A whole-body model for glycogen regulation reveals a critical role for substrate cycling in maintaining blood glucose homeostasis.

Timely, and sometimes rapid, metabolic adaptation to changes in food supply is critical for survival as an organism moves from the fasted to the fed state, and vice versa. These transitions necessitate major metabolic changes to maintain energy homeostasis as the source of blood glucose moves away from ingested carbohydrates, through hepatic glycogen stores, towards gluconeogenesis. The integration of hepatic glycogen regulation with extra-hepatic energetics is a key aspect of these adaptive mechanisms. Here we use computational modeling to explore hepatic glycogen regulation under fed and fasting conditions in the context of a whole-body model. The model was validated against previous experimental results concerning glycogen phosphorylase a (active) and glycogen synthase a dynamics. The model qualitatively reproduced physiological changes that occur during transition from the fed to the fasted state. Analysis of the model reveals a critical role for the inhibition of glycogen synthase phosphatase by glycogen phosphorylase a. This negative regulation leads to high levels of glycogen synthase activity during fasting conditions, which in turn increases substrate (futile) cycling, priming the system for a rapid response once an external source of glucose is restored. This work demonstrates that a mechanistic understanding of the design principles used by metabolic control circuits to maintain homeostasis can benefit from the incorporation of mathematical descriptions of these networks into "whole-body" contextual models that mimic in vivo conditions.

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts.

BACKGROUND: Simian Virus 40 (SV40) immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC) has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC) or SV40-immortalized and then 3-MC-transformed (HUC-TC). RESULTS: To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag) expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-gamma and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-gamma metabolic responses were functional in both cell lines, but IFN-gamma anti-proliferative responses functioned only in tumor cells. CONCLUSIONS: Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

Integrated disinfection by-products (DBP) mixtures research: gene expression alterations in primary rat hepatocyte cultures exposed to DBP mixtures formed by chlorination and ozonation/postchlorination.

Large-scale differential gene expression analysis was used to examine the biological effects of disinfected surface waters on cultured rat hepatocytes. Source water from East Fork Lake (Harsha Lake), a reservoir on the Little Miami River in Ohio, was spiked with iodide and bromide and disinfected by chlorination or ozonation/postchlorination. The chlorinated and ozonated/postchlorinated waters were concentrated, respectively, 136- and 124-fold (full strength) by reverse-osmosis membrane techniques. Volatile disinfection by-products (DBP) lost during concentration were restored to the extent possible. Primary rat hepatocytes were exposed to either full-strength or 1:10 or 1:20 dilutions of the concentrates for 24 h and assayed for cytotoxicity and gene expression alterations. The full-strength concentrates were cytotoxic, whereas the diluted samples exhibited no detectable cytotoxicity. Differential gene expression analysis provided evidence for the underlying causes of the severe cytotoxicity observed in rat hepatocytes treated with the full-strength ozonation/postchlorination concentrate (e.g., cell cycle arrest, metabolic stasis, oxidative stress). Many gene expression responses were shared among the hepatocyte cultures treated with dilutions of the ozonation/ postchlorination and chlorination concentrates. The shift in the character of the response between the full-strength concentrates and the diluted samples indicated a threshold for toxicity. A small subset of gene expression changes was identified that was observed in the response of hepatocytes to peroxisome proliferators, phthalate esters, and haloacetic acids, suggesting a peroxisome proliferative response.

Temporal gene expression profiling indicates early up-regulation of interleukin-6 in isoproterenol-induced myocardial necrosis in rat.

Gene expression was evaluated in the myocardium of male Wistar rats after a single subcutaneous administration of 0.5 mg of isoproterenol, a beta-adrenergic agonist that causes acute tachycardia with subsequent myocardial necrosis. Histology of the heart, clinical chemistry, and hematology were evaluated at 9 time points (0.5 hours to 14 days postinjection). Myocardial gene expression was evaluated at 4 time points (1 hour to 3 days). Contraction bands and loss of cross-striation were identified on phosphotungstic acid-hematoxylin-stained sections 0.5 hours postdosing. Plasma troponin I elevation was detected at 0.5 hours, peaked at 3 hours, and returned to baseline values at 3 days postdosing. Interleukin 6 (Il6) expression spiked at 1 to 3 hours and was followed by a short-lived, time-dependent dysregulation of its downstream targets. Concurrently and consistent with the kinetics of the histologic findings, many pathways indicative of necrosis/apoptosis (p38 mitogen-activated protein kinase [MAPK] signaling, NF-kappaB signaling) and adaptation to hypertension (PPAR signaling) were overrepresented at 3 hours. The 1-day and 3-day time points indicated an adaptive response, with down-regulation of the fatty acid metabolism pathway, up-regulation of the fetal gene program, and superimposed inflammation and repair at 3 days. These results suggest early involvement of Il6 in isoproterenol-induced myocardial necrosis and emphasize the value of early time points in transcriptomic studies.

The hepatic transcriptome as a window on whole-body physiology and pathophysiology.

Transcriptomics can be a valuable aid to pathologists. The information derived from microarray studies may soon include the entire transcriptomes of most cell types, tissues and organs for the major species used for toxicology and human disease risk assessment. Gene expression changes observed in such studies relate to every aspect of normal physiology and pathophysiology. When interpreting such data, one is forced to look "far from the lamp post:' and in so doing, face one's ignorance of many areas of biology. The central role of the liver in toxicology, as well as in many aspects of whole-body physiology, makes the hepatic transcriptome an excellent place to start your studies. This article provides data that reveals the effects of fasting and circadian rhythm on the rat hepatic transcriptome, both of which need to be kept in mind when interpreting large-scale gene expression in the liver. Once you become comfortable with evaluating mRNA expression profiles and learn to correlate these data with your clinical and morphological observations, you may wonder why you did not start your studies of transcriptomics sooner. Additional study data can be viewed at the journal website at (www.toxpath.org). Two data files are provided in Excel format, which contain the control animal data from each of the studies referred to in the text,including normalized signal intensity data for each animal (n=5) in the 6-hour, 24-hour, and 5-day time points. These files are briefly described in the associated 'Readme' file, and the complete list of GenBank numbers and Affymetrix IDs are provided in a separate txt file. These files are available at http://taylorandfrancis.metapress.comlopenurl.asp?genre=journal&issn=0192-6233. Click on the issue link for 33(1), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through (www.toxpath.org).

Complementary roles for toxicologic pathology and mathematics in toxicogenomics, with special reference to data interpretation and oscillatory dynamics.

Toxicogenomics is an emerging multidisciplinary science that will profoundly impact the practice of toxicology. New generations of biologists, using evolving toxicogenomics tools, will generate massive data sets in need of interpretation. Mathematical tools are necessary to cluster and otherwise find meaningful structure in such data. The linking of this structure to gene functions and disease processes, and finally the generation of useful data interpretation remains a significant challenge. The training and background of pathologists make them ideally suited to contribute to the field of toxicogenomics, from experimental design to data interpretation. Toxicologic pathology, a discipline based on pattern recognition, requires familiarity with the dynamics of disease processes and interactions between organs, tissues, and cell populations. Optimal involvement of toxicologic pathologists in toxicogenomics requires that they communicate effectively with the many other scientists critical for the effective application of this complex discipline to societal problems. As noted by Petricoin III et al (Nature Genetics 32, 474-479, 2002), cooperation among regulators, sponsors and experts will be essential for realizing the potential of microarrays for public health. Following a brief introduction to the role of mathematics in toxicogenomics, "data interpretation" from the perspective of a pathologist is briefly discussed. Based on oscillatory behavior in the liver, the importance of an understanding of mathematics is addressed, and an approach to learning mathematics "later in life" is provided. An understanding of pathology by mathematicians involved in toxicogenomics is equally critical, as both mathematics and pathology are essential for transforming toxicogenomics data sets into useful knowledge.

Frequent sampling reveals dynamic responses by the transcriptome to routine media replacement in HepG2 cells.

Cultured cell lines are employed extensively for biological research. Large-scale differential gene expression (LSDGE) is being used to study mechanisms of toxicity in such cultures. 'Normal' gene expression dynamics could have a major impact on the design and interpretation of these studies. In order to provide understanding of such dynamics, we investigated LSDGE responses to media replacement in human hepatoblastoma cells (HepG2) using 5-minute sampling frequencies for 6 hours post routine media replacement. Each mRNA transcript was found to exhibit a characteristic 'operating range' based on signal intensity. Following media replacement, which replenishes nutrients (eg, glucose and glutamate) and removes excretory products (eg, lactate), a complex set of gene expression changes was observed. Some transcripts appeared to switch on from a quiescent state to a very active one (eg, CYP1A1), others exhibited 'clocklike' oscillations (eg, asparagine synthetase), or a synchronous burst (chirp) of expression up regulation (eg, timeless). Mathematical analysis (Fourier Transform, Singular Value Decomposition, Wavelets, Phase Analysis) of oscillating expression patterns identified cycle lengths ranging from 11.8 to 210 minutes. There were prominent 36.5- and 17.4-minute cycles, for subsets of genes, and transcript-specific differences in phase angle with respect to these cycles. The functional consequences of these novel observations remain to be determined. It is clear that dense time-course studies provide a valuable approach to the investigation of physiological responses to nutrients, toxicants, and other environmental variables. This research also highlights the need for an understanding of biological dynamics when using cell culture systems. An Excel data file representing individual transcripts from the respective Clontech cDNA arrays referred to in this article is available at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. Rows represent data for individual transcripts and columns represent the time-points from 0 to 360 minutes. To access this file, click on the issue link for 31(4), then select this article. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Effects of minimally toxic levels of carbonyl cyanide P-(trifluoromethoxy) phenylhydrazone (FCCP), elucidated through differential gene expression with biochemical and morphological correlations.

Uncouplers of oxidative phosphorylation have relevance to bioenergetics and obesity. The mechanisms of action of chemical uncouplers of oxidative phosphorylation on biological systems were evaluated using differential gene expression. The transcriptional response in human rhabdomyosarcoma cell line (RD), was elucidated following treatment with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a classical uncoupling agent. Changes in mitochondrial membrane potential were used as the biological dosimeter. There was an increase in membrane depolarization with increasing concentrations of FCCP. The concentration at 75% uncoupling (20 microM) was chosen to study gene expression changes, using cDNA-based large-scale differential gene expression (LSDGE) platforms. At the above concentration, subtle light microscopic and clear gene expression changes were observed at 1, 2, and 10 h. Statistically significant transcriptional changes were largely associated with protein synthesis, cell cycle regulation, cytoskeletal proteins, energy metabolism, apoptosis, and inflammatory mediators. Bromodeoxyuridine (BrdU) and propidium iodide (PI) assays revealed cell cycle arrest to occur in the G1 and S phases. There was a significant initial decrease in the intracellular adenosine triphosphate (ATP) concentrations. The following seven genes were selected as potential molecular markers for chemical uncouplers: seryl-tRNA synthetase (Ser-tRS), glutamine-hydrolyzing asparagine synthetase (Glut-HAS), mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase (Mit BMD), mitochondrial heat shock 10-kDa protein (Mit HSP 10), proliferating cyclic nuclear antigen (PCNA), cytoplasmic beta-actin (Act B), and growth arrest and DNA damage-inducible protein 153 (GADD153). Transcriptional changes of all seven genes were later confirmed with reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that gene expression changes may provide a sensitive indicator of uncoupling in response to chemical exposure.

Formaldehyde-induced gene expression in F344 rat nasal respiratory epithelium.

Formaldehyde (FA), an occupational and environmental toxicant used extensively in the manufacturing of many household and personal use products, is known to induce squamous cell carcinomas in the nasal turbinates of rats and mice and squamous metaplasia in monkey noses. Tissue responses to FA include a dose dependent epithelial degeneration, respiratory cell hypertrophy, and squamous metaplasia. The primary target for FA-induced toxicity in both rodents and monkeys is the respiratory nasal epithelium. FA increases nasal epithelial cell proliferation and DNA-protein crosslinks (DPX) that are associated with subsequent nasal cancer development. To address the acute effects of FA exposure that might contribute to known pathological changes, cDNA gene expression analysis was used. Two groups of male F344 rats received either 40 ul of distilled water or FA (400 mM) instilled into each nostril. Twenty-four hours following treatment, nasal epithelium was recovered from which total RNA was used to generate cDNA probes. Significance analysis of microarrays (SAM) hybridization data using Clontech Rat Atlas 1.2 arrays revealed that 24 of the 1185 genes queried were significantly up-regulated and 22 genes were significantly downregulated. Results for ten of the differentially expressed genes were confirmed by quantitative real time RT PCR. The identified genes with FA-induced change in expression belong to the functional gene categories xenobiotic metabolism, cell cycle, apoptosis, and DNA repair. These data suggest that multiple pathways are dysregulated by FA exposure, including those involved in DNA synthesis/repair and regulation of cell proliferation. Differential gene expression profiles may provide clues that could be used to define mechanisms involved in FA-induced nasal cancer.

Use of a hybrid computational fluid dynamics and physiologically based inhalation model for interspecies dosimetry comparisons of ester vapors.

Numerous inhalation studies have demonstrated that exposure to high concentrations of a wide range of volatile acids and esters results in cytotoxicity to the nasal olfactory epithelium. Previously, a hybrid computational fluid dynamics (CFD) and physiologically based pharmacokinetic (PBPK) dosimetry model was constructed to estimate the regional tissue dose of organic acids in the rodent and human nasal cavity. This study extends this methodology to a representative volatile organic ester, ethyl acrylate (EA). An in vitro exposure of explants of rat olfactory epithelium to EA with and without an esterase inhibitor demonstrated that the organic acid, acrylic acid, released by nasal esterases is primarily responsible for the olfactory cytotoxicity. Estimates of the steady-state concentration of acrylic acid in olfactory tissue were made for the rat nasal cavity by using data from a series of short-term in vivo studies and from the results of CFD-PBPK computer modeling. Appropriate parameterization of the CFD-PBPK model for the human nasal cavity and to accommodate human systemic anatomy, metabolism, and physiology allowed interspecies dose comparisons. The CFD-PBPK model simulations indicate that the olfactory epithelium of the human nasal cavity is exposed to at least 18-fold lower tissue concentrations of acid released from EA than the olfactory epithelium of the rat nasal cavity under the same exposure conditions. The magnitude of this difference varies with the specific exposure scenario that is simulated and with the specific dataset of human esterase activity used for the simulations. The increased olfactory tissue dose in rats relative to humans may be attributed to both the vulnerable location of the rodent olfactory tissue (comprising greater than 50% of the nasal cavity) and the high concentration of rat olfactory esterase activity (comparable to liver esterase activity) relative to human olfactory tissue. These studies suggest that the human olfactory epithelium is protected from vapors of organic esters significantly better than rat olfactory epithelium due to substantive differences in nasal anatomy, nasal and systemic metabolism, systemic physiology, and air flow. Although the accumulation of acrylic acid in the nasal tissues may be a primary concern for nasal irritation and human risk assessment, acute animal inhalation studies to evaluate lethality (LD50-type studies) conducted at very high vapor concentrations of ethyl acrylate indicated that a different mechanism is primarily responsible for mortality. The rodent studies demonstrated that systemic tissue nonprotein sulfhydryl depletion is a primary cause of death at exposure concentrations more than two orders of magnitude above the concentrations that induce nasal irritation. The CFD-PBPK model adequately simulated the severe depletion of glutathione in systemic tissues (e.g., liver and lung) associated with acute inhalation exposures in the 500-1000 ppm range. These results indicate that the CFD-PBPK model can simulate both the low-dose nasal tissue dosimetry associated with irritation and the high-dose systemic tissue dosimetry associated with mortality. In addition, the comparison of simulation results for ethyl acetate and acetone to nasal deposition data suggests that the CFD-PBPK model has general utility as a tool for dosimetry estimates for a wide range of other esters and slowly metabolized vapors.

Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real-time RT-PCR-based screen for oxidative stress in Hep-G2 cells.

Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology. Tables 1-15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Correlation of simultaneous differential gene expression in the blood and heart with known mechanisms of adriamycin-induced cardiomyopathy in the rat.

As the genomes of mammalian species become sequenced and gene functions are ascribed, the use of differential gene expression (DGE) to evaluate organ function will become common in the experimental evaluation of new drug therapies. The ability to translate this technology into useful information for human exposures depends on tissue sampling that is impractical or generally not possible in man. The possibility that the DGE of nucleated cells, reticulocytes, or platelets in blood may present the necessary link with target organ toxicity provides an opportunity to correlate preclinical with clinical outcomes. Adriamycin is highly effective alone and more frequently in combination with other chemotherapeutic agents in the treatment of a variety of susceptible malignancies. Adriamycin-induced cardiomyopathy was examined as an endpoint to measure the utility of DOE on whole blood as a predictor of cardiac toxicity. Statistically significant gene changes were observed between relevant blood and cardiac gene profiles that corroborated the accepted mechanisms of toxicity (oxidative stress, effects on carnitine transport, DNA intercalation). There were, however, clear indications that other target organs (bone marrow and intestinal tract) were affected. The divergent expression of some genes between the blood and the heart on day 7 may also indicate the timing and mechanism of development of the cardiomyopathy and confirm current therapeutic approaches for its prevention. The data demonstrate that whole blood gene expression particularly in relation to oxidative stress, in conjunction with standard hematology and clinical chemistry, may be useful in monitoring and predicting cardiac damage secondary to adriamycin administration. Appendices A & B, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Normalization and analysis of DNA microarray data by self-consistency and local regression.

BACKGROUND: With the advent of DNA hybridization microarrays comes the remarkable ability, in principle, to simultaneously monitor the expression levels of thousands of genes. The quantitative comparison of two or more microarrays can reveal, for example, the distinct patterns of gene expression that define different cellular phenotypes or the genes induced in the cellular response to insult or changing environmental conditions. Normalization of the measured intensities is a prerequisite of such comparisons, and indeed, of any statistical analysis, yet insufficient attention has been paid to its systematic study. The most straightforward normalization techniques in use rest on the implicit assumption of linear response between true expression level and output intensity. We find that these assumptions are not generally met, and that these simple methods can be improved. RESULTS: We have developed a robust semi-parametric normalization technique based on the assumption that the large majority of genes will not have their relative expression levels changed from one treatment group to the next, and on the assumption that departures of the response from linearity are small and slowly varying. We use local regression to estimate the normalized expression levels as well as the expression level-dependent error variance. CONCLUSIONS: We illustrate the use of this technique in a comparison of the expression profiles of cultured rat mesothelioma cells under control and under treatment with potassium bromate, validated using quantitative PCR on a selected set of genes. We tested the method using data simulated under various error models and find that it performs well.

Normal gene expression in male F344 rat nasal transitional and respiratory epithelium.

The nasal epithelium is an important target site for chemically-induced toxicity and carcinogenicity in rodents. Gene expression profiles were determined in order to provide normal baseline data for nasal transitional/respiratory epithelium from healthy rats. Cells lining the rat nasal passages were collected and gene expression analysis was performed using Clontech cDNA Rat Atlas 1.2 arrays (1185 genes). The percentages of genes within specific average expression ranges were 4.2% at 45,000-1000, 14.8% at 1000-200, 25.0% at 200-68, and 56.0% below 68. Nine out of a subset of ten genes were confirmed for relative signal intensity using quantitative real-time RT-PCR. The most highly expressed genes included those involved in phase I (e.g. cytochrome P450s) and phase II (e.g. glutathione S-transferases) xenobiotic metabolism, bioenergetics (e.g. cytochrome oxidase), osmotic balance (e.g. Na(+)/K(+) ATPase) and epithelial ionic homeostasis (e.g. ion channels). Such baseline data will contribute to further understanding the normal physiology of these cells and facilitate the interpretation of responses by the nasal epithelial cells to xenobiotic treatment or disease.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.

Gene expression analysis reveals chemical-specific profiles.

The articles highlighted in this issue are "Gene Expression Analysis Reveals Chemical-Specific Profiles" by Hisham K. Hamadeh, Pierre R. Bushel, Supriya Jayadev, Karla Martin, Olimpia DiSorbo, Stella Sieber, Lee Bennett, Raymond Tennant, Raymond Stoll, J. Carl Barrett, Kerry Blanchard, Richard S. Paules, and Cynthia A. Afshari (pp. 219-231) and "Prediction of Compound Signature Using High Density Gene Expression Profiling" by Hisham K. Hamadeh, Pierre R. Bushel, Supriya Jayadev, Olimpia DiSorbo, Leping Li, Raymond Tennant, Raymond Stoll, J. Carl Barrett, Richard S. Paules, Kerry Blanchard, and Cynthia A. Afshari (pp. 232-240).

Toxicogenomics, drug discovery, and the pathologist.

The field of toxicogenomics, which currently focuses on the application of large-scale differential gene expression (DGE) data to toxicology, is starting to influence drug discovery and development in the pharmaceutical industry. Toxicological pathologists, who play key roles in the development of therapeutic agents, have much to contribute to DGE studies, especially in the experimental design and interpretation phases. The intelligent application of DGE to drug discovery can reveal the potential for both desired (therapeutic) and undesired (toxic) responses. The pathologist's understanding of anatomic, physiologic, biochemical, immune, and other underlying factors that drive mechanisms of tissue responses to noxious agents turns a bewildering array of gene expression data into focused research programs. The latter process is critical for the successful application of DGE to toxicology. Pattern recognition is a useful first step, but mechanistically based DGE interpretation is where the long-term future of these new technologies lies. Pathologists trained to carry out such interpretations will become important members of the research teams needed to successfully apply these technologies to drug discovery and safety assessment. As a pathologist using DGE, you will need to learn to read DGE data in the same way you learned to read glass slides, patiently and with a desire to learn and, later, to teach. In return, you will gain a greater depth of understanding of cell and tissue function, both in health and disease.