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Biomarkers of effect in toads and frogs

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DOI: 10.1080/1354700031000120116 · Source: PubMed

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BIOMARKERS, VOL. 8, NOS. 3 /4 (MAY  AUGUST 2003), 167 /186
/

REVIEW
Biomarkers of effect in toads and frogs

ANDRÉS VENTURINO1,2*, ENRIQUE ROSENBAUM2,

ADRIANA CABALLERO DE CASTRO2,
OLGA LILIANA ANGUIANO2, LIDIA GAUNA2,3,
TERESA FONOVICH DE SCHROEDER2,4 and
ANA MARÍA PECHEN DE D’ANGELO2
1
Cátedra de Quı́mica Biológica, Facultad de Ciencias Agrarias, Universidad Nacional del
Comahue, Buenos Aires 1400-8300 Neuquén, Argentina
2
LIBIQUIMA, Facultad de Ingenierı́a, Universidad Nacional del Comahue, Buenos Aires
1400-8300 Neuquén, Argentina
3
IUCS, Universidad Nacional del Comahue, Buenos Aires 1400-8300 Neuquén,
Argentina
4
Escuela de Ciencia y Tecnologı́a, Universidad Nacional de General San Martı́n, Alem
3901-1653 Villa Ballester, Buenos Aires, Argentina

Received 22 November 2002, revised form accepted 24 March 2003

Amphibians are good bioindicators of environmental pollution due to their suscept-

ibility to chemicals during their freshwater cycles. The effects of environmental pollution,
together with changes in human activity and climate, have contributed to the reduction in
the amphibian population over recent decades. However, toxicological research on
amphibians has been rather scarce compared with that on other vertebrates. In this article
we review the biochemical alterations underlying xenobiotic action and/or the detoxifying
responses described for anuran species, with the aim of establishing possible biomarkers of
effect. During the embryonic development of anurans, morphological and behavioural
alterations are the effects most frequently cited in connection with chemical exposures.
However, such biomarkers have a low sensitivity and are unspecific compared with
biochemical alterations. Some primary pesticide targets, in particular cholinesterases for
organophosphates and carbamates, have been evaluated. Esterases change seasonally and
with the stage of development, and their sensitivity to anticholinesterase agents varies
between species. Thus their use as biomarkers in anurans must be carefully analysed.
Enzymes and endogenous compounds related to oxidative metabolism may also be used as
biomarkers of effect. Glutathione pool, glutathione-S -transferases and metallothioneins
respond in different ways to pesticides and heavy metals in anuran embryos and tadpoles.
Mixed-function oxidases, in turn, are less developed in amphibians, and show a reduced
induction in response to pesticide exposures. Endogenous polyamine levels are also
proposed as good age-related biomarkers of damage. Finally, molecular biomarkers related
to receptor binding, signal transduction and genetic response have gained increasing
relevance, as they have been implicated in the fertilisation process and the earliest events in
anuran development. The identification of transcription factors associated with the
exposure of amphibians to xenobiotics as well as other alterations in hormone signalling
appears highly promising. However, these techniques are likely to complement other
methods. In conclusion, the use of several biomarkers with multiple endpoints is needed to
link exposure to response and to provide better predictive tools for the environmental
protection of endangered anuran species.

Keywords: amphibian, anuran, teratogens, cholinesterases, oxidative stress, molecular

biomarkers.

* Corresponding author: Andrés Venturino, LIBIQUIMA, Facultad de Ingenierı́a, Universidad

Nacional del Comahue, Buenos Aires 1400, 8300 Neuquén, Argentina. Tel: (
/54) 299 4490385; fax:
(
/54) 299 4490385; e-mail: [email protected]
Biomarkers ISSN 1354-750X print/ISSN 1366-5804 online # 2003 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/1354700031000120116
168 A. Venturino et al.

Ecotoxicology of amphibians and the decline of global populations: the need

for biomarkers
Amphibian populations have been declining globally over recent decades
(Barinaga 1990, Wake 1991, Houlahan et al. 2000). The decline in South-
American species has been even faster than the global mean, independently of a
single worldwide (Houlahan et al . 2001) or a spatially and temporarily varying
trend (Alford et al . 2001). Several reasons have been put forward to explain such
declines, some arising directly or indirectly from human activities and others
emerging from global and local climatic changes. These include the following:
direct destruction of amphibian habitats by humans, chemical pollution, acid rain,
fungal and bacterial infection, which may be in turn related to ozone depletion and
an increase in ultraviolet exposure (Blaustein et al . 1994) and relative drought
seasons in high altitude sites related to El Niño/Southern Oscillation cycles
(Klesecker et al . 2001).
The ecotoxicology of amphibians has received scarce attention compared with
that of other vertebrates. Nevertheless, a complete review on the topic has been
recently published (Sparling et al . 2000). The authors report that only 2.7% of the
literature covering aspects of ecotoxicology in the last 25 years (up to 1998)
concerned amphibians. The reasons for this disparity are not clear, since the
biomass of amphibians, their importance in the trophic chain and their relevance in
the loss of biodiversity justify ecotoxicological concern. Amphibians are found in
quite different habitats such as desert and forest, in varying climates and different
altitudes. Because amphibians pass their first life stages in water and their adult life
span as terrestrials, they are exposed to a wide range of contaminants. This,
together with their feeding habits, the continuous processing of water through their
gills, and their particular sensitivity to chemicals during their freshwater cycles,
makes amphibians good bioindicators of the overall conditions of the environment
(Dumpert and Zeitz 1984, Lefcort et al . 1998). Most amphibian species are
susceptible to xenobiotics during fertilization and early development (Cooke 1972,
Devillers and Exbrayat 1992).
Over the last decade, biomonitoring has been increasing used to track
environmental pollution (Whitfield 2001). Chemical analysis of water and soils
represents a direct proof of the nature and degree of contamination, but sensor
organisms can reveal the status of the dynamic scenario. Biomarkers of exposure,
effect and susceptibility are needed to connect the presence of pollutants in the
environment with their action in an organism. In this context, biomarkers can aid in
assessing the health status of amphibian populations by acting as sublethal
endpoints of intoxication.
We have been studying the effects and mechanisms of action of pesticides in the
embryonic and larval ontogenesis of the South American toad Bufo arenarum,
evaluating acute and chronic toxicology on recognized primary as well as secondary
molecular targets. In the following section we review the biochemical alterations
underlying xenobiotic action and/or the detoxifying responses described for anuran
species, discussing their use as possible biomarkers of effect in amphibians in
general and Bufo arenarum in particular, mainly in the embryonic and larval stages.
 Biomarkers in toads and frogs 169

Morphological, physiological and biochemical changes caused by xenobiotics

in anurans as biomarkers of effect
Morphological alterations induced in embryogenesis
Morphological alterations and impairment of normal growth are the most
frequently described biomarkers of exposure to pesticides and other contaminants
such as heavy metals. Table 1 summarizes the main developmental alterations
described in the literature for anurans. Early work by Kaplan and Overpeck (1964)
and Cooke (1970, 1972) described hyperactivity in frog and toad tadpoles exposed
to organochlorine (OC) pesticides. We have evaluated the malformations elicited by
OCs during the embryonic development of Bufo arenarum . Exposures were in
general characterized by the appearance of body twisting and progressive dropsy,
notocord alteration, defective gills, reduced weight and size, and shortening of the
time to reach metamorphosis (De Llamas et al . 1985, Gauna et al . 1991, Caballero
de Castro et al . 1997, Anguiano et al . 2001). These alterations have also been
described in other exposed species (Schuytema et al . 1991). Organophosphates
(OPs) and carbamates also cause body shortening with caudal fin folding, notocord
bending and abnormal pigmentation in Bufo arenarum (Rosenbaum et al . 1988,
Anguiano et al . 2001). Impaired growth, reduced body size, haemorrhage,
abnormal organogenesis and paralysis have been described in other OP-exposed
anuran embryos and tadpoles (Snawder and Chambers 1989, Alvarez et al . 1995,
Schuytema et al . 1995, Berrill et al . 1997, Harris et al . 1998, Fordham et al . 2001).
Heavy metals affect notocord, head, gill and caudal fin formation in Bufo arenarum,
causing a decrease in growth and size and delaying development (Pérez-Coll et al .
1985, 1988). Similar alterations were observed in Rana catesbeiana , Rana
luteiventris and Xenopus laevis (Rowe et al . 1996, Herkovits et al . 1997, Lefcort et
al . 1998). Polychlorinated biphenyls (PCBs), pyrethroids, herbicides, fungicides
and inclusive ultraviolet radiation are able to produce some of these developmental
alterations in different anuran species (Table 1).
It would seem that the alterations in morphology and development are not
specific for any class of compound. Most of the alterations are encountered after
gastrulation has been completed. Table 2 summarizes the different effects described
for the different classes of compounds in Table 1. The time needed for
metamorphosis is increased for nearly all types of compounds, except for some
OCs, which may produce a shortening of the development period. Both tadpole
weight and size are commonly reduced by xenobiotics. The larval body is generally
curved by chemical exposure, presenting a bent notocord, with defective formation
of gills, gut and caudal fin, and generalized oedema. Haemorrhage was only
described with OP exposure. Abnormal pigmentation in tadpoles is also a defect
common to various classes of xenobiotics. The behavioural alterations caused by
pesticide target impact include paralysis-hyperexcitability, body twisting, spasms,
convulsions, limited movement and altered swimming.
Amphibians are massive reproducers that frequently display a higher number of
malformations in normal populations (about 1%) than other species (Crawshaw
2000). Thus, distinguishing malformations due to xenobiotic exposure in the field
from the normal background level may be rather difficult. Malformations have been
detected in a few studies using caged tadpoles exposed to OCs, OPs, carbamates
 170
Table 1. Anatomical and physiological alterations in growth induced by xenobiotics and physical agents during anuran development.

Compound Species and stage Effect Reference

OCs
Aldrin Rana pipiens Hyperactivity Kaplan and Overpeck 1964
Chlordane Rana pipiens Hyperactivity Kaplan and Overpeck 1964
DDT Bufo arenarum larva Body twisting; defective gills; progres- Caballero de Castro et al . 1997
sive dropsy; reduced weight; shortening
of metamorphosis time; erratic swim-
ming; hyperexcitability
Rana temporaria tadpole Reduced weight; notocord alteration; Cooke 1970, 1972
deformed snout; hyperactivity
Rana sylvatica embryo Hyperactivity Licht 1985
Dieldrin Bufo arenarum embryo Exogastrulation; arrest in gastrula Anguiano et al . 2001
Bufo arenarum larva Reduced size; abnormal pigmentation; De Llamas et al . 1985

A. Venturino et al.
defective gills; progressive dropsy;
shortening of metamorphosis time
Tail lashing; body twisting; erratic Gauna et al . 1991; Caballero de Castro
swimming; hyperexcitability et al . 1997
Bufo bufo; Rana temporaria Hyperactivity Cooke 1970, 1972
Rana pipiens Hyperactivity Kaplan and Overpeck 1964
Xenopus laevis; Rana catesbeiana Notocord deformity Schuytema et al . 1991
Endosulfan Rana sylvatica tadpole; Rana clami- Paralysis (low levels), hyperactivity (high Berrill et al . 1998
tans tadpole levels)
Bufo americanus tadpole Also impaired metamorphosis Berrill et al . 1998
Endrin Rana sphenocephala tadpole Hyperactivity Hall and Swineford 1980
Bufo americanus; Rana catesbeiana; Erratic swimming; disturbed equili- Hall and Swineford 1981
Rana sylvatica brium; abnormal posture
Lindane Bufo arenarum embryo Arrest in gastrula Anguiano et al . 2001
Bufo arenarum larva Body twisting; defective gills; progres- Caballero de Castro et al . 1997
sive dropsy; reduced weight; shortening
of metamorphosis time; erratic swim-
ming
Caudal fin bending; hyperactivity Anguiano et al . 2001
Toxaphene Rana pipiens Hyperactivity Kaplan and Overpeck 1964
Rana sphenocephala tadpole Hyperactivity Hall and Swineford 1980
Rana catesbeiana; Rana sylvatica; Bufo Erratic swimming; disturbed equili- Hall and Swineford 1981
americanus brium; abnormal posture
Table 1 (Continued )
Compound Species and stage Effect Reference

PCBs
Arochlors Bufo americanus; Bufo fowleri Lordosis; scoliosis; oedema Eisler and Beslisle 1996
Clophen; PCB 126 Xenopus laevis; Rana temporaria Oedema; depigmentation; tail defor- Gutleb et al . 2000
mity; increased weight; delayed meta-
morphosis

OPs
Azinphos methyl Bufo arenarum larva Reduced size; notocord bending; ab- Caballero de Castro et al . 1997
normal pigmentation; defective gut and

Biomarkers in toads and frogs

gills; swimming in circles
Rana clamitans embryo Body shortening Harris et al . 1998
Hyla regilla tadpole; Xenopus laevis Impaired growth Schuytema et al . 1995
tadpole
Fenitrothion Bufo americanus tadpole; Rana cates- Paralysis Berrill et al . 1997
beiana tadpole; Rana clamitans tad-
pole; Rana pipiens tadpole; Rana
sylvatica tadpole
Malathion Bufo arenarum larva Body twisting; tail lashing; limited De Llamas et al . 1985
movement
Reduced size; tail folding; notocord Rosenbaum et al . 1988
bending; oedema
Haemorrhage; reduced pigmentation; Caballero de Castro et al . 1997
defective gut
Reduced gills; swimming in circles Anguiano et al . 2001
Rana catesbeiana tadpole Abnormal gills; haemorrhage; altered Fordham et al . 2001
equilibrium; paralysis
(also malaoxon) Xenopus laevis embryo Reduced size; abnormal pigmentation; Snawder and Chambers 1989
abnormal gut; notocord bending
Methyl parathion Rana perezi larva Notocord bending; scoliosis; tail folding Alvarez et al . 1995
Parathion Bufo arenarum larva Reduced size; notocord bending; oede- Caballero de Castro et al . 1997
ma; haemorrhage
Reduced pigmentation; defective gut Anguiano et al . 2001
and gills; swimming in circles

171
 172
Table 1 (Continued )
Compound Species and stage Effect Reference
Carbamates
Carbaryl Bufo arenarum larva Reduced size; notocord bending; ab- Caballero de Castro et al . 1997
normal pigmentation; defective gut and
gills; swimming in circles
Rana tigrina tadpole Reduced weight and growth Marian et al . 1983
Rana blairi tadpole Reduced motility Bridges 1997
Carbofuran Microhyla ornata tadpole Tail bending; abnormal swimming Pawar and Katdare 1984
Oxamyl Rana temporaria tadpole Body twisting; tail tip; malformations Cooke 1981
reduced development
Pirimicarb Rana perezi tadpole Notocord bending; limb malformation Alvarez et al . 1995
Propoxur Rana hexadactyla tadpole Tail kinking; increased length; limb Raj et al . 1988
malformation

A. Venturino et al.
Pyrethroids
Esfenvalerate Rana blairi larva; Rana sphenocephala Spasm; convulsive twisting; weight loss Materna et al. 1995
larva
Fenvalerate; permethrin Rana clamitans embryo and larva Slower growth; body bending Berrill et al . 1993
Rana pipiens embryo and larva Paralysis; weakness Berrill et al . 1997
Rana sylvatica embryo and larva; Bufo Delayed metamorphosis Berrill et al . 1993
americanus embryo and larva

Herbicides
Cyanatryn Rana temporaria tadpole Lethargy; spasm; reduced weight Scorgie and Cooke 1979
Diuron Hyla regilla; Xenopus laevis Deformities; delayed growth Schuytema and Nebeker 1998
Rana aurora Limb malformations Schuytema and Nebeker 1998
Paraquat Xenopus laevis embryo Tail folding; abnormal gut; oedema Visnara et al . 2000

Fungicides
Chloranil; dichlone; nabam Xenopus laevis embryo Disrupted cephalic development; re- Anderson and Prahlad 1976
duced size
Triphenyltin Rana lessonae tadpole; Rana esculenta Slower growth; delayed metamorphosis Fioramonti et al . 1997
tadpole

Heavy metals
Lead Bufo arenarum embryo and tadpole Delayed development; body bending; Pérez-Coll et al. 1988
microcephaly; defective gills; stunted tail
Table 1 (Continued )
Compound Species and stage Effect Reference
Cadmium Bufo arenarum embryo Skeletal malformations; delayed devel- Pérez-Coll et al. 1985
opment; size reduction
Xenopus laevis embryo and tadpole Body bending and shortening; micro- Herkovits et al . 1997
cephaly; eye and tail malformation;
reduced pigmentation
Arsenic; barium; cadmium; chromium; Rana catesbaiana tadpole Oral deformities; decreased growth Rowe et al . 1996
selenium
Lead; zinc Rana luteiventris tadpole Reduced weight; reduced motility and Lefcort et al . 1998
fright response

Biomarkers in toads and frogs

Others
Histamine; imidazole acetic acid Bufo arenarum embryo and tadpole Delayed development; oedema; organ Scolnik et al . 1987
displacement; abnormal intestine; de-
fective gills; tail bending; heart beat
reduction; reduced reaction; swimming
in circles
Ultraviolet radiation Bufo arboreas Lordosis; abnormal cornea; hyperplasia Worrest and Kimeldorf 1976
Hyla regilla; Rana cascadae Lordosis Hays et al . 1996

173
174 A. Venturino et al.

Table 2. Comparative summary of developmental alterations caused by classes of xenobiotics in anuran

embryos and larvae.

Target Effect Type of compound

Developmental rate Acceleration OCs

Delay PCBs, OPs, carbamates, pyrethroids, herbicides,
fungicides, heavy metals
Weight Reduction OCs, OPs, carbamates, pyrethroids, herbicides,
heavy metals
Increase PCBs
Size Reduction OCs, OPs, carbamates, fungicides, heavy metals
Increase Carbamates
Body shape Twisting OCs, PCBs, OPs, carbamates, pyrethroids, heavy
metals
Dropsy or oedema OCs, PCBs, OPs, herbicides
Subcutaneous circulation Haemorrhage OPs
Tegument Depigmentation OCs, PCBs, OPs, carbamates, heavy metals
Notocord Bending OCs, PCBs, OPs, carbamates, heavy metals
Gills Stunted OCs, PCBs, OPs, carbamates, heavy metals
Snout Deformities OCs, heavy metals
Caudal fin Folding/bending OCs, PCBs, OPs, carbamates, herbicides, heavy
metals
Gut Protrusion PCBs, OPs, carbamates, herbicides
Behaviour and locomotion Hyperexcitability OCs, pyrethroids, herbicides
(nervous system) Spasm Pyrethroids, herbicides
Paralysis OCs, OPs, pyrethroids
Weakness/lethargy OPs, carbamates, pyrethroids, herbicides, heavy
metals
Altered equilibrium OCs, OPs
Circular swimming OCs, OPs, carbamates

and herbicides (Mulla et al . 1963, Cooke 1973, 1977, 1981). Genotoxic alterations
in amphibian development have been described in a few studies with pyrethroids
(Rudek and Rozek 1992) and herbicides (Clements et al . 1997), although many
other organic compounds such as polycyclic aromatic hydrocarbons (PAHs) also
cause genotoxicity (Sparling 2000).
We may conclude that developmental growth alterations are relatively easy to
record as macroscopic biomarkers of effect. However, they are not specific for
pesticide class, as are also caused by other organic chemicals and heavy metals.

Signalling, transduction and genetic responses as molecular toxicology biomarkers

The development in molecular genomics and proteomics linked to the
recognition of molecular targets provides the basis for more powerful biomonitor-
ing techniques to protect the environment (Adams et al . 2001). The subacute and
long-term effects of pesticide exposures leading to fertilization impairment,
abnormal development and sexual dysfunction in adults involve molecular effectors
in the signalling, transduction and genetic response to the stress. Receptors such as
the aryl hydrocarbon receptor (Rowlands and Gustafsson 1997), the protein kinase/
phosphatase cascades (Matsumura et al . 1984) and nuclear transcription factors
(Ashida and Matsumura 1998) play essential roles in the toxicity of pesticides and
organic pollutants.
 Biomarkers in toads and frogs 175

A central factor in initiating egg activation at fertilization is a rise in free Ca2
in

the egg cytosol, which occurs as a result of its inositol triphosphate-mediated
release from the endoplasmic reticulum. Muscarinic receptors coupled to phos-
pholipase C are thought to mediate second messenger release by phosphoinositide
breakdown. In amphibians, lipophilic compounds coming in contact with laid
oocytes easily pass though the tiny jelly coat and are then able to diffuse into plasma
membranes. Membrane-associated processes such as second messenger signalling
during fertilization (Kusano et al . 1977, Miyazaki 1988) might be then disrupted by
lipophilic toxicants. Some compounds intercalate in the bilayer of oocyte
membranes, causing phosphoinositide breakdown (Bernard et al . 1988) (Table
3). We have studied some of these processes, and found an increased turnover of
phospholipids in Bufo arenarum oocytes as a consequence of exposure to dieldrin
and azinphos methyl. Associated with these changes, blockade of an agonist-gated
response via phospholipase C was observed. In addition, there was a retailoring of
phospholipids through phospholipase A2 deacylation and lysophospholipid reacy-
lation (Fonovich de Schroeder and Pechen de D’Angelo 1995a, 2000, Caballero de
Castro et al . 1997). In vitro studies showed that dieldrin in fact activates
phospholipase C, leading to a desensitized state of the enzyme (Fonovich de
Schroeder and Pechen de D’Angelo 1995b). In addition, the free fatty acid pool
was increased in dieldrin exposure, mainly as oleic acid (Fonovich de Schroeder
and Pechen de D’Angelo 2000), which is the major acyl component in phosphatidyl
choline and phosphatidyl ethanolamine from Bufo arenarum oocytes. As a
consequence of oocyte exposures to OCs, OPs and carbamates, significant
decreases in fertilization success have been observed in Bufo arenarum (Table 3).
Preliminary studies on molecular biomarkers in Bufo arenarum during early
development indicate that exposure to OPs affects protein phosphorylation.
Related to these changes, activator protein-1 response element (AP-1RE) binding
transcription factors are downregulated, while cAMP response element (CRE)-
related transcription factors show a biphasic response as determined by electro-
phoretic gel mobility shift assay (EMSA) (Venturino et al. 2001c). c-fos repression
and AP-1RE binding downregulation by pyrethroid exposure has been recently
reported (Imanura et al . 2000). In turn, c-Jun is regulated by c-Jun amino terminal
kinase (JNK), which is downregulated by glutathione-S -transferase (GST) hetero-
dimerization (Finkel and Holbrook 2000). cAMP-dependent protein kinases and
two nuclear transcription factors binding CRE are affected by dioxins (Matsumura
et al. 1984, Ashida and Matsumura 1998). These results are encouraging in the
search for early molecular biomarkers of response to pesticides in amphibians.
Endocrine disrupting chemicals (EDCs) and endocrine active chemicals
interfere with signalling systems through hormone receptor binding, altering
endocrine and sex determination processes. PCBs and PAHs cause sex reversal
in male reptiles, but their effects in amphibians are largely unknown (Sparling
2000). Recent reports have described feminization of male frogs exposed to the
herbicide atrazine in the laboratory and in the wild (Hayes et al . 2002a,b). Several
key developmental and physiological processes are steroid- and thyroid hormone-
dependent throughout the amphibian life cycle. Thyroid hormones are essential for
metamorphosis and are corticoid-modulated. Both testosterone and oestradiol
 176
Table 3. Compounds affecting receptor binding, second messenger signalling and/or genetic responses as molecular biomarkers in anurans.

Species and stage Compound Effect Reference

Bufo arenarum oocyte Dieldrin Increased phosphoinositide turnover; blockade of phospholipase C response Fonovich de Schroeder and Pechen de
D’Angelo 1995a
Reduced fertilisation rate; activation/desensitization of phospholipase C; Fonovich de Schroeder and Pechen de
phospholipase A2 activation; lysophospholipid acyltransferase activation D’Angelo 1995b, 2000

A. Venturino et al.
Azinphos methyl Increased phosphoinositide turnover; blockade of phospholipase C response; Caballero de Castro et al . 1997
reduced fertilization rate
Bufo arenarum Malathion; azinphos Alterations in protein phosphorylation; downregulation of AP-1RE and CRE Venturino et al . 2001c
embryo methyl binding transcription factors
Kassina senegalensis DDT Developmental abnormalities Hayes 2000
larva
Rana pipiens adult Atrazine Aromatase induction; decrease in testosterone levels and hermaphroditism in Hayes et al . 2002b
males
Xenopus laevis oocyte Maitoxin Phosphoinositide breakdown Bernard et al . 1988
Xenopus laevis adult DDT; toxaphene; Vitellogenin induction Palmer et al . 1998
dieldrin
Tetrachlorbiphenyl; Oestrogen receptor binding Lutz and Kloas 1999
DDT
Atrazine Aromatase induction; decrease in testosterone levels and hermaphroditism in Hayes et al . 2002a
males
 Biomarkers in toads and frogs 177

inhibit amphibian metamorphosis (Hayes 2000). Few effects of EDCs have been
reported in amphibians to date. Dioxins affect metamorphosis by targeting the
thyroid system. Developmental abnormalities were observed in Kassina senegalensis
exposed to dichlorodiphenyltrichloroethane (DDT) (Hayes 2000) (Table 3). In
adult Xenopus laevis females, thyroid hormones stimulate oestrogen receptors,
enhancing oestrogen-mediated induction of vitellogenin (May and Knowland
1980). Vitellogenin has become a recognized biomarker of effect for EDCs. OCs
such as DDT, toxaphene and dieldrin may act as pro-oestrogens, inducing
vitellogenin in male adults (Palmer et al . 1998). The level of binding to the liver
oestrogen receptor has been effectively assessed for several aromatic compounds,
including PCBs and DDT, in Xenopus laevis adults (Lutz and Kloas 1999).
Another proposed mechanism of feminization in male Xenopus laevis and Rana
pipiens adults exposed to atrazine is aromatase induction and testosterone
conversion to oestrogen (Hayes et al . 2002a,b).

Acetylcholinesterase and other esterases

Acetylcholinesterase (AChE) activity is crucial at cholinergic synapses and
muscular plates, contributing to the cessation of acetylcholine stimulation at the
postsynaptic membrane once the nervous signal has been transmitted. AChE is
considered the main target of OP insecticides, which bind irreversibly to the
enzyme active site as suicide inactivators, and the reduction in its activity is
generally associated with lethality. Nevertheless, AChE and butyrylcholinesterase
(BChE) have another relevant role in the development of the brain and nervous
system during early embryogenesis (Drews 1975, Krejci et al . 1991). AChE is
recognized as a specific biomarker of effect in pesticide exposures (Adams 2001).
Both BChE and the detoxifying activity of carboxylesterases (CEs) have been
proposed as biomarkers of effect for OP and carbamate pesticides (Bartowiak and
Wilson 1995, Sánchez et al . 1997). However, the use of AChE as a biomarker in
carbamate and OP exposure is less well quantified in amphibians than in other
vertebrate classes (Henry 2000).
We have evaluated the developmental pattern and seasonal variations in the
activities of AChE, BChE and CEs in Bufo arenarum (Caballero de Castro et al .
1991, Caballero de Castro 2000). AChE and BChE activities were detectable from
gastrulation on, and peaked at 5 days of development (complete operculum stage)
in summer embryos. CE activity was detected in oocytes and the earliest stages of
embryonic development, and also peaked at day 5 (Figure 1). Winter clutches
showed delayed patterns of these enzymes, associated with a slower embryonic
development.
OPs suppress the activity of these three enzymes or delay their appearance and
progression in embryos recovered from transient exposures (Rosenbaum et al .
1988, Caballero de Castro et al . 1991). Embryos treated with malathion for 5 days
recover between 25 and 40% of the peak activity after a delay of 2 /3 days. Embryos
treated for 3 days recover between 40 and 65% of the peak activity after a delay of 1
day. In Bufo arenarum larvae, the recovery times from exposure to different OPs
(malathion, parathion and azinphos methyl) ranged from 1 to 7 days to achieve 70 /
100% AChE activity (Venturino et al . 1992, 1993, 2001b, Anguiano et al . 1994).
178 A. Venturino et al.

Figure 1. Developmental pattern and seasonal changes in esterase activities during Bufo arenarum
embryogenesis. Esterase activities were determined in embryos obtained by in vitro
fertilization at different stages of development during the summer (a) or winter season
(b). The esterases analysed were acetylcholinesterase (AChE), butyrylcholinesterase (BChE)
and carboxylesterases (CEs). Stages achieved at days of development are indicated by
arrows: LG, late gastrulae; MR, muscular response; GC, gill circulation; CO, complete
operculum. Data from Caballero de Castro 2000.

Significant inhibition and a fast recovery were found with carbamate carbaryl. The
OP temephos inhibited BChE in Rana clamitans larvae, but increased AChE
activity (Sparling et al . 1996). This effect was attributed to inefficient oxidation of
the OP to the oxon form, the active inactivator of AChE. Other organic compounds
such as OCs may also affect cholinesterases; for instance, dieldrin decreases both
AChE and BChE activities in Bufo arenarum embryos (De Llamas et al . 1985).
Determination of the effects of exposure to OPs or carbamate, as well as those
of other toxicants, needs to take into account the species-specific variations
associated with development and season. In some species such as Xenopus laevis
tadpoles, AChE is highly resilient to anticholinesterase agents (Shapira et al . 1998).
The important fluctuations in AChE, BChE and CEs due to seasonal and
developmental changes observed in Bufo arenarum embryos make the assessment
of control reference values difficult. The relatively fast recovery of the activities after
an episodic exposure to pesticides may also complicate the assessment of field
 Biomarkers in toads and frogs 179

effects. Consequently, the use of these enzymes as biomarkers in anuran species

requires particular caution.

Biochemical responses related to pesticide oxidative metabolism and detoxification

Pesticide metabolism is generally linked to oxidative stress directly by genera-
tion of reactive oxygen species (ROS) through enzymatic transformation or by
consumption of reduced co-substrates such as reduced nicotinamide adenine
dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate
(NADPH) (Finkel and Holbrook 2000). Mixed-function oxidases (MFOs) are
involved in the oxidative metabolism of practically all pesticides and contribute to
ROS generation. Amphibians appear to have a less developed P450-dependent
MFO system than that found in mammals (Sparling 2000). P450 induction is low
in amphibians compared with other classes, reducing its effectiveness as a
biomarker of effect (Eartl and Winston 1998, Huang et al. 1998). Moreover, a
very large natural variation in the activity of several MFO components has been
found in Rana temporaria adults, probably resulting from the hormonal changes
associated with spawning (Harri 1980). MFO induction was demonstrated in
malathion-exposed Bufo arenarum larvae (Venturino et al . 2001b) (Table 4).
Reduced glutathione (GSH) is perhaps the most important ROS scavenger
participating in the control of the intracellular redox status (Finkel and Holbrook
2000). Some pesticides are detoxified by GSH conjugation or demethylation
through a pathway involving GST activity, and alterations in the cellular antioxidant
defence have been associated with OP exposures (Hai et al . 1997). Antioxidant
enzymes, glutathione and lipid peroxidation levels are relevant biomarkers in
aquatic toxicology (Doyotte et al . 1997). We have evaluated these different
pathways related to pesticide oxidative metabolism in exposed Bufo arenarum
embryos and larvae. Different OC and OP insecticides affect the reduced GSH
pool, measured as acid-soluble thiols (ASTs), in toad embryos and larvae
(Anguiano et al . 2001, Venturino et al . 2001a). Malathion, azinphos methyl and
lindane decrease the reduced AST level to nearly 50% in embryos, whereas
parathion and dieldrin do not affect it (Table 4). Larval stages are mainly affected
by malathion and azinphos methyl, which deplete AST by up to a 30% of control
levels. Nevertheless, all the pesticides tested induce GST activity in Bufo arenarum
larvae, suggesting the involvement of this enzyme in their metabolism. Malathion
also decreases total (reduced plus oxidized) AST, and methyl GSH has been
detected in exposed toad larvae, corroborating the participation of the GST
pathway in the detoxification of this insecticide (Venturino et al . 2001a,b). Thus,
these biochemical parameters are biomarkers of the oxidative metabolism of some
insecticides in exposed Bufo arenarum embryos and larvae. It is worth noting that
mild exposures to these insecticides do not lead to oxidative stress, as lipid
peroxidation levels remain similar to those in control tadpoles. GSH and
metallothioneins, commonly used as biomarkers in metal exposure, have also
been evaluated in exposed amphibians (Suzuki and Akitomi 1983, Suzuki and
Kawamura 1984, Vogiatzis and Loumbourdis 1998). Other metabolic alterations
have been studied in the ovary of adult Bufo arenarum females in the search for new
biomarkers for metal exposure. The enzyme glucose 6-phosphate dehydrogenase is
 180
Table 4. Biomarkers of oxidative stress, antioxidant defence and enzyme detoxification in anurans.

Biomarker Species and stage Compound Effect Reference

MFO Bufo arenarum larva Malathion Induction Venturino et al . 2001b

Rana pipiens Pentachlorobiphenyl Induction Huang et al. 1998
Rana temporaria adult DDT; seasonal variations No effect Harri 1980
GST Bufo arenarum embryo and larva Malathion; parathion; azinphos methyl; Induction Anguiano et al . 2001; Venturino et al .
lindane; dieldrin 2001c
Metallothionein Rana ridibunda adult Cadmium Induction Vogiatzis and Loumbourdis 1998

A. Venturino et al.
Rana catesbeiana larva and adult Cadmium Induction Suzuki and Akitomi 1983
Bombina orientalis adult; Bufo bufo japonicus Copper; zinc Induction Suzuki and Kawamura 1984
adult; Hyla arborea japonica adult
Glucose 6-phosphate Bufo arenarum adult Zinc Inhibition Fonovich de Schroeder et al . 2000; Naab
dehydrogenase et al. 2001
GSH Bufo arenarum embryo Malathion; azinphos methyl; lindane Decrease Anguiano et al . 2001
Bufo arenarum larva Malathion Decrease Venturino et al . 2001a
Bufo arenarum adult Zinc Increase Fonovich de Schroeder et al . 2000; Naab
et al. 2001
Rana ridibunda Cadmium Increase Vogiatzis and Loumbourdis 1998
GSH
/GSSG (total) Bufo arenarum larva Malathion Decrease Venturino et al . 2001a
Lipid peroxidation Bufo arenarum larva Malathion; parathion; dieldrin; lindane No effect Venturino et al . 2001a,c
Polyamines Bufo arenarum embryo Malathion Decrease Venturino et al . 2001c
Malathion (sublethal) Increase Venturino et al . 2001a
Bufo arenarum larva Malathion No effect Venturino et al . 2001a

GSH, reduced glutathione; GSSG, oxidized glutathione.

 Biomarkers in toads and frogs 181

able to bind zinc, which is considered a micronutrient that does not normally
accumulate in the tissues. This enzyme is inhibited by long-term exposure of
females to zinc in Ringer solution. As the result of this inhibition, the oocytes are
subjected to oxidative stress and respond with an increase in GSH content
(Fonovich de Schroeder et al . 2000, Naab et al . 2001) (Table 4).
The levels of polyamines, which are essential for a wide range of biological
processes, are altered in severe cellular stress and toxicosis associated with
apoptosis via hydrogen peroxide generation and GSH depletion during embryonic
development (Coffino and Poznanski 1991). We examined polyamine levels as
biomarkers of the effects of pesticide on the embryogenesis of Bufo arenarum.
Malathion concentrations causing acute toxicity decreased polyamine levels in
middle and late embryonic stages (Venturino et al . 2001c), and this effect was
related to impaired development and abnormal morphogenesis (Table 4). Sublethal
exposures to malathion caused an increase in putrescine concentration at the end of
embryonic development as a recovery response, while no effects were observed in
larvae (Venturino et al . 2001a). Thus, polyamines may be useful biomarkers of
pesticide effect and recovery responses during amphibian development, depending
on the stage and level of exposure.

Conclusions
Different biochemical, physiological and morphological parameters may be
needed at different stages in anuran development in order to assess exposure and
response to contaminants.
Reductions in fertilization are partially associated with changes in the oocyte
membrane phospholipid turnover, and alterations in the muscarinic acetylcholine
receptor signalling pathways and other membrane-associated enzyme activities.
During early embryonic development, different chemicals may affect the
nuclear transcription factors regulating gene expression, thus altering a cascade
of responses. The identification of transcription factors associated with the
exposure of amphibians to xenobiotics is highly promising. However, these
methods are likely to complement other biomarkers. Among them, GSTs are
induced by pesticides, in some instances lowering reduced AST pools (generally
associated with GSH) and total GSH.
After gastrulation, malformations are commonly found following chemical
exposure. However, developmental alterations in anurans are not specific to the
chemicals involved and may be difficult to assess in the field.
Esterases show seasonal and developmental patterns that are abolished or
delayed by insecticide exposures. Esterase activities show relatively fast recoveries
after brief exposures, and their sensitivity to anticholinesterase agents vary among
species, reducing their usefulness as biomarkers.
GSH, GSTs and metallothioneins respond to oxidative and metal stress in
exposed embryos and tadpoles. MFO activities in tadpoles are low and are poorly
induced by xenobiotics. Polyamines are depleted at the end of embryonic
development by lethal exposures, and are related to teratogenesis, reduced growth
182 A. Venturino et al.

and reduced survival. On the other hand, sublethal exposures cause stress-related
putrescine increases, associated with MFO induction.
Finally, in adult anurans a few specific biomarkers such as vitellogenin for
EDCs and metallothioneins and glucose 6-phosphate dehydrogenase inhibition for
heavy metals have been described.
In conclusion, the use of several biomarkers with different endpoints is needed
to link exposure to response, and to provide better predictive tools for the
environmental protection of endangered anuran species. These biomarkers, among
others coming into use, provide a range and diversity of biological responses in
toads and frogs that may be useful in environmental risk assessment after being
validated in the field (Adams et al . 2001). Applying a variety of biomarkers in
predictive ecotoxicology will improve the interpretation of effects and will help to
ensure their significance in impact assessment.

Acknowledgements
We thank Professor Claudia Herczeg for her helpful assistance in manuscript
language processing.
This work was supported by the National University of Comahue (grant I940)
and ANPCyT-Argentina (grant 01-03729).

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