Cadmium neurotoxicity on a freshwater planarian

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Archives of EnvironmentalContamination and Toxicology ISSN 0090-4341Volume 67Number 4 Arch Environ Contam Toxicol (2014)67:639-650DOI 10.1007/s00244-014-0056-0

Cadmium Neurotoxicity to a FreshwaterPlanarian

Jui-Pin Wu, Hui-Ling Lee & Mei-Hui Li

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Cadmium Neurotoxicity to a Freshwater Planarian

Jui-Pin Wu • Hui-Ling Lee • Mei-Hui Li

Received: 27 January 2014 / Accepted: 26 May 2014 / Published online: 5 July 2014

� Springer Science+Business Media New York 2014

Abstract Although freshwater planarians are evolution-

arily primitive, they are some of the simplest bilateral ani-

mals possessing integrated neural networks similar to those

in vertebrates. We attempted to develop planarian Dugesia

japonica as a model for investigating the neurotoxicity of

environmental pollutants such as cadmium (Cd). This study

was therefore designed to study the effects of Cd on the

locomotor activity, neurobehavior, and neurological

enzymes of D. japonica. After planarians were exposed to Cd

at high concentrations, altered neurobehavior was observed

that exhibited concentration-dependent patterns. Morpho-

logical alterations in Cd-treated planarians included irregu-

lar shape, body elongation, screw-like hyperkinesia, and

bridge-like position. To study the direct effects of Cd on

neurological enzymes, tissue homogenates of planarians

were incubated in vitro with Cd before their activity was

measured. Results showed that acetylcholinesterase

(AChE), adenosine triphosphatase (ATPase), and mono-

amine oxidase A (MAO-A) activities were inhibited in a

concentration-dependent manner. MAO-B activity was sig-

nificantly induced by Cd at low concentrations and inhibited

at high concentrations. Changes in the in vivo activity of

AChE and ATPase were also found after planarians were

treated with Cd at a sublethal concentration (5.56 lM).

These observations indicate that neurotransmission systems

in planarians are disturbed after Cd exposure.

Freshwater free-living flatworms, or planarians, of the

phylum Platyhelminthes, are evolutionarily primitive

invertebrates that have one obvious neuronal cluster loca-

ted at the head end and two symmetric longitudinal cords

with ladder-like interconnections (Nishimura et al. 2008).

Although some investigators prefer to designate the ante-

rior neuronal cluster in planarians as a ‘‘cephalic gan-

glion,’’ it actually fulfills all of the criteria for the definition

of a brain in animals (Sarnat & Netsky 2002). It had been

even considered that the evolution of the vertebrate central

nervous system (CNS) may have begun with planarians

(Sarnat & Netsky 2002), although this is controversial in

light of the fact that planarians do not seem to be in the

direct evolutionary lineage to vertebrates (Northcutt 2012).

Biochemical substances identical to those used by mam-

mals as neurotransmitters and second messengers, as well

as their receptors, also occur in freshwater planarians

(Buttarelli et al. 2008). In addition, they have a relatively

sophisticated repertoire of behaviors (e.g., learning and

memory) and the capacity for tissue regeneration (Raffa &

Rawls 2008). Specific behavioral patterns can be clearly

observed after treating planarians with drugs that act on

neural transmission (Buttarelli et al. 2000; Carolei et al.

1975; Palladini et al. 1996; Venturini et al. 1989). These

biological characteristics and advantages make the fresh-

water planarian a simple but powerful and practical model

for research into drug action and abuse (Raffa & Rawls

2008) as well as neurological diseases (Kitamura et al.

1998).

When exposed to neurotoxic chemicals, the normal

neural activities of humans and other animals will be

altered and/or their structures damaged. The most well-

known and widely distributed neurotoxic chemicals

include, but are not limited to, certain heavy metals such as

cadmium (Cd) (Wright & Baccarelli 2007). Cd

J.-P. Wu � M.-H. Li (&)

Environmental Toxicology Laboratory, Department of

Geography, National Taiwan University, 1, Section 4, Roosevelt

Road, Taipei 106, Taiwan

e-mail: [email protected]

H.-L. Lee

Department of Chemistry, Fu Jen Catholic University, 510,

Zhongzheng Road, New Taipei City 24205, Taiwan

123

Arch Environ Contam Toxicol (2014) 67:639–650

DOI 10.1007/s00244-014-0056-0

Author's personal copy

neurotoxicity has been reported in other experimentally

treated mammals (Antonio et al. 1998; Gabbiani et al.

1967; Lafuente et al. 2001, 2005; Pari & Murugavel 2007),

fish (Beauvais et al. 2001; Pretto et al. 2010; Richetti et al.

2011; Silva & Pathiratne 2008), and taxonomically higher

invertebrates (Cunha et al. 2007). However, related infor-

mation for acoelomates and pseudocoelomates, which

constitute a large proportion of the world’s invertebrate

biota, is still limited (Du & Wang 2009). Previously, we

found Cd distributed in the head of treated D. japonica

Ichikawa and Kawakatsu 1964 at a significantly greater

concentration than the tail, and suggested that the head

might be the target of Cd toxicity after exposure (Wu et al.

2011). Although head dissolution (Calevro et al. 1998) and

some neurobehavioral changes (Grebe & Schaeffer 1991a;

Plusquin et al. 2012) after Cd treatments have been

reported in some planarian species other than D. japonica,

further investigation of the neurotoxic effects of Cd to them

at the biochemical level has never been reported.

The present study was designed to investigate the neu-

rotoxic effects of Cd to D. japonica on the neurobehavioral

and biochemical levels. First, locomotor activity and spe-

cific behavioral patterns were tested in D. japonica treated

with Cd. Second, the effects of Cd on neurological enzyme

activities, including acetylcholinesterase (AChE), adeno-

sine triphosphatase (ATPase), and monoamine oxidase

(MAO), in D. japonica were determined after in vitro and

in vivo exposures. Finally, specific behavioral patterns

observed in Cd-treated planarians were linked to alterations

of their neurological enzyme activities. The results of this

study not only provide a mechanistic understanding of Cd

neurotoxicity, but also provide helpful information on

establishing D. japonica as a research model for neuro-

toxicity of other environmental contaminants.

Materials and Methods

Chemicals

Cadmium sulfate (3CdSO4�8H2O) was purchased from

Mallinckrodt (Hazelwood, Missouri, USA). Amplex Red

reagent was obtained from Life Technologies Corporation

(Carlsbad, California, USA). Other chemicals used in this

study were purchased from Sigma-Aldrich (St Louis,

Missouri, USA).

Animals

The stock of D. japonica used in this study was obtained

from Taikong Corporation (Taipei, Taiwan) and acclimated

in our laboratory for [2 weeks. In laboratory, planarians

were maintained in dechlorinated tap water at room tem-

perature (23–28 �C) and fed fresh chicken liver once per

week followed by immediate renewal of culture waters

(Wu et al. 2012). Size-selected intact planarians (approxi-

mately 1 cm in length) were used for this study. Planarians

were starved for 3–5 days to create a uniform metabolic

state before being used in the experiments.

Locomotor Activity Test

To study the effects of Cd on locomotor activity of treated

D. japonica, planarians were divided into five groups, each

containing one individual, and exposed to 3 mL formulated

artificial freshwater (ISO water) (Wu et al. 2012) with

various concentrations of Cd at room temperature. The Cd

concentrations used in this study were 0 (control), 0.5, 5,

50, and 500 lM. After exposure for 1 h, locomotor

Table 1 Behavioral responses of planarians categorized by response

group that was adapted according to Grebe and Schaeffer (1991b)

Response

group

Behavioral

response

Description

Morphology Head/nose twist Tip of nose curls up or down

Body elongated Body length is extremely

elongated

Dumbbell-

shape/irregular

shape

Body is contracted irregularly or

becomes dumbbell-shaped

Screw-like Planarian twists around anterior–

posterior axis, and corkscrew-

shaped movement appears

Bridge-like Head is brought closer to tail

ventrally leading to the body

being shaped like a bridge

C-like Head is brought closer to tail

laterally leading to a body

shapes like the letter ‘‘C’’

Walnut-shape Planarian is usually stationary,

moving only slowly with its

body contracted and with an

obvious decrease in its length,

leading to the body being

walnut-shaped

Ornamentation Edges of planarian become

crenelated

Neurological Convulsions Violent twitching or snake-like

movement

Morbidity Labored

movement

Planarian is stationary or moving

slowly

Depression Planarian barely moves when

poked

Unconsciousness Planarian does not move even

when poked

Death Planarian does not move when

poked, and rapid tissue necrosis

is apparent.

640 Arch Environ Contam Toxicol (2014) 67:639–650

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activities of control and treated planarians were quantified

as previously described (Raffa et al. 2001; Pagan et al.

2006). Briefly, planarians were placed individually into a

plastic petri dish (10-cm diameter) containing 10 mL ISO

water and located over graph paper with grid lines spaced

0.5 cm apart. Locomotor activity was quantified as the

number of grid lines each planarian crossed over a

10-minute observation period. Each planarian was used

only once. The experiment was repeated four times.

Neurobehavioral Test

Neurobehavioral testing of planarians after Cd treatment

was performed in 6-well cell culture plates at room tem-

perature with 1 planarian/well. Well culture waters were

replaced by 5 mL ISO water. No obvious abnormalities in

morphology or behavior were apparent in planarians after

culture waters were replaced. The test was started by

replacing the plain ISO water with 5 mL ISO water con-

taining Cd at concentrations of 0, 1.25, 2.5, 5, 10, and

20 mM. The concentrations of Cd used in neurobehavioral

test were based on the results of a preliminary test. Neu-

robehavioral responses in treated planarians was continu-

ously observed and recorded for 60 min. The experiment

was independently repeated ten times. Distinct neurobe-

havioral responses of planarians after Cd-treatment were

categorized based on Grebe and Schaeffer (1991b) with

modifications (Table 1).

Effects of Cd on In Vitro Neurological Enzyme

Activity

Planarians were homogenized with homogenizing buffer

(25 mM Tris-HCl [pH 7.0]) for AChE or 50 mM Tris-HCl

containing 250 mM sucrose and 1 mM ethylene diamine

tetraacetic acid (EDTA) (pH 7.4) for ATPase analysis.

After centrifuging at 12,0009g for 20 min at 4 �C, the

pellets were discarded and the supernatants used for

in vitro assays. For MAO, planarians were homogenized

with 250 mM sucrose (pH 7.6, adjusted by 1 M K2HPO4)

and centrifuged at 1,5009g for 20 min at 4 �C followed by

mitochondria extraction (Tipton & Dawson 1968). Mito-

chondria were resuspended in 50 mM potassium phosphate

buffer (pH 7.4) and kept on ice until in vitro assays were

performed. Supernatants from planarian tissue homoge-

nates for AChE and ATPase assays and mitochondria

extracted for MAO assay were incubated with Cd at con-

centrations of 0, 0.625, 1.25, 2.5, and 5 mM for 30 min at

25 �C before determinations of enzyme activities. These

Cd concentrations were chosen for the in vitro study based

on the neurobehavioral study, but they were lower. For

each enzyme, the experiment was repeated at least three

times.

Effects of Cd on In Vivo Neurological Enzyme Activity

To study the effects of Cd on the in vivo AChE, ATPase,

and MAO activity of treated D. japonica, 20 planarians

were divided into 2 groups, each containing 10 animals,

and exposed to 10 mL ISO water with Cd as the treatment

group or without Cd as controls. The Cd concentration used

in this study was 5.56 lM (=0.63 mg/L), a concentration

equivalent to half of the 48-hour median lethal concen-

tration (LC50) of Cd to D. japonica. This caused significant

alterations in several toxicological end points without the

death of animals during the experimental period (at least

7 days) (Wu et al. 2012). Each sample containing two

animals was killed after exposure for 1, 2, 4, and 7 days to

determine the enzyme activity of AChE, ATPase, and

MAO. This experiment was repeated six times.

Measurement of Neurological Enzyme Activity

To determine AChE activity, planarians were homogenized

with 25 mM Tris-HCl (pH 7.0) and centrifuged at

12,0009g for 20 min at 4 �C. The supernatant was used for

AChE activity measurement according to the method pre-

viously described for determining planarian ChE activity

(Wu et al. 2011) after applying a selective irreversible

butyrylcholinesterase inhibitor, tetraisopropyl pyrophos-

phoramide (iso-OMPA) before the assay was performed.

Briefly, enzymes were incubated with 10 mM iso-OMPA

for 30 min at room temperature before adding dithiobis-

nitrobenzoic acid and acetylthiocholine iodide followed by

recording the absorbance at 414 nm.

To determine ATPase activity, planarians were

homogenized with 50 mM Tris-HCl containing 250 mM

sucrose and 1 mM EDTA (pH 7.4). After centrifugation at

12,0009g for 20 min at 4 �C, the ATPase activity of the

supernatant was measured as previously described (Li

2012).

MAO activity in mitochondria extracted from planarians

was measured according to the method described by Zhou

and Panchuk-Voloshina (1997) with some modifications.

Briefly, 20 lL of resuspended mitochondria were added to

a 96-well microplate and incubated with 160 lL of work-

ing solution containing 200 lM Amplex Red reagent and

1 U/ml horseradish peroxidase in 50 mM potassium

phosphate buffer (pH 7.4) for three minutes at 25 �C.

After 20 lL of amine substrate was added into the samples,

fluorescence was measured with a fluorescence microplate

reader with its filter set for excitation and emission at 560

and 580 nm, respectively, for 30 min and corrected by

Arch Environ Contam Toxicol (2014) 67:639–650 641

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subtracting the values derived from the blank without the

amine substrate. Amine substrates used for the determi-

nation of MAO-A and -B activity were 0.1 mM serotonin

and 20 mM phenethylamine, respectively. Serial concen-

trations of resorufin standards were prepared to establish

the standard curve. MAO activity was expressed as the

amount of resorufin (fmol) produced per mg of mito-

chondria protein per minute. Protein concentrations in the

homogenate and mitochondria extracts were measured

using Bradford’s method (Bradford 1976).

Determination of Actual Cd Concentrations in Test

Solutions

To confirm the actual Cd concentrations in test solutions,

1 mL of each solution was sampled, combined with 9 mL

0.5 % HNO3 in distilled water, and stored at 4 �C until

analysis. Cd levels were measured using an atomic

absorption spectrophotometer (932 AA, GBC Scientific

Equipment, Braeside, Australia). Before measuring the test

solution, standard curves with high correlation coefficients

(R2 = 0.9884 to 0.9975) were established after the certified

Cd standard solution (Merck KGaA, Darmstadt, Germany)

was serially diluted and determined. The detection limit of

the AAS for Cd was 0.01 mg/L. If necessary, test solutions

were diluted with 0.5 % HNO3 in double-distilled water.

Statistical Analysis

Statistical evaluations were performed with the Minitab

Statistical Program (ver. 15) (Minitab; State College, PA,

USA). Data normality was tested and confirmed with the

Anderson-Darling test. Data that met the assumptions of

normality were analyzed with one-way analysis of variance

to determine the effects of Cd treatment on locomotor

activity, neural behavior patterns, and neurological enzyme

activities. Post hoc Duncan’s multiple-range test was used

to evaluate mean differences between control and treatment

groups. Unless noted, all comparisons were considered

significantly different at p \ 0.05. Results are presented as

the mean ± SD.

Results

Cd Concentrations Measured in Test Solutions

The actual concentrations of Cd in the test solutions were

analytically determined, and the average percent nominal

concentration calculated to be 121.38 ± 13.89 %. This

indicates that measured Cd concentrations were close to

those of nominal values.

Locomotor Activity in Cd-treated D. japonica

Locomotor activity of untreated D. japonica is shown in

Fig. 1a. Untreated planarians showed a characteristic and

stable locomotor velocity during the course of the

10-minute observation period (28.58 ± 1.07 crosses/min).

In planarians exposed to Cd, inhibition of locomotor

velocity was observed at C5 lM Cd (Fig. 1b).

Neural Behavioral Patterns in Cd-treated D. japonica

We modified the categories, and only three groups of

behavioral responses were recorded: morphological

responses, neurological responses, and morbidity

(Table 1). The morphology group contained eight different

responses: head/nose twist, body elongated, dumbbell-

shape/irregular shape, screw-like, bridge-like, C-like, wal-

nut-shape, and ornamentation. The neurological group

contained only convulsions response. Morbidity group

contained four different responses: labored movement,

depression, unconsciousness, and death.

Fig. 1 a Locomotor activity of untreated D. japonica expressed as

the mean ± SD of the cumulative number of grid lines crossed per

minute. b Locomotor velocity of planarians after exposure to various

concentrations of Cd for 1 h. After exposure, locomotor velocity was

quantified over a 10-minute observation period and expressed as

mean ± SD of the number grid lines crossed per minute. *p \ 0.05

and #p \ 0.1 compared with controls


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Neural behavioral responses in D. japonica after expo-

sure to Cd are shown in Figs. 2, 3, 4. After planarians were

exposed to Cd, alterations in morphological (Fig. 2a),

neurological (Fig. 3a), and morbidity (Fig. 4a) responses

were apparent. Incidence times were concentration-

dependent resulting in wave-like patterns in our figures.

Morphological alterations in planarians immediately

appeared after the animals were exposed to Cd at 5, 10, and

20 mM, and these responses lasted for 15 (10 and 20 mM

treatment groups) to 40 (5 mM treatment group) minutes

(Fig. 2a). In planarians exposed to Cd at lower concen-

trations (1.25 and 2.5 mM), morphological alterations

appeared 1–5 min after the beginning of exposure and

lasted until experiments ended. Morphological alterations

in Cd-treated planarians included irregular-shaped, elon-

gated, screw-like, and bridge-like positions (Fig. 2b). In

addition, the latency time of morphological alterations in

Cd-treated planarians decreased in a concentration-depen-

dent manner (Fig. 2c). In treatment groups exposed to 2.5,

5, 10, and 20 mM Cd, latency times of the bridge-like

position were greater than those for other morphological

responses. This indicated that irregular-shaped, elongated,

and screw-like positions in treated planarians appeared

earlier than the bridge-like position on exposure.

Similarly, neurological responses in planarians were

immediately observed after the animals were exposed to

Cd at 5, 10, and 20 mM, and these responses lasted for 10

(20 mM treatment group) to 15 (5 and 10 mM treatment

groups) minutes (Fig. 3a). In planarians exposed to 1.25

and 2.5 mM Cd, although the appearance of neurological

responses was delayed, these responses also lasted for

approximately 10–15 min. Neurological responses in Cd-

treated planarians that could be clearly identified included

head movement, violent twitching, and snake-like move-

ment (Fig. 3b).

Behavioral responses categorized in morbidity group

were observed in planarians exposed to Cd at concen-

trations of 2.5, 5, 10, and 20 mM 3–7 min after the

beginning of exposure (Fig. 4a). These responses in

1.25 mM Cd–treated planarians were observed 20 min

after the beginning of exposure. In treatment groups

exposed to 5, 10, and 20 mM Cd, behavioral responses of

the morbidity group lasted until the animals died. In

groups exposed to 1.25 and 2.5 mM Cd, these responses

lasted until the end of the experiment. Observed behav-

ioral responses of morbidity group in treated planarians

included labored movement, body contraction/depression,

unconsciousness, and death (Fig. 4b). Tissue necrosis

always followed the death of planarians. In addition, the

latency time of behavioral responses of morbidity group

in Cd-treated planarians decreased in a concentration-

dependent manner (Fig. 4c).

Fig. 2 Morphological responses of freshwater planarian D. japonica

treated with Cd at different concentrations. a Incidence rates of total

morphological alterations in Cd-treated planarians during the

experimental period B60 min. b Photographs of and c latency times

for different morphological alterations appearing in Cd-treated

planarians


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Effects of Cd Treatments on the In Vitro Activity

of Neurological Enzymes

Planarian neurological enzymes were prepared and

exposed in vitro to Cd at different concentrations followed

by measuring their activity (Fig. 5). After incubation with

Cd, AChE and ATPase activities decreased significantly

and in a concentration-dependent manner (p \ 0.1 or 0.05)

(Fig. 5a, b). In addition, a significant decrease in MAO-A

activity was apparent after the enzyme was incubated with

Cd at concentrations of 2.5 and 5 mM Cd (p \ 0.1 and

0.05, respectively) (Fig. 5c). It was interesting that pla-

narian MAO-B activity was significantly increased after

the enzyme was incubated with lower concentrations of Cd

(0.63 and 1.25 mM) (p \ 0.05) but was inhibited at a

greater concentration (5 mM) (p \ 0.1) (Fig. 5d).

In Vivo Activity of Neurological Enzymes in D.

japonica After Exposure to Sublethal Concentrations

of Cd

To investigate the effects of Cd on the in vivo activity of

neurological enzymes in planarians, animals were exposed

to sublethal concentrations of Cd for 1, 2, 4, and 7 days

followed by measurements of neurological enzyme activ-

ity. Significant decreases of AChE activity in treated pla-

narians were observed on days 4 and 7 after exposure

(Fig. 6a). Although Cd exposure did not affect planarian

ATPase activity when animals were exposed for 1 and

4 days, a significant increase in activity was observed when

the exposure was prolonged to 7 days (Fig. 6b). An obvi-

ous decrease in MAO-A activity was apparent in Cd-trea-

ted planarians after exposure for 4 days, but no significant

difference was found between the enzyme activity of

controls and treatment groups 7 days after exposure

(Fig. 6c). Cd exposure did not influence the activity of

planarian MAO-B during the entire experimental period

(Fig. 6d).

Discussion

Effects of Cd on Neural Behavior of D. japonica

We found that the differences between some of the

responses in D. japonica after exposure to Cd were not as

distinct as described by Grebe and Schaeffer (1991b). For

example, the ‘‘inch-worming’’ (hyperkinesia) response

categorized in their locomotive group is actually difficult to

clearly distinguish from the ‘‘shape change’’ response of

the morphological group. Under these circumstances,

experimental results could be influenced by observer bias.

To make the response categories more practical and the

results more consistent, we modified the categories by

discarding some that we considered equivocal and recorded

only those responses that were distinctive and clearly

identifiable.

Using the behavioral responses in our modified catego-

ries as end points, the effects of Cd on the neural behavior

of D. japonica were exhibited in a dose-dependent manner.

This was expected because, in the early 1990s, some Du-

gesia dorotocephala behavior was found to be altered by

metal exposure. This included prolonged righting (or thi-

gmotactic) times, decreased prey capture ability, decreased

motility, locomotive disorders, and changes in body shape

(Best & Morita 1991; Kapu & Shaeffer 1991). In this

study, inhibited locomotor activity in D. japonica after Cd

Fig. 3 Neurological responses of freshwater planarian D. japonica


neurological alterations in Cd-treated planarians during the experi-

mental period B60 min. b Photographs of morphological alterations

appearing in Cd-treated planarians


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treatment was also observed. Altered neural behaviors and

morphological changes in Cd-treated D. dorotocephala

were reported by Grebe and Schaeffer (1991a). However,

they did not further discuss the relationships between

altered neural behaviors and potential disorder/dysfunction

of the neural system.

Observations from previous pharmacological studies

revealed that planarians show specific behavioral patterns

when normal neural transmission is altered by chemicals.

Stimulations of dopamine receptors D1 (D1R) and D2

(D2R) induce hyperkinesia in planarians leading to the

appearances of screw-like and C-like morphologies,

respectively (Palladini et al. 1996; Venturini et al. 1989). In

contrast, stimulation of ACh transmission in planarians is

followed by hypokinesia of animals in bridge-like poses

(Carolei et al. 1975). More recently, the interaction of ACh

and dopamine transmission in planarians was investigated,

and it was found that the effects of its dopaminergic

transmission appear to be quite the opposite of those of

ACh (Buttarelli et al. 2000, 2008). Neural behaviors—

including the C-like position, screw-like hyperkinesia,

writhing, and head-swinging—were observed in planarians

treated with serotonin-receptor agonists and serotonin

(Farrell et al. 2008). The results of these pharmacological

studies indicate that ACh, dopamine, and serotonin are

crucial for mediating the regulation of neuromuscular

functions in planarians.

In this study, we found that both screw-like hyperkinesia

and bridge-like position occurred in Cd-treated D. japonica

suggesting that Cd possibly exerts its effect on cholinergic,

dopaminergic, and/or serotonergic transmission in planar-

ians. Irregular-shaped and elongated body morphologies

were also observed in Cd-treated D. japonica. Although the

correlations between these two neural behavioral responses

in planarians and neural transmission were not reported and

discussed previously, they were also apparently the results

of abnormal contractions in muscular systems. The C-like

position was not observed in Cd-treated D. japonica, sug-

gesting that Cd possibly did not stimulate its D2R. Previ-

ously, the loss of the D2R was found in rat striatal

homogenate pretreated with Cd (Scheuhammer & Cherian

1985).

The occurrences of neurological responses in Cd-treated

planarians were within the time periods when their mor-

phological responses occurred. This confirmed that treated

planarians were still alive, conscious, and responsive and

that their neurotransmission and neuromuscular system

were severely disturbed during these periods. When

exposure time was prolonged, however, Cd-treated pla-

narians died, especially those exposed to Cd at

Fig. 4 Morbidity responses of freshwater planarian D. japonica


morbidity responses in Cd-treated planarians during the experimental

period B60 min. b Photographs of and c latency times for different

morbidity responses appearing in Cd-treated planarians. Arrow

indicates the pharynx extended outside the body of the planarian


123


concentrations [5 mM. Before death, morbidity responses

occurred after the neurological and morphological

responses, occurring in the order of labored movement,

depression, and unconsciousness.

Effects of Cd on Neurological Enzymes of D. japonica

In mammals, although the entrance of Cd into the brain is

limited by the blood–brain barrier (BBB), it disturbs CNS

functioning by altering brain tissue levels of trace elements

and provoking disturbances to crucial neural enzymes

involved in neurotransmitter release and neurotransmission

(Carageorgiou & Katramadou 2012). Planarians lack the

equivalent structure of a BBB (Raffa et al. 2007), so it is

possible that Cd enters the brain directly and causes some

influences on its function and structure. Nevertheless, we

showed that AChE, ATPase, and MAO-A and -B activities

of D. japonica were altered after these enzymes were

directly exposed to Cd in vitro. In addition, altered in vivo

activities of these neurological enzymes were observed in

planarians treated with Cd.

AChE is a crucial enzyme for the regulation of cholin-

ergic transmission by catalyzing the hydrolysis of ACh to

acetate and choline to terminate the transmission. Thus,

any fluctuation of AChE activity could cause a change in

cholinergic transmission (Carageorgiou & Katramadou

2012). Our in vitro and in vivo studies both showed that

planarian AChE activity was inhibited by Cd exposure,

which was presumably followed by uninterrupted cholin-

ergic signal transmission.

Na?, K?-ATPase is a membrane-bound enzyme

involved in several neural functions of animals such as

neural excitability (Sastry & Phillis 1977) and penetration

of neurotransmitters across membranes (Hernandez 1989;

Swann 1984). We found that the activity of planarian

ATPase was inhibited in a concentration-dependent man-

ner after in vitro incubation with Cd. Inhibited Na?, K?-

ATPase activities after exposure to high concentrations of

Cd (0.01–1 mM) were also reported in in vitro systems of

other animals (Carageorgiou et al. 2004; Chandra et al.

1984; Kinne-Saffran et al. 1993). The inhibitory mecha-

nisms of Cd on Na?, K?-ATPase show species specificity

(Kinne-Saffran et al. 1993), so further investigation is still

needed to understand the molecular mechanism in planar-

ians. Regardless of the mechanism, Na?, K?-ATPase

activity inhibited by high concentrations of Cd (0.1 mM) is

Fig. 5 Activity of a AChE, b ATPase, c MAO-A, and d MAO-B in

tissue homogenates of planarian D. japonica after in vitro pre

incubations with different concentrations of Cd for 30 min. The

activity of the enzymes in treatment groups is presented as

percentages of corresponding controls. *p \ 0.05 and #p \ 0.1

compared with controls


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accompanied by decreases in dopamine and noradrenalin

uptakes in rat brain synaptosomes (Chandra et al. 1984).

This suggests that dopamine reuptake was possibly

decreased in D. japonica after treatment with Cd at high

concentrations. In addition, increased Na?, K?-ATPase

activity was observed in brains of rats treated with 1–5 mg

Cd/kg and in in vitro brain homogenate incubated with Cd

at low concentrations (Carageorgiou et al. 2004). These

findings suggest that compared with the effect caused by

Cd at high levels, low levels of Cd increase Na?, K?-

ATPase activity. A similar phenomenon was also observed

in planarians exposed to Cd at sublethal concentrations for

7 days with their in vivo ATPase activity significantly

increasing (p \ 0.05).

MAO is a key enzyme catalyzing the metabolism of

biogenic amines, including catecholamine neurotransmit-

ters. Two isoforms of MAO, including MAO-A and -B,

were characterized according to substrate specificity and

inhibitor sensitivity (Youdim et al. 1969). The differential

effects of Cd on the activity of these two MAO isoforms

were found in rat (Leung et al. 1992). Similarly, we found

that in vitro activities of planarian MAO isoforms were

affected by Cd with different patterns. Presumably, fluc-

tuations of MAO-A and -B activity caused by Cd might

alter the levels of serotonin and dopamine, which in turn

influence serotonergic and dopaminergic neurotransmis-

sions. In contrast, the in vivo MAO activity of D.

japonica was insensitive to Cd at sublethal concentra-

tions. Because the direct effect of Cd to planarian MAO

was shown through in vitro study, the insusceptibility of

in vivo MAO activity to Cd might be a result of pro-

tection by intrinsic physiological mechanisms. Indeed, we

previously showed that metallothionein, a well-known

inducible protein possessing the ability to sequester

divalent metals in animals, was largely induced in Cd-

treated D. japonica under an identical experimental

design (Wu et al. 2012). This suggested that Cd was

sequestered and that the amount of free Cd ions within

the planarian body might be insufficient to have direct

effects on MAO enzymes.

Fig. 6 Activity of a AChE, b ATPase, c MAO-A, and d MAO-B in the planarian D. japonica treated with Cd at a concentration of 5.56 lM for

1, 2, 4, and 7 days. *p \ 0.05 compared with controls


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Correlations Between the Altered Activity

of Neurological Enzymes and Neural Behavior

Responses in Cd-treated D. japonica

Apparently all of the enzymes investigated in this study

were susceptible to direct Cd exposure and potentially

contributed to altered neurotransmission in Cd-treated D.

japonica The susceptibility to Cd of these planarian neu-

rological enzymes was on the order of ATPase = MAO-B

[ AChE [ MAO-A according to Cd concentrations that

caused significant differences in their activity. It is also

interesting that in D. japonica treated with Cd at concen-

tration [2.5 mM, screw-like hyperkinesia in planarians

appeared earlier than the bridge-like position, which was

soon followed by depression and other morbidity respon-

ses. With these observations, we propose a hypothetic

model containing three stages to link the changes on altered

activities of neurological enzymes in treated D. japonica to

those of its neurobehavioral responses (Fig. 7).

According to the model, although initial body levels of

Cd are relatively low after exposure, the first stage ATPase

activity is inhibited due to its greater susceptibility to Cd

accompanying decreased dopamine reuptake (Chandra

et al. 1984). It might be followed immediately by the

accumulation of dopamine in the synaptic cleft and con-

tinuous stimulation on dopaminergic neurotransmission,

thus leading to the induction of hyperkinesia (screw-like

morphology) in this stage. At the second stage, however, as

exposure continues, the level of Cd in the treated planarian

body is gradually increased and causes an increase in ACh

release (Meyer & Cooper 1981), inhibition of AChE

activity, and enhancement of MAO-B activity. These

alterations might be followed by stimulation of cholinergic

neurotransmission and decrease of dopaminergic

neurotransmission. That would account for the bridge-like

position in treated planarians appearing at this stage.

Finally, at the third stage, accumulated body levels of Cd

are so high that disorders in other essential physiological

functions also occur in parallel. At this stage, the responses

of planarians related to disturbed neurotransmissions (e.g.,

morphological and neurological responses) are almost

completely replaced by morbidity responses, tissue necro-

sis, and death. Therefore, although decreases in MAO-A

and -B activity observed in planarian homogenate after

in vitro exposure to Cd at high concentrations (2.5 and

5 mM) are shown, no corresponding neurobehavioral

effects are presented at this stage.

Conclusion

In this study, Cd at high concentrations was used to explore

its direct effect on neurotransmission systems and the

neural behavior of the freshwater planarian D. japonica.

Clearly, concentration-dependent patterns in behavioral

responses of Cd-treated planarians were shown. In addi-

tion, through in vitro and in vivo studies, effects of Cd on

planarian AChE, ATPase, and MAO-A, and MAO-B

activity were revealed and shown to be comparable with

those of mammalian systems. Thus, disturbed neurotrans-

mission in Cd-treated planarians is suggested and used to

propose a hypothetic model connecting the results from

behavioral and enzyme studies. However, some key

mechanistic details about the effects of Cd on planarian

neurotransmission systems—such as the molecular mech-

anisms of alterations in neurological enzymes, the patterns

of changes in neurotransmitter levels, and the ranges of

body Cd levels that separate the three response stages that

Fig. 7 Hypothetical model connecting the changes in neurological enzyme activity and neurobehavioral responses in the Cd-treated planarian D.

japonica


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we propose—are still unknown. In addition, future study of

the long-term exposure of planarians to Cd at sublethal

and/or environmentally relevant concentrations is desired

for understanding the neurotoxic effects of this metal under

real situations.

Acknowledgments The authors are grateful for funding support

under Grants No. NSC101-2811-B-002-055 and NSC101-2321-B-

002-069 from the National Science Council, Taiwan.

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Cadmium neurotoxicity on a freshwater planarian

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