Cardiac output affects the response to pulsed inhaled nitric oxide in mechanically ventilated anesthetized ponies determined by CT angiography of the lung.

OBJECTIVE: To measure changes in regional lung perfusion using CT angiography in mechanically ventilated, anesthetized ponies administered pulsed inhaled nitric oxide (PiNO) during hypotension and normotension. ANIMALS: 6 ponies for anesthetic 1 and 5 ponies for anesthetic 2. PROCEDURES: Ponies were anesthetized on 2 separate occasions, mechanically ventilated, and placed in dorsal recumbency within the CT gantry. Pulmonary arterial, right atrial, and facial arterial catheters were placed. During both anesthetics, PiNO was delivered for 60 minutes and then discontinued. Anesthetic 1: hypotension (mean arterial pressure < 70 mmHg) was treated using dobutamine after 30 minutes of PiNO delivery. Following the discontinuation of PiNO, dobutamine administration was discontinued in 3 ponies and was continued in 3 ponies. The lung was imaged at 30, 60, and 105 minutes. Anesthetic 2: hypotension persisted throughout anesthesia. The lung was imaged at 30, 60, and 90 minutes. At all time points, arterial and mixed venous blood samples were analyzed and cardiac output (Q˙t) was measured. Pulmonary perfusion was calculated from CT image analysis. RESULTS: During PiNO delivery, perfusion to well-ventilated lungs increased if ponies were normotensive, leading to increased arterial oxygenation, reduced alveolar dead space, and reduced alveolar to arterial oxygen tension gradient. When PiNO was stopped and dobutamine administration continued, alveolar dead space and venous admixture increased, in contrast to when dobutamine and PiNO were both discontinued. CLINICAL RELEVANCE: If PiNO is administered to mechanically ventilated, anesthetized ponies with concurrent hypotension and low Q˙t, this must be supported to achieve favorable redistribution of pulmonary perfusion to improve pulmonary gas exchange.

Effects of pulsed inhaled nitric oxide delivery on the distribution of pulmonary perfusion in spontaneously breathing and mechanically ventilated anesthetized ponies.

OBJECTIVE: To measure changes in pulmonary perfusion during pulsed inhaled nitric oxide (PiNO) delivery in anesthetized, spontaneously breathing and mechanically ventilated ponies positioned in dorsal recumbency. ANIMALS: 6 adult ponies. PROCEDURES: Ponies were anesthetized, positioned in dorsal recumbency in a CT gantry, and allowed to breathe spontaneously. Pulmonary artery, right atrial, and facial artery catheters were placed. Analysis time points were baseline, after 30 minutes of PiNO, and 30 minutes after discontinuation of PiNO. At each time point, iodinated contrast medium was injected, and CT angiography was used to measure pulmonary perfusion. Thermodilution was used to measure cardiac output, and arterial and mixed venous blood samples were collected simultaneously and analyzed. Analyses were repeated while ponies were mechanically ventilated. RESULTS: During PiNO delivery, perfusion to aerated lung regions increased, perfusion to atelectatic lung regions decreased, arterial partial pressure of oxygen increased, and venous admixture and the alveolar-arterial difference in partial pressure of oxygen decreased. Changes in regional perfusion during PiNO delivery were more pronounced when ponies were spontaneously breathing than when they were mechanically ventilated. CLINICAL RELEVANCE: In anesthetized, dorsally recumbent ponies, PiNO delivery resulted in redistribution of pulmonary perfusion from dependent, atelectatic lung regions to nondependent aerated lung regions, leading to improvements in oxygenation. PiNO may offer a treatment option for impaired oxygenation induced by recumbency.

Strategies for extended lifetime of implantable intraperitoneal insulin catheters.

Implantable insulin infusion systems using the intra-peritoneal route have dramatically changed the management of diabetes paving the way toward the realization of the potential "holy grail" of a fully implantable artificial pancreas. However, the wear duration of delivery catheters is compromised by the foreign body-mediated immune response. Both occlusion material present at the distal catheter tip end and fibrotic encapsulation surrounding the catheters influence the controlled and precise delivery of insulin, which eventually leads to the need for surgical intervention. The novel part of the current work is the investigation of the roles of implant physical properties (catheter size and tip configuration), as well as local inflammation control (through utilization of an anti-inflammatory agent) on the host fibrotic response using a previously developed animal model. The cellular and molecular response, the medication delivery efficacy as well as the ability to flush the catheters were examined and further compared among the different mitigation strategies. Reduction in catheter size as well as tuning the tip configuration from a cone shape to a round shape showed delayed host recognition and delayed propagation of the fibrotic response. However, the round shaped tips had an increased occurrence of lumen occlusion as a result of flow change. It became apparent that changing the physical properties of the catheters was not a long-term solution to catheter obstructions caused by the foreign body reaction. In comparison, control of the local inflammatory response through the use of an anti-inflammatory agent demonstrated a promising strategy for maintenance of catheter functionality without any type of obstructions. These finding will have a large impact toward the development of long-term use catheters for continuous intraperitoneal insulin infusion.

Development of a method to measure regional perfusion of the lung in anesthetized ponies using computed tomography angiography and the maximum slope model.

OBJECTIVE: To develop a method based on CT angiography and the maximum slope model (MSM) to measure regional lung perfusion in anesthetized ponies. ANIMALS: 6 ponies. PROCEDURES: Anesthetized ponies were positioned in dorsal recumbency in the CT gantry. Contrast was injected, and the lungs were imaged while ponies were breathing spontaneously and while they were mechanically ventilated. Two observers delineated regions of interest in aerated and atelectatic lung, and perfusion in those regions was calculated with the MSM. Measurements obtained with a computerized method were compared with manual measurements, and computerized measurements were compared with previously reported measurements obtained with microspheres. RESULTS: Perfusion measurements obtained with the MSM were similar to previously reported values obtained with the microsphere method. While ponies were spontaneously breathing, mean ± SD perfusion for aerated and atelectatic lung regions were 4.0 ± 1.9 and 5.0 ± 1.2 mL/min/g of lung tissue, respectively. During mechanical ventilation, values were 4.6 ± 1.2 and 2.7 ± 0.7 mL/min/g of lung tissue at end expiration and 4.1 ± 0.5 and 2.7 ± 0.6 mL/min/g of lung tissue at peak inspiration. Intraobserver agreement was acceptable, but interobserver agreement was lower. Computerized measurements compared well with manual measurements. CLINICAL RELEVANCE: Findings showed that CT angiography and the MSM could be used to measure regional lung perfusion in dorsally recumbent anesthetized ponies. Measurements are repeatable, suggesting that the method could be used to determine efficacy of therapeutic interventions to improve ventilation-perfusion matching and for other studies for which measurement of regional lung perfusion is necessary.

Root cause determination of intraperitoneal catheter obstructions: Insulin amyloid aggregates vs foreign body reaction.

Continuous intraperitoneal insulin infusion, from an implanted insulin pump connected to a catheter that delivers insulin directly to the peritoneal cavity has many clinical advantages for patients with Type 1 diabetes. However, the ongoing incidence of catheter obstructions remains a barrier to the widespread use of this therapy. To date, the root cause of these obstructions remains unknown. Here, a two-year clinical investigation was conducted, along with the development of an animal model to enable a mechanistic investigation into this issue. This novel animal model was able to mimic the catheter obstructions that occur in patients and, fortuitously, at an accelerated rate. This model allowed for independent assessment of each potential cause associated with catheter obstructions to help identify the root cause. Both macroscopic and microscopic analysis were conducted with regards to the onset and progression of catheter obstructions, along with monitoring of insulin delivery. Interestingly, although insulin aggregation occurs in insulin pumps and insulin aggregates were found in some catheter obstructions, insulin is unlikely to be the root cause, since obstructions also occurred in the control groups where only diluent (no insulin) was administered to the animals. Inflammatory cells, different phenotypes of fibroblasts, as well as collagen were observed in all obstructed catheters explanted from the patients and the animals. The presence of these cells and collagen is indicative of a typical foreign body reaction. In addition, the dynamic change in the fibroblasts with respect to morphology, phenotype, and spatial distribution suggests that tissue irritation-mediated epithelial to mesenchymal transition plays a role in catheter obstructions.

Fibrotic Encapsulation Is the Dominant Source of Continuous Glucose Monitor Delays.

Continuous glucose monitor (CGM) readings are delayed relative to blood glucose, and this delay is usually attributed to the latency of interstitial glucose levels. However, CGM-independent data suggest rapid equilibration of interstitial glucose. This study sought to determine the loci of CGM delays. Electrical current was measured directly from CGM electrodes to define sensor kinetics in the absence of smoothing algorithms. CGMs were implanted in mice, and sensor versus blood glucose responses were measured after an intravenous glucose challenge. Dispersion of a fluorescent glucose analog (2-NBDG) into the CGM microenvironment was observed in vivo using intravital microscopy. Tissue deposited on the sensor and nonimplanted subcutaneous adipose tissue was then collected for histological analysis. The time to half-maximum CGM response in vitro was 35 ± 2 s. In vivo, CGMs took 24 ± 7 min to reach maximum current versus 2 ± 1 min to maximum blood glucose (P = 0.0017). 2-NBDG took 21 ± 7 min to reach maximum fluorescence at the sensor versus 6 ± 6 min in adipose tissue (P = 0.0011). Collagen content was closely correlated with 2-NBDG latency (R = 0.96, P = 0.0004). Diffusion of glucose into the tissue deposited on a CGM is substantially delayed relative to interstitial fluid. A CGM that resists fibrous encapsulation would better approximate real-time deviations in blood glucose.

A New Animal Model of Insulin-Glucose Dynamics in the Intraperitoneal Space Enhances Closed-Loop Control Performance.

Current artificial pancreas systems (AP) operate via subcutaneous (SC) glucose sensing and SC insulin delivery. Due to slow diffusion and transport dynamics across the interstitial space, even the most sophisticated control algorithms in on-body AP systems cannot react fast enough to maintain tight glycemic control under the effect of exogenous glucose disturbances caused by ingesting meals or performing physical activity. Recent efforts made towards the development of an implantable AP have explored the utility of insulin infusion in the intraperitoneal (IP) space: a region within the abdominal cavity where the insulin-glucose kinetics are observed to be much more rapid than the SC space. In this paper, a series of canine experiments are used to determine the dynamic association between IP insulin boluses and plasma glucose levels. Data from these experiments are employed to construct a new mathematical model and to formulate a closed-loop control strategy to be deployed on an implantable AP. The potential of the proposed controller is demonstrated via in-silico experiments on an FDA-accepted benchmark cohort: the proposed design significantly outperforms a previous controller designed using artificial data (time in clinically acceptable glucose range: 97.3±1.5% vs. 90.1±5.6%). Furthermore, the robustness of the proposed closed-loop system to delays and noise in the measurement signal (for example, when glucose is sensed subcutaneously) and deleterious glycemic changes (such as sudden glucose decline due to physical activity) is investigated. The proposed model based on experimental canine data leads to the generation of more effective control algorithms and is a promising step towards fully automated and implantable artificial pancreas systems.

Release of (and lessons learned from mining) a pioneering large toxicogenomics database.

AIM: We release the Janssen Toxicogenomics database. This rat liver gene-expression database was generated using Codelink microarrays, and has been used over the past years within Janssen to derive signatures for multiple end points and to classify proprietary compounds. MATERIALS & METHODS: The release consists of gene-expression responses to 124 compounds, selected to give a broad coverage of liver-active compounds. A selection of the compounds were also analyzed on Affymetrix microarrays. RESULTS: The release includes results of an in-house reannotation pipeline to Entrez gene annotations, to classify probes into different confidence classes. High confidence unambiguously annotated probes were used to create gene-level data which served as starting point for cross-platform comparisons. Connectivity map-based similarity methods show excellent agreement between Codelink and Affymetrix runs of the same samples. We also compared our dataset with the Japanese Toxicogenomics Project and observed reasonable agreement, especially for compounds with stronger gene signatures. We describe an R-package containing the gene-level data and show how it can be used for expression-based similarity searches. CONCLUSION: Comparing the same biological samples run on the Affymetrix and the Codelink platform, good correspondence is observed using connectivity mapping approaches. As expected, this correspondence is smaller when the data are compared with an independent dataset such as TG-GATE. We hope that this collection of gene-expression profiles will be incorporated in toxicogenomics pipelines of users.

Effects of pulse-delivered inhaled nitric oxide administration on pulmonary perfusion and arterial oxygenation in dorsally recumbent isoflurane-anesthetized horses.

OBJECTIVE: To image the spatial distribution of pulmonary blood flow by means of scintigraphy, evaluate ventilation-perfusion (VA/Q) matching and pulmonary blood shunting (Qs/Qt) by means of the multiple inert gas elimination technique (MIGET), and measure arterial oxygenation and plasma endothelin-1 concentrations before, during, and after pulse-delivered inhaled nitric oxide (PiNO) administration to isoflurane-anesthetized horses in dorsal recumbency. ANIMALS: 3 healthy adult Standardbreds. PROCEDURES: Nitric oxide was pulsed into the inspired gases in dorsally recumbent isoflurane-anesthetized horses. Assessment of VA/Q matching, Qs/Qt, and Pao2 content was performed by use of the MIGET, and spatial distribution of pulmonary blood flow was measured by perfusion scintigraphy following IV injection of technetium Tc 99m-labeled macroaggregated human albumin before, during, and 30 minutes after cessation of PiNO administration. RESULTS: During PiNO administration, significant redistribution of blood flow from the dependent regions to the nondependent regions of the lungs was found and was reflected by improvements in VA/Q matching, decreases in Qs/Qt, and increases in Pao2 content, all of which reverted to baseline values at 30 minutes after PiNO administration. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of PiNO in anesthetized dorsally recumbent horses resulted in redistribution of pulmonary blood flow from dependent atelectatic lung regions to nondependent aerated lung regions. Because hypoxemia is commonly the result of atelectasis in anesthetized dorsally recumbent horses, the addition of nitric oxide to inhaled gases could be used clinically to alleviate hypoxemia in horses during anesthesia.

Effects of hydration on scintigraphic glomerular filtration rate measured using integral and plasma volume methods in dogs with suspected renal disease.

The current standard scintigraphic method for estimating glomerular filtration rate (GFR) in dogs is the integral method, which normalizes renal GFR to body weight. The plasma volume method, that is normalizing GFR to plasma volume, has been reported to be more physiologically correct. The aim of this prospective study was to test the effect of hydration status on GFR measured by these two methods in a group of dogs with suspected renal disease. Eleven dogs were recruited. All dogs underwent standardized scintigraphic examinations before and after 15 ml/kg of fluid was administered intravenously at 5-7 ml/kg/min. Individual kidney GFR estimates (n = 22) were calculated using both methods and a consensus of two observers who were unaware of clinical findings. Individual kidney GFR increased significantly (P = 0.0008) after fluid administration using the integral method and individual kidney GFR using the plasma volume method remained constant. Percentage differences for individual kidney GFR before and after fluid administration were 31.4 ± 58.1% (change ± 95% CI) for the integral method and 0.1 ± 70% (change ± 95% CI) for the plasma volume method. Intravenously administered fluid increased individual kidney GFR from low to normal in 10 of 22 kidneys using the integral method and in 1 of 22 kidneys using the plasma volume method. Findings supported the use of the plasma volume method for scintigraphic calculation of GFR in dogs with suspected renal disease and indicated that errors of kidney status classification may more likely occur when the integral method is used.

Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants.

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds-chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates.

Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.

BACKGROUND: In this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: The consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: The participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.

Radiographic features of cardiogenic pulmonary edema in dogs with mitral regurgitation: 61 cases (1998-2007).

OBJECTIVE: To evaluate radiographic distribution of pulmonary edema (PE) in dogs with mitral regurgitation (MR) and investigate the association between location of radiographic findings and direction of the mitral regurgitant jet (MRJ). DESIGN: Retrospective case series. ANIMALS: 61 dogs with cardiogenic PE and MR resulting from mitral valve disease (MVD; 51 dogs), dilated cardiomyopathy (9), and hypertrophic cardiomyopathy (1). PROCEDURES: Thoracic radiographs of dogs with Doppler echocardiographic evidence of MR were reviewed for location (diffuse, perihilar, or focal) of PE. Also, direction (central or eccentric) of the MRJ, as evaluated by Doppler color flow mapping (DCFM), and distribution (symmetric or asymmetric) of radiographic findings were evaluated. RESULTS: Diffuse, perihilar, and focal increases in pulmonary opacity were observed in 11 (18.0%), 7 (11.5%), and 43 (70.5%) of 61 dogs, respectively. Radiographic evidence of asymmetric PE in a single lung lobe or 2 ipsilateral lobes was found in 21 dogs, with involvement of only the right caudal lung lobe in 17 dogs. Doppler color flow mapping of the MRJ was available for 46 dogs. Of 31 dogs with a central MRJ, 28 had radiographic findings indicative of symmetric PE. Of 15 dogs with eccentric MRJ, 11 had radiographic evidence of asymmetric PE, and all of these dogs had MVD. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs with cardiogenic PE, a symmetric radiographic distribution of increased pulmonary opacity was predominantly associated with a central MRJ, whereas an asymmetric radiographic distribution was usually associated with eccentric MRJ, especially in dogs with MVD.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Effects of measurement of plasma activity input on normalization of glomerular filtration rate to plasma volume in dogs.

Glomerular filtration rate (GFR) normalized to body fluid volumes to adjust for differing body size and conformation is more physiologically correct than a relationship with body weight (BW). GFR can be normalized to plasma volume by a renographic method that uses the Rutland-Patlak plot with plasma activity and kidney activity inputs. A plasma time-activity curve is obtained from a region of interest (ROI) of the left ventricle (LV), the size of which is in theory not critical. The aims of the study were to evaluate the effect of different LV ROI sizes, the effect of extravascular activity in the thorax over the LV ROI, and different time intervals for the semilogarithmic LV plot. Seventy-two scintigrams were used, with three different-sized automatic and a manual LV ROI, all with and without subtracting extravascular activity, and with LV curve time intervals of 30-120 s and 60-240 s. GFR/plasma volume was not affected by LV ROI sizes but significantly affected by extravascular activity subtraction and different time intervals. Subtracting extravascular activity from the LV ROI did not improve precision, but increased variability caused by different LV ROI sizes and time intervals chosen for the LV plot. The ROI for measuring extravascular activity apparently contained a considerable and variable intravascular component, which when subtracted, created noisy and unreliable LV curves. Manual LV ROI, without extravascular subtraction, and a time interval for LV input between 1 and 4 min are recommended as they gave the least variability determined by statistical analysis. With these methods, normal individual GFR/plasma volume in normal beagle dogs was 29.2 +/- 6.5 ml/min/l.

Toward a checklist for exchange and interpretation of data from a toxicology study.

Data from toxicology and toxicogenomics studies are valuable, and can be combined for meta-analysis using public data repositories such as Chemical Effects in Biological Systems Knowledgebase, ArrayExpress, and Gene Expression Omnibus. In order to fully utilize the data for secondary analysis, it is necessary to have a description of the study and good annotation of the accompanying data. This study annotation permits sophisticated cross-study comparison and analysis, and allows data from comparable subjects to be identified and fully understood. The Minimal Information About a Microarray Experiment Standard was proposed to permit deposition and sharing of microarray data. We propose the first step toward an analogous standard for a toxicogenomics/toxicology study, by describing a checklist of information that best practices would suggest be included with the study data. When the information in this checklist is deposited together with the study data, the checklist information helps the public explore the study data in context of time, or identify data from similarly treated subjects, and also explore/identify potential sources of experimental variability. The proposed checklist summarizes useful information to include when sharing study data for publication, deposition into a database, or electronic exchange with collaborators. It is not a description of how to carry out an experiment, but a definition of how to describe an experiment. It is anticipated that once a toxicology checklist is accepted and put into use, then toxicology databases can be configured to require and output these fields, making it straightforward to annotate data for interpretation by others.

Evaluation of felbamate and other antiepileptic drug toxicity potential based on hepatic protein covalent binding and gene expression.

Felbamate is an antiepileptic drug that is associated with minimal toxicity in preclinical species such as rat and dog but has an unacceptable incidence of serious idiosyncratic reactions in man. Idiosyncratic reactions account for over half of toxicity-related drug failures in the marketplace, and improving the preclinical detection of idiosyncratic toxicities is thus of paramount importance to the pharmaceutical industry. The formation of reactive metabolites is common among most drugs associated with idiosyncratic drug reactions and may cause deleterious effects through covalent binding and/or oxidative stress. In the present study, felbamate was compared to several other antiepileptic drugs (valproic acid, carbamazepine, phenobarbital, and phenytoin), using covalent binding of radiolabeled drugs and hepatic gene expression responses to evaluate oxidative stress/reactive metabolite potential. Despite causing only very mild effects on covalent binding parameters, felbamate produced robust effects on a previously established oxidative stress/reactive metabolite gene expression signature. The other antiepileptic drugs and acetaminophen are known hepatotoxicants at high doses in the rat, and all increased covalent binding to liver proteins in vivo and/or to liver microsomes from human and rat. With the exception of acetaminophen, valproic acid exhibited the highest covalent binding in vivo, whereas carbamazepine exhibited the highest levels in vitro. Pronounced effects on oxidative stress/reactive metabolite-responsive gene expression were observed after carbamazepine, phenobarbital, and phenytoin administration. Valproic acid had only minor effects on the oxidative stress/reactive metabolite indicator genes. The relative ease of detection of felbamate based on gene expression results in rat liver as having potential oxidative stressor/reactive metabolites indicates that this approach may be useful in screening for potential idiosyncratic toxicity. Together, measurements of gene expression along with covalent binding should improve the safety assessment of candidate drugs.

Predictive toxicogenomics approaches reveal underlying molecular mechanisms of nongenotoxic carcinogenicity.

Toxicogenomics technology defines toxicity gene expression signatures for early predictions and hypotheses generation for mechanistic studies, which are important approaches for evaluating toxicity of drug candidate compounds. A large gene expression database built using cDNA microarrays and liver samples treated with over one hundred paradigm compounds was mined to determine gene expression signatures for nongenotoxic carcinogens (NGTCs). Data were obtained from male rats treated for 24 h. Training/testing sets of 24 NGTCs and 28 noncarcinogens were used to select genes. A semiexhaustive, nonredundant gene selection algorithm yielded six genes (nuclear transport factor 2, NUTF2; progesterone receptor membrane component 1, Pgrmc1; liver uridine diphosphate glucuronyltransferase, phenobarbital-inducible form, UDPGTr2; metallothionein 1A, MT1A; suppressor of lin-12 homolog, Sel1h; and methionine adenosyltransferase 1, alpha, Mat1a), which identified NGTCs with 88.5% prediction accuracy estimated by cross-validation. This six genes signature set also predicted NGTCs with 84% accuracy when samples were hybridized to commercially available CodeLink oligo-based microarrays. To unveil molecular mechanisms of nongenotoxic carcinogenesis, 125 differentially expressed genes (P<0.01) were selected by Student's t-test. These genes appear biologically relevant, of 71 well-annotated genes from these 125 genes, 62 were overrepresented in five biochemical pathway networks (most linked to cancer), and all of these networks were linked by one gene, c-myc. Gene expression profiling at early time points accurately predicts NGTC potential of compounds, and the same data can be mined effectively for other toxicity signatures. Predictive genes confirm prior work and suggest pathways critical for early stages of carcinogenesis.

Hepatic expression of heme oxygenase-1 and antioxidant response element-mediated genes following administration of ethinyl estradiol to rats.

Heme oxygenase-1 (HO-1) is one of several enzymes induced by hepatotoxicants, and is thought to have an important protective role against cellular stress during liver inflammation and injury. The objective of the present study was to evaluate the role of HO-1 in estradiol-induced liver injury. A single dose of ethinyl estradiol (500 mg/kg, po) resulted in mild liver injury. Repeated administration of ethinyl estradiol (500 mg/kg/day for 4 days, po) resulted in no detectable liver injury or dysfunction. Using RT-PCR analysis, we demonstrate that HO-1 gene expression in whole liver tissue is elevated (>20-fold) after the single dose of ethinyl estradiol. The number and intensity of HO-1 immunoreactive macrophages were increased after the single dose of ethinyl estradiol. HO-1 expression was undetectable in hepatic parenchymal cells from rats receiving Methocel control or a single dose of ethinyl estradiol, however cytosolic HO-1 immunoreactivity in these cells after repeated dosing of ethinyl estradiol was pronounced. The increases in HO-1 mRNA and HO-1 immunoreactivity following administration of a single dose of ethinyl estradiol suggested that this enzyme might be responsible for the observed protection of the liver during repeated dosing. To investigate the effect of HO-1 expression on ethinyl estradiol-induced hepatotoxicity, rats were pretreated with hemin (50 micromol/kg, ip, a substrate and inducer of HO-1), with tin protoporphyrin IX (60 micromol/kg, ip, an HO-1 inhibitor), or with gadolinium chloride (10 mg/kg, iv, an inhibitor/toxin of Kupffer cells) 24 h before ethinyl estradiol treatment. Pretreatment with modulators of HO-1 expression and activity had generally minimal effects on ethinyl estradiol-induced liver injury. These data suggest that HO-1 plays a limited role in antioxidant defense against ethinyl estradiol-induced oxidative stress and hepatotoxicity, and suggests that other coordinately induced enzymes are responsible for protection observed with repeated administration of high doses of this compound.

Application of genomics in preclinical drug safety evaluation.

Understanding the response of biological systems to xenobiotics is fundamental to the evaluation of drug safety. Toxicologists have traditionally gathered pathological, morphological, chemical and biochemical information from in vivo studies of preclinical species in order to assess drug safety and to determine how new drugs can be safely administered to the human patient population. In recent years the emerging "-omics" technologies have been developed and integrated into preclinical studies in order to better assess drug safety by gaining information on the cellular and molecular events underlying adverse drug reactions. Genomics approaches in particular have become readily available and are being applied in several stages of drug development. The burgeoning literature on what has become known as "toxicogenomics" has for the most part highlighted successful applications of gene expression profiling in predictive toxicology, enabling decisions to be made on the developability of a compound early in the drug development process. It is also becoming apparent that toxicogenomic approaches are good starting points to develop experiments designed to gain a mechanistic insight into drug toxicities within and across species. Gene expression arrays permit the measurement of responses of essentially all the genes in the entire genome to be monitored, and knowledge of the function of the genes affected can identify the potential mechanisms to then be confirmed using conventional biochemical, toxicological and pathological approaches. As toxicologists put these technologies into practice they build up a knowledge base to better characterize toxicities at the molecular level and to make the search for much needed, novel biomarkers of toxicity more achievable.