Sandra M. Ward

Blood gene expression signatures predict exposure levels

To respond to potential adverse exposures properly, health care providers need accurate indicators of exposure levels. The indicators are particularly important in the case of acetaminophen (APAP) intoxication, the leading cause of liver failure in the U.S. We hypothesized that gene expression patterns derived from blood cells would provide useful indicators of acute exposure levels. To test this hypothesis, we used a blood gene expression data set from rats exposed to APAP to train classifiers in two prediction algorithms and to extract patterns for prediction using a profiling algorithm. Prediction accuracy was tested on a blinded, independent rat blood test data set and ranged from 88.9% to 95.8%. Genomic markers outperformed predictions based on traditional clinical parameters. The expression profiles of the predictor genes from the patterns extracted from the blood exhibited remarkable (97% accuracy) transtissue APAP exposure prediction when liver gene expression data were used as a test set. Analysis of human samples revealed separation of APAP-intoxicated patients from control individuals based on blood expression levels of human orthologs of the rat discriminatory genes. The major biological signal in the discriminating genes was activation of an inflammatory response after exposure to toxic doses of APAP. These results support the hypothesis that gene expression data from peripheral blood cells can provide valuable information about exposure levels, well before liver damage is detected by classical parameters. It also supports the potential use of genomic markers in the blood as surrogates for clinical markers of potential acute liver damage.

Gene expression response in target organ and whole blood varies as a function of target organ injury phenotype

This report details the standardized experimental design and the different data streams that were collected (histopathology, clinical chemistry, hematology and gene expression from the target tissue (liver) and a bio-available tissue (blood)) after treatment with eight known hepatotoxicants (at multiple time points and doses with multiple biological replicates). The results of the study demonstrate the classification of histopathological differences, likely reflecting differences in mechanisms of cell-specific toxicity, using either liver tissue or blood transcriptomic data.

Hemolytic anemia, thrombosis, and infarction in male and female F344 rats following gavage exposure to 2-butoxyethanol

2-butoxyethanol (BE; ethylene glycol monobutyl ether) is used extensively in the manufacture of a wide range of domestic and industrial products which may result in human exposure and toxicity. BE causes severe hemolytic anemia in male and female rats and mice. In a recent report, female F344 rats exposed to 500 ppm BE by inhalation and sacrificed moribund on day 4 of treatment exhibited disseminated thrombosis associated with infarction in several organs. In contrast, no such lesions were observed in male rats similarly exposed to BE. Additional studies were therefore undertaken to compare the effects of BE in rats of both sexes. Rats received 250 mg BE/kg/day by gavage for 1, 2 or 3 days and were sacrificed 24 or 48 hr after the last dose. Control rats received 5 ml/kg water. Progressive time-dependent hemolytic anemia--macrocytic, hypochromic, and regenerative--was observed in both sexes of rats exposed to BE. Additionally, BE caused significant morphological changes in erythrocytes, first observed 24 hr after a single dose, including stomatocytosis, macrocytosis with moderate rouleaux formation, and spherocytosis. These morphological changes became progressively more severe as BE dosing continued and included the occasional occurrence of schistocytes and ghost cells, rouleaux formation in rats of both sexes, and an increased number of red blood cells with micronuclei in female rats. Overall, the progression of hemolytic anemia and morphological changes as a function of the number of days of exposure varied with gender and suggested a faster onset of hemolysis in female rats. The range of BE-related histopathological changes noted in both sexes was comparable; however, while these lesions were observed in female rats following a single dose, similar effects were first observed in males after 3 consecutive days of exposure to BE. Pathological changes involved disseminated thrombosis in the lungs, nasal submucosa, eyes, liver, heart, bones and teeth, with evidence of infarction in the heart, eyes, teeth and bones. Hemoglobinuric nephrosis and splenic extramedullary hematopoiesis were also noted. An apparent correlation between the severity of hemolytic anemia and subsequent disseminated thrombosis in BE-treated rats is proposed. Thrombosis may be related to intravascular hemolysis, which could be triggered by procoagulant release and/or alterations in erythrocyte morphology, as well as increased rigidity.

Comparison of the hematologic effects of 2-butoxyethanol using two types of hematology analyzers

Earlier reports from this laboratory indicated that 2-butoxyethanol (BE) causes acute hemolytic anemia in rats as evidenced by a time- and dose-dependent decrease in the number of red blood cells, in hemoglobin concentrations, and in hematocrits (HCT). Subsequent studies showed that treatment with BE causes an early increase in HCT and mean cell volume (MCV). Since this effect went undetected in our early work and resulted in the publication of inaccurate information, present studies were designed to reinvestigate the hematologic effects of BE using the laser-based hematology analyzer which was used in the early studies and an impedance-based hematology analyzer, simultaneously. Packed cell volumes (PCV; spun HCT) were also performed on all blood samples. Male F344 rats were treated with 0, 125, 250, or 500 mg BE/kg (po) and blood was collected from the retro-orbital venous plexus at 1, 2, 4, 8, and 24 hr after dosing. Hematology profiles of BE-treated rats obtained from the impedance-based analyzer showed an early dose- and time-dependent increase in HCTs and MCVs. In contrast, analysis of the same blood samples using the laser-based analyzer showed a dose- and time-dependent decrease in HCTs with little or no change in MCVs. Changes observed in PCVs were consistent with results obtained from the impedance-based analyzer. Therefore, under the experimental conditions of this and previous studies, the laser-based analyzer was unable to detect early increases in HCTs and MCVs in rats treated with BE. Finally, these data explain the different principles utilized to measure cell size by both instruments as they relate to the hematologic effects of BE. Moreover, present data show that BE-induced hemolysis of erythrocytes is preceded by a quantitatively unique and massive swelling suggesting the erythrocyte membrane as the target.

Inhalation Toxicity of Phosphine for Fischer 344 Rats and B6C3F1 Mice

AbstractBecause of the potential increased use of phosphine (PH3) as a fumigant and the lack of adequate toxicity data, short-term inhalation studies were conducted to characterize the toxicity of PH3 for Fischer 344 (F344) rats and B6C3F1 mice. Male rats and mice were exposed to 0, 1, 5, or 10 ppm PH3 for up to 4 days, and males and females to 0, 1.25, 2.5, or 5 ppm for 2 wk. In the 4-day study, all rats died by the end of the third exposure to 10 ppm, and all mice were euthanized in moribund condition after the fourth exposure to 10 ppm. Clinical pathology data were obtained only for mice, due to early mortality of rats. There were no significant treatment-related effects in hematological indices in mice exposed to 1 or 5 ppm; at 10 ppm there was a moderate anemia, and leukocyte counts were significantly decreased. There were significant biologically relevant increases in serum activity of alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) and in the concentration of urine nitrogen (UN) at ...

INHALATION TOXICITY STUDIES OF THE α,β-UNSATURATED KETONES: Ethyl Vinyl Ketone

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity ofα,β -unsaturated ketones. Ethyl vinyl ketone (EVK) was selected for study because it is a representative straight-chain aliphaticα,β -unsaturated ketone with extensive use and widespread exposure. Short-term inhalation studies of EVK were conducted to provide toxicity data for comparison with the relatedα,β -unsaturated ketones 2-cyclohexene-1-one (CHX) and methyl vinyl ketone (MVK). These data will be used in designing chronic toxicity and carcinogenicity studies of these ketones. Male and female F344 rats and B6C3F1 mice were exposed to 0, 2, 4, or 8 ppm EVK 6 h/day, 5 days/wk for 13 wk. The nasal cavity was the major target organ of EVK in both rats and mice. Pathologic findings in both the olfactory and respiratory epithelium were observed. Lesions consisted primarily of olfactory epithelial necrosis, atrophy and regeneration, and/or hyperplasia and squamous metaplasia of the respiratory epithelium. Squamous metaplasia of the respiratory epithelium was present in all rats and mice exposed to 4 and 8 ppm EVK, and these lesions were more severe in rats than in mice. Few systemic effects were observed in rats and mice exposed to EVK. A transient decrease in total leukocytes due to decrements in lymphocyte and monocyte populations was present in male rats after exposure to 8 ppm for 3 and 21 days; however, this effect was not present after exposure for 13 wk. There were no chemicalrelated effects on micronucleus formation in mice, or on sperm motility and vaginal cytology in either species. EVK, like otherα,β -unsaturated ketones, is a reactive, directacting gaseous irritant with toxicity limited primarily to the upper respiratory tract.