Stella O. Sieber

Methapyrilene Toxicity: Anchorage of Pathologic Observations to Gene Expression Alterations

Methapyrilene (MP) exposure of animals can result in an array of adverse pathological responses including hepatotoxicity. This study investigates gene expression and histopathological alterations in response to MP treatment in order to 1) utilize computational approaches to classify samples derived from livers of MP treated rats based on severity of toxicity incurred in the corresponding tissue, 2) to phenotypically anchor gene expression patterns, and 3) to gain insight into mechanism(s) of methapyrilene hepatotoxicity. Large-scale differential gene expression levels associated with the exposure of male Sprague—Dawley rats to the rodent hepatic carcinogen MP for 1, 3, or 7 days after daily dosage with 10 or 100 mg/kg/day were monitored. Hierarchical clustering and principal component analysis were successful in classifying samples in agreement with microscopic observations and revealed low-dose effects that were not observed histopathologically. Data from cDNA microarray analysis corroborated observed histopathological alterations such as hepatocellular necrosis, bile duct hyperplasia, microvesicular vacuolization, and portal inflammation observed in the livers of MP exposed rats and provided insight into the role of specific genes in the studied toxicological processes.

Acetaminophen dosing of humans results in blood transcriptome and metabolome changes consistent with impaired oxidative phosphorylation

The diagnosis and management of drug‐induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound‐specific changes in the peripheral blood (PB) transcriptome in rodents, suggesting that the blood transcriptome might provide new biomarkers of DILI. To investigate in humans, we used DNA microarrays as well as serum metabolomic methods to characterize changes in the transcriptome and metabolome in serial PB samples obtained from six healthy adults treated with a 4‐g bolus dose of acetaminophen (APAP) and from three receiving placebo. Treatment did not cause liver injury as assessed by traditional liver chemistries. However, 48 hours after exposure, treated subjects showed marked down‐regulation of genes involved in oxidative phosphorylation/mitochondrial function that was not observed in the placebos (P < 1.66E‐19). The magnitude of down‐regulation was positively correlated with the percent of APAP converted to the reactive metabolite N‐acetyl‐p‐benzoquinone‐imide (NAPQI) (r= 0.739;P= 0.058). In addition, unbiased analysis of the serum metabolome revealed an increase in serum lactate from 24 to 72 hours postdosing in the treated subjects alone (P< 0.005). Similar PB transcriptome changes were observed in human overdose patients and rats receiving toxic doses. Conclusion: The single 4‐g APAP dose produced a transcriptome signature in PB cells characterized by down‐regulation of oxidative phosphorylation genes accompanied by increased serum lactate. Similar gene expression changes were observed in rats and several patients after consuming hepatotoxic doses of APAP. The timing of the changes and the correlation with NAPQI production are consistent with mechanisms known to underlie APAP hepatoxicity. These studies support the further exploration of the blood transcriptome for biomarkers of DILI. (HEPATOLOGY 2010.)

ATM-Dependent and -Independent Gene Expression Changes in Response to Oxidative Stress, Gamma Irradiation, and UV Irradiation

Abstract Heinloth, A. N., Shackelford, R. E., Innes, C. L., Bennett, L., Li, L., Amin, R. P., Sieber, S. O., Flores, K. G., Bushel, P. R. and Paules, R. S. ATM-Dependent and -Independent Gene Expression Changes in Response to Oxidative Stress, Gamma Irradiation, and UV Irradiation. Radiat. Res. 160, 273–290 (2003). Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G1-, S- and G2-phase cell cycle checkpoint functions in response to γ irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to γ irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G1 checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either γ radiation (5 Gy), oxidative stress (75 µM t-butyl-hydroperoxide), or UV radiation (7.5 J/m2), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1.

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.

Identification of Distinct and Common Gene Expression Changes After Oxidative Stress and Gamma and Ultraviolet Radiation

The human genome is exposed to many different kinds of DNA‐damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (γ) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G1 checkpoint induction and slow down in early S‐phase progression, as reflected by a comparable reduction in cyclin E–associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either γ radiation (5 Gy), oxidative stress (75 μM of tert‐butyl hydroperoxide (t‐butyl‐OOH)), or UV radiation (UVC) (7.5 J/m2) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to γ radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E–associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after γ radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, γ radiation had its major influence on cell‐cycle progression in S‐phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage. Published 2003 Wiley‐Liss, Inc.

MAPS: a microarray project system for gene expression experiment information and data validation

SUMMARY MAPS is a MicroArray Project System for management and interpretation of microarray gene expression experiment information and data. Microarray project information is organized to track experiments and results that are: (1) validated by performing analysis on stored replicate gene expression data; and (2) queried according to the biological classifications of genes deposited on microarray chips.

AhR-mediated gene expression in the developing mouse telencephalon.

We hypothesize that TCDD-induced developmental neurotoxicity is modulated through an AhR-dependent interaction with key regulatory neuronal differentiation pathways during telencephalon development. To test this hypothesis we examined global gene expression in both dorsal and ventral telencephalon tissues in E13.5 AhR-/- and wildtype mice exposed to TCDD or vehicle. Consistent with previous biochemical, pathological and behavioral studies, our results suggest TCDD initiated changes in gene expression in the developing telencephalon are primarily AhR-dependent, as no statistically significant gene expression changes are evident after TCDD exposure in AhR-/- mice. Based on a gene regulatory network for neuronal specification in the developing telencephalon, the present analysis suggests differentiation of GABAergic neurons in the ventral telencephalon is compromised in TCDD exposed and AhR-/- mice. In addition, our analysis suggests Sox11 may be directly regulated by AhR based on gene expression and comparative genomics analyses. In conclusion, this analysis supports the hypothesis that AhR has a specific role in the normal development of the telencephalon and provides a mechanistic framework for neurodevelopmental toxicity of chemicals that perturb AhR signaling.

ATM Requirement in Gene Expression Responses to Ionizing Radiation in Human Lymphoblasts and Fibroblasts

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function. (Mol Cancer Res 2006;4(3):197–207)

Blood transcript immune signatures distinguish a subset of people with elevated serum ALT from others given acetaminophen.

The diagnosis of drug‐induced liver injury is hindered by the limited utility of clinical chemistries. We have shown that hepatotoxicants can produce peripheral blood transcriptome “signatures” (PBTS) in rodents and humans. In this study, 42 adults were treated with acetaminophen (APAP; 1 g every 6 hours) for seven days, followed by three days of placebo. Eleven subjects received only placebo. After five days, 12 subjects (30%) had increases in serum alanine aminotransferase (ALT) levels (“responders”). PBTS of 707 and 760 genes, respectively, could distinguish responders and nonresponders from placebos. Functional analysis of the responder PBTS revealed increased expression of genes involved in TH2‐mediated and innate immune responses, whereas the nonresponders demonstrated increased gene expression consistent with a tolerogenic immune response. Taken together, these observations suggest that the clinical subjects with transient increases in serum ALT failed to maintain or intensify a hepatic tolerogenic immune response.