3-Methylcholanthrene (3-MC) and 4-chlorobiphenyl (PCB3) genotoxicity is gender-related in Fischer...

3-Methylcholanthrene (3-MC) and 4-Chlorobiphenyl (PCB3)genotoxicity is gender-related in Fischer 344 transgenic rats

J.A. Jacobus1,2, B. Wang1,2, C. Maddox1, H. Esch1, L. Lehmann1, L.W. Robertson1,2, K.Wang3, P. Kirby4, and G. Ludewig1,2,*1Department of Occupational and Environmental Health, University of Iowa, 100 Oakdale Campus# 124 IREH, Iowa City, IA 52242-50002Interdisciplinary Graduate Program in Human Toxicology, University of Iowa, 100 Oakdale Campus# 124 IREH, Iowa City, IA 52242-50003Department of Biostatistics, University of Iowa, 100 Oakdale Campus # 124 IREH, Iowa City, IA52242-50004Department of Pathology; University of Iowa, 100 Oakdale Campus # 124 IREH, Iowa City, IA52242-5000

AbstractPolychlorinated biphenyls (PCBs) are a class of persistent organic pollutants with myriad biologicaleffects, including carcinogenicity. We present data showing gender-specific genotoxicity in Fischer344 transgenic BigBlue rodents exposed to 4-chlorobiphenyl (PCB3), a hydroxylated metabolite,and the positive control 3-methylcholanthrene (3-MC) where female rats are more resistant to thegenotoxic effects of the test compounds compared to their male counterparts. This difference isfurther highlighted through our examination of gene expression, organ-specific weight changes, andtissue morphology. The purpose of the present study was to explores the complex and multifacetedissues of lower molecular weight PCBs as initiators of carcinogenesis, by examining the mutagenicityof PCB3, a hydroxylated metabolite (4′-OH-PCB3), and 3-methylcholanthrene (3-MC, positivecontrol) in a transgenic rodent model. Previous findings indicated that PCB3 is mutagenic in the liverof male BigBlue transgenic rats under identical exposure conditions. We expected that female ratswould be equally, if not more sensitive than male rats, since a 2-year carcinogenesis bioassay withSprague-Dawley rats and commercial PCB mixtures reported much higher liver cancer rates infemale than in male rats. The current study, however, revealed a similar trend in the mutationfrequencies across all four treatment groups in females as reported previously in males, but increasedvariability among animals within each group and a lower overall effect, led to non significantdifferences in mutation frequencies. A closer analysis of the possible reasons for this negative resultusing microarray, organ weight and histology data comparisons shows that female Fischer 344 rats1) had a higher baseline mutation frequency in the corn oil control group and greater variability thanmale rats; 2) responded with robust gene expression changes, which may also play a role in ourobservation of 3) highly increased liver, spleen, and lung weight in 3-MC and PCB3-treated animalsand thus changed distribution and kinetics of the test compounds. Our analysis indicates that femaletransgenic BigBlue Fischer 344 rats are more resistant to PCB3 and 3-MC genotoxicity comparedto their male counterparts.

*Corresponding author: G. Ludewig, phone: 319/335-4650, fax: 319/335-4290, [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptEnviron Int. Author manuscript; available in PMC 2011 November 1.

Published in final edited form as:Environ Int. 2010 November ; 36(8): 970–979. doi:10.1016/j.envint.2010.07.006.

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KeywordsPolychlorinated biphenyl; PCB3; 3-methylcholanthrene; 3-MC; gender difference; genotoxicity;biotransformation; carcinogenicity

1. IntroductionModern society has inherited a global legacy of persistent pollution with polychlorinatedbiphenyls (PCBS) from their industrial and commercial use. PCBs, a class of 209 indiviudalcongeners, are classified as probable human carcinogens (ATSDR 2000). A variety of humanepidemiology studies have concluded that PCBs may be linked to the formation of tumors atvarious sites. For example, Engel and colleagues found a statistically significant trend of higherodds ratios for non-Hodgkin lymphoma with higher total serum PCB-levels in three differentpopulations (Engel et al. 2007; Rothman et al. 1997). Recent reviews of the epidemiologicliterature are available (Faroon et al. 2003; Faroon et al. 2001; Golden and Kimbrough 2009).The cohort results reflect a lack of congruity that may be due to varying routes of exposure,and exposure to highly variable PCB congener mixtures (Hopf et al. 2009). Because invidualPCB congeners have differing modes of action, it seems plausible that a variety of tumor typescould arise from exposure to various congeners, or their metabolites.

The evidence for carcinogenicity in rodents is much clearer. Using the Sprague Dawley ratstrain a comprehensive chronic toxicity and carcinogenicity study analyzed the effects of fourdifferent commercial PCB mixtures (Aroclor 1016, 1242, 1254, and 1260) and multiple dietaryconcentrations, ranging from 25 to 200 ppm, during 24 months of exposure. In males ratsstatistically significant increases in hepatocellular carcinomas was noted only for the higher-chlorinated mixture Aroclor 1260, while all four commercial products caused an elevatedincidence of hepatocellular carcinomas in female rats. It should be noted that Aroclor 1016averages only three chlorines per biphenyl. These data indicate that commercial mixtures ofchlorinated biphenyls are complete carcinogens, especially in the female Sprague Dawley rat(Mayes et al., 1998). Likewise chronic carcinogenicity studies with individual PCB congeners,PCB 118 (2,3′,4,4′,5-pentachloro-biphenyl) and PCB 126 (3,3′,4,4′,5-pentachlorobiphenyl)found “clear evidence” of carcinogenicity in female Sprague Dawley rats(http://ntp.niehs.nih.gov/). Much less is known about the long term risks of exposure to lowerchlorinated PCBs.

Exposure to lower molecular weight PCBs occurs mainly via inhalation of indoor air pollutionin buildings constructed with PCB-containing materials in the 1940s to 1970s, and ofatmospheric pollution in large urban population centers with a deep-rooted industrial heritage,such as Chicago, Illinois (Ishikawa et al. 2007). Thus, while biomagnification of PCBs in thestaples of our diet, such as fish, provides a well-known, primary route for exposure to highermolecular weight PCBs, volatile, lower chlorianted PCB congeners represent a largelyunknown, and unavoidable, inhalation hazard (Hu et al. 2008).

4-chlorobiphenyl (PCB3) is a major constituent of Aroclor 1221, a predominant semi-volatilecongener in indoor air, and is released as a byproduct in certain industrial processes (Davis2002; Ishikawa et al. 2007). PCB3 is metabolized by cytochromes P-450 to electrophilic areneoxide metabolites, and also to dihydroxy species which can be oxidized to quinones (McLeanet al. 1996). Notably, male hepatic microsomes were found to be superior to female microsomesfor this bioactivation (McLean et al. 1996). Bioactivation is presumed vital to PCB3 toxicityand manifestation of genotoxic effects (Ludewig et al. 2008). In fact, employing severaldifferent in vitro genotoxicity assays PCB3 itself was found to be inactive, whereas several ofits metabolites were genotoxic in one or several of these assays (Zettner et al. 2007).

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Gender-based differential toxicity of xenobiotics is a well-established phenomenon acrossmultiple rodent species (Kobliakov et al. 1991). For example, Sprague-Dawley female ratsoverall are more sensitive to the development of cancers from xenobiotic exposure than theirmale counterparts (Brown et al. 2007). Fischer 344 rats, the genetic background employed forthe current study, demonstrate the opposite effect whereby female rats seem more resistant tohepatocarcinogenic effects of various polyaromatic compounds than males (Yang et al.2002).

Previous findings show that PCB3 is genotoxic in vivo, inducing a significant increase in LacImutations in male F344 BigBlue rats (Lehmann et al. 2007). The present study examinesgenotoxicity, pathology, gene expression and organ specific effects of PCB3 in female BigBlueF344 transgenic rats. In particular, we examine the differential toxicity of the study compoundsin male rats versus female rats. Our results show that although a weak trend towardsgenotoxicity is visible in the liver of female rats, it does not reach significance as in the malerat liver and we suggest possible explanations.

2. Materials and Methods2.1. Chemicals and Reagents

PCB3 and 4′-OH-PCB3 were synthesized and purified as described (Espandiari et al. 2003;Tampal et al. 2002). 3-Methylcholanthrene (Cat # M-6501) was obtained from Sigma, Inc. (StLouis, MO) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) from ResearchProducts International (Prospect, IL). Recoverease® DNA isolation kits and lambda phagetranspack kits were purchased from Stratagene, Inc. (La Jolla, CA). All other reagents wereobtained from Fischer Scientific (Pittsburgh, PA), if not indicated otherwise.

2.2. Animals and Treatment ProtocolThirty-two F344 BigBlue® rats (16 each gender), homozygous for a lacI transgene, werepurchased from Stratagene (La Jolla, CA) via Taconic Laboratories, (Germantown, NY) atpostnatal day (PND) 30. The animals were maintained on a 12-hr light/dark cycle and providedwith a standard 7013-NIH-13 Modified Open Formula Rat diet and water ad libitum. Animalsof each gender were randomly distributed into 4 groups of 4 animals each after one week ofacclimatization. A group size of n = 4 was chosen following the examples in the literature(Chen et al. 2005; Rihn et al. 2000). Weekly i.p. injections were administered to each animalon PND 37, 44, 51 and 58 consisting of the test compounds in corn oil or corn oil vehicle alone.Male and female animals were exposed concurrently, with the same preparation of studycompounds. Animals were treated at the following doses: 298 μmol (80 mg)/kg bodyweight(BW) 3-MC (positive control), 600 μmol (113 mg)/kg BW PCB3, 400 μmol (82 mg)/kg BW4′-OH-PCB3 or 5 ml/kg BW corn oil alone (negative control). Dosage of PCB3 and itsmetabolite were selected based on previous studies (Espandiari et al. 2004), the dose of 3-MCwas adopted from Rihn et al., 2000. Animals were monitored on a daily basis and weighedtwice per week during the exposure period and sacrificed on PND75, giving a total fixationtime of 38 days from first injection. The experiments detailed in this paper were approved bythe University of Iowa Institutional Animal Care and Use Committee.

2.3. Organ collection, sample preparation, and histological analysisAnimals were euthanized by CO2 asphyxiation followed by cervical dislocation seventeen days(PND 75) after the final injection. Livers were excised, weighed, and immediately a smalltissue sample was excised from the central portion of the large liver for mutation analysis, snapfrozen in liquid N2, and stored at -80°C for analysis.. Other organs were removed and weighed,and all organs were stored at -80°C. Organ weights were normalized to total body weight ofthe animal and depicted in the figures below as grams of organ weight per 100 grams total

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body weight. Frozen tissue samples were used to prepare four micron sections which werestained with hematoxylin/eosin, and examined using an Olympus BX40 light microscope forhistological anomalities.

2.4. Mutant Frequency AnalysisMutant analysis was performed in a blocked manner to control for variability, with each cycleof DNA extraction, packaging, and plating containing one animal from each treatment groupfollowing the procedures outlined in the BigBlue® instruction manual supplied by Stratagene,Inc. (La Jolla, CA). Experimental procedures, including mutant selection verification criteria,and mutant frequency calculations were followed as previously described (Lehmann et al.2007). Briefly, genomic DNA was isolated from 50 mg of female liver tissue using theBigBlue® RecoverEase kit and packaged into a lambda phage with the Lambda PhageTranspack® kit. The activated phage was then used to infect E. coli, which were plated in anX-gal-containing top agar and incubated overnight, after which the plaque density wasdetermined and blue-colored mutants, indicative of a mutation in the LacI gene, were manuallypicked. Positive control mutants purchased from Stratagene were analyzed alongsideexperimental plates to ensure optimal selection conditions.

A total of over 100,000 plaque forming units (pfu) were obtained for each animal. Each platecontained approximately 12,500 pfu. Mutant frequency was determined by dividing the totalnumber of mutant (blue) plaques by the pfu for that treatment and expressed as mutants per106 pfu ± standard error.

2.5. LacI Gene SequencingMutant plaques identified during the first phase of plating were replated twice at lower densityto ensure the isolation of a single mutant. These single mutant clone phages were then processedand stored at -80°C for later DNA sequence analysis. The primers used for amplification ofthe lacI gene, chosen according to the manufacturer's instructions, were as follows:

5′-GTATTACCGCCATGCATACTAG-3′ (Forward PCR primer)

5′-CGTAATCATGGTCATAGCTG-3′ (Reverse PCR primer)

PCR amplification, characterization of products via agarose gel electrophoresis, and productpurification was performed as previously described (Lehmann et al. 2007). The amplificationproducts were used to sequence the entire lacI gene (1083 bp) in both directions with the helpof the forward and reverse PCR primer above and an additional primer set:

Primer #5: 5′-TCTGGTCGCATTGGGTC-3′

Primer #12: 5′AGAACTTAATGGGCCCG-3′

Sequences were determined by the University of Iowa DNA Facility using an AppliedBiosystems (Forester City, CA) automated capillary DNA sequencer Model 3700. LacIsequences that were found to have no mutation were eliminated from mutation frequencyanalysis. Mutational events, such as transition or transversion, were verified by matching themutant on both the forward and reverse strand sequence. If identical mutations were found inmore than one plaque from the same animal we assumed that it may be due to the same initialmutational event and counted it therefore as one mutation. The number of independentmutations divided by the pfu was calculated to obtain the mutation frequency per 106 pfu ±standard error.

2.6. Gene Expression AnalysisRNA was isolated from both male and female liver sections using a Qiagen Total RNA MiniKit. RNA concentration and purity was determined using A260/A280 spectrophotometric

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measurements. Pooled RNA from all 4 animals per group, 80 μg from each animal, wasanalyzed by the University of Wisconsin EDGE2 program following standard protocols, usingeither an EDGE (male rodents) or Agilent (female rodents) microarray chip, with allcomparisons made against the corn oil control pooled sample. Male array results are notdiscussed in this publication, since the use of different chips makes a direct comparison of theresults inappropriate.

2.6.1 Agilent Microarray—First strand cDNA was synthesized from 1 μg of total RNAusing a SuperScript Indirect cDNA labeling Kit (Invitrogen, Cergy-Pontoise, France) labeledwith cyanine 3-dCTP (Cy3) (Amersham Biosciences) and an in-house referece sample waslabeled with cyanine 5-dCTP (Cy5) (Amersham Biosciences). Specific activity and yield ofboth labeled samples were determined by utilizing the NanoDrop ND-1000 spectrophotometer.All hybridizations were performed using Agilent Whole Rat Genome Oligo Micrarrays(G4131F). Each array was hybridized with 200 ng of Cy3-labeled cDNA (sample) and 200 ngof Cy5-labeled cDNA (reference) using the in situ Hybridization Kit Plus protocol (Agilent,Santa Clara, CA). Hybridizations were performed for 17 h at 65 °C in a rotating hybridizationoven (Agilent Technologies). After hybridization, slides were scanned on an AgilentTechnologies Scanner G2505B (Agilent) at five micron resolution. Images were processed,extracted and normalized (LOWESS method) using Feature Extraction software version9.1.3.1 (Agilent) with the GE2-v5_95_Feb07 protocol.

2.6.2 Agilent Microarray Data Analysis—Expression data of treated and corn oil controlsamples versus reference historical controls were converted to treated samples versus corn oilcontrol samples using the R statistical computing environment (R Development Core Team2008). Individual data points that have p values representing signal quality smaller than 0.01in all samples were selected. These data were further filtered to select only genes that werechanged more than two folds in at least one of the three treatment groups. Hierachical clusteringwas then performed.(Dennis et al. 2003)Genes in each treatment group were consideredsignificantly changed if their p values are smaller than 0.0001 and fold changes bigger than 5compared to corn oil control. These gene lists were then uploaded to DAVID (Huang et al,2009).(Dennis et al. 2003) for functional clustering.

2.7. Statistical AnalysisStatistical analysis was conducted using the R statistical computing environment (RDevelopment Core Team 2008). Poisson regression was used for the comparison of themutation frequencies. ANOVA models were fit for various organ weights using treatment andgender as independent variables. Interaction between treatment and gender is included in theanalysis. P-values of these analyses are reported in Table 1 and Supplemental Material TableS2 and S3. Cariello et al was used to analyze mutation spectra across classes (Cariello et al.1994).

3. Results3.1. Mutant frequency and mutation frequency after sequencing in female liver tissue

The mutant frequency in the liver of female rats was determined by examining more than100,000 pfu per animal, resulting in no less than 495,000 pfu per four-animal test group (Table1). The mutant frequency per 106 plaques was 24.1 (control), 48.4 (3-MC), 30.3 (PCB3) and27.3 (4′-OH-PCB3). A total of 78 mutants were isolated and sequenced which resulted in theexclusion of 5 mutants due to duplication of a mutation in the same animal (2) and lack ofmutation in the sequence (3) (see Supplemental Material Table S1). The resulting correctedmutation frequencies ranged from 22.1 in the control to 43.5 in the 3-MC group (Table 1). Thevariation in mutation frequency among the four treatment groups is not significant (chi-square

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p-value 0.2425). Although more mutations were found with all three test compounds, neitherPCB3 (p=0.429) nor 4′-OH-PCB3 (p=0.581) increased the mutation frequency significantlycompared to control. Even the 2-fold increase in mutation frequency in the positive control 3-MC failed marginally to reach statistical significance (p=0.0687).

The spontaneous mutations in the control group were mostly transversions and transitions,followed by a small percentage of frameshift mutations (Table 2). Using data presented inTable 2, the statistical test described by Cariello et al. (1994) was used to test the mutationspectra across classes “Transition”, “Transversion”, “Insertion”, and “Deletion” among thefour treatment groups. This test has been implemented in a computer program available atauthor's website (http://www.ibiblio.org/pub/academic/biology/dna-mutations/hyperg/).Mutation spectra of treatment 3-MC is not different from Corn Oil (p = 0.846), nor PCB3 (p= 0.6545) and 4′-HO-PCB3 (p = 0.851). A comparison with the other groups shows that 3-MC-treatment increased the proportion of frameshift mutations, i.e. deletions and insertions.The expected number, based on mutation type proportions in solvent control animals wouldbe 5, whereas 9 (32% of all mutations) were observed. Similarly the percent of insertions inthe PCB3 and 4′-OH-PCB3 group was higher than in the control group and the PCB3 grouphad a higher percent of transition mutations. However, the small number of mutants isolateddue to low mutant frequencies, along with a large intra-group variability in mutations peranimal (Fig 1), limits meaningful interpretation of these data.

3.2. Body weight gainAs expected, gender had a large impact on weight gain throughout the experiment, regardlessof the treatment (p-values are 7.652e-06 for 3-MC, 9.344e-11 for PCB 3 and 9.281e-09 for 4′-OH-PCB-3). The average weight gain in the 39 days of study period was about 160g in maleand 81g in female control rats (Table 3). Neither PCB3 nor 4′-OH-PCB3 had a significanteffect on weight gain in either gender compared to the current control group. 3-MC reducedweight gain during the treatment period by 23% in females and by 43% in males. This genderdifference for 3-MC-effect on weight gain, where male animals were more highly impacted atthe same dose versus female animals, was significant (p < 0.005). As reported previously forthe males (Lehmann et al. 2007), the reduction in weight gain in 3-MC animals occurred duringthe last 2 weeks of treatment.

3.3. Organ weights3-MC had an influence on several organ weights in female rats (Fig. 2). Particularly the liver,lung, and spleen increased in mass by over 30%, compared to those organs in corn oil controls.This effect was not (liver, lung) or barely (spleen) visible in male rats. PCB3 and 4′-OH-PCB-3-treatment had no statistically significant impact on these organ weights. A combination ofgender and treatment with 3-MC caused significant numbers with the Anova Model Effect testfor liver (p=0.0037), lung (p=0.0339), and heart (p=0.032), as well as weight gain (p=0.00439)(Table S2).

3.4. HistologyLiver, lung, and spleen were analyzed for pathological changes. Table 4 describes the observedchanges for each individual animal in detail; Figure 4 shows some typical histological findings.

The livers of control males were normal; two females had mild steatosis and one had scatteredacidophilic bodies and minimal centrilobular lymphocytes, overall minor changes. Male PCB3livers were very similar to female control animals, however, all female PCB3 livers hadacidophilic bodies and two animals also showed patchy hepatocellular necrosis. Female 4′-OH-PCB3 animals had rare acidophilic bodies, but 3 of 4 animals had patchy hepatocellularnecrosis and chronic inflammation; in contrast, all four male 4′-OH-PCB3 livers showed

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dilated sinosoids, a pathological change that was only observed in this group. 3-MC producedthe most severe changes. In male livers, centrilobular necrosis, in some cases severe, was thedominat finding. The portal tracts were normal. In female rats 3-MC produced only scatteredfoci of necrosis, prominent Kupffer cells and focal perilobular lymphocytes in two of the fouranimals.

3.5. Gene ExpressionSeventyfive unique genes were significantly changed by 3-MC treatment, 86 by PCB3 and 157by 4OH-PCB3. Hierachical clustering (Figure S8) shows that the expression profile of PCB3bears the closest resemblance to that of 4′OH-PCB3 and that all treatment groups have a verydistinct profile from the corn oil control.

Even though the female rats were sacrificed seventeen days after the last treatment, the effectsof 3-MC were still evident in terms of the expression of the Ah receptor gene battery. On theother hand, PCB3 and 4OH-PCB3 don't appear to have exerted their toxicity through longlasting effects on this receptor. The actual gene lists for males and females can be found insupplemental materials (Tables S4 to S6).

The Ah-receptor agonist 3-MC increased CYP1Al, CYP1A2 and UGT1A2 gene expression524-, 58- and 13-fold respectively (Table S4); all three genes belong to the known AhR genebattery. Other genes related to xenobiotic metabolism include ephx1 (epoxide hydrolase 1, 9.9-fold up), mgst2 (microsomal glutathione S-transferase 2, 7.9-fold up), ptgs1 (prostaglandin-endoperoxide synthase 1, 27.8-fold up), hmox1 (heme oxygenase 1, 16.3-fold up), glrx1(glutaredoxin 1, 10.2-fold up), and akr7a3 (aldo-keto reductases 7a3, 16-fold up). Functionalclustering of the most significantly changed genes (Table S7) not only confirmed this responseby the group named “metabolism of xenobiotics by CYP450, benzene and derivative metabolicprocess”, but also further revealed systematic responses to this compound. For example, theclusters of “response to bacterium” and “regulation of binding, transcription and apoptosis”indicate a regulation of general immune and defense mechanisms. Genes that belong to thiscluster include smarcb1(SWI/SNF related, matrix associated, actin dependent regulator ofchromatin b1, 8.5-fold up) which binds p53 and negatively regulate cell proliferation, bcl10(B-cell CLL/lymphoma 10, 16.3-fold up) which binds to NF-kB and regulates apoptosis, gdnf(glial cell derived neurotrophic factor, 6.0-fold up) which has anti-apoptosis function, calca(calcitonin/calcitonin-related polypeptide a), csda (cold shock domain protein A), trib3(tribbles homolog 3) and hmox1 which are all up regulated and negatively regulatetranscription. The cluster of homeostatic process contains genes involved in calcium, pH, cellvolume, cell number, erythrocyte and redox homeostasis.

No CYP mRNA increase was seen in the liver of female PCB3-treated rats (Table S5). Generaldefense response functional clusters affected by PCB3 include “homeostatic process”,“response to extracellular stimulus” and others. The “mitochondrial lumen” cluster contains aprotein similar to peroxiredoxin 1 (LOC678748, 7.7-fold up), cps1 (carbamoyl-phosphatesynthetase 1, 6.7-fold up), lrrc59 (leucine rich repeat containing 59, 33.1-fold up) and mrpl23(mitochondrial ribosomal protein L23, 223.3-fold up). The cluster of “regulation of apoptosisand cell proliferation” contains peroxiredoxin 1, Jun (55.9 fold-up), hmox1 (5.8-fold up) whichis involved in induction of apoptosis, cfdp1 (craniofacial development protein 1, 6.2-fold up)which is anti-apoptotic, mbd2 (Methyl-CpG-binding protein, 6.9-fold up) which negativelyregulates transcription, kdr (kinase insert domain protein receptor, 5.5-fold up) and avpr1a(arginine vasopressin receptor 1A, 7.3-fold up) which positively regulate cell proliferation.

4′-OH-PCB3 treatment induced greater changes in mRNA levels of female liver compared tothe parent compound PCB3 examplified by both the number of genes affected and themagnitude of expression changes (Table S6). Cyp2j4, a constitutively expressed gene in lung,

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was up-regulated 8.8 fold while cyp2b2 was down-regulated almost 19 fold. Clustering analysisalso shows general defense response such as “response to organic substance” and “immuneeffector process”. The cluster of “apoptosis and its regulation” also exists in 4′-OH-PCB3treatement group. Genes of interest in this cluster are peroxiredoxin 1 (5.5-fold up), kdr (6.4fold up), rock 1 (Rho-associated coiled-coil containing protein kinase 1, 5-fold up) whichnegatively regulate neuron apoptosis, stat1 (signal transducer and activator of transcription 1,10.6-fold up) which plays a role in the induction of apoptosis, bnip3l (BCL2/adenovirus E1Binteracting protein 3-like, 5.8-fold up) which may be involved in regulation of apoptosis, theanti-apoptotic sh3glb1 (SH3-domain GRB2-like endophilin B1, 6.3 fold up), ei24 (etoposideinduced 2.4 mRNA, 28.1 fold down) which negatively regulate cell growth and inducesapoptosis, syvn1 (synovial apoptosis inhibitor 1, 26.7-fold up), pigt (phosphatidylinositolglycan anchor biosynthesis, class T, 39.4-fold down) which is responsible for neuron apoptosis,aqp2 (aquaporin 2, 6.6-fold up) which regulates water reabsorption and cul1 (cullin 1, 36.3-fold down) that positively regulates ubiquitin-protein ligase activity during mitotic cell cycle.

Besides shared functional clusters between PCB3 and its metabolite 4′OH-PCB3, bothtreatments up-regulated phase II enzyme sult1b1 (sulfotransforase 1b1) to a similar extent (7-fold), indicating xenobiotic metabolism response after exposure to these organic compounds.

4. DiscussionThe goal of this study was to evaluate the mutagenicity of PCB3 and a hydroxylated metabolite4′-OH-PCB3 in the livers of female rats using transgenic animals. This specific metabolite waschosen, since it is a major metabolite of PCB3 (McLean et al. 1996) and it was positive in themodified Solt-Farber Initiation Protocol (Espandiari et al. 2004). 3-MC was chosen as positivecontrol since it is a known carcinogen and shown to be a mutagen in the male BigBlue ratmodel (Rihn et al. 2000). We did not expect our female BigBlue animals to be less affectedthan males from 3-MC- and PCB-induced genotoxicity, as female rats had been more proneto the development of hepatocellular carcinoma after chronic treatment with commercial PCBmixtures (Mayes et al. 1998). However, results from the current study ran contrary to our initialhypothesis, indicating the importance of three likely contributing factors: 1) Mayes andcoworkers used Sprague-Dawley rats, while the current study's BigBlue rats are on a F344background, 2) In the Mayes study rats were fed diets spiked with commercial Aroclor mixtureswhich were composed mostly of higher chlorinated congeners, not a single, monochlorinatedPCB congener such as PCB3, and the route of exposure for the current study was via i.p.injection, 3) The current study examined what is thought to be the initial stage ofcarcinogenesis, DNA mutations, while the Mayes study's endpoint was tumors. In fact, an NCIstudy using F344 animals in the 1970's concluded that “under the conditions of this bioassay,Aroclor® 1254 was not carcinogenic in Fischer 344 rats; however, a high incidence ofhepatocellular proliferative lesions in both male and female rats was related to administrationof the chemical” (NTP, 1978). Careful analysis of our data and the literature now indicate thatgender differences as cause for differet susceptibility may be more common than expected.

4.1. Mutant and Mutation FrequencyIn contrast to male BigBlue rodents, the present study indicates that neither PCB3 nor itshydroxylated metabolite significantly increased the mutant or mutation frequency of theLac1 transgene in female BigBlue rats. The genotoxic effect of our positive control, 3-MC,was also attenuated compared to the male rodents, where an about 5-fold increase had beenseen (Lehmann et al. 2007). Nevertheless, the mutation frequency was approximately doubledin 3-MC exposed female rats.

Differential response to xenobiotic exposure based both on the strain, and on the gender withinone strain, was noted over thirty years ago (Kato 1974). Often, these differences are based on

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specific cytochrome P450 (CYP) isoforms expressed differentially between strains and genders(Kato and Yamazoe 1992; Larsen et al. 1994). Phase II metabolism, which is mostly involvedin detoxification of a phase I metabolite, can also differ greatly between the genders, and thishas been shown in specifically for F344 rats (Ganem and Jefcoate 1998). As bioactivation ofPCB3 is thought to be the critical process in the manifestation of its cancer initiating activity(Ludewig et al. 2008), differences in the biotransformation potentials of male liver versusfemale liver become of paramount importance for the interpretation of our results.Unfortunately, it was not feasible to undertake an analysis of metabolic products and gender-specific toxicokinetics of PCB3 during this study, which therefore remains a task for the future.

An exhaustive comparison of gender effects in our rat model is hampered by the fact that manyBigBlue studies use only male or female animals probably because of the costs and difficultyof this assay system. However, one recent study examining a carcinogen was performedsimultaneously with both male and female BigBlue rats: Yang and coworkers studied thegenotoxic potential of 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and foundthat male BigBlue rats had a higher mutation frequency in the kidneys than female rats (Yanget al. 2002). The data reported by Yang and coworkers are very similar to our data in terms ofmutant frequency in control animals of both genders, intra-group variability, and gender-basedchemical susceptibility, despite having assayed tissue from the kidney whereas our currentstudy examined liver tissue (Yang et al. 2002). Another interesting study used intact andovariectomized female BigBlue rats to study the effect of endogenous hormones, particularlyestrogens, on mutation induction in the liver by 7,12-dimethylbenz[a]anthracene (Chen et al.2005). Similar to our gender difference they observed significantly more mutations inovariectomized than in intact female livers, pointing towards an endogenous modulator, Theauthors speculate that the antioxidant activity of estrogen may play a role in this protection.

Other factors could also account for the differences seen in our female rodent study. Notably,our sample sizes were quite small, with only four animals per treatment group. While thissample size is quite normal and frequently used (for example in Rihn et al., 2000 and Chen etal., 2005) and was the same number of animals used in the Lehmann et al. (2007) study whichresulted in statistically significantly elevated mutant frequencies with 3-MC and PCB3, it wasnot sufficient in this study to provide statistical significance in part because one female controlanimal had five verified mutations. This greatly increased not only the overall mean for themutation frequency in the control group, but also magnified the standard deviation, a criticalparameter in the statistical analysis, even though the overall mutation frequency for the femalecontrol group did not exceed the general range of mutation frequencies for normal BigBluerats, which is 4-6 × 10-5. However, it has been stated earlier that mutational events should beevaluated at the DNA sequence level and not only by mutant number, because weak mutagensmay only induce few mutations but of a different type (Heddle et al., 2000).

4.2. Mutation Spectrum AnalysisMutation spectra data for our corn oil treatment were surprising. Most mutations isolated fromcontrol animals in in vivo mutagenicity studies in transgenic animal models have been reportedto be transitions, i.e. a purine or pyrimidine DNA base is replaced by another nucleoside of thesame class (Lambert et al. 2005). However, we found four transitions (36% of mutations) andfive transversions out of eleven total mutants recovered from 496,967 pfu in the corn oil controlanimals. The remaining two mutations were an insertion and a deletion. Although this percentof transitions is still within the range described in the literature, Thornton and coworkers found22.2% in males and 44% in females, it was lower than expected (Thornton et al. 2001. Lehmannand coworkers found mostly transitions after sequencing mutants from male solvent controlanimals done in parallel with the current study, particularly G:C → A:T transitions (Lehmannet al. 2007). This is in agreement with spontaneous deamination as a possible mechanism for

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these mutations in control animals (Ehrlich et al. 1990). Transversions are believed to bederived more by exogenous chemicals or reactive oxygen species (ROS) interacting with DNAnucleosides. Despite this ‘rule of thumb’, our current female BigBlue dataset displayed a highnumber of transversions and low number of transitions in female control animals. The exactmechanism and importance remains elusive.

Although no significant change in percentage of base mutations due to chemical treatmentcouldbe observed, there seem to be an increase in frameshift mutations (deletions andinsertions) due to chemical exposure, since this number increased from only 18% in the solventcontrols to 32% in 3-MC, 27% in PCB3, and 36% in 4′-OH-PCB3 animals. This indicates apossible shift in the mutation spectrum. Although the number of sequenced mutations is toosmall to make a definite statement these changes are in agreement with the finding in malelivers, where 19%, 27%, 26%, and 22% of mutations in control, 3-MC, PCB3, and 4′-OH-PCB3 animals, respectively were frameshift mutations (Lehmann et al. 2007). This change inmutation spectrum could indicate that the test compounds are weak mutagens, as suggested byHeddle and coworkers (Heddle et al., 2000).

4.3. Gene ExpressionWe attempted to gain better understanding of the effects of our test compounds on female liversby performing gene array studies.

Besides inducing genes in the AhR gene battery, exposure to the positive control 3MC causedthe female rats to change their gene expression profile in order to maintain homeostasis inmany aspects such as calcium, pH, cell volume, cell number, erythrocyte and redox status.Notably quite a few genes encoding antioxidant enzymes and proteins were highly up-regulatedincluding epoxide hydrolase 1, glutathione S transferase 2, prostaglandin endoperoxidesynthase 1, glutaredoxin 1 and aldo-keto reductase7a3. Such response could render the femalerats more resistant to 3MC-induced oxidative stress and/or endogenous stressors and henceprotect them from 3-MC's or other compounds mutagenic effect. Moreover, the apoptosiscluster indicated a trend toward repression of cell proliferation and induction of apoptosis,which could reduce the likelihood of fixation of DNA damage. These facts could all contributeto our observation that the positive control failed to cause mutations in these female SD rats.

PCB3 and 4′-OH-PCB3 also elicited dramatic changes in gene expression, albeit throughdifferent mechanisms from 3-MC, which is AhR-mediated. Microarray data from thesetreatments shared the functional cluster of apoptosis and cell proliferation, containing bothanti-apoptotic and apoptotic genes, most of which were up-regulated. Although it is impossibleto conclude at this point whether cells were going toward apoptosis or proliferation due to thesechanges, the presence of this cluster suggests adverse effects and disruption of homeostasiscaused by these chemicals. In addition, both treatements caused expression changes in fattyacid metabolism and in circulatory system. Despite the similarities of responses to these twocompounds, 4′OH-PCB3 appeared to have a more profound impact compared to the parentcompound PCB3, at least at the messenger level, judging by the number of significantlyaffected genes in each treatment group. This could be explained by the fact that the metaboliteis more bio-chemically active than the parent compound.

These findings from the microarray study await further verification since the rat RNA samplesin each treatment group were pooled into one sample for microarray hybridization due tofinancial constraints. This makes it impossible to perform statistical tests on the data, althoughthe pooling step was also designed to reduce data variation. Another point worth noticing isthat the fixation time required for mutation induction makes this experimental design, wheremutation frequency is the primary endpoint, less optimal for detecting changes in geneexpression. PCB3 is rapidly metabolized and excreted (Kania-Korwel et al.; McLean et al.

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1996). The animals in the current study were sacrificed seventeen days after the last injection,providing ample time for metabolism and clearance of PCB3 and for gene expression to returnto baseline levels. 3-MC, on the other hand, has a longer half-life in the body and can beexpected to persist longer and cause persistent changes in gene expression. It was reported that3MC persists in the liver for 2 weeks (Li et al. 1997). Also, ip injected polycyclic aromatichydrocarbons (PAHs) dissolved in corn oil were reported to sequester into the peritoneum fromwhere they were constantly released (van Delft et al. 1998). This could very well be themechanistic reason for the long-lasting effect of 3-MC on gene regulation.

4.4. Body and Organ WeightsMale transgenic rats did not continue to gain weight after the fourth injection of 3-MC(Lehmann et al. 2007). In contrast to this, 3-MC-treated female rats' overall body weight wasonly slightly decreased in comparison to controls. This was the first sign of considerablegender-differences in these studies. The largest effect, however, was seen in the change in liverweight (Figure 2). Transgenic Fischer 344 female rats greatly increased their liver weight inresponse to 3-MC, and a small increase was even seen after PCB3-exposure. Compared to maleBigBlue rats, that lost gross liver weight in response to 3-MC, these changes are quite dramatic.Not only did female livers enlarge in response to 3-MC while male livers decreased in weight,but females also fared much better in regards to body weight gain than males, and thereforethere liver-to-body ratio increased even more dramatically, their livers nearly doubled in weightper grams of body weight compared to corn oil treated rats. The increased weight seen in thefemale liver and concomitant maintenance of growth through four 3-MC injections highlightsthe female gender's greater ability to defend against this xenobiotic insult compared to maleBigBlue rats administered an identical dose.

While only correlative, these results suggest that an adaptive response in female animals couldaccount for the lower mutation frequency compared to male F344 rats seen in our study. EvenPCB3-treatment slightly increased liver weights in female rats, producing significantly largerlivers in treated animals than control animals. The larger organs could result in a largerdistribution volume for the xenobiotic compounds, thereby lowering their toxic impact as isclearly visible in the sustained growth curves of female 3-MC rats compared to males. Theenourmosly enlarged liver would also decrease the number of mutants if the mutation was alate event. Not only livers were affected. 3-MC also increased spleen weights and decreasedthe thymus and adrenal gland weights relative to controls in females. No such changes wereobserved in male 3-MC animals. Clearly male and female F344 rats react very differently atboth the macroscopic and microscopic level to the toxic insult of 3-MC.

4.5. HistologyThe histological findings suggest that overt toxicity was not involved in any changes seen inthe treatment groups. Nevertheless some interesting observations were made. The leastpathology of all chemical treatment goups was seen in animals exposed to PCB3 in bothgenders, reflecting the relatively small changes in organ weight and gene transcription. 3-MCtreatment induced some signs of acute toxicity, including lymphocyte infiltration, acuteinflammation, and necrosis, and these changes were more pronounced in male rats than infemales, which is in agreement with the resistance against toxicity of females in this study.

4′-OH-PCB3 treatment caused pathological changes including chronic inflammation andpatchy necrosis in female rats. The gene expression changes seen with 4′-OH-PCB3 includedtranscripts involved in leukocyte differentiation (the cluster of differentiation, or CD proteins)and function (LAMP3), indicating the presence of these immune cells in response to treatmentwith hydroxylated PCB3. All four male livers of 4′-OH-PCB3 rats had dilated sinosoids, achange that cannot be explained at this time.

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Female total lung weights in 3-MC-treated rats markedly increased resulting in a more thandoubling of the relative lung weight. It could be speculated that the ARDS (acute respiratorydistress syndrome), which was observed only in females, may be the cause of the increase inthe weight of lungs. In 3-MC males the total lung weights decreased less than the body weightresulting in an increase in the relative lung weight. No histological abnormalities were seen inthe male 3-MC-lungs. Otherwise lung pathology was indiscriminate for treatment and gender.The spleens had no overt changes with the exception of 4′-OH-PCB3-treated animals where areduced mantle of the white pulp was seen in both genders. The significance of this change isnot known at this time.

Thus the histological data support the observation of gender differences in susceptibility to 3-MC induced damage and also provide first interesting indications that the metabolite of PCB3may have unknown effects in the liver and spleen of animals.

4.6. ConclusionsThe current study presents data indicating that female transgenic F344 rodents are less sensitiveto increases in mutation frequency of the liver caused by i.p. administration of 3-MC, PCB3and 4′-OH-PCB3. Differences in gene expression of female rodents compared to male rodentsmay be responsible for this difference in sensitivity. Organ weights also responded to PCB and3-MC treatments in a dichotomous manner according to gender. Female F344 have previouslybeen shown to be more resistant to various toxic agents than their male counterparts, and ourcurrent investigation supports these previous findings (Espandiari et al. 2003; Larsen et al.1994; Yang et al. 2002). It should be noted that changes in female gene expression werecalculated by comparison to female corn oil controls only, and comparison to male geneexpression was hampered due to the different microchips used. Basal and induced geneexpression patterns in female versus male F344 rats might provide more insight into possiblymechanisms causing gender differences, including already observed gender differences in CYPregulation in F344 rats (Ikegwuonu et al. 1996; Larsen et al. 1994). It should be mentioned thatrecent gene expression studies with several PCB congeners found profound congener specificeffects for males and femal mice, with by far more genes affected in female mice compared tomales and only a very small overlap in affected genes between both genders (Heimer et al, oralpresentation at the 11 International PCB Workshop in Visby, Sweden, May 30 – June 2, 2010;www.pcb-workshop-2010.se; see PCB Workshop Portal: http://www.pcbworkshop.org/ formore informationn about all PCB workshops).

Evidence is accumulating that some lower chlorinated biphenyls, especially PCB3, possessgenotoxic activity, most likely through metabolic bioactivation pathways (Espandiari et al.2003; Espandiari et al. 2004; Lehmann et al. 2007; Zettner et al. 2007). While these effects arenot always consistent and dramatic, as shown with our inconclusive results with female rats,research should continue in this arena. The higher mutant frequency in mlaes on the one handand the pronounced gene regulation changes in females on the other may suggest that DNAdamage may be a more important factor for hepatocarcinogenesis of these compounds in maleswhereas promotion should be considered in females. This possibility as well points towards aneed for gender-related research in toxicology.

Since exposure to lower chlorinated biphenyls may occur through different routes of exposure,particulalry inhalation, top priority should be placed on studies that more closely mimicenvironmental human exposures and the lung as possible target organ. Finally, it has beenestablished that PCB3 exposure in animal and cell culture models results in gene mutationsand preneoplastic foci, but the progression of these effects is poorly understood and needsfurther studies.

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http://www.pcb-workshop-2010.se

http://www.pcbworkshop.org/

Our finding about different sensitivity of male and female rats to mutagenic effects of our testcompounds highlight the importance of gender-influenced biotransformation and response inPCB-mediated genotoxicity and initiation processes and the need to understand and considerthese factors if we want to perform accurate risk assessment.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThe authors express their gratitude to Dr. Hans Lehmler for providing the PCBs and to the University of Iowa DNAfacility for mutant sequencing. We also thank Dr. Chris Bradfield and the EDGE project at the University of Wisconsinfor microarray analysis. This publication was made possible by grant number P42 ES 013661 from the NationalInstitute of Environmental Health Sciences (NIEHS). Funds were also available from P30 ES 05605. The contents ofthis publication are solely the responsibility of the authors and do not necessarily represent the official views of theNIEHS.

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Figure 1. Female Liver Mutation FrequencyVerified mutation frequency in female BigBlue livers per 106 plaque forming units. Althoughthe mutation frequencies of the chemical treatments were increased, these increases did notreach statistical significance.

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Figure 2. Relative Organ WeightOrgan weights are depicted as % of body weight. * statistically significantly inceasd comparedto solvent control, P < 0.05.

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Figure 3. Histology of the LiverDepicted are liver areas and structures (central vein, portal tract) as well as pathological changesobserved in treatment groups.

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Tabl

e 1

Fem

ale

Liv

er M

utan

t Fre

quen

cy

Tre

atm

ent g

roup

Tot

al n

umbe

r of

mut

ant

colo

nies

Tot

al n

umbe

r of

pla

ques

Mut

ants

per

106 p

laqu

esN

umbe

r of

Mut

atio

ns(c

orre

cted

)M

utat

ion

Freq

uenc

y(M

F) ±

SE

(cor

rect

ed)

P va

lues

of M

F(c

orre

cted

)

Cor

n O

il12

496,

967

24.1

1122

.1 ±

12.1

3-M

C32

661,

818

48.4

2843

.5 ±

14.

80.

0687

PCB

315

495,

039

30.3

1530

.3 ±

17.

90.

4290

4′-O

H-P

CB

319

696,

607

27.3

1927

.2 ±

6.6

0.58

10

Tot

al78

2,35

0,43

173


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Table 2Female liver type of mutations

Distinct Mutations (% of total)

Mutational Event Corn oil 3-MC PCB3 4′-HO-PCB3

Transitions 4 (36) 9 (32) 8 (53) 6 (35)

Transversions 5 (45) 10 (36) 3 (20) 5 (29)

Insertions 1 (9) 4 (14) 3 (20) 4 (24)

Deletions 1 (9) 5 (18) 1 (7) 2 (12)

Total Mutations 11 28 15 19


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Table 3Weight Gain (average ± StDev) in gram

Gender control 3-MC PCB3 4′-OH-PCB3

Male 159.7 ± 7.63 90.4 ± 25.6* 146.1 ± 7.99 129.6 ± 17.50

Female 80.7 ± 7.42 61.8 ± 7.78*,# 79.6 ± 4.51 85.3 ± 4.13

*Statistically significantly different from the control,

#significantly different from males


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Table 4Organ Pathology (animal 1 – 4 in each group)

Treatmentz Male Female

SPLEEN

Controls No abnormality No abnormality

3-MC No abnormality No abnormality

PCB3 No abnormality No abnormality

4′-OH-PCB3 1: No abnormality2 + 3: Reduced mantle with focally increased neutrophils.4: Reduced mantle with focally increased neutrophils withred pulp congestion.

1 – 4: Reduced mantle

LUNG

Controls 1: One focus of acute inflammation2: Focal acute inflammation with focal increase inperivascular lymphocytes.3 + 4: Minimal perivascular lymphocytes.

1: Patchy acute inflammation with increase in perivascularlymphocytes.2: Mild increase in intra-alveolar macrophages and fluid.3: Patchy acute inflammation with increase in perivascularlymphocytes.4: No abnormality.

3-MC 1: No abnormality2 + 3: Focal perivascular lymphocytes.4: Focal perivascular lymphocytes with increased intra-alveolar macrophages.

1: Bronchopneumonia with what looks like early ARDS.Pulmonary edema2: Bronchopneumonia with what looks like early ARDS more sothan #1. Pulmonary edema.3: Focal acute inflammation with increased intra-alveolarmacrophages4: No abnormality.

PCB3 1: Increased perivascular lymphocytes2: Peribronchial lymphocytes3: Peribronchial lymphocytes and increased intra-alveolarmacrophages.4: Perivascular lymphocytes.

1: Focal acute inflammation2: Minimal acute inflammation with areas of atelectasis3: Minimal acute inflammation4: Patchy bronchopneumonia with early ARDS. Perivascularlymphocytes increased.

4′-OH-PCB3 1 + 2: No abnormality.3 + 4: Minimal perivascular lymphocytes.

1: Minimal acute inflammation2: Almost normal with focal perivascular lymphocytes.3: No abnormality.4: Patchy bronchopneumonia with early ARDS. Perivascularlymphocytes increased

LIVER

Controls 1, 2, and 4: normal architecture.3: normal architecture with rare lymphoid aggregates.

1: Scattered acidophil bodies with minimal centrilobularlymphocytes2 + 3: Mild steatosis4: No abnormality

3-MC 1: centrilobular necrosis, acidophil bodies and Kupffer cellclusters. Sinusoids are dilated and there are clusters oflymphocytes in the parenchyma. The portal tracts areunremarkable.2: severe hepatocellular necrosis with sparing of theperilobular region. Abundant neutrophils at rim and withinnecrotic foci. Lymphocytes within the necrosis. Portaltracts well preserved with rare foci of piecemeal necrosis.3: centrilobular necrosis with areas of panlobular necrosis.Neutrophils and lymphocytes associated with the necrosis.Portal tracts OK.4: milder disease than above with focal centrilobularnecrosis. Acidophil bodies present with patchylymphocytes. Focal piecemeal necrosis.

1: Scattered acidophil bodies with minimal centrilobularlymphocytes2: Mild steatosis, patchy acute inflammation with acidophil bodiesand focal increase in centrilobular lymphocytes.3: Scattered foci of hepatocellular necrosis with acuteinflammation. Kupffer cells prominent. Focal perilobularlymphocytes.4: Scattered foci of hepatocellular necrosis with acuteinflammation. Kupffer cells prominent. Focal perilobularlymphocytes.

PCB3 1, 3, and 4: minimal changes with small clusters oflymphocytes. Rare acidophil body.2: as for #1 but with no lymphocytes.

1 + 2: Scattered acidophilic bodies3: Centrilobular acidophilic bodies and patchy hepatocellularnecrosis.4: Centrilobular acidophilic bodies and patchy hepatocellularnecrosis.


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Jacobus et al.

Treatmentz Male Female

4′-OH-PCB3 1: dilated sinusoids, rare acidophil body. No obviousinflammation.2, 3, and 4: dilated sinusoids,. Focal lymphocyte clusterwith rare acidophil body. Minimal portal tractinflammation.

1: Rare acidophilic bodies with patchy hepatocellular necrosis andchronic inflammation.2 + 3: Rare acidophilic bodies with patchy hepatocellular necrosisand chronic inflammation.4: Scattered acidophilic bodies with dilated sinusoids.

Pathology of liver and lung in male rats have been previously reported (Lehmann et al. 2007; Maddox et al. 2008) and are included here to allow foreasy comparison with female pathology.


3-Methylcholanthrene (3-MC) and 4-chlorobiphenyl (PCB3) genotoxicity is gender-related in Fischer...

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Transcript of 3-Methylcholanthrene (3-MC) and 4-chlorobiphenyl (PCB3) genotoxicity is gender-related in Fischer...