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.
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Looking at livers. The Tox/Path
group is using rat liver studies to elucidate whether phenotypic alterations
can be associated with differential gene expression changes.
image
credit: MRPath |
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.
Chemical and toxicokinetic parameters are also important to toxicogenomics
studies. Toxicokinetic data help in the selection of time points depending on
peak chemical or metabolite concentration in the target tissue (time points
are often selected when the chemical can be expected to be cleared from the
tissue). Understanding when pathological lesions may appear and the development
of the pathological response to injury is also important in determining the
time intervals for sampling for DGE.
The route of exposure is critical because it can influence serum and tissue
concentration levels, and also affects the kinetics of distribution and elimination
of the compound. Intravenous and intraperitoneal exposure results in faster
and higher plasma and tissue concentrations than oral gavage, dietary, or drinking
water exposure. Unlike oral exposure, in which the liver is exposed primarily
through the first pass portal venous blood, intravenous and intraperitoneal
routes expose the liver by arterial perfusion. Enzymes within the stomach, the
intestine, and the cells of the intestinal wall may modify many chemicals. Selection
of the route and duration of exposure to match expected human exposure is the
choice for most studies.
Variables in the animal model also may influence toxicogenomics studies. Strain
differences between commercially available rodents may, in some cases, reflect
differences in metabolic rates. Murine viruses and pathogenic bacteria confound
experiments, so there is an emphasis on specific pathogen-free rodent sources
and an active sentinel animal program.
Selection of the appropriate indicators of toxicity is highly important. If
the purpose of the toxicogenomics study is to evaluate the gene changes during
progression of a toxic effect (such as necrosis or apoptosis), then adequate
documentation of that altered phenotype is critical. For many experiments, the
standard battery of clinical chemistry and histopathology assays is sufficient
to document the desired phenotypic changes. However, hematological evaluations
may be necessary if alterations in blood cell number or composition are suspected.
Special histopathological or immunohistochemistry stains may be needed to document
apoptosis, increased cell proliferation, or other end points. Subtle changes
that occur at low doses may require ultrastructure analysis. For subtle changes,
immersion fixation of tissue samples for ultrastructure analysis may not be
adequate, and a special study with fixation by liver perfusion may be necessary
to avoid artifacts of fixation.
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It’s all in the details. Tox/Path researchers are designing toxicogenomics studies to control for
a diverse array of variables such as animal strain, circadian cycle, time
of tissue sampling, animal housing, and others, all of which may affect
differential gene expression.
image credit: Digital Vision |
When the goal of the study is to translate the results to clinical practice,
selection of surrogate tissues should be considered. Blood sampling for DGE,
which offers the advantage of multiple sampling over time with minimal distress
to the animal, is feasible, although not fully validated. Proteomic analysis
of serum or plasma may also be performed on these samples, and may prove to
be as useful as DGE analysis. Extensive validation is still required at the
present time. However, the value of being able to follow gene changes in an
easily acquired human sample makes the effort to develop blood sampling worthwhile.
Challenge and Promise
One challenge for correlating pathology diagnoses with DGE analysis involves
standardizing pathology terminology. Especially with acute toxicities, multiple
morphological diagnoses can be accurate and convey essentially the same information,
and yet use different terms. To address this communication problem, the NCT
is utilizing the NTP approach whereby each study has a study pathologist, a
reviewing pathologist, and a pathology panel (known as a pathology working group)
to ensure consistency and uniformity of diagnoses within and across studies.
Tox/Path researcher Dave Malarkey is also working with the International Life
Sciences Institute on an effort to standardize pathology terminology for toxicogenomics
studies.
Another means of informing this process may be to include a description of
the diagnostic process in the published study results and in databases. For
example, with acute acetaminophen exposure, the process is acute hepatic centrilobular
necrosis and repair; however, specific morphological diagnoses vary with time
and dose.
Another challenge for toxicogenomics studies lies in dealing with the rapidly
evolving technology, the extensive literature on each chemical, and the vast
amount of data generated by even a modest experiment. But including multiple
disciplines in study design, study conduct, data mining, and data interpretation
is proving useful to the NCT teams, and provides an added benefit of a camaraderie
that is helpful in combining resources to face a daunting list of differentially
expressed genes. Both toxicologists and pathologists have the background and
training to contribute to toxicogenomics. The NCT Tox/Path team allows both
disciplines to bring their strengths to bear on the issues.
The task of the Tox/Path team is exciting, if at times overwhelming. The potential
advantage of adding genomic technology to toxicology evaluations is vast. Shorter
studies, fewer animals, and less expense are among the obvious advantages. Far
greater potential benefits include the ability to recognize precursor lesions
or biomarkers of effect that may be applicable to humans exposed in the workplace
or the environment. Furthermore, understanding the pathways and mechanisms of
toxicity may lead to better therapeutic interventions and treatment of diseases.
Michael L. Cunningham, Richard Irwin, and Gary Boorman