BACKGROUND
pyrrolizidine alkaloids  are common constituents of thousands of plant species around the world and pa-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans.
thus, the human health risk posed by exposure to pas has been a concern  <cit> .
out of more than  <dig> plants, about  <dig> pas and their n-oxide derivatives have been identified, and at least half of them are genotoxic and many are tumorigenic  <cit> .
riddelliine is a representative genotoxic pa, and is present in plants growing in the rangelands of the western united states  <cit> .
these plants containing riddelliine appear to enter the human food chain since riddelliine residues have been detected in meat, milk, and honey  <cit> .
riddelliine was nominated by the u.s. food and drug administration to the national toxicology program  for genotoxicity and carcinogenicity testing due to the potential for human exposure  <cit> .
riddelliine is a 12-membered macrocyclic diester pa with an α, β-unsaturated double bond linked to the ester group at the c- <dig> position of the necine base.
riddelliine is completely absorbed within  <dig> minutes after gavage dosing to rodents and metabolized to the major metabolites,  <dig> -dihydro-1-hydroxymethyl-5h-pyrrolizine  and riddelliine n-oxide, by mammalian microsomes  <cit> .
32p-postlabeling-hplc analysis has identified a set of dhp-derived dna adducts from rat and human liver microsomal metabolism of riddelliine in vitro  <cit>  and in the livers of rats treated in vivo  <cit> .
a linear dose-dependent formation of dhp-derived dna adducts was observed in riddelliine-treated rats  <cit> .
riddelliine is genotoxic both in vitro and in vivo, inducing increases in sister chromatid exchange, chromosomal aberrations, unscheduled dna synthesis, and micronucleated erythrocyte frequencies  <cit> .
in the ntp carcinogenicity studies, riddelliine was tumorigenic, causing liver tumors in male mice and both sexes of rats, mononuclear cell leukemia in rats, and lung neoplasms in female mice.
riddelliine induced a high incidence of liver hemangiosarcomas  and lower incidences of hepatocellular carcinoma  in rat liver  <cit> .
in our previous studies, we observed that riddelliine is mutagenic in the liver of riddelliine-treated rats and that the mutant frequencies  increased in a linear dose-dependent manner.
riddelliine also produced a unique mutational spectrum in the liver cii gene of big blue rats with g:c → t:a transversions being the major type of mutation  <cit> .
moreover, we found that the cii mf in liver endothelial cells from riddelliine-treated rats was significantly greater than the cii mf in endothelial cells from control rats, suggesting that the relatively high mutagenicity of riddelliine in rat liver endothelial cells may be partially responsible for the tumorigenic specificity of this agent  <cit> .
it has been reported that riddelliine-treated mice and rats have higher and more persistent dna adduct levels in liver endothelial cells than in parenchymal cells  <cit> .
the sensitivity of tissues and cell types to the mutagenicity of carcinogens may be an important factor in the tissue- and cell-specificity of tumorigenesis.
microarray technology has a profound impact on gene expression research because of its ability to examine the expression of thousands of genes at a time.
the differentially expressed genes that are identified may be used to develop potential biomarkers, elucidate molecular mechanisms, and create gene signatures that identify classes of samples  <cit> .
this technology, as one of the core technologies for pharmacogenomics and toxicogenomics, provides new insights into the effects of botanical chemicals on biological systems and allow the macrodissection of molecular events in botanical carcinogenesis  <cit> .
identification of unique gene expression patterns produced by botanical carcinogens may allow us to elucidate the mechanisms of action.
in this study, we treated rats with a tumorigenic dose of riddelliine and conducted microarray analysis of gene expression in the target tissue liver.
we found that the gene expression profiles were significantly altered by riddelliine treatment, and many of the differentially expressed genes were involved in metabolism, injury of liver endothelial cells, liver abnormalities, and cancer development.
RESULTS
samples and dna microarray data analysis
liver samples used in this study were from a previous report  <cit>  in which female big blue rats were treated with a carcinogenic dose  of riddelliine for  <dig> weeks.
the mf in the liver cii gene was about 3-fold higher than those in the untreated group and the mutation spectra in the riddelliine-treated rats was significantly different from those in the control rats.
in the present study, gene expression profiles were determined for the livers of control and riddelliine-treated rats using the applied biosystems' rat genome survey microarray which contains  <dig>  verified rat genes.
because liver tumors had not developed at the  <dig> week sacrifice time, the gene expression changes reflected early events in the carcinogenesis process.
after data normalized by quantile normalization which is recommended by the manufacturer, the intensities of the whole rat gene data were analyzed by principal components analysis .
a separation between control and riddelliine-treated groups was observed, indicating that there was a riddelliine-treatment effect on liver gene expression.
pca analysis also demonstrates that sample # <dig> from the control group appears to be different from the rest of the five sample arrays.
figure  <dig> shows the pair-wise log <dig> intensity pearson's correlation coefficients for the  <dig> controls samples.
the correlation coefficient numbers containing samples # <dig> were lower than the others.
therefore, this array was excluded from further data analysis.
differentially expressed genes were identified based on the criteria of fold-change greater than  <dig>  and p-value less than  <dig>  in comparison to the control group .
a total of  <dig> genes satisfied the requirements, of which  <dig> genes were up-regulated and  <dig> genes were down-regulated in response to riddelliine treatment.
among the differentially expressed genes,  <dig> were in the ingenuity pathway analysis  database, and  <dig> genes were mapped to the networks.
figure  <dig> shows the top  <dig> networks; each network was associated with specific genes and involved in different functions.
the major relevant functions altered by riddelliine treatment in rat liver are listed in table  <dig>  these functions include cancer, cell death, tissue development, cell morphology, cell-to-cell signaling and interaction, and cellular development.
because riddelliine treatments induce relatively high dna adduct formation, mutation induction, and tumor incidence in the liver endothelial cells  <cit> , we focused our analysis on genes involved in carcinogenesis, mainly metabolism, injury of endothelial cells, liver abnormalities, and cancer development using ingenuity pathway analysis.
alteration of metabolizing genes
since metabolic activation of riddelliine is required for liver tumor induction  <cit> , we investigated the gene expression changes of drug metabolizing genes.
table  <dig> shows phases i, ii, and iii drug metabolizing genes whose expression was significantly changed by riddelliine treatment.
four phase  <dig> cytochrome p <dig> genes  were up-regulated.
phase  <dig> glutathione s-transferase  and phase  <dig> atp-binding cassette transporters  were also up-regulated.
in addition, there were many down-regulated genes involved in these  <dig> subgroups .
phase i metabolism
phase ii metabolism
phase iii metabolism
the hepatic cytochrome p <dig>  metabolizing enzymes are involved in the oxidation of the necine base of pas with the cyp3a's being the major enzymes catalyzing the metabolism of retronecine-based pas to form the genotoxic pyrrolic ester dhp and n-oxide derivatives  <cit> .
in addition, cyp3a enzyme inducers  and inhibitors  cause increased and decreased riddelliine-induced dhp formation, respectively  <cit> .
we observed that cyp3a <dig> gene expression was increased 6-fold after riddelliine treatment which is consistent with the metabolic activation findings and suggests that cyp3a <dig> is the major rat liver enzyme involved in riddelliine's metabolic activation.
in human liver, it has been shown that cyp3a <dig>  which is equivalent to rat cyp3a <dig>  catalyzes the bioactivation of pas  <cit> .
our finding of increase expression of cyp2e <dig> is consistent with data from gordon et al.
who demonstrated that retrorsine  caused increased expression of hepatic cyp2e <dig> in rats  <cit> .
the observation that the expression of many p <dig> genes were down-regulated may be related to the finding that many herbal/dietary constitutes form reactive intermediates capable of irreversibly inhibiting some cyp <dig> enzymes  <cit> .
the down-regulated p <dig> genes in this study may also contribute to decreased formation of n-oxide derivatives, the detoxification pathway.
thus, the biological relations of these genes related to riddelliine toxicities warrant further investigations.
the glutathione pathway plays a critical role in the detoxification of many drugs and xenobiotics.
however, there is a lack of information on the types and isozymes of glutathione s-transferases  that mediate glutathione conjugation of different pas  <cit> .
in this study, we observed that gsta <dig> was increased 11-fold and gstm <dig> and gstm <dig> were decreased about 2-fold after riddelliine treatment .
these results imply that these particular types of gsts may therefore be involved in the conjugative detoxification of riddelliine electrophiles and play an essential role in the cellular oxidative defense mechanisms.
in addition, there were four atp binding cassette transporter genes altered  .
these phase iii transporters, localized to the cell membrane, play key physiological roles in drug availability, metabolism and toxicity resulting in protection of cells and tissues against xenobiotics  <cit> .
injury of liver endothelial cells
riddelliine treatments induce relatively higher dna adduct formation, mutation induction, and tumor incidence in the liver endothelial cell than in liver parenchymal cells  <cit> .
in this study, the gene expression changes of a number of genes involved in the injury of liver endothelial cell were detected .
they were mainly related to cell death , cell movement , and cell-to-cell signaling and interaction .
we confirmed the up-regulation  of previously reported hgf, itga <dig>  tnfrsf <dig>  and tnfsf <dig>  <cit> , and also identified the novel up-regulated genes of lepr, prkca, slc7a <dig>  and src .
* genes also involved in cancer development.
leptin  has been consistently associated with angiogenesis and tumor growth.
leptin exerts its physiological action through its specific receptor .
protein kinase c alpha  is a serine/threonine protein kinase that has been implicated in the regulation of a variety of cellular functions in response to a diverse range of stimuli, and has recently become a target for anti-cancer therapies  <cit> .
it has been reported that solute carrier family  <dig> number  <dig>  is translated into the heavy chain of the cell surface antigen 4f <dig> and l-type amino acid transporter  <dig> and is induced by many oxidation products  <cit> .
src encodes non-receptor tyrosine kinases that are the intermediates of information transfer, and control pathways as diverse as cell growth, migration, death, and genome maintenance  <cit> .
the overexpression of these genes may be responsible to the detrimental effects of riddelliine.
also, the decreased expression of some genes, such as adm, edg <dig>  and f11r, suggests a role in riddelliine-induced toxicity.
the g-protein-coupled receptors were originally termed endothelial-cell-differentiation genes  that are upregulated during endothelial cell differentiation  <cit> .
adrenomedullin  is a vasodilator peptide having a wide range of biological actions such as reduction of oxidative stress and inhibition of endothelial cell apoptosis  <cit> .
the f <dig> receptor  plays a critical role in the function of endothelial cells and in platelet adhesion to inflamed endothelium  <cit> .
thus, reduction in the expression of these genes may result in less protection against oxidative stress leading to injury of liver endothelial cells.
genes involved in liver abnormalities
in our previous study, we determined mfs in the liver cii gene of big blue transgenic rats treated with  <dig>  to  <dig> mg/kg riddelliine for  <dig> weeks, and observed increases of mf in a linear dose-dependent manner  <cit> .
the increase in mf was consistent with dose-dependent dhp-derived dna adduct formation  <cit> .
chronic exposure to  <dig> mg/kg of riddelliine resulted in the alteration of a number of genes involved in liver injury and abnormalities .
significantly changed genes were divided into subsets based on functionality, and categories included cell death , cellular growth and proliferation , oxidative stress , and liver morphology .
* genes also involved in cancer development.
the decreased expressions of mthfd <dig>  pparg, tgfa, and tnf indicated that hepatic system development and function were harmed by riddelliine exposure, whereas the elevated ahr, igfbp <dig>  il <dig>  prkcz, tnfrsf <dig>  and tnfsf <dig> were responsible for the cell death.
riddelliine treatment also resulted in 2- to 11-fold up-regulation of mt1a, nqo <dig>  and ren, suggesting the induction of oxidative stress.
the metallothioneins , a family of proteins with antioxidant activity, are upregulated in response to zinc and oxidative stress  <cit> .
nqo <dig> gene expression is coordinately induced with other detoxifying enzyme genes in response to xenobiotics, antioxidants, oxidants, heavy metals, and radiations  <cit> .
renin  is involved in the renin-angiotensin-aldosterone system  which plays a major role in progressive liver fibrosis, and the blockade of the ras could be effective in preventing fibrosis progression in chronic liver diseases  <cit> .
regulation of cancer development
the 2-year ntp carcinogenicity study showed that riddelliine induced liver tumors in rats and male mice, lung tumors in female mice, and leukemia in rats, and that liver tumors were the cause of death for the most of these animals  <cit> .
in the present study, pathway and function analysis indicated  <dig> genes involved in liver cancer development , including  <dig> genes coding for proteins located in nucleus,  <dig> genes coding for proteins in the cytoplasm,  <dig> genes encoding plasma membrane proteins, and  <dig> genes encoding proteins in the extracellular space.
these significantly up- and down-regulated genes were also categorized into subgroups, including ligand-dependent nuclear receptor , transcription regulator , phosphatase , enzyme , kinase , transmembrane receptor , g-protein coupled receptor , growth factor , and cytokine .
consequently, these genes may affect multiple cellular events that contribute to riddelliine-induced toxicological pathways.
the early gene expression changes in the carcinogenic process may involve different genes or groups of genes, depending on the carcinogen  <cit> .
riddelliine-induced gene expression changes appeared to involve morphology, cell death or apoptosis, growth, proliferation, and binding-related genes.
among  <dig> genes,  <dig> genes were also related to injury of liver endothelial cells  and  <dig> genes were related to liver injury and abnormalities .
riddelliine treatment increased the level of prlr, ncr <dig>  igfbp <dig>  mt1a, mt <dig>  ppp2r1b, and timp <dig> about  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and 2-fold, respectively.
it has been reported that prolactin is involved in the pathogenesis of liver cirrhosis and an accumulation of the prolactin receptor  is observed in hepatocytes damaged by cirrhosis and fibrosis  <cit> .
the surface density of the triggering receptors  responsible for natural killer  cell-mediated cytotoxicity determines the ability of nk cells to kill susceptible target cells  <cit> .
the dramatic increase in ncr <dig> may, therefore, indicate an increase in cells susceptible to cell-mediated toxicity.
insulin-like growth factor-binding proteins  are important modulators of igf actions, and overexpression of igfbp <dig> is observed in a variety of pathological conditions.
in addition, igfbp <dig> is expressed in many malignant tissues including liver, and igfbp <dig> appears to be a suitable marker for the evaluation of the serological status of hcc patients  <cit> .
metallothioneins  are a group of low-molecular weight and cysteine rich intracellular proteins.
the expression and induction of these genes  have been associated with protection against dna damage, oxidative stress and apoptosis.
a number of studies have shown an increased expression of mt in various human tumors including hepatoma  <cit> .
ppp2r1b has been implicated as a tumor suppressor gene, and somatic alterations of ppp2r1b have been detected in several cancers.
most recently, chou et al.
reported that aberrant transcripts of ppp2r1b might be associated with the development of hcc  <cit> .
tumor-necrosis factor  is a pleiotropic cytokine that triggers physiological and pathological responses in several organs.
timp <dig> is a crucial innate negative regulator of tnf in both tissue homeostasis and tissue response to injury  <cit> .
thus, the overexpression of these genes supports a critical role of them in the toxicology of riddelliine.
CONCLUSIONS
the present study represents the first comprehensive in vivo examination of the chronic transcriptional response of the liver to riddelliine exposure.
the available evidence on the metabolism and target-tissue specificity for riddelliine's tumorigenesis suggests that active metabolites of riddelliine interact with endothelial cells in the liver, which causes cell toxicity, followed by compensatory proliferation of dna-damaged endothelial cells, 'fixation' of the adducts into mutations in these cells, and eventual development of hemangiosarcoma and hcc.
we have identified  <dig> genes in the livers of riddelliine-treated rats that were differentially expressed and related to these physiological and pathological outcomes.
relating the gene expression changes to phenotypic anchors such as metabolism, injury of liver endothelial cells, liver abnormalities, and cancer development, has helped in the interpretation of these data.
although the significance of all of the hundreds of gene expression changes is not fully understood, the genome-wide global information obtained herein will contribute to an improved understanding of the molecular alterations that occur after exposure to riddelliine, and provide further insight into the mechanisms involved in toxicity and carcinogenesis.
materials and methods
chemical and animals
riddelliine  was obtained from the ntp and dissolved in  <dig> % sodium chloride.
female big blue fisher  <dig> transgenic rats were obtained from taconic laboratories  through purchase from stratagene .
all animal procedures followed the recommendations of the nctr institutional animal care and use committee for the handling, maintenance, treatment, and sacrifice.
riddelliine treatment
the treatment schedule was based on the preliminary results from the ntp two-year chronic tumorigenicity bioassay  <cit> .
six-week-old big blue rats were treated with riddelliine at the dose of  <dig> mg/kg body weight by gavage five times a week for  <dig> weeks.
vehicle control rats were gavaged with  <dig> % sodium chloride.
six rats from treatment and control groups were sacrificed one day after the last treatment.
the livers were isolated, frozen quickly in liquid nitrogen, and stored at -80°c.
tumors had not developed at the  <dig> week sacrifice time so that gene expression changes reflected early events in the carcinogenesis process.
rna isolation and quality control
total rna was isolated from liver tissues of  <dig> control and  <dig> riddelliine-treated rats using an rneasy system .
the yield of the extracted rna was determined spectrophotometrically by measuring the optical density at  <dig> nm.
the purity and quality of extracted rna were evaluated using the rna  <dig> labchip and agilent  <dig> bioanalyzer .
rna samples with rna integrity numbers  greater than  <dig>  were used for microarray experiments performed using applied biosystems' rat genome survey microarray platform, which is a one channel microarray with chemiluminescence detection, and contains  <dig>  probes  for the interrogation of  <dig>  genes and  <dig> controls that track system performance through each experiment.
preparation of digoxigenin labeled in vitro transcribed crna
all rna targets were labeled using the applied biosystems rt-ivt labeling kit version  <dig> .
briefly,  <dig>  μg of total rna was reverse transcribed via  <dig> h incubation at 42°c with arrayscript rt enzyme  and oligo dt-t <dig> primer.
double stranded cdna was produced following  <dig> h incubation with e. coli dna polymerase and rnase h at 16°c.
double-stranded cdna was purified according to the rt-ivt kit protocol.
in vitro transcription was performed by incubation of the cdna product with t <dig> rna polymerase,  <dig>  mm digoxigenin-11-utp  and all other ntps for  <dig> h. labeled crna was purified according to the rt-ivt kit protocol and analyzed for quality and quantity using standard uv spectrometry and the bioanalyzer.
hybridization of labeled crna to microarrays and microarray imaging
digoxigenin labeled crna targets were hybridized to applied biosystems rat whole genome survey microarrays using the applied biosystems chemiluminescent detection kit.
briefly,  <dig> μg of labeled crna targets were fragmented via incubation with fragmentation buffer provided in the kit for  <dig> min at 60°c.
fragmented targets were hybridized to microarrays during a  <dig> h incubation at 55°c with buffers and reagents from the chemiluminescent detection kit.
post-hybridization washes and anti-digoxigenin-alkaline phosphatase binding were performed according to the protocol of the kit.
chemiluminescence detection, image acquisition and analysis were performed using applied biosystems chemiluminescence detection kit and applied biosystems  <dig> chemiluminescent microarray analyzer following the manufacturer's protocols.
images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the applied biosystems  <dig> chemiluminescent microarray analyzer software version  <dig> .
microarray data analysis
raw microarray intensity data from the applied biosystems' rat genome survey microarray were normalized with quantile normalization which is recommended by the manufacturer.
the normalized data were then input to arraytrack, a software system developed by the fda's national center for toxicological research for the management, analysis, visualization and interpretation of microarray data  <cit> .
chemiluminescent signals from  <dig> control probes that track system performance through each experiment were not used in normalization.
the identification of differentially expressed genes based on fold-change and t-tests cutoffs, and principal component analysis were conducted within arraytrack.
ingenuity pathway analysis  was used for network and function analysis.
competing interests
the authors declare that they have no competing interests.
authors' contributions
nm performed the animal treatment and was involved in the analysis of microarray data, and wrote the manuscript.
lg, ql, and jcf helped conceive the experiments, analyze the data, and write the manuscript.
tc was involved in designing the experiment and writing the manuscript.
all authors approved the final version of manuscript.

 pyrrolizidine alkaloids are common constituents of thousands of plant species around the world and pa-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. thus, the human health risk posed by exposure to pas has been a concern. out of more than plants, about pas and their n-oxide derivatives have been identified, and at least half of them are genotoxic and many are tumorigenic. riddelliine is a representative genotoxic pa, and is present in plants growing in the rangelands of the western united states. these plants containing riddelliine appear to enter the human food chain since riddelliine residues have been detected in meat, milk, and honey. riddelliine was nominated by the u.s. food and drug administration to the national toxicology program for genotoxicity and carcinogenicity testing due to the potential for human exposure. riddelliine is a 12-membered macrocyclic diester pa with an α, β-unsaturated double bond linked to the ester group at the c- position of the necine base. riddelliine is completely absorbed within minutes after gavage dosing to rodents and metabolized to the major metabolites, -dihydro-1-hydroxymethyl-5h-pyrrolizine and riddelliine n-oxide, by mammalian microsomes. 32p-postlabeling-hplc analysis has identified a set of dhp-derived dna adducts from rat and human liver microsomal metabolism of riddelliine in vitro and in the livers of rats treated in vivo. a linear dose-dependent formation of dhp-derived dna adducts was observed in riddelliine-treated rats. riddelliine is genotoxic both in vitro and in vivo, inducing increases in sister chromatid exchange, chromosomal aberrations, unscheduled dna synthesis, and micronucleated erythrocyte frequencies. in the ntp carcinogenicity studies, riddelliine was tumorigenic, causing liver tumors in male mice and both sexes of rats, mononuclear cell leukemia in rats, and lung neoplasms in female mice. riddelliine induced a high incidence of liver hemangiosarcomas and lower incidences of hepatocellular carcinoma in rat liver. in our previous studies, we observed that riddelliine is mutagenic in the liver of riddelliine-treated rats and that the mutant frequencies increased in a linear dose-dependent manner. riddelliine also produced a unique mutational spectrum in the liver cii gene of big blue rats with g:c → t:a transversions being the major type of mutation. moreover, we found that the cii mf in liver endothelial cells from riddelliine-treated rats was significantly greater than the cii mf in endothelial cells from control rats, suggesting that the relatively high mutagenicity of riddelliine in rat liver endothelial cells may be partially responsible for the tumorigenic specificity of this agent. it has been reported that riddelliine-treated mice and rats have higher and more persistent dna adduct levels in liver endothelial cells than in parenchymal cells. the sensitivity of tissues and cell types to the mutagenicity of carcinogens may be an important factor in the tissue- and cell-specificity of tumorigenesis. microarray technology has a profound impact on gene expression research because of its ability to examine the expression of thousands of genes at a time. the differentially expressed genes that are identified may be used to develop potential biomarkers, elucidate molecular mechanisms, and create gene signatures that identify classes of samples. this technology, as one of the core technologies for pharmacogenomics and toxicogenomics, provides new insights into the effects of botanical chemicals on biological systems and allow the macrodissection of molecular events in botanical carcinogenesis. identification of unique gene expression patterns produced by botanical carcinogens may allow us to elucidate the mechanisms of action. in this study, we treated rats with a tumorigenic dose of riddelliine and conducted microarray analysis of gene expression in the target tissue liver. we found that the gene expression profiles were significantly altered by riddelliine treatment, and many of the differentially expressed genes were involved in metabolism, injury of liver endothelial cells, liver abnormalities, and cancer development.  samples and dna microarray data analysis liver samples used in this study were from a previous report in which female big blue rats were treated with a carcinogenic dose of riddelliine for weeks. the mf in the liver cii gene was about 3-fold higher than those in the untreated group and the mutation spectra in the riddelliine-treated rats was significantly different from those in the control rats. in the present study, gene expression profiles were determined for the livers of control and riddelliine-treated rats using the applied biosystems' rat genome survey microarray which contains verified rat genes. because liver tumors had not developed at the week sacrifice time, the gene expression changes reflected early events in the carcinogenesis process. after data normalized by quantile normalization which is recommended by the manufacturer, the intensities of the whole rat gene data were analyzed by principal components analysis. a separation between control and riddelliine-treated groups was observed, indicating that there was a riddelliine-treatment effect on liver gene expression. pca analysis also demonstrates that sample # from the control group appears to be different from the rest of the five sample arrays. figure shows the pair-wise log intensity pearson's correlation coefficients for the controls samples. the correlation coefficient numbers containing samples # were lower than the others. therefore, this array was excluded from further data analysis. differentially expressed genes were identified based on the criteria of fold-change greater than and p-value less than in comparison to the control group. a total of genes satisfied the requirements, of which genes were up-regulated and genes were down-regulated in response to riddelliine treatment. among the differentially expressed genes, were in the ingenuity pathway analysis database, and genes were mapped to the networks. figure shows the top networks; each network was associated with specific genes and involved in different functions. the major relevant functions altered by riddelliine treatment in rat liver are listed in table these functions include cancer, cell death, tissue development, cell morphology, cell-to-cell signaling and interaction, and cellular development. because riddelliine treatments induce relatively high dna adduct formation, mutation induction, and tumor incidence in the liver endothelial cells, we focused our analysis on genes involved in carcinogenesis, mainly metabolism, injury of endothelial cells, liver abnormalities, and cancer development using ingenuity pathway analysis. alteration of metabolizing genes since metabolic activation of riddelliine is required for liver tumor induction, we investigated the gene expression changes of drug metabolizing genes. table shows phases i, ii, and iii drug metabolizing genes whose expression was significantly changed by riddelliine treatment. four phase cytochrome p genes were up-regulated. phase glutathione s-transferase and phase atp-binding cassette transporters were also up-regulated. in addition, there were many down-regulated genes involved in these subgroups. phase i metabolism phase ii metabolism phase iii metabolism the hepatic cytochrome p metabolizing enzymes are involved in the oxidation of the necine base of pas with the cyp3a's being the major enzymes catalyzing the metabolism of retronecine-based pas to form the genotoxic pyrrolic ester dhp and n-oxide derivatives. in addition, cyp3a enzyme inducers and inhibitors cause increased and decreased riddelliine-induced dhp formation, respectively. we observed that cyp3a gene expression was increased 6-fold after riddelliine treatment which is consistent with the metabolic activation findings and suggests that cyp3a is the major rat liver enzyme involved in riddelliine's metabolic activation. in human liver, it has been shown that cyp3a which is equivalent to rat cyp3a catalyzes the bioactivation of pas. our finding of increase expression of cyp2e is consistent with data from gordon et al. who demonstrated that retrorsine caused increased expression of hepatic cyp2e in rats. the observation that the expression of many p genes were down-regulated may be related to the finding that many herbal/dietary constitutes form reactive intermediates capable of irreversibly inhibiting some cyp enzymes. the down-regulated p genes in this study may also contribute to decreased formation of n-oxide derivatives, the detoxification pathway. thus, the biological relations of these genes related to riddelliine toxicities warrant further investigations. the glutathione pathway plays a critical role in the detoxification of many drugs and xenobiotics. however, there is a lack of information on the types and isozymes of glutathione s-transferases that mediate glutathione conjugation of different pas. in this study, we observed that gsta was increased 11-fold and gstm and gstm were decreased about 2-fold after riddelliine treatment. these results imply that these particular types of gsts may therefore be involved in the conjugative detoxification of riddelliine electrophiles and play an essential role in the cellular oxidative defense mechanisms. in addition, there were four atp binding cassette transporter genes altered. these phase iii transporters, localized to the cell membrane, play key physiological roles in drug availability, metabolism and toxicity resulting in protection of cells and tissues against xenobiotics. injury of liver endothelial cells riddelliine treatments induce relatively higher dna adduct formation, mutation induction, and tumor incidence in the liver endothelial cell than in liver parenchymal cells. in this study, the gene expression changes of a number of genes involved in the injury of liver endothelial cell were detected. they were mainly related to cell death, cell movement, and cell-to-cell signaling and interaction. we confirmed the up-regulation of previously reported hgf, itga tnfrsf and tnfsf, and also identified the novel up-regulated genes of lepr, prkca, slc7a and src. * genes also involved in cancer development. leptin has been consistently associated with angiogenesis and tumor growth. leptin exerts its physiological action through its specific receptor. protein kinase c alpha is a serine/threonine protein kinase that has been implicated in the regulation of a variety of cellular functions in response to a diverse range of stimuli, and has recently become a target for anti-cancer therapies. it has been reported that solute carrier family number is translated into the heavy chain of the cell surface antigen 4f and l-type amino acid transporter and is induced by many oxidation products. src encodes non-receptor tyrosine kinases that are the intermediates of information transfer, and control pathways as diverse as cell growth, migration, death, and genome maintenance. the overexpression of these genes may be responsible to the detrimental effects of riddelliine. also, the decreased expression of some genes, such as adm, edg and f11r, suggests a role in riddelliine-induced toxicity. the g-protein-coupled receptors were originally termed endothelial-cell-differentiation genes that are upregulated during endothelial cell differentiation. adrenomedullin is a vasodilator peptide having a wide range of biological actions such as reduction of oxidative stress and inhibition of endothelial cell apoptosis. the f receptor plays a critical role in the function of endothelial cells and in platelet adhesion to inflamed endothelium. thus, reduction in the expression of these genes may result in less protection against oxidative stress leading to injury of liver endothelial cells. genes involved in liver abnormalities in our previous study, we determined mfs in the liver cii gene of big blue transgenic rats treated with to mg/kg riddelliine for weeks, and observed increases of mf in a linear dose-dependent manner. the increase in mf was consistent with dose-dependent dhp-derived dna adduct formation. chronic exposure to mg/kg of riddelliine resulted in the alteration of a number of genes involved in liver injury and abnormalities. significantly changed genes were divided into subsets based on functionality, and categories included cell death, cellular growth and proliferation, oxidative stress, and liver morphology. * genes also involved in cancer development. the decreased expressions of mthfd pparg, tgfa, and tnf indicated that hepatic system development and function were harmed by riddelliine exposure, whereas the elevated ahr, igfbp il prkcz, tnfrsf and tnfsf were responsible for the cell death. riddelliine treatment also resulted in 2- to 11-fold up-regulation of mt1a, nqo and ren, suggesting the induction of oxidative stress. the metallothioneins, a family of proteins with antioxidant activity, are upregulated in response to zinc and oxidative stress. nqo gene expression is coordinately induced with other detoxifying enzyme genes in response to xenobiotics, antioxidants, oxidants, heavy metals, and radiations. renin is involved in the renin-angiotensin-aldosterone system which plays a major role in progressive liver fibrosis, and the blockade of the ras could be effective in preventing fibrosis progression in chronic liver diseases. regulation of cancer development the 2-year ntp carcinogenicity study showed that riddelliine induced liver tumors in rats and male mice, lung tumors in female mice, and leukemia in rats, and that liver tumors were the cause of death for the most of these animals. in the present study, pathway and function analysis indicated genes involved in liver cancer development, including genes coding for proteins located in nucleus, genes coding for proteins in the cytoplasm, genes encoding plasma membrane proteins, and genes encoding proteins in the extracellular space. these significantly up- and down-regulated genes were also categorized into subgroups, including ligand-dependent nuclear receptor, transcription regulator, phosphatase, enzyme, kinase, transmembrane receptor, g-protein coupled receptor, growth factor, and cytokine. consequently, these genes may affect multiple cellular events that contribute to riddelliine-induced toxicological pathways. the early gene expression changes in the carcinogenic process may involve different genes or groups of genes, depending on the carcinogen. riddelliine-induced gene expression changes appeared to involve morphology, cell death or apoptosis, growth, proliferation, and binding-related genes. among genes, genes were also related to injury of liver endothelial cells and genes were related to liver injury and abnormalities. riddelliine treatment increased the level of prlr, ncr igfbp mt1a, mt ppp2r1b, and timp about and 2-fold, respectively. it has been reported that prolactin is involved in the pathogenesis of liver cirrhosis and an accumulation of the prolactin receptor is observed in hepatocytes damaged by cirrhosis and fibrosis. the surface density of the triggering receptors responsible for natural killer cell-mediated cytotoxicity determines the ability of nk cells to kill susceptible target cells. the dramatic increase in ncr may, therefore, indicate an increase in cells susceptible to cell-mediated toxicity. insulin-like growth factor-binding proteins are important modulators of igf actions, and overexpression of igfbp is observed in a variety of pathological conditions. in addition, igfbp is expressed in many malignant tissues including liver, and igfbp appears to be a suitable marker for the evaluation of the serological status of hcc patients. metallothioneins are a group of low-molecular weight and cysteine rich intracellular proteins. the expression and induction of these genes have been associated with protection against dna damage, oxidative stress and apoptosis. a number of studies have shown an increased expression of mt in various human tumors including hepatoma. ppp2r1b has been implicated as a tumor suppressor gene, and somatic alterations of ppp2r1b have been detected in several cancers. most recently, chou et al. reported that aberrant transcripts of ppp2r1b might be associated with the development of hcc. tumor-necrosis factor is a pleiotropic cytokine that triggers physiological and pathological responses in several organs. timp is a crucial innate negative regulator of tnf in both tissue homeostasis and tissue response to injury. thus, the overexpression of these genes supports a critical role of them in the toxicology of riddelliine.  the present study represents the first comprehensive in vivo examination of the chronic transcriptional response of the liver to riddelliine exposure. the available evidence on the metabolism and target-tissue specificity for riddelliine's tumorigenesis suggests that active metabolites of riddelliine interact with endothelial cells in the liver, which causes cell toxicity, followed by compensatory proliferation of dna-damaged endothelial cells,'fixation' of the adducts into mutations in these cells, and eventual development of hemangiosarcoma and hcc. we have identified genes in the livers of riddelliine-treated rats that were differentially expressed and related to these physiological and pathological outcomes. relating the gene expression changes to phenotypic anchors such as metabolism, injury of liver endothelial cells, liver abnormalities, and cancer development, has helped in the interpretation of these data. although the significance of all of the hundreds of gene expression changes is not fully understood, the genome-wide global information obtained herein will contribute to an improved understanding of the molecular alterations that occur after exposure to riddelliine, and provide further insight into the mechanisms involved in toxicity and carcinogenesis. materials and methods chemical and animals riddelliine was obtained from the ntp and dissolved in % sodium chloride. female big blue fisher transgenic rats were obtained from taconic laboratories through purchase from stratagene. all animal procedures followed the recommendations of the nctr institutional animal care and use committee for the handling, maintenance, treatment, and sacrifice. riddelliine treatment the treatment schedule was based on the preliminary results from the ntp two-year chronic tumorigenicity bioassay. six-week-old big blue rats were treated with riddelliine at the dose of mg/kg body weight by gavage five times a week for weeks. vehicle control rats were gavaged with % sodium chloride. six rats from treatment and control groups were sacrificed one day after the last treatment. the livers were isolated, frozen quickly in liquid nitrogen, and stored at -80°c. tumors had not developed at the week sacrifice time so that gene expression changes reflected early events in the carcinogenesis process. rna isolation and quality control total rna was isolated from liver tissues of control and riddelliine-treated rats using an rneasy system. the yield of the extracted rna was determined spectrophotometrically by measuring the optical density at nm. the purity and quality of extracted rna were evaluated using the rna labchip and agilent bioanalyzer. rna samples with rna integrity numbers greater than were used for microarray experiments performed using applied biosystems' rat genome survey microarray platform, which is a one channel microarray with chemiluminescence detection, and contains probes for the interrogation of genes and controls that track system performance through each experiment. preparation of digoxigenin labeled in vitro transcribed crna all rna targets were labeled using the applied biosystems rt-ivt labeling kit version. briefly, μg of total rna was reverse transcribed via h incubation at 42°c with arrayscript rt enzyme and oligo dt-t primer. double stranded cdna was produced following h incubation with e. coli dna polymerase and rnase h at 16°c. double-stranded cdna was purified according to the rt-ivt kit protocol. in vitro transcription was performed by incubation of the cdna product with t rna polymerase, mm digoxigenin-11-utp and all other ntps for h. labeled crna was purified according to the rt-ivt kit protocol and analyzed for quality and quantity using standard uv spectrometry and the bioanalyzer. hybridization of labeled crna to microarrays and microarray imaging digoxigenin labeled crna targets were hybridized to applied biosystems rat whole genome survey microarrays using the applied biosystems chemiluminescent detection kit. briefly, μg of labeled crna targets were fragmented via incubation with fragmentation buffer provided in the kit for min at 60°c. fragmented targets were hybridized to microarrays during a h incubation at 55°c with buffers and reagents from the chemiluminescent detection kit. post-hybridization washes and anti-digoxigenin-alkaline phosphatase binding were performed according to the protocol of the kit. chemiluminescence detection, image acquisition and analysis were performed using applied biosystems chemiluminescence detection kit and applied biosystems chemiluminescent microarray analyzer following the manufacturer's protocols. images were auto-gridded and the chemiluminescent signals were quantified, corrected for background, and finally, spot- and spatially-normalized using the applied biosystems chemiluminescent microarray analyzer software version. microarray data analysis raw microarray intensity data from the applied biosystems' rat genome survey microarray were normalized with quantile normalization which is recommended by the manufacturer. the normalized data were then input to arraytrack, a software system developed by the fda's national center for toxicological research for the management, analysis, visualization and interpretation of microarray data. chemiluminescent signals from control probes that track system performance through each experiment were not used in normalization. the identification of differentially expressed genes based on fold-change and t-tests cutoffs, and principal component analysis were conducted within arraytrack. ingenuity pathway analysis was used for network and function analysis. competing interests the authors declare that they have no competing interests. authors' contributions nm performed the animal treatment and was involved in the analysis of microarray data, and wrote the manuscript. lg, ql, and jcf helped conceive the experiments, analyze the data, and write the manuscript. tc was involved in designing the experiment and writing the manuscript. all authors approved the final version of manuscript. 
previously we observed that the mutant frequency in the liver of rats gavaged with riddelliine was 3-fold higher than that in the control group. pyrrolizidine alkaloids are probably the most common plant constituents that poison livestock, wildlife, and humans worldwide. riddelliine-induced gene expression profiles in livers of big blue transgenic rats were determined. these results provided further insight into the mechanisms involved in toxicity and carcinogenesis after exposure to riddelliine, and permitted us to investigate the interaction of gene products inside the signaling networks. molecular analysis of the mutants indicated that there was a statistically significant difference between the mutational spectra from riddelliine-treated and control rats. by analysis with the ingenuity pathway analysis network, we found that these significantly changed genes were mainly involved in cancer, cell death, tissue development, cellular movement, tissue morphology, cell-to-cell signaling and interaction, and cellular growth and proliferation. we further analyzed the genes involved in metabolism, injury of endothelial cells, liver abnormalities, and cancer development in detail. the alterations in gene expression were directly related to the pathological outcomes reported previously. riddelliine was used to investigate the genotoxic effects of pas via analysis of gene expression in the target tissue of rats in this study. rat whole genome microarray was used to perform genome-wide gene expression studies.
 pyrrolizidine alkaloids are probably the most common plant constituents that poison livestock, wildlife, and humans worldwide. riddelliine is isolated from plants grown in the western united states and is a prototype of genotoxic pas. riddelliine was used to investigate the genotoxic effects of pas via analysis of gene expression in the target tissue of rats in this study. previously we observed that the mutant frequency in the liver of rats gavaged with riddelliine was 3-fold higher than that in the control group. molecular analysis of the mutants indicated that there was a statistically significant difference between the mutational spectra from riddelliine-treated and control rats.  riddelliine-induced gene expression profiles in livers of big blue transgenic rats were determined. the female rats were gavaged with riddelliine at a dose of mg/kg body weight days a week for weeks. rat whole genome microarray was used to perform genome-wide gene expression studies. when a cutoff value of a two-fold change and a p-value less than were used as gene selection criteria, genes were identified as differentially expressed in riddelliine-treated rats compared to the control animals. by analysis with the ingenuity pathway analysis network, we found that these significantly changed genes were mainly involved in cancer, cell death, tissue development, cellular movement, tissue morphology, cell-to-cell signaling and interaction, and cellular growth and proliferation. we further analyzed the genes involved in metabolism, injury of endothelial cells, liver abnormalities, and cancer development in detail.  the alterations in gene expression were directly related to the pathological outcomes reported previously. these results provided further insight into the mechanisms involved in toxicity and carcinogenesis after exposure to riddelliine, and permitted us to investigate the interaction of gene products inside the signaling networks. 1– february fourth annual mcbios conference. computational frontiers in biomedicine new orleans, la, usa 