Analysis of Genetic and Epigenetic Mechanisms of Toxicity: Potential Roles of Toxicogenomics and Proteomics in Toxicology

Scott W. Burchiel*,{dagger},1, Cindy M. Knall*,{dagger}, John W. Davis, II*, Richard S. Paules{ddagger}, Susan E. Boggs{dagger} and Cynthia A. Afshari{ddagger}

* Toxicology Program, College of Pharmacy, University of New Mexico, 2502 Marble N. E., Albuquerque, New Mexico 87131-5691; {dagger} Lovelace Respiratory Research Institute, Albuquerque, New Mexico; and {ddagger} National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27711

ABSTRACT

The article highlighted in this issue is "An Aryl Hydrocarbon Receptor Independent Mechanism of JP-8 Jet Fuel Immunotoxicity in Ah-Responsive and Ah-Nonresponsive Mice" by Andrew C. Dudley, Margie M. Peden-Adams, Jackie EuDaly, Richard S. Pollenz, and Deborah E. Keil (pp. 251–259).

The recent completion of the human genome sequencing project and the push to finish the mouse genome have raised the stakes in science with predictions of disease cures, more effective and safer pharmaceuticals, and a greater understanding of environmental effects on human health. The impact of these projects on toxicological research is high, as demonstrated by recent articles in Science (Lovett, 2000Go), and The New York Times (Pollack, 2000Go) heralding the emerging technologies of toxicogenomics, proteomics, and bioinformatics. These articles highlight the future use of these technologies and their impact on drug discovery, safety evaluation, elucidation of pathways of toxicity, and risk assessment. The utilization of these new technologies along with more established genetic approaches such as quantitative polymerase chain reaction (PCR), and the use of genetically altered animals will dramatically move the field of toxicology forward.

Genetic approaches allow toxicologists to address mechanistic questions concerning the effects of toxicants. Transfection systems to introduce plasmids containing cDNAs encoding mutated forms of proteins of interest, anti-sense oligonucleotides to inhibit endogenous proteins, and reporter genes to measure gene promoter induction are all useful in examining mechanisms of toxicity. Transgenic and knockout mice can be used to address the role of selected gene products in the response to toxicants in vivo. For example, cancer prone transgenic mice may be valuable in identification of carcinogenic chemicals (Gulezian et al., 2000Go).

In the highlighted paper by Dudley et al., the utility of another genetic approach, analysis of responsive and nonresponsive mouse strains, was demonstrated. Genetically responsive (B6C3F1) and nonresponsive (DBA/2) strains of mice were utilized to assess the role of AhR in immunotoxicity produced by JP-8 jet fuel. The rationale for the study was twofold: (1) JP-8 jet fuel is known to produce immunotoxicity in mice, and its pattern of immunotoxicity may be similar to that produced by AhR ligands; (2) AhR ligands, particularly polycyclic aromatic hydrocarbons (PAHs), are known to be present in JP-8. Since PAHs are known to produce immunotoxicity by both AhR-dependent and AhR-independent mechanisms (Burchiel and Luster, 2001Go), it is a reasonable hypothesis to assess the role of the AhR in immunotoxicity. Because the immunotoxicity was essentially equivalent in Ah responsive and nonresponsive strains of mice, the conclusion of the study is that JP-8 jet fuel produces immunotoxicity by AhR-independent mechanisms. However, while there appears to be strong evidence for this conclusion, there is still a possibility that this receptor plays some role in the immunotoxicity of JP-8, since nonresponsive mice are known to express low affinity AhR. The use of AhR transgenic knockout mice (AhR–/–) that are available would definitively address this issue (Lahvis and Bradfield, 1998Go).

The highlighted paper illustrates two important points relevant to examining mechanisms of action of immunotoxicants and indeed all toxicants. First, a common pattern of toxicity does not necessarily equate to a common mechanism. Second, while the use of susceptible mouse strains gives important information on the mechanism of action when positive correlations can be found, negative results do not shed light on the mechanism of action of the toxicant. Thus, in this case, there is a need to evaluate other genetic and epigenetic mechanisms of JP-8 jet fuel immunotoxicity. While a transgenic mouse in vivo approach to assessing immunotoxicity is valid and important, one must ensure that models are employed with consideration that the model is appropriate for the hypotheses and mechanisms to be addressed in the study. Furthermore, if the goal is to characterize the toxicologic significance of xenobiotic effects on a broad range of genes and gene products, other technologies should also be employed. Recent advances in gene expression arrays and bioinformatics allow toxicologists to assess the effects of xenobiotics on literally thousands of genes simultaneously (Afshari et al., 1999Go; Nuwaysir et al., 1999Go). Expression arrays may be utilized in combination with analyses of allelic and single nucleotide polymorphisms to characterize responses to individual chemicals and complex mixtures, as well as to evaluate the effectiveness of drugs in treating disease. This information will then allow investigators to target selected genes and to generate mechanistic hypotheses based upon the results obtained. Information obtained from expression arrays can be complemented by analysis of alterations in protein levels, posttranslational modifications, and function using functional proteomics (Banks et al., 2000Go). Proteomics, in comparison to genomics, is still in its infancy. However, rapid progress in proteomics will undoubtedly begin to answer the questions that genomics cannot. An example of the progress being made in proteomics is the work of MacBeath and Schreiber (2000) that was highlighted recently in Science (Service, 2000Go).

There are both advantages and limitations to the use of gene array and proteomics technologies in toxicologic screening (Table 1Go). The main advantage is a global approach to understanding the complex mechanisms involved in toxicology. Genomics and proteomics technologies may provide useful "biosignatures" that will be particularly valuable biomarkers in characterizing novel chemicals and complex mixtures. Genomics cannot answer questions about protein levels or posttranslational modifications, which must be addressed using proteomic technologies. Gene arrays have been costly and limited in availability, but the past year has shown a commitment by the scientific community to the general use and availability of gene arrays. Consequently, cost has been reduced by increased supply and demand. Furthermore, the availability and cost is substantially improving with many universities and research centers establishing genomic and proteomic facilities. Therefore, genomics and proteomics will certainly become generally used technologies in the near future.


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TABLE 1 Genomics and Proteomics in Toxicology
 
An example of how this new technology can be applied is in the field of immunotoxicology. Chemicals are usually screened for immunotoxicity using a tiered approach (Luster et al., 1988Go). In Tier I screening assays, descriptive general toxicology-pathology analyses are made and certain functional assays are performed before focusing on specific target cells and organs of the immune system. Tier II involves a more complex and specialized set of assays that are performed pending the outcome of Tier I studies. We must now ask whether gene array and proteomics studies fit in the first or second tier of studies. Arguments supporting the use of these technologies in both Tier I and Tier II could be made.

The use of gene expression array and proteomics technology has a bright future in toxicology research. However, as these highly technical and complex fields evolve, the scientific and nonscientific communities must keep in mind that there can be quite different goals and applications in the research and regulatory communities. Whereas scientists pursuing mechanistic data may be interested in identifying a broad range of genes that are affected by drugs, chemicals, and complex mixtures, the regulatory communities will be interested in only those genes that are indicative of a critical health effect. Therefore, the interpretation and application of findings may be difficult until sufficient databases are developed and defined to clearly make this distinction.

NOTES

1 To whom correspondence should be addressed. Fax: (505) 272-6749. E-mail: sburchiel{at}salud.unm.edu. Back

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