BACKGROUND
high-throughput toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo  <cit> .
recent microarray studies have shown that the in vitro gene expression profiles in liver slices treated with various compounds could predict the toxicity and pathology observed in vivo  <cit> .
however, the degree to which in vitro results can be extended to in vivo activity and possible mechanisms of action remains to be fully addressed.
compared with animal models, primary cell cultures have advantages for investigating mechanisms of chemical toxicity.
primary cultured cells, such as hepatocytes, can offer a convenient system that is easily genetically manipulated and can be used to test various throughput screens using different molecular and biochemical methods.
use of primary cell cultures can also reduce cost and mitigate animal welfare concerns inherent in in vivo studies  <cit> .
in vitro systems have a long history of use in screening new drugs for human diseases such as cancer and in studying cellular and molecular events of different molecules   <cit> .
in this study, we used the nitroaromatic  <dig> , <dig> -rinitrotoluene  as a model chemical to compare and determine how we might extrapolate in vitro data to in vivo systems.
we compared the gene expression profiles of in vitro primary liver cells with the gene expression profiles of in vivo liver tissue of rats exposed to tnt.
tnt is a munitions compound which is released to the environment as a result of military training activities and manufacturing processes  <cit> .
the major toxic effects to rats in response to tnt exposure are methemoglobinemia, anemia, hypercholesterolemia, and hepatomegaly, splenomegaly, and testicular atrophy with their accompanying histologic lesions  <cit> .
cytotoxic and genotoxic effects are also caused by tnt  <cit> .
our recent toxicity studies in rats found that tnt decreased body weight, increased liver weight, and induced erythrocytosis .
although a variety of studies have focused on toxicity aspects of tnt, the underlying mechanisms of toxicity induced by this compound are largely unknown.
several laboratories have started to use microarray analysis to understand the molecular mechanisms of invertebrate ecotoxicity.
the exposure of the earthworm eisenia fetida with tnt regulates the expression of genes involved in multiple biological processes including muscle contraction, neuronal signaling and growth, ubiquitinylation, fibrinolysis and coagulation, iron and calcium homeostasis, oxygen transport, and immunity  <cit> .
microarray analysis of tnt exposed arabidopsis thaliana  reveals the induced expression of oxophytodienoate reductases , the protein responsible for tnt detoxification in arabidopsis.
the same team also found that bifunctional o- and c-glucosyltransferases is involved in combating tnt induced toxicity in arabidopsis  <cit> .
eisentraeger et al.
<cit>  investigated the mechanism of human liver cell line hepg <dig> treated with tnt using the toxicology cdna microarray.
they found that the detoxification metabolism in the hepg <dig> cells by tnt induced genes encoded phase i and phase ii enzymes.
in the current study, we examined gene transcriptional responses to short term  tnt exposures in rat in both in vitro hepatocytes and in vivo liver.
we observed that a number of genes were commonly regulated by both in vitro and in vivo tnt treatments.
moreover, we found commonly affected pathways in both in vivo and in vitro systems exposed with tnt.
functional analysis indicates that both tnt treatments in vivo and in vitro impact genes involved in cell cycle, cell growth and cell death signaling, detoxification response, lipid metabolism and immune response, which can reasonably account for the physiological dysfunctions induced by tnt.
in addition, we identified conserved gene networks inferred from in vivo and in vitro networks.
our results suggest that in vitro system can predict in vivo functions and mechanisms based on gene expression profiles, and gene network is a valuable approach for predicting in vivo function using in vitro data.
RESULTS
commonly regulated genes by in vivo and in vitro tnt treatments
to determine if in vitro tnt exposure could be used to predict in vivo tnt exposure, the first aim of this experiment was to determine if there were common genes differentially regulated by both in vivo and in vitro tnt treatments.
for the in vivo experiment, rats were gavaged with tnt at:  <dig> ,  <dig> ,  <dig>   <dig> or  <dig> mg/kg and sacrificed after  <dig> h or  <dig> h. rna was isolated for microarray hybridization.
agilent rat whole genome array  was employed in the study.
four biological replicates were used for each unique condition as described in the material and methods.
for the in vitro experiments, primary rat hepatocytes  were cultured and treated with tnt at  <dig> ppm or vehicle control for  <dig> h, after which rna was isolated for microarray experiments.
the in vivo and in vitro micorarray data have been deposited in the geo databases with assigned numbers gse <dig> and gse <dig>  respectively.
to identify genes differentially expressed in vivo, a one-way anova was applied to identify differentiated transcripts across different doses of tnt treatments at each of the  <dig> h or  <dig> h time points.
a p value cut-off of  <dig>  and a  <dig>  fold gene transcript change by comparing at least one pair of conditions between different doses including control were used as filters to identify differentially expressed genes.
a total of  <dig> gene transcripts were significantly and differentially expressed  in in vivo tnt treatment for all doses and time points.
an un-paired t-test with a p value cut-off of  <dig>  and  <dig>  fold change filter was used to test for differential expression between controls and tnt treated hepatocytes in the in vitro assay yielding  <dig> differentially expressed transcripts.
a venn diagram shows that there were  <dig> transcripts that were commonly regulated by both in vivo and in vitro tnt treatments .
the common differentiated genes between in vivo and in vitro exposures suggest that a common mechanism exists between in vitro tnt and in vivo tnt treated liver systems.
similar gene expression pattern shared by commonly regulated in vivo and in vitro genes
we examined the expression pattern of the  <dig> common transcripts from in vivo and in vitro tnt exposures.
to reach the goal, each sample was normalized by the mean intensity of a gene of relative control samples, and the samples under the same condition were averaged into one condition.
a two-way hierarchical clustering resulted in two cluster dendrograms of the  <dig> transcripts across  <dig> in vivo conditions and  <dig> in vitro conditions .
the controls and  <dig> lower in vivo dose conditions  at  <dig> h were in a cluster, indicating that these doses at  <dig> h are closer to controls and have less genes affected than other conditions.
the remaining in vivo and in vitro tnt exposed hepatocytes and livers formed the second cluster.
overall, we found the conditons were more likely to group together according to exposure duration than dose concentration, implying that the time effect is stronger than the dose effect.
interestingly, we found that  <dig> conditions  fell into one subgroup.
moreover, we noticed that the gene expression patterns for the  <dig> mg/kg,  <dig> h in vivo tnt treatment and the  <dig> h in vitro tnt treatment were similar.
the genes are upregulated  by the in vitro treatment, and they were usually upregulated in vivo treatment.
most downregulated genes in in vitro were also repressed in vivo.
since these two conditions behaved closely, we directly compared commonly upregulated and downregulated genes based on these two conditions.
out of these  <dig> transcripts,  <dig> transcripts were upregulated by the in vivo tnt treatment, and  <dig> transcripts were upregulated by the in vitro tnt treatment.
one hundred sixty-four transcripts  were commonly upregulated under both conditions .
one hundred forty in vivo tnt transcripts and  <dig> in vitro tnt transcripts were downregulated.
furthermore,  <dig> transcripts  were reduced by both conditions .
another common phenomenon was that both tnt exposed liver systems had more upregulated than downregulated genes.
our results demonstrate that tnt treatment in in vivo and in vitro liver systems results in genes regulated in the same direction, providing more evidence that in vitro hepatocytes act as a suitable surrogate for in vivo liver exposures.
functional analysis of commonly regulated genes
to understand the functions of the commonly regulated genes, we identified most significantly functional categories using the ingenuity function analysis tool .
these functional groups mainly included cell cycle, carbohydrate metabolism, molecular transport, cell growth and proliferation, cell death, dna replication, recombination and repair, lipid metabolism, cellular assembly and organization, and immune response .
the most significant functional category regulated by tnt was cell cycle, which possessed  <dig> genes, of which,  <dig> genes were commonly downregulated by in vivo and in vitro tnt exposures.
the most heavily downregulated genes involved in cell cycle by both in vivo and in vitro tnt treatments included lgals <dig>  cyp26b <dig>  and pttg <dig>  highly induced genes in this cell cycle category included adm, dusp <dig>  and pparg.
more genes participating in cell cycle were downregulated, indicating that tnt appears to interfere with cell cycle progress.
* full names of the genes are listed in additional file  <dig>  table s  <dig>
other commonly significantly affected functional terms were cell growth and proliferation , cell death , dna replication , and recombination and repair .
interestingly, there were  <dig> genes that were repeatedly present in all four functional terms: adm, ccnb <dig>  ccnd <dig>  cxcl <dig>  lgals, myc, nrg <dig>  nrg <dig>  pparg, and pttg <dig>  in addition, these genes were consistently regulated by both in vivo tnt and in vitro tnt in the same directions.
the expression of ccnb <dig>  ccnd <dig>  pttg <dig>  cxc <dig>  and lgals <dig> was decreased by tnt, and the expression of adm, myc, ngr <dig>  ngr <dig>  and pparg was elevated by tnt.
there were  <dig> commonly regulated genes that were involved in molecular transport .
nineteen of the  <dig> genes  were upregulated, and only  <dig> of them were downregulated by both in vitro and in vivo tnt treatments.
some significant upregulated genes included nqo <dig>  adm, abcc <dig> and abcc <dig>  our results suggest that tnt can enhance molecular transport both in vivo and in vitro liver systems.
twenty-three genes contributing to carbohydrate metabolism were commonly regulated by both in vivo and in vitro tnt treatments.
among them, only  <dig> genes were repressed and  <dig> genes were induced.
the expression of  <dig> genes participating in lipid metabolism was significantly changed in response to both in vivo and in vitro tnt exposures.
similar to carbohydrate metabolism, we saw more upregulated genes  than downregulated genes  in this category.
several cytochrome p <dig> family members such as cyp1a <dig>  cyp3a <dig> and cyp2c <dig> were in the upregulated gene list.
interestingly,  <dig> genes  belong to the three functional categories: molecular transport, carbohydrate metabolism and lipid metabolism.
three of these  <dig> genes  were downregulated and the other  <dig> genes were upregulated by both in vivo and in vitro tnt treatments.
there were  <dig> commonly regulated genes that were involved in cellular assembly and organization.
the expression of  <dig> genes was decreased, while expression of  <dig> genes were increased.
eight genes played a role in immune response,  <dig> of which were downregulated and  <dig> genes upregulated.
more immune response genes were downregulated, indicating that tnt may interfere with normal immune function to induce its toxicity.
overall, some commonly regulated genes were highly represented.
for instance, the gene myc falls into all the functional categories listed in table  <dig>  cxcl <dig> belongs to all the functional groups except cellular assembly and organization.
adm and lgals <dig> genes pparg were absent in only two functional categories, cellular assembly and organization, and immune response.
pathway analysis
to further understand the gene function influenced by tnt exposure to in vivo and in vitro liver cells, we conducted a canonical pathway analysis using the ingenuity pathway analysis tool.
three separate gene lists were used to run the pathway analysis: the list of genes most significantly regulated by in vivo tnt treatment, the list of genes most significantly regulated by in vitro tnt exposure, and the list of the  <dig> commonly regulated genes.
in order to identify the most significantly influenced pathways in vivo, a more stringent p value  was applied, which resulted in  <dig> significantly regulated transcripts for the in vivo tnt experiment.
the top significantly impacted pathways from each gene list are depicted in figure  <dig>  using a threshold p value of  <dig> , a total of  <dig> pathways were regulated by in vivo tnt treatment,  <dig> pathways were evidently affected by in vitro tnt exposure, and  <dig> significantly pathways were obtained by analyzing the common  <dig> genes alone .
twenty commonly regulated pathways were shared by the in vivo and in vitro pathway lists, which is 50% of the pathways impacted by in vitro tnt treatment .
the percentage of overlapping pathways was much higher than that of overlapping genes illustrated in figure  <dig>  indicating that the in vitro system perturbed by tnt could reflect more biological truth for an in vivo system from a mechanism related pathway view.
sixteen consensus pathways were shared by the three significant pathway lists .
the top regulated pathways with relative genes are listed in table  <dig>  several gene families that were highly represented in these pathways are related to xenobiotic metabolism and transport.
these families were cytochrome p <dig> , glutathione s-transferase , udp glucuronosyltransferase , aldehyde dehydrogenase  <dig> and atp-binding cassette  families.
the regulated genes in the cyp family included cypa <dig>  cyp2c <dig>  cyp2f <dig>  and cyp3a <dig>  the genes listed in table  <dig> belonging to gst family included gst <dig> and gsta <dig>  the ugt family included ugt1a <dig>  ugt2b <dig>  and ugt2b <dig>  the regulated gene aldehyde dehydrogenase  <dig> family, member l <dig>  in the family aldh <dig> was involved in many pathways listed in table  <dig>  the regulated genes in the abc family included abcc <dig>  abcc <dig>  and abcg <dig>
* full names of the genes are listed in additional file  <dig>  table s 1
interestingly, all the genes in table  <dig>  except for ccnd <dig> and retinoid acid receptor beta , were commonly upregulated by both in vivo and in vitro tnt treatments.
mapk <dig>  a critical intracellular signaling protein involved in multiple cellular functions  was also presented in multiple pathways such as nrf- <dig> mediated oxidative stress response, xenobiotic metabolism signaling, and aryl hydrocarbon signaling pathways.
inferring in vitro and in vivo gene networks
motivated by commonly regulated genes, functional terms and pathways, we then investigated whether a common gene network was regulated by both the in vitro and in vivo tnt treatments.
to achieve this goal, we used a reverse engineering algorithm called context likelihood of relatedness .
clr is based on mutual information of any pair of genes and is a static network.
a static gene network reflects all possible gene interactions for a given gene list.
therefore, the more samples used to construct a gene network using this algorithm make the built network more trustworthy.
in order to build in vivo gene networks,  <dig> arrays from rat liver tissues treated with one of  <dig> compounds tnt, 2-amino- <dig> -dinitrotoluene , 4-amino- <dig> -dinitrotoulene ,  <dig> -dinitrotoluene  and  <dig> -dinitrotoluene  or vehicle controls were used .
for in vitro gene network modeling,  <dig> arrays resulted from liver primary cultured cells treated by  <dig> distinctive compounds with relative controls were employed .
using the gene expression data of the commonly regulated  <dig> transcripts across the  <dig> arrays, an in vivo gene network was constructed with the clr algorithm.
figure 6a depicts the network which included  <dig> nodes and  <dig> edges.
in vitro gene network was built using the same  <dig> genes and the same clr algorithm, but expression data were generated from the  <dig> arrays.
the gene network from the in vitro data had  <dig> nodes and  <dig> edges .
since transcriptional regulated gene networks are trigged by transcription factors, we then counted the connections of the transcription factors  in the  <dig> transcripts for the both in vivo and in vitro gene networks.
top tfs with most connections in the in vitro gene network were cellular repressor of e1a-stimulated genes  <dig> , pirin  , btb  domain containing  <dig>  sam pointed domain containing ets transcription factor  and basic helix-loop-helix family, member e <dig> .
tfs with most connections in the in vivo gene network included hiv- <dig> tat interactive protein  <dig>  30kda , transcription factor dp- <dig>  , leucine zipper, putative tumor suppressor  <dig> , zinc finger protein  <dig> , pir, and myc.
the transcription factors that had over  <dig> connections in the both gene networks included creg <dig>  pir, btbd <dig>  lzts <dig>  asb <dig>  hes <dig>  and sqstm <dig>  two tfs, spdef and bhlhe <dig>  had at least twice more connections in the in vitro gene network than that in the in vivo gene network.
seven tfs including htatip <dig>  myc, neq <dig>  znf <dig>  e2f <dig>  dnajb <dig> and tfdp <dig> possessed over twice as many connections in the in vivo gene network than that in the in vitro gene network.
conserved in vitro and in vivo gene networks
by comparing the in vivo and in vitro gene networks , we identified several conserved sub-networks that were common in both gene networks .
the tf pttg <dig> centered sub-gene network consisted of  <dig> genes , all of which are involved in cell cycle processes.
moreover, all the genes in the cell cycle sub-network were consistently repressed by both in vivo and in vitro tnt treatments.
the conserved sub-network provides further evidence that the cell cycle process may be impaired in both in vivo and in vitro systems treated with tnt.
the tfs pir, btbd <dig>  htatip <dig>  and creg <dig> were colocalized in the biggest conserved sub-network .
eight genes were connected with the tf pir centered sub-gene network module , and contained highly enriched genes associated with oxidoreductase activity .
akr7a <dig> and txnrd <dig> also participate in nrf2-mediated oxidative stress pathway.
all the genes in this network module were upregulated by both in vivo and in vitro tnt exposures.
this result suggests that oxidative stress is activated by tnt  <cit> .
the tf creg <dig> centered sub-network module included  <dig> genes ,  <dig> of which were also cell cycle related genes .
in contrast to pttg <dig> centered sub-network, all the genes in the creg sub-network module were upregulated by both in vivo and in vitro tnt treatments.
glutathione reductase , a gene related to oxidoreductase activity, and txnrd <dig> provided the connections between the creg <dig>  htatip <dig>  and pir sub-networks , creating a larger  <dig> gene sub-network.
in addition, gsr also participated in the nrf2-mediated oxidative stress pathway.
indeed, we found that all the genes in the biggest conserved sub-network formed by creg <dig>  htatip <dig>  btbd <dig>  and pir mediated modules were consistently upregulated by both in vivo and in vitro tnt additions.
verification of microarray responses using real time qrt-pcr
to verify the credibility of microarray and gene network modeling results, we selected  <dig> genes to perform real time quantitative pcr  both in vitro and in vivo.
the genes chosen consistently played key roles in functional categories and pathways altered by tnt exposures in vivo and in vivo.
as illustrated in figure  <dig>  we observed good consistency between microarray and qrt-pcr results.
similar upregulation and downregulation trends were seen with both microarray data and qrt-pcr data for all  <dig> genes.
the overall regression coefficient of the two methods across various conditions was  <dig>  .
our results indicate the microarray data quality is good and our interpretation and analysis should be convincing.
specific comparisons between microarray and qrt-pcr are presented in additional file  <dig>  figures s 1a-1f.
for instance, the gene abcc <dig> was shown in microarray  to be upregulated at both  <dig> h and  <dig> h with a clear dose response to in vivo tnt treatment, and was also induced by tnt in vitro, and the same pattern was exhibited by qrt-pcr .
in both microarray and qrt-pcr results, the gene akr7a <dig> was upregulated at both time points in vivo as well as upregulated in vitro.
interestingly, akr7a <dig> was significantly upregulated at the highest dose  in vivo at  <dig> h but not at other lower doses, which was shown consistently by both microarray and qrt-pcr .
for the gene cyp1a <dig>  we saw it was upregulated more in response to in vitro tnt than in vivo tnt treatment, and was induced much more at  <dig> h than  <dig> h in vivo, which was consistently revealed by both microarray and qrt-pcr results .
three genes pttg <dig> , ccnb <dig> and ccnd <dig> that were in pttg <dig> centered subnetwork were consistently shown to be downregulated by tnt in vivo and in vitro, with both approaches .
the qrt-pcr results could exactly confirm our two observations obtained from the microarray data: the genes up or downregulated in the same directions in vitro and in vivo, and a clear dose response in vivo.
discussion
in this study, we compared gene expression profiles regulated by tnt exposure in vivo and in vitro from shared gene lists, functional terms, common pathways, and conserved networks.
overall, good consistency existed between in vitro and in vivo exposures.
the venn diagram  shows that a only small percentage of the in vivo transcripts were also regulated in the in vitro experiments.
because the regulated gene number in vivo is the total number from all tnt treatments including multiple doses and two time points , the gene number is much larger than that of in vitro exposures.
however, the in vitro gene number comes from only one dose.
the reason we used one dose is that the in vitro experiment is a large one with a total of over  <dig> chemicals and  <dig> arrays.
but if we compare the overlapped transcripts with the in vitro transcripts , the number of overlapped transcripts is not small, with over one third of the total in vitro transcripts.
this observation is found more than in overlaps examined in other publications , the number is comparable to that of the  <dig> h in vitro tnt treatment.
in addition, in vivo and in vitro are two different systems, we cannot expect that they are exactly the same.
our purpose is to identify common transcripts between these two systems, thereby to understand the in vivo function through the in vitro system.
from the perspective of functional analysis, both tnt treatments in vivo and in vitro influence genes involved in cell cycle, cell growth and cell death signaling, detoxification response, lipid metabolism and immune reponse, which can reasonably explain the physiological dysfunctions induced by tnt.
we also found conserved sub-networks between inferred networks from in vivo and in vitro tnt regulated gene expression profiles.
cell cycle, cell growth, and cell death signaling
a large number of genes involved in cell cycle, cell growth and cell death were commonly regulated in both in vitro and in vivo systems perturbed by tnt.
the highly represented downregulated genes were ccnb <dig>  ccnd <dig>  pttg <dig>  cxc <dig>  and lgals <dig>  and the highly represented upregulated genes were adm, myc, nrg <dig>  nrg <dig>  and pparg.
the cyclin b <dig>  gene product complexes with p <dig> to form the maturation-promoting factor .
ccnb <dig> is expressed predominantly during g2/m phase because of its critical role in cell mitosis  <cit> .
the protein encoded by ccnd <dig> belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle.
cyclins function as regulators of cdk kinases.
different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event.
the ccnd2-translated cyclin forms a complex with and functions as a regulatory subunit of cdk <dig> or cdk <dig>  whose activity is essential for cell cycle g1/s transition.
pttg <dig> encoded protein is a homolog of yeast securin proteins, which prevent separins from promoting sister chromatid separation.
the gene product contains  <dig> pxxp motifs, which are essential for its transforming and tumorigenic activities, as well as for its stimulation of basic fibroblast growth factor expression  <cit> .
besides the functions of cell cycle and growth, the major role of cxc <dig> is involved in immune response.
the protein encoded by lgals <dig> is a member of beta-galactoside-binding protein family implicated in modulating cell-cell and cell-matrix interactions, which may act as an autocrine negative growth factor that regulates cell proliferation  <cit> .
the protein product of the gene adrenomedullin  is a multifunctional peptide vasodilator that carries out its functions through calcitonin receptor-like receptor/receptor activity modifying protein- <dig> and - <dig> .
it can positively enhance cell proliferation  <cit> .
myc is a well known multifunctional, nuclear phosphoprotein that contributes to cell cycle progression, apoptosis and cellular transformation  <cit> .
it acts as a transcription factor that regulates transcription of specific target genes.
it has been implicated as an oncogene to facilitate cell growth and survival.
the protein encoded by nrg <dig> was initially characterized as a 44-kd glycoprotein that interacts with the neu/erbb <dig> receptor tyrosine kinase to increase its phosphorylation on tyrosine residues.
this protein is a signaling protein that regulates cell-cell interactions and is involved in the promotion of growth and development of multiple organ systems  <cit> .
neuregulin  <dig>  is a new member of the neuregulin family of growth and differentiation factors.
by interacting with the erbb family of receptors, nrg <dig> induces the growth and differentiation of epithelial, neuronal, glial, and other cell types  <cit> .
the gene pparg encodes a member of the peroxisome proliferator-activated receptor  subfamily of nuclear receptors.
ppars form heterodimers with retinoid x receptors  and these heterodimers regulate transcription of various genes to execute various functions including lipid metabolism and cell growth  <cit> .
the gene bcl2a <dig>  encodes a member of the bcl- <dig> protein family, a well known protein family functioning as anti- and pro-apoptotic regulators <cit> .
the protein encoded by this bcl2a <dig> is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation, thereby enhance cell survival.
the reduction of the expression of this gene could partially account for the cell damage induced by tnt.
interestingly, the downregulated genes involved in cell growth and cell death also play a role in the cell cycle process.
therefore, the cell growth and other possible cell death related phenotypes may occur primarily through interrupting cell cycle progression of cells.
recently we found that there was weight loss in rats treated with tnt .
the possible reason for the body weight loss is that cell growth is reduced by tnt.
tnt could inhibit the growth of v <dig> and tk <dig> human lymphoblastic cells  <cit> .
it could explain cytotoxic effects caused by tnt  <cit> .
detoxification response
our results revealed a couple of pathways that were commonly regulated by both in vivo and in vitro tnt treatments.
from the pathways listed in table  <dig>  we identified xenobiotic metabolism signaling as the key of all the pathways because all the other pathways are involved in xenobiotic metabolism signaling.
certainly, several phase i and ii metabolizing enzymes with their family members that take part in multiple pathways play a pivotal role in the detoxification process.
these enzymes mainly cover cyps, gsts, ugts, akr7a <dig>  aldhl <dig>  nqo <dig>  and ephx <dig>  as expected, all these enzymes were consistently elevated by both in vitro and in vivo tnt treatments.
cyp1a <dig> has been a hallmark for the treatments of many toxins and carcinogens  <cit> .
cyp1a <dig> was strongly induced in both in vivo and in vitro liver cells by tnt.
due to its catalytic function, it could play dual roles in the cells in the presence of tnt.
its activation leads to the production of reactive oxygen species   <cit>  which induces oxidative stress, lipid peroxidation , protein modification and denaturation, and dna damage, and thus induces toxicity.
meanwhile, it may also directly metabolize tnt or its metabolites, resulting in cell survival.
glutathione transferases  catalyze the conjunction of reduced glutathione  to electrophiles and oxidative stress products, thus facilitating their removal  <cit> .
ugts catalyze the generation of glucuronide conjugates of dihydrodiols to execute their detoxification function  <cit> .
akr7a <dig> catalyzes aflatoxin b <dig>  and suppresses afb <dig> dialdehyde metabolite to its corresponding mono and dialcohols.
hence, the activation of akr7a <dig> attenuates the toxicity of the afb1-dialdehyde that reacts with proteins, and thus decreases afb1-induced toxicity  <cit> .
aldh1l <dig> is well recognized as protectors against ros caused oxidative damage.
it metabolizes reactive products of toxic lpo, which include 4-hydroxy-2-nonenal  and malondialdehyde   <cit> .
this protective mechanism could explain how aldh responds to the cellular recovery to tnt exposure.
as a cytosolic flavoenzyme, nqo <dig> catalyzes the two-electron reduction of diversified substrates  <cit> .
nqo <dig> is characterized as a detoxification enzyme mainly because of its capability of degenerating quinone substrates to their less toxic hydroquinone metabolites, bypassing the redox-cycling semiquinone radical  <cit> .
nqo <dig> can also transform ubiquinone and vitamin e quinone to their antioxidant forms  <cit> .
ephx <dig> is one member of epoxide hydrolases  that catalyze the hydrolysis of electrophiles such as epoxides to the less reactive vicinal diols, which can explain the mechanism of epoxide hydrolases as classical detoxifying enzymes.
eph is able to inactivate highly diversified reactive epoxides with different structures, and therefore plays a critical role in the enzymatic defense against adverse effects of xenobiotic compounds  <cit> .
many upregulated genes involved in xenobiotic metabolism signaling also participate in cell cycle, cell growth, and cell death.
our results suggest that, upon exposure to tnt, cells arrest the cell cycle and increase detoxification and oxidative stress enzymes to remove the xenobiotic and eliminate any cell damage caused by the xenobiotic..
lipid metabolism
several genes participating in lipid metabolism were commonly regulated by both in vivo and in vitro tnt exposures.
most of these lipid metabolism genes showed elevated expression, while only a small number of genes were repressed.
the downregulated genes included lgals <dig>  pnpla <dig>  cxcl <dig>  hsd11b <dig> and inpp <dig> d. one well known gene, inositol polyphosphate-5-phosphatase, 145kda , encodes a protein that is largely confined to hematopoietic cells.
it is a well characterized inhibitory molecule that is recruited by engagement of the inhibitory fcγ type iib receptor in b cells and mast cells or by engagement of fcε type i or fcγ type iii, cytokine, and growth factor receptors in myeloid cells  <cit> .
once recruited to the plasma membrane by signaling complexes, its enzymatic activity depletes ptdinsp <dig> and prevents membrane localization of some ph domain-containing effectors, eventually leading to impaired pi3k-dependent signaling events.
the protein encoded by the gene pnpla <dig> is a member of the adiponutrin family complement the hormone sensitive lipase  as responsible for adipocyte triacylglycerol lipase activity.
mice lacking hsl reveal a lean phenotype and accumulate diglycerides suggesting that hsl is the main enzyme for the second step of lipolysis  <cit> .
lgals <dig> has been shown to be involved in many other functions in table  <dig>  also plays a role in lipid metabolism, by increasing the induction of levels of phosphatidylserine as well as the mobilization of phosphatidylserine  <cit> .
hydroxysteroid  dehydrogenase  <dig>  has been reported to contribute to the metabolism of aldosterone, cortisone, glucorticoid and hydrocortisone  <cit> .
the downregulated genes involved in lipid metabolism may indicate that tnt may interrupt lipid metabolism to some degree.
the lipid metabolism was also impaired in the liver of quail exposed to  <dig> -dnt  <cit> .
meanwhile, we saw many other genes involved in lipid metabolism upregulated by tnt, many of which overlap with xenobiotic signaling genes , suggesting that they also function for detoxification by trying to recover normal lipid metabolism.
immune response
as described earlier in the results,  <dig> of  <dig> genes associated with immune response were downregulated by tnt both in vivo and in vitro.
these  <dig> genes are cxcl <dig>  col1a <dig>  bcl2a <dig>  inpp <dig> d, and cytip.
chemokine  ligand  <dig>  was strongly downregulated by tnt.
it is a ligand for the g-coupled receptor protein chemokine  receptor cxcr <dig>  activation of cxcr <dig> by cxcl <dig> is involved in many biological functions such as cell migration, growth, and survival  <cit> .
besides enhancing cell survival, bcl2a <dig> is a direct transcription target of nf-κb in response to inflammatory mediators, and is upregulated by different extracellular signals such as granulocyte-macrophage colony-stimulating factor , cd <dig>  phorbol ester and inflammatory cytokines tnf and il- <dig>  which suggests a cytoprotective function that is essential for lymphocyte activation, and plays a role in immune response  <cit> .
col1a <dig> encodes the pro-alpha  <dig> chains of type i collagen whose triple helix is composed of two alpha  <dig> chains and one alpha  <dig> chain.
type i collagen is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon.
thus this gene is critical for cell aggregation, migration, proliferation, binding, adhesion and growth  <cit> .
this gene carries out its immune response by primarily affecting transmigration of t lymphocytes.
as a gene primarily functioning in lipid metabolism, inpp <dig> d participating in immune response by mainly maintaining the quantity of b lymphocytes and participating in phospholipid metabolism  <cit> .
cytohesin  <dig> interacting protein  encodes a protein containing  <dig> leucine zipper domains and a putative c-terminal nuclear targeting signal.
its major role is involved in the quantity maintenance, migration of leukocytes and lymphocytes  <cit> .
the decreased above gene expression could explain the interference of normal immune response by tnt both in vitro and in vivo, which could lead to inflammation and other immune related toxicities caused by tnt.
network as a valuable approach for predicting in vivo function using in vitro data
finally, not only could we find commonly regulated genes, functional terms, and pathways, but we were also able to identify conserved gene networks between in vitro and in vivo liver systems perturbed by tnt.
there were two big conserved sub-networks, the tf pttg <dig> centered gene network and the tfs pir, btbd <dig>  htatip <dig> and creg1-connected gene network .
interestingly, all the genes in the pttg <dig> sub-network were associated with cell cycle function.
for example, cdc <dig> and cyclin b  form a complex which is responsible for the onset of metaphase  <cit> .
pttg <dig>  cdc <dig>  and ccnb <dig> all participated in the mitotic roles of polo-like kinase pathway  <cit> .
furthermore, all the genes in the pttg sub-network were downregulated by tnt in vitro and in vivo.
since cell cycle is the most significant functional term affected by tnt both in vitro and in vivo , this conserved cell cycle relating network could well explain the common mechanism of tnt treated liver systems both in vivo and in vitro, which is that cell cycle progression is interfered by tnt, thereby leading to cell growth inhibition and cell death, ultimately cause toxicity  <cit> .
we did observe liver cell death in rats exposed to high dose tnt .
in the pbhc sub-network, some genes such as creg <dig>  ntrk <dig>  dusp <dig>  gsr, and txnrd <dig> play a role in cell cycle and/or cell death.
many genes possess oxidoreductase activity  <cit> , and are involved in oxidative stress process and play a crucial role in metabolizing toxic compounds and their metabolites.
the tf pir encodes a tf which is a member of the cupin superfamily.
the encoded protein is a fe-containing nuclear protein expressed in all tissues of the body and concentrated within dot-like subnuclear structures  <cit> .
interactions with nuclear factor i/ccaat box transcription factor as well as b cell lymphoma 3-encoded oncoprotein suggest the encoded protein may function as a transcriptional cofactor and be involved in the regulation of dna transcription and replication.
interestingly, the tf htatip <dig> also has oxidoreductase activity  <cit> , and connects two genes gsr and txnrd <dig> that both possess oxidoreductase activity as well as both take part in nrf2-mediated oxidative stress pathway.
htatip was reported to be regulated by nrf <dig>  too  <cit> .
overall, we can see that the genes in the pbhc sub-network are involved in cell cycle growth, and/or oxidoreductase activity.
surprisingly,  <dig> genes in the pbhc sub-network  were all consistently upregulated by tnt both in vivo and in vitro.
the induction of these gene expression lead to cell survival and removal of toxic products, therefore, this conserved sub-network may well explain the common detoxification mechanism both in vivo and in vitro liver systems treated with tnt.
we did not use only tnt-exposed samples but included all samples in the pool to construct static gene networks, which aim to uncover any gene interactions.
in so doing, the more samples and conditions included make the network analyses more robust.
for instance, faith et al.
used  <dig> arrays with over  <dig> various conditions to build an e. coli static gene network.
because the genes we used are tnt-regulated genes, the constructed gene networks are specifically tnt-regulated networks.
we built separate gene networks using in vitro and in vivo samples and found commonly conserved networks.
these common networks are ideal because they validate both in vivo and in vitro conditions.
CONCLUSIONS
our results indicate that tnt perturbed similar gene regulated networks when in vitro effects on hepatocyte cells are compared with in vitro effects on liver.
thus, gene regulatory networks obtained from an in vitro system can be predictive of in vivo function in the liver when perturbed by a chemical stressor such as tnt.
furthermore, pttg <dig> regulated cell cycle may be a key targeted process indicator for tnt induced toxicity.
this study is the first report to use an in vitro transcriptional regulatory gene network to predict in vivo toxicity and mechanism induced by a chemical.
knowledge achieved from this innovative study can provide an efficient way to assess whether a soldier or civilian has been exposed to tnt and to find possible ways to prevent, treat, and reduce tnt induced adverse effects.

 high-throughput toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. recent microarray studies have shown that the in vitro gene expression profiles in liver slices treated with various compounds could predict the toxicity and pathology observed in vivo. however, the degree to which in vitro results can be extended to in vivo activity and possible mechanisms of action remains to be fully addressed. compared with animal models, primary cell cultures have advantages for investigating mechanisms of chemical toxicity. primary cultured cells, such as hepatocytes, can offer a convenient system that is easily genetically manipulated and can be used to test various throughput screens using different molecular and biochemical methods. use of primary cell cultures can also reduce cost and mitigate animal welfare concerns inherent in in vivo studies. in vitro systems have a long history of use in screening new drugs for human diseases such as cancer and in studying cellular and molecular events of different molecules. in this study, we used the nitroaromatic, -rinitrotoluene as a model chemical to compare and determine how we might extrapolate in vitro data to in vivo systems. we compared the gene expression profiles of in vitro primary liver cells with the gene expression profiles of in vivo liver tissue of rats exposed to tnt. tnt is a munitions compound which is released to the environment as a result of military training activities and manufacturing processes. the major toxic effects to rats in response to tnt exposure are methemoglobinemia, anemia, hypercholesterolemia, and hepatomegaly, splenomegaly, and testicular atrophy with their accompanying histologic lesions. cytotoxic and genotoxic effects are also caused by tnt. our recent toxicity studies in rats found that tnt decreased body weight, increased liver weight, and induced erythrocytosis. although a variety of studies have focused on toxicity aspects of tnt, the underlying mechanisms of toxicity induced by this compound are largely unknown. several laboratories have started to use microarray analysis to understand the molecular mechanisms of invertebrate ecotoxicity. the exposure of the earthworm eisenia fetida with tnt regulates the expression of genes involved in multiple biological processes including muscle contraction, neuronal signaling and growth, ubiquitinylation, fibrinolysis and coagulation, iron and calcium homeostasis, oxygen transport, and immunity. microarray analysis of tnt exposed arabidopsis thaliana reveals the induced expression of oxophytodienoate reductases, the protein responsible for tnt detoxification in arabidopsis. the same team also found that bifunctional o- and c-glucosyltransferases is involved in combating tnt induced toxicity in arabidopsis. eisentraeger et al. investigated the mechanism of human liver cell line hepg treated with tnt using the toxicology cdna microarray. they found that the detoxification metabolism in the hepg cells by tnt induced genes encoded phase i and phase ii enzymes. in the current study, we examined gene transcriptional responses to short term tnt exposures in rat in both in vitro hepatocytes and in vivo liver. we observed that a number of genes were commonly regulated by both in vitro and in vivo tnt treatments. moreover, we found commonly affected pathways in both in vivo and in vitro systems exposed with tnt. functional analysis indicates that both tnt treatments in vivo and in vitro impact genes involved in cell cycle, cell growth and cell death signaling, detoxification response, lipid metabolism and immune response, which can reasonably account for the physiological dysfunctions induced by tnt. in addition, we identified conserved gene networks inferred from in vivo and in vitro networks. our results suggest that in vitro system can predict in vivo functions and mechanisms based on gene expression profiles, and gene network is a valuable approach for predicting in vivo function using in vitro data.  commonly regulated genes by in vivo and in vitro tnt treatments to determine if in vitro tnt exposure could be used to predict in vivo tnt exposure, the first aim of this experiment was to determine if there were common genes differentially regulated by both in vivo and in vitro tnt treatments. for the in vivo experiment, rats were gavaged with tnt at:,, or mg/kg and sacrificed after h or h. rna was isolated for microarray hybridization. agilent rat whole genome array was employed in the study. four biological replicates were used for each unique condition as described in the material and methods. for the in vitro experiments, primary rat hepatocytes were cultured and treated with tnt at ppm or vehicle control for h, after which rna was isolated for microarray experiments. the in vivo and in vitro micorarray data have been deposited in the geo databases with assigned numbers gse and gse respectively. to identify genes differentially expressed in vivo, a one-way anova was applied to identify differentiated transcripts across different doses of tnt treatments at each of the h or h time points. a p value cut-off of and a fold gene transcript change by comparing at least one pair of conditions between different doses including control were used as filters to identify differentially expressed genes. a total of gene transcripts were significantly and differentially expressed in in vivo tnt treatment for all doses and time points. an un-paired t-test with a p value cut-off of and fold change filter was used to test for differential expression between controls and tnt treated hepatocytes in the in vitro assay yielding differentially expressed transcripts. a venn diagram shows that there were transcripts that were commonly regulated by both in vivo and in vitro tnt treatments. the common differentiated genes between in vivo and in vitro exposures suggest that a common mechanism exists between in vitro tnt and in vivo tnt treated liver systems. similar gene expression pattern shared by commonly regulated in vivo and in vitro genes we examined the expression pattern of the common transcripts from in vivo and in vitro tnt exposures. to reach the goal, each sample was normalized by the mean intensity of a gene of relative control samples, and the samples under the same condition were averaged into one condition. a two-way hierarchical clustering resulted in two cluster dendrograms of the transcripts across in vivo conditions and in vitro conditions. the controls and lower in vivo dose conditions at h were in a cluster, indicating that these doses at h are closer to controls and have less genes affected than other conditions. the remaining in vivo and in vitro tnt exposed hepatocytes and livers formed the second cluster. overall, we found the conditons were more likely to group together according to exposure duration than dose concentration, implying that the time effect is stronger than the dose effect. interestingly, we found that conditions fell into one subgroup. moreover, we noticed that the gene expression patterns for the mg/kg, h in vivo tnt treatment and the h in vitro tnt treatment were similar. the genes are upregulated by the in vitro treatment, and they were usually upregulated in vivo treatment. most downregulated genes in in vitro were also repressed in vivo. since these two conditions behaved closely, we directly compared commonly upregulated and downregulated genes based on these two conditions. out of these transcripts, transcripts were upregulated by the in vivo tnt treatment, and transcripts were upregulated by the in vitro tnt treatment. one hundred sixty-four transcripts were commonly upregulated under both conditions. one hundred forty in vivo tnt transcripts and in vitro tnt transcripts were downregulated. furthermore, transcripts were reduced by both conditions. another common phenomenon was that both tnt exposed liver systems had more upregulated than downregulated genes. our results demonstrate that tnt treatment in in vivo and in vitro liver systems results in genes regulated in the same direction, providing more evidence that in vitro hepatocytes act as a suitable surrogate for in vivo liver exposures. functional analysis of commonly regulated genes to understand the functions of the commonly regulated genes, we identified most significantly functional categories using the ingenuity function analysis tool. these functional groups mainly included cell cycle, carbohydrate metabolism, molecular transport, cell growth and proliferation, cell death, dna replication, recombination and repair, lipid metabolism, cellular assembly and organization, and immune response. the most significant functional category regulated by tnt was cell cycle, which possessed genes, of which, genes were commonly downregulated by in vivo and in vitro tnt exposures. the most heavily downregulated genes involved in cell cycle by both in vivo and in vitro tnt treatments included lgals cyp26b and pttg highly induced genes in this cell cycle category included adm, dusp and pparg. more genes participating in cell cycle were downregulated, indicating that tnt appears to interfere with cell cycle progress. * full names of the genes are listed in additional file table s other commonly significantly affected functional terms were cell growth and proliferation, cell death, dna replication, and recombination and repair. interestingly, there were genes that were repeatedly present in all four functional terms: adm, ccnb ccnd cxcl lgals, myc, nrg nrg pparg, and pttg in addition, these genes were consistently regulated by both in vivo tnt and in vitro tnt in the same directions. the expression of ccnb ccnd pttg cxc and lgals was decreased by tnt, and the expression of adm, myc, ngr ngr and pparg was elevated by tnt. there were commonly regulated genes that were involved in molecular transport. nineteen of the genes were upregulated, and only of them were downregulated by both in vitro and in vivo tnt treatments. some significant upregulated genes included nqo adm, abcc and abcc our results suggest that tnt can enhance molecular transport both in vivo and in vitro liver systems. twenty-three genes contributing to carbohydrate metabolism were commonly regulated by both in vivo and in vitro tnt treatments. among them, only genes were repressed and genes were induced. the expression of genes participating in lipid metabolism was significantly changed in response to both in vivo and in vitro tnt exposures. similar to carbohydrate metabolism, we saw more upregulated genes than downregulated genes in this category. several cytochrome p family members such as cyp1a cyp3a and cyp2c were in the upregulated gene list. interestingly, genes belong to the three functional categories: molecular transport, carbohydrate metabolism and lipid metabolism. three of these genes were downregulated and the other genes were upregulated by both in vivo and in vitro tnt treatments. there were commonly regulated genes that were involved in cellular assembly and organization. the expression of genes was decreased, while expression of genes were increased. eight genes played a role in immune response, of which were downregulated and genes upregulated. more immune response genes were downregulated, indicating that tnt may interfere with normal immune function to induce its toxicity. overall, some commonly regulated genes were highly represented. for instance, the gene myc falls into all the functional categories listed in table cxcl belongs to all the functional groups except cellular assembly and organization. adm and lgals genes pparg were absent in only two functional categories, cellular assembly and organization, and immune response. pathway analysis to further understand the gene function influenced by tnt exposure to in vivo and in vitro liver cells, we conducted a canonical pathway analysis using the ingenuity pathway analysis tool. three separate gene lists were used to run the pathway analysis: the list of genes most significantly regulated by in vivo tnt treatment, the list of genes most significantly regulated by in vitro tnt exposure, and the list of the commonly regulated genes. in order to identify the most significantly influenced pathways in vivo, a more stringent p value was applied, which resulted in significantly regulated transcripts for the in vivo tnt experiment. the top significantly impacted pathways from each gene list are depicted in figure using a threshold p value of, a total of pathways were regulated by in vivo tnt treatment, pathways were evidently affected by in vitro tnt exposure, and significantly pathways were obtained by analyzing the common genes alone. twenty commonly regulated pathways were shared by the in vivo and in vitro pathway lists, which is 50% of the pathways impacted by in vitro tnt treatment. the percentage of overlapping pathways was much higher than that of overlapping genes illustrated in figure indicating that the in vitro system perturbed by tnt could reflect more biological truth for an in vivo system from a mechanism related pathway view. sixteen consensus pathways were shared by the three significant pathway lists. the top regulated pathways with relative genes are listed in table several gene families that were highly represented in these pathways are related to xenobiotic metabolism and transport. these families were cytochrome p, glutathione s-transferase, udp glucuronosyltransferase, aldehyde dehydrogenase and atp-binding cassette families. the regulated genes in the cyp family included cypa cyp2c cyp2f and cyp3a the genes listed in table belonging to gst family included gst and gsta the ugt family included ugt1a ugt2b and ugt2b the regulated gene aldehyde dehydrogenase family, member l in the family aldh was involved in many pathways listed in table the regulated genes in the abc family included abcc abcc and abcg * full names of the genes are listed in additional file table s 1 interestingly, all the genes in table except for ccnd and retinoid acid receptor beta, were commonly upregulated by both in vivo and in vitro tnt treatments. mapk a critical intracellular signaling protein involved in multiple cellular functions was also presented in multiple pathways such as nrf- mediated oxidative stress response, xenobiotic metabolism signaling, and aryl hydrocarbon signaling pathways. inferring in vitro and in vivo gene networks motivated by commonly regulated genes, functional terms and pathways, we then investigated whether a common gene network was regulated by both the in vitro and in vivo tnt treatments. to achieve this goal, we used a reverse engineering algorithm called context likelihood of relatedness. clr is based on mutual information of any pair of genes and is a static network. a static gene network reflects all possible gene interactions for a given gene list. therefore, the more samples used to construct a gene network using this algorithm make the built network more trustworthy. in order to build in vivo gene networks, arrays from rat liver tissues treated with one of compounds tnt, 2-amino- -dinitrotoluene, 4-amino- -dinitrotoulene, -dinitrotoluene and -dinitrotoluene or vehicle controls were used. for in vitro gene network modeling, arrays resulted from liver primary cultured cells treated by distinctive compounds with relative controls were employed. using the gene expression data of the commonly regulated transcripts across the arrays, an in vivo gene network was constructed with the clr algorithm. figure 6a depicts the network which included nodes and edges. in vitro gene network was built using the same genes and the same clr algorithm, but expression data were generated from the arrays. the gene network from the in vitro data had nodes and edges. since transcriptional regulated gene networks are trigged by transcription factors, we then counted the connections of the transcription factors in the transcripts for the both in vivo and in vitro gene networks. top tfs with most connections in the in vitro gene network were cellular repressor of e1a-stimulated genes, pirin, btb domain containing sam pointed domain containing ets transcription factor and basic helix-loop-helix family, member e. tfs with most connections in the in vivo gene network included hiv- tat interactive protein 30kda, transcription factor dp-, leucine zipper, putative tumor suppressor, zinc finger protein, pir, and myc. the transcription factors that had over connections in the both gene networks included creg pir, btbd lzts asb hes and sqstm two tfs, spdef and bhlhe had at least twice more connections in the in vitro gene network than that in the in vivo gene network. seven tfs including htatip myc, neq znf e2f dnajb and tfdp possessed over twice as many connections in the in vivo gene network than that in the in vitro gene network. conserved in vitro and in vivo gene networks by comparing the in vivo and in vitro gene networks, we identified several conserved sub-networks that were common in both gene networks. the tf pttg centered sub-gene network consisted of genes, all of which are involved in cell cycle processes. moreover, all the genes in the cell cycle sub-network were consistently repressed by both in vivo and in vitro tnt treatments. the conserved sub-network provides further evidence that the cell cycle process may be impaired in both in vivo and in vitro systems treated with tnt. the tfs pir, btbd htatip and creg were colocalized in the biggest conserved sub-network. eight genes were connected with the tf pir centered sub-gene network module, and contained highly enriched genes associated with oxidoreductase activity. akr7a and txnrd also participate in nrf2-mediated oxidative stress pathway. all the genes in this network module were upregulated by both in vivo and in vitro tnt exposures. this result suggests that oxidative stress is activated by tnt. the tf creg centered sub-network module included genes, of which were also cell cycle related genes. in contrast to pttg centered sub-network, all the genes in the creg sub-network module were upregulated by both in vivo and in vitro tnt treatments. glutathione reductase, a gene related to oxidoreductase activity, and txnrd provided the connections between the creg htatip and pir sub-networks, creating a larger gene sub-network. in addition, gsr also participated in the nrf2-mediated oxidative stress pathway. indeed, we found that all the genes in the biggest conserved sub-network formed by creg htatip btbd and pir mediated modules were consistently upregulated by both in vivo and in vitro tnt additions. verification of microarray responses using real time qrt-pcr to verify the credibility of microarray and gene network modeling results, we selected genes to perform real time quantitative pcr both in vitro and in vivo. the genes chosen consistently played key roles in functional categories and pathways altered by tnt exposures in vivo and in vivo. as illustrated in figure we observed good consistency between microarray and qrt-pcr results. similar upregulation and downregulation trends were seen with both microarray data and qrt-pcr data for all genes. the overall regression coefficient of the two methods across various conditions was. our results indicate the microarray data quality is good and our interpretation and analysis should be convincing. specific comparisons between microarray and qrt-pcr are presented in additional file figures s 1a-1f. for instance, the gene abcc was shown in microarray to be upregulated at both h and h with a clear dose response to in vivo tnt treatment, and was also induced by tnt in vitro, and the same pattern was exhibited by qrt-pcr. in both microarray and qrt-pcr results, the gene akr7a was upregulated at both time points in vivo as well as upregulated in vitro. interestingly, akr7a was significantly upregulated at the highest dose in vivo at h but not at other lower doses, which was shown consistently by both microarray and qrt-pcr. for the gene cyp1a we saw it was upregulated more in response to in vitro tnt than in vivo tnt treatment, and was induced much more at h than h in vivo, which was consistently revealed by both microarray and qrt-pcr results. three genes pttg, ccnb and ccnd that were in pttg centered subnetwork were consistently shown to be downregulated by tnt in vivo and in vitro, with both approaches. the qrt-pcr results could exactly confirm our two observations obtained from the microarray data: the genes up or downregulated in the same directions in vitro and in vivo, and a clear dose response in vivo. discussion in this study, we compared gene expression profiles regulated by tnt exposure in vivo and in vitro from shared gene lists, functional terms, common pathways, and conserved networks. overall, good consistency existed between in vitro and in vivo exposures. the venn diagram shows that a only small percentage of the in vivo transcripts were also regulated in the in vitro experiments. because the regulated gene number in vivo is the total number from all tnt treatments including multiple doses and two time points, the gene number is much larger than that of in vitro exposures. however, the in vitro gene number comes from only one dose. the reason we used one dose is that the in vitro experiment is a large one with a total of over chemicals and arrays. but if we compare the overlapped transcripts with the in vitro transcripts, the number of overlapped transcripts is not small, with over one third of the total in vitro transcripts. this observation is found more than in overlaps examined in other publications, the number is comparable to that of the h in vitro tnt treatment. in addition, in vivo and in vitro are two different systems, we cannot expect that they are exactly the same. our purpose is to identify common transcripts between these two systems, thereby to understand the in vivo function through the in vitro system. from the perspective of functional analysis, both tnt treatments in vivo and in vitro influence genes involved in cell cycle, cell growth and cell death signaling, detoxification response, lipid metabolism and immune reponse, which can reasonably explain the physiological dysfunctions induced by tnt. we also found conserved sub-networks between inferred networks from in vivo and in vitro tnt regulated gene expression profiles. cell cycle, cell growth, and cell death signaling a large number of genes involved in cell cycle, cell growth and cell death were commonly regulated in both in vitro and in vivo systems perturbed by tnt. the highly represented downregulated genes were ccnb ccnd pttg cxc and lgals and the highly represented upregulated genes were adm, myc, nrg nrg and pparg. the cyclin b gene product complexes with p to form the maturation-promoting factor. ccnb is expressed predominantly during g2/m phase because of its critical role in cell mitosis. the protein encoded by ccnd belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. cyclins function as regulators of cdk kinases. different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. the ccnd2-translated cyclin forms a complex with and functions as a regulatory subunit of cdk or cdk whose activity is essential for cell cycle g1/s transition. pttg encoded protein is a homolog of yeast securin proteins, which prevent separins from promoting sister chromatid separation. the gene product contains pxxp motifs, which are essential for its transforming and tumorigenic activities, as well as for its stimulation of basic fibroblast growth factor expression. besides the functions of cell cycle and growth, the major role of cxc is involved in immune response. the protein encoded by lgals is a member of beta-galactoside-binding protein family implicated in modulating cell-cell and cell-matrix interactions, which may act as an autocrine negative growth factor that regulates cell proliferation. the protein product of the gene adrenomedullin is a multifunctional peptide vasodilator that carries out its functions through calcitonin receptor-like receptor/receptor activity modifying protein- and -. it can positively enhance cell proliferation. myc is a well known multifunctional, nuclear phosphoprotein that contributes to cell cycle progression, apoptosis and cellular transformation. it acts as a transcription factor that regulates transcription of specific target genes. it has been implicated as an oncogene to facilitate cell growth and survival. the protein encoded by nrg was initially characterized as a 44-kd glycoprotein that interacts with the neu/erbb receptor tyrosine kinase to increase its phosphorylation on tyrosine residues. this protein is a signaling protein that regulates cell-cell interactions and is involved in the promotion of growth and development of multiple organ systems. neuregulin is a new member of the neuregulin family of growth and differentiation factors. by interacting with the erbb family of receptors, nrg induces the growth and differentiation of epithelial, neuronal, glial, and other cell types. the gene pparg encodes a member of the peroxisome proliferator-activated receptor subfamily of nuclear receptors. ppars form heterodimers with retinoid x receptors and these heterodimers regulate transcription of various genes to execute various functions including lipid metabolism and cell growth. the gene bcl2a encodes a member of the bcl- protein family, a well known protein family functioning as anti- and pro-apoptotic regulators. the protein encoded by this bcl2a is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation, thereby enhance cell survival. the reduction of the expression of this gene could partially account for the cell damage induced by tnt. interestingly, the downregulated genes involved in cell growth and cell death also play a role in the cell cycle process. therefore, the cell growth and other possible cell death related phenotypes may occur primarily through interrupting cell cycle progression of cells. recently we found that there was weight loss in rats treated with tnt. the possible reason for the body weight loss is that cell growth is reduced by tnt. tnt could inhibit the growth of v and tk human lymphoblastic cells. it could explain cytotoxic effects caused by tnt. detoxification response our results revealed a couple of pathways that were commonly regulated by both in vivo and in vitro tnt treatments. from the pathways listed in table we identified xenobiotic metabolism signaling as the key of all the pathways because all the other pathways are involved in xenobiotic metabolism signaling. certainly, several phase i and ii metabolizing enzymes with their family members that take part in multiple pathways play a pivotal role in the detoxification process. these enzymes mainly cover cyps, gsts, ugts, akr7a aldhl nqo and ephx as expected, all these enzymes were consistently elevated by both in vitro and in vivo tnt treatments. cyp1a has been a hallmark for the treatments of many toxins and carcinogens. cyp1a was strongly induced in both in vivo and in vitro liver cells by tnt. due to its catalytic function, it could play dual roles in the cells in the presence of tnt. its activation leads to the production of reactive oxygen species which induces oxidative stress, lipid peroxidation, protein modification and denaturation, and dna damage, and thus induces toxicity. meanwhile, it may also directly metabolize tnt or its metabolites, resulting in cell survival. glutathione transferases catalyze the conjunction of reduced glutathione to electrophiles and oxidative stress products, thus facilitating their removal. ugts catalyze the generation of glucuronide conjugates of dihydrodiols to execute their detoxification function. akr7a catalyzes aflatoxin b and suppresses afb dialdehyde metabolite to its corresponding mono and dialcohols. hence, the activation of akr7a attenuates the toxicity of the afb1-dialdehyde that reacts with proteins, and thus decreases afb1-induced toxicity. aldh1l is well recognized as protectors against ros caused oxidative damage. it metabolizes reactive products of toxic lpo, which include 4-hydroxy-2-nonenal and malondialdehyde. this protective mechanism could explain how aldh responds to the cellular recovery to tnt exposure. as a cytosolic flavoenzyme, nqo catalyzes the two-electron reduction of diversified substrates. nqo is characterized as a detoxification enzyme mainly because of its capability of degenerating quinone substrates to their less toxic hydroquinone metabolites, bypassing the redox-cycling semiquinone radical. nqo can also transform ubiquinone and vitamin e quinone to their antioxidant forms. ephx is one member of epoxide hydrolases that catalyze the hydrolysis of electrophiles such as epoxides to the less reactive vicinal diols, which can explain the mechanism of epoxide hydrolases as classical detoxifying enzymes. eph is able to inactivate highly diversified reactive epoxides with different structures, and therefore plays a critical role in the enzymatic defense against adverse effects of xenobiotic compounds. many upregulated genes involved in xenobiotic metabolism signaling also participate in cell cycle, cell growth, and cell death. our results suggest that, upon exposure to tnt, cells arrest the cell cycle and increase detoxification and oxidative stress enzymes to remove the xenobiotic and eliminate any cell damage caused by the xenobiotic.. lipid metabolism several genes participating in lipid metabolism were commonly regulated by both in vivo and in vitro tnt exposures. most of these lipid metabolism genes showed elevated expression, while only a small number of genes were repressed. the downregulated genes included lgals pnpla cxcl hsd11b and inpp d. one well known gene, inositol polyphosphate-5-phosphatase, 145kda, encodes a protein that is largely confined to hematopoietic cells. it is a well characterized inhibitory molecule that is recruited by engagement of the inhibitory fcγ type iib receptor in b cells and mast cells or by engagement of fcε type i or fcγ type iii, cytokine, and growth factor receptors in myeloid cells. once recruited to the plasma membrane by signaling complexes, its enzymatic activity depletes ptdinsp and prevents membrane localization of some ph domain-containing effectors, eventually leading to impaired pi3k-dependent signaling events. the protein encoded by the gene pnpla is a member of the adiponutrin family complement the hormone sensitive lipase as responsible for adipocyte triacylglycerol lipase activity. mice lacking hsl reveal a lean phenotype and accumulate diglycerides suggesting that hsl is the main enzyme for the second step of lipolysis. lgals has been shown to be involved in many other functions in table also plays a role in lipid metabolism, by increasing the induction of levels of phosphatidylserine as well as the mobilization of phosphatidylserine. hydroxysteroid dehydrogenase has been reported to contribute to the metabolism of aldosterone, cortisone, glucorticoid and hydrocortisone. the downregulated genes involved in lipid metabolism may indicate that tnt may interrupt lipid metabolism to some degree. the lipid metabolism was also impaired in the liver of quail exposed to -dnt. meanwhile, we saw many other genes involved in lipid metabolism upregulated by tnt, many of which overlap with xenobiotic signaling genes, suggesting that they also function for detoxification by trying to recover normal lipid metabolism. immune response as described earlier in the results, of genes associated with immune response were downregulated by tnt both in vivo and in vitro. these genes are cxcl col1a bcl2a inpp d, and cytip. chemokine ligand was strongly downregulated by tnt. it is a ligand for the g-coupled receptor protein chemokine receptor cxcr activation of cxcr by cxcl is involved in many biological functions such as cell migration, growth, and survival. besides enhancing cell survival, bcl2a is a direct transcription target of nf-κb in response to inflammatory mediators, and is upregulated by different extracellular signals such as granulocyte-macrophage colony-stimulating factor, cd phorbol ester and inflammatory cytokines tnf and il- which suggests a cytoprotective function that is essential for lymphocyte activation, and plays a role in immune response. col1a encodes the pro-alpha chains of type i collagen whose triple helix is composed of two alpha chains and one alpha chain. type i collagen is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon. thus this gene is critical for cell aggregation, migration, proliferation, binding, adhesion and growth. this gene carries out its immune response by primarily affecting transmigration of t lymphocytes. as a gene primarily functioning in lipid metabolism, inpp d participating in immune response by mainly maintaining the quantity of b lymphocytes and participating in phospholipid metabolism. cytohesin interacting protein encodes a protein containing leucine zipper domains and a putative c-terminal nuclear targeting signal. its major role is involved in the quantity maintenance, migration of leukocytes and lymphocytes. the decreased above gene expression could explain the interference of normal immune response by tnt both in vitro and in vivo, which could lead to inflammation and other immune related toxicities caused by tnt. network as a valuable approach for predicting in vivo function using in vitro data finally, not only could we find commonly regulated genes, functional terms, and pathways, but we were also able to identify conserved gene networks between in vitro and in vivo liver systems perturbed by tnt. there were two big conserved sub-networks, the tf pttg centered gene network and the tfs pir, btbd htatip and creg1-connected gene network. interestingly, all the genes in the pttg sub-network were associated with cell cycle function. for example, cdc and cyclin b form a complex which is responsible for the onset of metaphase. pttg cdc and ccnb all participated in the mitotic roles of polo-like kinase pathway. furthermore, all the genes in the pttg sub-network were downregulated by tnt in vitro and in vivo. since cell cycle is the most significant functional term affected by tnt both in vitro and in vivo, this conserved cell cycle relating network could well explain the common mechanism of tnt treated liver systems both in vivo and in vitro, which is that cell cycle progression is interfered by tnt, thereby leading to cell growth inhibition and cell death, ultimately cause toxicity. we did observe liver cell death in rats exposed to high dose tnt. in the pbhc sub-network, some genes such as creg ntrk dusp gsr, and txnrd play a role in cell cycle and/or cell death. many genes possess oxidoreductase activity, and are involved in oxidative stress process and play a crucial role in metabolizing toxic compounds and their metabolites. the tf pir encodes a tf which is a member of the cupin superfamily. the encoded protein is a fe-containing nuclear protein expressed in all tissues of the body and concentrated within dot-like subnuclear structures. interactions with nuclear factor i/ccaat box transcription factor as well as b cell lymphoma 3-encoded oncoprotein suggest the encoded protein may function as a transcriptional cofactor and be involved in the regulation of dna transcription and replication. interestingly, the tf htatip also has oxidoreductase activity, and connects two genes gsr and txnrd that both possess oxidoreductase activity as well as both take part in nrf2-mediated oxidative stress pathway. htatip was reported to be regulated by nrf too. overall, we can see that the genes in the pbhc sub-network are involved in cell cycle growth, and/or oxidoreductase activity. surprisingly, genes in the pbhc sub-network were all consistently upregulated by tnt both in vivo and in vitro. the induction of these gene expression lead to cell survival and removal of toxic products, therefore, this conserved sub-network may well explain the common detoxification mechanism both in vivo and in vitro liver systems treated with tnt. we did not use only tnt-exposed samples but included all samples in the pool to construct static gene networks, which aim to uncover any gene interactions. in so doing, the more samples and conditions included make the network analyses more robust. for instance, faith et al. used arrays with over various conditions to build an e. coli static gene network. because the genes we used are tnt-regulated genes, the constructed gene networks are specifically tnt-regulated networks. we built separate gene networks using in vitro and in vivo samples and found commonly conserved networks. these common networks are ideal because they validate both in vivo and in vitro conditions.  our results indicate that tnt perturbed similar gene regulated networks when in vitro effects on hepatocyte cells are compared with in vitro effects on liver. thus, gene regulatory networks obtained from an in vitro system can be predictive of in vivo function in the liver when perturbed by a chemical stressor such as tnt. furthermore, pttg regulated cell cycle may be a key targeted process indicator for tnt induced toxicity. this study is the first report to use an in vitro transcriptional regulatory gene network to predict in vivo toxicity and mechanism induced by a chemical. knowledge achieved from this innovative study can provide an efficient way to assess whether a soldier or civilian has been exposed to tnt and to find possible ways to prevent, treat, and reduce tnt induced adverse effects. 
however, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. similarly modulated common pathways were identified between in vitro and in vivo. the results of our investigation of tnt effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. here we use the nitroaromatic,6-trinitrotoluene as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. the major functional term corresponding to these transcripts was cell cycle. interestingly, all genes in the pttg module were involved in cell cycle and downregulated by tnt both in vitro and in vivo. furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to tnt exposure, which mainly contain subnetwork modules: pttg and pir centered networks.
 evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. however, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed.  here we use the nitroaromatic,6-trinitrotoluene as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. we found transcripts differentially expressed in common among in vitro and in vivo assays in response to tnt. the major functional term corresponding to these transcripts was cell cycle. similarly modulated common pathways were identified between in vitro and in vivo. furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to tnt exposure, which mainly contain subnetwork modules: pttg and pir centered networks. interestingly, all genes in the pttg module were involved in cell cycle and downregulated by tnt both in vitro and in vivo.  the results of our investigation of tnt effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. inhibiting pttg and its targeted cell cyle related genes could be key machanism for tnt induced liver toxicity. 