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Evaluation of microsomal and cytosolic biomarkers in a seven-day larval trout sediment toxicity test
Aquatic
Toxicology
3 1 (1995) 189-202
Evaluation of microsomal and cytosolic biomarkers in a seven-day larval trout sediment toxicity test L. ViganS*,
Received
21 February
A. Arillob, S. De Flora”, J. Lazorchakd
1994; revision
received 29 June 1994; accepted
7 July 1994
Abstract Rainbow trout (0ncorh~~1chu.s fn$riss) sac fry (larvae) were exposed to River PO sediments for 7 days. The sediments were collected in the River PO at two sites located upstream and downstream of the confluence of a polluted tributary, the River Lambro. An additional sediment treatment was also tested, in which trout larvae were kept from direct contact with the downstream sediment by interposing a Teflon net. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-0-deethylase (EROD), aminopyrine-N-demethylase (APDM) and UDP glucuronyl transferase (UDPGT) activities were found to be significantly induced in whole-body microsomal preparations of sac fry exposed to the downstream sediment. No significant modification was evident in any of the tested cytosolic biomarkers, i.e. glutathione reductase (GR) and glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PD). 6-phosphogluconate dehydrogenase (6PGD) and the content of nonprotein thiols (SH). With the exception of a slight induction of AHH enzyme activity, no difference could be found between fry exposed to control sediment and those screened from the downstream sediment, suggesting that direct contact with sediment was the major route of exposure to contaminants. This study demonstrates that several enzyme activities, which are known to occur in juvenile and adult rainbow trout, are also detectable at the sac-fry (larval) stage, and some of these activities can be induced by a short-term exposure to a contaminated sediment. K~~ysord.~:
exposure:
Oncorh~whus
Biotransformation
*Corresponding
m$ciss;
Sac fry; River sediment;
enzymes;
Sediment
Biomarkers
author.
0166-445X/95/$09.50 0 1995 Eisevier Science B.V. All rights reserved SSDI 0 166-445X(94)00063-8
toxicity;
Short-term
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1. Introduction Bottom sediments have a very large capacity for storage of chemicals and represent a major sink for pollutants in the aquatic environment (Karickhoff et al., 1978; Pavlou and Dexter, 1979; Means et al., 1980; Hamdy and Post, 1985). Sedimentassociated chemicals and their availability to aquatic organisms, mainly benthic species, have received much attention in recent years. In addition to those groups of animals that are typically considered benthic organisms, some fish species can represent appropriate targets for assessing the biological effects of pollutants sorbed to sediments since their early-life stages live in direct contact with bottom sediments (Blaxter, 1969, 1988). Many studies have documented that fish possess cytochrome P450 dependent monooxygenases and that induction of these enzyme activities can provide a useful early warning marker to assess the exposure to some important classes of organic pollutants (for reviews see Payne et al., 1987; Kleinow et al., 1987; Stegeman and Klopper-Sams, 1987; De Flora et al., 1991; Andersson and Fiirlin, 1992). However, only a few studies have examined these enzyme activities in early-life stages of fish and their potential for inducibility. Binder and Stegeman (1983) demonstrated that early life stages of brook trout (Salvelinus fontinalis) and killifish (Fundulus heteroclitus) (Binder and Stegeman, 1984) have measurable aryl hydrocarbon hydroxylase (AHH) activity. Of great interest was also the observation that lake trout (Salvelinus namaycush) embryos from parents collected in contaminated sites had elevated AHH activities (Binder and Lech, 1984). In early-life stages of rainbow trout (Oncorhynchus mykiss), measurable AHH enzyme activity was found to be related to the developmental stage (Stahl and Kocan, 1986) and, more recently, similar results were obtained with cod larvae (G&us morhua) exposed to a water soluble fraction of North Sea crude oil. Exposure was initiated at the embryo stage, but an increase of P4501Al was not evident until after the first days after hatching (Goksoyr et al., 1991). The current investigation was designed in order to achieve the following objectives: (a) to assess whether some enzymes, which are known to occur in juvenile and adult rainbow trout (0. mykiss), are also detectable at the sac-fry stage; (b) to assess whether some of these activities can be used as an early warning probe of pollutant exposure; (c) to assess whether this potential can be used in a short-term test aimed at discriminating contaminated sediments. To this aim, early sac fry specimens were exposed for 7 days to two bottom river sediments, and a range of potential biomarkers was measured in whole-body microsomal and cytosolic fractions. Some of them were chosen because directly involved in Phase I or Phase II of xenobiotic metabolism, i.e., the monooxygenase activities associated to CYPl Al induction, measured as ethoxyresorufin-O-deethylase (EROD) and AHH, and the conjugase UDP glucuronyl transferase (UDPGT). Another CYP isoenzyme was measured using the prototype monooxygenase activity aminopyrine-N-demethylase (APDM). Previous results showed that juvenile rainbow trout had modified APDM activity when exposed to River PO water (Vigano et al., 1991, 1994b), thus, although the induction response of this Phase I activity in fish is not yet clearly understood (Kleinow et al., 1990; Goksoyr and Forlin, 1992), the availability of further results and in the earlier sac-fry
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stage was considered positively. Nonprotein thiols (SH), whose major constituent is glutathione (GSH) (Wallace, 1989), and the enzyme glutathione peroxidase (GPx) were tested because of their important role as antioxidant defenses (Winston and Di Giulio, 1991). In addition, the enzyme glutathione reductase (GR) was also measured since it regenerates the active thiol GSH, catalyzing the reduction of the oxidized form (GSSG), thereby contributing to the maintenance of redox status (Wallace, 1989; Di Giulio et al., 1989). The range of potential biomarkers measured in sac-fry, was completed by the two enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They generate reducing power in the extramitochondrial cytoplasm in the form of NADPH, a cofactor involved, for example, in the regeneration of reduced GSH (Di Giulio et al., 1989) and in function of monooxygenase system (Goksoyr and Forlin, 1992) or depleted by redox cycling of xenobiotic compounds (Winston and Di Giulio, 1991). G6PD and 6PGD have been used as markers of metabolic and cytotoxic alteration, and of oxidative stress in fish liver (Jtirss et al., 1986; Hinton et al., 1988; Bagnasco et al.. 1991; Vigano et al., 1993). The sediments used in the present study were collected in the River PO, which is the main Italian river, at two sites located upstream and downstream, respectively, from the confluence of the River Lambro which is one of the most polluted tributaries of the PO. In sediments sampled at the mouth of the River Lambro, De Simone and Damiani (1983) found concentrations of Aroclor 1242 and 1260 of 3.5 and 10.9 ppm. respectively, with traces of Aroclor 1254. Recent studies demonstrated that the River Lambro is a source in the River PO of both polychlorinated biphenyls (PCB) (Vigano et al., 1994a) and polycyclic aromatic hydrocarbons (PAH). Water concentrations of between 0.05 and 0.35 pg/l were measured for some PAHs in the River Lambro and in the downstream stretch of the River PO (Ciceri et al., 1991). Moreover, this same stretch of the river has shown mutagenic and clastogenic potential (De Flora et al., 1993) and induced some biotransformation enzyme activities in fish liver (Vigano et al., 1994b).
2. Materials and methods 2. I. E,xperimental
animak
Newly fertilized eggs of rainbow trout (0. mykiss) obtained from a fish hatchery (Foglio, Trento, Italy), were incubated at the laboratory in flow-through incubation tanks supplied with flowing aerated unchlorinated tap water (temperature 13 f 1°C; pH 7.66 + 0.02; hardness 283.9 + 6.9 mg/l as CaCO,; alkalinity 235.7 _+4.4 mg/l as CaCO,). 2.2. Sediment
collection
Composite samples of bottom sediments were collected from two sites of the River PO. The first one is located about 2 km upstream the emission of the River Lambro
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and the second one is about 20 km downstream of this tributary. Sediments were sampled using a hand-pushed core sampler and taking only the top 2 cm of each core. Subsamples of the two sediments were freeze-dried and analysed for the content in total organic carbon (Carlo Erba C: N : S analyzer). Triplicate analyses showed a mean content (& SD) of 0.63% (+ 0.03) for the upstream sediment and 1.73% (? 0.12) for the downstream one. The water contents, based on weight loss after lyophilization, were about 33% and 49%, respectively. 2.3. E.xposure of trout larvae Sediments were tested in triplicate in borosilicate glass vessels, each containing 700 ml of thoroughly mixed sediment and 2,800 ml (1:4) of the same tap water used for eggs incubation. Test vessels were prepared the day before the test was started and then allowed to stabilize for about 24 h. Each exposure chamber was gently aerated with a Pasteur pipette during the test. Both eggs incubation and sediment tests were run under dim light. In this experiment, the sac-fry stage was used. At the start of the test, 150 larvae, hatched 2448 h before, were placed in each vessel. As a control, sac fry specimens were exposed in triplicate to river sand that was collected from a clean area (River Ticino) and thoroughly washed in flowing tap water for some days before the test. River sand and overlying tap water had been previously checked as free of inducers in both fry and juvenile tests (Vigano et al., 1991, 1993, 1994b, unpublished data). To assess whether enzyme induction might result from direct contact with the sediment surface, a fourth treatment group was used. In the fourth treatment, trout larvae were exposed to the downstream sediment but direct contact of sac fry with sediment was prevented by interposing a Teflon net (# 1 mm) suspended 4-5 cm over the surface of the sediment. The sac fry specimens were placed on this net. The four exposure groups, referred to as river sand, upstream, downstream and suspended, were tested in parallel at 13 & 1“C with water renewals every 48 h. Daily measurements gave mean results (+SD) of 8.35 & 0.06 for pH, 9.1 & 0.7 mg/l for dissolved oxygen and 289.5 k 8.7 mg/l CaCO, for hardness, without any significant difference among the four treatment groups. The observed mortality was lower than 5% for each exposure group and was not significantly related to treatment as checked by ANOVA. 2.4. Preparation
of subcellular fractions
After 7 days of exposure, trout larvae were collected in a net, rinsed with tap water, immediately blotted on filter paper and frozen in liquid nitrogen. Fish were weighed, the yolk sac was excised and discarded and the remaining larvae were slowly thawed and minced in KC1 (150 mM), Tris-HCl (10 mM), pH 7.4. Larvae were then homogenized in sucrose (250 mM), EDTA-Na (1 mM), Tris-HCl (50 mM), pH 7.4, by means of a Potter-Elvehjem type glass-Teflon homogenizer. Homogenates were subsequently centrifuged at 12 000 g for 20 min and at 105 000 g for 60 min. The resulting supernatants were stored at -80°C in small aliquots to be used for the analysis of
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cytosolic biochemical parameters. In order to prevent sulphydryl group oxidation, portions of homogenate were supplemented with 20 mM EDTA and the corresponding 105 000 g supernatants were used for SH-group analysis. The pellet resulting from the 105 000 g centrifugation was resuspended in washing buffer (KCl, Tris-HCl, EDTA) and centrifuged again at 105 000 g for 60 min. The microsomal pellet was then suspended in Tris-HCl (100 mM), KC1 (150 mM), EDTANa (0.2 mM), glycerol (20%) pH 7.4. For this final suspension, a ratio of 0.5 ml buffer per 3 g original tissue was adopted to obtain a microsomal suspension about three fold more concentrated than that usually prepared from liver of juvenile rainbow trout (0.5 ml buffer per g original tissue). Microsomal suspensions were stored at -80°C in small aliquots until analyses of the biochemical parameters. 2.5. Biochemical
assays
The protein content of the microsomal fraction was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. 3-Hydroxylation of benzo[a]pyrene was evaluated by means of the spectrofluorometric method described by Dehnen et al. (1973) with some modifications. Microsomes (0.3 mg protein per ml) were incubated at 25°C with benzo[a]pyrene (20 ,uM) and a NADPH-generating mixture composed of NADP (2 mM), glucose 6-phosphate (20 mM), glucose 6-phosphate dehydrogenase (3.5 U), nicotinamide (20 mM), MgCl, (20 mM) and KC1 (40 mM) in Tris-HCl (50 mM, pH 7.4). The reaction was stopped after 10 min by adding a solution of 10% Triton x-100 and 1% EDTA (disodium salt) in 0.1 N NaOH, and fluorescence was read at excitation and emission wavelengths of 475 and 522 nm, respectively. The fluorescence produced by an identical reaction mixture of the same sample, stopped at time 0, was subtracted, and the value thus obtained was compared with a standard 3-hydroxy-benzo[a]pyrene (3-OH-BP) curve. The results were expressed as pmol 3-OH-BP mini’ mg-’ microsomal protein. Benzo[a]pyrene
hydroxylase
(AHH)
activity.
Etlzo.~:resor~~n-0-deetl~?~la.~e (EROD) activity. 0-Deethylation of ethoxyresorufin was evaluated by means of the spectrofluorometric method described by Burke and Mayer (1974). Briefly, microsomes (0.3 mg protein per ml) were mixed with ethoxyresorufin (0.5 yM) in Tris-HCl (100 mM, pH 7.8) kept a 20°C. The reaction was started by adding NADPH (50 PM), and fluorescence was recorded for 2 min at excitation and emission wavelengths of 530 and 585 nm, respectively. Increases in relative fluorescence were compared with a standard curve with resorufin. The results were expressed as pmol resorufin min-’ rng-’ microsomal protein. Aminopyrine-N-demethylase (APDM) activity. Microsomes (4 mg protein per ml) were incubated at 20°C in presence of MgCl, (10 mM), NADP (0.3 mM), glucose 6-phosphate (10 mM) glucose 6-phosphate dehydrogenase (7 U), MOPS (100 mM. pH 7.4). Addition of 5 mM aminopyrine started the reaction and APDM activity was determined following the production of formaldehyde (Arillo et al., 1984). After
194
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Toxicology 31 (1995) 189-202
45-min incubation, formaldehyde was measured spectrophotometrically action at 412 nm (Werringloer, 1978).
by Nash re-
UDP-glucuronosyltransferase (UDPGT) activity. This conjugating enzyme was determined spectrophotometrically according to Hanninen (1968). Microsomes (2 mg protein per ml) were incubated at 20°C in a reaction mixture containing potassium phosphate buffer (0.5 M, pH 7.5), p-nitrophenol as substrate (0.35 mM), UDP glucuronic acid (4.5 mM), EDTA (dipotassium salt, 20 mM). After 30 min incubation, the reaction was stopped by adding trichloroacetic acid (TCA) (3%, final concentration), and samples were centrifuged. The pH of the supernatant was brought to alkaline by adding NaOH (0.48 M, final concentration) and absorbance was measured at 400 nm. Nonprotein thiol (SH)
groups. Samples of homogenized larvae, obtained as described above, were deproteinized by TCA (lo%, final concentration) and centrifuged. Then, SH groups were measured spectrophotometrically (412 nm) in an assay mixture containing Tris-HCl (320 mM, pH 8.9) and 5,5’-dithiobis-(2-nitrobenzoic acid) (0.1 mM), according to Sedlak and Lindsay (1968). Glutathione reductase (GR) activity. The enzymatic reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) was evaluated according to the spectrophotometric method described by Veiga Salles and Ochoa (1950). Cytosolic fractions were mixed with GSSG (3.3 mM) and NADPH (0.1 mM) in Tris-HCl (1 M, pH 8.0). The progressive decrease of absorbance at 340 nm was recorded for 2 min at 20°C and GR activity was expressed as nmol min-’ mg-’ cytosolic protein. Glutathione peroxidase (GPx) activity. The enzymatic conversion of H,O, to water, coupled to the oxidation of GSH to GSSG, was evaluated according to the spectrophotometric method described by Beutler (1975). Cytosolic fractions were mixed with GR (3.0 U), GSH (0.5 mM), NADPH (0.1 mM) and tert-butyl hydroperoxide (0.1 PM) in Tris-HCl (1 M, pH 8.0). The progressive decrease of absorbance at 340 nm was recorded for 2 min at 20°C and GPx activity was expressed as nmol min-’ mg-’ cytosolic protein. Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. G6PD and 6PGD were measured according to the spectro-
photometric method described by Rudack et al. (1971). Cytosolyc fractions were mixed with NADP (0.2 mM), 6-phosphoglucuronic acid (1 mM), glucose 6-P (1 mM) and MgCl, (10 mM) in Tris-HCl(O.1 M, pH 8.0). The progressive increase of absorbance at 340 nm was recorded for 2 min at 2O”C, thus yielding the cumulative G6PD plus 6PGD activities. 6PGD activity was evaluated by testing an identical reaction mixture but lacking glucose 6-P. G6PD activity was calculated by subtracting 6PGD activity from cumulative G6PD plus 6PGD activity. Enzyme activities were expressed as nmol min-’ mg-’ cytosolic protein. All biochemical reagents were purchased from Sigma Chemical Co (St Louis, MO, USA), Boehringer Mannheim Italia (Milan, Italy) and BDH (Poole, Dorset, UK).
L. Vigund etal.lAquatic
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Statistics. All data presented in this study are mean 5 SD of values obtained from three pools of trout fry, each pool consisting of about 150 organisms. Significant differences among treatments for each parameter were analyzed by one-way ANOVA and Duncan’s multiple range test (SAS, 1988).
3. Results 3. I. Mcrosomal hiomarkers The four enzyme activities were all measurable in microsomes prepared from whole
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Fig. 1. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD), demethylase (APDM), UDP glucuronyl transferase (UDPGT) enzyme activities measured sac fry after T-day test. Larvae were exposed to clean river sand (R), bottom sediments River PO, upstream (U) and downstream (D) its tributary River Lambro, or downstream from direct contact by interposing a Teflon net (S). Bars represent mean values k SD (n the same letter are not significantly different (Duncan test).
aminopyrine-Nin rainbow trout collected in the sediment isolated = 3). Values with
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larvae. Moreover, after 7 days of treatment, i.e. 8-9 days after hatching, AHH, EROD, APDM and UDPGT activities were significantly different among the exposure groups. Sediment collected downstream the River Lambro, induced significantly higher activities than the sediment from the upstream site (Fig. 1). In addition, the sac fry exposed to the latter sediment showed microsomal activities that were not significantly different from control fry. In particular, in the downstream larvae, AHH and EROD enzyme activities were about 3- and 4-fold higher, respectively, than those of the upstream larvae. Similarly, the ability to conjugate with glucuronic acid (UDPGT) was also markedly induced in sac fry exposed to downstream sediment, showing almost twice the activity measured in control group. The response of APDM parallelled the described patterns, with a limited yet significant increase of activity in the downstream treatment, whereas upstream and control organisms were not significantly different. The microsomal activities determined in sac fry exposed on Teflon nets placed over the downstream sediment were not significantly different from those of fry maintained in direct contact with both upstream and control sediment. The only exception was AHH enzyme activity that had an intermediate level between downstream and upstream treatment (Fig. 1). 3.2. Cytosofic bioma~kers After 7 days of exposure, i.e., 8-9 days after hatching, glutathione peroxidase (GPx) and reductase (GR), were measurable in trout larvae cytosolic fractions (Table 1). Despite a consistent trend towards slightly higher values for both upstream and downstream sediments, none of the treatments differed for either of these antioxidant defenses to a significant extent. Similarly, the contents of nonprotein sulphydryl groups (SH), G6PD and 6PGD activities were not influenced by the different exposure conditions (Table 1). Since none of the cytosolic markers was significantly modified by treatments, grand Table 1 Rates of glutathione reduction (GR) and peroxidation (GPx), contents of nonprotein thiol groups (SH) and rates of dehydrogenase activity of glucose 6-phosphate (G6PD) and 6-phosphogluconate (6PGD) measured in sac fry of rainbow trout after 7-day test. Larvae were exposed to clean river sand (control). bottom sediments collected in the River PO, either upstream or downstream its tributary River Lambro, and downstream sediment prevented from direct contact by a Teflon net (suspended) Treatment
GR
River sand Upstream Downstream Suspended
6.4 7.0 7.2 5.7
GPx k f + It
0.5 0.5 1.4 0.4
3.6 4.8 4.1 4.0
G6PD
SH f 0.6 f 1.1 Zk0.5 f 1.3
7.4 8.3 7.7 6.6
f f + f
2.1 1.2 1.0 1.3
4.1 4.5 4.9 4.6
f f + f
6PGD 0.9 1.3 1.6 1.2
Values are mean + SD of three replicates. SH contents are reported as nmollmg protein; as nmol min-’ mg-’ protein. No significant differences were detected (Duncan test).
8.2 9.1 9.3 8.5
f f * k
1.1 1.1 1.2 0.4
enzyme activities
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mean values were also calculated for each one of the five parameters. The following values (&SD) were obtained: GR 6.6 ? 0.6 and GPx 4.1 + 0.5 nmol min-’ mg-’ protein; SH 7.5 ? 0.7 nmol/mg protein; G6PD 4.5 + 0.3 and 6PGD 8.8 ? 0.5 nmol mini’ mg-’ protein. These data can be considered to be representative of the normal biochemical pattern of rainbow trout sac fry, 889 days after hatching.
4. Discussion 4. I. Level
of enzyme
activities in trout lavvur
Our results demonstrate that several biotransformation enzyme activities are present and measurable in rainbow trout sac fry, although most of them are lower than in later developmental stages. In fact, when microsomal basal activities of sac fry are compared to those detected in juvenile rainbow trout (about 1 year old) (Vigano et al., 1991, 1993) it can be inferred that the former organisms have levels of UDPGT enzyme and CYPlAl isoenzyme, measured as AHH and EROD enzyme activities. which are approximately one order of magnitude lower. Binder and Stegeman (1983) also found measurable AHH activity in early life stages of a salmonid, i.e., in both embryo and sac fry of brook trout (S. j&tinalis). They tested hepatic tissue preparations and did not detect any enzyme activity in extrahepatic tissues. Assuming a similar tissue distribution in rainbow trout larvae. the low values observed in fry in the present study could be partially explained by an extrahepatic tissue dilution occurring in whole-body preparations. In addition, the ontogeny of the enzyme systems may also contribute to the differences between larval and juvenile trout. The few data available on development of biotransformation in fish almost exclusively refer to AHH and suggest an increase of activity either with the developmental stage (Binder and Lech, 1984; Stahl and Kocan, 1986; Goksoyr et al.. 1991) or limited to the around-hatching period (Binder and Stegeman, 1983, 1984). Our data seem to be more consistent with the former trend and suggest it may be applicable to EROD, another typical CYPlAl activity, and to a conjugating enzyme such as UDPGT. The extrahepatic dilution and stage-related development of activity are likely to account for the inability to detect AHH activity in rainbow trout fry until 21 days after hatching, as reported by Stahl and Kocan (1986). The analysis of more concentrated microsomal preparation in the present study permitted to detect not only AHH but also other biotransformation activities, demonstrating that they are already present in early sac-fry stage (8-9 days after hatching). This is consistent with the excretion capability of benzo[a]pyrene metabolites observed in the period elapsing before hatching and later stages of rainbow trout development (Stahl and Kocan, 1986). Despite extrahepatic dilution and ontogenic effects, the APDM activity measured in rainbow trout sac fry is surprisingly well comparable to that of juvenile hepatic microsomes (Vigano et al., 1991, 1993). This seems to suggest that APDM activity follows a different developmental pattern and also that this enzyme is regulated by mechanisms different from those regulating AHH, EROD and UDPGT. The litera-
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ture seems to support these differences, at least for the mentioned monooxygenase activities, which should be expression of different cytochrome P-450 genes. In fact, in mammals APDM enzyme activity is catalyzed by cytochrome member of CYP2B subfamily, and recent studies have shown that in trout genome there are sequences similar to mammalian CYP2B (Kleinow et al., 1990). Prototype activities AHH and EROD, on the other hand, are catalytic activities of a different isoenzyme identified in rainbow trout as CYPlAl, which, in turn, has shown great similarity with CYPlAl of mammals (Goksoyr and Forlin, 1992). The comparison of sac fry and juvenile trout for cytosolic biomarkers again reveals lower activities of GR and GPx enzymes as well as lower contents of SH groups in the earlier stage (Vigano et al., 1993). These differences are however less pronounced than those recorded for AHH or EROD activities, i.e. about 20% ofjuvenile fish value, and might be related to the developmental stage. In contrast, G6PD and 6PGD activities show markedly lower levels in larvae (about 2%) as compared to juvenile fish. However, the known relationships of these enzymes with food composition and nutritional status (Jtirss et al., 1986; Vigano et al., 1993) make comparisons difficult since larvae utilize yolk components with a precise sequence and likely have different nutritional requirements than older fish (Heming and Buddington, 1988). 4.2. Induction responses and discriminatory potential Limited information is available regarding the induction potential of biotransformation in early life stages of fish. In a previous study, an increased content of cytochrome P-450 was found in the liver of rainbow trout sac fry obtained from female exposed to dietary PCBs (Hendricks et al., 1981). Sac fry from treated females had higher PCBs content and showed an increased capability of producing aflatoxinepoxide; however, some components of the MFO system, such as epoxide hydrase and glutathione-S-epoxide transferase, although measurable, were unaltered or even reduced. Stahl and Kocan (1986) observed that the excretion of benzo[a]pyrene metabolites can be stimulated by MFO inducers in early life stages of rainbow trout. However, the direct evidence of a microsomal enzyme induction in early life stage of salmonid was reported by Binder and Stegeman (1983) who measured increased level of AHH in PCB-treated brook trout (S. fontinalis) embryos. Our results provide evidence that the microsomal enzymes of rainbow trout larvae, are responsive to inducers as shown by the enhancement of both Phase I AHH, EROD, APDM and Phase II UDPGT activities in fry exposed to downstream sediment, collected from the stretch of River PO receiving the pollutants from the River Lambro. The potential of fry responses to discriminate contaminated sediments can be better appreciated when comparing the results of present study with those obtained with juvenile rainbow trout caged in the River PO upstream and downstream from the confluence of the River Lambro, i.e. exposed to the same sites of sediments collection (Vigano et al., 1994). Like in present sediment test, microsomal activities of caged trout (AHH, EROD, APDM, UDPGT) were markedly induced only at the downstream site. In both studies, AHH and EROD showed the highest induction responses, thereby confirming to be the most sensitive catalytic biomarkers in both juvenile
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and sac-fry stage. It can be mentioned that APDM activity, which was found to be induced both in juvenile trout maintained downstream of the River Lambro confluence and in sac fry exposed to downstream sediments, was also found to be induced in lake trout swim-up fry (45-55 days after hatching) treated with Aroclor 1254 (Binder and Lech, 1984). In addition, the field-caging experiment showed that the enzymes GPx and Gr responded very slowly to River PO water, showing modified activity only after 30 days of exposure. If a similar behavior can be expected from fry antioxidant defences, this may account for their unaltered levels after only 7 days of sediment test. A number of recent studies have reported the induction of antioxidant enzymes in fish exposed to contaminated sediments or collected from polluted areas. Livingstone et al. (1992) have found induced activity of SOD, catalase and GPx enzymes in Limanda limandu caught near the German Bight in the North Sea. Comparable responses have been found in dab (L. limandu) when exposed to a contaminated sediment in a laboratory study (Livingstone et al., 1993). The enhancement of SOD and catalase activities has also been observed in channel catfish (Ictalurus purzctatus) exposed to Black Rock Harbor sediment, highly contaminated with various aromatic hydrocarbons (Di Giulio et al., 1993). Finally, it is noteworthy that grey mullet (Mugil sp.), collected from Spanish littoral areas, have shown a wide range of increased antioxidant defences including the enzymes GPx, SOD, catalase, GR and G6PD (Rodriguez-Ariza et al., 1993). In contrast, both in the above mentioned study with caged trout and in the present study with sac-fry, the two enzymes G6PD and 6PGD were unaffected by River PO water (unpublished results). Probably, longer time of exposure and/or higher levels of contamination are necessary to modify these two enzymes, as seems to be suggested by their induced activities found in another study on native free-swimming fish (Bagnasco et al., 1991) The proposed comparisons suggest that the responses of sac-fry obtained by applying a short-term test are consistent and qualitatively similar to those pointed out in an older life stage. Furthermore, in agreement with previous findings, they confirm that the stretch downstream of the River Lambro is more polluted than the upstream site in the River PO (De Simone and Damiani, 1983; Ciceri et al., 1991; Marchetti, 1991; Vigano et al., 1994a,b). In particular, other biomarkers had been found to become positive in juvenile trout exposed in situ downstream the Lambro confluence, including mutagenicity of bile, liver activation of promutagens to mutagenic metabolites and frequency of micronuclei in peripheral blood erythrocytes (De Flora et al., 1993). 4.3. Route of’exposure
and temporary
benthic niche
The negligible response to the fourth treatment, in which trout larvae were prevented from direct contact with downstream sediment, indicates the importance of the route of exposure and, on the other hand, the risk deriving to some fish species from their temporary niche of benthic organism. The hydrophobic nature of the most common inducers makes them partitioning between solid phase and interstitial water of sediment, higher concentrations occurring in pore water than in the upper water column. In accordance with the present results, suggesting a negligible availability of
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inducers over the narrow zone of the sediment-water interface, several investigators supported the importance of interstitial water and/or water at the sediment/water interface as the primary routes of exposure for a benthic organisms (for a review, see Adams, 1987). Similarly, adult fathead minnows (Pimephales prom&s), exposed to sediment spiked with PCBs, accumulated much more residue than did fish screened from the sediment and likely prevented from mouthing the interstitial water (Halter and Johnson, 1977). More recently, it has been reported that a demersal fish (Leiostomus xanthurus) isolated from direct contact with a contaminated sediment did not significantly accumulate PCB residues when compared with fish in intimate contact with the sediment (Rubinstein et al., 1984). 5. Conclusions Despite a general picture displaying lower enzyme activities than juvenile trout, rainbow trout sac fry exhibited an appreciable biochemical pattern for both Phase I and Phase II biotransformation activities and for antioxidant defences. Some of these activities have shown to be induced by short-term exposure to bottom river sediment.
Acknowledgements
The authors thank Dr F. Melodia and C. Bennicelli for the collaboration in biochemical analyses, F. Bacciu for the measurements of sediment organic carbon content and L. Previtali for technical assistance. This study was supported by MURST (40% project).
References Adams, W.J. (1987) Bioavailability of neutral lipophilic chemicals contained in sediments: a review. In: K.L. Dickson, A.W. Maki and W.A. Brungs (editors), Fate and Effects of Sediment bound Chemicals in Aquatic Systems. SETAC Special Publications Series, pp. 219-244. Andersson, T and L. Fiirlin (1992) Regulation of the cytochrome P450 enzyme system in fish. Aquat. Toxicol. 24, l-20. Arillo, A., P. Mensi and G. Pirozzi (1984) Nitrite binding to cytochrome P-450 from liver microsomes of trout (Saltno gairdneri) and effects on two microsomal enzymes. Toxicol. Lett. 21, 69974. Bagnasco, M., A. Camoirano, S. De Flora, F. Melodia and A. Arillo (1991) Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. Mutat. Res. 262, 1299137. Beutler, E. (1975) Red cell metabolism. In: Manual of Biochemical Methods, 2nd edition. Grune and Stratton, New York, pp. 69-73. Binder, R.L. and J.J. Lech (1984) Xenobiotics in gametes of Lake Michigan lake trout (Salvelinus numaycush) induce hepatic monooxygenase activity in their offspring. Fund. Appl. Toxicol. 4, 1042-1054. Binder, R.L. and J.J. Stegeman (1983) Basal levels and induction of hepatic aryl hydrocarbon hydroxylase activity during the embryonic period of development in brook trout. Biochem. Pharmacol. 32, 1324 1327. Binder, R.L. and J.J. Stegeman (1984) Microsomal electron transport and xenobiotic monooxygenase activities during embryonic period of development in the killifish, Fundulus heteroclitus. Toxicol. Appl. Pharmacol. 73.432443.
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Blaxter, J.H.S. (1969) Development: eggs and larvae. In: W.S. Hoar and D.J. Randall (editors), Fish Physiology. III. Reproduction and Growth. Bioluminescence. Pigments and Poisons. Academic Press. London, pp. 111-252. Blaxter. J.H.S. (1988) Pattern and variety in development. In: W.S. Hoar and D.J. Randall (editors), Fish Physiology. XI. The Physiology of Developing Fish. A. Eggs and Larvae. Academic Press, London, pp. 148. Burke. M.D. and R.T. Mayer (1974) Ethoxyresorufin: direct fluorometric assay of a microsomal O-deakylation which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Disp. 2, 583. 588. Ciceri, G.. D. Heltai, M. Maspero and W. Martinotti (1991) Determinazione di microinquinanti organici in componenti ecologicamente significative de1 fiume PO: approccio metodologico e risultati preliminari. ENEL-CRTN Rel. E5/91/13. De Flora, S., M. Bagnasco and P. Zanacchi (1991) Genotoxic, carcinogenic and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea. Mutat. Res. 258, 285-320. De Flora, S., L. Vigano, M. Bagnasco, C. Bennicelli. F. Melodia and A. Arillo t 1993) Multiple genotoxicity biomarkers in fish exposed in situ to polluted river water. Mutat. Res. 319. 1677177. Dehnen, W., R. Tomingas and J. Roos (1973) A modified method for the assay of benzo[a]pyrene hydroxylase. Anal. Biochem. 53, 3733383. De Simone, R. and V. Damiani (1983) Fiume Lambro. PCBs, pesticidi organoclorurati e mercurio nei sedimenti fluviali sottoposti a piene eccezionali. Acqua Aria 9. 9655971. Di Giulio. R.T., P.C. Washburn. R.J. Wenning, G.W. Winston and C.S. Jewel1 (1989) Biochemical responses in aquatic animals: a review of determinants of oxidative stress, Environ, Toxicol. Chem. 8, 1103~1123. Di Giulio, R.T.. C. Habig and E.P. Gallagher (1993) Effects of Black Rock Harbor sediments on indices of biotransformation. oxidative stress and DNA integrity in channel catfish. Aquat. Toxicol. 26, ll22. Goksoyr. A., T.S. Solberg and B. Serigstad (1991) Immunochemical detection of cytochrome P45OlAl induction in cod larvae and juveniles exposed to a water soluble fraction of North Sea crude oil. Mar. Poll. Bull. 22, 1222127. Goksoyr, A. and L. Forlin (1992) The cytochrome P-450 system in hsh. aquatic toxicology and envnonmental monitoring. Aquat. Toxicol. 22. 2877312. Halter, M.T. and H.E. Johnson (1977) A model system to study the desorption and biological availability of PCB in hydrosoils. In: F.L. Mayer and J.L. Hamelink (editors), Aquatic Toxicology and Hazard Evaluation. ASTM-STP 634, American Society for Testing and Materials. Philadelphia, PA, pp. I78 195. Hamdy. Y. and L. Post (I 985) Distribution of mercury, trace organics and other heavy metals in Detroit River sediments. J. Great Lakes Res. I I, 3533365. Hanninen. 0. (1968) On the metabolic regulation in the glucuronic acid pathway in the rat tissue. Ann. Acad. Sci. Fenn. A.11. 142. l-92. Heming. T.A. and R.K. Buddington (1988) Yolk absorption in embryonic and larval fishes. In: W.S. Hoar and D.J. Randall (editors), Fish Physiology. XI. The Physiology of Developing Fish. A. Eggs and Larvae. Academic Press, London, pp. 407446. Hendricks, J.D.. W.T. Stott. T.P. Putnam and R.O. Sinnhuber (1981) Enhancement of Aflatoxin Bl hepatocarcinogenesis in rainbow trout (Sdmo goirdneri) embryos by prior exposure of gravid females to dietary Arocior 1254. In: D.R. Branson and K.L. Dickson (editors), 4th Conf. Aquatic Toxicology and Hazard Assessment. ASTM STP 737, American Society for Testing and Mateinns, r’tiua&ipl&i, PA. pp. 203-m214. Hinton. D.E., J.A. Couch, S.J. Teh and L.A. Courtney (1988) Cytological changes during progression of neoplasia in selected fish species. Aquat. Toxicol. II, 72-I 12. Jtirss, K., Th. Bittorf and Th. Volkler (1986) Influence of salinity and food deprivation on growth, RNA/ DNA ratio and certain enzyme activities in rainbow trout (Sdmo g~ird~7wi Richardson). Comp. Biothem. Physiol. 83B, 425433. Karickhoff, S.W.. D.S. Brown and T.A. Scott (1979) Sorption of hydrophobic pollutants on natural sediments. Water Res. 13. 241-248. Kleinow, K.M., M.J. Melancon. and J.J. Lech (1987) Biotransformation and induction: implication for
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toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish. Environ. Health Persp. 71, 105-l 19. Kleinow, K.M., M.L. Haasch, D.E. Williams and J.J. Lech (1990) A comparison of hepatic induction in rat and trout (Oncorhynchus mykiss): delineation of the site of resistance of fish to phenobarbital-type of inducers. Comp. Biochem. Physiol. 96C, 25c-270. Livingstone, D.R., S. Archibald, J.K. Chipman and J.W. Marsh (1992) Antioxidant enzymes in the liver of the dab, Limanda limandu, from the North Sea. Mar. Ecol. Prog. Ser. 91, 97-104. Livingstone, D.R., P. Lemarie, A. Matthews, L. Peters, D. Bucke and R.J. Law (1993) Pro-oxidant, antioxidant and 7-ethoxyresorufin O-deethylase (EROD) activity responses in liver of dab (Limanda lima&a) exposed to sediment contaminated with hydrocarbons and other chemicals. Mar. Pollut. Bull. 26,602-606. Lowry, O.H., N.I. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 19,265-275. Marchetti, R. (1991) Quadro complessivo delle condizioni delle acque de1 fiume PO negli anni ‘90 e tendenze evolutive. Quad. 1st. Ric. Acque 92, 16.1-16.26. Means, J.C., S.G. Wood, J.J. Hassett and W.L. Banwart (1980) Sorption of polynuclear aromatic hydrocarbons by sediments and soils. Environ. Sci. Technol. 14, 1524-1528. Pavlou, S.P. and R.N. Dexter (1979) Distribution of polychlorinated biphenyls (PCB) in estuarine ecosystems. Testing the concept of equilibrium partitioning in the marine environment. Environ. Sci. Technol. 13, 65-71. Payne, J.F., L.L. Fancey, A.D. Rahimtula and E.L. Porter (1987) Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245. Rodriguez-Ariza, A., J. Peinado, C. Pueyo and J. Lbpez-Barea (1993) Biochemical indicators of oxidative stress in fish from polluted littoral areas. Can. J. Fish. Aquat. Sci. 50, 2568-2573. Rubinstein, N.I., W.T. Gilliam and N.R. Gregory (1984) Dietary accumulation of PCBs from a contaminated sediment source by a demersal fish (Leiostomus xanthurus). Aquat. Toxicol. 5, 331-342. Rudack, D., E.M. Chrisholm and D. Holten (1971) Rat liver glucose 6-phosphate dehydrogenase. Regulation by carbohydrate diet and insulin. J. Biol. Chem. 246, 1249-1254. SAS (1988) SASlSTAT User’s Guide, Release 6.03 edition. SAS Institute Inc., Cary, NC. Sedlack, J. and R.H. Lindsay (1968) Estimation of total, protein-bound and nonprotein sulphydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205. Stahl, R.G. and R.M. Kocan (1986) Influence of age on patterns of uptake and excretion of polycyclic aromatic hydrocarbons in the rainbow trout embryo. In: T.M. Poston and R. Purdy (editors), Aquatic Toxicology and Environmental Fate, 9. ASTM STP 92 1, American Society for Testing and Materials, Philadelphia, PA, pp. 287-303. Stegeman, J.J. and P.J. Klopper-Sams (1987) Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals. Environ. Health Persp. 71, 87-95. Veiga Salles, J.B. and S. Ochoa (1950) Biosynthesis of dicarboxylic acids by carbon dioxide fixation. J. Biol. Chem. 187,849-861. Viganb, L., A. Arillo, M. Bagnasco, F. Melodia and S. Galassi (1991) Xenobiotic metabolizing enzymes in rainbow trout exposed to river water. Comp. Biochem. Physiol. lOOC, 169-172. Viganb, L., A. Arillo, M. Bagnasco, C. Bennicelli and F. Melodia (1993) Xenobiotic metabolizing enzymes in uninduced and induced rainbow trout: effects of diets and food deprivation. Comp. Biochem. Physiol. 104c, 51-55. Vigand, L., S. Galassi and A. Arillo (1994a) Bioconcentration of polychlorinated biphenyls (PCBs) in rainbow trout caged in the River PO. Ecotoxicol. Environ. Saf. 28, 287-297. Viganb, L., A. Arillo, M. Bagnasco, C. Bennicelli and F. Melodia (1994b) Time course of xenobiotic biotransformation enzyme activities of rainbow trout caged in the River PO. Sci. Total Environ. 151, 3746. Winston, G.W. and R.T. Di Giulio (1991) Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137-161. Werringloer, J. (1978) Assay of formaldehyde generated during microsomal oxidation reactions. In: S. Fleisher and L. Packer (editors), Methods in Enzymology, 52. Academic Press, New York, pp. 142-l 5 1.
Toxicology
3 1 (1995) 189-202
Evaluation of microsomal and cytosolic biomarkers in a seven-day larval trout sediment toxicity test L. ViganS*,
Received
21 February
A. Arillob, S. De Flora”, J. Lazorchakd
1994; revision
received 29 June 1994; accepted
7 July 1994
Abstract Rainbow trout (0ncorh~~1chu.s fn$riss) sac fry (larvae) were exposed to River PO sediments for 7 days. The sediments were collected in the River PO at two sites located upstream and downstream of the confluence of a polluted tributary, the River Lambro. An additional sediment treatment was also tested, in which trout larvae were kept from direct contact with the downstream sediment by interposing a Teflon net. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-0-deethylase (EROD), aminopyrine-N-demethylase (APDM) and UDP glucuronyl transferase (UDPGT) activities were found to be significantly induced in whole-body microsomal preparations of sac fry exposed to the downstream sediment. No significant modification was evident in any of the tested cytosolic biomarkers, i.e. glutathione reductase (GR) and glutathione peroxidase (GPx), glucose 6-phosphate dehydrogenase (G6PD). 6-phosphogluconate dehydrogenase (6PGD) and the content of nonprotein thiols (SH). With the exception of a slight induction of AHH enzyme activity, no difference could be found between fry exposed to control sediment and those screened from the downstream sediment, suggesting that direct contact with sediment was the major route of exposure to contaminants. This study demonstrates that several enzyme activities, which are known to occur in juvenile and adult rainbow trout, are also detectable at the sac-fry (larval) stage, and some of these activities can be induced by a short-term exposure to a contaminated sediment. K~~ysord.~:
exposure:
Oncorh~whus
Biotransformation
*Corresponding
m$ciss;
Sac fry; River sediment;
enzymes;
Sediment
Biomarkers
author.
0166-445X/95/$09.50 0 1995 Eisevier Science B.V. All rights reserved SSDI 0 166-445X(94)00063-8
toxicity;
Short-term
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Toxicology 31 (1995) 189-202
1. Introduction Bottom sediments have a very large capacity for storage of chemicals and represent a major sink for pollutants in the aquatic environment (Karickhoff et al., 1978; Pavlou and Dexter, 1979; Means et al., 1980; Hamdy and Post, 1985). Sedimentassociated chemicals and their availability to aquatic organisms, mainly benthic species, have received much attention in recent years. In addition to those groups of animals that are typically considered benthic organisms, some fish species can represent appropriate targets for assessing the biological effects of pollutants sorbed to sediments since their early-life stages live in direct contact with bottom sediments (Blaxter, 1969, 1988). Many studies have documented that fish possess cytochrome P450 dependent monooxygenases and that induction of these enzyme activities can provide a useful early warning marker to assess the exposure to some important classes of organic pollutants (for reviews see Payne et al., 1987; Kleinow et al., 1987; Stegeman and Klopper-Sams, 1987; De Flora et al., 1991; Andersson and Fiirlin, 1992). However, only a few studies have examined these enzyme activities in early-life stages of fish and their potential for inducibility. Binder and Stegeman (1983) demonstrated that early life stages of brook trout (Salvelinus fontinalis) and killifish (Fundulus heteroclitus) (Binder and Stegeman, 1984) have measurable aryl hydrocarbon hydroxylase (AHH) activity. Of great interest was also the observation that lake trout (Salvelinus namaycush) embryos from parents collected in contaminated sites had elevated AHH activities (Binder and Lech, 1984). In early-life stages of rainbow trout (Oncorhynchus mykiss), measurable AHH enzyme activity was found to be related to the developmental stage (Stahl and Kocan, 1986) and, more recently, similar results were obtained with cod larvae (G&us morhua) exposed to a water soluble fraction of North Sea crude oil. Exposure was initiated at the embryo stage, but an increase of P4501Al was not evident until after the first days after hatching (Goksoyr et al., 1991). The current investigation was designed in order to achieve the following objectives: (a) to assess whether some enzymes, which are known to occur in juvenile and adult rainbow trout (0. mykiss), are also detectable at the sac-fry stage; (b) to assess whether some of these activities can be used as an early warning probe of pollutant exposure; (c) to assess whether this potential can be used in a short-term test aimed at discriminating contaminated sediments. To this aim, early sac fry specimens were exposed for 7 days to two bottom river sediments, and a range of potential biomarkers was measured in whole-body microsomal and cytosolic fractions. Some of them were chosen because directly involved in Phase I or Phase II of xenobiotic metabolism, i.e., the monooxygenase activities associated to CYPl Al induction, measured as ethoxyresorufin-O-deethylase (EROD) and AHH, and the conjugase UDP glucuronyl transferase (UDPGT). Another CYP isoenzyme was measured using the prototype monooxygenase activity aminopyrine-N-demethylase (APDM). Previous results showed that juvenile rainbow trout had modified APDM activity when exposed to River PO water (Vigano et al., 1991, 1994b), thus, although the induction response of this Phase I activity in fish is not yet clearly understood (Kleinow et al., 1990; Goksoyr and Forlin, 1992), the availability of further results and in the earlier sac-fry
L. Viganb et al. I Aquatic
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191
stage was considered positively. Nonprotein thiols (SH), whose major constituent is glutathione (GSH) (Wallace, 1989), and the enzyme glutathione peroxidase (GPx) were tested because of their important role as antioxidant defenses (Winston and Di Giulio, 1991). In addition, the enzyme glutathione reductase (GR) was also measured since it regenerates the active thiol GSH, catalyzing the reduction of the oxidized form (GSSG), thereby contributing to the maintenance of redox status (Wallace, 1989; Di Giulio et al., 1989). The range of potential biomarkers measured in sac-fry, was completed by the two enzymes glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They generate reducing power in the extramitochondrial cytoplasm in the form of NADPH, a cofactor involved, for example, in the regeneration of reduced GSH (Di Giulio et al., 1989) and in function of monooxygenase system (Goksoyr and Forlin, 1992) or depleted by redox cycling of xenobiotic compounds (Winston and Di Giulio, 1991). G6PD and 6PGD have been used as markers of metabolic and cytotoxic alteration, and of oxidative stress in fish liver (Jtirss et al., 1986; Hinton et al., 1988; Bagnasco et al.. 1991; Vigano et al., 1993). The sediments used in the present study were collected in the River PO, which is the main Italian river, at two sites located upstream and downstream, respectively, from the confluence of the River Lambro which is one of the most polluted tributaries of the PO. In sediments sampled at the mouth of the River Lambro, De Simone and Damiani (1983) found concentrations of Aroclor 1242 and 1260 of 3.5 and 10.9 ppm. respectively, with traces of Aroclor 1254. Recent studies demonstrated that the River Lambro is a source in the River PO of both polychlorinated biphenyls (PCB) (Vigano et al., 1994a) and polycyclic aromatic hydrocarbons (PAH). Water concentrations of between 0.05 and 0.35 pg/l were measured for some PAHs in the River Lambro and in the downstream stretch of the River PO (Ciceri et al., 1991). Moreover, this same stretch of the river has shown mutagenic and clastogenic potential (De Flora et al., 1993) and induced some biotransformation enzyme activities in fish liver (Vigano et al., 1994b).
2. Materials and methods 2. I. E,xperimental
animak
Newly fertilized eggs of rainbow trout (0. mykiss) obtained from a fish hatchery (Foglio, Trento, Italy), were incubated at the laboratory in flow-through incubation tanks supplied with flowing aerated unchlorinated tap water (temperature 13 f 1°C; pH 7.66 + 0.02; hardness 283.9 + 6.9 mg/l as CaCO,; alkalinity 235.7 _+4.4 mg/l as CaCO,). 2.2. Sediment
collection
Composite samples of bottom sediments were collected from two sites of the River PO. The first one is located about 2 km upstream the emission of the River Lambro
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and the second one is about 20 km downstream of this tributary. Sediments were sampled using a hand-pushed core sampler and taking only the top 2 cm of each core. Subsamples of the two sediments were freeze-dried and analysed for the content in total organic carbon (Carlo Erba C: N : S analyzer). Triplicate analyses showed a mean content (& SD) of 0.63% (+ 0.03) for the upstream sediment and 1.73% (? 0.12) for the downstream one. The water contents, based on weight loss after lyophilization, were about 33% and 49%, respectively. 2.3. E.xposure of trout larvae Sediments were tested in triplicate in borosilicate glass vessels, each containing 700 ml of thoroughly mixed sediment and 2,800 ml (1:4) of the same tap water used for eggs incubation. Test vessels were prepared the day before the test was started and then allowed to stabilize for about 24 h. Each exposure chamber was gently aerated with a Pasteur pipette during the test. Both eggs incubation and sediment tests were run under dim light. In this experiment, the sac-fry stage was used. At the start of the test, 150 larvae, hatched 2448 h before, were placed in each vessel. As a control, sac fry specimens were exposed in triplicate to river sand that was collected from a clean area (River Ticino) and thoroughly washed in flowing tap water for some days before the test. River sand and overlying tap water had been previously checked as free of inducers in both fry and juvenile tests (Vigano et al., 1991, 1993, 1994b, unpublished data). To assess whether enzyme induction might result from direct contact with the sediment surface, a fourth treatment group was used. In the fourth treatment, trout larvae were exposed to the downstream sediment but direct contact of sac fry with sediment was prevented by interposing a Teflon net (# 1 mm) suspended 4-5 cm over the surface of the sediment. The sac fry specimens were placed on this net. The four exposure groups, referred to as river sand, upstream, downstream and suspended, were tested in parallel at 13 & 1“C with water renewals every 48 h. Daily measurements gave mean results (+SD) of 8.35 & 0.06 for pH, 9.1 & 0.7 mg/l for dissolved oxygen and 289.5 k 8.7 mg/l CaCO, for hardness, without any significant difference among the four treatment groups. The observed mortality was lower than 5% for each exposure group and was not significantly related to treatment as checked by ANOVA. 2.4. Preparation
of subcellular fractions
After 7 days of exposure, trout larvae were collected in a net, rinsed with tap water, immediately blotted on filter paper and frozen in liquid nitrogen. Fish were weighed, the yolk sac was excised and discarded and the remaining larvae were slowly thawed and minced in KC1 (150 mM), Tris-HCl (10 mM), pH 7.4. Larvae were then homogenized in sucrose (250 mM), EDTA-Na (1 mM), Tris-HCl (50 mM), pH 7.4, by means of a Potter-Elvehjem type glass-Teflon homogenizer. Homogenates were subsequently centrifuged at 12 000 g for 20 min and at 105 000 g for 60 min. The resulting supernatants were stored at -80°C in small aliquots to be used for the analysis of
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cytosolic biochemical parameters. In order to prevent sulphydryl group oxidation, portions of homogenate were supplemented with 20 mM EDTA and the corresponding 105 000 g supernatants were used for SH-group analysis. The pellet resulting from the 105 000 g centrifugation was resuspended in washing buffer (KCl, Tris-HCl, EDTA) and centrifuged again at 105 000 g for 60 min. The microsomal pellet was then suspended in Tris-HCl (100 mM), KC1 (150 mM), EDTANa (0.2 mM), glycerol (20%) pH 7.4. For this final suspension, a ratio of 0.5 ml buffer per 3 g original tissue was adopted to obtain a microsomal suspension about three fold more concentrated than that usually prepared from liver of juvenile rainbow trout (0.5 ml buffer per g original tissue). Microsomal suspensions were stored at -80°C in small aliquots until analyses of the biochemical parameters. 2.5. Biochemical
assays
The protein content of the microsomal fraction was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. 3-Hydroxylation of benzo[a]pyrene was evaluated by means of the spectrofluorometric method described by Dehnen et al. (1973) with some modifications. Microsomes (0.3 mg protein per ml) were incubated at 25°C with benzo[a]pyrene (20 ,uM) and a NADPH-generating mixture composed of NADP (2 mM), glucose 6-phosphate (20 mM), glucose 6-phosphate dehydrogenase (3.5 U), nicotinamide (20 mM), MgCl, (20 mM) and KC1 (40 mM) in Tris-HCl (50 mM, pH 7.4). The reaction was stopped after 10 min by adding a solution of 10% Triton x-100 and 1% EDTA (disodium salt) in 0.1 N NaOH, and fluorescence was read at excitation and emission wavelengths of 475 and 522 nm, respectively. The fluorescence produced by an identical reaction mixture of the same sample, stopped at time 0, was subtracted, and the value thus obtained was compared with a standard 3-hydroxy-benzo[a]pyrene (3-OH-BP) curve. The results were expressed as pmol 3-OH-BP mini’ mg-’ microsomal protein. Benzo[a]pyrene
hydroxylase
(AHH)
activity.
Etlzo.~:resor~~n-0-deetl~?~la.~e (EROD) activity. 0-Deethylation of ethoxyresorufin was evaluated by means of the spectrofluorometric method described by Burke and Mayer (1974). Briefly, microsomes (0.3 mg protein per ml) were mixed with ethoxyresorufin (0.5 yM) in Tris-HCl (100 mM, pH 7.8) kept a 20°C. The reaction was started by adding NADPH (50 PM), and fluorescence was recorded for 2 min at excitation and emission wavelengths of 530 and 585 nm, respectively. Increases in relative fluorescence were compared with a standard curve with resorufin. The results were expressed as pmol resorufin min-’ rng-’ microsomal protein. Aminopyrine-N-demethylase (APDM) activity. Microsomes (4 mg protein per ml) were incubated at 20°C in presence of MgCl, (10 mM), NADP (0.3 mM), glucose 6-phosphate (10 mM) glucose 6-phosphate dehydrogenase (7 U), MOPS (100 mM. pH 7.4). Addition of 5 mM aminopyrine started the reaction and APDM activity was determined following the production of formaldehyde (Arillo et al., 1984). After
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Toxicology 31 (1995) 189-202
45-min incubation, formaldehyde was measured spectrophotometrically action at 412 nm (Werringloer, 1978).
by Nash re-
UDP-glucuronosyltransferase (UDPGT) activity. This conjugating enzyme was determined spectrophotometrically according to Hanninen (1968). Microsomes (2 mg protein per ml) were incubated at 20°C in a reaction mixture containing potassium phosphate buffer (0.5 M, pH 7.5), p-nitrophenol as substrate (0.35 mM), UDP glucuronic acid (4.5 mM), EDTA (dipotassium salt, 20 mM). After 30 min incubation, the reaction was stopped by adding trichloroacetic acid (TCA) (3%, final concentration), and samples were centrifuged. The pH of the supernatant was brought to alkaline by adding NaOH (0.48 M, final concentration) and absorbance was measured at 400 nm. Nonprotein thiol (SH)
groups. Samples of homogenized larvae, obtained as described above, were deproteinized by TCA (lo%, final concentration) and centrifuged. Then, SH groups were measured spectrophotometrically (412 nm) in an assay mixture containing Tris-HCl (320 mM, pH 8.9) and 5,5’-dithiobis-(2-nitrobenzoic acid) (0.1 mM), according to Sedlak and Lindsay (1968). Glutathione reductase (GR) activity. The enzymatic reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) was evaluated according to the spectrophotometric method described by Veiga Salles and Ochoa (1950). Cytosolic fractions were mixed with GSSG (3.3 mM) and NADPH (0.1 mM) in Tris-HCl (1 M, pH 8.0). The progressive decrease of absorbance at 340 nm was recorded for 2 min at 20°C and GR activity was expressed as nmol min-’ mg-’ cytosolic protein. Glutathione peroxidase (GPx) activity. The enzymatic conversion of H,O, to water, coupled to the oxidation of GSH to GSSG, was evaluated according to the spectrophotometric method described by Beutler (1975). Cytosolic fractions were mixed with GR (3.0 U), GSH (0.5 mM), NADPH (0.1 mM) and tert-butyl hydroperoxide (0.1 PM) in Tris-HCl (1 M, pH 8.0). The progressive decrease of absorbance at 340 nm was recorded for 2 min at 20°C and GPx activity was expressed as nmol min-’ mg-’ cytosolic protein. Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. G6PD and 6PGD were measured according to the spectro-
photometric method described by Rudack et al. (1971). Cytosolyc fractions were mixed with NADP (0.2 mM), 6-phosphoglucuronic acid (1 mM), glucose 6-P (1 mM) and MgCl, (10 mM) in Tris-HCl(O.1 M, pH 8.0). The progressive increase of absorbance at 340 nm was recorded for 2 min at 2O”C, thus yielding the cumulative G6PD plus 6PGD activities. 6PGD activity was evaluated by testing an identical reaction mixture but lacking glucose 6-P. G6PD activity was calculated by subtracting 6PGD activity from cumulative G6PD plus 6PGD activity. Enzyme activities were expressed as nmol min-’ mg-’ cytosolic protein. All biochemical reagents were purchased from Sigma Chemical Co (St Louis, MO, USA), Boehringer Mannheim Italia (Milan, Italy) and BDH (Poole, Dorset, UK).
L. Vigund etal.lAquatic
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Statistics. All data presented in this study are mean 5 SD of values obtained from three pools of trout fry, each pool consisting of about 150 organisms. Significant differences among treatments for each parameter were analyzed by one-way ANOVA and Duncan’s multiple range test (SAS, 1988).
3. Results 3. I. Mcrosomal hiomarkers The four enzyme activities were all measurable in microsomes prepared from whole
EROD
AHH 1.60
g
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Fig. 1. Benzo[a]pyrene hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD), demethylase (APDM), UDP glucuronyl transferase (UDPGT) enzyme activities measured sac fry after T-day test. Larvae were exposed to clean river sand (R), bottom sediments River PO, upstream (U) and downstream (D) its tributary River Lambro, or downstream from direct contact by interposing a Teflon net (S). Bars represent mean values k SD (n the same letter are not significantly different (Duncan test).
aminopyrine-Nin rainbow trout collected in the sediment isolated = 3). Values with
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31 (1995) 189-202
larvae. Moreover, after 7 days of treatment, i.e. 8-9 days after hatching, AHH, EROD, APDM and UDPGT activities were significantly different among the exposure groups. Sediment collected downstream the River Lambro, induced significantly higher activities than the sediment from the upstream site (Fig. 1). In addition, the sac fry exposed to the latter sediment showed microsomal activities that were not significantly different from control fry. In particular, in the downstream larvae, AHH and EROD enzyme activities were about 3- and 4-fold higher, respectively, than those of the upstream larvae. Similarly, the ability to conjugate with glucuronic acid (UDPGT) was also markedly induced in sac fry exposed to downstream sediment, showing almost twice the activity measured in control group. The response of APDM parallelled the described patterns, with a limited yet significant increase of activity in the downstream treatment, whereas upstream and control organisms were not significantly different. The microsomal activities determined in sac fry exposed on Teflon nets placed over the downstream sediment were not significantly different from those of fry maintained in direct contact with both upstream and control sediment. The only exception was AHH enzyme activity that had an intermediate level between downstream and upstream treatment (Fig. 1). 3.2. Cytosofic bioma~kers After 7 days of exposure, i.e., 8-9 days after hatching, glutathione peroxidase (GPx) and reductase (GR), were measurable in trout larvae cytosolic fractions (Table 1). Despite a consistent trend towards slightly higher values for both upstream and downstream sediments, none of the treatments differed for either of these antioxidant defenses to a significant extent. Similarly, the contents of nonprotein sulphydryl groups (SH), G6PD and 6PGD activities were not influenced by the different exposure conditions (Table 1). Since none of the cytosolic markers was significantly modified by treatments, grand Table 1 Rates of glutathione reduction (GR) and peroxidation (GPx), contents of nonprotein thiol groups (SH) and rates of dehydrogenase activity of glucose 6-phosphate (G6PD) and 6-phosphogluconate (6PGD) measured in sac fry of rainbow trout after 7-day test. Larvae were exposed to clean river sand (control). bottom sediments collected in the River PO, either upstream or downstream its tributary River Lambro, and downstream sediment prevented from direct contact by a Teflon net (suspended) Treatment
GR
River sand Upstream Downstream Suspended
6.4 7.0 7.2 5.7
GPx k f + It
0.5 0.5 1.4 0.4
3.6 4.8 4.1 4.0
G6PD
SH f 0.6 f 1.1 Zk0.5 f 1.3
7.4 8.3 7.7 6.6
f f + f
2.1 1.2 1.0 1.3
4.1 4.5 4.9 4.6
f f + f
6PGD 0.9 1.3 1.6 1.2
Values are mean + SD of three replicates. SH contents are reported as nmollmg protein; as nmol min-’ mg-’ protein. No significant differences were detected (Duncan test).
8.2 9.1 9.3 8.5
f f * k
1.1 1.1 1.2 0.4
enzyme activities
L. Vigund et al. l Aquatic Toxicology 31 (1995) 189-202
197
mean values were also calculated for each one of the five parameters. The following values (&SD) were obtained: GR 6.6 ? 0.6 and GPx 4.1 + 0.5 nmol min-’ mg-’ protein; SH 7.5 ? 0.7 nmol/mg protein; G6PD 4.5 + 0.3 and 6PGD 8.8 ? 0.5 nmol mini’ mg-’ protein. These data can be considered to be representative of the normal biochemical pattern of rainbow trout sac fry, 889 days after hatching.
4. Discussion 4. I. Level
of enzyme
activities in trout lavvur
Our results demonstrate that several biotransformation enzyme activities are present and measurable in rainbow trout sac fry, although most of them are lower than in later developmental stages. In fact, when microsomal basal activities of sac fry are compared to those detected in juvenile rainbow trout (about 1 year old) (Vigano et al., 1991, 1993) it can be inferred that the former organisms have levels of UDPGT enzyme and CYPlAl isoenzyme, measured as AHH and EROD enzyme activities. which are approximately one order of magnitude lower. Binder and Stegeman (1983) also found measurable AHH activity in early life stages of a salmonid, i.e., in both embryo and sac fry of brook trout (S. j&tinalis). They tested hepatic tissue preparations and did not detect any enzyme activity in extrahepatic tissues. Assuming a similar tissue distribution in rainbow trout larvae. the low values observed in fry in the present study could be partially explained by an extrahepatic tissue dilution occurring in whole-body preparations. In addition, the ontogeny of the enzyme systems may also contribute to the differences between larval and juvenile trout. The few data available on development of biotransformation in fish almost exclusively refer to AHH and suggest an increase of activity either with the developmental stage (Binder and Lech, 1984; Stahl and Kocan, 1986; Goksoyr et al.. 1991) or limited to the around-hatching period (Binder and Stegeman, 1983, 1984). Our data seem to be more consistent with the former trend and suggest it may be applicable to EROD, another typical CYPlAl activity, and to a conjugating enzyme such as UDPGT. The extrahepatic dilution and stage-related development of activity are likely to account for the inability to detect AHH activity in rainbow trout fry until 21 days after hatching, as reported by Stahl and Kocan (1986). The analysis of more concentrated microsomal preparation in the present study permitted to detect not only AHH but also other biotransformation activities, demonstrating that they are already present in early sac-fry stage (8-9 days after hatching). This is consistent with the excretion capability of benzo[a]pyrene metabolites observed in the period elapsing before hatching and later stages of rainbow trout development (Stahl and Kocan, 1986). Despite extrahepatic dilution and ontogenic effects, the APDM activity measured in rainbow trout sac fry is surprisingly well comparable to that of juvenile hepatic microsomes (Vigano et al., 1991, 1993). This seems to suggest that APDM activity follows a different developmental pattern and also that this enzyme is regulated by mechanisms different from those regulating AHH, EROD and UDPGT. The litera-
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ture seems to support these differences, at least for the mentioned monooxygenase activities, which should be expression of different cytochrome P-450 genes. In fact, in mammals APDM enzyme activity is catalyzed by cytochrome member of CYP2B subfamily, and recent studies have shown that in trout genome there are sequences similar to mammalian CYP2B (Kleinow et al., 1990). Prototype activities AHH and EROD, on the other hand, are catalytic activities of a different isoenzyme identified in rainbow trout as CYPlAl, which, in turn, has shown great similarity with CYPlAl of mammals (Goksoyr and Forlin, 1992). The comparison of sac fry and juvenile trout for cytosolic biomarkers again reveals lower activities of GR and GPx enzymes as well as lower contents of SH groups in the earlier stage (Vigano et al., 1993). These differences are however less pronounced than those recorded for AHH or EROD activities, i.e. about 20% ofjuvenile fish value, and might be related to the developmental stage. In contrast, G6PD and 6PGD activities show markedly lower levels in larvae (about 2%) as compared to juvenile fish. However, the known relationships of these enzymes with food composition and nutritional status (Jtirss et al., 1986; Vigano et al., 1993) make comparisons difficult since larvae utilize yolk components with a precise sequence and likely have different nutritional requirements than older fish (Heming and Buddington, 1988). 4.2. Induction responses and discriminatory potential Limited information is available regarding the induction potential of biotransformation in early life stages of fish. In a previous study, an increased content of cytochrome P-450 was found in the liver of rainbow trout sac fry obtained from female exposed to dietary PCBs (Hendricks et al., 1981). Sac fry from treated females had higher PCBs content and showed an increased capability of producing aflatoxinepoxide; however, some components of the MFO system, such as epoxide hydrase and glutathione-S-epoxide transferase, although measurable, were unaltered or even reduced. Stahl and Kocan (1986) observed that the excretion of benzo[a]pyrene metabolites can be stimulated by MFO inducers in early life stages of rainbow trout. However, the direct evidence of a microsomal enzyme induction in early life stage of salmonid was reported by Binder and Stegeman (1983) who measured increased level of AHH in PCB-treated brook trout (S. fontinalis) embryos. Our results provide evidence that the microsomal enzymes of rainbow trout larvae, are responsive to inducers as shown by the enhancement of both Phase I AHH, EROD, APDM and Phase II UDPGT activities in fry exposed to downstream sediment, collected from the stretch of River PO receiving the pollutants from the River Lambro. The potential of fry responses to discriminate contaminated sediments can be better appreciated when comparing the results of present study with those obtained with juvenile rainbow trout caged in the River PO upstream and downstream from the confluence of the River Lambro, i.e. exposed to the same sites of sediments collection (Vigano et al., 1994). Like in present sediment test, microsomal activities of caged trout (AHH, EROD, APDM, UDPGT) were markedly induced only at the downstream site. In both studies, AHH and EROD showed the highest induction responses, thereby confirming to be the most sensitive catalytic biomarkers in both juvenile
L. Vigunb et al. iAqualic Toxicology 31 (1995) 189-202
199
and sac-fry stage. It can be mentioned that APDM activity, which was found to be induced both in juvenile trout maintained downstream of the River Lambro confluence and in sac fry exposed to downstream sediments, was also found to be induced in lake trout swim-up fry (45-55 days after hatching) treated with Aroclor 1254 (Binder and Lech, 1984). In addition, the field-caging experiment showed that the enzymes GPx and Gr responded very slowly to River PO water, showing modified activity only after 30 days of exposure. If a similar behavior can be expected from fry antioxidant defences, this may account for their unaltered levels after only 7 days of sediment test. A number of recent studies have reported the induction of antioxidant enzymes in fish exposed to contaminated sediments or collected from polluted areas. Livingstone et al. (1992) have found induced activity of SOD, catalase and GPx enzymes in Limanda limandu caught near the German Bight in the North Sea. Comparable responses have been found in dab (L. limandu) when exposed to a contaminated sediment in a laboratory study (Livingstone et al., 1993). The enhancement of SOD and catalase activities has also been observed in channel catfish (Ictalurus purzctatus) exposed to Black Rock Harbor sediment, highly contaminated with various aromatic hydrocarbons (Di Giulio et al., 1993). Finally, it is noteworthy that grey mullet (Mugil sp.), collected from Spanish littoral areas, have shown a wide range of increased antioxidant defences including the enzymes GPx, SOD, catalase, GR and G6PD (Rodriguez-Ariza et al., 1993). In contrast, both in the above mentioned study with caged trout and in the present study with sac-fry, the two enzymes G6PD and 6PGD were unaffected by River PO water (unpublished results). Probably, longer time of exposure and/or higher levels of contamination are necessary to modify these two enzymes, as seems to be suggested by their induced activities found in another study on native free-swimming fish (Bagnasco et al., 1991) The proposed comparisons suggest that the responses of sac-fry obtained by applying a short-term test are consistent and qualitatively similar to those pointed out in an older life stage. Furthermore, in agreement with previous findings, they confirm that the stretch downstream of the River Lambro is more polluted than the upstream site in the River PO (De Simone and Damiani, 1983; Ciceri et al., 1991; Marchetti, 1991; Vigano et al., 1994a,b). In particular, other biomarkers had been found to become positive in juvenile trout exposed in situ downstream the Lambro confluence, including mutagenicity of bile, liver activation of promutagens to mutagenic metabolites and frequency of micronuclei in peripheral blood erythrocytes (De Flora et al., 1993). 4.3. Route of’exposure
and temporary
benthic niche
The negligible response to the fourth treatment, in which trout larvae were prevented from direct contact with downstream sediment, indicates the importance of the route of exposure and, on the other hand, the risk deriving to some fish species from their temporary niche of benthic organism. The hydrophobic nature of the most common inducers makes them partitioning between solid phase and interstitial water of sediment, higher concentrations occurring in pore water than in the upper water column. In accordance with the present results, suggesting a negligible availability of
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inducers over the narrow zone of the sediment-water interface, several investigators supported the importance of interstitial water and/or water at the sediment/water interface as the primary routes of exposure for a benthic organisms (for a review, see Adams, 1987). Similarly, adult fathead minnows (Pimephales prom&s), exposed to sediment spiked with PCBs, accumulated much more residue than did fish screened from the sediment and likely prevented from mouthing the interstitial water (Halter and Johnson, 1977). More recently, it has been reported that a demersal fish (Leiostomus xanthurus) isolated from direct contact with a contaminated sediment did not significantly accumulate PCB residues when compared with fish in intimate contact with the sediment (Rubinstein et al., 1984). 5. Conclusions Despite a general picture displaying lower enzyme activities than juvenile trout, rainbow trout sac fry exhibited an appreciable biochemical pattern for both Phase I and Phase II biotransformation activities and for antioxidant defences. Some of these activities have shown to be induced by short-term exposure to bottom river sediment.
Acknowledgements
The authors thank Dr F. Melodia and C. Bennicelli for the collaboration in biochemical analyses, F. Bacciu for the measurements of sediment organic carbon content and L. Previtali for technical assistance. This study was supported by MURST (40% project).
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