Toxicokinetic and metabolism studies are considered crucial to dose setting and interpretation of data from rodent carcinogenicity studies. The metabolism and distribution studies are usually conducted in 6 to 8 week old rodents. However cancer studies generally involve exposures from 6 weeks of age to 108 weeks or more of age. It is recognized that age-related differences in sensitivities to some xenobiotics occur in several rodent models including the rat. However, the potential for changes in metabolism with age has received little attention in the toxicology community especially in regard to extrapolation of rodent data to human risk assessment. The advent of microarray technology has allowed a more global assessment of genes whose products are associated with xenobiotic metabolism. We studied hepatic gene expression in 38 untreated F344/N rats at approximately 32, 58 and 84 weeks of age corresponding to 6, 12, and 18 months on study. Statistical analysis of microarray (SAM) identified differential expression between the youngest and the oldest rats for genes whose products are involved in phase I and phase II metabolic pathways. Fifteen of 30 cytochrome P450 genes from CYP families 1 – 4 showed marked decrease in expression at 84 weeks while only two showed increased expression. The results of this study demonstrate prominent decreased expression in CYP family genes involved in xenobiotic metabolism in untreated rats as they age. The assumption of a constant metabolic profile when extrapolating results from long-term chemical exposures needs to be reconsidered.
Keywords: : Liver, rat, differential gene expression, microarray, transcriptome, aging, cytochrome P450, xenobiotic metabolism
Overall design: Animals and Study Design. Male Fischer 344 rats approximately 36 ± 3 days old were supplied by Taconic laboratory animals (Germantown, NY) and were approximately six weeks old when placed in study rooms as sentinel animals for two-year studies. All studies were conducted at Battelle Science and Technology International (Columbus, Ohio). The livers from the male rat sentinels from three studies at six months (15 total rats), two studies at twelve months (10 total rats) and three studies at eighteen months (13 total rats) were used in this study. The sampling times corresponded to approximately 32, 58 and 84 weeks of age for the rats. The studies in which the sentinels were a part were approved by Battelle’s Institute Animal Care and Use Committee and were conducted in accordance with the guide for the care and use of laboratory animals (ILAR 1996). Details of the animal care and handling for studies conducted at Battelle have been reported previously (Boorman et al. 2005).
The rats were kept in a cage until necropsy. The rats were anesthetized with CO2/O2, blood samples obtained for serology by cardiac puncture, the abdominal cavity was opened and the portal vein was severed. The left liver lobe was promptly removed and a cross section was placed in 10% neutral buffered formalin (NFB). The liver section was embedded, sectioned and stained with H&E and examined. The remainder of the left lobe was processed for subsequent RNA isolation.
RNA isolation. The left hepatic lobe was cut into 0.5 cm cubes or smaller and immersed in RNALater® (Ambion, Austin, TX) within 4 minutes of necropsy. The tissues were stored in RNALater® overnight at 4+/-30C, then stored at - 20+/-10C until RNA isolation (within 60 days). Details of the RNA isolation procedures have been previous published (Boorman et al. 2005). Briefly, the RNA samples were frozen at - 700 C and shipped to the NTP repository until transfer to Paradigm Array Labs (Icoria, Inc., A Clinical Data Inc. Company, RTP, NC) for microarray analysis. RNA was isolated from individual rats. Equal amounts of RNA from sixty-three male sentinel rats (25 rats at 32 weeks of age, 25 rats at 58 weeks of age and 13 rats at 84 weeks of age) were used to form a composite control pool for comparison with the individual rats at each age.
Microarray Hybridizations. One µg of total RNA from either an individual rat or from the pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA).
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