Hepatic metabolism of contaminants in the terapontid fish, yellowtail trumpeter (Amniataba...

Hepatic Metabolism of Contaminants in theTerapontid Fish, Yellowtail Trumpeter (Amniatabacaudavittata Richardson)

Diane Webb,1 Marthe Monique Gagnon,1 Tom Rose2

1Department of Environmental Biology, Curtin University of Technology, Bentley,Western Australia 6102, Australia

2Department of Water, SGIO Atrium, 49 St Georges Terrace, Perth, Western Australia 6000,Australia

Received 24 April 2007; revised 18 June 2007; accepted 21 June 2007

ABSTRACT: The yellowtail trumpeter (Amniataba caudavittata) is an estuarine-dependent omnivorous fishfound in the Swan-Canning Estuary, Western Australia. Thirty five fish were injected with either the poly-cyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), the synthetic flavenoid b-naphthoflavone (BNF), orused as controls. The fish were then sampled at 3 and 7 days postinjection. Induction of the enzymeethoxyresorufin O-deethylase (EROD) activity was nonsignificant while ethoxycoumarin O-deethylase(ECOD) activity induction differed amongst treatments. A high interindividual variability in the EROD activ-ity was observed. The measurement of sorbitol dehydrogenase in the serum (s-SDH) was elevated (BNF2.2 times and B[a]P 3.2 times the control fish) demonstrating that liver cell damage had occurred.Increases in biliary metabolites of both B[a]P-type and pyrene-type (19 times and 3.4 times the controlsrespectively) indicated that detoxification of pyrene-type compounds had taken place. Fish of the Tera-pontidae family, such as the yellowtail trumpeter, were found to be suitable for biomonitoring the health ofthe Swan-Canning Estuary. A combination of ECOD activity, s-SDH, and the measurement of biliarymetabolites represents a suitable suite of biomarkers for environmental monitoring of the sublethal effectsof PAH pollution in these fish. # 2008 Wiley Periodicals, Inc. Environ Toxicol 23: 68–76, 2008.

Keywords: benzo[a]pyrene; biomarker; biomonitoring; bile metabolites; b-naphthoflavone; environmentalmonitoring; EROD; ECOD; serum sorbitol dehydrogenase (s-SDH); Western Australia

INTRODUCTION

The yellowtail trumpeter (Amniataba caudavittata Richard-

son, 1845) is a member of the Terapontidae family of fishes

that are native to Australia and New Guinea (Indo-West

Pacific). The fish can attain a maximum size of 300 mm,

however in the Swan-Canning Estuary (SCE), Western

Australia, they rarely exceed 220 mm in size (Allen et al.,

2002). According to Wise et al. (1994), the yellowtail trum-

peter cannot be aged past 21 to 24 months because of con-

founding changes in the growth patterns on annuli rings

found in the otolith. However, based on catches of wild fish

it is assumed many of the fish live until at least the begin-

ning of their fourth year of life. In south-western Australia

yellowtail trumpeter are essentially confined to estuaries,

including the SCE (Potter et al., 1994; Wise et al., 1994;

Hoeksema and Potter, 2006). In the SCE the main dietary

items of juvenile yellowtail trumpeter include planktonic

crustaceans such as the calanoid copepod Gladioferansimparipes and crab zoea. Adult fish are benthic omnivores

and consume a variety of food items including the western

Correspondence to: D. Webb; e-mail: [email protected]

Contract grant sponsor: Australian Research Council.

Published online 23 January 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI 10.1002/tox.20307

�C 2008 Wiley Periodicals, Inc.

68

king prawn (Melicertus latisculatus), carid shrimps, iso-

pods, Halicarcinus spp. crabs, polychaetes, molluscs, small

teleost fish and algae (Wise et al., 1994).

The life history of this fish in the SCE suggests that the

yellowtail trumpeter could be a suitable species to use in

the environmental monitoring of the estuary using bio-

chemical markers (biomarkers) of fish health. These bio-

markers are ‘biological responses to environmental chemi-

cals which give a measure of exposure and sometimes,

also, of toxic effect’ (Walker et al., 1996). They are mea-

surements of body fluids, cells or tissues indicating bio-

chemical or cellular modifications due to the presence and/

or concentration of pollutants (van der Oost et al., 2003).

Detoxification of xenobiotics is a function of the liver and

involves enzymes that metabolise compounds to facilitate

their excretion from the body. Specific forms of cyto-

chrome P450-dependent enzyme activities, known as

mixed-function oxygenase (MFO) enzymes, are induced by

exposure to a variety of lipophilic compounds such as orga-

nochlorines, polychlorinated compounds, polycyclic aro-

matic hydrocarbons (PAHs) and polychlorinated-biphenyls

(PCBs) (Hodson et al., 1991; Connell et al., 1999). Fish

exposed to PAHs, PCBs and organochlorine pesticides all

show increased levels of MFO enzyme activity in the liver

(Collier and Varanasi, 1991; Holdway et al., 1994; Arcand-

Hoy and Metcalfe, 1999). Persistent organic chemicals

have been found to be present in the SCE (Swan River

Trust, 1999a), and Webb et al. (2005a,b) has identified

metabolites of the naphthalene, pyrene, and benzo[a]pyrene(B[a]P) in the bile of black bream (Acanthopagrus butch-eri) captured in the estuary although chemical analysis of

water, sediments and fish flesh consistently fail to identify

the presence of these PAHs in the SCE.

Webb and Gagnon (2002), in a previous laboratory

study, suggested that the yellowtail trumpeter was not a

suitable biomonitor of estuarine health after the fish were

intraperitoneally (i.p.) injected with 3,30,4,40,5-pentachloro-biphenyl (PCB-126). They only measured the induction of

MFO enzyme activity, using ethoxyresorufin-O-deethylase

(EROD) after 10 days of exposure. In that study, the induc-

tion of EROD activity in the yellowtail trumpeter was

appreciably lower than in another two species that were

also investigated, the black bream and the sea mullet (Mugilcephalus).

There are a number of factors (either singly or in combi-

nation) that could have affected this initial assessment of

the utility of this fish for biomonitoring. MFO enzymes in

the yellowtail trumpeter could have been highly efficient in

metabolising contaminants with maximum induction occur-

ring before day 10, which then could have rapidly declined

before sampling. Different results may have been obtained

if the fish had been analysed for EROD activity induction,

in three or seven days post-injection. Alternately, the cyto-

chrome P450-dependent enzyme activities in the yellowtail

trumpeter may have been non-responsive to PCB exposure,

as Celander et al. (1996) had found in the rainbow trout

(Oncorhynchus mykiss). The yellowtail trumpeter may also

be susceptible to hepatic cellular injury on exposure to

PCB-126 with the injection concentration (10 lg kg21 fish

weight) potentially high enough to cause liver damage. Fish

livers with cellular injuries are less capable of MFO induc-

tion than are fish with non-injured livers (Holdway et al.,

1994). Lastly, a different cytochrome P450 pattern of MFO

induction could be used in the Terapontidae family of fishes

for metabolising and the elimination of compound, rather

than CYP1A as suggested by Machala et al. (1997) in com-

mon carp from the Cyrinidae family, and also by Stegeman

et al. (1997) in species from the Haemulidae, Kyphosidae,

Lutjanidae and Pomacentridae families of fishes.

These points provided the basis for this study to re-

assess the utility of the Terapontid fish, A. caudavittata, asa suitable local estuarine fish species to investigate non-nu-

trient contaminant effects in the estuary using biochemical

markers of fish health. To achieve this central aim, two

MFO enzymes [EROD and ethoxycoumarin O-deethylase(ECOD) activity] in the liver of the yellowtail trumpeter

were determined after i.p. injection with either B[a]P (a

known inducer of EROD) or b-naphthoflavone (BNF; a

synthetic PAH) in order to examine any differences in

induction of the two enzyme activities. Further, the level of

sorbitol dehydrogenase enzyme activity in the serum of the

fish (s-SDH) was analysed to investigate whether MFO

induction was influenced by hepatocellular damage follow-

ing injection. This enzyme is specific to the liver and only

detected in the serum following hepatocellular damage

(Wiesner et al., 1965; Dixon et al., 1987). Finally, the level

of pyrene-type and B[a]P-type biliary metabolites were

measured to confirm that absorption, metabolism and elimi-

nation of PAHs had occurred (Connell et al., 1999).

METHODS

Chemicals

All chemicals were purchased from Sigma–Aldrich, Castle

Hill, NSW, Australia, unless otherwise indicated.

Fish Capture and Acclimation

Yellowtail trumpeter were captured in March 2005 (N 535) from the upper reaches of the Swan River (Fig. 1) using

a 250 m seine net with a mesh size of 100 mm. The sizes of

the fish ranged between 106 and 178 mm standard length

and were of adult size (Potter et al., 1994). The fish were

transferred to the laboratory and acclimated for ten days in

100 L aquariums at about 30 ppt saline water, similar to the

salinity of the estuarine water at time of collection. The fish

were randomly allocated to one of six aquariums to give a

biomass loading in each of 3–5 g of fish per liter. Each

HEPATIC METABOLISM IN YELLOWTAIL TRUMPETER 69

Environmental Toxicology DOI 10.1002/tox

aquarium was provided with a triangular corner sponge

filter and airstone connected via airline to a Precision

SR-12000 air pump. Aeration was maintained at �100%

oxygen saturation of the water throughout the experiment.

A 12-h light/12-h dark regime was used. During the dark

regime, a 15-Watt lamp was reflected against the laboratory

wall to simulate moonlit conditions. Detritus was syphoned

from the bottom of the aquariums daily and a 20% water

change was done every second day. Water physico-chemis-

try (salinity, temperature, pH, and dissolved oxygen) was

measured daily during acclimation and experimental peri-

ods to ensure constant conditions. The fish were fed each

day with frozen bloodworm, Bio-PoreTM Mega-Marine

Mix, or commercial fish pellets at a maintenance level of

1% body weight per day. The fish were feeding well and

appeared healthy.

Fish Injection and Sample Collection

After 10 days acclimation, each fish was immersed in an

anesthetic solution of tricaine methanesulfonate (MS-222;

70 mg L21) for �2 min prior to injection. The fish were

weighed, and total, standard, and fork lengths were

recorded for identification purposes. One group of yellow-

tail trumpeter (N 5 5) was given an intraperitoneal (i.p.)

injection with corn oil only at 1 mL kg body wt21 (control

group). A needle attached to an empty syringe was inserted

into the intraperitoneal cavity of a second group of fish

(sham injected group; N 5 6). The fish from a further two

aquariums (N 5 12) were injected with 20 lM kg body

wt21 of B[a]P dissolved in corn oil and the remaining 12

fish were injected with 40 lM kg body wt21 of b-naphtho-flavone (BNF) in corn oil. All injections were standardised

to ensure a final injection volume of 1 mL of corn oil kg

body wt21.

Food was withheld from the fish 24 h prior to sampling.

On each of days 3 and 7 postinjection, six yellowtail trum-

peter from both the B[a]P and the BNF treatments were

euthanized using Ike Jime (spiking through the brain). A

sample of blood was taken from the caudal vein using a

vaccutainer. The blood samples were allowed to clot on ice

for 15 min, then centrifuged (Jouan CR3i centrifuge) for

10 min at 3000 rpm. The gall bladder was excised from

each fish and the bile harvested. Livers were removed,

quickly examined for any visible anomaly, weighed, and

rinsed in ice-cold KCl. All samples were placed in cryo-

vials, immediately immersed in liquid nitrogen, then later

transferred to a freezer and held at 2808C until analysis.

The corn oil control and sham injected fish were sacrificed

on day 7 and biopsies collected.

Gonads were removed and assessed for maturity using

the scale of gonad development of Nikolskii (1969). The

gonads and remaining abdominal organs were discarded

and the fish weighed to record the carcass weight.

The liver somatic index (LSI) and condition factor (CF)

were calculated according to the following equations:

CF ¼ ½ðBW� GWÞ=SL3� 3 100 ð1Þ

LSI ¼ ðLW=CWÞ 3 100 ð2Þ

where BW 5 total body weight, GW 5 gonad weight, SL

5 standard length, LW 5 liver weight, and CW 5 carcass

weight. The condition factor is based on gonad-free weight

to avoid any bias due to variations in sexual maturation and

stomach contents, and the LSI is based on carcass weight to

avoid bias due to variable levels of fat in the gonads and

intestines, and variable gonad weight (Hodson et al., 1991).

Mixed-Function Oxygenase Enzymes

Postmitochondrial Supernatant Preparation

Individual liver samples were thawed on ice then homoge-

nized in HEPES pH 7.5 using a Heidolph DIAX 900

homogeniser. The homogenate was centrifuged at 9800

rpm for 20 min at 48C and the S9 postmitochondrial super-

natant (PMS) collected for immediate use. Protein content

of the PMS was based on the method of Lowry et al.

(1951).

EROD Assay

EROD activity was measured using a modified method of

Hodson et al. (1991). The reaction mixture contained

HEPES pH 7.8, MgSO4, bovine serum albumin (BSA),

NADPH (b-nicotinamide adenine dinucleotide phosphate,

reduced form) solution, and PMS. The reaction was initi-

ated by adding ethoxyresorufin, incubated at room tempera-

ture for 2 min, and then terminated by adding HPLC grade

methanol. Resorufin standards (0.000 to 0.085 mM) and

Fig. 1. Field collection site, Swan-Canning Estuary (mapadapted from Swan River Trust, 1999b).

70 WEBB, GAGNON, AND ROSE


samples were centrifuged to precipitate proteins and the flu-

orescence of the supernatant read on a Perkin–Elmer LS-45

Luminescence Spectrometer at excitation/emission wave-

lengths of 535/585 nm (slit 10 ex/10 em). EROD activity

was expressed as picomoles of resorufin produced per mg

of total protein per minute (pmol R mg Pr21 min21).

ECOD Assay

ECOD activity was assessed using the method of Webb

et al. (2005b), optimized for yellowtail trumpeter. The reac-

tion mixture containing 0.1 M Tris(hydroxymethyl amino-

methane) buffer pH 7.4, KCl, MgCl2, NADPH solution,

and PMS was incubated for 2 min in a water bath at 308C.The reaction was initiated by adding 2 mM ethoxycou-

marin, incubated for a further 10 min at 308C, and then

terminated by the addition of 5% ZnSO4 and saturated

Ba(OH)2. Umbelliferone (C9H6O3; 7-hydroxycoumarin)

standards (0.000 to 0.093 nM) and samples were centri-

fuged to precipitate proteins and 1 mL of the supernatant

was transferred to a test tube. 0.5 M glycine-NaOH buffer

pH 10.4 was added to each tube and the fluorescence of the

buffered supernatant was read on a Perkin–Elmer LS-45

Luminescence Spectrometer at excitation/emission wave-

lengths of 380/452 nm (slit 10 nm ex/10 nm em). ECOD

activity was expressed as picomoles of 7-hydroxycoumarin

produced per mg of total protein per minute (pmol H mg

Pr21 min21).

Serum Sorbitol Dehydrogenase Assay

The methods for the s-SDH assay were adapted from the

Sigma Diagnostics Sorbitol Dehydrogenase Kit (No. 50-

UV) procedure. Serum was thawed on ice before a 50 lLaliquot was placed in a cuvette with 450 lL of b-NADH(b-Nicotinamide Adenine Dinucleotide, reduced form) -

Tris Buffer, pH7.5, solution. This was then incubated at

room temperature for 10 min to allow for the reaction of

keto acids in the serum. Following incubation, 100 lL of D-

fructose solution was added to commence the reaction and

the decrease in the rate of absorbance (DA) over 1 min was

immediately read on a Pharmacia UV-Visible Spectropho-

tometer at 340 nm. The s-SDH activity expressed as milli-

International Units (mIU) in the serum of the yellowtail

trumpeter was calculated using the following equation:

milli-International Units (mIU) of SDH activity

¼ DA=min 3 0:6

0:00622 3 0:05

where 0.6 5 reaction volume (mL); 0.00622 5 micromolar

absorptivity of NADH at 340 nm (McComb et al., 1976);

and 0.05 5 sample volume (mL).

Bile Metabolite Measurement

Two biliary metabolite-types were measured, B[a]P and py-

rene, by fixed wavelength fluorescence (FF). The term ‘‘metab-

olite-type’’ is used as groups of compounds that fluoresce at

specific wavelengths are detected. This offers the advantage of

a particularly sensitive detection of a group of metabolites orig-

inating from a common parent compound (Lin et al., 1996).

For example, nearly all ‘‘B[a]P-type’’ metabolites fluoresce at

380/430 nm (Lin et al., 1996) and ‘‘pyrene-type’’ metabolites

fluoresce at 340/380 nm (Krahn et al., 1986). The concentra-

tions of PAH bile metabolites are reported as metabolite equiv-

alents to their respective standards, representing the amount of

a metabolite that would be present if the group of metabolites

originated from a parent compound (Krahn et al., 1986). Nei-

ther B[a]P-type nor pyrene-type metabolites were expected to

occur in the BNF-treated fish.

Bile samples were thawed on ice and diluted to 1:4000 in

50% HPLC grade methanol/H2O for FF determination of

pyrene-type metabolites and measured on a Perkin–Elmer

LS-45 Luminescence Spectrometer at excitation/emission

wavelengths of 340/380 nm (slit 10 ex/10 em) (Aas et al.,

1998). The bile was further diluted to 1:8000 for the deter-

mination of B[a]P-type metabolites and read at excitation/

emission wavelengths of 380/430 nm (slit 10 ex/10 em)

(Lin et al., 1996). The concentration of PAH metabolites in

the bile was determined using 1-hydroxypyrene (98%; 1-

OH pyrenol) as the external standard with the fluorescence

reading of bile converted to 1-OH pyrenol equivalents from

the linear regression curves (pyrene, 0.000 to 0.046 lM;

B[a]P, 0.000 to 0.458 lM).

The protein content of the bile was determined using the

Lowry et al. (1951) method to account for changes in the

level of PAHs due to differences in the feeding status of fish

(Collier and Varanasi, 1991). The protein content of the bile

reflects the amount of water in the bile, or the dilution of the

bile, when collected from the gall bladder. Biliary PAHs

were standardized to biliary protein (metabolite mg Pr21).

Statistical Analysis

For each biomarker, the data were tested for normality and

homoscedasticity and, where necessary, log10-transformed

to achieve normality. Statistical analysis was undertaken

using the SPSS 14 statistical package. Student t-tests werecarried out to determine whether any sexual differences

were present for each biomarker (a 5 0.05). A two-way

analysis of variance (ANOVA) was run to investigate if the

data was affected by tank/treatment interaction. As no inter-

actions were found in the data sets, main effects were ana-

lyzed using one-way ANOVAs. Where significant differen-

ces between treatments were found (p\ 0.05), a Dunnett’s

(two-sided) test was run to compare the treatment groups

with the corn oil control group. Data are presented as mean

6 standard error (SEM).



RESULTS

Water quality parameters were similar in all treatment

groups throughout the experimental period (Table I). The

fish were fed well and appeared well adapted to laboratory

conditions. Length and weights of the yellowtail trumpeter

are shown in Table II. All individuals had gonads of a simi-

lar size (p � 0.05) that were regressed. Therefore, as

expected based on gonadal status and comparison with

other species, no sex differences were evident in any of the

physiological indices or biomarkers; consequently, the data

for males and females were pooled. No abnormalities were

observed in the liver (parasitic or mechanical injury) on vis-

ual inspection. In addition no statistical differences were

identified when comparing the sham injected yellowtail

trumpeter group to the corn oil control group in any indice

or biomarker.

Significant differences between treatments were identi-

fied in the LSI of the chemically treated yellowtail trum-

peter (Table II). The fish in the BNF 3 day treatment had a

significantly higher LSI than all other fish (p � 0.001). The

fish in the BNF 7 day also had a significantly higher LSI

compared to the B[a]P-treated fish and both the sham-

injected and corn oil control groups (p 5 0.04). No effect

on LSI was detected in the B[a]P-treated fish compared to

the sham-injected and corn oil control fish (p5 0.9). Exam-

ination of the condition factor did not identify any differen-

ces (p � 0.05) between the groups.

Mixed-Function Oxygenase Enzyme Activities

EROD activity in the yellowtail trumpeter injected with

B[a]P was induced 1.8 times relative to the corn oil control

levels 3 days postinjection, and declined by day 7 to 1.4

times the corn oil control levels. BNF-injected fish showed

no increased in EROD activity induction over corn oil con-

trol fish at day 3 rising to 1.5 times induction over the con-

trol fish by day 7. These measured differences in EROD ac-

tivity were not statistically significant from the corn oil

control group [p 5 0.30; Fig. 2(a)].

Some significant differences were recorded in ECOD ac-

tivity in the chemically injected yellowtail trumpeter

between treatments (p 5 0.001). Neither B[a]P- nor BNF-injected fish demonstrated any induction over the corn oil

control fish on day 3; however, on the 7th day postinjection,

the B[a]P group had 2.0 times the level of ECOD activity

compared to the control fish and the BNF-injected fish were

1.9 times induced compared to the corn oil controls [Fig.

2(b)].

Serum Sorbitol Dehydrogenase Activity

Serum SDH activities in both B[a]P-injected and BNF-

injected fish were significantly higher than the corn oil

control fish (p\ 0.001). In both treatment groups, maxi-

mum activity was recorded at 3 days, being a factor of 3.2

for B[a]P-injected fish and 2.2 times in the BNF-injected

TABLE I. Mean (6SE) water parameters in aquaria during experimental period

Corn Oil

Control

Sham

Injection B[a]P B[a]P BNF BNF p

Time (days)a 17 17 13 17 13 17

Salinity (ppt) 29.76 0.2 29.9 6 0.2 30.26 0.2 29.9 6 0.2 30.26 0.3 30.26 0.3 0.61

pH 7.846 0.01 7.83 6 0.01 7.816 0.01 7.82 6 0.01 7.826 0.01 7.826 0.01 0.41

Temperature (8C) 22.46 0.1 22.4 6 0.1 21.96 0.1 22.3 6 0.1 22.46 0.1 22.46 0.1 0.18

Note: B[a]P5 benzo[a]pyrene; BNF5 b-napthoflavone.a Includes acclimation time (10 days) plus postinjection period.

TABLE II. Mean (6SE) morphological measurements and physiological indices of the yellowtail trumpeter

Corn Oil Control Sham-Injected B[a]P B[a]P bNF bNF p

Time (days) 7 7 3 7 3 7

N 5 6 6 6 6 6

Standard length (cm) 14.56 1.1 14.1 6 1.0 15.56 0.9 14.16 0.7 15.1 6 0.8 13.86 0.7 0.73

Final body weight (g) 69 76 13.8 69.3 6 13.3 89.56 12.7 68.06 8.0 70.6 6 8.3 64.16 9.5 0.65

Carcass weight (g) 63.26 12.9 62.1 6 12.0 80.96 11.8 62.06 7.6 62.4 6 7.5 57.76 8.5 0.65

Liver weight (g) 0.416 0.07 0.42 6 0.08 0.536 0.12 0.756 0.08 0.43 6 0.04 0.566 0.08 0.07

Liver somatic indexa 0.686 0.03 0.69 6 0.05 0.736 0.06 0.706 0.03 1.20* 6 0.02 0.91*6 0.08 \0.001

Condition factorb 2.186 0.06 2.31 6 0.05 2.316 0.04 2.346 0.05 2.01 6 0.08 2.356 0.05 0.12

Note: Within rows, treatments marked with an asterisk (*) are significantly different from the corn oil control group (p � 0.05).aLiver Somatic Index5 (Liver Weight/Carcass Weight)3 100.bCondition Factor5 [(Total Body Weight – Gonad Weight)/Standard Length3] 3 100.



fish. These elevated levels were maintained until the

experiment was terminated 7 days postinjection (Fig. 3).

Bile Metabolites

Both B[a]P-type and pyrene-type metabolites were signifi-

cantly higher in the B[a]P-injected fish than either the corn

oil control, sham-injected fish or the BNF-injected fish (p �0.05). Metabolites of B[a]P-type in the bile of the B[a]P-injected yellowtail trumpeter reached their maximum level

at day 3 being 19 times higher than the corn oil control

group before declining to be 11 times higher than these

control fish at day 7 [p � 0.001; Fig. 4(a)]. Pyrene-type

metabolites in the bile on the B[a]P-injected fish were 3.4

above the level in the bile of the corn oil control fish at day

3 (p 5 0.001), falling to be only 1.7 times the level

observed in the control fish by the 7th day postinjection.

The pyrene-type metabolites in B[a]P-injected fish at day 7

were not significantly higher than the corn oil control fish

Fig. 3. SDH activity (mIU mL21; mean 6 SEM) in the serumof yellowtail trumpeter following injection with benzo[a]pyr-ene or b-naphthoflavone. Treatment groups significantly dif-ferent from corn oil controls (p\ 0.05) are denoted by aster-isks. Numbers within the bars represent the number of fish.

Fig. 2. Mixed function oxygenase enzyme induction (mean6 SE) in the yellowtail trumpeter following injection withbenzo[a]pyrene or b-naphthoflavone: (A) EROD activity inpmol R mg Pr21 min21; (B) ECOD activity in pmol H mg Pr21

min21. Treatment groups significantly different from corn oilcontrols (p\0.05) are denoted by asterisks. Numbers withinthe bars represent the number of fish.

Fig. 4. Biliary metabolites (lg metabolite mg Pr21; mean 6SEM) in the yellowtail trumpeter following injection with ben-zo[a]pyrene or b-naphthoflavone: (A) B[a]P-type metabolites;(B) pyrene-type metabolites. For each metabolite type, treat-ment groups significantly different from corn oil controls (p\ 0.05) are denoted by asterisks. Numbers within the barson graph (B) represent the number of fish for eachmetabolite.



[Fig. 4(b)]. As expected, neither B[a]P-type nor pyrene-

type metabolites were elevated in the BNF-injected fish

over the corn oil control or sham-injected fish levels

(B[a]P-type metabolites: p 5 0.33; pyrene-type: p 5 0.29;

Fig. 4).

DISCUSSION

The present study has investigated the relationship between

changes in EROD and ECOD activities, s-SDH and biliary

metabolites in yellowtail trumpeter i.p. injected with either

B[a]P or BNF. As no significant differences were detected

between the physiological indices or the measured bio-

markers of the sham injected yellowtail trumpeter com-

pared to the corn oil control group, it can be concluded that

the corn oil itself did not have an effect on the results in

this study.

All yellowtail trumpeters in this study lacked developing

gonads. It is assumed that, because the fish were not repro-

ductively active, hormonal inhibition/induction was not a

confounding factor in the measured MFO enzyme activity.

This lack of sexual difference in MFO activities in fish with

sexually regressed gonads was also described by Stegeman

et al. (1997) after measuring EROD, ECOD, pentoxyresor-

ufin O-dealkylase, and aryl hydrocarbon (benzo[a]pyrene)hydroxylase activities in several different fish species.

Results indicate that maximum induction of EROD ac-

tivity was measured on day 3 in the B[a]P-injected fish, and

it then had started to decline by day 7. EROD activity

induction following BNF injection was not noted until day

7. Neither of these chemicals resulted in induction of

EROD activity notably higher than corn oil control values,

which agrees with the results of Webb and Gagnon (2002),

following i.p. injection of PCB-126 in yellowtail trumpeter.

However, it is noted that, within the B[a]P-injected groups

there was a high interindividual variability in the data sets

when compared to the control and BNF-injected groups.

This variability may be due to the dose-response being non-

linear, and to the rates at which individuals take up and

metabolise aromatic compounds (Krahn et al., 1986;

Vignier et al., 1996). The high interindividual variability

may have masked statistical differences between the treat-

ment groups, and makes EROD activity induction an

unsuitable biomarker in this species.

Unlike EROD activity, both B[a]P and BNF injections

had an effect on ECOD activity induction, although an

increase was not detected until the 7th day. ECOD activity

appeared to be suppressed in the BNF-injected group on

day 3, even though the reduction in ECOD activity was not

statistically significant. A high variability in ECOD activity

induction was apparent in both the B[a]P- and BNF-

injected groups at day 7. However, the increase in ECOD

activity induction on exposure to both chemicals was suffi-

ciently high to overcome this high level of inter-individual

variation, with the result that the higher ECOD activities on

day 7 could be statistically identified.

BNF is considered the most potent P-450 MFO inducer

among a number of synthetic flavenoid compounds. It is

considered to display strong EROD activity responses simi-

lar to PAHs such as B[a]P (Stegeman et al., 1997; Teles

et al., 2003). In the yellowtail trumpeter, the similarity in

pattern of induction to the two chemicals only occurred in

ECOD activity response and not in EROD activity, and

BNF induction of EROD activity was not significant. A

study by Lange et al. (1999) suggests EROD and ECOD are

both catalyzed by one P-450 MFO family (CYP1A) in the

dab (Limanda limanda), but a second ECOD isozyme exists

so that each MFO activity exhibits species- and xenobiotic-

specific differences. Goksoyr and Forlin (1992) propose

typical CYP1A activities may be differentially induced by

different chemicals. These authors also advocate that there

may be a second inducible CYP1A gene present in fish to

explain the difference in induction response of EROD and

ECOD activities in Atlantic salmon (Salmo salar) to BNF

exposure. It is possible that the yellowtail trumpeter is simi-

larly influenced by the presence of more than one inducible

CYP gene. Results of the present study clearly demonstrate

that in the yellowtail trumpeter, ECOD activity measure-

ments provides better discriminatory powers between

B[a]P- or BNF-treated fish, than does EROD activity.

The SDH enzyme is highly concentrated in the cyto-

plasm of liver cells and is not found in other tissues other

than small quantities in kidney and testes cells (Wiesner

et al., 1965; Dixon et al., 1987). The breakdown of liver

cells causes the cytoplasm to discharge into the blood-

stream resulting in the detection of SDH in the serum. The

measured s-SDH levels in the yellowtail trumpeter point to-

ward leakage of this liver specific enzyme into the blood-

stream in both the B[a]P- and BNF-injected fish. This

increase suggests chemically induced liver damage has

occurred as s-SDH was elevated by day 3 in the serum by a

factor of 3.2 in B[a]P-injected fish, and 2.2 times in the

BNF-injected fish compared to corn oil control fish. At day

7 postinjection, the level of s-SDH remained at these ele-

vated levels.

Injection concentration of both chemicals was low com-

pared to many other laboratory exposure studies. For exam-

ple, Au et al. (1999) used injection concentrations ranging

from 100 lg to 10 mg kg21 of B[a]P in the fish Solea ovata,and did not detect any observable cytological changes in

the liver at doses lower than 1 mg kg21. Injections with

BNF in other studies ranged from 40 mg to 100 mg kg21

(Raza et al., 1995; Stegeman et al., 1997; Novi et al., 1998;

Weber and Janz, 2001; Teles et al., 2003). Our study used

injection concentrations of only 5 lg kg21 of B[a]P, and10 lg kg21 of BNF, in the yellowtail trumpeter. Despite

the low strength of the injections, they seem to have been

sufficient to cause hepatocellular damage. Shailaja and

D’Silva (2003) similarly found liver damage in tilapia



(Oreochromis mossambicus) measured by s-SDH following

injections with low concentrations of the PAH, phenanthrene,

compared to higher concentration injections. They suggest

that in the Tilapia, cell injury occurred because of poor induc-

tion of Phase II detoxification of Phase I metabolites. An ex-

amination of the LSI indicates some changes in the liver in

the BNF injected fish had occurred but this was not apparent

in B[a]P-treated fish. The LSI of the yellowtail trumpeter in

the BNF-injected groups increased significantly indicating ei-

ther hyperplasia (increased cell numbers) or hypertrophy

(increased cell volume) or both had occurred following treat-

ment (Slooff et al., 1983; Heath, 1995).

Even though EROD activity was not significantly higher

than corn oil control values, and liver damage appears to

have occurred, biliary metabolite analysis shows high levels

of both B[a]P-type and pyrene-type present in the bile by day

3 (1905 % and 343% higher respectively). In the B[a]P-injected group, pyrene-type biliary metabolites had fallen

close to control levels by day 7 while B[a]P-type metabolites,

although lower than day 3, remained much higher than corn

oil controls on the 7th day. This suggests that CYP1A activity

induction as measured by EROD is the major metabolic route

for the detoxification of B[a]P in the yellowtail trumpeter

and, despite the apparent hepatocellular damage, phase II me-

tabolism of B[a]P-type had taken place.The yellowtail trumpeter appears to be highly sensitive

to xenobiotics and susceptible to hepatocellular injury. De-

spite this, detoxification of B[a]P has occurred as evidenced

by the high level of metabolites in the bile. This study con-

firms Webb and Gagnon’s (2002) study that, using EROD

activity induction as a biomarker of exposure, the yellow-

tail trumpeter has limitations for environmental monitoring.

On the other hand, ECOD activity induction supported by

s-SDH determination and biliary metabolite measurement

appears to be a sensitive and useful tool for environmental

monitoring of the sublethal effects of PAHs in this bioindi-

cator fish species in the Swan-Canning Estuary. Further

investigation is required to assess the suitability of other

biomarkers of fish health (e.g., DNA strand breakage, stress

protein expression, metabolic enzyme activity, endocrine

disruption and others) in this fish species.

The treatment of animals was in accordance with Curtin Uni-

versity Animal Experimentation Ethics N-55-05.

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