Tox/Path team takes on differential gene expression - Environews: NCT update

Environmental Health Perspectives, Nov 15, 2003 by Michael L. Cunningham, Richard Irwin, Gary Boorman

Toxicology and pathology are critical elements in toxicogenomics studies. The National Center for Toxicogenomics (NCT) has established a Tox/Path team that includes both NCT scientists and toxicologists and pathologists from the National Toxicology Program (NTP). The Tox/Path team advises the NCT by formulating research questions, designing studies, and mining databases for information. NTP members of the Tox/Path team also bring their toxicogenomics experience to bear on study design and assessment of proposed NTP toxicogenomics evaluations.

One of the goals of the NCT is to determine whether phenotypic alterations can be associated with differential gene expression (DGE) changes. To help meet this goal, the Tox/Path team has designed a series of studies to elicit different responses within the liver to determine whether DGE can distinguish specific pathological processes.

Correlating Phenotypic Alterations with DGE Changes

Initial Tox/Path liver studies focused on acetaminophen, a compound that causes centrilobular hepatic necrosis. Acetaminophen has been widely studied both because of its importance as a drug for humans (misuse of acetaminophen is the most common reason for admission to emergency rooms with acute liver toxicity) and because it exerts a specific regional acute centrilobular (zone 3) necrosis in the liver.

Pathological evaluations awaiting publication have revealed that acetaminophen-induced hepatic necrosis is not uniformly distributed throughout the liver. Further study has revealed differences in the extent of lesions among the liver lobules. The Tox/Path team has designed studies to evaluate the distribution of lesions throughout the liver. These studies will use magnetic resonance imaging to obtain a three-dimensional view of the liver. This technology may also allow the researchers to follow the development of lesions using noninvasive techniques, and possibly to correlate data obtained by noninvasive techniques with the development of lesions.

In addition to the acetaminophen studies, a second compound under study is the industrial chemical carbon tetrachloride, a known liver carcinogen also known to cause acute hepatic centrilobular necrosis. Comparison of acetaminophen and carbon tetrachloride may help to identify DGE changes that are specific to centrilobular hepatic necrosis and possibly differentiate between pathways to toxicity.

Allyl alcohol, also a large-scale industrial chemical, causes a different form of liver toxicity: acute hepatic periportal (zone 1) necrosis. Allyl alcohol will be contrasted with acetaminophen and carbon tetrachloride to further probe variable genetic pathways to toxicity. Other chemicals that target specific subpopulations in the liver such as biliary epithelial or endothelial cells are under consideration for study.

One issue in associating phenotypic alterations with DGE changes involves histological sampling relative to sampling for gene expression. In some cancer studies, a frozen-tissue histological analysis is performed on each sample before is it subjected to RNA isolation for gene expression. Although this provides a direct morphological diagnosis for each DGE sample, such sampling is too time-consuming and expensive for most toxicogenomics studies. The Tox/Path team is exploring means for taking histological samples immediately adjacent to the samples taken for DGE analysis to ensure the least amount of variance in the tissue samples used for different assays

Julie Foley, a researcher in the Laboratory of Experimental Pathology, is investigating yet another sampling technology: laser-capture microscopy coupled with RNA amplification for gene expression This method would allow regional sampling of the liver, for example of centrilobular hepatocytes versus periportal hepatocytes. Comparing such samples is critical for hepatic toxicants where the lesions appear regionally. Laser-capture microscopy would also allow NCT researchers to target specific cell populations within the liver, taking DGE from the tissue to the cellular level.

Controlling the Variables

There are many parameters that may affect toxicogenomics study results, and the experimental details are crucial to DGE interpretation. For example, the composition of the test animal diet and circadian rhythms can profoundly affect gene expression. Due to their nocturnal nature, rodents will naturally eat during the night and sleep during the day, resulting in diurnal differences in liver glycogen and glutathione content that affect metabolism and toxicity of compounds. This circadian cycle has profound effects on DGE in the liver.

NCT protocols have been designed to control for time of dosing, light/dark cycles, feeding schedules, time of tissue collection, and other factors that may influence DGE. Where the palatability of the feed may induce changes in the time or amount of feed consumed, appropriate controls are included. The decision to fast animals overnight prior to morning dosing is questionable for DGE studies, because fasting is a powerful stressor. Room temperature, humidity, number of animals per cage, and even the person conducting the experiment have all been suggested to contribute to differences in transcription. The Tox/Path team is still considering how to control for these variables.