Download Applications for Toxicogenomics in Risk Assess

TOXICOLOGICAL SCIENCES 105(2), 233–234 (2008)
doi:10.1093/toxsci/kfn138
Advance Access publication August 6, 2008
TOXICOLOGICAL HIGHLIGHT
Gene Expression, Dose-Response, and Phenotypic Anchoring:
Applications for Toxicogenomics in Risk Assessment
George P. Daston1
Central Product Safety, Procter & Gamble, Cincinnati, Ohio 45253
Received July 3, 2008; revised July 3, 2008; accepted July 5, 2008
There are a number of ways in which global analysis of gene
expression may be useful in improving the way we assess the
risk of chemical agents, especially at the concentrations to
which people are likely to be exposed. The methodology for
assessing changes in gene expression is quite sensitive: most of
the methods in use today have attomolar or even subattomolar
sensitivity. That sensitivity, coupled with the observation that
virtually all toxic responses are accompanied by changes in gene
expression, suggests that it is possible to use gene expression
analysis to determine whether responses at the molecular level
continue to occur at dose levels below those that produce frankly
adverse effects. This kind of information will be extremely
important in addressing controversies about the likelihood of
occurrence of those adverse effects at ambient exposure levels.
This issue of Toxicological Sciences includes a seminal paper
on the use of global gene expression analysis to support
conclusions about dose-dependent transitions in toxic
responses, in this case, the response of the rodent nasal
epithelium to formaldehyde (Andersen et al., 2008). These
investigators correlate histological and gene expression changes
in the nose of the formaldehyde-exposed rat. The study design
had both time and dose dimensions. The exposure levels were
chosen such that the two lowest dosages did not produce
discernible effects at the histological level or changes in cell
proliferation, nor were they tumorigenic in a cancer bioassay.
The lack of effect at a cellular level suggests that there are
thresholds for the adverse responses; the gene expression data
confirm that the responses are not linear at low doses. This
paper provides a novel way to use microarray data in doseresponse assessment, the grouping of genes according to gene
ontology terms, such that groups of genes that control particular
functions are considered together.
Changes in gene expression accompany virtually every toxic
response (Nuwaysir et al., 1999). Several years of research in
many laboratories has demonstrated that specific mechanisms
1
To whom correspondence should be addressed at Central Product Safety,
Procter & Gamble, Cincinnati, OH 45253. E-mail: [email protected].
Fax: 1-513-627-0323.
of toxicity elicit characteristic profiles of gene expression,
suggesting that gene expression may be integral to the toxic
response or to the cell’s attempts to repair toxicant-induced
damage. Perhaps the most obvious examples of gene expression driving the toxic response are the responses to ligands of
nuclear receptors, as much of the signal transduction for this
pathway depends on gene expression (Boverhof et al., 2008;
Naciff et al., 2002). Evaluations of the temporal response of the
uterus to an estrogenic stimulus illustrate this point: the
families of genes responsible for eliciting each stage of the
estrous cycle are expressed prior to the appearance of cellular
and morphological changes in the tissue; for example, the
genes that control cell division and suppress apoptosis are
expressed a few hours before the onset of measurable cell
proliferation, transcription factors appear a few hours before
changes in cellular differentiation, etc. (Naciff et al., 2007).
The ability of gene expression analysis to identify mechanisms of action has primarily been used for predicting potential
toxicity, especially in the pharmaceutical industry. Doseresponse assessment is another application of gene expression
analysis in toxicology and risk assessment. The sensitivity of
the methodology is good enough that it is possible to detect
small effects at low levels of exposure, if they exist. The global
nature of microarray analysis (i.e., all genes in the genome can
be evaluated) means that these experiments can be unbiased. If
there are pleiotropic effects at lower exposure levels that would
elicit a different profile of gene expression, those genes would
not go unnoticed.
The ability to detect effects at levels lower than the lowestobserved effect level for traditional toxicity endpoints makes it
possible to address questions about the linearity of the doseresponse curve at these lower exposure levels. From a mechanistic standpoint, a comparison of gene expression results at
doses at or above the LOAEL with those below the LOAEL can
address hypothesized dose-dependent transitions in toxic
response. Dose-dependent transitions are inflection points in
the dose-response curve that occur when the concentration of
the exogenous agent is sufficiently high to alter normal
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234
DASTON
physiological function (Slikker et al., 2004). There is evidence
for a dose-dependent transition for formaldehyde, in that
inflammatory and proliferative responses in the nasal epithelium do not occur at doses lower than those that are tumorigenic
(Conolly et al., 2003). However, it is possible that there were
effects produced at lower levels that were simply not detected
by the methods used previously.
The present study correlates the gene expression changes
that occur at three dose levels and over three time points
following short-term exposure to formaldehyde. The three dose
levels were similar to the lowest levels used in the chronic
bioassay. The highest of the three, 6 ppm, was the lowest
tumorigenic concentration, a dose that also induces cell
proliferation. The gene expression analysis indicated no changes
in gene expression at the lowest exposure concentration. Fifteen
genes were changed at the intermediate concentration level
after 5 days of exposure, but the effect had resolved by day 15 of
the experiment and no genes were changed versus control.
Approximately twice as many genes were changed in expression
on day 5 in the 6 ppm group and about four times as many on day
15. These results clearly indicate dose- and time-dependent
changes in gene expression and support the notion of a dosedependent transition for cell proliferation, thought to be the
precursor event for formaldehyde-induced tumorigenesis. The
nature of the gene expression changes is consistent with the
phenotypic effects at the various dose levels.
These investigators derived dose-response curves and
estimated benchmark doses for sets of affected genes that
were based on gene ontology terms for cellular/extracellular
location. This grouping of genes for dose-response analysis
acknowledges that genes typically do not change in isolation
and that one would expect any change that is causally related to
toxicity to be in a set of related genes rather than a single gene.
It is interesting to note that excluding the lowest benchmark
dose, all were in the range of 3.1–4.1 ppm, comparable to the
benchmark dose for formaldehyde-induced proliferation (4.9
ppm) reported previously (Schlosser et al., 2003), and even the
lowest BMD of 1.6 ppm, for genes associated with basal
plasma membrane, were only three-fold lower than the BMD
for cell proliferation. This is good evidence for a tight
correlation in gene expression changes and changes at higher
levels of biological organization.
The results of this study should be useful for the risk
assessment for formaldehyde. At present, there is still
uncertainty about the mode of action for formaldehyde and
the nature of the dose-response relationship at exposure levels
below the experimental LOAEL. The research presented by
Andersen et al. (2008) supports the notion that there is a clear,
dose-dependent transition in the effects of formaldehyde at the
level of transcription, as well as at the cellular and
histopathological levels. One hopes that this information is
taken into consideration in establishing regulatory guidance for
formaldehyde.
More importantly, Andersen et al. (2008) have provided
a model for the use of gene expression in dose-response
analysis. The use of gene ontology terms as a means of
grouping genes for dose-response analysis is innovative. It
should be noted that these experiments are neither simple nor
inexpensive. Dose-response studies cannot be done without
multiple dose levels. To this was added multiple time points,
a necessary consideration because the histopathological
changes to formaldehyde occur over a period of time. Enough
animals needed to be evaluated, and microarrays run, at each
dose- and time-point to provide reasonable statistical power.
Still, given the societal importance of a less uncertain risk
assessment for formaldehyde, the results more than justify the
expense.
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Conolly, R. B., Kimbel, J. S., Janszen, D., Schlosser, P. M., Kalisak, D.,
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