Benzo[a]pyrene-induced transcriptomic responses in ... - Springer Link

Arch Toxicol (2013) 87:505–515 DOI 10.1007/s00204-012-0949-5

IN VITRO SYSTEMS

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: Toxicokinetics is essential for in vivo–in vitro comparisons P. C. E. van Kesteren • P. E. Zwart • M. M. Schaap • T. E. Pronk • M. H. M. van Herwijnen • J. C. S. Kleinjans • B. G. H. Bokkers • R. W. L. Godschalk • M. J. Zeilmaker • H. van Steeg • M. Luijten

Received: 20 July 2012 / Accepted: 18 September 2012 / Published online: 2 October 2012 Ó Springer-Verlag Berlin Heidelberg 2012

Abstract The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa-/-p53?/- primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancerrelated pathways in Xpa-/-p53?/- hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P

Electronic supplementary material The online version of this article (doi:10.1007/s00204-012-0949-5) contains supplementary material, which is available to authorized users. P. C. E. van Kesteren  P. E. Zwart  M. M. Schaap  T. E. Pronk  H. van Steeg  M. Luijten (&) Laboratory for Health Protection Research, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands e-mail: [email protected] P. C. E. van Kesteren  T. E. Pronk  M. H. M. van Herwijnen  J. C. S. Kleinjans Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands M. M. Schaap  H. van Steeg Department of Toxicogenetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands B. G. H. Bokkers  M. J. Zeilmaker Centre for Substances and Integral Risk Assessment, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands R. W. L. Godschalk Department of Toxicology, School for Nutrition, Toxicology and Metabolism (NUTRIM), Maastricht University, Maastricht, The Netherlands

exposure of wild-type and Xpa-/-p53?/- mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa-/-p53?/- livers only. In order to be able to make a meaningful in vivo–in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa-/-p53?/exposed livers, which were previously found to be induced by B[a]P in Xpa-/-p53?/- primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models. Keywords Toxicogenomics  Carcinogenesis  Benzo[a]pyrene  Xpa-/-p53?/-  Physiologically based kinetic modeling Abbreviations ANOVA Analysis of variance B[a]P Benzo[a]pyrene BPDE Benzo[a]pyrene-7,8-diol-9,10-epoxide GI Gastrointestinal FDR False discovery rate GenMAPP Gene Map Annotator and Pathway Profiler GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes

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PBK PCA WT Xpa Xpa/p53

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Physiologically based kinetic Principal component analysis Wild-type Xeroderma pigmentosum A Xpa-/-p53?/-

Introduction Existing and newly developed substances require human risk assessment to guarantee safe use. The first step in risk assessment is hazard identification, which aims to determine whether exposure to a substance may increase the incidence of adverse effects. In the second step, the potency of a substance is evaluated by means of a dose– response analysis. Genotoxicity assays are currently used for the hazard identification of large numbers of chemicals with respect to carcinogenic properties. A substance is suspected to be carcinogenic based on the results of in vitro and in vivo genotoxicity tests. The results of a subsequent two-year cancer bioassay are used to confirm hazard and to inform on cancer potency. However, a cancer bioassay needs a long exposure time and a high number of laboratory animals, is costly, and may result in a high percentage of false-positive results (Ames and Gold 1990; JacobsonKram et al. 2004). In addition, the mode of action of a substance is not clarified using this assay. Therefore, many efforts have been taken to develop improved methods for the identification of carcinogens and their modes of action (Benigni 2012; Waters et al. 2010). The use of transgenic mouse models has shown a number of potential advantages. Tumors develop faster, less animals are needed, and results are more predictive for a human cancer response (Pritchard et al. 2003). One of these promising transgenic mouse models is the DNA repair-deficient Xpa-/-p53?/- mouse (further referred to as Xpa/p53) (van Steeg et al. 2001). Xpa/p53 mice have low levels of spontaneous tumors, but are more cancerprone than wild-type (WT) mice upon exposure to genotoxic and non-genotoxic carcinogens (van Kreijl et al. 2001). Furthermore, the overall accuracy of a 39-week exposure assay with Xpa/p53 mice was much better than the accuracy of the traditional bioassay (van Kreijl et al. 2001; Pritchard et al. 2003). The increased susceptibility of this mouse model to carcinogens might not only be due to impaired DNA damage responses and repair due to Xpa and p53 deficiencies; also divergent regulation of pathways involved in earlystage carcinogenesis may play an essential role. A powerful tool to investigate the early processes triggered in Xpa/p53 mice by carcinogens is the application of toxicogenomics.

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Identification of cancer-related processes can be of great importance in providing mechanistic information on substances or more specifically in early detection of carcinogenic features of substances. The use of a transcriptomics approach in an in vivo setting in the field of carcinogenesis has been promising so far. Researchers were able to get mechanistic insight into early cellular alterations after carcinogenic exposure, and they derived gene signatures that display a high sensitivity and specificity during validation with a panel of known liver carcinogens and non-carcinogens (Ellinger-Ziegelbauer et al. 2008; Fielden et al. 2008; Auerbach et al. 2010; Uehara et al. 2011; Park et al. 2011). Additionally, transcriptomics applied to in vitro systems has been explored, since this approach is more feasible in a highthroughput setting. Previous in vitro studies displayed gene signatures that are predictive for a set of tested substances or were used to discriminate between carcinogens and noncarcinogens, resulting in a concordance of up to 85 % (van Delft et al. 2004; Tsujimura et al. 2006; Rohrbeck et al. 2010). We have previously studied the effect of genotoxic exposures on gene expression in primary hepatocytes derived from both WT and Xpa/p53 mice. Cancer-related pathways were observed in primary hepatocytes derived from Xpa/p53 mice after treatment with benzo[a]pyrene (B[a]P), demonstrating the possible usefulness of these hepatocytes for identifying genotoxic carcinogens (van Kesteren et al. 2011). Our ultimate goal is to develop an in vitro screening method for carcinogenicity and to discern the modes of action of carcinogens. In this context, one of the major issues is the quantitative comparison of the exposure of cells in vitro (i.e., the concentration added to the cells) with the exposure of cells after in vivo administration of the carcinogen, the latter concentration being required for risk assessment. Here, physiologically based kinetic (PBK) modeling may provide a valuable tool to perform such a comparison (Verwei et al. 2006; Blaauboer et al. 2010; Adler et al. 2011; Mielke et al. 2011). In this study, we therefore used PBK modeling to estimate the B[a]P concentration in the mouse liver after in vivo exposure and compared this with the B[a]P concentration to which primary hepatocytes were exposed in vitro. PBK models for B[a]P have been shown to be a suitable tool to describe the toxicokinetics of B[a]P in rats and mice (Zeilmaker et al. 1999; Crowell et al. 2011). Here, PBK simulations, together with DNA adduct analysis, were used to compare in vivo and in vitro exposure levels. Subsequently, gene expression responses in WT and Xpa/p53 livers were analyzed and compared with our previously obtained in vitro transcriptomics data. Our findings show that determination of internal in vivo doses is a prerequisite for further selection of biological meaningful in vitro concentrations.

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Methods Mice All mice used in this study were kept in a C57BL/6 J background. Xpa-/- mice (de Vries et al. 1995) were crossed with p53?/- mice (Jacks et al. 1994) in order to obtain Xpa-/-p53?/- double-mutant mice. To confirm genotypes, PCR analyses were performed as described previously (de Vries et al. 1997). All mice were housed in a climate-controlled room with a 12-h on/off light cycle. The health status of the mice was monitored daily, beginning at the day of weaning. Feed and water were available ad libitum. The study was agreed upon by the institute’s ethical committee on experimental animals, in accordance with national legislation. In vivo exposure Six-week-old male WT and Xpa/p53 mice (n = 4) were acclimated for 2 weeks. At 8 weeks old, animals were treated by oral gavage on day 0, 2, 4 and 6 with 13 mg B[a]P/kg bodyweight in sunflower oil or were given only the solvent. Administration of this dose for 13 weeks has been shown to induce tumors in both WT and Xpa/p53 mice, with Xpa/p53 mice being more susceptible to the carcinogenic effects (van Steeg et al. 2001; de Vries et al. 1997). During acclimation and the duration of the experiment, animals were weighed weekly. On day 7, mice were sacrificed, and the liver was isolated and stored according to the protocol using RNAlater (Qiagen, Valencia, CA, USA). Isolation, culture and exposure of primary mouse hepatocytes Primary mouse hepatocytes were isolated, maintained in vitro and exposed to B[a]P as described previously (van Kesteren et al. 2011). In short, primary hepatocytes were isolated from WT and Xpa/p53 C57BL/6 mice by a twostep liver perfusion and treated for 24 h in vitro with 5, 15, 30 or 60 lM B[a]P or the solvent control. The concentrations corresponding to 80 % cellular viability, measured with an MTT test, were 30 lM for WT hepatocytes and 15 lM for Xpa/p53 hepatocytes (van Kesteren et al. 2011). RNA isolation, labeling and hybridization RNA from mouse livers was extracted and purified as described previously (van Kesteren et al. 2011). Four biological replicates were used for each dose. RNA concentrations were measured using the NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies), and RNA

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quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies). Labeled RNA was prepared using the Affymetrix gene chip 30 IVT express kit and hybridized to Affymetrix Mouse Genome 430 2.0 GeneChip arrays according to the manufacturer’s instructions. After hybridization arrays were washed and stained with a GeneChip Fluidics Station 450 and scanned using the Affymetrix gene chip scanner 3000. Data analysis Quality controls, including RNA degradation control, correlation and clustering, were all within acceptable limits according to Affymetrix standards. Affymetrix CEL files were normalized by robust multichip average (Irizarry et al. 2003) using the custom chip description files as described by de Leeuw et al. (2008). Of the hybrid probeset definitions included in the custom annotation, only the 16,331 probe sets selected according to Dai et al. (2005) and the 4,648 Affymetrix probe sets corresponding to an Entrez Gene ID were used in further analysis, giving a total of 20,979 probe sets. The data set has been deposited in ArrayExpress under accession number E-MTAB-1247. Significant changes in gene expression after B[a]P treatment (as compared to controls) were calculated for each genotype and dose with one-way analysis of variance (ANOVA), using a false discovery rate (FDR) of \0.05. Data were analyzed by principal component analysis (PCA) in R. The pathway-finding tool T-Profiler (Boorsma et al. 2005) was used to identify overrepresentation of biological pathways in the total gene set without setting thresholds to the level of single gene expression changes. Biological pathways were based on Gene Ontology (GO; ontology biological process and molecular function), Kyoto encyclopedia of genes and genomes (KEGG) and Gene Map Annotator and Pathway Profiler (GenMAPP), and pathways with an E-value (Bonferroni-corrected P value) of \ 0.05 were considered statistically significantly regulated. Leading genes within significantly changed pathways were selected based on fold change, with a threshold of C1.2-fold up- or downregulation. PBK modeling The in vivo B[a]P concentration in the portal vein was estimated by applying a PBK mouse model, based on Mielke et al. (2011). An overview of the structure of this B[a]P PBK model, corresponding model equations and model parameters, based on physiological and estimated values from mice and rats, is given in Supplementary Document 1 (Brown et al. 1997; Clewell et al. 2000; Pery et al. 2011; Foth et al. 1988; Klaassen et al. 1996; Ramesh et al. 2001; Uno et al. 2004; Hecht et al. 1979). In the PBK

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simulations, a value of 0.50 was taken as most likely representative for the absorbed fraction after oral exposure to B[a]P. For the absorption rate constant, a value of 0.05 min-1 was used, which is equivalent to an absorption half-life of 15 min. In concordance with Zeilmaker et al. (1999) and Uno et al. (2004), the metabolic rate constant was set such that 99 % of the absorbed dose was metabolized by the liver within 6 h after administration. To correct for the ability of the liver to adapt to repeated B[a]P exposures by means of aryl hydrocarbon (Ah) receptordependent induction of P450 metabolism, a reduction factor was applied based on Zeilmaker et al. (1999). They showed a fourfold reduction in the peak B[a]P concentration in blood after repeated oral exposure to 30 mg/kg bw B[a]P. In the Zeilmaker study, repeated exposure did not lead to any accumulation of B[a]P in the blood as exposure proceeded. In concordance with Mielke et al. (2011), the in vivo B[a]P concentration in the portal vein was used as surrogate for the peak exposure of the liver immediately after oral exposure. DNA adduct analysis DNA was isolated using TRIzol reagent according to the manufacturer’s protocol. DNA adduct levels were determined according to Reddy and Randerath (1986) with modifications as described by Godschalk et al. (1998). Three standards of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-modified DNA with known modification levels (1 adduct/106, 107 and 108 unmodified nucleotides) were run in parallel for quantification. Adduct spots on the chromatograms were located and quantified using a phosphor imager (FLA-3000, Fuji, Paris, France) and AIDA/2D densitometry software. The average and standard deviation of the four replicate samples were calculated. The independent Student’s t-test (SPSS Statistics, version 19) was used to analyze the difference between the two genotypes per dose. Additionally, the difference between the various treatments compared with the corresponding controls was analyzed using a one-way ANOVA with Dunnett’s post hoc analysis. The level of significance was set at P \ 0.05.

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approach, because such a concentration is expected to result in measurable effects at a gene expression level, inducing only limited toxicity. The in vivo study was performed for 7 days at a dose that is carcinogenic in Xpa/p53 mice and WT mice, when given for 13 weeks in a 39-week study (van Steeg et al. 2001; de Vries et al. 1997). To compare gene expression responses, we first examined the in vitro concentrations as related to the carcinogenic in vivo dose used. For this, we used two procedures: estimation of internal B[a]P doses in vivo by PBK modeling and measurement of DNA adduct levels. PBK modeling of in vivo B[a]P concentrations We applied a PBK model to simulate the B[a]P concentration over time in the hepatic portal vein of mice after a single oral exposure to B[a]P. The results of this simulation are displayed in Fig. 1 and indicate a maximum level of 12 lM in the portal vein, 2.0 lM in the hepatic vein and 0.94 lM in the mixed venous blood. However, in vivo–in vitro differences during the passage from initial peak concentration in the portal vein or culture medium to the actual exposure of the hepatocytes should be taken into account. The exposure level in the liver will be lower after repeated exposure due to induced cytochrome P450 enzyme levels that increase B[a]P biotransformation. Based on the findings of Zeilmaker et al. (1999) and given the fact that B[a]P was repeatedly administered to the mice, we estimated the actual effective dose in the liver cells to be up to a fourfold lower (* 3 lM) than the simulated level in the portal vein. This indicates that the

Results To investigate the relevance of in vitro findings for cancer risk, we compared in vivo and in vitro transcriptomics responses upon exposure to B[a]P. The in vitro study we previously performed comprised 24-h exposure of primary hepatocytes to a concentration range (0-60 lM) of B[a]P. This range was based on an initial dose level corresponding to 80 % cellular viability (30 lM in WT hepatocytes, 15 lM in Xpa/p53 hepatocytes). This is a widely used

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Fig. 1 PBK simulation of the time-course of the concentration of B[a]P in the portal vein, the hepatic vein and mixed venous blood of mice exposed to a single oral dose of 13 mg B[a]P/kg bodyweight

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lowest concentration tested in vitro (5 lM) corresponds best with the estimated internal in vivo concentration. B[a]P-induced DNA adducts in mouse livers and primary hepatocytes To analyze B[a]P-induced DNA damage, we measured DNA adduct levels after in vivo and in vitro exposure to B[a]P. The profiles showed a DNA adduct spot that co-migrates with the benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adduct standard, the major B[a]P DNA adduct (data not shown). Average BPDE-DNA adduct levels were 24 and 37 adducts/107 nucleotides in WT and Xpa/p53 livers, respectively (Fig. 2), while DNA adduct levels in untreated mice were very low. The levels in primary hepatocytes ranged from 126 (WT) and 239 (Xpa/p53) adducts/107 nucleotides at low dose (5 lM) up to 4617 (WT) and 7090 (Xpa/p53) adducts/107 nucleotides at the highest dose (60 lM) tested. A clear dose–response was observed for both genotypes (Fig. 2). The average BPDE-DNA adduct levels in Xpa/p53 hepatocytes were, as expected, higher than those found in WT hepatocytes, although a statistically significant difference was only obtained for a concentration of 30 lM. Although the DNA adduct levels measured after exposure to the lowest B[a]P concentration in vitro (5 lM) were significantly higher than those measured in vivo, they were in the same order of magnitude.

Fig. 2 BPDE-DNA adduct levels in total DNA isolated from livers and primary hepatocytes from WT and Xpa/p53 mice. Mice were treated with B[a]P (13 mg/kg bw) for 7 days; primary hepatocytes were treated with 5, 15, 30 or 60 lM B[a]P for 24 h. Bars represent the average DNA adduct levels. *Significantly increased compared to untreated controls, based on one-way ANOVA with Dunnett’s post hoc analysis.  Significant difference between Xpa/p53 hepatocytes and WT hepatocytes, based on independent Students t test

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These findings together with the PBK modeling data indicate that the lowest concentration tested in vitro (5 lM) corresponds best with the estimated internal in vivo concentration. Therefore, those in vitro data were used for the in vivo—–in vitro comparison. Changes in gene expression profiles upon B[a]P exposure in WT and Xpa/p53 mouse livers To visualize the overall in vivo gene expression response upon B[a]P exposure, we performed principal component analysis on the complete data set. Analysis of the gene expression in mouse livers revealed distinction between B[a]P-exposed livers and control livers (Fig. 3), indicating B[a]P-induced gene expression changes. However, determination of differentially expressed genes after treatment with B[a]P as compared to control samples (FDR \ 0.05) showed no significantly differentially expressed genes in WT livers and only one differentially expressed gene (Cyp1a2) in Xpa/p53 livers. Therefore, further analyses were performed with the total gene set using threshold-free pathway analyses with T-Profiler. Pathways (from the databases KEGG, GO and GenMAPP) that were significantly changed in expression upon in vivo exposure to B[a]P were identified and grouped into seven different categories (Table 1). A total overview of the individual pathways is presented in Supplementary Table S1. B[a]P exposure resulted in induction of xenobiotic metabolism, immune-related processes and lipid metabolism in both WT and Xpa/p53 livers. Identification

Fig. 3 Principal component analysis of the complete gene expression data set of WT and Xpa/p53 mouse livers upon exposure to B[a]P. WT and Xpa/p53 mice were treated on day 0, 2, 4 and 6 with 13 mg/kg bw B[a]P in sunflower oil or were given solvent only. Each symbol represents an independent experiment

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Table 1 Regulated pathways and leading genes in livers from WT and Xpa/p53 mice upon B[a]P exposure Xpa/p53

Processes

WT

Xenobiotic metabolism

?

Cyp1a2, Cyp2b10, Cyp2b9, Cyp2c37, Cyp2c54, Cyp4a14, Cyp26a1, Fmo3, Gstt2

?

Akr1b7, Cbr1, Cyp17a1, Cyp1a2, Cyp2b10, Cyp2b9, Cyp2c38, Cyp4a10, Cyp4a14, Dhtkd1, Fmo2, Fmo3, Fmo4, Gstm2, Gstm3, Gstt2, Hsd17b6, Htatip2, Mgst2, Mtmr11, Nqo1

Immune response

?

Apoa4, Cd274,Cd74, Gadd45g, Gbp1, Gbp2, Gbp3, H2Aa, H2Ab1, H2Dmb1, H2Eb1, Id2, Il15, Il1a, Vnn1

?

Apoa4, Clec7a, Cxcl1, Defb1, Gbp1, Gbp2, Gbp3, H2Aa, H2Ab1, H2Dmb1, Id2, Il15, Il1b, Irf1, Klf10, Oasl2, S100a9, Serpina3g,Vnn1

Lipid metabolism

?

Abcd2,Abcg5, Cyp1a2, Cyp2b10, Cyp2c37, Cyp2b9, Cyp2c54, Cyp4a14, Cyp26a1

?

Abcb1a, Abcd2, Abcg5, Acnat2, Acot1, Acot3, Acot4, Agpat9, Akr1b7, Atp6v0d2, Cbr1, Cyp17a1, Cyp1a2, Cyp2b9, Cyp2c38, Cyp2j9, Cyp4a10, Cyp4a14, Gstt2, Gstt3, Hsd17b6, Il1b, Nampt, Pla2g12a, Serpina6, Sult1e1, Vldlr

Angiogenesis

?

Lect1, Id1, Illa

Oxidative stress response

?

Gstt2, Mgst3, Gstt3, Gstm2, Gstm3, Junb, Mt1, Nqo1

Antiapoptosis

–

Hspa1b, Bcl6, Tsc22d3, Syvn1, Angptl4, Cebpb, Cdkn1a

DNA replication

?

Cdt1, Gmnn

Results indicate upregulation (?) or downregulation (-) of biological pathways. Leading genes, whose expression was changed with a fold change of C1.2, are presented. Genes observed in both genotypes are indicated in bold. A complete overview of the significantly regulated pathways is given in Supplementary Table S1

of the leading genes revealed overlap between WT and Xpa/p53 livers. For example, cytochromes P450 enzymes Cyp1a2, Cyp2b9, Cyp2b10, Cyp4a14, monooxygenase Fmo3 and glutathione-S-transferase Gstt2 were responsible for induction of xenobiotic metabolism in both WT and Xpa/p53 mice, indicating a comparable B[a]P biotransformation mechanism in both genotypes. Angiogenesis was differentially regulated only in WT livers and revealed induction of the angiogenesis inhibitor Lect1 and the angiogenesis promoters Id1 and interleukin IL1a. More interestingly, four processes were perturbed significantly in Xpa/p53 mice by B[a]P, but were unaffected in WT mice. A response to oxidative stress was observed in Xpa/p53 livers, mainly represented by induction of glutathioneS-transferases, such as Gstt2, Gstt3, Gstm2 and Gstm3. Despite that B[a]P is known for its induction of reactive oxygen species under normal conditions, an oxidative stress response was not significantly induced in WT mice. Possibly, the dose used and/or the time of measurement were not suitable for detection of such a mechanism in WT livers. Next, several genes involved in anti-apoptosis regulation, including Cdkn1a (p21), Hspa1b, Tsc22d3, Angptl4, Bcl6, were downregulated, indicating induction of a significant DNA damage response by B[a]P in Xpa/p53 liver cells via pro-apoptotic regulation. The most interesting process that was statistically significantly triggered in Xpa/p53 but not in WT mice was DNA replication. The two leading genes were chromatin licensing and DNA

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replication factor 1 (Cdt1) and Geminin (Gmnn), which are involved in pre-initiation of DNA replication. Comparison of in vivo and in vitro pathway regulation after B[a]P exposure To investigate whether the B[a]P-induced processes in vivo (Table 1) can also be detected in vitro, we compared the in vivo findings with the gene expression response in WT and Xpa/p53 primary hepatocytes. The overall transcriptomic response of the total gene set in primary hepatocytes after exposure to 5 lM B[a]P was much more pronounced as compared with changes upon in vivo exposure, as visualized by PCA (Fig. 4). Both PC1 and PC2 show that the in vitro data account for a large part for the variance. The average transcriptomics response in exposed WT and Xpa/p53 hepatocytes seemed to have a similar comparability as to their corresponding in vivo results. Analysis of pathway regulation showed that Xpa/p53 livers and primary hepatocytes have slightly more similarities than WT livers and hepatocytes (Table 2; total overview of in vitro regulated pathways in Supplementary Table S2). It should be noted that the number of regulated pathways in WT mice was very limited, making comparison with in vitro results more difficult. Most of the pathways detected in vivo in both genotypes were also observed in WT and Xpa/p53 hepatocytes, that is, xenobiotic metabolism and lipid metabolism. However, the

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511 Table 2 B[a]P-induced pathway regulation in livers and primary hepatocytes from WT and Xpa/p53 mice In vivo

In vitro

WT

Xpa/p53

WT

Xpa/p53

Xenobiotic metabolism

?

?

?/-

?

Immune response

?

?

–

-

Lipid metabolism

?

?

?/-

?/-

Angiogenesis

?

Oxidative stress response

?

?

?

Anti-apoptosis

-

DNA replication

?

Pathway

Fig. 4 Principal component analysis of gene expression changes of WT and Xpa/p53 mouse livers and WT and Xpa/p53 primary mouse hepatocytes upon exposure to B[a]P as related to controls. WT and Xpa/p53 mice were treated on day 0, 2, 4 and 6 with 13 mg/kg bw B[a]P in sunflower oil or solvent only. WT and Xpa/p53 primary hepatocytes were treated for 24 h to 5 lM B[a]P in DMSO or solvent only. The gene expression changes from the whole data set as related to the controls are presented. Each symbol represents an independent experiment. The average of the controls is illustrated by 9

immune response was upregulated in livers of both genotypes, but was downregulated in vitro. Immune responses were upregulated in livers mainly due to upregulation of histocompatibility genes and genes involved in activation of immune cells via cytokines. In vitro, cytokines were downregulated, and histones were upregulated in Xpa/p53 primary hepatocytes, while in WT primary hepatocytes complement components and chemokines were downregulated. The pathways that were only significantly regulated in livers of Xpa/p53 mice, that is, oxidative stress response, anti-apoptosis and DNA replication, were not detected specifically in Xpa/p53 hepatocytes. An upregulated oxidative stress response was also observed in WT hepatocytes, and DNA replication was significantly regulated in WT hepatocytes, but not in Xpa/p53 hepatocytes. No significant change in apoptosis regulation was observed upon in vitro exposure. Importantly, many pathways induced upon in vitro exposure were not significantly changed upon in vivo exposure. These include general downregulation of metabolisms like those of amino acids and carbohydrates. Typical genotoxicity-related responses such as DNA damage response, cell cycle response and DNA repair as found in WT hepatocytes were absent in vivo. In addition, the cancer-related Mapk pathway, which was observed in Xpa/p53 hepatocytes after B[a]P exposure, was not affected in WT or Xpa/p53 mouse livers after B[a]P treatment.

?

Amino acid metabolism

-

-

Carbohydrate metabolism Proteolysis

?

?

DNA damage response/repair

?

Cell cycle

?

TGFb signaling

?

TNFa/NFjB signaling

?

PPAR signaling

-

Cancer-related pathways/Mapk

-

Calcium signaling

-

Cytoskeleton remodeling

-

Intercellular communication via junctions

-

G-proteins/GTPase activity

-

-

Results indicate upregulation (?), downregulation (-) or both (?/-) of biological pathways

Discussion The identification of carcinogenic features of substances by using the traditional cancer bioassay is accompanied with several disadvantages. Transgenic mice, including Xpa/p53 mice, have been shown to provide advantages due to their increased susceptibility to true human carcinogens. Here, we studied in vivo gene expression changes in WT and Xpa/p53 mice after treatment to B[a]P to identify early responses to this carcinogenic stimulus. The results were compared with previously obtained gene expression data upon in vitro exposure of primary hepatocytes isolated from both WT and Xpa/p53 mice. To determine the relevance of the in vitro concentrations in comparison to the carcinogenic in vivo dose tested, we estimated the internal in vivo peak concentration of B[a]P. Our PBK model showed that the lowest in vitro concentration tested approximated the simulated in vivo peak concentration. As previously mentioned by Mielke et al. (2011), in vitro concentrations in toxicogenomics studies are often chosen based on cellular toxicity tests and may not represent a similar biological response as related to the

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in vivo doses tested, which are based on long-term pathological effects. In contrast to the model used by Mielke et al. (2011), we included metabolism in our PBK model. Although the inclusion of metabolism had a negligible effect on the maximal concentrations of B[a]P in the portal vein, it gives a better representation of the in vivo situation and makes comparison to other models possible. The estimated peak concentration of B[a]P in venous blood was similar to the maximum level as measured by Uno et al. (2004) and as estimated by Crowell et al.(2011), thereby supporting our simulation. By using PBK modeling, initial B[a]P concentrations to which the liver cells are exposed can be estimated and directly compared to the initial in vitro concentrations in culture medium. As such, in future studies, this information can be used as guidance for the selection of equivalent B[a]P concentrations that are to be added to the cells in vitro, thereby improving the comparability of in vitro and in vivo transcriptomics data. DNA adduct analysis confirmed B[a]P-induced DNA damage in mouse livers and primary hepatocytes. Additionally, it displayed lower DNA adduct levels upon in vivo exposures than observed after in vitro exposure. It should be noted that direct correlation of DNA adduct levels in vivo and in vitro is hindered by differences between the two exposure settings. An important factor to take into account is DNA repair efficiency, but also the type of exposures (repeated exposure in vivo versus single exposure in vitro) and the time point of measurement (24 h after final exposure versus directly after exposure) can affect the outcome. Nevertheless, the DNA adduct levels observed in vitro upon treatment to 5 lM B[a]P most resembled the levels observed in vivo. Gene expression analyses revealed no differentially expressed genes (FDR \ 0.05) upon in vivo exposure to B[a]P, with the exception of significant upregulation of Cyp1a2 in livers from Xpa/p53 mice. Cyp1a2 is induced upon Ah receptor activation by polycyclic aromatic hydrocarbons, such as B[a]P (Bartosiewicz et al. 2001), and is one of the cytochrome P450 enzymes responsible for the first biotransformation step of B[a]P (Gelboin 1980). The biotransformation of B[a]P in vivo might explain the lack of significantly changed gene expression, since 99 % of B[a]P is metabolized and eliminated from the blood within 6 h (Zeilmaker et al. 1999; Crowell et al. 2011). This indicates that the 24-h time lapse between administration and tissue isolation may have been too long to detect initial gene expression changes. Furthermore, low numbers of differentially expressed genes upon short-term exposures is not an uncommon finding for B[a]P, as was also shown by Malik et al. (2012) who found only 6 and 7 genes significantly changed upon 28 days of exposure to 25 and 50 mg B[a]P/kg bodyweight per day, respectively. Application of an ANOVA with an FDR of \ 0.05 is a

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commonly used but conservative approach in detecting significant differential gene expression. We explicitly chose to analyze the in vivo data using the same approach as used for the interpretation of the in vitro data. When using in vivo data, this approach can result in incomplete detection of relevant gene expression changes. The use of threshold-free pathway analysis does not rely on single gene expression changes and, despite the low responsiveness, provided more insight into B[a]P-induced changes in pathway regulation. The results revealed significant changes in xenobiotic metabolism, immune responses and lipid metabolism in both WT and Xpa/p53 livers and are common B[a]P responses (Yauk et al. 2011; Malik et al. 2012). When focusing on comparison of the genotypes, we found Xpa/p53-specific regulation of an oxidative stress response, apoptosis and DNA replication. Amongst the metabolites formed during biotransformation of B[a]P are the quinones that can form reactive oxygen species during an oxidation reaction (Flowers et al. 1997). Oxidative stress response is therefore a commonly detected reaction to B[a]P exposure and would have been expected to be upregulated in WT mice too. The lack of a response to oxidative stress in WT mice is probably dose-related and not considered to be of high relevance for genotypic differences. Apoptosis was induced by inhibition of anti-apoptosis genes. Interestingly, most of these genes are not directly regulated downstream of p53 during a DNA damage response and can act independently of p53. In addition, expression of Cdkn1a (p21) and Bcl6 is normally upregulated by p53 activation, but these genes were downregulated in Xpa/p53 mice. Although absent in our study, induction of apoptosis was previously detected in WT mouse livers and consisted of well-known p53-induced genes, including Apaf1, Bax and Igf1 (Malik et al. 2012). These findings indicate a disturbed response to DNA damage in Xpa/p53 mice livers with regulation of apoptosis via an alternative route, probably due to p53 haplo-insufficiency in Xpa/p53 mice. Possibly, this alternative route of apoptosis regulation is less efficient as compared to regulation in cells carrying a fully functional p53 protein. The upregulated DNA replication pathway in Xpa/p53 mice could be mainly attributed to the induction of Cdt1 and Gmnn gene expression. DNA replication is required during the mammalian cell cycle and is pre-initiated during the late G1 phase by an assembly of proteins, including Cdt1 (Nishitani et al. 2000; Blow and Tada 2000). To prevent re-replication of DNA, Gmnn is induced to inhibit Cdt1 expression during the S-phase (Wohlschlegel et al. 2000; Tada et al. 2001). It has been previously described that Cdt1 can function as an oncogene when it is overexpressed (Arentson et al. 2002) and can overcome negative regulation by Gmnn (Wohlschlegel et al. 2000), resulting in

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accumulation of gene mutations and chromosomal damage (Seo et al. 2005; Tatsumi et al. 2006). Our data indicate that Cdt1 is stable in WT mice, but responded to B[a]P treatment in Xpa/p53 mice. These findings suggest that genomic instability in Xpa/p53 liver cells, due to re-replication of DNA, might contribute to the enhanced susceptibility of Xpa/p53 mice toward carcinogens. Further investigations of Xpa/p53 livers treated with a more extended set of genotoxic carcinogens should be performed to confirm the observed B[a]P-induced increase of Cdt1 gene expression. Although the number of regulated pathways in livers was low, comparison to the most relevant in vitro concentration tested (5 lM) revealed several similarities. Despite the fact that more pathways were significantly regulated in vitro upon 5 lM B[a]P exposure than in the in vivo experiments, our previously published in vitro data reveal that the number of regulated pathways rises with increasing concentrations (van Kesteren et al. 2011). This further strengthens that 5 lM is the most equivalent concentration in comparison to the in vivo dose tested. The most interesting in vivo findings were the regulation of apoptosis and DNA replication that were only observed in Xpa/p53 mice. Regulation of apoptosis was also observed in vitro in Xpa/p53 hepatocytes, but only at the highest, toxic, concentrations used (van Kesteren et al. 2011). We believe that in vitro regulation of apoptosis is not representative for the observed in vivo changes and is not indicative of a disturbed DNA damage response at non-toxic levels of B[a]P as observed in vivo. A differential regulation of DNA replication was only observed in WT hepatocytes and was not differentially regulated in Xpa/p53 hepatocytes. The initiation of DNA replication is attended with cell cycle and cellular proliferation. Primary hepatocytes maintained in a sandwich of collagen are known to sustain in a G1 cell cycle arrest and lack proliferation of cells (Fassett et al. 2003; Hansen et al. 2006). It is therefore understandable that some Xpa/p53 liverspecific in vivo findings might not be detectable in hepatocytes cultured in a sandwich conformation. Further, it was recently demonstrated that the highest DNA damage levels following B[a]P exposure occur in the S and G2/M phase of the cell cycle (Hamouchene et al. 2011), indicating that the effects observed in primary hepatocytes may underestimate the susceptibility to B[a]P. As was previously described (van Kesteren et al. 2011), Xpa/p53-specific cancer-related pathways were regulated in primary hepatocytes, with a main role for Mapk signaling. This pathway regulation was absent in mouse livers after treatment of mice to B[a]P. However, since we only tested one dose and one time point in vivo, it is plausible that this pathway regulation might be displayed in vivo upon a higher dose of B[a]P, longer exposure times or with a shorter time between the final treatment and the sampling time. The finding of apoptosis and DNA replication

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regulation in vivo and Mapk signaling in vitro display the possible advantage of using Xpa/p53 mice and hepatocytes in the search for early processes related to carcinogenesis. More research with other (genotoxic) carcinogens is required to further explore the possible added value of Xpa/p53 mice over WT mice. In conclusion, the in vivo data indicated differential regulation of DNA damage responses directly due to Xpa and/or p53 deficiency, demonstrated by alternative control of apoptosis and disturbed DNA replication. These and other pathways were also found in vitro, at a concentration that corresponded best with the estimated internal in vivo dose. One of the major challenges of in vitro research is the choice of substance concentrations that are relevant to in vivo dose levels. We showed the usefulness of PBK modeling and DNA adduct analysis in the comparison of in vitro and in vivo gene expression data and recommend inclusion of such approaches to improve further development of alternative screening methods. Acknowledgments We thank R. Vlug, J. Bos, H. Strootman and T. van de Kuil for their support with the hepatocytes isolations. We also thank Prof. Dr. J. Hengstler for his advice on hepatocyte isolation and culturing. This work was supported by the Technology Foundation STW [grant MFA6809]. Conflict of interest of interest.

The authors declare that they have no conflict

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