ToxSci Advance Access originally published online on May 21, 2007
Toxicological Sciences 2007 99(1):35-42; doi:10.1093/toxsci/kfm129
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Spatial Distribution of CYP2B1/2 Messenger RNA within the Rat Liver Acinus following Exposure to the Inducers Phenobarbital and Dieldrin
Center for Environmental Health Sciences and Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762
1 To whom correspondence should be addressed at Center for Environmental Health Sciences, College of Veterinary Medicine, Wise Center, Mississippi State University, PO Box 6100, Mississippi State, MS 39762-6100. Fax: (662) 325-1031. E-mail: chambers{at}cvm.msstate.edu.
Received May 15, 2007; accepted May 16, 2007
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ABSTRACT |
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Traditionally, the liver has been considered a homogeneous organ, but literature suggests that the cytochromes P450 are differentially distributed among the hepatocytes and that the pattern of this distribution is altered by various xenobiotics. In this study, the CYP2B1/2 messenger RNA (mRNA) in the hepatocytes was compared following treatment of rats with either of two inducers, phenobarbital (PB), or dieldrin. Adult male Sprague–Dawley–derived rats were treated with either ip PB in saline at 80 mg/kg/day for 5 days or dieldrin in corn oil by oral gavage at 2.5 mg/kg/day for 13 days. Control rats received ip saline or po corn oil for the same time. Laser capture microdissection (LCM) followed by duplex quantitative real-time reverse transcriptase PCR was used to measure the CYP2B1/2 mRNA produced in bands of hepatocytes isolated from three locations along the sinusoidal path. The amounts of mRNA present in whole liver subsamples were also analyzed. CYP2B1/2 enzyme activity was determined by assaying 16ß-hydroxytestosterone formation. Whole liver mRNA samples exhibited significant induction in CYP2B1/2 transcript levels: sixfold for PB and 2200-fold for dieldrin. All the LCM band samples also showed significant fold induction in CYP2B1/2 mRNA compared to controls. Dieldrin caused marked increases in CYP2B1/2 mRNA levels in the direction of blood flow through the acinus: periportal, 300-fold; midzonal, 600-fold; and centrilobular, 1700-fold. A different pattern of induction was observed in the PB-treated rats: periportal, 1800-fold; midzonal, 8800-fold; and centrilobular, 1600-fold. The present study indicates the differences in spatial responses that can be exhibited within the liver following exposure to various xenobiotics. It also indicates the importance of examining xenobiotic metabolism in the liver in light of its nonhomogeneous, zoned microenvironment.
Key Words: cytochrome P450 induction; CYP2B1/2; dieldrin; phenobarbital; CYP2B1/2 mRNA; spatial distribution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The cytochromes P450 (CYPs) are among the most important of all the xenobiotic metabolizing enzymes, catalyzing a diverse array of oxidative reactions (Parkinson, 2001
). These monooxygenases constitute the most important group of Phase 1 enzymes. Within the CYP family of enzymes, CYP2B1/2 is one of the inducible subfamilies of greatest importance in the metabolism of xenobiotics. It is readily induced by numerous drugs such as the barbiturate phenobarbital (PB) and organochlorine pollutants such as the persistent chlorinated alicyclic insecticide dieldrin (Campbell et al., 1983; Krampl and Hladka, 1975). This induction can lead to numerous downstream effects on the metabolism of environmental contaminants, pesticides, and drugs and may result in an alteration in normal physiological function (Kretschmer and Baldwin, 2005). Ingested xenobiotics enter the liver through the hepatic portal vein. As blood moves through the hepatic sinusoids, some of the xenobiotic molecules can enter the hepatocytes where they can be metabolized by CYP. The metabolites can be transported into the sinusoidal blood and travel to the central vein through which they exit the liver.
Traditionally, the liver has been considered a homogeneous, well-mixed organ in pharmacokinetic studies. Some literature, however, indicates that cytochrome P450 is differentially distributed among the hepatocytes lining the sinusoids of the liver acini (Baron et al., 1981, 1982, 1984; Bühler et al., 1992; Mino et al., 1998; Omiecinski et al., 1990). In livers of naïve animals, the hepatocytes closest to the portal vein (the periportal region) bound less antibody to CYP2B1/2 than those located adjacent to the central vein (the centrilobular region) (Baron et al., 1981, 1984; Bühler et al., 1992; Mino et al., 1998). Thus, theoretical maximal rates of xenobiotic metabolism would be expected to increase through the hepatic acinus periportally to centrilobularly. However, the absolute level of xenobiotic entry into hepatocytes should decrease as its concentration in the sinusoidal blood decreases following xenobiotic absorption by the more proximal (periportal) hepatocytes. Intuitively, this would seem to suggest that the amount of CYP should be highest in the area of greatest substrate concentration, i.e., near the portal vein, in order to more effectively accommodate the higher substrate concentrations. Baron et al. (1984) did report an increase in the level of CYP2B1/2 antibody binding in this area following PB exposure, but also demonstrated an increase in the midzonal area that indicated a CYP2B1/2 level greater than that found in either of the acinal termini. Research with mixtures of PB and 3-methylcholanthrene has shown that the centrilobular expression of CYP2B1/2 is suppressed while midzonal expression is promoted (Mino et al., 1998).
The autoradiographic tracking of parathion metabolites (Nakatsugawa, 1992; Tsuda et al., 1987) suggested a possible explanation for this apparent conundrum. Low doses of parathion were found to be totally absorbed and biotransformed by the periportal hepatocytes. As the dose increased, the parathion unable to be biotransformed by the limited amount of enzymes present in the periportal hepatocytes returned to the sinusoidal space and migrated to the midzonal hepatocytes where it was absorbed again. This "chromatographic migration" (Tsuda et al., 1987) down the acinus while moving in and out of the hepatocytes continued as long as some unmetabolized parathion was present. According to this hypothesis, the centrilobular hepatocytes have the highest metabolic rates and function only when higher doses are encountered.
Differences in xenobiotic concentration would result in different rates of metabolite production in different areas of the liver and point to a deficiency in the tradition of considering only time, and not space, following exposure to xenobiotics. Understanding the spatial variability of CYP2B1/2 expression may also more completely elucidate the causes of region-specific liver damage (Bühler et al., 1992), and may also lead to better predictions of saturation of P450-mediated pathways.
Most of the previous CYP isozyme localization work utilized immunohistochemistry (Baron et al., 1981, 1982, 1984; Bühler et al., 1992; Mino et al., 1998) or in situ hybridization (Omiecinski et al., 1990). The present study determined the spatial distribution of CYP2B1/2 expression in the acinus of rats, exposed to PB or dieldrin, by using duplex quantitative real-time reverse transcriptase PCR (QRT-PCR) to measure messenger RNA (mRNA) levels from acinar bands of cells extracted by laser capture microdissection (LCM). Using LCM coupled to QRT-PCR to compare mRNA abundance is less subjective than immunohistochemistry (Desaulniers et al., 2005) and less labor intensive than in situ hybridization. Others have previously measured CYP2B1/2 mRNA via QRT-PCR but they used simplex QRT-PCR on cultured rat hepatocytes (Burczynski et al., 2001) or precision-cut liver slices (Pan et al., 2002). The lack of an internal standard in simplex QRT-PCR allows for only approximate quantification of large changes, whereas the inclusion of primers and probes for an internal standard, such as 18S ribosomal RNA, in duplex QRT-PCR allows for the accurate determination of much smaller differences (Joyce, 2002). Recently, Cui et al. (2005) utilized duplex QRT-PCR to measure CYP2B1/2 mRNA, but again the authors used precision-cut liver slices and therefore they were not able to determine the changes seen in CYP expression in different areas of the liver acinus. The use of both LCM and duplex QRT-PCR in the present study was designed to determine how CYP2B1/2 mRNA is modulated spatially at the acinar level in response to xenobiotic exposure (i.e., PB and dieldrin) and should help to more accurately describe the kinetics of hepatic xenobiotic metabolism. In addition, in order to confirm whether the mRNA expression data reflected the overall levels of CYP2B1/2 enzyme function, CYP2B1/2 activities on select substrates were also analyzed in the same livers using high-performance liquid chromatography (HPLC) techniques. For an additional comparison, general CYP enzyme activity was also examined using ultra high-performance liquid chromatography (UPLC) and spectrophotometric methods.
PB was selected as a prototypical CYP2B1/2 inducer. Dieldrin, also a CYP inducer (Parkinson, 2001), is representative of the numerous legacy organochlorine insecticides and was selected because it was one of the most extensively used organochlorines in the 1950s and 1960s. It is lipophilic and is very stable to both environmental and metabolic degradation, leading to environmental persistence and bioaccumulation. Recent work in our laboratories confirmed that dieldrin still persists in the soil of highly agricultural regions at about 0.012 ppm (unpublished data), and may cause human exposure since it is efficiently absorbed through the skin (Ecobichon, 2001). Dieldrin is a high priority hazardous waste chemical, ranking 18 out of 275 on the Superfund National Priority List.
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MATERIALS AND METHODS |
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Chemicals.
Dieldrin was a generous gift from Shell Chemical LP (Houston, TX). Methyl parathion (MPS) was a generous gift from the Monsanto Company (St Louis, MO). Chlorpyrifos (CPS) was a generous gift from DowElanco Chemical (Indianapolis, IN). Hydroxyphthalimidyl diethyl phosphate (HPDEP) was provided by Dr Howard Chambers (Department of Entomology and Plant Pathology, Mississippi State University). HPLC grade ethyl acetate and acetonitrile were purchased from Burdick and Jackson (VWR International, West Chester, PA). Optima grade methanol was purchased from Fisher Scientific (Hampton, NH). The 16ß-hydroxytestosterone standard was purchased from Steraloids Inc. (Newport, RI). The 17ß-N,N-diethylcarbamoyl-4-methyl-4-aza-5
-androstan-3-one was a generous gift from Merck and Co., Inc. (Rahway, NJ). All other chemicals were purchased from Sigma Chemical Co (St Louis, MO).
Animal treatment.
Adult male Sprague–Dawley–derived rats were housed in an AAALAC-accredited facility in a temperature-controlled environment (22 ± 2°C) with a 12-h dark–light cycle with lights on between 0700 and 1900 h. LabDiet rodent chow and tap water were freely available. The Mississippi State University Animal Care and Use Committee approved all procedures. The rats were treated ip with PB in saline at 80 mg/kg body weight/day for 5 days as described previously (Ma and Chambers, 1995) or po with dieldrin in corn oil at 2.5 mg/kg body weight/day for 13 days following the method of Krampl and Hladka (1975). The dose of dieldrin selected was within the range described as eliciting subchronic effects by Kolaja et al. (1996). Control rats received ip saline or po corn oil for the same length of time. The number of animals per treatment ranged from 3 to 8.
Tissue processing.
Following sacrifice the livers were removed, weighed, and their gross morphology noted. The lobes were separated and snap frozen in liquid nitrogen and stored at – 80°C. Serum was also collected and stored at – 80°C.
Hepatic enzyme profile in serum.
The standard for the rapid screening of liver health in medical and veterinary settings is the hepatic enzyme panel. A hepatic profile was done on serum from six corn oil treated rats and six rats treated with dieldrin at 2.5 mg/kg/day by the Center for Veterinary Medicine Diagnostic Laboratory at Mississippi State University. The chemistry panel quantified the levels of total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), cholesterol, triglycerides, and creatinine. Student t-test comparisons were made between the corn oil and dieldrin treatment groups.
Total RNA isolation from whole livers.
Subsamples of about 100 mg were dissected from the livers while they were on dry ice and placed into 1.5 ml of RNAse free microcentrifuge tubes. One milliliter of TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) was added to each sample tube followed by homogenization using a Caframo, Ltd. (Wiarton, Ontario) motorized tissue grinder and a Teflon pestle. The rest of the isolation was done according to the protocol supplied with the TRI Reagent.
RNA isolation from LCM samples.
Liver cryosections (8 µm) were cut from frozen (– 80°C) tissues embedded in Jung Tissue Freezing Medium (Leica Microsystems, Inc., Bannockburn, IL). Cryosections were mounted onto aminoalkylsilane-coated, RNAse-free glass slides (Sigma-Aldrich, Inc., St Louis, MO) and stored at – 80°C for no longer than 1 week prior to use. Slides were removed to room temperature and processed for LCM using the Histo Gene Frozen Section Staining Kit as per the manufacturer's protocol (Molecular Devices Corp., Sunnyvale, CA) with the addition of a 30-s xylene step prior to the 5-min xylene step. Three equal-sized, individual bands of about 10 hepatocytes each were excised using a Pix Cell II LCM system with beam settings at 7.5 µm, 70 mW, and 5.0-ms duration as per the manufacturer's instructions (Molecular Devices Corp.). One band of cells was taken from the periportal area, the second from the centrilobular region, and the last was collected from a location equidistant between the first two bands. This resulted in samples from the beginning (periportal), middle (midzonal), and end (centrilobular) of the liver acinus in relationship to the direction of blood flow. Photomicrographs were also taken with the LCM to document the appearance of the liver sections. RNA was isolated from each band with the PicoPure RNA Isolation Kit following the manufacturer's directions (Molecular Devices Corp.).
Primer and probe sets.
Information from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and the CYP450 Homepage (http://drnelson.utmem.edu/CytochromeP450.html) indicated that the amino acid sequences of the two enzymes (CYP2B1: J00719
[GenBank]
and CYP2B2: J00728) are identical for the first 339 bases so the nucleotide sequence reported for this area (Fujii-Kuriyama et al., 1982) was subjected to Blast and Ensembl searches for introns and exons. Using the Beacon Designer 3.0 Program (BioRad, Hercules, CA) a primer/probe set was designed that would give an amplicon of 127 bps and span an intron/exon boundary to prevent the amplification of genomic DNA. The forward primer used for CYP2B1/2 was 5'-GTG GGC CAA GCT GAG GAT TT-3' and the reverse was 5'-TAG CCA GAG AGA ATC GCC GA-3'. The fluorogenic probe was 5'-TTG CCA ATG GGG AAC GCT GGA AGG-3'. The gene for 18S ribosomal RNA (GenBank: X01117
[GenBank]
) was chosen as the internal standard for the duplex quantitative reverse transcriptase PCR (QRT-PCR). The Beacon Designer 3.0 program (BioRad, Hercules, CA) was also used to design a primer/probe set for this gene. Intron/exon boundaries were not relevant, however, for the design of this amplicon (137 bps). The 18S forward primer was 5'-CGT TGA TTA AGT CCC TGC CCT T-3', the reverse primer was 5'-TCA AGT TCG ACC GTC TTC TCA-3', and the fluorogenic probe was 5'-ACA CAC CGC CCG TCG CTA CTA CCG-3'. All primers were synthesized by MWG Biotech (High Point, NC). Probes were synthesized by Sigma Genosys (The Woodlands, TX). Primer, probe, and template concentrations were optimized for 18S and CYP2B1/2 in duplex QRT-PCRs.
Real-time PCR.
Each duplex QRT-PCR was done in triplicate in a final reaction volume of 25 µl with 2.5 µl of template from either a LCM dissected band or a whole liver subsample using the Platinum Quantitative RT-PCR Thermoscript One-Step System (Invitrogen Life Technologies, Carlsbad, CA) following the manufacturer's protocol. The thermal cycler used was the iCycler iQ Real-Time PCR Detection System (BioRad, Hercules, CA). The conditions for the QRT-PCR reactions were 65°C 30 min (reverse transcription), then 95°C 5 min, followed by 40 cycles of 15 s at 95°C and 1 min at 65°C. In all experiments, three wells containing no template were included as negative controls. Standard curves of both the reference gene (18S) and the target gene (CYP2B1/2) were used to calculate the PCR efficiency (dilutions of a template mixture from pure to 1:1000). The threshold cycle values (Ct values) of CYP2B1/2 were normalized to the Ct values of 18S to correct for variations in template amount and pipetting. Following normalization, mean Ct values from each treatment and control group were compared statistically via the Student's t-test. Comparisons were made between control and treated samples within each treatment group for both whole liver and LCM samples. In addition, the LCM band samples were examined for changes in CYP2B1/2 mRNA production in the three regions of the liver acinus. For ease in graphing, mean Ct values were subtracted from the maximum possible Ct value of 40.
Fold differences were determined based on an assumed 100% PCR reaction efficiency as described in Maglich et al. (2002) by subtracting the mean, normalized Ct value for each treatment group from the corresponding value of its control group and raising 2 to the power of this number. Since fold changes compared to control greater than 3 have been reported to be above the signal to noise ratio of the Taqman assay (Cui et al., 2005), absolute values greater than this limit were considered to be significant.
Phosphorothionate desulfuration activity monitored indirectly.
As an index of a CYP-mediated reaction on a xenobiotic substrate relevant to our interests in pesticide toxicology, the desulfuration (bioactivation) of the phosphorothionate insecticides CPS and MPS to their respective anticholinesterase oxons (i.e., chlorpyrifos-oxon [CPXN] and methyl paraoxon [MPXN]) was studied (slight modification of Ma and Chambers, 1994). Our method uses a high concentration of exogenous cholinesterase in a bovine brain preparation present concurrently with the liver microsomes as a trap for the reactive oxon as it is generated during the incubation. The degree of cholinesterase inhibition is subsequently converted to concentration of oxons based on concentration-inhibition curves of CPXN or MPXN on the bovine brain cholinesterase. Hepatic microsomes were prepared and stored at – 80°C as previously described (Forsyth and Chambers, 1989). Briefly, resuspended microsomes were incubated for 30 min with 50µM CPS or MPS at 37°C with an NADPH (nicotinamide adenine dinucleotide phosphate, reduced)–generating system. Cholinesterase activity was subsequently assayed using a modification of the Ellman et al. (1961) technique as previously described in Chambers and Chambers (1989) with acetylthiocholine as the substrate and 5,5'-dithiobis(nitrobenzoic acid) as the chromogen. Desulfuration activities were measured every 3 days during the 2.5 mg/kg/day dieldrin dosing protocol to determine time to peak induction. They were also measured after 5 days in the PB dosed animals. Student t-test comparisons were made between control and treated samples.
CPS desulfuration and dearylation activities monitored directly.
Adapting the method of Tang et al. (2001), the CYP-mediated desulfuration of CPS to CPXN was studied concurrently with the parallel CYP-mediated dearylation (detoxication) of CPS to 3,5,6-trichloro-2-pyridinol (TCP). The assay mixture containing the microsomal suspension and an NADPH-generating system was first incubated for 15 min with 1.0µM HPDEP. HPDEP is a labile carboxylesterase inhibitor which allows the protection of the generated oxon from carboxylesterase-mediated degradation and thereby allows a more accurate quantification of the oxon generated. The reaction was initiated by the addition of 50µM CPS and stopped after 15 min by addition of an equal amount of ice cold methanol amended with 0.6% trichloroacetic acid. The resulting mixture was centrifuged for 5 min and filtered into an HPLC vial. A Waters Acquity UPLC system (Waters, Milford, MA) with a tunable UV detector set at 230 nm and an Acquity UPLC bridged ethylsiloxane/silica hybrid C18 column (1.7 µm, 2.1 x 50 mm) were used during a 4-min linear gradient of 80% (89 water:1 phosphoric acid:10 acetonitrile) and 20% (99 acetonitrile:1 phosphoric acid) to 0:100. At 5 min, the gradient was returned to the starting value and re-equilibrated for 1.5 min. A standard curve was calculated using pure standards of TCP and CPXN. Using the Detect Program, limits of detection of 1.09 µmol/ml for TCP and 3.551 µmol/ml for CPXN were calculated. Extraction efficiency was determined by spiking tissue samples with standards and comparing the recoveries with pure standards. Recoveries ranged from 89% to 92%. Student t-test comparisons were made between control and treated samples.
Hydroxylation of testosterone.
The rate of formation of 16ß-hydroxytestosterone was measured as an index of CYP2B1/2 enzymatic activity (Waxman, 1988; Wood et al., 1983) using a modification of Purdon and Lehman-McKeeman (1997). The assay mixture was incubated for 10 min at 37°C with 250µM testosterone. The reaction was terminated by the addition of ethyl acetate. Thrice the samples were vortexed for 20 s, iced to separate the phases, and the solvent saved. The combined solvent was dried under nitrogen at 45°C, resuspended in 500 µl of 1:1 water and methanol, vortexed for 30 s, and filtered into an autosampler vial. A Waters Alliance HPLC system (Waters, Milford, MA) with ultraviolet detection set at 247 nm and a Supelco (Bellefonte, PA) Supelcosil LC-18 column (reversed phase, 4.6 mm x 150 mm, 3 µm) were used during a 35-min linear gradient run beginning at a ratio of 80 water:10 methanol:10 acetonitrile and ending at 40:50:10. The flow rate was 1 ml/min and the column temperature 40°C. Extraction efficiency was determined by spiking tissue samples with standards and comparing the recoveries with pure standards. Recoveries ranged from 94% to 100%. Student t-test comparisons were made between control and treated samples.
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RESULTS |
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The LCM photomicrographs showed dramatic differences in the liver samples after the different treatments (Fig. 1). The corn oil controls showed no visible sinusoidal spaces and the stained nuclei are clearly visible, which was in contrast to the other three treatment groups. The number of sinusoidal spaces visible in the saline treatment was closest to that seen in the normal architecture. The number of spaces was higher still in the PB group and was greatest in the dieldrin treated animals. In spite of these latter histological changes, no statistically significant differences were observed in the hepatic biochemical panels for the dieldrin treatment group (Table 1).
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The average Ct values for the dieldrin samples showed a statistically significant difference from their corresponding controls in both the whole liver and LCM samples, but in the PB samples only the Ct values at the LCM level were significantly different from corresponding controls. The baseline values for both the saline and corn oil controls are much lower in the LCM samples than in the whole liver subsamples (Fig. 2).
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The whole liver samples showed a statistically significant increase in CYP2B1/2 mRNA expression with a sixfold increase following the PB treatment and a 2200-fold increase in CYP2B1/2 mRNA production after the exposure to dieldrin (Fig. 3).
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Examination of the fold change in CYP2B1/2 mRNA levels for each acinal band following xenobiotic exposure showed regional differences in induction with large statistically significant increases seen throughout the acinus (Fig. 3). Dieldrin caused CYP2B1/2 mRNA values to rise in the direction of blood flow through the acinus from the periportal region (300-fold increase) to the midzonal region (600-fold increase) to the central vein (1700-fold increase). A different pattern was observed in the bands following PB exposure where the largest increase was present in the midzonal area (8800-fold increase) with lower induction in both the periportal area (1800-fold increase) and the region near the central vein (1600-fold increase).
The desulfuration activities toward CPS and MPS measured indirectly were induced after exposure to dieldrin (Table 2). A significant increase in the CYP-mediated bioactivation of CPS was observed as early as 3 days into the dosing regimen and significant amounts of CYP-mediated MPS bioactivation were first observed at 6 days (data not shown). A maximum induction of sevenfold and threefold for desulfuration of CPS and MPS, respectively, was observed on day 13. Direct measurement using HPLC also showed that both PB and dieldrin caused significant increases in the levels of metabolites of both dearylation (TCP) and desulfuration (CPXN) (Table 3). Although the amounts of each metabolite were equal at the end, a greater total increase from the control level was seen for desulfuration than for dearylation with both inducers.
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Following the dieldrin treatment for 13 days, significant increases in 16ß-hydroxytestosterone metabolite production, reflecting CYP2B1/2 activity, were also observed (Table 4). Indeed, CYP2B1/2 activity was dramatically increased from no measurable activity in the control livers to 13,232 nmol 16ß-hydroxytestosterone formed/g tissue/min. Similar induction of testosterone hydroxylation was observed following treatment with 80 mg/kg/day PB.
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DISCUSSION |
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The photomicrographs suggest that the corn oil engorged the hepatocytes with storage byproducts of metabolism in the controls but not in the dieldrin treated livers, even though the pesticide was dissolved in corn oil (Fig. 1). This may suggest that the dieldrin altered the ability of the liver to process fats. The livers of the dieldrin treated rats were larger and much darker in color than the livers of the PB-exposed rats. While the dieldrin sections appeared to be generally less healthy than the PB sections, the hepatic blood biochemistry panel (Table 1) showed no significant differences between the dieldrin and control liver samples. Similar results were reported by Kolaja et al. (1996)
, who reported normal liver enzyme levels in spite of centrilobular hypertrophy in both dieldrin (10 mg/kg diet) and PB (500 mg/kg diet) treated rats.
The sixfold increase in whole liver CYP2B1/2 mRNA levels after PB exposure fell within the wide range reported in the literature. Previously reported CYP2B1 mRNA values, determined via competitive reverse transcriptase PCR, vary from an increase of fivefold (Glöckner et al., 2002) to an increase of 3700-fold (Martignoni et al., 2004). This wide variability is most likely the result of the tremendous variety of methods used as well as the exponential nature and inherent variability of PCR, especially when used in a simplex format. Both in vitro (Burczynski et al., 2001; Cui et al., 2005; Glöckner et al., 2002; Martignoni et al., 2004; Müller et al., 2000; Pan et al., 2002) and in vivo systems (Martignoni et al., 2004; Müller et al., 2000) combined with significant differences in both dosing paradigms and PCR techniques, make direct comparison of fold changes problematic.
Dieldrin yielded a much more dramatic induction of whole liver mRNA levels for CYP2B1/2 than did PB with a 2200-fold increase. This impressive induction supports earlier Northern blot work done by Wei et al. (2002). In their paper dieldrin was shown to be such a potent inducer of mouse CYP2B10 (homologue of rat CYP2B1/2) that 8-week-old pups, which normally have no CYP activity, strongly expressed the gene.
In addition to the mRNA measurements, CYP-mediated reactions were also studied to verify that typical induction of enzyme activities was occurring. Both dieldrin and PB exposure resulted in substantial induction of CYP2B1/2-mediated 16ß-hydroxytestosterone production (Table 4), as well as CYP-mediated desulfuration and dearylation reactions (Tables 2 and 3).
The LCM acinal band work also supports the concept that different chemicals induce in different ways. When CYP2B1/2 mRNA levels were examined in the periportal, midzonal, and centrilobular regions following dieldrin exposure, the pattern of expression was the same as that suggested previously for the livers of naïve rats via immunohistochemistry (Baron et al., 1981, 1984; Bühler et al., 1992; Mino et al., 1998) and in situ mRNA hybridization (Mino et al., 1998). The zonal expression of CYP2B1/2 in hepatocytes apparently increases as the blood carrying the dieldrin flows from the periportal acinal entrance to the centrilobular exit (Fig. 3).
A different pattern of expression was seen in the various acinal regions following PB treatment. In our study (Fig. 3) the pattern is similar to that observed after PB exposure by Baron et al. (1984) where CYP2B1/2 antibody binding was greater in the midzonal area than it was on either of the acinal ends. It should be noted, however, that only a single time point was investigated for each chemical and it is unknown at this point whether the time of maximal mRNA expression was being studied.
The picture of CYP2B1/2 induction derived from the whole liver samples is very different from that determined via LCM with the LCM values more closely mimicking the previous immunohistochemical research (Figs. 2 and 3). A comparison of the data derived from the whole liver samples to that from the acinal bands shows a large amount of variation. Expression was lower in PB-treated whole liver samples than LCM band samples. An opposite trend was seen after dieldrin exposure with higher expression in the whole liver data than the acinal band data. These results again suggest the importance of not treating the liver as a homogeneous "black box," but rather emphasize the importance of carefully examining the microenvironment where zonation affects liver function. The whole liver samples are unknown mixtures of hepatocytes and other cells in various stages of death, birth, and health. By utilizing LCM, a more accurate sampling of viable cells of interest can be selected for examination. LCM has the ability to select only hepatocytes rather than the mixture of cell types that are present in the whole liver subsamples. The lower LCM control (saline and corn oil) Ct values (Fig. 2) also support the idea that LCM gives a more accurate profile. Also the fact that dieldrin exposure caused higher mRNA levels in the whole liver subsamples (Figs. 2 and 3) suggests that the hepatotoxicity of dieldrin may be the result of its effects on liver cells other than hepatocytes which are the sole component of the LCM samples.
In conclusion, the present study indicates the variety of responses that can be exhibited by the liver following exposure to xenobiotics. It also indicates the importance of examining the liver in light of its nonhomogeneous zoned microenvironment and suggests that LCM can be successfully used to do this. The present work also shows that the classical inducer, PB, is a not a general model for all inducers of CYP2B1/2. The present data suggest that care should be taken not to oversimplify liver function or to make broad generalizations about metabolism in modeling and risk assessment.
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FUNDING |
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This study was funded by National Institutes of Health Center for Biomedical Research Excellence (# P20-RR17661); Mississippi State University Center for Environmental Health Sciences.
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ACKNOWLEDGMENTS |
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The authors wish to acknowledge the technical expertise and instruction received from the Life Sciences and Biotechnology Institute, the statistical advice of Dr. Carolyn Boyle, and the histological assistance of Dr. Frank Austin and Dr. Robert Read. The authors also wish to thank Mr. Shane Bennett and Mr. Kevin Black for their fine technical assistance. This paper is Center for Environmental Health Sciences publication # 114 and Mississippi Agricultural and Forestry Experiment Station publication # J-11123.
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REFERENCES |
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