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Thermal modulation of the toxicokinetics of benzo[a]pyrene in isolated hepatocytes of sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops), and chub mackerel (Scomber japonicus)
Comparative Biochemistry and Physiology Part C 124 (1999) 157 – 164 www.elsevier.com/locate/cbpc
Thermal modulation of the toxicokinetics of benzo[a]pyrene in isolated hepatocytes of sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops), and chub mackerel (Scomber japonicus) Blair D. Johnston, George Alexander, Christopher J. Kennedy * Department of Biological Sciences, 8888 Uni6ersity Dri6e, Simon Fraser Uni6ersity, Burnaby, BC V5A 1S6, Canada Received 28 January 1999; received in revised form 17 June 1999; accepted 21 June 1999
Abstract Hepatocytes from sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops) and chub mackerel (Scomber japonicus) were isolated from 11°C acclimated animals. The uptake, metabolism, and excretion of benzo[a]pyrene (B[a]P) in hepatocytes was measured at 6, 11 and 19°C. Chub mackerel hepatocyte uptake rates were significantly lower (0.012 9 0.003 mg/s per g cell) at 11°C than black rockfish (0.028 90.009 mg/s per g cell) or sablefish (0.032 90.012 mg/s per g cell) hepatocytes at all temperatures. Hepatocytes metabolized B[a]P to phase I (1–8%) and phase II (92 – 99%) metabolites. Accumulation of phase II metabolites was lower in chub mackerel hepatocytes (0.016 9 0.004 mg/h per g cell), than black rockfish (0.052 9 0.012 mg/h per g cell), or sablefish hepatocytes (0.060 90.015 mg/h per g cell). Phase II metabolite accumulation increased greatest with temperature in chub mackerel hepatocytes (Q10 =1.9490.30), followed by sablefish (Q10 = 1.6590.30), and rockfish (Q10 = 1.3890.30). Sablefish hepatocytes had higher excretion rates of phase II metabolites (0.010 9 0.0023 mg/h per g cell), than mackerel (0.0046 90.0009 mg/h per g cell) or rockfish hepatocytes (0.0029 90.0008 mg/h per g cell). Phase II metabolite excretion rates increased with temperature only in sablefish hepatocytes (Q10 =1.6790.76). These differences in toxicokinetics may indicate distinct consequences for various species exposed to xenobiotics. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Benzo[a]pyrene; Hepatocytes; Temperature; Toxicokinetics; Anoplopoma fimbria; Sebastes melanops; Scomber japonicus
1. Introduction As ectotherms, teleosts have adapted to their thermal environments by developing a host of physiological and biochemical alterations associated with acute and seasonal temperature change [9]. As well as altering the routine physiology and biochemistry of the animal, these adjustments to temperature can have a modifying influence on many events during the interaction between xenobiotic and organism. These events include xenobiotic exposure (initiation of interaction), toxicokinetics (uptake, distribution, biotransformation and excretion of xenobiotic) and toxicodynamics (xenobiotic –receptor interaction). Modification of xenobiotic– * Corresponding author. Tel.: +1-604-2915640; fax: + 1-6042913496. E-mail address: [email protected] (C.J. Kennedy)
organism interactions at any of these phases may alter the toxicity of the xenobiotic [12]. At present, no clear pattern emerges from the literature regarding the effects of temperature on xenobiotic toxicity. Because of the many pathways of chemical fate and the diverse number of mechanisms of action of xenobiotics, an acute change in temperature can result in an increase, a decrease, or in no change in toxicity [17]. In studies with the gulf toadfish, an acute increase in temperature leads to an elevation in the rate of benzo[a]pyrene (B[a]P) uptake due to increases in ventilation rate and possibly due to alteration in the organization of the plasma membrane of the gill cells [13,16]. In addition, several studies have demonstrated temperature compensation in xenobiotic metabolism, such that biotransformation rates in animals acclimated to different temperatures are similar, if they are exposed
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to the xenobiotic at their respective acclimation temperatures [22,1]. Acute temperature change, however, appears to alter xenobiotic metabolism approximately 2-fold [15], although much of this may be due to temperature effects on chemical uptake. Increased temperature may also increase the rate of excretion via the biliary route [4,11]. As temperature clearly affects the toxicokinetics of xenobiotics in fish, it is surmised that ectothermic organisms adapted to different thermal environments may exhibit different toxicological responses when confronted with acute temperature changes. The differences in organism – xenobiotic interactions due to temperature modulation may lead to altered impacts across communities, resulting from a single xenobiotic exposure. For this study, three marine teleosts from different thermal habitats were chosen in order to maximise any potential differences in the effects of temperature on xenobiotic toxicokinetics. Also, utilising isolated hepatocytes provided a rapid, precise and statistically powerful determination of toxicokinetics at the cellular level. Unfortunately, because the hepatocytes are removed from the complex effects of the entire system, conclusions about how these effects relate to the whole animal may not be possible. However, as a preliminary study investigating how thermal adaptation may confer differences in toxicokinetics, the objective is clear: to employ the use of isolated hepatocytes from three different marine teleosts to determine the species-related differences in the acute effects of temperature on the toxicokinetics of a model xenobiotic, benzo[a]pyrene.
2. Materials and methods
2.1. Experimental animals and acclimation Mature specimens of the sablefish, Anoplopoma fimbria (779.69107.0 g) were obtained by otter trawl in Trevor Channel near Bamfield, BC at a depth of 90 m ( : 8°C). Specimens of the black rockfish, Sebastes melanops, (144.3914.8 g) and chub mackerel, Scomber japonicus (396.8 9 30.3 g) were obtained in Bamfield Inlet by angling at depths of 5– 10 m (11°C) and 1 – 2 m (15°C), respectively. Fish were acclimated to 1191°C seawater (30%) under flow-through conditions and a natural photoperiod for :3 weeks prior to an experiment. Fish were fed frozen krill and herring until 48 h prior to commencement of an experiment. Survivability through holding was 100%. All animals used in experiments were males as determined by gonadal examination.
2.2. Chemicals [3H]-Benzo[a]pyrene (57 Ci/mmol) was purchased from Amersham (Arlingtion Heights, IL). B[a]P metabolite standards were obtained from the National Cancer Institute Chemical Repositories (Kansas City, MO). Collagenase, L-15 medium, sulphatase and b-glucuronidase were from Sigma (St Louis, MO).
2.3. Hepatocyte isolation Hepatocytes were isolated from each of the three marine species by a modification of procedures previously developed for rainbow trout and toadfish hepatocytes [22,15]. Livers were initially perfused with a Ca2 + -free Hanks salts solution for 5 min, and then collagenase (95 units/ml) was added to the perfusate and perfusion continued to 10–25 min depending upon the size of the liver. Hepatocytes were gently disaggregated through two nylon filters (250 mm followed by 80 mm) and concentrated by low speed centrifugation. Hepatocytes were resuspended in L-15 medium to a concentration of approximately 25 mg/100 ml. Viability, determined by Trypan Blue exclusion was \ 95% for all preparations. Temperature of isolation was 11°C.
2.4. Benzo[a]pyrene uptake Uptake rates of B[a]P were determined according to [17]. Briefly, the uptake suspension medium was a balanced Hanks salts solution which included 10 mM HEPES, 1 mM CaCl2, 4% BSA, 3 mM glucose, 5 mM sodium bicarbonate and 5 mg/ml [3H] B[a]P (0.25 mCi). A microcentrifuge tube (1.5 ml) containing 0.9 ml of suspension medium layered onto 0.4 ml of 1-bromododecane:dionyl pthalate (3:1 v/v), layered onto 0.1 ml of 1.2 N perchloric acid was incubated at 6, 11 or 19°C and immediately positioned in a Fisher 235B microcentrifuge. At time zero, 100 ml of cell suspension (containing approximately 25 mg of cells) was added to the suspension medium layer. Several time points thereafter (1, 2, 3, 5, 10, 15, 20, 30, 45, 60 and 120 s), the different tubes were centrifuged at 13 000× g to rapidly separate cells from the suspension medium and to allow only viable cells to pass through the oil to the acid layer. Following centrifugation, the suspension medium and the oil layer were carefully aspirated and the remaining acid layer removed and counted for radioactivity in a Beckman LS6500 Liquid Scintillation Counter. Cell wet mass was considered to be the weight of the pellet after centrifugation. A correction factor was applied to account for extracellular water remaining with the pellet [22]. Routine controls were performed including the injection of heat-killed cells and of suspension medium without cells [7]. Total radioactivity recovered was always \95% as indicated by mass balance on the three separate layers.
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2.5. Benzo[a]pyrene metabolism
2.8. Statistics and experimental design
Rates of metabolism in hepatocytes were determined by a modification of previously developed methods [16,7]. Hepatocytes (:50 mg in 200 ml) were added to microreaction vials containing 1.8 ml of L-15 containing 5 mg/ml of [3H] B[a]P (1.0 mCi) at 6, 11 or 19°C. Bottles were capped with serum stoppers and gently stirred and aerated. At the end of an 8 h incubation, metabolic reactions were terminated by the addition of 2 ml of ethyl acetate. The mixture was extracted twice with 2 ml ethyl acetate and the aqueous and organic fractions separated and stored at −86°C until further analysis.
The use of isolated cell cultures provides a simple and statistically powerful system for the study of direct effects of temperature on xenobiotic action at the cellular level [2]. All values are reported as means 9 SE. All measurements were performed in duplicate. Data from uptake, metabolism and excretion experiments were compared using a Model III, two factor ANOVA for significant differences (PB 0.05) [22]. Multiple comparisons were made with the Student–Newman–Keuls test [23]. Uptake rates were determined from the linear portion of the accumulation versus time curve (not shown) for hepatocytes from each animal [16]. The linear portion of the curve lasted for 3 s and steady-state was achieved between 1 and 1.5 min.
2.6. Benzo[a]pyrene excretion To determine the rates of excretion of B[a]P and its metabolites, 100 ml of hepatocyte suspension (25 mg cells) was added to a microcentrifuge tube containing 0.9 ml of L-15 containing 5 mg/ml of [3H] B[a]P (1.0 mCi). The cells were allowed to accumulate B[a]P for 1.5 min which was determined from the uptake experiments to be sufficient time to reach steady state. Cells were then gently centrifuged (100 ×g) and the supernatant removed. The cells were washed once with 1 ml of L-15 medium (no B[a]P) and centrifuged. The pellet was then resuspended in L-15 medium at the appropriate temperature in a microreaction vial. The vials were capped with serum stoppers and aerated. To determine the extent of B[a]P or metabolite excretion, vials were centrifuged (13 000×g) at various time points and an aliquot of supernatant was counted for total radioactivity by LSC. The remaining supernatant was extracted twice with ethyl acetate and the aqueous and organic fractions stored at − 86°C for further analysis.
3. Results
3.1. Uptake of B[a]P Chub mackerel hepatocytes had significantly lower uptake rates (0.01590.003, 0.01290.003 and 0.014 9 0.002 mg/s per g cell) than those of the sablefish (0.0269 0.005, 0.0329 0.005 and 0.03429 0.005 mg/s per g cell) and the black rockfish (0.029 9 0.005, 0.02990.006 and 0.0319 0.006) at 6, 11 and 19°C, respectively, (Fig. 1). There were no significant changes in rates of uptake in any of the species’ hepatocytes in response to an acute change in temperature as evidenced by the Q10 values of 1.199 0.11, 1.099 0.12 and 0.9290.07 for sablefish, rockfish and mackerel hepatocytes, respectively (Fig. 2).
2.7. Analysis of metabolites Quantitation of 12 isomeric B[a]P metabolites was achieved by a modification of the methods used by previous investigators [14,7,16]. HPLC was performed according to [5] with a Hewlett Packard 1050 quaternary pump system, and a Phenomenex C-18 silica column. Compounds were detected with a Hewlett Packard 1050 series fluorescence detector and identified by comparison with retention times for known standards. Fractions of HPLC eluate were collected in 2 min intervals with a Gilson FC 203 fraction collector and counted by LSC. The aqueous layer was enzymatically treated with ß-glucuronidase (pH 5) followed by sulphatase (pH 7), and then acidified to pH 2 with 1 N H2SO4 and heated for 24 h at 80°C. Ethyl acetate extraction after each treatment was used to determine the presence of glucuronic acid or sulphate conjugates or of an aqueous remainder [14,16].
Fig. 1. Rate of uptake of benzo[a]pyrene by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * Denotes statistical significance between species.
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Fig. 2. Q10 for the three phases of the toxicokinetics of benzo[a]pyrene in 11°C acclimated, isolated hepatocytes of black rockfish ( ), sablefish ( ) and chub mackerel (b). Q10, average of Q10 values from 11 to 19°C and from 11 to 6°C. * Denotes statistical significance between species.
3.2. B[a]P metabolite accumulation by isolated hepatocytes The majority of metabolites accumulated in 8 h were phase II (92–99%) as opposed to phase I (1 – 8%). The types of phase I metabolites accumulated and identified in each species were: r-7,t-8,9,c-10 hydroxy-benzo[a]pyrene and t-9,10 hydroxy-benzo[a]pyrene in mackerel hepatocytes; 9-hydroxy-B[a]P in rockfish hepatocytes; and t-9,10 hydroxy-B[a]P and 9-hydroxyB[a]P in sablefish hepatocytes (Table 1). The rate of phase II metabolite accumulation was highest in sablefish hepatocytes (0.040 90.011, 0.059 9 0.015 and 0.08090.021 mg/h per g cell), followed by rockfish hepatocytes (0.0399 0.010, 0.052 9 0.012 and 0.061 9 0.018 mg/h per g cell) and chub mackerel hepatocytes (0.01090.002, 0.016 90.004 and 0.027 90.008 mg/h
Table 1 Benzo[a]pyrene phase I metabolites identified (out of 12 detectable metabolites) accumulated by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C.) Species
Incubation temperature 6°C
11°C
Metabolite 19°C
% of total organic-fraction radioactivity Mackerel
n.d.
0.7490.42
1.079 0.52 r-7,t-8,9, c-10 tetrol 3.58 90.13 3.539 0.11 3.759 0.07 t-9,10 diol 94.21 9 4.18 90.919 6.78 92.609 3.78 B[a]P
Rockfish
1.07 90.24 2.149 1.23 1.419 0.48 9-OH 93.18 93.20 92.759 2.96 94.309 4.98 B[a]P
Sablefish
1.24 90.53 2.139 0.41 1.779 0.52 t-9,10 diol 7.84 93.28 10.019 5.25 6.119 4.62 9-OH 88.35 9 7.42 82.619 8.93 91.129 6.98 B[a]P
Fig. 3. Rate of accumulation of benzo[a]pyrene phase II metabolites by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * Denotes statistical significance between species. NB: all between-temperature variation is statistically significant.
per g cell) at 6, 11 and 19°C, respectively, (Fig. 3). The greatest increase in the rate of accumulation of aqueous soluble metabolites was in chub mackerel hepatocytes (Q10 = 1.949 0.53), followed by sablefish (Q10 =1.659 0.17), and then in rockfish hepatocytes (Q10 =1.389 0.10). Glucuronide conjugates comprise an increasing percentage of total phase II metabolites with increasing temperature in both sablefish and rockfish hepatocytes (Fig. 4).
3.3. B[a]P metabolite excretion The rate of excretion of phase II metabolites was significantly higher at all incubation temperatures in sablefish hepatocytes (0.00679 0.0008, 0.01090.002, 0.01590.004 mg/h per g cell) than in either chub mackerel (0.0047 9 0.001, 0.00469 0.0006, 0.00799 0.0018 mg/h per g cell) or rockfish hepatocytes (0.00569 0.0012, 0.002990.0003, 0.00509 0.009 mg/h per g cell) at 6, 11 and 19°C, respectively, (Fig. 5). The excretion rate in sablefish hepatocytes demonstrated the greatest change with temperature (Q10 = 1.679 0.76) across the incubation temperatures used in this experiment compared to either chub mackerel (Q10 = 1.419 0.54) or rockfish (Q10 = 0.929 0.11) hepatocytes. An examination of the pattern of excretion of different phase II
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Fig. 4. Benzo[a]pyrene phase II metabolites accumulated by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C expressed as percentage of total of aqueous-soluble, B[a]P-derived radioactivity and tentatively identified by enzymatic digestion as glucuronide conjugates (b), sulphate conjugates (a), other conjugates ( ) and remaining aqueous-soluble radioactivity ( ). * Denotes statistical significance between temperature treatments.
metabolites indicates that glucuronide conjugates comprised a greater percent of the total phase II metabolites excreted from mackerel hepatocytes. Rockfish and sablefish hepatocytes excreted more radioactivity in the aqueous remainder form (Fig. 6). 4. Discussion In this study, the thermal modulation of the toxicokinetics of B[a]P in isolated hepatocytes of three species
Fig. 5. Rate of excretion of benzo[a]pyrene phase II metabolites by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * denotes statistical significance between species. Letters denote statistical significance between temperatures.
Fig. 6. Concentration of various benzo[a]pyrene phase II metabolites excreted into extracellular L-15 medium by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C at 30, 120 and 240 min. Metabolites were tentatively identified by enzymatic digestion as glucuronide conjugates (), sulphate conjugates ( ), other conjugates (), and remaining aqueous-soluble radioactivity (). NB: the scale of the ordinal axis is different for each species.
of marine teleost was investigated in order to begin an examination of how biochemical adaptations to temperature might confer different characteristics on organism–xenobiotic interaction in different species. The intention of this study is not to argue that differences seen are strictly due to any ecophysiological adaptations (as species is a confounding factor) of the animals in question, but rather to start such an investigation by first trying to determine if there are any differences at all in these toxicokinetic traits. The results are therefore interpreted in terms of ‘species-related’ effects as opposed to ecophysiological effects. In this respect, the results show interesting differences in the rate of xenobiotic uptake, metabolism, and excretion between the three species, in response to acute temperature change. Benzo[a]pyrene is believed to enter cells transcellularly, and pass through the cell membrane by passive diffusion [17]. Therefore, species-differences in chemical uptake rates or rates of change with temperature will likely be due to differences within the plasma membrane itself. B[a]P uptake rates were significantly lower in chub mackerel hepatocytes compared to the hepat-
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cytes of either black rockfish or sablefish. This may, in part, be due to the larger size of the chub mackerel liver cells. The consequent reduction in the surfacearea:volume ratio may account for the differences seen. It is also possible that the plasma membrane of the mackerel hepatocytes is more viscous (less fluid) than that of the other two species at the incubation temperatures used in this experiment. Viscosity differences of this sort have been well characterised in plasma membranes of teleost species adapted to different thermal environments [8]. Also notable, is the relative insensitivity to acute temperature change of the uptake of benzo[a]pyrene into the hepatocytes of all three species. These results are in contrast to previous findings of an approximately 2-fold increase in the rate of uptake of B[a]P by isolated gill cells of the gulf toadfish across a 10°C temperature range [16]. It is unclear at present how or if the plasma membranes of different organ systems show differential temperature adaptation. Previous studies have demonstrated thermally-induced changes in the viscosity of plasma membranes in the liver cells of the rainbow trout, Oncorhynchus mykiss [8]. However, it is uncertain how viscosity measurements relate to the movement of a molecule such as benzo[a]pyrene, across the membrane which, due to its large, planar hydrodynamic size, may encounter a significant cavity transfer resistance as it crosses the plasma membrane [19] and therefore may not be affected by small changes in fluidity. After uptake, the next stage in the toxicokinetic process is biotransformation, which renders the lipophillic parent compound into a more readily excretable water-soluble form (phase I and phase II metabolites). The results of this experiment indicate that there are species-related differences in the type of phase I metabolites accumulated by isolated hepatocytes. This may have interesting consequences in terms of potential impacts of a single xenobiotic exposure on different species. The chub mackerel hepatocytes were the only cells studied which accumulated the B[a]P metabolite r-7,t-8,9,c-10 hydroxybenzo[a]pyrene to measurable concentrations which is significant as this metabolite is generally thought to be an indicator of a carcinogenic epoxide precursor. Overall, the effect of incubation temperature on changes in metabolite accumulation were: no detectable differences in rates of phase I metabolite accumulation, a clear indication of rate changes in the production of phase II metabolites, and a shift in the type of phase II metabolites towards greater glucuronide production in rockfish and sablefish hepatocytes. Measured rates of phase II metabolism are in the range found in other experiments employing the
use of isolated hepatocytes [7]. Relative rates of phase II metabolite accumulation mirror the results observed for B[a]P uptake rates; the mackerel hepatocytes have lower rates than either sablefish or rockfish hepatocytes. This may be due in part to species effects on the uptake rate as opposed to a reflection of biotransformation rates for each species. A simple comparison of the relative ratios of rates of uptake and metabolism at 11°C indicates that this is a strong possibility. An acute increase in the rate of phase II xenobiotic metabolism was seen in response to an acute temperature increase in all of the species studied. It is not possible to ascertain from the results of this experiment whether this is due to the effect of temperature on any one particular enzyme, as the process involved in producing phase II metabolites is complex and potentially involves many enzymatic pathways [6]. However, the pattern of phase II metabolite accumulation seen in this study clearly demonstrates temperature modulation in all three species examined, as was shown previously in toadfish [7]. The significant differences in the rate changes seen across the different species supports the possibility that species adapted to different thermal regimes may exhibit differences in their response to temperature with respect to the kinetics of xenobiotic metabolizing enzymes. However, until the response to temperature of these multiple pathways can be assessed by an examination of the temperature-related enzyme kinetics on the specific enzymes involved, the specific mechanism will remain unclear. Another variation in the response to temperature of phase II metabolite production appears to be in the accumulation of glucuronide conjugates. There is a significant increase in the percentage of glucuronide conjugates, in relation to total phase II metabolites produced, at the higher incubation temperatures in both sablefish and rockfish — this increase is only marginally not significant in mackerel hepatocytes (PB 0.055). This observation may support the previously mentioned temperature-related variation in enzyme kinetics or may be indicative of at least partial microenvironment influences as the enzyme responsible for glucuronide conjugation, UDP-glucuronosyltransferase (UDPGT) is predominantly located in the lipid-rich membrane of the smooth endoplasmic reticulum, whereas the remaining phase II enzymes including, the sulphotransferases and glutathione-S-transferase are mainly present in the aqueous-based compartment of the cytosol [17]. Although some progress has been made in the area of temperature effects on various cellular compartments [21,18], it is not yet clear what differences may exist between cytosolic and SER fluidity responses to acute temperature change. The final stage of the toxicokinetic process is the excretion of the aqueous-soluble metabolites out of the
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cell, possibly via a Na+/K+-ATPase driven, ion exchange mechanism, as has been shown in mammalian livers and fish kidneys [3,20]. A comparison of overall rates of uptake, accumulation of phase II metabolites, and of excretion of phase II metabolites in this study, appear to indicate that some aspect of the process of excretion (from metabolite formation to its release into extracellular fluid) is the rate-limiting step in the toxicokinetics of benzo[a]pyrene in isolated hepatocytes. This raises the possibility that phylogenetic variation in the biochemical mechanisms responsible for xenobiotic metabolite excretion may be a primary determinant of differences in species-specific xenobiotic clearance rates. Also notable, is the relatively low rate of excretion by the hepatocytes of the black rockfish, especially with increasing temperature. As temperature increased, metabolite accumulation increases were not being offset by comparable increases in excretion. This may increase the potential for toxicity, specifically in this case through the possibility that reactive metabolites of B[a]P are being retained within the cell for a longer duration. The overall pattern of the rates of change with temperature of benzo[a]pyrene toxicokinetics in all three species shows: no change in uptake, an increase in metabolism, and a variable response in excretion. As there were no changes in uptake with temperature, measurements of uptake and metabolism rates were considered to be accurate. However, due to the complicating influences of temperature on B[a]P metabolism, measured rates of excretion may not reflect true values. This study shows that changes in the toxicokinetics of a given xenobiotic can vary greatly among species, in response to acute changes in temperature. In toxicological terms, these findings suggest that different species may exhibit different xenobiotic – toxicant interactions based on differential biochemical adaptations, and that adaptations to different thermal regimes may be an important factor in determining the outcome of a xenobiotic exposure event. However, more general conclusions about which environmental and physiological factors determine these differences cannot be ascertained until comparisons can be made among closely related species which already have well characterised phylogenies [10]. By concentrating efforts on withintaxa comparisons of rate-limiting steps in the toxicokinetic processes, it may be possible to make such conclusions. Knowledge in this area may provide important insight for researchers in understanding how biochemical adaptation to environmental niches may determine the outcome of xenobiotic exposure.
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Acknowledgements The authors thank Sophie Campagna, Melissa Evanson, Joel Leech, Sherry McGregor, Malcolm Wyeth and the staff at the Bamfield Marine Station for their technical assistance during this study.
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Thermal modulation of the toxicokinetics of benzo[a]pyrene in isolated hepatocytes of sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops), and chub mackerel (Scomber japonicus) Blair D. Johnston, George Alexander, Christopher J. Kennedy * Department of Biological Sciences, 8888 Uni6ersity Dri6e, Simon Fraser Uni6ersity, Burnaby, BC V5A 1S6, Canada Received 28 January 1999; received in revised form 17 June 1999; accepted 21 June 1999
Abstract Hepatocytes from sablefish (Anoplopoma fimbria), black rockfish (Sebastes melanops) and chub mackerel (Scomber japonicus) were isolated from 11°C acclimated animals. The uptake, metabolism, and excretion of benzo[a]pyrene (B[a]P) in hepatocytes was measured at 6, 11 and 19°C. Chub mackerel hepatocyte uptake rates were significantly lower (0.012 9 0.003 mg/s per g cell) at 11°C than black rockfish (0.028 90.009 mg/s per g cell) or sablefish (0.032 90.012 mg/s per g cell) hepatocytes at all temperatures. Hepatocytes metabolized B[a]P to phase I (1–8%) and phase II (92 – 99%) metabolites. Accumulation of phase II metabolites was lower in chub mackerel hepatocytes (0.016 9 0.004 mg/h per g cell), than black rockfish (0.052 9 0.012 mg/h per g cell), or sablefish hepatocytes (0.060 90.015 mg/h per g cell). Phase II metabolite accumulation increased greatest with temperature in chub mackerel hepatocytes (Q10 =1.9490.30), followed by sablefish (Q10 = 1.6590.30), and rockfish (Q10 = 1.3890.30). Sablefish hepatocytes had higher excretion rates of phase II metabolites (0.010 9 0.0023 mg/h per g cell), than mackerel (0.0046 90.0009 mg/h per g cell) or rockfish hepatocytes (0.0029 90.0008 mg/h per g cell). Phase II metabolite excretion rates increased with temperature only in sablefish hepatocytes (Q10 =1.6790.76). These differences in toxicokinetics may indicate distinct consequences for various species exposed to xenobiotics. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Benzo[a]pyrene; Hepatocytes; Temperature; Toxicokinetics; Anoplopoma fimbria; Sebastes melanops; Scomber japonicus
1. Introduction As ectotherms, teleosts have adapted to their thermal environments by developing a host of physiological and biochemical alterations associated with acute and seasonal temperature change [9]. As well as altering the routine physiology and biochemistry of the animal, these adjustments to temperature can have a modifying influence on many events during the interaction between xenobiotic and organism. These events include xenobiotic exposure (initiation of interaction), toxicokinetics (uptake, distribution, biotransformation and excretion of xenobiotic) and toxicodynamics (xenobiotic –receptor interaction). Modification of xenobiotic– * Corresponding author. Tel.: +1-604-2915640; fax: + 1-6042913496. E-mail address: [email protected] (C.J. Kennedy)
organism interactions at any of these phases may alter the toxicity of the xenobiotic [12]. At present, no clear pattern emerges from the literature regarding the effects of temperature on xenobiotic toxicity. Because of the many pathways of chemical fate and the diverse number of mechanisms of action of xenobiotics, an acute change in temperature can result in an increase, a decrease, or in no change in toxicity [17]. In studies with the gulf toadfish, an acute increase in temperature leads to an elevation in the rate of benzo[a]pyrene (B[a]P) uptake due to increases in ventilation rate and possibly due to alteration in the organization of the plasma membrane of the gill cells [13,16]. In addition, several studies have demonstrated temperature compensation in xenobiotic metabolism, such that biotransformation rates in animals acclimated to different temperatures are similar, if they are exposed
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to the xenobiotic at their respective acclimation temperatures [22,1]. Acute temperature change, however, appears to alter xenobiotic metabolism approximately 2-fold [15], although much of this may be due to temperature effects on chemical uptake. Increased temperature may also increase the rate of excretion via the biliary route [4,11]. As temperature clearly affects the toxicokinetics of xenobiotics in fish, it is surmised that ectothermic organisms adapted to different thermal environments may exhibit different toxicological responses when confronted with acute temperature changes. The differences in organism – xenobiotic interactions due to temperature modulation may lead to altered impacts across communities, resulting from a single xenobiotic exposure. For this study, three marine teleosts from different thermal habitats were chosen in order to maximise any potential differences in the effects of temperature on xenobiotic toxicokinetics. Also, utilising isolated hepatocytes provided a rapid, precise and statistically powerful determination of toxicokinetics at the cellular level. Unfortunately, because the hepatocytes are removed from the complex effects of the entire system, conclusions about how these effects relate to the whole animal may not be possible. However, as a preliminary study investigating how thermal adaptation may confer differences in toxicokinetics, the objective is clear: to employ the use of isolated hepatocytes from three different marine teleosts to determine the species-related differences in the acute effects of temperature on the toxicokinetics of a model xenobiotic, benzo[a]pyrene.
2. Materials and methods
2.1. Experimental animals and acclimation Mature specimens of the sablefish, Anoplopoma fimbria (779.69107.0 g) were obtained by otter trawl in Trevor Channel near Bamfield, BC at a depth of 90 m ( : 8°C). Specimens of the black rockfish, Sebastes melanops, (144.3914.8 g) and chub mackerel, Scomber japonicus (396.8 9 30.3 g) were obtained in Bamfield Inlet by angling at depths of 5– 10 m (11°C) and 1 – 2 m (15°C), respectively. Fish were acclimated to 1191°C seawater (30%) under flow-through conditions and a natural photoperiod for :3 weeks prior to an experiment. Fish were fed frozen krill and herring until 48 h prior to commencement of an experiment. Survivability through holding was 100%. All animals used in experiments were males as determined by gonadal examination.
2.2. Chemicals [3H]-Benzo[a]pyrene (57 Ci/mmol) was purchased from Amersham (Arlingtion Heights, IL). B[a]P metabolite standards were obtained from the National Cancer Institute Chemical Repositories (Kansas City, MO). Collagenase, L-15 medium, sulphatase and b-glucuronidase were from Sigma (St Louis, MO).
2.3. Hepatocyte isolation Hepatocytes were isolated from each of the three marine species by a modification of procedures previously developed for rainbow trout and toadfish hepatocytes [22,15]. Livers were initially perfused with a Ca2 + -free Hanks salts solution for 5 min, and then collagenase (95 units/ml) was added to the perfusate and perfusion continued to 10–25 min depending upon the size of the liver. Hepatocytes were gently disaggregated through two nylon filters (250 mm followed by 80 mm) and concentrated by low speed centrifugation. Hepatocytes were resuspended in L-15 medium to a concentration of approximately 25 mg/100 ml. Viability, determined by Trypan Blue exclusion was \ 95% for all preparations. Temperature of isolation was 11°C.
2.4. Benzo[a]pyrene uptake Uptake rates of B[a]P were determined according to [17]. Briefly, the uptake suspension medium was a balanced Hanks salts solution which included 10 mM HEPES, 1 mM CaCl2, 4% BSA, 3 mM glucose, 5 mM sodium bicarbonate and 5 mg/ml [3H] B[a]P (0.25 mCi). A microcentrifuge tube (1.5 ml) containing 0.9 ml of suspension medium layered onto 0.4 ml of 1-bromododecane:dionyl pthalate (3:1 v/v), layered onto 0.1 ml of 1.2 N perchloric acid was incubated at 6, 11 or 19°C and immediately positioned in a Fisher 235B microcentrifuge. At time zero, 100 ml of cell suspension (containing approximately 25 mg of cells) was added to the suspension medium layer. Several time points thereafter (1, 2, 3, 5, 10, 15, 20, 30, 45, 60 and 120 s), the different tubes were centrifuged at 13 000× g to rapidly separate cells from the suspension medium and to allow only viable cells to pass through the oil to the acid layer. Following centrifugation, the suspension medium and the oil layer were carefully aspirated and the remaining acid layer removed and counted for radioactivity in a Beckman LS6500 Liquid Scintillation Counter. Cell wet mass was considered to be the weight of the pellet after centrifugation. A correction factor was applied to account for extracellular water remaining with the pellet [22]. Routine controls were performed including the injection of heat-killed cells and of suspension medium without cells [7]. Total radioactivity recovered was always \95% as indicated by mass balance on the three separate layers.
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2.5. Benzo[a]pyrene metabolism
2.8. Statistics and experimental design
Rates of metabolism in hepatocytes were determined by a modification of previously developed methods [16,7]. Hepatocytes (:50 mg in 200 ml) were added to microreaction vials containing 1.8 ml of L-15 containing 5 mg/ml of [3H] B[a]P (1.0 mCi) at 6, 11 or 19°C. Bottles were capped with serum stoppers and gently stirred and aerated. At the end of an 8 h incubation, metabolic reactions were terminated by the addition of 2 ml of ethyl acetate. The mixture was extracted twice with 2 ml ethyl acetate and the aqueous and organic fractions separated and stored at −86°C until further analysis.
The use of isolated cell cultures provides a simple and statistically powerful system for the study of direct effects of temperature on xenobiotic action at the cellular level [2]. All values are reported as means 9 SE. All measurements were performed in duplicate. Data from uptake, metabolism and excretion experiments were compared using a Model III, two factor ANOVA for significant differences (PB 0.05) [22]. Multiple comparisons were made with the Student–Newman–Keuls test [23]. Uptake rates were determined from the linear portion of the accumulation versus time curve (not shown) for hepatocytes from each animal [16]. The linear portion of the curve lasted for 3 s and steady-state was achieved between 1 and 1.5 min.
2.6. Benzo[a]pyrene excretion To determine the rates of excretion of B[a]P and its metabolites, 100 ml of hepatocyte suspension (25 mg cells) was added to a microcentrifuge tube containing 0.9 ml of L-15 containing 5 mg/ml of [3H] B[a]P (1.0 mCi). The cells were allowed to accumulate B[a]P for 1.5 min which was determined from the uptake experiments to be sufficient time to reach steady state. Cells were then gently centrifuged (100 ×g) and the supernatant removed. The cells were washed once with 1 ml of L-15 medium (no B[a]P) and centrifuged. The pellet was then resuspended in L-15 medium at the appropriate temperature in a microreaction vial. The vials were capped with serum stoppers and aerated. To determine the extent of B[a]P or metabolite excretion, vials were centrifuged (13 000×g) at various time points and an aliquot of supernatant was counted for total radioactivity by LSC. The remaining supernatant was extracted twice with ethyl acetate and the aqueous and organic fractions stored at − 86°C for further analysis.
3. Results
3.1. Uptake of B[a]P Chub mackerel hepatocytes had significantly lower uptake rates (0.01590.003, 0.01290.003 and 0.014 9 0.002 mg/s per g cell) than those of the sablefish (0.0269 0.005, 0.0329 0.005 and 0.03429 0.005 mg/s per g cell) and the black rockfish (0.029 9 0.005, 0.02990.006 and 0.0319 0.006) at 6, 11 and 19°C, respectively, (Fig. 1). There were no significant changes in rates of uptake in any of the species’ hepatocytes in response to an acute change in temperature as evidenced by the Q10 values of 1.199 0.11, 1.099 0.12 and 0.9290.07 for sablefish, rockfish and mackerel hepatocytes, respectively (Fig. 2).
2.7. Analysis of metabolites Quantitation of 12 isomeric B[a]P metabolites was achieved by a modification of the methods used by previous investigators [14,7,16]. HPLC was performed according to [5] with a Hewlett Packard 1050 quaternary pump system, and a Phenomenex C-18 silica column. Compounds were detected with a Hewlett Packard 1050 series fluorescence detector and identified by comparison with retention times for known standards. Fractions of HPLC eluate were collected in 2 min intervals with a Gilson FC 203 fraction collector and counted by LSC. The aqueous layer was enzymatically treated with ß-glucuronidase (pH 5) followed by sulphatase (pH 7), and then acidified to pH 2 with 1 N H2SO4 and heated for 24 h at 80°C. Ethyl acetate extraction after each treatment was used to determine the presence of glucuronic acid or sulphate conjugates or of an aqueous remainder [14,16].
Fig. 1. Rate of uptake of benzo[a]pyrene by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * Denotes statistical significance between species.
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Fig. 2. Q10 for the three phases of the toxicokinetics of benzo[a]pyrene in 11°C acclimated, isolated hepatocytes of black rockfish ( ), sablefish ( ) and chub mackerel (b). Q10, average of Q10 values from 11 to 19°C and from 11 to 6°C. * Denotes statistical significance between species.
3.2. B[a]P metabolite accumulation by isolated hepatocytes The majority of metabolites accumulated in 8 h were phase II (92–99%) as opposed to phase I (1 – 8%). The types of phase I metabolites accumulated and identified in each species were: r-7,t-8,9,c-10 hydroxy-benzo[a]pyrene and t-9,10 hydroxy-benzo[a]pyrene in mackerel hepatocytes; 9-hydroxy-B[a]P in rockfish hepatocytes; and t-9,10 hydroxy-B[a]P and 9-hydroxyB[a]P in sablefish hepatocytes (Table 1). The rate of phase II metabolite accumulation was highest in sablefish hepatocytes (0.040 90.011, 0.059 9 0.015 and 0.08090.021 mg/h per g cell), followed by rockfish hepatocytes (0.0399 0.010, 0.052 9 0.012 and 0.061 9 0.018 mg/h per g cell) and chub mackerel hepatocytes (0.01090.002, 0.016 90.004 and 0.027 90.008 mg/h
Table 1 Benzo[a]pyrene phase I metabolites identified (out of 12 detectable metabolites) accumulated by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C.) Species
Incubation temperature 6°C
11°C
Metabolite 19°C
% of total organic-fraction radioactivity Mackerel
n.d.
0.7490.42
1.079 0.52 r-7,t-8,9, c-10 tetrol 3.58 90.13 3.539 0.11 3.759 0.07 t-9,10 diol 94.21 9 4.18 90.919 6.78 92.609 3.78 B[a]P
Rockfish
1.07 90.24 2.149 1.23 1.419 0.48 9-OH 93.18 93.20 92.759 2.96 94.309 4.98 B[a]P
Sablefish
1.24 90.53 2.139 0.41 1.779 0.52 t-9,10 diol 7.84 93.28 10.019 5.25 6.119 4.62 9-OH 88.35 9 7.42 82.619 8.93 91.129 6.98 B[a]P
Fig. 3. Rate of accumulation of benzo[a]pyrene phase II metabolites by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * Denotes statistical significance between species. NB: all between-temperature variation is statistically significant.
per g cell) at 6, 11 and 19°C, respectively, (Fig. 3). The greatest increase in the rate of accumulation of aqueous soluble metabolites was in chub mackerel hepatocytes (Q10 = 1.949 0.53), followed by sablefish (Q10 =1.659 0.17), and then in rockfish hepatocytes (Q10 =1.389 0.10). Glucuronide conjugates comprise an increasing percentage of total phase II metabolites with increasing temperature in both sablefish and rockfish hepatocytes (Fig. 4).
3.3. B[a]P metabolite excretion The rate of excretion of phase II metabolites was significantly higher at all incubation temperatures in sablefish hepatocytes (0.00679 0.0008, 0.01090.002, 0.01590.004 mg/h per g cell) than in either chub mackerel (0.0047 9 0.001, 0.00469 0.0006, 0.00799 0.0018 mg/h per g cell) or rockfish hepatocytes (0.00569 0.0012, 0.002990.0003, 0.00509 0.009 mg/h per g cell) at 6, 11 and 19°C, respectively, (Fig. 5). The excretion rate in sablefish hepatocytes demonstrated the greatest change with temperature (Q10 = 1.679 0.76) across the incubation temperatures used in this experiment compared to either chub mackerel (Q10 = 1.419 0.54) or rockfish (Q10 = 0.929 0.11) hepatocytes. An examination of the pattern of excretion of different phase II
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Fig. 4. Benzo[a]pyrene phase II metabolites accumulated by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C expressed as percentage of total of aqueous-soluble, B[a]P-derived radioactivity and tentatively identified by enzymatic digestion as glucuronide conjugates (b), sulphate conjugates (a), other conjugates ( ) and remaining aqueous-soluble radioactivity ( ). * Denotes statistical significance between temperature treatments.
metabolites indicates that glucuronide conjugates comprised a greater percent of the total phase II metabolites excreted from mackerel hepatocytes. Rockfish and sablefish hepatocytes excreted more radioactivity in the aqueous remainder form (Fig. 6). 4. Discussion In this study, the thermal modulation of the toxicokinetics of B[a]P in isolated hepatocytes of three species
Fig. 5. Rate of excretion of benzo[a]pyrene phase II metabolites by 11°C acclimated, isolated hepatocytes of black rockfish ("), sablefish ( ) and chub mackerel () at 6, 11 and 19°C. * denotes statistical significance between species. Letters denote statistical significance between temperatures.
Fig. 6. Concentration of various benzo[a]pyrene phase II metabolites excreted into extracellular L-15 medium by 11°C acclimated, isolated hepatocytes of black rockfish, sablefish, and chub mackerel at 6, 11 and 19°C at 30, 120 and 240 min. Metabolites were tentatively identified by enzymatic digestion as glucuronide conjugates (), sulphate conjugates ( ), other conjugates (), and remaining aqueous-soluble radioactivity (). NB: the scale of the ordinal axis is different for each species.
of marine teleost was investigated in order to begin an examination of how biochemical adaptations to temperature might confer different characteristics on organism–xenobiotic interaction in different species. The intention of this study is not to argue that differences seen are strictly due to any ecophysiological adaptations (as species is a confounding factor) of the animals in question, but rather to start such an investigation by first trying to determine if there are any differences at all in these toxicokinetic traits. The results are therefore interpreted in terms of ‘species-related’ effects as opposed to ecophysiological effects. In this respect, the results show interesting differences in the rate of xenobiotic uptake, metabolism, and excretion between the three species, in response to acute temperature change. Benzo[a]pyrene is believed to enter cells transcellularly, and pass through the cell membrane by passive diffusion [17]. Therefore, species-differences in chemical uptake rates or rates of change with temperature will likely be due to differences within the plasma membrane itself. B[a]P uptake rates were significantly lower in chub mackerel hepatocytes compared to the hepat-
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cytes of either black rockfish or sablefish. This may, in part, be due to the larger size of the chub mackerel liver cells. The consequent reduction in the surfacearea:volume ratio may account for the differences seen. It is also possible that the plasma membrane of the mackerel hepatocytes is more viscous (less fluid) than that of the other two species at the incubation temperatures used in this experiment. Viscosity differences of this sort have been well characterised in plasma membranes of teleost species adapted to different thermal environments [8]. Also notable, is the relative insensitivity to acute temperature change of the uptake of benzo[a]pyrene into the hepatocytes of all three species. These results are in contrast to previous findings of an approximately 2-fold increase in the rate of uptake of B[a]P by isolated gill cells of the gulf toadfish across a 10°C temperature range [16]. It is unclear at present how or if the plasma membranes of different organ systems show differential temperature adaptation. Previous studies have demonstrated thermally-induced changes in the viscosity of plasma membranes in the liver cells of the rainbow trout, Oncorhynchus mykiss [8]. However, it is uncertain how viscosity measurements relate to the movement of a molecule such as benzo[a]pyrene, across the membrane which, due to its large, planar hydrodynamic size, may encounter a significant cavity transfer resistance as it crosses the plasma membrane [19] and therefore may not be affected by small changes in fluidity. After uptake, the next stage in the toxicokinetic process is biotransformation, which renders the lipophillic parent compound into a more readily excretable water-soluble form (phase I and phase II metabolites). The results of this experiment indicate that there are species-related differences in the type of phase I metabolites accumulated by isolated hepatocytes. This may have interesting consequences in terms of potential impacts of a single xenobiotic exposure on different species. The chub mackerel hepatocytes were the only cells studied which accumulated the B[a]P metabolite r-7,t-8,9,c-10 hydroxybenzo[a]pyrene to measurable concentrations which is significant as this metabolite is generally thought to be an indicator of a carcinogenic epoxide precursor. Overall, the effect of incubation temperature on changes in metabolite accumulation were: no detectable differences in rates of phase I metabolite accumulation, a clear indication of rate changes in the production of phase II metabolites, and a shift in the type of phase II metabolites towards greater glucuronide production in rockfish and sablefish hepatocytes. Measured rates of phase II metabolism are in the range found in other experiments employing the
use of isolated hepatocytes [7]. Relative rates of phase II metabolite accumulation mirror the results observed for B[a]P uptake rates; the mackerel hepatocytes have lower rates than either sablefish or rockfish hepatocytes. This may be due in part to species effects on the uptake rate as opposed to a reflection of biotransformation rates for each species. A simple comparison of the relative ratios of rates of uptake and metabolism at 11°C indicates that this is a strong possibility. An acute increase in the rate of phase II xenobiotic metabolism was seen in response to an acute temperature increase in all of the species studied. It is not possible to ascertain from the results of this experiment whether this is due to the effect of temperature on any one particular enzyme, as the process involved in producing phase II metabolites is complex and potentially involves many enzymatic pathways [6]. However, the pattern of phase II metabolite accumulation seen in this study clearly demonstrates temperature modulation in all three species examined, as was shown previously in toadfish [7]. The significant differences in the rate changes seen across the different species supports the possibility that species adapted to different thermal regimes may exhibit differences in their response to temperature with respect to the kinetics of xenobiotic metabolizing enzymes. However, until the response to temperature of these multiple pathways can be assessed by an examination of the temperature-related enzyme kinetics on the specific enzymes involved, the specific mechanism will remain unclear. Another variation in the response to temperature of phase II metabolite production appears to be in the accumulation of glucuronide conjugates. There is a significant increase in the percentage of glucuronide conjugates, in relation to total phase II metabolites produced, at the higher incubation temperatures in both sablefish and rockfish — this increase is only marginally not significant in mackerel hepatocytes (PB 0.055). This observation may support the previously mentioned temperature-related variation in enzyme kinetics or may be indicative of at least partial microenvironment influences as the enzyme responsible for glucuronide conjugation, UDP-glucuronosyltransferase (UDPGT) is predominantly located in the lipid-rich membrane of the smooth endoplasmic reticulum, whereas the remaining phase II enzymes including, the sulphotransferases and glutathione-S-transferase are mainly present in the aqueous-based compartment of the cytosol [17]. Although some progress has been made in the area of temperature effects on various cellular compartments [21,18], it is not yet clear what differences may exist between cytosolic and SER fluidity responses to acute temperature change. The final stage of the toxicokinetic process is the excretion of the aqueous-soluble metabolites out of the
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cell, possibly via a Na+/K+-ATPase driven, ion exchange mechanism, as has been shown in mammalian livers and fish kidneys [3,20]. A comparison of overall rates of uptake, accumulation of phase II metabolites, and of excretion of phase II metabolites in this study, appear to indicate that some aspect of the process of excretion (from metabolite formation to its release into extracellular fluid) is the rate-limiting step in the toxicokinetics of benzo[a]pyrene in isolated hepatocytes. This raises the possibility that phylogenetic variation in the biochemical mechanisms responsible for xenobiotic metabolite excretion may be a primary determinant of differences in species-specific xenobiotic clearance rates. Also notable, is the relatively low rate of excretion by the hepatocytes of the black rockfish, especially with increasing temperature. As temperature increased, metabolite accumulation increases were not being offset by comparable increases in excretion. This may increase the potential for toxicity, specifically in this case through the possibility that reactive metabolites of B[a]P are being retained within the cell for a longer duration. The overall pattern of the rates of change with temperature of benzo[a]pyrene toxicokinetics in all three species shows: no change in uptake, an increase in metabolism, and a variable response in excretion. As there were no changes in uptake with temperature, measurements of uptake and metabolism rates were considered to be accurate. However, due to the complicating influences of temperature on B[a]P metabolism, measured rates of excretion may not reflect true values. This study shows that changes in the toxicokinetics of a given xenobiotic can vary greatly among species, in response to acute changes in temperature. In toxicological terms, these findings suggest that different species may exhibit different xenobiotic – toxicant interactions based on differential biochemical adaptations, and that adaptations to different thermal regimes may be an important factor in determining the outcome of a xenobiotic exposure event. However, more general conclusions about which environmental and physiological factors determine these differences cannot be ascertained until comparisons can be made among closely related species which already have well characterised phylogenies [10]. By concentrating efforts on withintaxa comparisons of rate-limiting steps in the toxicokinetic processes, it may be possible to make such conclusions. Knowledge in this area may provide important insight for researchers in understanding how biochemical adaptation to environmental niches may determine the outcome of xenobiotic exposure.
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Acknowledgements The authors thank Sophie Campagna, Melissa Evanson, Joel Leech, Sherry McGregor, Malcolm Wyeth and the staff at the Bamfield Marine Station for their technical assistance during this study.
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