* National Institute of Environmental Health Sciences, P.O. Box 12233, MD2-04, Research Triangle Park, North Carolina 27709; and
Boehringer-Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877
Received November 26, 2001; accepted January 8, 2002
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ABSTRACT |
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Key Words: toxicogenomics; gene expression database; discriminant genes; prediction; algorithms; DNA arrays.
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INTRODUCTION |
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The genomics approach for predictive toxicology mandates the successful interrogation of databases populated with gene expression profiles corresponding to biological responses to well characterized, known compounds and comparing those with expression profiles from biological responses to exposures corresponding to unknown chemicals (Lovett, 2000; Nuwaysir et al., 1999
; Hamadeh et al., 2002a
). The hypothesis that underlies this approach is that similarities among profiles will indicate shared mechanisms of action and/or toxicological responses among the chemicals being compared. It has been demonstrated that compounds with similar pharmacological or toxicological effects produced similar gene expression profiles following either in vitro (Waring et al., 2001a
) or in vivo (Waring et al., 2001b
) exposure conditions. We have previously demonstrated that gene expression profiles corresponding to livers of rats exposed to either peroxisome proliferators or an enzyme inducer, clustered based on the mechanism of toxicant action (Hamadeh et al., 2002b
). Gene expression measurements corresponding to the in vitro response of rat hepatocytes to 15 known compounds revealed that profiles of chemicals with similar toxic mechanisms clustered together (Waring et al., 2001a
). Another study demonstrated a strong correlation between the histopathology, clinical chemistry, and gene expression profiles corresponding to livers derived from chemically exposed rats (Waring et al., 2001b
).
The use of gene expression profiles for classification and predictive purposes has been demonstrated in the field of oncology (Alaiya et al., 2000; Alizadeh et al., 2000
; Golub et al., 1999
; Perou et al., 1999
; Perou et al., 2000
). Tumor samples from human patients were classified in a blinded fashion, based on learning data sets that provided knowledge on the tumor categorization and allowed for objective classification of the unknown samples. This approach, however, has not been robustly applied toward the determination of the identity of biological samples derived from in vivo chemical exposure models. A challenging question facing the validity of the use of transcript profiling to reveal chemically induced responses in treated animals is whether profiles can be used to predict the classification of coded samples generated from exposures to compounds that have not been profiled before.
To test our hypothesis, we investigated, in a blinded study, gene expression profiles from liver samples of chemically treated Sprague-Dawley rats. Specific compound identities, mechanistic classes, or doses of the compounds were coded to the team members involved in the gene expression profiling and data interpretation throughout the analysis and prediction process. In addition, no grouping of samples was provided in cases in which multiple samples were derived from animals treated with the same agent. The only information provided was that the duration of exposure to the agents varied from 24 h, to 3 days, to 2 weeks. The study included 23 coded samples. The knowledge derived from previous studies about key discriminator genes that correlated highly with their mechanism of action (Bushel et al., unpublished data) was used to interpret the gene expression profiles of the blinded samples in order to generate predictions about the identity of these samples. Using these discriminative genes, we were able to predict that 13 of the samples were similar to either the class of enzyme inducers (phenobarbital-like) or to peroxisome proliferators. The remaining 10 compounds were classified as being not similar to profiles in our database. Upon completion of the prediction, the sample identifiers were decoded, and we found that "correct" statements were made regarding 22 of the 23 samples. These results provide strong evidence that the classification of unknown compounds, based on in vivo gene expression profiles by comparison to a limited known data set, is possible, and provides validation of the strategy that underlies a toxico- or pharmacogenomic approach to classification of agent action.
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MATERIALS AND METHODS |
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RNA isolation and DNA microarray hybridization and analysis.
RNA isolation protocols are identical to those reported earlier in (Hamadeh et al., 2002b). The cDNA Rat Chip software, v1.0, developed in-house at NIEHS, was used for gene expression profiling experiments. A complete listing of the genes on this chip is available at the following website: http://dir.niehs.nih.gov/microarray/chips.htm. cDNA microarray chips were prepared as previously described. (DeRisi et al., 1996
) and are also described in (Hamadeh et al., 2002b
). Each RNA pair, from coded control and treated livers, was hybridized to at least 2 arrays yielding at least 4 measurements on each gene. The raw pixel intensity images were analyzed using the ArraySuite, v1.3, extensions of the IPLab image processing software package (Scanalytics, Fairfax, VA) (Chen et al., 1997
). The ratio intensity data from all of the 1700 spots printed on the NIEHS Rat Chip, v1.0, was used to fit a probability distribution to the ratio intensity values and estimate the normalization constants that this distribution provides. Genes having normalized ratio intensity values outside of the 95% confidence interval were considered significantly differentially expressed and deposited into the NIEHS MAPS database (Bushel et al., 2001
). For each exposure condition, a query of the database yielded a list of genes that were differentially expressed in at least 3 of the 4 replicate measurements. A calculation using the binomial probability distribution indicated that the probability of a single gene appearing on this list when there was no real differential expression is approximately 0.0025.
Training set.
The training set used in this study comprised of RNA samples derived from livers of Sprague-Dawley rats exposed to one of 3 peroxisome proliferators (clofibrate, Wyeth 14,643, gemfibrozil), or an enzyme inducer (phenobarbital) for 24 h or 2 weeks. A detailed description of this set is provided in Hamadeh et al. (2002b).
Genetic algorithm/K-nearest neighbor (GA/KNN).
The GA/KNN method combines a genetic algorithm (GA) as a searching tool and the K-nearest neighbor (KNN) approach for nonparametric pattern recognition. The method not only selects a subset of informative genes that jointly discriminate among different classes of specimens but also assesses the relative predictive importance of all the genes for specimen classification. The methodology of GA/KNN is briefly described below; see Li et al., 2001 and the website http://dir.niehs.nih.gov/microarray/datamining/ for details. Let Gm = (g1m, g2m, ..., gim, ..., gqm), where gim is the log expression ratio of the ith gene in the mth specimen; m = 1,...,M (M = number of samples in the training set = 27; 9-clofibrate, 9-Wyeth, 9-gemfibrozil, and 9-phenobarbital). In the KNN method, one computes the Euclidean distance between each specimen, represented by its vector Gm, and each of the other specimens. Each specimen is classified according to the class membership of its k-nearest neighbors. In this study, we set q = 30 and k = 3 and required all of the 3 nearest neighbors to agree. If the 3 nearest neighbors was not of the same chemical class, the specimen was considered unclassified. A set of q (q = 30) of genes was considered discriminative when at least 25 of 27 specimens were correctly classified. A total of 10,000 such subsets of genes were obtained. Genes were then rank-ordered according to how many times they were selected into these subsets. The top 100 genes were subsequently used for prediction purposes.
Linear discriminant analysis (LDA).
Standard ANOVA models (Kerr and Churchill 2001), were used to identify genes that have significantly different mean expression values between classes of compounds in the training set of peroxisome proliferators and enzyme inducers (Hamadeh et al., 2002b
). Any genes that were identified by ANOVA but had a global standard deviation of 0.3 (log2 units) or higher were excluded. Linear discriminant analysis (LDA) was then used to test all pairs of genes to identify those that can jointly discriminate between the classes, again using a minimum variability criterion to reduce the number of pairs selected. Additional genes that had high similarity (r > 0.95) in their expression profile across known samples were determined using GeneSpring software (Silicon Genetics, Wood, CA) and added to this list of discriminatory genes.
Prediction criteria.
Genes, found to be highly discriminatory between peroxisome proliferators and the enzyme inducer in the training set, using LDA and the 100 top-ranked class discriminatory genes selected from the GA/KNN, were compared. The intersection of these gene lists was generated and resulted in a list of 22 genes. For each, the calibrated ratios (log-transformed) were then averaged across the replicate hybridizations in the training set. Next, a pairwise Pearson correlation coefficient was calculated for each of the training samples and each of the coded samples, according to the expression ratios of all 22 genes using JMP software (SAS, Cary NC). Samples were determined to be similar if r 0.8.
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RESULTS |
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In order to make a prediction on properties of the blinded samples, we used the gene expression profile data set (Hamadeh et al., 2002b) corresponding to livers from rats exposed to 4 known compounds (Wyeth 14,643, clofibrate, gemfibrozil, phenobarbital) as a training set. Multiple approaches were used to find highly discriminatory/informative genes whose expression pattern could distinguish RNA samples derived from livers exposed to different chemicals. LDA and GA/KNN were useful in revealing single genes or groups of genes that could separate known samples based on the class of chemical involved in the exposure. Table 1
lists 22 highly informative genes that clearly exhibited different patterns of expression between the 2 pharmacological/toxicological classes of compounds, peroxisome proliferators and enzyme inducers.
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In summary, we were successful in correctly making a positive prediction regarding the classes of 12 out of 13 of the blinded samples. We were also successful at noting that 10 other blinded samples did not belong to the class of peroxisome proliferators, as evidenced by the lack of similarity in pattern to compounds in that class. A summary of classifications is listed in Table 5, which shows the list of blinded samples, our corresponding prediction, and their actual identity. The results show that, using the approach described, we had a 92.3% accuracy rate in class prediction. The animals that had a gene expression profile similar to phenobarbital were treated with high-dose phenytoin, a drug in the same class as phenobarbital. Similarly, the coded samples that were identified as being similar to samples from clofibrate- and Wyeth 14,643-treated rats were from animals exposed to diethylhexylphthalate (DEHP), a known peroxisome proliferator. Finally, the samples that were noted to be definitely unrelated to peroxisome proliferators but weakly similar to phenobarbital were from animals treated with low doses of either phenytoin or hexobarbital. The one sample that appeared to be classified incorrectly was that of a hexobarbital-exposed animal as phenobarbital-exposed. Further investigation of interanimal variation is being conducted that might help to explain this inaccuracy.
After the decoding of the samples, we were interested in visualizing the gene expression profiles of all of the coded samples in the context of the discriminator genes. Cluster analysis was performed to visualize the grouping of known samples within unknowns according to the expression levels of the highly discriminant genes. Figure 2 shows the hierarchical dendrogram that clearly separated known and blinded samples into 2 major nodes according to their major mechanistic classes. One node (Fig. 2
, node I) contained all of the animals exposed to the barbiturates phenobarbital, phenytoin, and hexobarbital. Upon further inspection of this node, one can see that all of the high-dose phenytoin animals (616, 618, 674,672,676) were tightly clustered with the phenobarbital, and that animals treated with low-dose or short-time phenytoin or hexobarbital were clustered slightly apart. The second major node (Fig. 2
, node II) contained all of the animals that had been exposed to peroxisome proliferators.
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DISCUSSION |
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It is of interest to note that samples classified as phenobarbital-like (616, 618, 672, 674, 676, 678) were actually derived from rats exposed to high doses of phenytoin (5,5-diphenylhydantoin). Phenytoin and phenobarbital belong to the same pharmacological class of compounds that act as anticonvulsants with enzyme-inductive properties (Brodie 1992; Fitzsimmons et al., 1990
; Liu and Delgado 1995
; Patsalos and Duncan 1993
; Pichard et al., 1990
; Riva et al., 1996
), which attributes validity to the prediction made on the identity of these samples. Our findings corroborate numerous previous studies that have reported on the proximity of responses to phenytoin and phenobarbital in different biological models (Brodie 1992
; Fitzsimmons et al., 1990
). Likewise, coded samples that were correctly classified as similar to clofibrate or Wyeth 14,643 corresponded to rat livers exposed to DEHP (di-(2-ethylhexyl)phthalate). DEHP belongs to the peroxisome proliferator class of compounds (Lake et al., 1986
; Mitchell et al., 1985
) and produces a multitude of effects that are shared by other peroxisome proliferators such as clofibrate and Wyeth 14,643 (Crane et al., 1990
; Lake et al., 1984
).
We provided positive classification rather than the exact identity of the coded samples since no gene expression profiles corresponding to DEHP or phenytoin were present in our learning set/database. However, we were successful in classifying coded samples according to their pharmacological/toxicological effects or modes of action. A prediction on the classification of samples 630, 632, 634, 690, 692, 694, 3462, 3464, and 3468 was made by the definite assessment that none of these samples were similar to peroxisome proliferator compounds. The first 6 of these samples were derived from animals treated with hexobarbital, a compound that is structurally related to phenobarbital but is not carcinogenic in rodents and elicits a less potent enzyme-inducing response (Nims et al., 1987). The last 3 samples corresponded to a 2-week exposure to a low dose of phenytoin and were negatively correlated with known peroxisome proliferator samples, but did not meet our stringent criteria to be classified as potential phenobarbital samples. If our database had contained expression profiles of rat livers exposed to low-dose phenobarbital, positive identification of those samples may have been possible. This highlights the importance of building multiple doses and time points into studies designated to populate a database developed for the purpose of screening and prediction.
Another challenge for this study was the lack of information regarding biological replicates among the blinded set. In our previous studies (Hamadeh et al., 2002b) we utilized multiple animals for each dose and time group to determine what gene expression changes occurred robustly in all exposed animals vs. those that reflected variation between animals. In this prediction study, we did not know which samples represented biological replicates. This lack of replicate knowledge contributed to the difficulty for making our predictions. For example, we classified one of the hexobarbital-treated blinded samples (688) as being weakly similar to phenobarbital and did not positively classify sample 612 (phenytoin exposure) as similar to phenobarbital (Table 5
). These calls might be due to interanimal variation where a relatively higher- or lower-amplitude response in gene expression was evident in those particular samples, respectively, since their biological replicates were classified correctly.
Inherent in this data set are a variety of time- and dose-dependent, as well as independent, changes that might be exploited in further studies. By studying samples treated with phenytoin at the low and high dose, we found numerous genes that appeared to respond in a dose-dependent manner. GST Yb2 (AA998732), carboxylesterase precursor (AI070587), cytochrome p450 2C6 (AA858966), palmitoyl-protein thioesterase (AA818995) and cytochrome p450 2B2 (AA818412) were among genes that were apparently induced in a dose-dependent fashion by phenytoin. Likewise, the expression levels of diazepam-binding inhibitor (AA925794), parvalbumin (AA819345), growth hormone receptor (AA819745), p450 1A2 (AA924594), and cytochrome p450 2C7 (AA818043) were repressed in a dose-dependent manner as a result of phenytoin exposure. If, in additional studies, these genes continue to appear to have dose- and time-independent responses, they may provide valuable identifiers for classifying compounds at unique dose or time points.
There were several challenges we encountered in this study. Ideally, the information housed in a database should be large so that one can interrogate this large sum of information with a relatively small query, to derive more knowledge from the database. However, we were challenged to interrogate a database with a large data set that outweighed the relevant database set that we could query against. Our query of 24 gene expression profiles, potentially belonging to 24 different chemicals, exceeded the database set of 4 chemicals that was used for the purpose of this study. Our learning set contained gene expression profiles corresponding to 24 h or 2 weeks of chemical exposure, however, 4 of the blinded samples were generated in rats exposed for 3 days. This required us to reduce the dimension of the data set (Bushel et al., unpublished data) and to find time-independent, highly discriminative genes that helped to separate different compounds, so that we could query 24-h and 2-week data with 3-day profiles (Table 1). This investigation demonstrates the first example of a successful query of a database with gene expression profiles to predict classification of unknown compounds of this number.
In summary, this work illustrates the successful prediction of properties of blinded samples using gene expression profiling. It demonstrates that large gene expression profile databases will be able to be successfully queried to help classify unknown compounds from exposed tissues. It also highlights the importance for beginning, even at this early stage, to develop analysis models for these types of data. Our analyses informed us on important considerations for our experimental study designs in the future. It is now foreseeable that in the future, gene expression profiling or other high density genomics analyses will prove valuable in the screening of compounds for mechanistic classification in a high-throughput fashion. In turn, this will ultimately better our understanding in selecting chemicals for advanced stages of target testing in commercial settings, and allow the advancement of predictive information on uncharacterized human health hazards.
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NOTES |
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