ToxSci Advance Access originally published online on July 24, 2008
Toxicological Sciences 2008 106(1):263-283; doi:10.1093/toxsci/kfn144
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Coordinated Changes in Xenobiotic Metabolizing Enzyme Gene Expression in Aging Male Rats
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* NHEERL/ORD, US EPA, Research Triangle Park, North Carolina 27711
North Carolina Central University, Durham, North Carolina 27707
NHEERL Toxicogenomics Core, US EPA, Research Triangle Park, North Carolina 27711
1 To whom correspondence should be addressed at Chris Corton, Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, 109 T.W. Alexander Dr., MD-B143-06, Research Triangle Park, NC 27711. Fax: (919) 541-0694. E-mail: corton.chris{at}epa.gov.
Received April 25, 2008; accepted July 11, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In order to gain insight into the effects of aging on susceptibility to environmental toxins, we characterized the expression of xenobiotic metabolizing enzymes (XMEs) from the livers of male F344 and Brown Norway (BN) rats across the adult lifespan. Using full-genome Affymetrix arrays, principal component analysis showed a clear age-dependent separation between young and old animals in both rat strains. Out of 1135 or 1435 genes altered between the old and young groups in the F344 or BN rats, 7 or 3% were XMEs and included members of the phase I, II, and III classes of genes. There was a 20 or 32% overlap in the gene expression profile between the two strains for F344 or BN, respectively. Lipid, ergosterol, alcohol, and fatty acid metabolism genes were also altered with age in both strains. Some of the genes altered by age exhibited a gender-dependent expression pattern in young adult rats, suggesting an increasingly feminized pattern of gene expression with age in male rats. To examine transcriptional responses across lifespan after challenge with a xenobiotic compound, BN rats were exposed to toluene by oral gavage. Toluene exposure decreased the expression of glutathione synthetase, and dramatically increased the number of phase III genes being downregulated. The expression of CYP2B2 and glutathione-S-transferase decreased with age but increased in all age groups after toluene exposure. Decreased ability to detoxify and transport chemicals out of the body with age could result in increased susceptibility to some classes of chemicals in the aging population.
Key Words: xenobiotic metabolism; aging; liver; gene expression; susceptibility; microarrays.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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In the United States, more than 12% of the population is over the age of 65, and estimates indicate that this percentage will increase to nearly 20% by the year 2030 (He et al., 2005
). Exposures to environmental toxicants in older adults may be similar to those experienced by the general population, but there are a number of pharmacokinetic changes associated with aging that may increase susceptibility in this segment of the population (Geller and Zenick, 2005). This paper examines genomic indicators of changes in hepatic xenobiotic metabolizing enzymes (XMEs) in two strains of rats as a step toward defining potential mediators of susceptibility and identifying specific environmental compounds targeted by those XMEs.
The increased sensitivity of older adults to drugs is well known and a product of both pharmacokinetic and pharmacodynamic changes with aging (McLean and Le Couteur, 2004). Slower elimination of drugs in older adults compared with younger adults has been clearly demonstrated, and age-related differences in drug and toxicant responsiveness have been shown to be due to altered absorption, distribution, metabolism, and excretion (ADME) (Kinirons and O'Mahony, 2004). Many factors have been suggested as causes of age-dependent changes such as decreased blood flow to the liver, decreased liver mass, and decreased content of specific cytochrome P450s (CYPs), the combination of which could result in decreased hepatic clearance of chemicals in older adults (Ginsberg et al., 2005). For example, decreases in the metabolism of analgesics and anti-inflammatory, cardiovascular, and psychoactive drugs are due in part to a 30–40% decrease in drug clearance (Gurwitz, 2005). In addition to alterations in drug metabolism, some segments of the aging population may also be more sensitive to environmentally relevant chemicals compared with younger adults or children because of alterations in pharmacokinetic parameters (Birnbaum, 1991).
Detoxification and elimination of xenobiotics are major functions of the liver and are important in maintaining metabolic homeostasis. Xenobiotics are metabolized by a large number of XMEs which fall into three broad categories. Phase I enzymes are mainly monooxygenases that convert hydrophobic xenobiotics into hydrophilic molecules and include CYP family members, alcohol and aldehyde dehydrogenases, and amine oxidases. Phase II enzymes convert the products of phase I metabolism into amphiphilic anionic conjugates that are water soluble and include glutathione transferases, uridine diphosphate-glucuronosyltransferases, and sulfotransferases. Phase III transporters export conjugated xenobiotics out of the liver and include ATP-binding cassette subfamily members, organic anion and cation transporters, and solute carriers (Francis et al., 2003). A large number of genetic and biochemical studies have shown that expression and activity of individual XMEs in part determines the metabolic fate and toxicity of xenobiotics (Bleasby et al., 2006; Kohle and Bock, 2007).
Expression of many XMEs in mammals is regulated by a set of ligand-activated transcription factors (TFs). These include the nuclear receptors constitutive androstane/activated receptor (CAR), pregnane X receptor (PXR), and to a lesser extent, the peroxisome proliferator–activated receptor (PPAR-
). Overlapping sets of XMEs are also regulated by other TFs including the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2 p45–related factor (Nrf2), which contain helix-loop-helix-per/arnt/sim and cap-N-collar DNA binding domains, respectively. AhR regulates the expression of CYP1A1/2, CYP1B1, UGT1A1/6, and ABCG2 (Nakata et al., 2006). CAR activation increases the expression of CYP2A6, CYP2B1/6, CYP2C9/19, UGT1A1, ABCC2/3/4 (Nakata et al., 2006). Nrf2 regulates the expression of Phase II enzymes (
-GCS, GST, NQO1, UGT, HO-1) and Phase III transporters (ABCC2/3) (Nakata et al., 2006). PPAR- mainly regulates the expression of fatty acid metabolizing enzymes, CYP4A1 and CYP4A3, as well as UGT1A9, UGT2B4, ABCA1, ABCC2, and ABCD2/3 (Nakata et al., 2006). PXR is a key regulator of CYP3A4, responsible for metabolism and clearance of over 50% of clinically prescribed drugs (Guengerich, 1999). PXR also regulates a large number of Phase I (CYP1A2, CYP2B6, CYP2C9/19, CYP3A7, CYP7A1), Phase II (SULT2A1, UGT1A1/3/4), and Phase III (ABCA1, ABCB1/11, ABCC1/2/3, ABCG2) genes. The network of genes under control of these factors can also include those that determine hepatocyte fate. For the AhR, PPAR-, and CAR there is a clear relationship between activation of the receptor by a wide variety of environmental chemicals and the induction of liver cancer through increases in oxidative stress and alteration in hepatocyte fate (Klaunig et al., 2003; Walisser et al., 2005; Yamazaki et al., 2005).
Constitutive expression of many XMEs is determined by the gender-dependent pattern of growth hormone (GH) secretion (Waxman and O'Connor, 2006). GH regulates transcription of gender-dependent CYP genes, including members of the CYP2A, CYP2C, and CYP3A families. Up to 500-fold differences between males and females in CYP expression have been observed in rats and mice. Differences between genders were observed in human samples, but the magnitudes were much smaller (Waxman and O'Connor, 2006). A number of specific XMEs have been investigated for gender differences in rats including CYP2C11, which is strongly induced at puberty in male but not female rat liver, and CYP2C12 which is only expressed in the female rat liver. In addition to CYP family members, sulfotransferases (Klaassen et al., 1998) and glutathione-S-transferases (Srivastava and Waxman, 1993) are also sex-dependent and regulated by GH. The relationship between the expression of XMEs, age and gender-specificity has not been comprehensively examined.
Age-associated changes in gene expression of XMEs in the liver have been found in both rats and humans. In the aged male rat liver, the gene expression of CYP2C7, CYP2J3, and CYP3A1 increased by 13-, 4-, and 4-fold, respectively (Thomas et al., 2002). In their comparison between 32- and 84-week-old F344 rats, Mori et al. (2007) determined changes in the transcript profiles of the liver and found that 21 phase I and 13 phase II enzymes exhibited age-dependent differences in expression. They did not report changes in phase III genes. In the aged human liver, CYP1A1, CYP1A2, and CYP2C18 increased by nine-, five-, and threefold, respectively (Thomas et al., 2002). Further information is necessary to determine the global changes in expression of XMEs with age in the rodent liver.
There is some evidence that age impacts the metabolism and toxicity of aromatic hydrocarbons (Sukhodub and Padalko, 1999) such as toluene. Toluene is produced in the process of making gasoline and other fuels from crude oil and making coke from coal. It is widely used in commercial and industrial applications, is a solvent in paints, lacquers, thinners, glues, and nail polish remover, and is used in printing and leather tanning processes. The main route of human exposure is inhalation of vapor, but exposure can also occur by ingesting the liquid or via skin contact. Toluene depresses neuronal activity and reversibly enhances gamma-aminobutyric acid A receptor-mediated synaptic currents and 1-glycine receptor-activated ion channel function (Beckstead et al., 2000). Toluene also inhibits glutamatergic neurotransmission via N-methyl D-aspartate receptors and alters dopaminergic transmission (Cruz et al., 1998). This chemical is well-absorbed following oral ingestion and rapidly absorbed following inhalation. High concentrations are found in the liver, kidney, brain and blood. Toluene cannot exit the body via urine, feces or sweat because of its low water solubility, and therefore it must be metabolized for excretion. Ninety five percent of toluene is metabolized to benzyl alcohol by CYP2E1 and the rest is metabolized to benzaldehyde, o-cresol, and p-cresol by CYP2B6, CYP2C8, and CYP1A1/2 (Chapman et al., 1990; Hanioka et al., 1995; Nakajima et al., 1997). Benzyl alcohol is further oxidized by the sequential action of alcohol dehydrogenase and aldehyde dehydrogenase to produce benzaldehyde and then benzoic acid (Antti-Poika et al., 1987). Although most toluene metabolites are detoxified by conjugation to glutathione, those remaining may severely damage cells (van Doorn et al., 1981). There is no information about the effects of age in determining the transcriptional changes upon toluene exposure.
Despite the understanding of the biological mechanisms of aging and interventions that slow aging, remarkably little is known about the risks to older adults from exposure to toxic chemicals in the environment. The present study was designed to identify age-dependent differences in hepatic expression of phase I, II, and III xenobiotic metabolizing genes in F344 and Brown Norway (BN) rats. We also determined if these age-dependent differences affected toluene-induced changes in hepatic gene expression.
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MATERIALS AND METHODS |
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Animals and study design.
Male F344 rats were obtained from Charles River Laboratory and acclimated for 1 week. Control animals at 6, 11, 18, and 24 months of age were sacrificed using CO2 asphyxiation. Livers were removed and weighed, and sections from the left and median lobes were fixed in formalin, embedded, sectioned, and stained with H&E. The remainder of the liver was cubed and stored at –80°C until RNA isolation.
Male BN rats (4, 12, and 24 months old) (Harlan Laboratory and the National Institute of Aging) were acclimated for one week. Six animals per age and dose group were administered 1.0 g/kg body weight toluene by gavage (Burdick and Jackson Chemical, Muskegon, MI) in corn oil (4 ml/kg body weight) or corn oil alone. The animals were necropsied 4 h after dosing. Livers were removed and weighed, and sections from the left and median lobes were fixed in formalin. The remainder of the liver was cubed and stored at –80°C until RNA isolation. Necropsies started in the morning and were completed within a 3- to 4-h time window. Details of the effects of toluene exposure including toxicity will be published in separate manuscripts. All aspects of these studies were conducted in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care-International and approved by the United States Environmental Protection Agency/National Health and Environmental Effects Research Laboratory Institutional Animal Care and Use Committee.
RNA isolation.
Liver tissue for RNA isolation was selected based on minimal histological findings to eliminate incidental changes in gene expression unrelated to hepatocyte aging. Total RNA was isolated from rat livers according to the TriReagent procedure (Molecular Research Center, Cincinnati, OH) and purified using the Qiagen RNeasy mini RNA cleanup protocol (Qiagen, Valencia, CA). The integrity of each RNA sample was determined using an Agilent 2100 Bioanalyzer (Agilent, Foster City, CA), and RNA quantity was determined using a Nanodrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE).
Microarray hybridizations.
Liver gene expression analysis was performed according to the Affymetrix recommended protocol using Affymetrix Rat Genome 230 2.0 GeneChips containing probes for over 28,000 well-annotated genes. Total RNA (5 µg per sample) was labeled using the Affymetrix One-Cycle cDNA Synthesis protocol and hybridized to Affymetrix Rat 230 2.0 arrays as described by the manufacturer (Affymetrix, Santa Clara, CA). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After 16 h of hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450 according to the Affymetrix-recommended protocol. Arrays were scanned on an Affymetrix GeneChip scanner. Four F344 and three BN rats per age/dose group were examined. A detailed description of the microarray experiment is available through Gene Expression Omnibus at the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/geo/, as accession number GSE11097.
Analyses of microarray data.
Differentially expressed genes (DEGs) were identified using the following algorithm: background correction was performed using MAS 5.0 followed by a quantile normalization (robust multiarray analysis [RMA]), perfect match adjustment (MAS 5.0), median polish (RMA), LOESS normalization and Cyber-T statistics. This algorithm was adapted from Choe et al. (2005) because these procedures detect DEGs while minimizing the false discovery rate (FDR). A Benjamini–Hochberg multiple test correction (MTC) with p-value 
0.05 was applied during a global analysis of F344 rats. A fold change of ± 1.5 and a p-value 0.05 was used as the cutoff. Hierarchical clustering was performed using CLUSTER and visualized with TREEVIEW (Eisen et al., 1998).
The genes altered by aging in the present study were compared with those that are differentially expressed between male and female rats identified in a recent study examining baseline gene expression in the liver (Boedigheimer et al., 2008). Pathway and global function analyses were performed using Ingenuity (Mountain View, CA) and GATHER (Gene Annotation Tool to Help Explain Relationships) (Chang and Nevins, 2006). Gene Set Enrichment Analysis (GSEA; http://www.broad.mit.edu/gsea/) was used to evaluate whether a predefined set of genes shows statistically significant, concordant differences between two biological states (Subramanian et al., 2005). Expression profiles for combined young and old F344 and BN rats were submitted to GSEA using default settings and searched for enriched gene sets among C2 and C3 gene sets. Gene set C2 (curated gene sets) includes genes from online pathway databases, publications in PubMed, and knowledge from domain experts. Gene set C3 (motif gene sets) contains genes that share a TF binding site defined in TRANSFAC, a database for TFs and their genomic binding sites. TF analyses were run using Bibliosphere in Genomatix (Munich, Germany) to identify enriched binding sites in the promoters of genes significantly altered in our studies.
Real-time polymerase chain reaction.
Confirmation of gene expression was performed using the TaqMan procedure (Applied Biosystems, Foster City, CA). Nine XME genes were examined to confirm microarray results in F344 control rats, and five XME genes were examined to confirm results in the BN control and treated animals. Table 1 lists the TaqMan primers used. RNA was reverse transcribed into first-strand cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the AB protocol. RNA and 2X RT master mix containing random primers, dNTP mixture, and Multiscribe RT enzyme were placed in individual tubes. Tubes were incubated for 10 min at 25°C, 120 min at 37°C, and 85°C for 5 s in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). cDNA template and PCR Reaction Mix containing TaqMan Gene Expression Assay (20x) and TaqMan 2x Universal PCR Master Mix were prepared according to the TaqMan Gene Expression Assay Protocol (Applied Biosystems, Foster City, CA). Reaction volumes of 20 µl for each well on a 384-well reaction plate were used and run in duplicate.
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PCR reactions were performed in a Perkin-Elmer ABI PRISM 7700 Sequence Detection System (Perkin-Elmer Inc., Waltham, MA). Amplification was carried out following the standard run conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Manual threshold values were used and expression of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In aging rats, HPRT has been found to be the most stable housekeeping gene followed by GAPDH (Chen et al., 2006
). We tested hypoxanthine-guanine phosphoribosyltransferase (HPRT) and GAPDH on our samples and found GAPDH to be more stable with age than HPRT.
Hepatic enzyme activity assays.
Rat microsomal fractions were prepared as described previously (DeVito et al., 1996). Ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-deethylase (MROD), and pentoxyresorufin O-deethylase (PROD) are enzymatic markers for CYP2C6, CYP1A2, and CYP2B2 activity, respectively. Caffeine, a substrate for CYP1A2 activity, is metabolized to theophylline (13x), paraxanthine (17x), and theobromine (37x). These activities were detected spectrofluorimetrically using a method previously described (Staskal et al., 2005). The deltamethrin metabolism assay determines CYP2C6 and CYP2C11 activity and was determined according to the method of Godin et al. (2007). The T4 glucuronidation assay assesses UGT1A1 and UGT1A6 activities and is based on the method of Zhou et al. (2001). All data comparisons were performed by a one-way ANOVA (GraphPad Prizm 4.0). Differences between groups were considered significant when p < 0.05. All data are presented as mean ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Microarray Analysis of Gene Expression Profiles throughout the Rat Adult Life
We examined gene expression in the livers of F344 rats 6, 11, 18, and 24 months of age. Principal component analysis (PCA) was able to clearly separate young (6 month) and old (24 month) rats along PC1 (Fig. 1A). Although not shown, there is greater separation by age along PC2. A total of 1135 genes were found to be significantly different in the 24 versus 6 month comparison, and 155 genes were significantly different in the 18 versus 6 month comparison. No significant differences in expression were observed between 11- and 6-month-old rats. Xenobiotic metabolism genes that changed with age were identified by comparing our results to a microarray-based compendium of ADME genes, including a number that regulate XME expression or activity (Slatter et al., 2006
). In the 24- versus 6-month comparison, we found 30 phase I, 12 phase II, and 39 phase III metabolism genes that exhibited significant differences in expression (Table 2). In the 18- versus 6-month comparison, there were 7 phase I, 7 phase II, and 20 phase III metabolism genes significantly different (Table 2).
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We also examined the transcript profiles in the livers of 4-, 12-, and 24-month-old BN rats. PCA showed clear separation among the three age groups along PC1, with much less separation by age along PC2 or PC3 (Fig. 1B). There were 1435 genes significantly different in the 24 versus 4 month comparison, and 975 genes were significantly different in the 12 versus 4 month comparison. The top gene ontology (GO) categories identified in the old versus young BN rats were similar to those found in the aged F344 rats (Supplementary Tables 1–2). Xenobiotic metabolism genes significantly different in the 24 versus 4 month comparison included 17 phase I, 5 phase II, and 23 phase III metabolism genes (Table 2). In the 12- versus 4-month comparison, we found five phase I, one phase II, and seven phase III metabolism genes significantly different (Table 2).
Hierarchical clustering of XMEs in F344 and BN rats is shown in Figures 2A and 2B, respectively. Analogous to the PCA results for both strains, the 24-month-old animals segregate from the younger age groups. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of selected genes were in good agreement with microarray results (Table 3).
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Analysis of Enzymatic Activity
In the F344, EROD (CYP2C6) activity increased with age, with the 24-month-old group exhibiting the greatest activity and achieving statistical significance (Fig. 3). PROD (CYP2B) activity decreased for the 18- and 24-month-old groups but was not statistically significant. No significant changes in MROD (CYP1A1/2) activity were seen in F344. No significant changes were observed at the different ages for EROD (CYP2C6), MROD (CYP1A1/2), and PROD (CYP2B) for the BN rat. No consistent age-related changes were observed in the F344 rats in the caffeine biotransformation assay. For BN rats, although not statistically significant, minimal changes in CYP1A2 enzyme activity were seen at the different ages, and activity was the highest in the 24-month-old group for all three caffeine metabolites (theobromine [37X], paraxanthine [17X], and theophylline [13X]). Using thyroxine as a substrate no changes in UGT1A1 or UGT1A6 enzyme activity with age were observed in either strain. In both strains, deltamethrin metabolism (CYP2C6/CYP2C11) decreased with age, but did not attain statistical significance. These activity data are consistent with the trends from mRNA results for these specific genes, with the exception of CYP2C6 activity which increased in the 24-month-old F344 rats whereas gene expression was not significantly changed.
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Comparison of Age-Related Genes between F344 and BN Rats
A direct comparison of the significantly altered genes between youngest and oldest for the two rat strains revealed 462 genes that overlapped (Fig. 4). The six XMEs in common between strains were all Phase III genes and five out of six exhibited similar direction of change (Table 4). Based on 50% survival of aging rats, a 24-month-old F344 rat and a 32-month-old BN rat are at approximately equivalent physiological ages (Nadon, 2004
). Therefore, in our study we also compared 18-month F344 to 24-month BN (
90% survival for both strains). There were 303 genes in common between F344 (18 vs. 6 month) and BN (24 vs. 4 month), with 93% concordance in direction of change. Using significantly altered genes, the top networks identified by Ingenuity in old versus young male F344 and BN rats are shown in Table 5. Consistent functions identified between the two groups include inflammatory disease, dermatological diseases and conditions, immunological disease, cell-to-cell signaling and interaction, cell morphology, hematological system development and function, tissue development, and nervous system development and function.
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Using the program GATHER (Chang and Nevins, 2006
), the 462 genes significantly different by age that are in common between F344 and BN rats were involved in the following kyoto encyclopedia of genes and genomes pathways (Bayes Factor cutoff of 6): MAPK signaling pathway, focal adhesion, and cytokine-cytokine receptor interaction. GATHER also identified the top GO categories: carbohydrate metabolism, energy derivation by oxidation of organic compounds, alcohol metabolism, pyruvate metabolism, and cellular carbohydrate metabolism. In the old versus young group, many GO categories identified by GATHER fall under intermediary metabolism, including lipid metabolism, ergosterol metabolism, alcohol metabolism, and fatty acid metabolism.
Comparison of Genes Altered by Aging and Gender
From our analysis above, genes known to be regulated by GH in a male predominant manner (e.g., CYP2A1, CYP2A2, CYP2C13, CYP2C40) were downregulated with age. We hypothesized that many of the gene expression changes in old rats were due to changes in GH secretory pattern that determines gender expression in the liver (Legraverend et al., 1992; Sundseth et al., 1992). To test this hypothesis, we compared the expression of all genes altered by aging in the F344 and BN rats to genes that were consistently different between male and female rats identified in a study of baseline gene expression in the livers of control rats. Control rats were from eight different studies performed at two institutions (Boedigheimer et al., 2008). We compared 223 genes differentially expressed in at least one of the aging rat strains and in at least one of the two institutions from the baseline study. Gender differences impacted 19% (213/1135) and 8% (116/1435) of the genes significantly altered by aging in the F344 and BN rat, respectively. Remarkably, most genes upregulated in the aging rat were expressed at greater levels in young adult female rats compared with males, whereas most genes downregulated by aging were expressed at greater levels in young adult male rats (Fig. 5). These results suggest that the aging male rat liver exhibits a "feminized" pattern of gene expression which impacts the expression of 25 (31%) and 17 (38%) XMEs in the F344 and BN comparisons, respectively (Supplementary Table 3).
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GSEA Results
Expression profiles for combined young (4–6 months) and old (24 months) F344 and BN rats were analyzed using GSEA. Twelve gene sets were found to be significantly enriched in young versus old (FDR < 25%). Of these sets, six were related to Myc, two were related to androgen, and one was related to amino acid metabolism. There were 717 gene sets found to be significantly enriched in the old versus young comparison (FDR < 25%). Many of these gene sets were related to apoptosis in the liver, inflammation, and aging. We also used GSEA to search for TFs (C3 gene set) enriched in the old or young populations potentially regulating the gene expression profiles. There were zero and 258 significant gene sets enriched in the young and old phenotypes, respectively (FDR < 25%). Some top ranked motifs in the old phenotype included binding motifs for AhR and xenobiotic metabolism signaling (NF-kB, AhR-ARNT), as well as LXR/RXR activation (SREBF1).
Effect of Age on the Transcriptional Response to Toluene Exposure
The expression of CYP1A2 and CYP2D22, involved in organic solvent metabolism, decreased with age in both rat strains. The expression of glutathione-S-transferase M3 (GSTM3), an important phase II enzyme involved in detoxification, also decreased with age, whereas the expression of glutathione synthetase (GSS) increased with age. To test the hypothesis that older rats exhibit altered transcriptional responses to environmental chemicals, we exposed BN rats of three ages to 1 g/kg of toluene for 4 h. Control rats received corn oil alone. We found 1133, 946, and 1772 genes significantly altered in the 4-, 12-, and 24-month-old rats, respectively. The top GO categories for the age groups dosed with toluene are listed in Supplementary Tables 4–6. Toluene is mainly metabolized by CYP1A1/2, CYP2A1, CYP2B1/2, CYP2C6, CYP2C11, and CYP2E1 in the rat liver (Nakajima and Wang, 1994). The gene expression of CYP2B2 after acute toluene exposure increased 3.2-, 6.6-, and 7.1-fold in the 4-, 12-, and 24-month groups, respectively. Although age did not have an appreciable effect on the number of phase I genes that were altered by toluene exposure, there was a dramatic effect on the number of phase II and phase III genes altered, increasing the number altered from 14 and 15 in the 4- and 12-month age groups to 33 in the 24-month-old group (Table 6). After exposure to toluene, the expression of many glutathione-S-transferases increased in the 4-month (GSTA5), 12-month (GSTM2, YC2), and 24-month-old groups (GSTM3, GSTM2, GSTA5). Age being the only parameter, 30% (7/23) of the Phase III genes were downregulated in the control 24-month-old group compared with the control 4-month-old group, and this increased to 70% (19/27) after exposure to toluene. In the 24-month-old toluene exposed group the expression of glutathione synthetase (GSS) and glutathione S-transferase YC2 subunit decreased, suggesting a decreased production of glutathione with age. Hierarchical clustering of XMEs in BN rats exposed to 1 g/kg toluene is shown in Figure 6. As seen in the heat map, there is segregation of the exposed and nonexposed animals. Top functions identified by Ingenuity are shown in Table 7. In summary, toluene exposure mainly affected the expression of phase II and phase III genes in the aging rats.
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TF Analysis
Potential targets of aging are the TFs that regulate the changes in XME gene expression. TFs involved in XME expression that were altered in aging rats (F344 and BN) and rats exposed to toluene (BN) were clustered (Fig. 7). Most of the TFs (85%) altered by age were unique to F344 rats. TFs were almost always upregulated in BN rats after exposure to toluene. In F344 rats, Nr1i3 (CAR) expression decreased 2.4-fold at 24 months compared with the 6 month group. Expression of many CYPs and other XMEs regulated by CAR also decreased in the 24 versus 6 month F344 rats, including CYP1A2, CYP2C, CYP3A2, CYP2B2, CYP2B3, STE, SLC21A10, and SLC29A1. Toluene exposure increased the expression of CAR 1.9-fold in 24-month-old BN rats. The expression of CYP2B2, regulated by CAR, increased in all age groups exposed to toluene with the highest fold change (7.1-fold) observed in the 24-month-old group. The expression of CYP2C and GSTA5, also regulated by CAR, increased in the 24-month-old BN rats exposed to toluene. Thus, the expression changes in the XMEs regulated by CAR are in good agreement with the expression changes in CAR itself.
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As a complement to the analysis of TF expression, we also identified enriched binding sites in the promoters of genes significantly altered in our studies. Many of the XMEs altered by age, GH, or toluene contain AHR, FXR, RXR, PPAR, STAT5B, STAT3, STAT1, and STAT6 sites (Supplementary Figs. 1A and 1B). Many of these same TFs exhibited altered expression with age or toluene exposure or both.
Potential Effect of Age on Chemical Sensitivity
To predict classes of chemicals to which the aging population exhibit differential sensitivity, we used the Comparative Toxicogenomics Database (http://ctd.mdibl.org/) to identify chemicals that interact with individual CYPs affected by age in our study. We focused our search for chemicals that increase the expression of CYPs that were decreased with age in our study, that is, CYPs likely involved in metabolism of the chemical. CYP1A2, CYP2C, and CYP2B2 expression decreased in old F344 and BN rats. CYP3A2 expression decreased in old F344 rats only. Based on these gene expression changes, we identified a number of chemicals that may exhibit altered metabolism in the livers of aged rats. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a chemical by-product from incineration processes, induces CYP1A2 through activation of the Ah receptor. Insecticides (Mirex, methoxychlor, chlordecone), the pesticide dichloro-diphenyl-trichloroethane (DDT), the herbicide alachlor, some polychlorinated biphenyls, and the organic solvent dioxane are chemicals that induce CYP2B2 through activation of CAR. Aflatoxin B1, DDT, and propiconazole fungicides interact with CYP3A2. Thus, it follows that old male F344 and BN rats might be more sensitive to the effects of TCDD, insecticides, DDT, alachlor, some polychlorinated biphenyls (PCBs), and dioxane due to reduced metabolism. Old male F344 might also be more sensitive to the effects of aflatoxin B1 and propiconazole fungicides. In addition to gene expression data, protein and enzyme activity data would strengthen our predictions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The expression and activity of genes involved in chemical metabolism can have a profound impact on the biological fate of the chemical and whether exposure results in toxicity. Very little is known about the impact of aging on the transcript profile of XMEs and the relationships between age-dependent differences in expression and chemical susceptibility. In this study changes in XME expression in aging male rat livers were characterized to hypothesize altered responses to environmental chemicals with age. A comparison was performed between two rat strains commonly used for toxicology and aging studies (F344 and BN) to identify strain-dependent and -independent gene expression changes. We used this information to hypothesize that there would be differences in transcriptomic responses between young and old rats after exposure to an environmentally relevant chemical (toluene). Our study finds age-related changes in phase I, phase II, and phase III genes in two strains of rats up to 24 months old, as well as age-related differences in XME gene expression due to toluene exposure.
Aging had an impact on the overall transcript profile as well as the expression of XMEs in the liver. Almost 16 and 7% of the genome exhibited some change in expression in F344 and BN rats, respectively (p-value < 0.05). Many of the gene sets for the old phenotype, identified by GSEA, were related to inflammation and aging. Aging is associated with dysregulated inflammatory response, and increased inflammatory response has been implicated in the pathogenesis of several age-related diseases (Wu and Meydani, 2008). Out of the 491 XME genes examined in our profiling studies, 22 or 14% were altered by aging in F344 or BN rats, respectively. The greater number of age-related gene expression changes in F344 than BN rats at equivalent ages is not surprising, because survival data indicates BN rats age more slowly than F344 rats. The median survival for F344 rats is 24 months whereas for BN rats it is 32 months (Turturro et al., 1999). The 462 genes significantly altered in both strains between young and old were 89% concordant, that is, exhibited similar direction of change with age. Using Ingenuity, the top associated network functions for genes significantly altered in both strains were related to drug metabolism, free radical scavenging, and fatty acid metabolism. This is similar to the GO categories that fall under intermediary metabolism as identified by GATHER. Genes altered only in F344 with age were related to cancer, gastrointestinal disease, and gene expression. Genes altered only in BN rats with age were related to embryonic development, tissue development, and tissue morphology. It is not surprising that cancer is one of the top diseases identified in aged F344 rats (Table 5) because aged F344 rats have a greater incidence of certain diseases than other rat strains, including leukemia, which is rare in BN rats (Nadon, 2006). Reproductive system development and function is also uniquely identified in aged BN rats (Table 5), and this correlates with the high rate of testicular atrophy seen in aged males (Lipman et al., 1999). The incidence of many diseases increases with age, making it hard to separate the effects of aging from the effects of various diseases (Sprott and Ramirez, 1997). BN rats have far fewer strain-specific lesions with age compared with the F344, and these lesions are similar to the ones seen in the human population (Nadon, 2006). Further, there are physiological similarities to humans that make BN rats useful models for human senescence, particularly in the male reproductive system (Nadon, 2006). Our comparison of the gene expression profiles in the aging rat liver will be a useful resource for future studies in these rat strains to correlate changes in gene expression with age-dependent diseases in liver and other tissues.
Most of the age-dependent phase I genes in F344 rats are involved in oxidation-reduction reactions, including alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), flavin-containing monooxygenase (FMO), esterase (ES), heme oxygenase (HMOX), and the cytochrome P450 family (CYP) of enzymes. Hydrolases including epoxide hydrolase were not altered with age. We found increased expression of ADH1 and ALDH1A4, which play important roles in the metabolism of alcohols and aldehydes. ADH and ALDH family members are involved in the pathway leading to fatty acid biosynthesis; alterations in lipid homeostasis were indicated for 24-month F344 rats as top GO categories included lipid metabolism, sterol biosynthesis, sterol metabolism, and fatty acid metabolism. FMO converts lipophilic compounds to hydrophilic metabolites via oxidation. We found a greater than twofold decline in FMO1 transcript levels between 6 and 24 months of age, and increases in FMO5 gene expression between 6 and 24 months (2.4-fold) and 6 and 18 months of age (2.5-fold). HMOX1 transcript levels increased almost 2-fold with age. Most of the genes altered with age that encode phase I enzymes are members of the CYP1, CYP2, and CYP3 families (Table 2). Transcript levels for CYP2A1 and CYP2C40 increased with age, whereas transcript levels for CYP1A2, CYP2A2, CYP2B2, CYP2B3, CYP2C, CYP2C13, CYP3A2, and CYP3A18 all decreased with age. This pattern suggests an overall decline in Phase I reactions with increasing age. We also observed an increased age-related expression of carboxylesterase 2 (CES2) and a decreased age-related expression of CES3. Carboxylesterases are important in the detoxification of organophosphorous pesticides (Karanth and Pope, 2000) and pyrethroid insecticides (Huang et al., 2005), leading to the prediction of increased levels and responses to these chemicals with age.
In BN rats, ALDH, dehydrogenase/reductase (DHRS), and CYP enzymes were significantly altered with age. Transcript levels for ALDH1A1 increased 1.6-fold and decreased 4.3-fold for DHRS9 in the 24-month-old group. Transcript levels for all significantly altered members of CYP1, CYP2, and CYP3 families decreased with age (CYP1A2, CYP2A2, CYP2B2, CYP2C, CYP3A13, CYP3A18).
Phase II biotransformation processes altered with age in F344 rats included glucuronidation (UGT2A1), sulfation (SULT1A2, SULT1C2, SULT4A1), and glutathione conjugation (GPX2, GSTM2, GSTM3, GSTM4, GSTP1/2, GSTT1, YC2). Transcript profiles for glucuronidation genes decreased in 24-month-old F344 rats. Gene expression profiles for all sulfation genes, except for SULT4A1, also decreased in the oldest age group. Three of the glutathione conjugation genes were downregulated with age (GSTM2, GSTM3, GSTT1), whereas four were upregulated with age (GPX2, GSTM4, GSTP1/GSTP2, YC2). In 18-month-old BN rats, glutathione conjugation is the main process affected by age with glutathione S-transferase YC2 subunit decreasing by 2.1-fold. In 24-month-old BN rats, GSS was increased by 1.7-fold and GSTM3 was decreased by 2.9-fold. The biological impact of these complex age-related changes in XME expression will need to be determined using specific biochemical assays.
Mori et al. (2007) determined changes in the expression of phase I and II genes between 32- and 84-week-old F344 rats (Mori et al., 2007). Eleven out of the 21 phase I enzymes identified in our F344 study overlap with Mori's list (ADH1, CES2, CES3, CYP1A2, CYP2A1, CYP2A2, CYP2C, CYP2D22, CYP3A2, FMO1, CYP3A18), and exhibit similar direction of change. There were 4 out of 13 phase II enzymes from Mori's list in common with our study and they all showed the same direction of change; GSTP2 was upregulated, whereas GSST1, SULT1A2, and SULT1C2 were downregulated with age. In our study we showed that 7 efflux transporters were altered by age in either the F344 or BN rat (Table 2). ABCB1A, ABCC6, and ABCG5 were downregulated with age, whereas ABCB1, ABCC3, ABCG2, and TAP2 were upregulated with age. ABCB1A, ABCG5, ABCB1, and ABCG2 are transporters expressed on the canalicular membrane, responsible for biliary excretion of chemicals (Klaassen and Lu, 2008). ABCC6 and ABCC3 are present at high levels on the basolateral membrane of hepatocytes, responsible for efflux of substrates back into the blood (Klaassen and Lu, 2008). Two uptake transporters, SLC29A1 and SLCO1A4, which are expressed highly on the basolateral membrane of hepatocytes, were both downregulated with age. Overall these changes indicate complex effects of age on the expression of drug and xenobiotic transporters that may affect uptake and excretion.
For the most part, enzymatic activity data were consistent with the trends from mRNA results, with the exception of CYP2C6. Several factors, including the rate of transcription initiation, mRNA stability, the efficiency of translation, as well as protein stability and modification, may account for discrepancies between gene expression and enzyme activity levels (Glanemann et al., 2003). A lack of specificity in the assays may also help to explain the differences seen between enzyme activity and gene expression. For example, EROD activity measures CYP1A1 as well as CYP2C6 activity. A number of studies in the literature examine age-related changes in CYP activity in rodents and humans. There are inconsistencies in these data. For example, in the present study, no changes were observed with PROD activity, a marker for CYP2B. In contrast, Birnbaum and Baird (1978) using 28- to 30-month-old male Wistar rats, showed a 25% decrease in CYP2B activity using benzphetamine as a substrate. It is likely that these differences are due to either the use of older animals or a different probe substrate in the studies by Birnbaum and Baird.
In our F344 and BN rat comparison, we observed a limited overlap in the genes altered by age in the two strains (20 or 32% for F344 or BN, respectively). Out of these genes there was a remarkable concordance in the magnitude and direction of change due to age. One explanation for the number of nonoverlapping genes may originate in the way in which the control animals were treated. F344 rats were untreated whereas the BN rat controls received corn oil. There has been concern that oil vehicle used in administering chemicals by gavage may change the rate of ADME of a chemical, or may affect hormonal status, cell division or other factors that modify tumorigenic responses (Baker et al., 1981; Herzberg and Rogerson, 1981; Newberne et al., 1979). Vehicle controls from 2 year toxicological studies resulted in unusually high incidences of focal acinar hyperplasia and acinar adenoma in control F344/N rats receiving corn oil by gavage (Boorman and Eustis, 1984). Despite these limitations, data from gavage studies are valuable (Perera et al., 1989) and in our experiments show generally consistent age-dependent patterns of gene expression. Our study involved only a single gavage dose, and the dosing was completed four hours prior to the collection of tissues. Our age-dependent patterns of gene expression are also consistent with those found in previous studies (Mori et al., 2007; Thomas et al., 2002). Although we cannot rule out that corn oil may have affected gene expression, there is no indication that the effect is profound.
We identified genes that exhibited differences in expression between males and females that are also targets for age-dependent changes in expression. A comprehensive list of genes that exhibit gender differences in the young adult rat liver (Boedigheimer et al., 2008) was used to show that 9 and 8% of the aging genes in F344 and BN rats, respectively also exhibit differences in expression between genders. Most of the overlapping aging genes exhibited a more female-like expression pattern. This feminization of the gene expression pattern in the male rat liver with old age can be explained in part by decreases in plasma testosterone and increases in circulating estrogen levels with age (Fujita et al., 1990). Consistent with this, sulfotransferase 1E (STE) which metabolizes estrogen exhibited expression decreases by 23-fold in our study. These hormonal changes can affect gene expression directly or indirectly by altering the gender-dependent release of GH from the pituitary gland (Waxman and O'Connor, 2006). Results from GSEA were consistent with our studies as the young animals exhibited expression changes that significantly overlapped with gene sets for androgen and androgen metabolism
Based on the gene expression changes in the aged liver, we hypothesized that rats would exhibit altered transcriptional responses to xenobiotics including organic solvents. We tested this hypothesis by exposing young and old rats to toluene, with the understanding that baseline changes may not necessarily predict age-specific chemical induction responses. Toluene is metabolized to benzyl alcohol by CYPs, which is then oxidized by alcohol and aldehyde dehydrogenases to benzaldehyde and benzoic acid. Older rats exhibited a greater number of gene expression changes than younger rats after toluene exposure. There were four significantly altered genes in common among all age groups exposed to toluene, all of which were phase I genes (CYP26B1, CYP3A3, DHRS9, CYP2B2) with fold changes monotonically increasing. In a study by Nakajima and Wang (1994), six CYP isoenzymes were found to be involved in the metabolism of toluene in rat liver. They found toluene exposures induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A1, but decreased CYP2C11/6 and CYP2A1 in adult males. The inductive effect was more prominent in younger than in older animals and more prominent in males than in females. The male-specific CYP2C11 and CYP2E1 are the main P450 isoforms involved in toluene metabolism at high and low concentrations, respectively (Nakajima, 1997). Based on gene expression results in our study, CYP2B2 expression increased with increasing age and toluene exposure whereas CYP3A3 expression is similar in the 4 and 24 month groups exposed to 1 g/kg toluene (Fig. 8). Although involved in toluene metabolism, CYP2E1 expression may not have been detected because it is regulated at the protein level after ligand stabilization (Chien et al., 1997). Glutathione-S-transferase genes were altered to similar extents in all age groups by toluene exposure. However, a decrease in glutathione synthetase in the 24-month group was observed, suggesting increased toxicity with age due to diminished detoxification via conjugation by glutathione. The number of phase III genes being downregulated in the 24-month group increased from 7 to 19, (this includes efflux transporters ABCA1, ABCB1, ABCC1, ABCG2, ABCG5), whereas the number of phase III genes being upregulated decreased from 16 to 8, after exposure to toluene. Because efflux transporters were downregulated and no uptake transporters were altered after toluene exposure in aged rats, this suggests a reduced capacity to transport metabolites out of the body with age. Decreased amounts of glutathione, along with decreased levels of Phase III efflux transporters, may lead to increased levels of environmental chemicals in the livers of aging rats. These results suggest that an age-related decreased ability to detoxify and transport chemicals out of the body may lead to an increased susceptibility to chemicals in the aging population.
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In conclusion, we have comprehensively identified age-dependent changes in genes involved in xenobiotic metabolism in the livers of untreated F344 and BN rats, and BN rats treated with toluene. The long-term goal is to use this information to improve pharmacokinetic models of the aged by incorporating transcript profile information of the differences in XME gene expression between young and old populations and to predict the sensitivity of the aged to particular classes of chemicals. Based on gene expression changes, sulfation, glucuronidation, and glutathione conjugation pathways are affected by age. Age-related declines in many of the XMEs suggest decreased metabolic capacity with age in male rats. This finding has important implications for life stage susceptibility to environmental toxicants and impacts the interpretation of chronic studies when compared with short-term studies using young animals. An understanding of xenobiotic metabolism in young animals may not be relevant when analyzing gene expression or toxicity data from long-term studies, because internal dosimetry may change with the animal's metabolic capacity. This decreased metabolic capacity in aging rats also impacts the response to chemicals and could increase susceptibility to chemicals such as pesticides, insecticides, herbicides, fungicides, PCBs, and organic solvents in aged animals. However, it may be possible that in cases in which the metabolite is more toxic than the chemical itself, decreases in gene expression of certain XMEs may provide a protective effect. Because humans metabolize chemicals in a manner similar to rodents, our comprehensive assessment of XME expression provides scientific support for conducting risk assessments that consider the aged as a susceptible subpopulation.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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U.S. Environmental Protection Agency; and Cooperative Training Agreement between the United States Environmental Protection Agency and North Carolina Central University (CT 829460) funded C.M.
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NOTES |
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Disclaimer: It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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ACKNOWLEDGMENTS |
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We thank the NHEERL Toxicogenomics Core for their input and advice, as well as assistance in using their equipment. We thank Mr Geremy Knapp for his guidance on qPCR experiments and Mr David Ross and Ms Vicki Richardson for technical assistance with the enzymatic assays. We thank Drs Don Delker, Robert MacPhail, Linda Birnbaum, and Andrew Geller for review of the manuscript. We also thank Ms Tanya Moore and Mr Mike George for their help during necropsies.
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