Combinations of anticancer agents may have synergistic anti-tumor effects, but enhanced hematological toxicity often limit their clinical use. We examined whether “microarray profiles” could be used to compare early molecular responses following a single dose of agents administered individually with that of the agents administered in a combination. We compared the mRNA responses within bone marrow of Sprague-Dawley rats after a single 30 min treatment with topotecan at 4.7 mg/kg or oxaliplatin at 15 mg/kg alone to that of sequentially administered combination therapy or vehicle control for 1, 6, and 24 h. We also examined the histopathology of the bone marrow following all treatments. Drug-related histopathological lesions were limited to bone marrow hypocellularity for animals dosed with either agent alone or in combination. Lesions had an earlier onset and higher incidence for animals given topotecan alone or in combination with oxaliplatin. Severity increased from mild to moderate when topotecan was administered prior to oxaliplatin compared with administering oxaliplatin first. Notably, six patterns of co-expressed genes were detected at the 1 h time point that indicate regulatory expression of genes that are dependent on the order of the administration. These results suggest alterations in histone biology, chromatin remodeling, DNA repair, bone regeneration, and respiratory and oxidative phosphorylation are among the prominent pathways modulated in bone marrow from animals treated with an oxaliplatin/topotecan combination. These data also demonstrate the potential for early mRNA patterns derived from target organs of toxicity to inform toxicological risk and molecular mechanisms for agents given in combination.
In the area of toxicology, the subdiscipline of toxicogenomics has emerged, which is the use of genome-scale mRNA expression profiling to monitor responses to adverse xenobiotic exposure. Toxicogenomics is being investigated for use in the triage of compounds through predicting potential toxicity, defining mechanisms of toxicity, and identifying potential biomarkers of toxicity. Whereas various approaches have been reported for the development of algorithms predictive of toxicity and for the interpretation of gene expression data for deriving mechanisms of toxicity, there are no clearly defined methods for the discovery of biomarkers using gene expression technologies. Ways in which toxicogenomics may be used for biomarker discovery include analysis of large databases of gene expression profiles followed by in silico mining of the database for differentially expressed genes; the analysis of gene expression data from preclinical studies to find differentially expressed genes that correlate with pathology (coincident biomarker) or precede pathology (leading biomarker) within a lead series; or gene expression profiling can be performed directly on the blood from preclinical studies or clinical trials to find biomarkers that can be obtained noninvasively. This chapter broadly discusses the issues and the utility of applying toxicogenomics to biomarker discovery.
An improved three-step Percoll density gradient centrifugation technique is described for simultaneous isolation of glial plasmalemmal vesicles (GPV) and synaptosomal vesicles (SYN) from a rat brain homogenate. While electron microscopy revealed that fractions contained intact vesicles with markedly distinct morphological features, measures of high-affinity [3H]choline uptake, glutamine synthetase and carbonic anhydrase activities, as well as Western blot analyses for glial fibrillary acidic protein and neuron specific enolase, served to confirm the low level of neuronal contamination in GPV fractions as well as the low level of glial contamination in SYN fractions. In addition, GPV and SYN fractions were used to characterize the kinetic and pharmacological properties of sodium-dependent [3H]L-glutamate transport. In conclusion, these results demonstrate the usefulness of this method for obtaining highly-enriched, functionally viable populations of glial and neuronal elements which are suitable for studies of their respective cell functions in vitro.
The goal of this study was to compare and contrast the basal gene expression levels of the various enzymes involved in glutathione metabolism among tissues and genders of the rat, mouse and canine. The approach taken was to use Affymetrix GeneChip microarray data for rat, mouse and canine tissues, comparing intensity levels for individual probes between tissues and genders. As was hypothesized, the relative expression in liver, lung, heart, kidney and testis varied from gene to gene, with differences of expression between tissues sometimes greater than a 1000-fold. The pattern of differential expression was usually similar between male and female animals, but varied greatly between the three species. Gstp1 appears to be expressed at high levels in male mouse liver, reasonable levels in canine liver, but very low levels in male rat liver. In all species examined, Gstp1 expression was below detectable levels in testis. Gsta3/Yc2 expression appeared high in rodent liver and female canine liver, but not male canine liver. Finally, Mgst1 and Gpx3 expression appeared to be lower in canine heart and testis than seen in rodents. Given the critical role of the glutathione pathway in the detoxification of many drugs and xenobiotics, the observed differences in basal tissue distribution among mouse, rat and canine has far-reaching implications in comparing responses of these species in safety testing.
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