National Health and
Environmental Effects Laboratory, US Environmental Protection Agency, Duluth,
MN, USA
James M.McKim is at the Mid-continent Ecology Division, National Health and
Environmental Effects Laboratory, US Environmental Protection Agency, Duluth,
MN, USA
Everett J.Perkins Jr is at Eli Lilly and Company, Drug Disposition, Indianapolis, IN,
USA
Helmut Segner is at the Sektion Chemische Okotoxikologie, UFZ-
Umweltforschungszentrum Leipzig-Halle GmbH, Leipzig, Germany
Chris M.Wood is at the Department of Biology, McMaster University, West Hamilton,
ON, Canada
 Foreword

This book provides a timely reminder of how far the field of aquatic toxicology,
particularly with respect to fishes, has progressed in recent years. This field emerged only
two to three decades ago, largely in response to the need for scientifically valid data upon
which to base water quality regulations or criteria. To a considerable degree, therefore,
the field became synonymous with acute and chronic toxicity testing that focused upon
whole-organism effects. And while these remain important activities, many scientists
perceived the importance of elucidating mechanisms underlying interactions of chemicals
with aquatic organisms, including fishes. Such studies contribute to our basic
understanding of toxicological phenomena in these organisms, but also improve our
ability to establish regulations with confidence and provide useful tools for monitoring
(‘biomarkers’). Thus, elegant research is now being performed that is identifying
mechanisms of chemical metabolism, toxicity and adaptation in fish models. These
studies require an understanding of specific organ systems that chemicals interact with,
and cellular and subcellular components of target organs in these models.
Thus, this book provides an immensely useful reference text for scientists pursuing
mechanistic aspects of toxicology in teleost models. Each chapter, written by eminent
scientists in their field, provides a detailed synopsis of the state of the science regarding
our understanding of chemical interactions in a particular organ system. This information
constitutes a solid foundation and springboard for students and researchers to extend our
understanding of mechanistic toxicology in marine and freshwater teleosts. Such efforts
will foster both the maturation of this field and the wise management of chemical releases
into aquatic ecosystems.
Richard T.Di Giulio
Duke University
 Copyright acknowledgments

The authors and publishers would like to thank the following for granting permission to
reproduce material in this work:
John Wiley & Sons, Inc., New York, for permission to reproduce Figure 1.4,
originally published as Figure 16 in Pisam, M. et al. 1987. Two types of chloride cells in
the gill epithelium of a freshwater-adapted euryhaline fish: Lebistes reticulatus; their
modifications during adaptation to saltwater. American Journal of Anatomy 179:40–50.
NRC Research Press, Ottawa, for permission to reproduce Figure 1.8, originally
published as Figure 8 in Mallatt, J. 1985. Fish gill structural changes induced by toxicants
and other irritants: a statistical review. Canadian Journal of Fisheries and Aquatic
Sciences 42:630–648.
Springer-Verlag, Heidelberg, for permission to reproduce Figure 1.9, originally
published as Figures 1, 9 and 10 in Wendelaar Bonga, S.E. et al. 1990. The ultrastructure
of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to
acidified water. Cell and Tissue Research 259:575–585.
Springer-Verlag, Heidelberg, for permission to reproduce Figure 1.10, originally
published as Figure 4a and b in Greco A.M. et al. 1996. The effects of soft-water
acclimation on gill structure in the rainbow trout Oncorhynchus mykiss. Cell and Tissue
Research 285:75–82.
Springer-Verlag, Heidelberg, for permission to reproduce Figure 1.14, originally
published as Figure 7 in Play le, R.C. and Wood, C.M. 1989. Water pH and aluminum
chemistry in the gill micro-environment of rainbow trout during acid and aluminum
exposures. Journal of Comparative Physiology B 159:539–550.
Academic Press for permission to reproduce Figures 3.1, 3.2 and 3.11, originally
published as Figures 2, 3 and 7 in McKim et al. 1996. Dermal absorption of three
waterborne chloroethanes in rainbow trout (Oncorhynchus mykiss) and channel catfish
(Ictalurus punctatus). Fundamental and Applied Toxicology 31:218–228.
Elsevier Science, Oxford, for permission to reproduce Figure 3.6, originally published
as Figure 1A in Comparative Biochemistry and Physiology (1993) 105A: 625–641.
Elsevier Science, Oxford, for permission to reproduce Figure 3.8, originally published
as Figure 3 in Respiration Physiology (1978) 35:111–118.
Academic Press for permission to reproduce Figure 3.9, originally published as Figure
1 in General and Comparative Endocrinology 66:415–424.
Elsevier Science, Oxford, for permission to reproduce Figures 3.10 and 3.13,
originally published as Figures 3 and 4a in Aquatic Toxicology (1993) 27:15–32.
The Society of Environmental Toxicology and Chemistry for permission to reproduce
Figure 3.12, originally published as Figure 1 in Environmental Toxicology and Chemistry
(1994) 13:1195–1205.
Academic Press for permission to reproduce Figure 3.14, originally published as
Figure 1 in Fundamental and Applied Toxicology 31:229–242.
 University of Wisconsin Press, Madison, WI, for permission to reproduce Figure 3.15,
originally published as Figure 34.1 in Ribelin, W.E. and Migaki, G. 1975. The Pathology
of Fishes.
Elsevier Science, Oxford, for permission to reproduce Figure 3.16, originally
published as Figure 14 in Aquatic Toxicology (1988) 11:241–257.
Battele Press, Columbus, OH, for permission to reproduce Figures 3.17 and 3.18,
originally published as Figures 1A and B and 2A and B in Black, J.J. 1982. Polynuclear
Aromatic Hydrocarbons: Formation, Metabolism and Measurement. Cooke, M.W. and
Dennis, A.J. (eds), pp. 99–111.
 1
Toxic responses of the gill
Chris M.Wood

Introduction

The gills of a fish constitute a multifunctional organ (respiration, ionoregulation, acid-

base regulation, nitrogenous waste excretion) accounting for well over 50 percent of the
total surface area of the animal. The branchial epithelium is made up of multiple cell
types and is both delicate and geometrically complex. Because of the low oxygen
capacitance of water, the gills are irrigated externally with a massive ventilatory flow, in
the order of 20 L kg−1 h−1, whereas internally they are perfused with the entire cardiac
output, approximately 2 L kg−1 h−1. The diffusion distance between water and blood is
extremely thin (0.5–10 m), and the effectiveness of diffusive exchange is maximized by
the countercurrent flow of water versus blood at the exchange sites. Given these facts, it
is not surprising that that the gills are not only the major site of uptake for most
waterborne toxicants but also the first, and most important, site of toxic impact for many
of them. All four of the major physiologic processes performed by the gills are essential
for life and are sensitive to both structural and biochemical disturbance of the branchial
epithelium. Interruption of any of them will result in death, whereas sublethal
disturbances will chronically depress the fitness of the fish.

Functional anatomy

External anatomy
The basic anatomy of the gills is well described in the older literature (e.g. van Dam,
1938; Hughes, 1966, 1984; Hughes and Morgan, 1973; Wood, 1974; Laurent and Dunel,
1980; Laurent, 1984; Satchell, 1984). Typically, teleost fish have four gill arches (Figure
1.1 A), each bearing two hemibranchs comprising up to several hundred gill filaments
(Figure 1.1B; primary lamellae in the older literature). The gill filaments are long finger-
like processes (e.g. 5–15 mm) which may be seen with the naked eye. The internal
cartilaginous skeleton of the filaments and the filament abductor and adductor muscles
ensure that, during normal ventilation, the tips of adjacent filaments are held in close
apposition, forming a more or less complete curtain of water channels (see below)
through which the water flow is pumped. When ventilatory volume increases (exercise,
hypoxia, stress), the curtain
Target organ toxicity in marine and freshwater teleosts 2

Figure 1.1 Gross and fine anatomy of the gills of a

typical teleost fish. Drawing courtesy of
S.Wood. (A) Location of four gill arches
(g.a.). (B) Detail of portions of two gill
arches each bearing two hemibranchs (h.)
composed of many gill filaments (g.f.) in
close apposition. (C) Detail of a portion of
one gill arch with two attached gill
filaments, each bearing two rows of
respiratory lamellae (r.l.), illustrating
organization of afferent branchial artery
(a.b.a.), efferent branchial artery (e.b.a.),
 Toxic responses of the gill 3

afferent filamental artery (a.f.a.), and

efferent filamental artery (e.f.a.); arrows
indicate the directions of blood and water
flow. (D) External detail of epithelium of
respiratory lamellae and gill filament
illustrating predominant locations of
mucous cells (m.c.), chloride cells (c.c.),
and pavement cells (p.v.c.), the last
marked by a typical ‘fingerprint’ surface.
(E) Internal detail of respiratory lamellae,
gill filament, and gill arch illustrating
organization of blood vessels: afferent
lamellar arteriole (a.l.a.), efferent lamellar
arteriole (e.l.a.), pillar cell channels
(p.c.c.), nutritive vessels (n.v.),
arteriovenous anastomoses (a.v.a.; which
are much more frequent on the post-
lamellar blood flow side), central venous
sinus (c.v.s.), and venolymphatic drainage
(v.l.d.).

may be pulled apart, so that some of the water flow bypasses the channels and respiratory
gas exchange per unit water flow becomes less efficient. Because of the dense, viscous
nature of water, the cost of ventilation is high even at rest (most estimates are in the range
of 10 percent of resting metabolic rate) and increases greatly during hyperventilation
(Perry and McDonald, 1993).
On each surface of a filament, there are rows of regularly spaced, leaf-like structures,
the respiratory lamellae (or secondary lamellae; Figure 1.1C) which are the actual sites of
gas exchange, analogous to the alveoli of the human lung. The respiratory lamellae are
generally quite thin (10–25 m), but vary greatly among species in both spacing (20–
100 m) and shape (oblong to triangular). Typical lamellar heights and lengths are 100–
500 m and 500–1500 m respectively; the leading edge is generally the highest point
of the structure (Figure 1.1D). The spaces between the respiratory lamellae constitute the
water channels through which the ventilatory flow occurs in a direction opposite to that
of the internal blood flow. In general, more active, pelagic-type species have more
filaments, more closely spaced lamellae, more numerous but smaller individual lamellae,
and greater total lamellar surface area. In a 1-kg fish, the total number of secondary
lamellae might range from 0.5 million in the relatively inactive toadfish to about 6
million in highly active tuna, with corresponding total lamellar surface areas of 1300–13
000 cm2. Lamellar surface area increases and the mean blood-to-water diffusion distance
decreases in euryhaline fish after transfer to seawater (Laurent and Hebibi, 1989). Total
lamellar area is normally taken as the area available for gas exchange, although it should
be realized that water-to-blood diffusion distance will be quite heterogeneous within
different parts of individual lamellae because of their complex internal structure.
 Target organ toxicity in marine and freshwater teleosts 4

Internal anatomy
Internally, the respiratory lamellae are composed of a web-like anastomosis of blood
channels (pillar cell channels; Figure 1.1E) separated and indeed formed by the bodies of
individual pillar cells; these cells run laterally across the width of the lamella, thereby
supporting the two opposite epithelial layers in a brace-like fashion. Bundles of collagen
traverse the lamellae and are anchored to the basement membrane underlying the
epithelium on each side, creating circular depressions in this membrane (Penrice and
Eddy, 1993). In parallel to the collagen columns, the pillar cells contain fibrils of an
actomyosin-like contractile protein, whose function remains conjectural (autoregulation?)
in light of the general belief that the pillar cells are not innervated. The flanges of
individual pillar cells abut, forming a pseudoendothelium which contains the blood space.
Pillar cell channels are so narrow (e.g. 4 m diameter) that erythrocytes must deform as
they transit. Possible pathways for shunting blood flow within the lamellae include a
wider marginal channel around the upper border of each lamellae, through which the
erythrocytes appear to move more quickly, and pillar cell channels at the base of the
lamellae, which are largely buried in the body of the filament and are therefore
unavailable for gas exchange (Tuurala et al., 1984).
Deoxygenated ‘venous’ blood travels along the ventral aorta under arterial pressure
from the heart, then via individual afferent branchial arteries in each arch, individual
afferent filamental arteries running down the trailing margin of each filament, and finally
short afferent lamellar arterioles leading into each respiratory lamella (Figure 1.1C–E).
Oxygenated ‘arterial’ blood leaves the lamellae along short efferent lamellar arterioles
into efferent filamental arteries which course along the leading margin of each filament
and then drain into the efferent branchial artery of each gill arch. The branchial arteries
coalesce to form the dorsal aorta. Thus, the millions of respiratory lamellae are perfused
in parallel with one another at virtually the highest blood pressures (30–60 mmHg)
present in the fish. Together with their associated arteries and arterioles, the lamellae
constitute the branchial vascular resistance which is arranged in series with the
vasculature of the rest of the fish, the systemic resistance. Considering its anatomic
complexity, the overall branchial resistance is amazingly low under normal
circumstances, such that 60–80 percent of the arterial pressure ‘survives’ post-gill in the
dorsal aorta to allow effective perfusion of the much higher systemic resistance with
oxygenated blood.
The preceding paragraph describes the traditional ‘arterioarterial pathway’. However,
much debate has centered on the functional importance of a recurrent ‘arteriovenous’
pathway (also variously called ‘nutritive’, ‘venolymphatic’, ‘secondary’, or ‘Fromm’s
arteries’) which leads post-lamellar blood flow back through a network of vessels or
sinuses (‘central venous sinus’) in the body of the filament and eventually drains into
filamental veins and then branchial veins (Figure 1.1E; Fromm, 1974; Randall, 1985;
Vogel, 1985; Satchell, 1991). The origin of the arteriovenous pathway is usually ascribed
to short arteriovenous anastomoses (AVAs) leaving the efferent filamental arteries and to
longer thin arteries leaving both the efferent filamental and efferent branchial arteries.
However, in a few species, additional AVAs have been reported on the afferental
filamental arteries, which would allow blood flow to bypass the respiratory lamellae all
together. Although early work with perfused gill and head preparations suggested that
arteriovenous blood flow could be very high, these findings were likely biased by
 Toxic responses of the gill 5

abnormal outflow resistances in the in vitro preparations. In vivo, the arteriovenous flow
appears to be less than 10 percent of the arterioarterial flow, and the hematocrit of blood
collected from the branchial vein is only about 15 percent of that in the dorsal aorta,
which indicates considerable ‘plasma skimming’ at the point of entry into the
arteriovenous circulation (Ishimatsu et al., 1988). These measurements indicate that the
ability of this pathway to deliver O2 to the body of the filament is quite limited. Instead,
the overlying filamental epithelium probably gets most of its O2 directly from the water,
and the more important function of the arteriovenous flow is supplying and removing
ions, acid–base equivalents, nutrients, and wastes to and from this epithelium which
appears to be heavily involved in active transport processes (see below).
The possibilities for shunting blood flow within the gills are numerous (Wood, 1974;
Booth, 1979a, b; Farrell et al., 1979a, b; Soivio and Tuurala, 1981; Pettersson, 1983;
Nilsson, 1984; Bailly et al., 1989). Under resting conditions, only the more proximally
located lamellae on the filaments appear to be completely perfused, with the distal
lamellae providing reserve capacity which may be opened up during exercise, hypoxia, or
stress. This may be achieved by increases in inflow (ventral aortic) pressure, outflow
pressure (dorsal aortic) and pressure pulsatility (via greater cardiac stroke volume) which
provide transmural pressures which surpass the critical opening pressures of afferent and
efferent lamellar arterioles or pillar channels themselves in more distal lamellae. At the
same time, higher transmural pressure will increase the thickness of the blood sheet in
individual lamellae, may stretch and thereby thin the epithelium, and may favor flow
through central pillar cell channels and marginal channels where overall blood-to-water
diffusion distances are lower and countercurrent exchange is more efficient.
Catecholamines circulating in the blood plasma will tend to augment these effects by -
adrenergically dilating the afferent arterioles and -adrenergically constricting the
efferent arterioles, although the overall effect is vasodilatory. On the outflow side, the -
constrictory response may also be reinforced by catecholamines released from
sympathetic neural activity. Serotonin, released from indoleaminergic neurons
innervating the efferent filamental sphincters, similarly constricts the outflow of the
arterioarterial pathway and may favor filling of more distal lamellae by the back-pressure
effect. Additionally, catecholamines from either neural or hormonal sources tend to
constrict AVAs so as to favor arterioarterial flow over arteriovenous flow. In contrast,
cholinergic stimulation, probably arising in vivo only from parasympathetic nerves, tends
to reduce the number of lamellae perfused by an unknown mechanism, causes muscarinic
vasoconstriction of the sphincters on efferent filamental arteries, and has an overall
vasoconstrictory effect on the arterioarterial pathway.

The branchial epithelium

The branchial epithelium (Figure 1.1D) covers the lamellar surfaces, the filamental
surface including the region between each respiratory lamella, and extends over the
surface of the gill arches themselves, and even to the neighboring inner opercular surface
in a few species. The epithelium lies on top of a prominent basement membrane (basal
lamina) which is thought to provide important structural support to both filaments and
lamellae (Penrice and Eddy, 1993). The branchial epithelium is made up of four or five
 Target organ toxicity in marine and freshwater teleosts 6

cell types, only the first of which is abundant on the lamellar surfaces (Laurent and
Dunel, 1980; Laurent, 1984).
Pavement cells (respiratory or squamous epithelial cells in the older literature) are
generally large, polygonal in shape and thin, and cover the majority of the gills, including
virtually all of the lamellar surface in healthy, non-stressed fish (Figures 1.1D and 1.2A).
Traditionally, the pavement cells were considered the sites of diffusive gas exchange and
passive ion and water movements, but more recently an additional role in active acid-base
and ion transport has been proposed, specifically in H+ excretion and coupled Na+ uptake
(Goss et al., 1992, 1994, 1995; Laurent et al., 1994a; Perry, 1997). The apical membrane
of the pavement cell is overlaid with glycocalyx and decorated with characteristic
‘fingerprint’ ridges, whorls or microvilli, structures which may have some role in
increasing the surface area available for gas exchange, trapping mucus, and/or in creating
an unstirred boundary layer (Figure 1.2A and B). Pavement cells are closely joined to one
another and to neighboring chloride cells by numerous desmosomes and deep tight
junctions at the apical surface (Figure 1.2B). Based on their impermeability to lanthanum
and their multistranded nature, these junctions are thought to have a high electrical
resistance and low ionic conductance. Internally, the cells have relatively sparse
mitochondria but are rich in other organelles, including well-developed Golgi, abundant
rough endoplasmic reticulum, and numerous vesicles. These last structures are especially
prominent under conditions in which the pavement cells are thought to become ‘active’ in
ion and acid–base regulation (Figure 1.2C). Often, several layers of pavement cells
overlap (generally two in the respiratory lamellae), with underlying ones being more
columnar in shape and less well differentiated (Figure 1.3A). Lacunae between pavement
cell layers appear to communicate with the central venous sinus.
 Toxic responses of the gill 7

Figure 1.2 Electron micrographs of the branchial

epithelium on the trailing edge of the gill
filament in a freshwater rainbow trout.
Plate courtesy of P.Laurent. (A)
Representative scanning electron
micrograph of the epithelium, illustrating
typical surface morphology of pavement
cells (pvc), chloride cells (cc), and mucous
cells (mc). Note the characteristic
Target organ toxicity in marine and freshwater teleosts 8

‘fingerprint’ whorls on pavement cells

compared with the smoother surface of the
chloride cells decorated only with
microvilli, and the similar but smaller
surface exposure of the mucous cell
(1500×). (B ) Representative transmission
electron micrograph of a freshwater-type
chloride cell and its relationship to
neighboring pavement cells, marked by
long, presumably very tight, junctions
(small arrows) and close contact below
(broken line). Note also the density of
mitochondria (m) closely associated with
the internal network of tubules (t) and the
microvilli (mv) in contact with the external
water (w). The long arrow indicates the
faint glycocalyx coat on the pavement
cells (18 000×). (C) TEM of an ‘active’
pavement cell in freshwater completely
overlying a chloride cell (on the left). In
this case, the pavement cell contains larger
mitochondria and a rich complement of
Golgi apparatus (g), the latter giving rise
to vesicles (v) (25000×).
 Toxic responses of the gill 9

Figure 1.3 Electron micrographs of the branchial

epithelium. Plate courtesy of P.Laurent.
(A) Representative TEM of the trailing
edge region of the gill filament in a
freshwater rainbow trout, illustrating
typical internal structure of mucous cells
(mc), surface pavement cells (pvc), less-
differentiated subsurface pavement cells
(nc), and an electron-dense apoptotic cell
(apc). In the mucous cell, note the
abundant droplets (d) of mucus ready to be
 Target organ toxicity in marine and freshwater teleosts 10

released, the small smooth apical contact

with the water (w), and the rich
endoplasmic reticulum (er) and Golgi
apparatus (g) (5000×). (B) Representative
TEM of a neuroepithelial cell (nec) resting
on the basement membrane (bm), very
close to endings (arrow heads) of nerve
fibers (nf) within the filament epithelium
of a freshwater perch. Note the
characteristic dense core vesicles (cv)
(9000×). (C) TEM illustrating the
seawater-type organization of a chloride
cell (cc) relative to a neighboring
accessory cell (ac) and a prominent apical
crypt (c) in a tilapia living in a
hyperosmotic medium. The edge of a
pavement cell (pvc), which curves around
(out of field) to cover partially the apical
crypt, can also be seen. Note the
cytoplasmic processes (asterisks) of the
accessory cell which invade the chloride
cell apex with multiple very short,
presumably leaky, junctions (long arrows),
in contrast to the long, presumably tight,
junction (arrow head) between the
accessory cell and the pavement cell. Note
the very different organization from that
seen in the freshwater situation (see Figure
1.2B) (23000×).

Chloride cells (also called mitochondrial-rich cells or ionocytes) are largely restricted
to the interlamellar region (Figure 1.1D) and trailing edge of the filamental epithelium
and the base of the respiratory lamellae, but in freshwater fish may proliferate up onto the
surfaces of the respiratory lamellae under a variety of stressful conditions (e.g. Figure
1.10B). In seawater fish, their occurrence on the lamellae is rare. The chloride cells
generally make up less than 10 percent of the total branchial epithelial cells, but are
nevertheless the second most numerous type after the pavement cell. Under the light
microscope (LM), chloride cells are recognized by their rounded morphology and their
ability to take up zinc and silver stains, and under phase contrast by their granular
appearance. The accumulation of mitochondrial-specific fluorochromes, such as DASPEI
(dimethylaminostyrylethylpyridinium iodide) or DASPMI (dimethylamino-
styrylmethylpyridinium iodide), as seen under epifluorescence, is a standard diagnostic
marker for chloride cells. Fluorescent derivatives of ouabain (e.g. anthroylouabain;
McCormick, 1990), radioactive ouabain (e.g. Karnaky et al., 1976) or antibodies against