* Department of Biomolecular Sciences, TNO Nutrition and Food Research, PO box 360, 3700 AJ Zeist, The Netherlands; Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160; and
Department of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands
Received December 15, 2003; accepted March 2, 2004
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ABSTRACT |
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Key Words: toxicogenomics; bromobenzene; transcriptomics; hepatotoxicity; rat; cDNA microarray.
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INTRODUCTION |
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A new transcriptomics study was designed to investigate the sequence of events in time in hepatotoxicity, after oral exposure to BB, and to show the dose dependency of the observed effects at the transcriptome level. Measuring the expression of thousands of genes allowed more in-depth investigations in the hepatic changes at the molecular level in response to BB administration. Moreover, we expected to detect changes in gene expression at lower dose levels and earlier time points compared to the routine toxicity examinations. Rats were given BB by po gavage, at three dose levels and liver gene expression profiles were determined 6, 24, and 48 h later. Histopathology and clinical chemistry parameters in plasma were determined, as well as glutathione contents of the liver. Expression of several genes, selected based on the cDNA microarray results, was analyzed in more detail using the branched DNA signal amplification (bDNA) assay.
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MATERIALS AND METHODS |
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GSH and clinical chemistry parameters.
Glutathione ([GSH + GSSG] and [GSSG]) levels in liver homogenate were determined spectrophotometrically according to Anderson (1985), and from these values, reduced glutathione, ([GSH]), levels were calculated. Lactate dehydrogenase (LDH), alkaline phosphatase (ALP), ASAT (asparate aminotransferase), ALAT (alanine aminotransferase), bilirubin, cholesterol, phospholipids, triglycerides, glucose, GGT (gamma-gluramyl transpeptidase), creatin, albumin, urea and albumin to globulin (A/G) ratio in plasma were determined using a Hitachi-911 Bioanalyzer, using Boehringer reagents, according to the manufacturer's protocols. One-way analysis of variance (ANOVA) was used to assess the level of statistical significance of the changes.
RNA extraction.
Total RNA was extracted from liver homogenate using Trizol (Life Technologies S.A., Merelbeke, Belgium) according to the manufacturer's protocol, and further purified using the RNEasy kit (Qiagen, Westburg B.V., Leusden, Netherlands), including a DNA digestion by RNAse-free DNAseI incubation. RNA was checked for purity and integrity by agarose gel electrophoresis and the concentration was determined spectrophotometrically.
cDNA Microarray preparation and labeling.
cDNA microarray preparation was described previously (Heijne et al., 2003). Briefly, about 3000 different sequence-verified rat cDNA fragments were arrayed on glass slides, and control spots were included. A typical labeling reaction was performed using 25 µg of total RNA, using the Cyscribe (Amersham Biosciences, Freiburg, Germany) fluorescence labeling kit. Cy3 or Cy5 fluorophore dUTP nucleotides were directly incorporated in the cDNA during the in vitro transcription reaction. RNA was degraded by hydrolysis in NaOH (30 min at 37°C). Labelled cDNA was purified using an Autoseq G-50 (Amersham Biosciences, Freiburg, Germany) chromatography column. Hybridization of labeled cDNA to the slides was performed as described before (Heijne et al., 2003
).
Image capture and analysis.
Slides were scanned using a (Packard Biosciences) ScanArray Express confocal laser scanner, at wavelength 550 nm (cy3 signal) and 650 nm (cy5). TIFF images were analyzed using Imagene (Biodiscovery Inc., El Segundo, CA USA), and settings were applied to automatically flag weak or negative signals and spots with a nonhomogenous signal. Excel (Microsoft Corporation, Redmond, WA USA) and SAS (SAS, Cary, NC) were used to further process and analyze the data.
Transcriptomics experimental design.
In order to compare all samples of individual rats to each other, and to other studies, an external reference sample was used. Thus, for each gene fragment, the amount of mRNA in the sample relative to the amount in the reference was determined. The complete set of hybridizations was duplicated with swapping of the fluorophore incorporation in the sample and reference RNA. The value of this reference RNA has been described before (Heijne et al., 2003). A good correlation was found between the changes in gene expression determined in direct hybridizations and in indirect hybridizations using the reference.
DNA microarray data preprocessing.
After image analysis, the local background intensity was subtracted from the signal for each spot. Background intensities outside the cDNA spots were very low and homogeneous. Control spots and background fluorescence were used to determine a minimal signal to noise ratio threshold value of 1.5 for the two channels. Flagged spots and controls were excluded from further interpretation, as well as genes for which less than 75% of the microarrays yielded an acceptable signal. To account for technical variations introduced during labeling or hybridization, data were normalized assuming that the majority of the transcripts was equally present in both samples. Since a relationship was found between the intensity of the signals and the variation in the ratio of gene, the lowest normalization algorithm, according to Yang and Speed (2002), was applied in SAS software. This procedure fits the expression ratios to an intensity-dependent curve by locally weighted regression. After normalization and logarithm transformation of the ratios tester/reference, a set of about 2700 rat cDNAs was used for further analysis. Excluded values were replaced by 0, and averages were calculated of the logarithms of fold changes between treatment groups.
DNA microarray data analysis.
Pairwise comparisons of the expression ratios were made between samples of the different time and dose groups and controls. In these comparisons, untreated and corn oil control samples were considered as one control group, since preliminary analyses revealed only minor changes induced by single oral corn oil dosage. Statistics (two-sided, unpaired t-tests) was applied assuming unequal variance and changes were considered significant if the tests resulted in a p-value less than 0.01. In the tables, p-values were denoted as follows: ***p < 0.001; **p < 0.01. The genes significantly differentially expressed upon treatment were explored in the context of biological mechanisms and pathways. Genes were categorized based on biological processes using literature and gene information databases.
Gene expression measurement by branched DNA signal amplification assay.
mRNA levels of microsomal epoxide hydrolase (mEH), heme oxygenase 1(HO-1), NQO1, Mt-1, gamma cysteine ligase catalytic (heavy) chain (or gamma-glutamyl cysteine synthethase) (GCLC), CYP2B1/2, CYP4A2/3, multidrug resistance protein 1 (Mrp1), Mrp2, and Mrp3, and glyceraldehyde phosphate dehydrogenas (GAPDH) were analyzed for all samples individually by the bDNA assay using probes specific to each transcript (Quantigene HV10 kits, Genospectra, www.genospectra.com) as described in Cherrington et al. (2002) and Slitt et al. (2003)
. Oligonucleotide probesets that detect NQO1, CYP2B1/2, CYP4A2/3, Mt-1, Mrp1, Mrp2, and Mrp3 were previously described (Cherrington et al., 2002
; Hartley and Klaassen, 2000
; Li et al., 2002
) Oligonucleotide probesets for mEH, HO-1, and GCLC are described in Table 5 (Supplementary Material)
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RESULTS |
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(Histo)pathology
No significant changes in body weights or macroscopic changes were seen. Neither the livers of the controls nor of the low or mid dose groups displayed pathological aberrations. The livers isolated 24 h after a high dose of BB revealed a patchy appearance and gross lesions, while the livers of rats sacrificed 48 h after a high dose had focal discoloration. Six h after dosing of mid and high levels of BB, relative liver weights were around 10% lower than the controls (ANOVA + Dunnett's test, p < 0.05 for high and p < 0.01 for mid). At 24 or 48 h, controls as well as low dosed rats had relative liver weights of 7585% of the rats sacrificed after 6 h. In all the high BB treated rats, a significant increase to around 130% of vehicle controls (p < 0.01) was observed. Microscopic examination of the livers showed slight presence of mononuclear cell aggregates and/or necrotic hepatocytes in several rats regardless of the treatment. After 24 and 48 h, pronounced centrilobular necrosis was found in all rats of the high dose groups, with interindividual variation from very slight to very severe necrosis.
Clinical Chemistry
Clinical chemistry parameters in plasma are represented in Figure 2. Bilirubin levels (panel A) in the mid dose group were increased after 24 h, but not after 48 h. In the high dose group, bilirubin levels were slightly up at 6 h, and highly elevated after 24 h and 48 h. Only upon high dose, the rats showed highly elevated alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels after 24 h and 48 h (panels B and C). Plasma lactate dehydrogenase (LDH, not shown) activity was increased 100-fold 24 h after the high dose, but showed normal levels at 48 h. Alkaline phosphatase (ALP; panel D) activity in plasma gradually increased with dose at 24 h (not significant), and was elevated 48 h after the high dose. Plasma glucose (panel E) decreased equally by mid and high dose after 24 and 48 h. Cholesterol (not statistically significant [n.s.]) and phospholipid levels (panel F) in the high dose group were increased at 6 h and remained elevated. The creatin content decreased upon the high dose after 6 h (n.s.). Regardless of the treatment, urea levels were higher and triglyceride levels lower at 24 and 48 h compared to 6 h. No significant changes were observed in plasma levels of gamma-glutamyl transpeptidase, total protein, albumin, and the albumin to globulin ratio (A/G).
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Six h
Table 1 lists the genes that were significantly differentially expressed after 6 h in the BB dosed rats. A marked (28 fold) increase in gene expression was observed for several metallothioneins (Mt). Both the flavin-containing mono-oxygenases FMO1 and FMO3 were upregulated by the high dose, as well as cathepsin L and ASAT. The enzymes cysteine dioxygenase 1 and betaine homocysteine methyltransferase were upregulated. A two-fold upregulation was found for mRNA encoding Rho-interacting protein 3. Genes downregulated by the high dose include LDH B, tubulin, and cholesterol metabolism enzymes farnesyl diphosphate synthase and farnesyl diphosphate farnesyl transferase 1, "sterol-C4-methyl oxidase-like," HMG-CoA synthase 1. Several genes, HO-1, TIMP1, aflatoxin B1 aldehyde reductase (AFAR), and serine protease 15, changed significantly with the mid or the low dose after 6 h but not with the high dose. Three genes were down regulated by both the mid and high dose, namely the "EST similar to GSTP," HSP70, and calpain.
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Forty-eight h
The differential expression of genes 48 h after the high concentration of BB was similar to the expression pattern after 24 h (Table 3, in Supplementary Material) Many of these 175 genes were also differentially expressed after 24 h, although frequently the difference was less marked and not statistically significant. Only two genes, interferon-inducible transmembrane protein and steroid sulfatase were found to be subtly but significantly differentially expressed at the low BB dose. The mid dose slightly induced three genes, also induced by the high dose.
Intraday Variation
The control rats were used to analyze the intraday variation in liver gene expression as well as in other parameters. Intraday differences were observed in GSH content and relative liver weights as well as in levels of parameters in plasma. Triglyceride levels were lower at 24 h (and 48 h) than at 6 h. This was also the case for ALP, phospholipids, cholesterol, and urea Also at the gene expression level, the 6 h time point was found to be clearly distinct from the 24 and 48 h time points. Profiles obtained from untreated rats were very similar to those obtained from corn oil treated rats. No genes were recognized with a marked change in expression between 24 and 48 h in the controls. Table 4 (Supplementary Material) shows genes in which expression changed significantly from 6 to 24 h in controls. The most distinctly higher expressed genes were metallothioneins. Moreover, various CYP isozymes (CYP4A1 and CYP4A2/3), arginosuccinate lyase, cathepsin L, and various genes involved in fatty acid metabolism were higher expressed at 24 h than at the 6 h time point. Genes in which expression was lower after 24 h compared to 6 h included several GSTs, transferrin, tubulin, lysyl hydroxylase, and GSH peroxidase.
Confirmation of Gene Expression Changes
cDNA microarrays results for selected genes were analyzed for all rat samples using the bDNA assay (Fig. 4). This method was shown to very specifically determine mRNA levels in a wide concentration range. The gene expression levels for mEH, GAPDH, HO-1, Mt-1, NQO1, and CYPs in the bDNA assay largely overlapped with the cDNA microarray results. For example, between both methods, a coefficient of correlation of 0.94, 0.89 and 0.86 was found for the individual rats' levels of mEH, HO-1, and Mt-1, respectively. Also, genes were analyzed that were not (conclusively) measured in the microarrays. We hypothesized these genes (Mrp1, Mrp2 or Mrp3, GCLC) could be modulated based on other changes observed using the microarrays, for instance indicating EpRE-mediated transcriptional regulation. GAPDH, which is frequently regarded as a so-called housekeeping gene with stable expression levels, was measured using both the microarrays and the bDNA assay. By both methods, GAPDH was found to be upregulated more than two-fold at high dose levels after 24 h.
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DISCUSSION |
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Paradoxically, several genes including mEH, AFAR, HO-1, and TIMP1 were markedly upregulated by the low and mid dose at 6 h, but not by the high dose. At later stages, these genes were highly upregulated by BB in a dose-dependent manner. The effects measured by cDNA microarrays were confirmed using the bDNA assay. The initial lack of response with the high dose contradicts the dose-dependency usually assumed in toxicology. No explanation was found for this phenomenon, and no such observations were found in literature.
Pathways and Mechanisms
Genes with statistically significant differential expression upon BB administration were categorized according to biological processes in the cell, putatively relevant in BB-induced hepatotoxicity. The most relevant changes in BB-induced hepatotoxicity were schematically displayed in Figure 5.
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GSH Metabolism
GSH is believed to be of crucial importance in the detoxification of xenobiotics like BB. A dose-dependent lowering of the liver GSH levels was observed 6 h after BB administration. Compared to our previous study with ip injection of BB (Heijne et al., 2003), total GSH depletion was not observed in this study, 24 h after an oral administration. On the contrary, after 24 h GSH concentration was higher upon BB administration than in the controls. The increased level of GSH after the initial depletion has been suggested as a recovery mechanism (Chakrabarti, 1991
). Also, a significant upregulation of GSTA and GSTM was found after 24 h even by the low dose. The rate-limiting enzyme in GSH synthesis is GCLC, transcriptionally regulated by electrophile response element (EpRE). Using the bDNA assay, we detected a pronounced upregulation of GCLC as early as 6 h after the low dose (Fig. 6). Recently, we also found GSH synthase protein to be induced by BB (Heijne et al., 2003
). The upregulation of the enzymes in GSH metabolism can be explained as an adaptive response to restore the depleted GSH levels in liver.
Oxidative Stress
Highly reactive metabolites such as formed from BB can induce oxidative stress. Additionally, the depletion of GSH diminishes the intracellular protective mechanism against ROS and electrophilic metabolites The early induction of key markers, the mRNA levels of HO-1 and TIMP1 suggests the induction of oxidative stress by BB. After 24 h, HO-1 mRNA levels were elevated only in the high dose group, while returned to normal in the mid dose group. HO-1 catalyzes the degradation of heme to CO2 and biliverdin, which is subsequently catabolized to bilirubin. Bilirubin may serve as an intracellular antioxidant. In line with this, we observed bilirubin levels in plasma to be slightly increased in the high dose group at 6 h, while largely increased after 24 h. Also peroxiredoxin 1 and the ferritin light and heavy subunit transcripts were elevated significantly by BB. Ferritins sequester free iron molecules thus preventing formation of ROS, hydroxyl radicals, through the Fenton reaction. Peroxiredoxins are antioxidant enzymes with a role in signal transduction. Metallothionein transcripts were highly induced at 6 h after all doses of BB. At 24 and 48 h an elevation of the Mt mRNA levels was observed independent of the treatment. Metallothioneins are cysteine-rich proteins that function in the sequestration of the metals Cu2+, Cd2+, Zn2+, while also ROS can be scavenged. The BB-induced rapid increase of Mt mRNA corresponds with reported 40% increased protein concentrations in the liver and kidneys 6 h after BB (Wong and Klaassen, 1981; Szymanska et al., 1991
). The protein Vdup1 was induced and has been reported to interact with thioredoxin and to be associated with oxidative stress (Wang et al., 2002
). GSH peroxidase, selenoprotein P, and synaptojanin 2, genes possibly related to oxidative stress, were down regulated. GAPDH was induced more than two-fold 24 h after the high dose of BB. GAPDH has been reported to be regulated by hypoxia and induced by insulin and glutamine. Responsive elements for hypoxia-inducible factor, and C/EBP have been identified in the GAPDH gene promotor (Claeyssens et al., 2003
; Graven et al., 1999
; Rolland et al., 1995
); The induction of GAPDH might be in concordance with its function in glycolysis, upregulated to meet the energy requirements of the regenerating liver.
Acute Phase Response
The acute phase response is elicited by various types of stress like mechanical damage and inflammation (Ramadori and Christ, 1999; Suffredini et al., 1999
). Especially in the liver, changes in many genes and proteins provide a protective response and re-establish cellular homeostasis. We previously identified that the acute phase response was elicited upon an intraperitoneal administration of BB (Heijne et al., 2003
). Present experiments showed that also upon oral gavage, BB was able to elicit changes in transcript levels of many acute phase proteins in liver. Orosomucoid 1 (former AGP), cytokeratin-18, and apolipoprotein A1 were induced. Negative acute phasetranscripts including alpha-1-inhibitor 3 and pre-alpha-inhibitor heavy chain 3, serine protease inhibitor, complement component 1 and 4, L-FABP 1 and fibrinogen B were down regulated by BB.
Fatty Acid and Cholesterol Metabolism
Plasma levels of cholesterol increased upon high dose after 6 h and remained elevated at 24 and 48 h. Several enzymes involved in cholesterol metabolism were significantly and dose-dependently down regulated after 6 h by BB. Cholesterol is biosynthesized from acetyl-CoA, the product of fatty acid degradation, and enzymes in both pathways are down regulated by BB, including HMG-CoA synthase, Acetyl-CoA acetyltransferase 1, hydroxylacyl-CoA dehydrogenase, acyl-coA oxidase, trifunctional protein. Many of the genes coordinately downregulated are transcriptionally induced through binding of the sterol responsive element binding protein (SREBP) to a sterol-responsive element in the upstream DNA sequence of those genes (Osborne, 2000; Shimano, 2002
). Cholesterol levels play a role in the regulation of the SREBP pathway. The downregulation of the fatty acid and cholesterol metabolism could be due to the requirement of energy for these processes. In this situation of distress, the cell might have to dedicate all energy supplies to cope with the toxicity induced by BB, and restore homeostasis. Energy-requiring processes should be down regulated, while energy-generating processes are upregulated. In line with this, the decrease of plasma glucose levels could be ascribed to increased catabolism of glucose in the glycolysis.
Protein Synthesis and Proteolysis
Genes involved in protein synthesis, including many ribosomal subunits, eukaryotic initiation factors, and elongation factor, were overexpressed one day after dosage. Furthermore, an induction was observed for several components of the proteasome and the proteolytic enzyme cathepsin L. Dose and time related changes in gene expression were observed for serine protease, dipeptidyl peptidase, polyubiquitin and calpain. The histopathologically observed slight nucleolar enlargement and the mitotic increase in the BB-treated livers correspond with the induction of proteins required for transcription and translation.
Structure and Cytoskeleton
Genes encoding cell-structure proteins increased upon the high dose of BB. Stongly upregulated were actin and "weakly similar to pervin," a protein with high homology to a human cytoskeleton-interacting protein. Also actinin, cortactin, "EST highly similar to actin," keratin, dynein, tubulins, and others were differentially expressed. Thymosin beta-4 is an actin binding protein and is upregulated 48 h after a high dose of BB. Rho-interacting protein is involved in cytoskeleton rearrangement, and increases after 6 h. Oxidative modifications of the microfilaments are suggested to cause cytotoxicity in the form of blebs on plasma membranes, when polymerized actin is disrupted by oxidation of its sulfhydryl groups. Upon rupture of the blebs, cellular ion gradients and intracellular components are lost, leading to necrotic cell death. The change of cellular calcium (Ca2+) levels may play a role in the cytotoxicity. Increasing Ca2+ levels promote dissociation of actin from -actinin, and activation of the calpain protease, which cleaves actin-binding proteins. When the anchoring of the cytoskeleton to the plasma membrane is disturbed, membrane blebbing may occur (Boelsterli, 2003
; Dalle-Donne et al., 2001
). BB caused down regulation of calpain gene expression after 6 h. The induction of both the protein synthesis and the cell-structure genes suggests that enhanced protein synthesis and/or proliferation occur, which aids in hepatic tissue remodelling and recovery after BB-induced hepatocellular injury. Corroborating evidence is found in the histopathological observation of a mitotic increase and nucleolar enlargement in the hepatocytes upon high BB dosage.
Recovery
The gene expression profiles obtained from livers isolated 48 h after a mid BB dose resembled the profiles of the controls and the low dose livers. We suggest that the mid dose still effectuates a marked response, especially detectable 24 h after oral ingestion of BB, but is not high enough to induce irreversible damage detectable by histopathology or clinical chemistry. This suggests that the rats in the mid dose group did not suffer, or recovered from the toxic stimulus. From the time-series, it was clear that most pronounced changes in gene expression were observed 24 h after BB administration. Previously, Lind and Gandolfi (1999) suggested that irreversible changes occurred after 24 h, when the limited centrilobular lesions progressed to a more widespread pattern, and DMSO could no longer attenuate the BB-induced necrosis.
Coordinate Expression Mediated by the Electrophile Response Element
The coordinate induction of HO-1, ferritins as well as GSTA, and NQO1 by BB is consistent with the reported presence of an EpRE, formerly named antioxidant response element (ARE), in those genes (Friling et al., 1990; Kong et al., 2001
; Tsuji et al., 2000
). Rat GSTA, NQO1, and ferritin were known to be transcriptionally regulated by binding of Nrf2 to this EpRE. More Nrf2-regulated genes were identified in mice upon induction by the isothiocyanate sulforaphane (Thimmulappa et al., 2002
). These included AFAR, glucose-6-phosphate dehydrogenase, carboxylesterase, transketolase, and aldehyde dehydrogenase. In our studies in rats, BB induced HO-1, ferritins, GSTA, GSTM, and NQO1, concurrently with TIMP1, AFAR, GSTM, peroxiredoxin1, aldo-keto reductase, transketolase, and also Mrp3. The rat Mrp3 was recently found to be induced by CAR and EpRE activators in liver (Cherrington et al., 2002
). The presence of the EpRE in rat AFAR, as suggested by our data, was confirmed recently (Ellis et al., 2003
).
Intraday Variation in Gene Expression
Control rats sacrificed 6 h after the start of the study had higher relative liver weights and also the levels of GSH differed. Urea, triglycerides, and phospholipids levels in plasma varied on an intraday basis. These changes were accompanied by altered liver gene expression. Intraday variation of GSH levels (and of other plasma parameters) might not be a negligible effect on the outcome of pharmacology and toxicity studies. Our results show that also expression levels of certain genes change considerably during the day.
Interstudy Comparison and Route of Administration
Previously, we reported rat liver genes and proteins with altered expression upon ip administration of BB (Heijne et al., 2003). The majority of the genes that changed in the previous study with ip administration of BB again was identified to change in the present study with oral administration of BB. The overlap between the two independent studies demonstrates the robustness of the methods and confirms that our data analysis approach did not allow the introduction of many false positives, frequently raised as a point of concern for cDNA microarray experiments. We conclude that with both routes of administration, ip and po, the same hepatic response was induced at the transcriptome level. The ip injection of corn oil induced some subtle effects, while changes induced by the oral administration of corn oil were not readily detected.
Concluding Remarks
In summary, gene expression measurements in liver 6, 24, and 48 h after dosage of several doses of BB yielded a more comprehensive insight into different cellular pathways that are activated when rats are given BB, leading to hepatotoxicity. Results expanded the findings of our earlier experiments (Heijne et al., 2003). Many changes were in line with the observations from routine toxicological assessments, while also new hypotheses on mechanisms of BB-induced hepatotoxicity were postulated. Recovery of the liver was suggested in response to BB with the altered expression of genes involved in protein synthesis and cytoskeleton rearrangement. After 48 h, the rats in the mid dose group showed no signs of toxicity, concurrent with a gene expression patterns that largely resembled the controls. We identified genes responding to dose levels below 5.0 mmol/kg BW, with some genes responding to oral administration of as low as 0.5 mmol/kg. Thus, we were able to detect significant effects at 2 to 10-fold lower doses with transcriptomics compared to clinical chemistry or histopathology. Genes that could serve as early biomarkers of hepatotoxicity at lower BB exposure were revealed, such as HO-1, mEH, AFAR, and Mt-1. A sample of results from the cDNA microarrays were confirmed by the bDNA assay. Future research will have to establish further whether the changes in gene expression are adverse or protective, and whether they are reversible or irreversible effects.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence can be addressed at PO box 360, 3700 AJ Zeist, The Netherlands. Fax: + 31 30 696 02 64. E-mail: Heijne{at}voeding.TNO.nl
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