Metabonomic identification of two distinct phenotypes in Sprague-Dawley (Crl:CD(SD)) rats.

Genetic drift in animal populations has been a recognized concern for many years. Less understood is the potential for phenotypic "drift" or variation that is not related to any genetic change. Recently, stock Sprague-Dawley (Crl:CD(SD)) rats obtained from the Charles River Raleigh facility demonstrated a distinct endogenous urinary metabonomic profile that differed from historical control SD urine spectral profiles obtained over the past several years in our laboratory. In follow-up studies, the origin of the variant phenotype was narrowed down to animals of both sexes that were housed in one specific room (Room 9) in the Raleigh facility. It is likely that the two phenotypes are related to distinct populations of gut flora that particularly impact the metabolism of aromatic molecules. The most pronounced difference between the two phenotypes is the relative amounts of hippuric acid versus other aromatic acid metabolites of chlorogenic acid. Though both molecular species are present in either phenotype, the marked variation in levels of these molecules between the two phenotypes has led to the designation of high hippuric acid (HIP) and high chlorogenic acid metabolites (CA) phenotypes. Specific urinary components that distinguish the phenotypes have been thoroughly characterized by NMR spectroscopy with additional, limited characterization by LC-MS (high performance liquid chromatography coupled with mass spectrometry). Co-habitation of rats from the two phenotypes rapidly facilitated a switch of the CA phenotype to the historical Sprague-Dawley phenotype (HIP). The impact of these variant phenotypes on drug metabolism and long-term safety assessment studies (e.g., carcinogenicity bioassays) is unknown.

Metabonomic evaluation of metabolic dysregulation in rats induced by PF 376304, a novel inhibitor of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3K) is an enzyme fundamental to the regulation of various metabolic processes. Metabonomic studies were undertaken in order to gain mechanistic insight into significant, yet unexplained, toxicity issues associated with PF 376304, a nonspecific PI3K inhibitor under development for anti-inflammatory indications. Two experiments were conducted in which rats were given daily doses of up to 1000 mg of PF 376304/kg for as long as 7 days. Mortality rapidly ensued (within 72 h) at doses of >or=300 mg/kg. Doses of >or=100 mg/kg were associated with a profound but transient glucosuria. Despite the magnitude of this effect, within 72 h urinary glucose excretion in surviving animals returned to control levels even with continued dosing. Other metabolic effects associated with drug treatment included increased urinary beta-hydroxybutyrate and creatine and decreased citrate. A time-course study revealed elevated serum glucose within 1 h, followed by increases in serum insulin and decreases in serum triglycerides. Serum corticosterone was also significantly elevated within 1 h of treatment. All metabolic effects were largely reversed within 24 h of administration of the third daily dose and remained that way through day 7. The likely explanation for the onset of effects involves the role of PI3K in regulation of glucose at multiple points, but the reversal of the effects in the presence of continued exposure to the drug has not been explained. Finally, the data demonstrate the power of metabonomics technology in mechanistic toxicology investigations.

Metabonomic evaluation of Schaedler altered microflora rats.

Previously, we identified two distinct metabonomic phenotypes in Sprague-Dawley rats sourced from two different rooms (colonies) in the Charles River, Raleigh facility [Robosky, L. C., Wells, D. F., Egnash, L. A., Manning, M. L., Reily, M. D., and Robertson, D. G. (2005) Metabonomic identification of two distinct phenotypes in Sprague-Dawley (Crl:CD(SD)) rats. Toxicol. Sci. 87, 277-284]. On the basis of literature reports and cohabitation experiments, we concluded that the differing phenotypes were due to different gut flora populations. One hypothesis explaining this phenomenon was attributed to the practice of initiating new colonies with rats derived from foundation colonies that had limited gut floral populations, the Charles River altered Schaedler flora (CRASF) rats. We hypothesized that the lack of differentiation of CRASF rats to the full complement of microflora was responsible for the altered phenotype characterized by increased urinary chlorogenic acid metabolites and decreased hippurate (CA rats) as opposed to the prevalent phenotype characterized by the inverse ratio of these metabolites (HIP rats). Upon receipt, it was confirmed that the CRASF rats exhibited a metabonomic profile similar to CA rats that remained constant while animals were housed individually in a dedicated animal room. However, exposure of CRASF rats to HIP rats, or their bedding, led to a relatively rapid but variable rate of reversion to the historic HIP type metabolic profile. On the basis of the results, we conclude that CRASF rats have a unique metabolic profile due to their limited gut flora constitution. If rigorous isolation procedures are not employed, the CRASF phenotype will eventually differentiate into the more typical HIP phenotype with a time course that may be quite variable. Given the marked metabolic heterogeneity between the phenotypes, this work highlights the importance of monitoring rat metabolic profiles.