Environ. Sci. Technol. 2010, 44, 3052–3058
Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss): A Validation Study to Support Their Application in Bioaccumulation Assessment ROBERT T. MINGOIA, KYLE P. GLOVER, DIANE L. NABB, CHING-HUI YANG, SUZANNE I. SNAJDR, AND XING HAN* DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, Delaware
Received January 1, 2010. Revised manuscript received February 15, 2010. Accepted February 17, 2010.
Determination of biotransformation rates of xenobiotics in freshly isolated trout hepatocytes has been demonstrated to significantly improve the performance of bioaccumulation assessment models. In order to promote this in vitro approach, trout hepatocytes need to be cryopreserved to facilitate their availability while ensuring their metabolic competency. In the present study, we obtained basal level metabolic enzyme activities for cytochrome P450 (CYP) 1A, CYP3A, glutathione-Stransferase, and uridine 5′-diphospho-glucuronosyltransferase from trout hepatocytes cryopreserved for various periods of time up to three months and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance (CLint) values determined in cryopreserved trout hepatocytes to those determined in freshly isolated hepatocytes for reference compounds molinate, michler’s ketone, 4-nonylphenol, 2,4-ditert-butylphenol, benzo(a)pyrene, and pyrene. Our results show that cryopreserved trout hepatocytes maintained greater than 75% of their basal level enzyme activities and greater than 72% of xenobiotic biotransformation capabilities, regardless of the length of cryostorage. As a result, bioconcentration factors of the reference compounds were adequately predicted based on the CLint values. We simulated the condition for shipping cryopreserved trout hepatocytes and demonstrated that 24 h dry ice storage did not negatively affect the rates of xenobiotic biotransformation. We conclude that cryopreserved trout hepatocytes are suitable for biotransformation rate determination of xenobiotics in vitro, and therefore, are an acceptable alternative to freshly isolated trout hepatocytes in the application in bioaccumulation assessment.
Introduction Biotransformation reduces bioaccumulation potential of xenobiotics. Determination of biotransformation rates of xenobiotics is considered one of the most important steps to improve our current practice in bioaccumulation assessment (1). As a proof of concept, several laboratories, including ours, have shown that fish biotransformation rates can be * Corresponding author phone: (302) 451-5808; fax: (302) 4513568; e-mail:
[email protected]. 3052
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adequately predicted using in vitro incubations of freshly isolated hepatocytes, and the accuracy of modeled bioconcentration factors (BCFs) has been dramatically improved with the input from this in vitro assay (2-6). Despite the recognized importance of biotransformation rate information in bioaccumulation assessment, adoption of the in vitro hepatocyte approach has been challenging due to the requirement for freshly isolated hepatocytes in each assay. There are many obstacles to obtaining fresh trout hepatocytes: first, a nearby rainbow trout farm is preferred to provide live fish; second, an on-site acclimation period is required prior to hepatocyte isolation to reduce variability as a result of animal stress during shipping; and third, the technique of trout hepatocyte isolation is time-consuming and requires significant training and expertise. In addition to the above-mentioned technical challenges, more troublingly, a plethora of viable hepatocytes can be isolated from a single rainbow trout, which means that a large number of unused cells have to be discarded after each assay, resulting in significant waste. Recently, there have been efforts to develop in vitro approaches using fish liver subcellular fractions such as S9 and microsomes as alternatives to hepatocytes (4-6). However, available evidence from studies with fish suggests that liver subcellular fractions are not as competent as hepatocytes in the prediction of biotransformation rate in vivo (4, 6). Cryopreservation of isolated fish hepatocytes for use in biotransformation assays is a potential solution that would allow any lab to have access to hepatocytes (e.g., from commercial or other lab sources). Cryopreserved hepatocytes from humans and other mammalian species are routinely used in pharmaceutical research for their proven metabolic competencies (7-12). Significant effort has been spent on the optimization of cryopreservation conditions and validation of cryopreserved hepatocytes from mammalian species (7-12). Unfortunately, such effort with fish hepatocytes has been very limited. Ferraris et al. (13) have shown that cryopreserved trout hepatocytes can be successfully cultured in plates and were responsive to cytochrome P450 (CYP) 1A1 inducers. However, for cryopreserved trout hepatocytes to be used for the purpose of bioaccumulation assessment, it is necessary to demonstrate that cryopreserved trout hepatocytes maintain metabolic activities that are comparable to those from freshly isolated hepatocytes. In the present study, we obtained basal level enzyme activities for CYP1A, CYP3A, glutathione-S-transferase (GST), and uridine 5′-diphospho-glucuronosyltransferase (UGT) from rainbow trout hepatocytes cryopreserved for various periods of time, up to three months, and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance values of the reference compounds molinate, michler’s ketone (MK), 4-nonylphenol (4NP), 2,4-ditert-butylphenol (DTBP), benzo(a)pyrene (BaP), and pyrene that were determined in cryopreserved trout hepatocytes with those determined in freshly isolated hepatocytes. In order to simulate expected shipping conditions, we stored the cryopreserved trout hepatocytes on dry ice for 24 h and verified their metabolic competency using the reference compounds. The objective of our study is to demonstrate that cryopreservation is a practical approach for long-term storage of trout hepatocytes and cryopreserved trout hepatocytes are adequate in generating biotransformation rate information for the purpose of bioaccumulation assessment of xenobiotics. 10.1021/es903909g
2010 American Chemical Society
Published on Web 03/02/2010
FIGURE 1. Sampling and assay scheme for cryopreserved trout hepatocytes.
Experimental Section Materials. Molinate was obtained from Chem Service (West Chester, PA). MK, 4NP, Pyrene, and DTBP were obtained from Sigma-Aldrich (St. Louis, MO). BaP was obtained from MolTox (Boone, NC). The purities of these chemicals were between 98 and 99.5%. Perfusion media and buffers were obtained from Invitrogen (Carlsbad, CA). All other chemicals, if not specified, were obtained from Sigma-Aldrich. Animals. Male rainbow trout (Oncorhynchus mykiss), approximately one and a half years old and approximately 10-12 in. in length, were purchased from Limestone Springs Fishing Preserve, Richland, Pennsylvania. The fish were held for at least one week in continuously flowing well water at a water temperature of approximately 10 °C under a 16:8 light/dark cycle, fed with AquaMax Starter Fingerling 300 5D03 (PMI Nutrition International, LLC) once daily, and fasted for 24 h prior to surgical manipulation. Study Design. Figure 1 describes the design of our study, which included three individual batches of pooled trout hepatocytes (three fish per isolation batch) that were isolated on three different days. A small portion of the freshly isolated hepatocytes for each batch was assayed on the isolation day (Day 0, Figure 1) and the remaining cells were cryopreserved. At different intervals during cryostorage (shown in Figure 1), trout hepatocytes were thawed and assayed for the comparison of their basal level metabolic enzyme activities and xenobiotic clearance rates to those obtained on Day 0. At the end of an approximate three-month cryostorage period, the cells were stored on dry ice for 24 h before being thawed to simulate potential shipping conditions. Isolation of Trout Hepatocytes. The procedures for isolation of trout hepatocytes were described previously (14, 15). The cells were suspended in Leibovitz L-15 medium at pH 7.8 and were counted in the presence of 0.04% trypan blue (Beckman Coulture, ViCell XR) to evaluate cell viability. Hepatocytes were isolated in three different batches according to Figure 1 and cryopreserved separately by batch. Cryopreservation of Trout Hepatocytes. Hepatocytes were centrifuged at 50g for three minutes at 4 °C and suspended in ice-cold cryopreservation medium (Dulbecco’s Modified Eagle Medium (DMEM) with 20% fetal bovine serum and 0.25% bovine serum albumin, pH 7.8) at 10 × 106 cells/mL. The cells were centrifuged at 50g to remove the supernatant and then suspended in cryopreservation medium at a volume equal to 1/3 of the original volume that yielded 10 × 106 cells/mL. Cryopreservation medium containing DMSO was gradually added to the cell suspension as described previously (16). Briefly, cryopreservation medium containing 12% (v/v) of DMSO was slowly added to the cell suspension up to 50% of the original volume, resulting in a DMSO concentration of 4% and 20 × 106 cells/mL. After five minutes on ice, cryopreservation medium containing 16% (v/v) of DMSO was slowly added up to the original volume of the cell suspension. After these steps, the final DMSO concentration was 10% and the final cell concentration was 10 × 106 cells/mL. After five minutes on ice, the cells were suspended by gentle shaking and 1.5 mL of hepatocyte suspension was transferred to 1.8 mL cryovials. The hepa-
tocytes were kept at 4 °C for thirty minutes before the cooling process began. A Thermo CryoMed 7450 controlled rate freezer (Rolling Meadows, IL) was used to freeze the hepatocytes based on the following cooling protocol: initial cooling at -1 °C/min from sample temperature to -4 °C, chamber temperature to -40 at -25 °C/min, chamber temperature to -12 at 10 °C/min, chamber temperature to -40 at -1 °C/min, and chamber temperature to -140 at -10 °C/min. The cooling protocol was designed to include a shock cooling step to minimize effects of the latent heat of fusion (17). At the end of the freezing cycle, the cryovials were placed in the vapor phase of liquid nitrogen for long-term storage. Thawing of Trout Hepatocytes. Hepatocytes were thawed in a room temperature water bath until only a small ice crystal remained and then quickly added to a 15-fold volume of DMEM (containing 10% FBS and 0.25% BSA, pH 7.8) at room temperature. The cells were washed twice with ice-cold L-15 medium, suspended in ice-cold L-15 medium at pH 7.8, and then were counted for recovery and viability in the presence of 0.04% trypan blue. Dry Ice Storage. In order to simulate the shipping condition, trout hepatocytes were removed from liquid nitrogen storage and quickly placed onto dry ice after approximately three months cryostorage period. After 24 h storage on dry ice, the cells were thawed as described above. Incubation of Hepatocytes. Freshly isolated and cryopreserved trout hepatocytes in L-15 medium at 2 × 106 cells/ mL were preincubated at 10 °C and pH 7.8 for 10 min. The reaction was initiated by adding the compound (final concentrations 4 µM for molinate, MK, and BaP; 10 µM for 4NP, Pyrene, and DTBP; final solvent (acetonitrile) concentration was 1%). Reactions were terminated at regular time intervals with 9-fold ice-cold acetonitrile. The samples were centrifuged briefly to remove the precipitates, and the supernatants were transferred to HPLC vials for LC analysis. Enzyme Activities. Freshly isolated and cryopreserved trout hepatocytes were lysed by brief sonication on a Branson Sonifier S-250 (Danbury, CT) before the assays. Testosterone 6β-hydroxylase (for CYP3A), 7-ethoxyresorufin O-dealkylase (EROD, for CYP1A), uridine 5′-diphospho-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) activities in fresh and cryopreserved hepatocytes were determined according to previously published procedures (4, 14). The cell concentration of the lysate was 2 × 106 cells/ mL, except in the UGT activity assay where 7.5 × 106 cells/ mL was used. CYP1A and CYP3A activity assays were done in Hank’s Balanced Salt Solution (HBSS, Invitrogen) at pH 7.4. GST activity assay was done in 0.1 M phosphate buffer at pH 6.5 (18). UGT activity assay was done in a buffer containing 66 mM Tris-HCl, 10 mM MgCl2, 0.05% (w/w) Brij 58, and 10 mM EDTA, pH 7.4 (19). Sample Analysis. Molinate, MK, 4NP, DTBP, and BaP samples were analyzed by LC/MS/MS or LC/fluorescence methods according to previously published procedures (2). Pyrene samples were analyzed on an LC/fluorescence system (Waters 2795 HPLC system (Milford, MA) equipped with a Waters 2475 Multi λ fluorescence detector). Separation occurred on an Agilent Zorbax SB-C18 column (4.6 × 75 VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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mm, 3.5 µm) within 10.5 min by linear gradient from 70% A (water) to 100% B (acetonitrile) at a flow rate of 1 mL/min. Fluorescence was measured with excitation at 338 nm and emission at 375 nm. Data Analysis. Substrate depletion data were analyzed by using either the monoexponential or biexponential decay model according to reference (3) to obtain the intrinsic clearance (CLint, mL/h/106 cells) values. Biexponential decay curves were reported in the past in substrate depletion studies (3, 20). The actual causes of this nonlinear behavior were not completely understood, but were suggested to be possibly related to loss of enzyme activities over time, enzyme inhibition from the metabolites, or nonspecific protein binding (20). Using a biexponential decay model sufficiently described the substrate depletion curves and provided good estimation of the CLint values in the previous studies (3, 20) as well as in the present study. Trout hepatic clearance (CLH, mL/h/kg) was predicted based on a “well-stirred” liver model using the values of CLint, trout hepatic blood flow (327.6 mL/ h/kg, (21)), trout hepatocellularity (540 × 106 cells/g, (3)), trout liver weight (12.7 g/kg, (22)), and the unbound fractions of the chemical in blood and in hepatocyte incubation (2). Bioconcentration factor (BCF) was predicted using a kinetic model that was proposed by Arnot and Gobas (23, 24) using a previously described calculation procedure (2).
Results and Discussion Cryopreservation Conditions. Conditions for cryopreservation of isolated hepatocytes have been explored extensively, especially for the purpose of preserving human hepatocytes (9-11, 25-31). Further optimization of those conditions is an ongoing area that attracts active research (32, 33). We expended considerable effort at the beginning of this study optimizing our cryopreservation conditions, which included the composition of freezing and thawing media, freezing and thawing protocols, and the post-thaw washing step by centrifugation. The principles for our choices of cryopreservation conditions were mainly derived from mammalian hepatocyte research (8, 11), which we assumed to be valid for fish hepatocytes as well. During this condition optimization phase, we learned that (1) DMEM in freezing and thawing media improved recovery if compared to either Leibovitz’s L-15 medium or Kreb-Henseleit buffer; (2) freezing rate needs to be computer-controlled to allow slow cooling in general and a shock cooling step to minimize effects of the latent heat of fusion (17); (3) cells need to be thawed at room temperature instead of at 37 °C to avoid reduction in enzyme activities; and (4) the number of washes and the centrifugation speed need to be fine-tuned at the post-thawing step because less washes and higher speed would allow higher yield but cause lower enzyme activities (possibly as a result of an increased proportion of metabolically incompetent cells (8)), while more washes at lower speed would result in poor yield with higher enzyme activities. Our final choices for cryopreservation conditions (see Experimental Section) aimed at the highest yield and enzyme activities we could achieve with a preference for maintaining enzyme activities in cases where we had to sacrifice one in exchange for another (condition optimization step 4 as stated above). Cell Viability and Yield after Cryopreservation. Resuspension of trout hepatocytes from cryopreservation requires a thawing procedure and a washing step (Experimental Section). Cell death is inevitable during the thawing procedure and the washing step was used to remove the dead cells. Viability of the resuspended trout hepatocytes from cryopreservation was not significantly different than that of the freshly isolated hepatocytes, and the values were between 98.2 and 99.5% based on the trypan blue exclusion approach. Under our cryopreservation conditions, the yield after cryopreservation (percentage of the number of hepatocytes 3054
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FIGURE 2. The percentage of yield of cryopreserved trout hepatocytes that were in cryostorage for up to 90 days. The dotted line represents the average yield (37%) of hepatocytes from all three batches and from all time points. The number of freshly isolated trout hepatocytes that were frozen for cryostorage was considered 100% for each batch. after being resuspended from cryostorage relative to the number of hepatocytes being put into cryostorage) fluctuated between 25 and 45% for all three batches (mean ) 37%) and was independent of the duration of cryostorage (Figure 2). The yield of the trout hepatocyte isolation was normally very high, which has been a consistent observation in our lab with an average yield of more than 300 million hepatocytes per fish for a 300-500 g rainbow trout. Since we normally pool hepatocytes from three fish, this would yield approximately one billion cells per isolation. Without cryopreservation, most of the cells would be discarded at the end of the assay day. With cryopreservation, we could have approximately 300 million viable hepatocytes available for future assays assuming a 30% recovery rate from cryopreservation. Three hundred million hepatocytes are adequate for 150 individual incubations in a typical clearance assay in one milliliter of incubated hepatocytes at 2 × 106 cells/mL. If we further assume triplicate incubations per compound, a single isolation of trout hepatocytes would allow future screening of approximately 50 compounds. This will result in a significant cost saving, and more importantly, a dramatic reduction in the demand of trout for hepatocyte isolation. Another benefit of cryopreservation is that one can easily test a compound in trout hepatocytes cryopreserved from different isolation batches to understand interbatch variability (see more in the discussion below) and to obtain an average of the biotransformation rates. Obviously, this would be a rather tedious task if only freshly isolated hepatocytes are to be used. Basal Level Metabolic Enzyme Activities. The first step in our validation effort was to verify basal level phase I and II metabolic enzyme activities in cryopreserved trout hepatocytes. Reactions of 7-ethoxyresorufin (ER)-O-dealkylation, testosterone 6β-hydroxylation, 1-chloro-2,4-dinitrobenzene (CDNB)-glutathione conjugation, and p-nitrophenol-glucuronidation were measured to represent CYP1A, CYP3A, GST, and UGT activities, respectively (values are in Supporting Information (SI) Table S1). In general, CYP1A and CYP3A activities (0.69-3.65) and 16.8-31.0 pmol/min/mg, respectively, SI Table S1) measured in the present study are comparable to the values we published previously (14). However, GST activities (24.1-45.2 nmol/min/mg, SI Table S1) were significantly lower than values previously reported (14). In the present study, we conducted the CDNBglutathione conjugation assay in 0.1 M phosphate buffer at pH 6.5 (4, 18). In contrast, we did the same assay in HBSS at pH 7.4 in the previous work (14). This could explain the differences in GST activities between these two studies. Basal level UGT activities in isolated trout hepatocytes were quite
FIGURE 3. Percentage of basal level enzyme activities in cryopreserved trout hepatocytes for metabolic enzymes CYP1A (A), CYP3A (B), GST (C), and UGT (D). Trout hepatocytes were isolated in three different batches. The enzyme activities obtained from freshly isolated hepatocytes of each respective batch were considered 100% (dotted line). UGT activities were too low to detect at 10 °C for batch 1 and 2 and therefore the data were only available for batch 3 samples that were measured at room temperature (D). low (supported by personal communication with Dr. Helmut Figure 4 shows the percentages of in vitro-determined Segner, University of Bern, Switzerland). Attempts at meaCLint values and model-predicted BCF values of all reference compounds in cryopreserved trout hepatocytes from all suring UGT activities at 10 °C in trout hepatocytes from batch batches. The CLint and BCF values obtained from freshly 1 and 2 failed. In batch 3, we raised the assay temperature isolated hepatocytes of each respective batch were considered to room temperature and also increased the number of cells 100% (dotted line in the figure). For compounds molinate, from 2 × 106 cells/mL to 7.5 × 106 cells/mL. These changes resulted in measurable UGT activities (5.75-13.6 nmol/min/ MK, DTBP, pyrene, and BaP, their CLint percentages generally fluctuated along the 100% line due to assay variability. The mg, SI Table S1). CLint values for 4NP, however, were all lower than 100%. Figure 3 shows the percentage values of enzyme activities Because there was no cryostorage duration-dependent trend measured in cryopreserved trout hepatocytes relative to those for any compound, we averaged CLint data obtained from measured in freshly isolated hepatocytes. We did not observe cryopreserved trout hepatocytes from all batches and from a time-dependent trend indicating reduction of enzyme all cryostorage time periods. Table 1 shows that the average activities over the duration of cryostorage (Figure 3), and we CLint percentage values for molinate, MK, DTBP, pyrene, and therefore averaged the percentage values for all batches and BaP were between 93 to 106%. These results suggest that for all measured time points. The recovered CYP1A, CYP3A, cryopreserved trout hepatocytes performed comparably to GST, and UGT activities in cryopreserved trout hepatocytes freshly isolated hepatocytes in biotransforming most of our were 75 ( 23%, 85 ( 14%, 90 ( 20%, and 82 ( 33%, reference compounds. The average CLint percentage for 4NP respectively. These values suggest that, on average, the was the lowest among the reference compounds with a value cryopreservation process itself (freezing and thawing) may of 72%. Table 1 also shows that the average percent BCF have caused a 10-25% reduction in metabolic enzyme values of the three hepatocyte batches were between 97 and activities, but the enzyme activities were not affected by the 111% for molinate, MK, DTBP, pyrene, and BaP and 126% duration of hepatocyte cryostorage. for 4NP. Intrinsic Clearance and Bioconcentration Factor. The Dry Ice Storage. One of the main goals of cryopreserving capabilities of cryopreserved trout hepatocytes to biotranstrout hepatocytes is to allow shipment to other laboratories form xenobiotics was evaluated subsequently. Our previous to facilitate wide acceptance of this in vitro approach in research involved determining biotransformation rates of bioaccumulation assessment. Therefore, we simulated the molinate, MK, 4NP, DTBP, and BaP in freshly isolated trout conditions required for overnight shipping of cryopreserved hepatocytes and in trout liver S9 and microsomes (2-4). trout hepatocytes on dry ice (Figure 1 for the assay scheme). Thus, we used these same compounds, with the addition of After approximately three months cryostorage period, all pyrene, as our reference compounds in the present study. three hepatocyte batches were left on dry ice for 24 h before Representative depletion curves of the reference combeing thawed and assayed for 4NP, DTBP, and BaP clearance. pounds in freshly isolated trout hepatocytes from batch 2 Figure 5 shows the comparison of the CLint values in trout are included in SI Figure S1. The intrinsic clearance (CLint) values of these compounds were derived from the depletion hepatocytes with and without dry ice storage. It is apparent curves by using either the monoexponential or biexponential that 24-h dry ice storage did not affect the capabilities of decay model (Experimental Section). CLint values that were cryopreserved trout hepatocytes to biotransform 4NP, DTBP, determined in both freshly isolated and cryopreserved trout and BaP. hepatocytes from all three batches are included in SI Table Experimental Variability in the Determination of XeS2. Bioconcentration factor (BCF) values of the reference nobiotic Intrinsic Clearance. Cryopreservation did not compounds were predicted using a kinetic model that significantly affect CLint determination in isolated trout hepatocytes for most of our reference compounds (Figure 4 benefited from the input of CLint (for model details, see ref 2) and are also included in SI Table S2. and SI Table S2). The only compound whose CLint values VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Percentage of intrinsic clearance (CLint, left panel) and BCF (right panel) of reference compounds molinate (A), MK (B), 4NP (C), pyrene (D), DTBP (E), and BaP (F) in cryopreserved trout hepatocytes. Trout hepatocytes were isolated in three different batches. The CLint and BCF values obtained from freshly isolated hepatocytes of each respective batch are considered 100% (dotted line). Pyrene data are represented from batch 1 and 2 hepatocytes only. CLint value for pyrene in freshly isolated trout hepatocytes from batch 3 was not determined due to the high noise level in the depletion curves. were consistently lower after cryopreservation was 4NP. The average CLint value for 4NP in cryopreserved trout hepatocytes was 28% lower than that determined in freshly isolated trout hepatocytes, which translated to a 26% higher BCF value if predicted using cryopreserved trout hepatocytes (Table 1). In order to evaluate if the 28% lower CLint value would have any significant consequence in the prediction of BCF, one needs to understand the types and levels of variability of this in vitro hepatocyte-based approach. Values of the coefficient of variation (CV) to represent intra-assay, interassay, and interbatch variability are provided in SI Table S3. In general, we observed that replicates in one assay (intra-assay) would generate a 1-10% CV; repeats of multiple assays using the same batch of isolated trout hepatocytes (interassay) would have CVs’ of approximately 10-20%; and repeats using 3056
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different batches of isolated trout hepatocytes (interbatch) would have a 30-50% CV which is consistent with the findings from our previous study (3). Therefore, a 28% lower CLint due to cryopreservation would not generate a significant impact in BCF prediction, considering the inherent variability associated with the in vitro clearance assay. In addition, one could argue that a BCF prediction by incorporating conservative biotransformation rate information would still be superior to a BCF estimation that does not consider metabolism at all. Comparison to Trout Liver S9 and Microsomes. Previously, we demonstrated that the CLint values of a similar set of reference compounds, if determined in isolated trout liver S9 and microsomes, were only 6.3-22.4% of those determined in freshly isolated trout hepatocytes (4). Our results in the
TABLE 1. Percentage Recovery of CLint and BCF Values of the Reference Compounds in Cryopreserved Trout Hepatocytes compounds
logKOWa
CLint (%)b
BCF (%)b
molinate MK 4NP pyrene DTBP BaP
2.90 3.87 4.48 4.93 5.19 6.13
106 ( 14 101 ( 15 72 ( 11 106 ( 13 94 ( 14 93 ( 24
100 ( 1 100 ( 7 126 ( 12 97 ( 9 106 ( 12 111 ( 28
a
LogKOW values were obtained from a previous report (2) for molinate, MK, 4NP, DTBP, and BaP, or calculated using EPI Suite software KOWWIN version 1.67 for pyrene. b Percentage values were averaged from all three batches of trout hepatocytes and from all time points due to the observation that there was not a cryostorage duration-dependent trend (Figure 4).
limited to 90 days in the present study, the fact that we did not observe any time-dependent loss of enzyme activities suggests trout hepatocytes could be cryopreserved for a much longer period of time. This assumption is supported by studies of cryopreserved human hepatocytes in which human hepatocytes have been cryopreserved for years without losing viability or cellular functions (9, 10, 31, 35).
Acknowledgments We thank Alan Samel, Barbra Ferrell, and Jeff Rivenbark for their general assistance with fish maintenance during the course of this study and Dr. Robert Hoke for commenting on the manuscript.
Supporting Information Available Three tables and one figure. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited
FIGURE 5. Comparison of intrinsic clearance (CLint) values of 4NP (A), DTBP (B), and BaP (C) in trout hepatocytes before dry ice storage (filled bars, averaged from all time points with standard deviations) to those obtained in trout hepatocytes that have been stored in dry ice for 24 h (open bars). present study suggest that cryopreserved trout hepatocytes would be superior to trout liver subcellular fractions in predicting bioaccumulation potential of xenobiotics.
Summary It has been repeatedly shown that cryopreservation of isolated hepatocytes from mammalian species is a practical, viable approach to maintain cellular functions of the hepatocytes (9, 10, 12, 30, 34-36). Our present study has extended the previous work to rainbow trout hepatocytes and has successfully demonstrated the acceptable performance of cryopreserved trout hepatocytes in their application in bioaccumulation assessment. Even though the test duration was
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