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Environmental Factor, January 2008, National Institute of Environmental Health Sciences

Intramural Papers of the Month

By Robin Arnette
January 2008

Genes in Blood Can Predict Harmful Levels of Acetaminophen

A new study, published in a recent issue of PNAS and funded by the NIH and the NIEHS, reports that gene expression data obtained from blood cells can predict harmful levels of acetaminophen exposure.

Millions of people around the world use acetaminophen to reduce fever and relieve pain, but too much of it or heavy prolonged use may lead to acetaminophen intoxication and eventually severe liver damage. With this information in mind, the team set out to determine whether gene expression patterns derived from blood cells could indicate levels of acetaminophen exposure.

Researchers used a rat model to generate a training data set consisting of genomic, clinical chemistry, histopathology and hematology data. They then determined the gene sets that would act as indicators of subtoxic/nontoxic and toxic dose levels of acetaminophen and used microarray analysis to screen thousands of genes simultaneously. Genetic markers derived from a rat blood test data set indicated from several prediction methodologies that the level of accuracy was 88.9–95.8 percent. After determining the human gene orthologs, they found that the blood from overdose victims contained different patterns of expression from the cluster of indicator genes.

This work represents a possible first step in the development of a diagnostic test that can identify patients who are at risk of an acetaminophen overdose.

Citation: Bushel PR, Heinloth AN, Li J, Huang L, Chou JW, Boorman GA, Malarkey DE, Houle CD, Ward SM, Wilson RE, et al. (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17984051&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) 2007. Blood gene expression signatures predict exposure levels. PNAS 104(46):18211-18216.

DNA Polymerase Gamma Mutant Causes Enhanced Oxidative Stress

NIEHS scientists have found a biochemical link that explains the enhanced oxidative stress and DNA mutagenesis that occurs in patients suffering from progressive external ophthalmoplegia (PEO) with Parkinson symptoms. The research was funded by NIEHS and published in Human Molecular Genetics.

Previous work had demonstrated that the DNA polymerase gamma (pol γ) present in these patients carried a tyrosine to cysteine mutation (Y955C) that, in turn, induced replication stalling and DNA deletions. Because these patients also develop Parkinson symptoms, and animal models show enhanced incorporation of 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-oxo-dG), a common oxidative lesion into mitochondrial DNA, the team wanted to determine why the Y955C pol g allowed more 8-oxo-dG in DNA and oxidative stress.

They performed DNA polymerase reactions with both wild type and mutant polymerases and then developed molecular models with 8-oxo-dG in the active site and the incorporation of different dNTPs.

The results indicated that the Y955C pol γ was more likely to insert the 8-oxo-dG lesion instead of the normal nucleotide dGTP. Once the 8-oxo-dG was present in DNA, the Y955C pol γ was also more likely to incorporate a dATP opposite this lesion which results in a mutation. The research explains why these PEO patients present phenotypes normally associated with oxidative stress and Parkinson disease.

Citation: Graziewicz MA, Bienstock RJ, Copeland WC. (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17725985&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) 2007. The DNA polymerase γ Y955C disease variant associated with PEO and parkinsonism mediates the incorporation and translesion synthesis opposite 7,8-dihydro-8-oxo-2’-deoxyguanosine. Hum Mol Genet 16(22):2729-2739.

Regulatory Roles of Two Retinoid-related Orphan Receptor Isoforms

In a study funded by NIEHS and the Clinical Nutrition Research Unit Center and published in Physiological Genomics, researchers reported that retinoid-related orphan receptors alpha (RORα) and gamma (RORγ), expressed in the liver, have multiple physiological roles — including regulating the genes encoding several phase I and phase II enzymes, such as cytochrome P450.

The researchers had previously shown that RORα and RORγ were coexpressed in a variety of tissues including fat, kidney and liver. The team set out to identify their physiological roles in liver and, in addition, wanted to determine whether these two receptors exhibited redundant functions as has been shown for several other nuclear receptors. They used RORα-deficient staggerer (sg) (RORαsg/sg) mice, RORγ-/- mice and RORαsg/sgRORγ-/- double knockout mice and microarray analysis to compare the gene expression profiles from the livers of these mice to those of wild-type mice.

Gene expression analysis indicated that some liver genes were specifically regulated by either RORα or RORγ, while other genes were regulated by both receptors indicating functional redundancy. Most importantly, lack of RORα and RORγ affected the expression of phase I and phase II enzymes, suggesting these receptors play a role in the regulation of metabolic homeostasis.

Citation: Kang HS, Angers M, Beak JY, Wu X, Gimble JM, Wada T, Xie W, Collins JB, Grissom SF, Jetten AM. (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17666523&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) 2007. Gene expression profiling reveals a regulatory role for RORα and RORγ in phase I and phase II metabolism. Physiol Genomics 31(2):281-294.

Glucocorticoid Receptor Isoforms Regulate Bone Cell Apoptosis

A newly published study found that the glucocorticoid receptor D (GR-D) isoform, one of eight translational glucocorticoid receptor isoforms, exhibited reduced cell-killing capacity in bone cells while still repressing nuclear factor kappa-beta, which is involved in many inflammatory diseases. This data is important because glucocorticoids — generally prescribed to treat a variety of illnesses such as cancer, inflammation and autoimmune disorders — selectively kill bone cells while protecting other cells such as liver and skin. NIEHS researchers published this data in Molecular and Cellular Biology. The work was supported by NIEHS and a division grant from the Department of Medicine, Northwestern University.

Glucocorticoids provide a medical benefit, but patients who use them chronically suffer debilitating and sometimes life-threatening side effects, such as muscle wasting and osteoporosis. To determine the enzymes important in glucocorticoid-induced apoptosis in bone, the team treated human osteoblastic cells U-2 OS with dexamethasone and used small interfering RNA (siRNA) and PCR to determine that granzyme A and caspase-6 were involved. Results from microarray analysis indicated that each GR isoform had a distinct set of glucocorticoid target genes and specific functions.

These findings may lead to the development of safe glucocorticoids that exhibit reduced side effects.

Citation: Lu NZ, Collins JB, Grissom SF, Cidlowski JA. (http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17682054&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum) 2007. Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor. Mol Cell Biol 27(20):7143-7160.

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