Environ. Sci. Technol. 2010, 44, 1854–1859
Enantioselective Behavior of r-HCH in Mouse and Quail Tissues D A I B I N Y A N G , † X I Q I N G L I , † S H U T A O , * ,† YAQIN WANG,† YONG CHENG,‡ DIYU ZHANG,† AND LONGCHUAN YU‡ Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China, and Neurobiology Laboratory and National Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China
Received October 3, 2009. Revised manuscript received December 16, 2009. Accepted January 26, 2010.
R-HCH (hexachlorocyclohexane) is chiral and can still be detected in almost all environmental media. In this study, the enantioselective behavior of R-HCH in mice (CD1) and quail (Coturnix japonica) was investigated and compared after a single dose of exposure. The primary nerve cell culture was conducted to evaluate the enantioselective metabolic capacity of nerve cells of mouse and quail for R-HCH. In various tissues of the mice and quail, the R-HCH concentrations showed a typical pattern of first-order dynamics after exposure. The enantiomeric fractions (EFs) in nonbrain tissues of mice decreased substantially, indicating continuous depletion of (+)R-HCH in mice. Tissue-specific EF trends in quail and enantioselective degradation of (-)-R-HCH in quail liver were observed. These observations indicated that the dynamic changes of EFs in mice and quail were independent of concentration changes in the same tissues. In brain tissues, the enantioenrichment of (+)-enantiomer was totally independent of their concentrations in blood. The in vitro metabolism of R-HCH in the primary nerve cells were negligible, and the slight EF changes in primary nerve cells demonstrated that metabolism, uptake, and excretion in the brain cells would not lead to the observed dramatic enantioenrichment of (+)-RHCH in the brain tissues of the two animals. The enantioselective transport across the blood-brain barrier was the primary cause for the enantioenrichment of (+)-R-HCH in the brain tissues.
Introduction Hexachlorocyclohexanes (HCHs) are among the most public concerned persistent organic pollutants. They can still be detected in almost all environmental media, including air (1, 2), seawater (3), soil (4), plant (5), animals (6, 7), and even human tissues (8-10). Technical HCH is a mixture of various isomers, of which R-HCH is a sole chiral isomer. HCHs have been found to produce various harmful effects in tissues of animals and induce neurotoxic effects on the nervous system (11-13). It was reported that (+)-R-HCH was more toxic than the (-)-enantiomer to primary rat hepatocyte (14). It * Corresponding author phone and fax: 0086-10-62751938; email:
[email protected]. † Laboratory for Earth Surface Processes. ‡ Neurobiology Laboratory and National Laboratory of Biomembrane and Membrane Biotechnology. 1854
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has been reported that R-HCH can be transported across the blood-brain barrier and accumulate in the brain (15-17). The blood-brain barrier in vertebrates is the main barrier responsible for separating blood from the brain. It acts as a barrier to large compounds between the central nervous system and blood to maintain the functions of the central nervous system. Likewise, this barrier can exclude toxic xenobiotics into the brain from the blood (18, 19). It was generally accepted that the blood-brain barrier plays important roles in the intriguingly different enantioselective distribution of R-HCH in brain tissues from other tissues. For example, enantiomeric ratios (ER: (+)-enantiomer/(-)enantiomer) of R-HCH in brain tissues of harbor seals (Phoca vitulina L.) were above 7.9 (enantiomeric fraction (EF): 0.89). In some cases, only (+)-R-HCH was found in brain tissue, while the ERs were below 2.0 (EF: 0.67) in blubber and liver tissues from the same animals (20, 21). Nearly pure (+)-RHCH was also detected in common Eider duck brain (Somateria mollissima L.) (22, 23). In liver and muscle tissues of double-crested cormorants (Phalacrocorax auritus), the ERs were 1.08-1.24 (EF: 0.52-0.55) while the ERs were above 3.6 (EF: 0.78) in the brain tissues (24). Likewise, the enantioenrichment of (+)-R-HCH was found in rat brain as compared to other tissues (15). The pattern of ERs in different brain sections of humans who died of various diseases was detected. Interestingly, in all five cerebellum samples, the ERs were below 1.0 (EF: 0.5), indicating more abundance of (-)-R-HCH, while the ERs did not have a consistent pattern in white and gray matter; ERs were below 1.0 in two samples and above 1.0 in the other three. Malfunction of the blood-brain barrier was thought to be the reason leading to such a difference (25). At present, there is still a lack of full understanding of the mechanism of enantioselective distribution of chiral persistent organic pollutants in animals. In the present study, mice and quail were selected as test animals to compare the enantioselective biotransformation and distribution of R-HCH in different tissues by in vivo exposure. The primary nerve cell culture was conducted to assess the enantioselective capacities of nerve cells to accumulate and deplete R-HCH enantiomers. The goal of this study is to improve the understanding of the role of the blood-brain barrier in the enantioenrichment of chiral persistent organic pollutants in brain tissues.
Materials and Methods Animals and Reagents. Eighty one male mice (CD1, ca. 30 g each) were purchased from Peking University Health Center. Upon arrival at the laboratory, the mice were divided in groups of 9 and each group was kept in a stainless steel cage. The mice were allowed to acclimate for 1 week and access food and water ad libitum (standard diet provided by Peking University Health Center) until 24 h before the exposure experiment. Eighty one male quail (Coturnix japonica), ca. 120 g each, were purchased from Beijing Deling Quail Farm and were grouped and fed similarly to mice. HCHs (R-HCH, 64.23%) were obtained from China Standard Chemical Centre. R-HCH in HCHs was measured and found to be racemic before exposure. R-HCH and aldrin standards were provided by AccuStandard (New Haven, CT). Pure enantiomer of R-HCH was from Dr. Ehrensrorfer (Augsburd, Germany). In Vivo Exposure Experiment. In the exposure experiment, 9 mice and 9 quail were randomly selected to serve as controls, which were intubated with 0.3 mL of corn oil, while the remaining 72 mice and 72 quail received an oral 10.1021/es9030134
2010 American Chemical Society
Published on Web 02/09/2010
intubation of 0.3 mL of 50 µg mL-1 HCHs in corn oil. The test groups of 9 dosed mice and 9 dosed quail were euthanized at 20, 40, 80, 160, 320, 500, 800, and 1280 min after intubation. Immediately after euthanization, samples of blood, brain, heart, liver, intestine, kidney, muscle, fat, and spleen tissues were collected from each subject. For each group including the control group, tissue samples from three randomly selected mice and three randomly selected quail were composited to form three replicates for each time interval. The composite tissue samples were stored at -18 °C prior to further treatment. Sample Extraction and Cleanup. The tissue samples were freeze-dried, and approximately 1 g of dried sample was homogenized with 5 g of anhydrous sodium sulfate. The mixtures were Soxhlet extracted for 12 h with 100 mL of acetone and dichloromethane (1:1 v/v). Extract cleanup followed the method in ref 26 with minor modification. Briefly, after spiking with 1 mL of dimethyl sulfoxide (DMSO), the extracts were evaporated in a rotary evaporator and passed through a column packed with 0.5 g silica gel (80-100 mesh). The column was eluted with 3 mL of DMSO, and the eluate passed through a second column packed with 8 g of florisil (60-100 mesh, deactivated by adding 15% water). The second column was eluted using 80 mL of hexane. The eluate was concentrated into 1 mL of hexane for GC analysis. The samples of control group were further reduced to 10 µL by a gentle stream of nitrogen to bring the concentrations above the detection limits. Primary Nerve Cell Cultures. The procedure of primary cultures of mouse nerve cells followed the method of Park et al. (27) and Theuns et al. (28). Briefly, the brain tissues of postnatal day 1 mice were digested in trypsin solution (1×, Hyclone) at 37 °C for 20 min, titrated, centrifuged and resuspended. The dissociated nerve cells in Dulbecco’s Modified Eagle Media (DMEM) containing 10% FBS (fetal bovine serum), 2 g L-1 HEPES (N-2-hydroxyethylpiperazineN-ethane-sulphonicacid), 2 g L-1 NaHCO3, and 100 U mL-1 streptomycin and penicillin were plated on 15 tissue culture flasks coated with poly-L-lysine (Sigma). The medium was exchanged with 5 mL of fresh medium containing 40 ng mL-1 HCHs and 60 ng mL-1 o,p’-DDT after the primary nerve cells were cultured for 4 days. The number of nerve cells was approximately 120 000 cells per flask. Since the evaporation of R-HCH and water during the culture period could not be avoided, a control culture with the same medium but without the primary nerve cells was performed simultaneously. Three flasks of primary mouse nerve cells and three control flasks were sampled on days 1, 2, 3, 5, 7 after exposure. After sampling, the media were collected and the nerve cells were washed three times with 2 mL of fresh DMEM. The sampled medium together with the rinse solution were combined and extracted three times with 30 mL of dichloromethane each time. The extractants were combined and concentrated into 1 mL of hexane by rotary evaporation. The nerve cells at the bottom of the flasks were extracted twice with 2 mL of acetonitrile in a sonicator. Primary quail nerve cells were taken from 7 day quail embryos from the same supplier. The dissociation, culture, and sampling methods were the same as those for mouse nerve cells. The capacity of the primary nerve cells to degrade the chlorinated pesticides was checked by Trypan Blue (Chroma) staining and DDT spiking experiments. It was demonstrated that the Trypan Blue added to the primary culture system on the day of exposure was excluded by the nerve cell. Approximately 14.4% and 49.6% of o,p’-DDT spiked in 5 mL of medium containing primary mouse or quail nerve cells were degraded within 7 days, respectively, while the loss was not observed in the control medium without primary nerve cells. Sample Analysis. The cleaned samples were analyzed on an Agilent 6890 Plus GC with 63Ni-electron capture detector
(ECD) with nitrogen as carrier gas. The achiral analysis was performed using a DB-5 column (30 m, 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific). Samples were injected in splitless mode. The oven temperature was held at 50 °C for 1 min, ramped to 250 at 15 °C/min, and held for 20 min. The injector and detector temperature were 220 and 280 °C, respectively. The chiral analysis was carried out using a Chiraldex G-PT column (12 m, 0.25 mm i.d., Astec, Whippany, NJ) tandemed with a shortened column DB-5 (15 m, 0.25 mm i. d., 0.25 µm film thickness; J&W Scientific). The samples were injected in split mode with a split ratio of 10:1 (5:1 for control samples), and the oven temperature was set at 160 °C. The injector and detector temperatures were 220 and 280 °C, respectively. Both EF and ER (EF ) ER/(1 + ER)) were used in the paper for easy comparison between our results and those reported in the literature. Quality Control. The homogenized samples (with anhydrous sodium sulfate) of the same tissues collected at different time points were processed as a single batch. One blank was included in each batch. The R-HCH levels in control samples were all below 5% of the lowest concentrations of the samples. Therefore, no blank corrections were made. The samples were spiked with aldrin (1 mg kg-1, 0.2 mL) as internal standard. The average recovery was 78.5 ( 3.1%. To ensure correct identification of R-HCH, selected samples were analyzed using Agilent 6890 GC/5973 Mass Spectroscopy with the same column and oven temperature program to confirm R-HCH peaks detected on GC/ECD. The EF of racemic R-HCH standard was 0.499 ( 0.002 (n ) 3). Data Analysis. Statistical analysis was conducted using SPSS 10.0 (SPSS Inc., Chicago). The mean concentrations or EF values in a given tissue were compared by one-way ANOVA at R ) 0.05.
Results and Discussion Accumulation Dynamics of r-HCH in Mouse and Quail Tissues. Concentrations of R-HCH in intestine, liver, blood, kidney, muscle, brain, and fat tissues from mice and quail were measured at various time intervals after a single dose exposure. Figure 1 shows the typical examples of R-HCH in blood, liver, and brain tissues while other data are presented in the Supporting Information, Figures S1and S2. Similar patterns of R-HCH concentrations were observed in other tissues. The R-HCH concentrations increased sharply immediately after exposure, reached maxima, and decreased gradually afterward, showing a typical pattern of first-order dynamics. As expected, R-HCH in blood reached peak values quickly at 40 min for mice and 50 min for quail, respectively, followed by those in intestine and liver. However, the maximum concentrations in brain tissues appeared at different times for the two animals. The concentration in quail brain reached the peak value at almost the same time when the maximum was observed in blood, while the peak in mice brain appeared around 500 min, which was much later than the peaking time in mouse blood. The potentials of the brain tissue of the two animals for accumulating R-HCH were evaluated by calculating the ratios of peak concentrations in brain over blood. The average ratios were 77.8 and 44.4 for quail and mice, respectively. Although they were both greater than 1, they were significantly different statistically (t-test, R ) 0.05). This result indicates that quail had twice as strong a potential to accumulate R-HCH in brain than mice. Strong accumulation of R-HCH in brain tissues was also observed in rats (15, 29). It was generally accepted that the abundances of pollutants accumulated in vertebrate brains were under the control of the blood-brain barrier (30). Enantioenrichment of r-HCH in Mouse and Quail Tissues. On the basis of the measured concentrations of (+)and (-)-enantiomers of R-HCH in tissues, EF values were VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. r-HCH concentrations in blood (left), liver (middle), and brain (right) tissues of mice (top) and quail (bottom) after single-dose exposure of HCHs (15 µg of HCHs/mouse, 15 µg of HCHs/quail. HCHs: r-HCH 64.2%). The results are presented as means and standard deviations (n ) 3).
FIGURE 2. EFs of r-HCH in the blood (left), liver (middle), and brain (right) of mice (top) and quail (bottom) after a single dose exposure to HCHs containing racemic r-HCH (EF ) 0.50, the dash line). The results are presented as means and standard deviations (n ) 3). calculated. The EFs in blood, liver, and brain are presented in Figure 2, whereas EFs in other tissues are provided in the Supporting Information, Figures S3 and S4. For mice, EF background values in blood of the controls were racemic. Nearly racemic storage of R-HCH in male Sprague-Dawley rat blood was also reported in the literature (15). For intestine, spleen, muscle, fat, and liver of the control mice, the measured EFs were 0.48 ( 0.02, 0.49 ( 0.01, 0.45 ( 0.01, 0.45 ( 0.01, and 0.46 ( 0.03, respectively, all of which were significantly lower than 0.5 (t-test, R ) 0.05). Enantioenrichment of (-)-R-HCH was also reported in liver tissues of other mammals including roe deer, sheep, and pig, which was explained as the result of the enantioselective enzymatic degradation (15, 23). After exposure, the EFs in all mouse nonbrain tissues decreased substantially (Figure 2 and Figure S3 in the Supporting Information) to a range between 0.17 (liver) and 0.31 (fat) over the course of the experiment, indicating continuously enantioselective depletion of (+)R-HCH in mice. However, an opposite trend was observed in brain. The EF value of R-HCH in the control mice was as high as 0.84 and increased even higher up to 0.96 after 1856
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exposure, showing a strong enantioenrichment of (+)-RHCH in brain. Similar enantioenrichment of (+)-R-HCH has also been reported in rat brains (15). Unlike mouse, the enantioenrichment of (()-R-HCH and temporal trends of EFs over the experimental period were different among the nonbrain tissues of quail. Twenty minutes after exposure, EFs in liver, heart, and kidney were 0.72 ( 0.05, 0.54 ( 0.04, and 0.60 ( 0.03, respectively, indicating the enantioenrichment of (+)-R-HCH; while EFs in blood and fat were 0.47 ( 0.00 and 0.45 ( 0.01, respectively, suggesting a slight preference of (-)-R-HCH. Muscle, intestine, and stomach EFs were statistically racemic indicating no enantioenrichment occurred. Similar tissue specificity in EFs was also observed in male Sprague-Dawley rats (15) and was believed to be the result of different physiological properties of various tissues (31). Forty minutes after exposure, EFs in quail nonbrain tissues were well above 0.5, which resulted from the enantioselective biotransformation that occurred in quail and (-)-R-HCH was enantioselectively depleted. In addition, dominant enantiomer was always reported to be (+)-R-HCH in other
FIGURE 3. Dynamic changes of the r-HCH quantities (nanograms) in the medium without (control, left) or with (middle) primary mouse nerve cells and the separated primary nerve cells alone (right) after exposure to HCHs containing 128.5 ng of r-HCH. The curves in the left and middle plots are first-order kinetic fitting curves. birds including common Eider duck (32), double-crested cormorant (24), and seabirds in northern Baffin Bay (33). In quail liver tissues, EFs were always greater than 0.5 and greater than EFs in the other tissues. It appears that liver tissues were the primary enantioselective biotransformation site for quail and the primary force driving the change of EF in other nonbrain tissues. Over the course of the experiment, EF values and the dynamic variations of EFs were different among nonbrain tissues of quail. EFs in quail blood, fat, and intestine tissues increased over time until they leveled off at 500 min. In liver, however, the EFs decreased from 0.72 at 20 min to 0.58 at 40 min and then increased gradually over time and finally reached 0.78 at the end of the experiment. In muscle tissues, the EFs first increased with time, reached peak value at 500 min, and then declined gradually (Figure S4 in the Supporting Information). The difference in enantioenrichment of R-HCH enantiomers in the nonbrain tissues in the mice and quail suggested that mice and quail have different enzymatic systems to degrade the two R-HCH enantiomers. With similar temporal trends of concentration, the kinetics of EF was drastically different in mouse and quail tissues. It appears that the dynamic change of EF of chiral pollutants in these animals was independent of the concentration changes. On the other hand, in brain, the patterns of the enantioselective biological processes were much different from those of their nonbrain tissues. Considerable enantioenrichment of (+)-R-HCH occurred in brain cells of both mice and quail, though the enantioenrichment of (()-R-HCH in blood of the two animals were in opposite directions. Obviously, the blood-brain barriers of both mice and quail worked effectively. In addition to EF values in brain, the dynamic changes in the concentrations of individual enantiomers in brain and blood provided further information on how the EF values changed in brain tissues. The result of a simple calculation of R-HCH mass in mouse tissues, based on the R-HCH concentration and mouse tissue mass, demonstrated that the high EF (0.843) in mouse brain was likely resulted from the accumulation of (+)-R-HCH, rather than the depletion of (-)-R-HCH, even though the mouse blood was (-)-R-HCH dominated. In quail, although there were excesses of (+)-R-HCH in all tissues, the increase of (+)-R-HCH in brain was much faster than the increases in other tissues including blood. EF values in quail brain quickly increased to 0.981 within a period of 160 min. Obviously, the enantioenrichment of R-HCH enantiomers in brain was totally independent of their concentrations in blood. To this stage of discussion, the possible reasons for the enantioenrichment of R-HCH enantiomers in brain tissues were (1) enantioselective transport of (+)-R-HCH across the bloodbrain barrier (18, 19) and/or (2) the rapid enantioselective metabolism of (-)-R-HCH in brain tissues. In the following
section, we provide further evidence to explain the observed EF behavior in brain. Metabolism of r-HCH in Primary Nerve Cells of Mice and Quail. The possible metabolism and transport of R-HCH in brain tissues of mice and quail were examined on the basis of primary cultured nerve cells treated with R-HCH. During the culture period, the medium volume decreased from 5.0 to 4.4 mL. The concentrations of R-HCH in the medium and primary nerve cells were monitored daily for a period of 7 days. Because of the different volatility of R-HCH from that of water, the results are presented as the total quantities, instead of concentrations of R-HCH. Figure 3 shows temporal variations of R-HCH in the culture systems. Because of its volatility, R-HCH evaporated at a relatively steady rate, leading to gradual decreases in the quantities of R-HCH in the control medium (Figure 3, left). However, there appeared to be no difference in the loss rates of R-HCH between the control and the primary nerve cell culture systems of the two animals. The total quantities of R-HCH decreased initially from 129 ng to 12.2 ( 3.0 ng (day 7) in the control and from 107 ( 1.9 ng (day 1) to 12.2 ( 3.0 ng (day 7) in the medium with primary mouse nerve cells (quail: from 105 ( 2.0 ng to 13.1 ( 1.0 ng). For the three culture systems, the loss rates were calculated by fitting the data to first-order kinetics. The calculated loss rate constants were 0.34 ( 0.04 ng/d (control), 0.32 ( 0.01 ng/d (mice), and 0.35 ( 0.01 ng/d (quail), respectively. Statistically, the calculated rates were not significant (p > 0.05), indicating that the metabolism of R-HCH in the primary nerve cells were negligible. The quantity of R-HCH in the medium (Figure 3, middle) was much greater than that in the primary nerve cells (Figure 3, right), indicating that only small fractions of R-HCH entered the nerve cells. However, the concentrations (w/v) of R-HCH in the nerve cells were much higher than in the medium. For instance, the total volume of primary mouse nerve cells on the fifth day after exposure was ca. 9 µL (determined by measuring the sedimentation volume of cells after centrifugation). The concentration in medium at the same time was 7.26 ng/mL, whereas the concentration in the cells was much higher (148.9 ng/mL). This result demonstrates that the primary nerve cells have strong capacity to accumulate R-HCH, consistent with the high concentrations of R-HCH observed in brain after in vivo exposure. The EFs of R-HCH in the medium and the primary nerve cells were also measured (Table 1). Although some measured EFs seem to be slightly higher than the EF of the control medium without the primary nerve cells (0.498 ( 0.001), they were not statistically different at p > 0.05 (t-test). Further analysis using a paired t-test indicated that there were statistically significant (p < 0.05) enrichment of (+)-R-HCH in both medium and the isolated nerve cells for mice, VOL. 44, NO. 5, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Temporal Variations in EFs of r-HCH in the Culture Systems for Primary Mouse and Quail Nerve Cells in 7 Days after Exposure to HCHs Containing Racemic r-HCH mice
quail
time (day)
medium
cell
medium
cell
1 2 3 5 7 control
0.501 0.505 0.506 0.509 0.508 0.498
0.510 0.516 0.506 0.511 0.517
0.504 0.503 0.503 0.507 0.514
0.499 0.495 0.497 0.494 0.500
particularly the latter. However, such a slight change could not account for the dramatic increase in the EF values observed in mouse brain (Figure 2). Negligible metabolism and slight enantioenrichment of (+)-R-HCH unequivocally revealed that metabolism, uptake, and excretion in the brain cells themselves would not have led to the observed enantioenrichment of (+)-R-HCH in the brain tissues of both mice and quail. Therefore, the enantioselective transport across the blood-brain barrier should be the predominant process for such enantioenrichment. Those experiments have demonstrated that mice and quail have different enantioselective biotransformation of R-HCH. Although R-HCH had similar concentration trends in mice and quail, the EF trends were different. Interestingly, EF trends in brain tissues of both animals were quite different from those in other tissues. In the brain tissues of both animals, substantial enantioenrichment of (+)-R-HCH was observed, primarily due to enantioselective accumulation rather than depletion. The results of the primary nerve cell culture showed that there was no significant metabolism or enantioenrichment induced by nerve cells in the brain tissues, indicating that the enantioselective transport of (()-R-HCH across the blood-brain barrier is the primary cause for the enantioenrichment of R-HCH enantiomers in the brain tissues.
Acknowledgments The funding of this study was provided by the National Science Foundation of China (Grants 140710019001 and 40730737) and National Basic Research Program (2007CB407301). We are grateful to Dr. Jian Peng for his valuable comments.
Supporting Information Available Detailed information on temporal variations of R-HCH concentrations in the other tissues of mice and quail and temporal variations of EF values of R-HCH in the other tissues of mice and quail. This material is available free of charge via the Internet at http://pubs.acs.org.
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