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Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants with myriad biological effects, including carcinogenicity. We present data showing gender-specific genotoxicity in Fischer 344 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 the genotoxic effects of the test compounds compared to their male counterparts. This difference is further highlighted through our examination of gene expression, organ-specific weight changes, and tissue morphology. The purpose of the present study was to explores the complex and multifaceted issues of lower molecular weight PCBs as initiators of carcinogenesis, by examining the mutagenicity of PCB3, a hydroxylated metabolite (4′-OH-PCB3), and 3-methylcholanthrene (3-MC, positive control) in a transgenic rodent model. Previous findings indicated that PCB3 is mutagenic in the liver of male BigBlue transgenic rats under identical exposure conditions. We expected that female rats would be equally, if not more sensitive than male rats, since a 2-year carcinogenesis bioassay with Sprague-Dawley rats and commercial PCB mixtures reported much higher liver cancer rates in female than in male rats. The current study, however, revealed a similar trend in the mutation frequencies across all four treatment groups in females as reported previously in males, but increased variability among animals within each group and a lower overall effect, led to non significant differences in mutation frequencies. A closer analysis of the possible reasons for this negative result using microarray, organ weight and histology data comparisons shows that female Fischer 344 rats 1) had a higher baseline mutation frequency in the corn oil control group and greater variability than male rats; 2) responded with robust gene expression changes, which may also play a role in our observation of 3) highly increased liver, spleen, and lung weight in 3-MC and PCB3-treated animals and thus changed distribution and kinetics of the test compounds. Our analysis indicates that female transgenic BigBlue Fischer 344 rats are more resistant to PCB3 and 3-MC genotoxicity compared to their male counterparts.
Modern society has inherited a global legacy of persistent pollution with polychlorinated biphenyls (PCBS) from their industrial and commercial use. PCBs, a class of 209 indiviudal congeners, are classified as probable human carcinogens (ATSDR 2000). A variety of human epidemiology studies have concluded that PCBs may be linked to the formation of tumors at various sites. For example, Engel and colleagues found a statistically significant trend of higher odds ratios for non-Hodgkin lymphoma with higher total serum PCB-levels in three different populations (Engel et al. 2007; Rothman et al. 1997). Recent reviews of the epidemiologic literature 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 invidual PCB congeners have differing modes of action, it seems plausible that a variety of tumor types could arise from exposure to various congeners, or their metabolites.
The evidence for carcinogenicity in rodents is much clearer. Using the Sprague Dawley rat strain a comprehensive chronic toxicity and carcinogenicity study analyzed the effects of four different commercial PCB mixtures (Aroclor 1016, 1242, 1254, and 1260) and multiple dietary concentrations, ranging from 25 to 200 ppm, during 24 months of exposure. In males rats statistically significant increases in hepatocellular carcinomas was noted only for the higher-chlorinated mixture Aroclor 1260, while all four commercial products caused an elevated incidence of hepatocellular carcinomas in female rats. It should be noted that Aroclor 1016 averages only three chlorines per biphenyl. These data indicate that commercial mixtures of chlorinated 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 lower chlorinated PCBs.
Exposure to lower molecular weight PCBs occurs mainly via inhalation of indoor air pollution in buildings constructed with PCB-containing materials in the 1940s to 1970s, and of atmospheric 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 the staples of our diet, such as fish, provides a well-known, primary route for exposure to higher molecular weight PCBs, volatile, lower chlorianted PCB congeners represent a largely unknown, and unavoidable, inhalation hazard (Hu et al. 2008).
4-chlorobiphenyl (PCB3) is a major constituent of Aroclor 1221, a predominant semi-volatile congener in indoor air, and is released as a byproduct in certain industrial processes (Davis 2002; Ishikawa et al. 2007). PCB3 is metabolized by cytochromes P-450 to electrophilic arene oxide metabolites, and also to dihydroxy species which can be oxidized to quinones (McLean et al. 1996). Notably, male hepatic microsomes were found to be superior to female microsomes for this bioactivation (McLean et al. 1996). Bioactivation is presumed vital to PCB3 toxicity and manifestation of genotoxic effects (Ludewig et al. 2008). In fact, employing several different in vitro genotoxicity assays PCB3 itself was found to be inactive, whereas several of its metabolites were genotoxic in one or several of these assays (Zettner et al. 2007).
Gender-based differential toxicity of xenobiotics is a well-established phenomenon across multiple rodent species (Kobliakov et al. 1991). For example, Sprague-Dawley female rats overall are more sensitive to the development of cancers from xenobiotic exposure than their male counterparts (Brown et al. 2007). Fischer 344 rats, the genetic background employed for the current study, demonstrate the opposite effect whereby female rats seem more resistant to hepatocarcinogenic 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 LacI mutations in male F344 BigBlue rats (Lehmann et al. 2007). The present study examines genotoxicity, pathology, gene expression and organ specific effects of PCB3 in female BigBlue F344 transgenic rats. In particular, we examine the differential toxicity of the study compounds in male rats versus female rats. Our results show that although a weak trend towards genotoxicity is visible in the liver of female rats, it does not reach significance as in the male rat liver and we suggest possible explanations.
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. (St Louis, MO) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) from Research Products International (Prospect, IL). Recoverease® DNA isolation kits and lambda phage transpack kits were purchased from Stratagene, Inc. (La Jolla, CA). All other reagents were obtained from Fischer Scientific (Pittsburgh, PA), if not indicated otherwise.
Thirty-two F344 BigBlue® rats (16 each gender), homozygous for a lacI transgene, were purchased from Stratagene (La Jolla, CA) via Taconic Laboratories, (Germantown, NY) at postnatal day (PND) 30. The animals were maintained on a 12-hr light/dark cycle and provided with a standard 7013-NIH-13 Modified Open Formula Rat diet and water ad libitum. Animals of each gender were randomly distributed into 4 groups of 4 animals each after one week of acclimatization. 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 animal on 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 study compounds. 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 BW 4′-OH-PCB3 or 5 ml/kg BW corn oil alone (negative control). Dosage of PCB3 and its metabolite were selected based on previous studies (Espandiari et al. 2004), the dose of 3-MC was adopted from Rihn et al., 2000. Animals were monitored on a daily basis and weighed twice per week during the exposure period and sacrificed on PND75, giving a total fixation time of 38 days from first injection. The experiments detailed in this paper were approved by the University of Iowa Institutional Animal Care and Use Committee.
Animals were euthanized by CO2 asphyxiation followed by cervical dislocation seventeen days (PND 75) after the final injection. Livers were excised, weighed, and immediately a small tissue sample was excised from the central portion of the large liver for mutation analysis, snap frozen 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 of the animal and depicted in the figures below as grams of organ weight per 100 grams total body weight. Frozen tissue samples were used to prepare four micron sections which were stained with hematoxylin/eosin, and examined using an Olympus BX40 light microscope for histological anomalities.
Mutant analysis was performed in a blocked manner to control for variability, with each cycle of DNA extraction, packaging, and plating containing one animal from each treatment group following 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 the BigBlue® RecoverEase kit and packaged into a lambda phage with the Lambda Phage Transpack® kit. The activated phage was then used to infect E. coli, which were plated in an X-gal-containing top agar and incubated overnight, after which the plaque density was determined and blue-colored mutants, indicative of a mutation in the LacI gene, were manually picked. Positive control mutants purchased from Stratagene were analyzed alongside experimental plates to ensure optimal selection conditions.
A total of over 100,000 plaque forming units (pfu) were obtained for each animal. Each plate contained approximately 12,500 pfu. Mutant frequency was determined by dividing the total number of mutant (blue) plaques by the pfu for that treatment and expressed as mutants per 106 pfu ± standard error.
Mutant plaques identified during the first phase of plating were replated twice at lower density to ensure the isolation of a single mutant. These single mutant clone phages were then processed and stored at -80°C for later DNA sequence analysis. The primers used for amplification of the lacI gene, chosen according to the manufacturer's instructions, were as follows:
PCR amplification, characterization of products via agarose gel electrophoresis, and product purification was performed as previously described (Lehmann et al. 2007). The amplification products were used to sequence the entire lacI gene (1083 bp) in both directions with the help of the forward and reverse PCR primer above and an additional primer set:
Sequences were determined by the University of Iowa DNA Facility using an Applied Biosystems (Forester City, CA) automated capillary DNA sequencer Model 3700. LacI sequences that were found to have no mutation were eliminated from mutation frequency analysis. Mutational events, such as transition or transversion, were verified by matching the mutant on both the forward and reverse strand sequence. If identical mutations were found in more than one plaque from the same animal we assumed that it may be due to the same initial mutational event and counted it therefore as one mutation. The number of independent mutations divided by the pfu was calculated to obtain the mutation frequency per 106 pfu ± standard error.
RNA was isolated from both male and female liver sections using a Qiagen Total RNA Mini Kit. RNA concentration and purity was determined using A260/A280 spectrophotometric measurements. Pooled RNA from all 4 animals per group, 80 μg from each animal, was analyzed by the University of Wisconsin EDGE2 program following standard protocols, using either an EDGE (male rodents) or Agilent (female rodents) microarray chip, with all comparisons made against the corn oil control pooled sample. Male array results are not discussed in this publication, since the use of different chips makes a direct comparison of the results inappropriate.
First strand cDNA was synthesized from 1 μg of total RNA using a SuperScript Indirect cDNA labeling Kit (Invitrogen, Cergy-Pontoise, France) labeled with cyanine 3-dCTP (Cy3) (Amersham Biosciences) and an in-house referece sample was labeled with cyanine 5-dCTP (Cy5) (Amersham Biosciences). Specific activity and yield of both 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 ng of 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 hybridization oven (Agilent Technologies). After hybridization, slides were scanned on an Agilent Technologies Scanner G2505B (Agilent) at five micron resolution. Images were processed, extracted and normalized (LOWESS method) using Feature Extraction software version 18.104.22.168 (Agilent) with the GE2-v5_95_Feb07 protocol.
Expression data of treated and corn oil control samples versus reference historical controls were converted to treated samples versus corn oil control samples using the R statistical computing environment (R Development Core Team 2008). Individual data points that have p values representing signal quality smaller than 0.01 in all samples were selected. These data were further filtered to select only genes that were changed more than two folds in at least one of the three treatment groups. Hierachical clustering was then performed.(Dennis et al. 2003)Genes in each treatment group were considered significantly changed if their p values are smaller than 0.0001 and fold changes bigger than 5 compared to corn oil control. These gene lists were then uploaded to DAVID (Huang et al, 2009).(Dennis et al. 2003) for functional clustering.
Statistical analysis was conducted using the R statistical computing environment (R Development Core Team 2008). Poisson regression was used for the comparison of the mutation frequencies. ANOVA models were fit for various organ weights using treatment and gender as independent variables. Interaction between treatment and gender is included in the analysis. P-values of these analyses are reported in Table 1 and Supplemental Material Table S2 and S3. Cariello et al was used to analyze mutation spectra across classes (Cariello et al. 1994).
The mutant frequency in the liver of female rats was determined by examining more than 100,000 pfu per animal, resulting in no less than 495,000 pfu per four-animal test group (Table 1). The mutant frequency per 106 plaques was 24.1 (control), 48.4 (3-MC), 30.3 (PCB3) and 27.3 (4′-OH-PCB3). A total of 78 mutants were isolated and sequenced which resulted in the exclusion of 5 mutants due to duplication of a mutation in the same animal (2) and lack of mutation in the sequence (3) (see Supplemental Material Table S1). The resulting corrected mutation frequencies ranged from 22.1 in the control to 43.5 in the 3-MC group (Table 1). The variation in mutation frequency among the four treatment groups is not significant (chi-square p-value 0.2425). Although more mutations were found with all three test compounds, neither PCB3 (p=0.429) nor 4′-OH-PCB3 (p=0.581) increased the mutation frequency significantly compared 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 in Table 2, the statistical test described by Cariello et al. (1994) was used to test the mutation spectra across classes “Transition”, “Transversion”, “Insertion”, and “Deletion” among the four treatment groups. This test has been implemented in a computer program available at author'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 would be 5, whereas 9 (32% of all mutations) were observed. Similarly the percent of insertions in the PCB3 and 4′-OH-PCB3 group was higher than in the control group and the PCB3 group had a higher percent of transition mutations. However, the small number of mutants isolated due to low mutant frequencies, along with a large intra-group variability in mutations per animal (Fig 1), limits meaningful interpretation of these data.
As expected, gender had a large impact on weight gain throughout the experiment, regardless of 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 male and 81g in female control rats (Table 3). Neither PCB3 nor 4′-OH-PCB3 had a significant effect on weight gain in either gender compared to the current control group. 3-MC reduced weight gain during the treatment period by 23% in females and by 43% in males. This gender difference for 3-MC-effect on weight gain, where male animals were more highly impacted at the same dose versus female animals, was significant (p < 0.005). As reported previously for the males (Lehmann et al. 2007), the reduction in weight gain in 3-MC animals occurred during the last 2 weeks of treatment.
3-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 of gender and treatment with 3-MC caused significant numbers with the Anova Model Effect test for liver (p=0.0037), lung (p=0.0339), and heart (p=0.032), as well as weight gain (p=0.00439) (Table S2).
Liver, lung, and spleen were analyzed for pathological changes. Table 4 describes the observed changes 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 scattered acidophilic bodies and minimal centrilobular lymphocytes, overall minor changes. Male PCB3 livers were very similar to female control animals, however, all female PCB3 livers had acidophilic 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 hepatocellular necrosis and chronic inflammation; in contrast, all four male 4′-OH-PCB3 livers showed dilated sinosoids, a pathological change that was only observed in this group. 3-MC produced the most severe changes. In male livers, centrilobular necrosis, in some cases severe, was the dominat finding. The portal tracts were normal. In female rats 3-MC produced only scattered foci of necrosis, prominent Kupffer cells and focal perilobular lymphocytes in two of the four animals.
Seventyfive unique genes were significantly changed by 3-MC treatment, 86 by PCB3 and 157 by 4OH-PCB3. Hierachical clustering (Figure S8) shows that the expression profile of PCB3 bears the closest resemblance to that of 4′OH-PCB3 and that all treatment groups have a very distinct profile from the corn oil control.
Even though the female rats were sacrificed seventeen days after the last treatment, the effects of 3-MC were still evident in terms of the expression of the Ah receptor gene battery. On the other hand, PCB3 and 4OH-PCB3 don't appear to have exerted their toxicity through long lasting effects on this receptor. The actual gene lists for males and females can be found in supplemental materials (Tables S4 to S6).
The Ah-receptor agonist 3-MC increased CYP1Al, CYP1A2 and UGT1A2 gene expression 524-, 58- and 13-fold respectively (Table S4); all three genes belong to the known AhR gene battery. 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). Functional clustering of the most significantly changed genes (Table S7) not only confirmed this response by the group named “metabolism of xenobiotics by CYP450, benzene and derivative metabolic process”, but also further revealed systematic responses to this compound. For example, the clusters of “response to bacterium” and “regulation of binding, transcription and apoptosis” indicate a regulation of general immune and defense mechanisms. Genes that belong to this cluster include smarcb1(SWI/SNF related, matrix associated, actin dependent regulator of chromatin 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 regulate transcription. The cluster of homeostatic process contains genes involved in calcium, pH, cell volume, cell number, erythrocyte and redox homeostasis.
No CYP mRNA increase was seen in the liver of female PCB3-treated rats (Table S5). General defense response functional clusters affected by PCB3 include “homeostatic process”, “response to extracellular stimulus” and others. The “mitochondrial lumen” cluster contains a protein similar to peroxiredoxin 1 (LOC678748, 7.7-fold up), cps1 (carbamoyl-phosphate synthetase 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 apoptosis and cell proliferation” contains peroxiredoxin 1, Jun (55.9 fold-up), hmox1 (5.8-fold up) which is 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 negatively regulates 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 to the parent compound PCB3 examplified by both the number of genes affected and the magnitude of expression changes (Table S6). Cyp2j4, a constitutively expressed gene in lung, was up-regulated 8.8 fold while cyp2b2 was down-regulated almost 19 fold. Clustering analysis also shows general defense response such as “response to organic substance” and “immune effector process”. The cluster of “apoptosis and its regulation” also exists in 4′-OH-PCB3 treatement group. Genes of interest in this cluster are peroxiredoxin 1 (5.5-fold up), kdr (6.4 fold up), rock 1 (Rho-associated coiled-coil containing protein kinase 1, 5-fold up) which negatively 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 E1B interacting protein 3-like, 5.8-fold up) which may be involved in regulation of apoptosis, the anti-apoptotic sh3glb1 (SH3-domain GRB2-like endophilin B1, 6.3 fold up), ei24 (etoposide induced 2.4 mRNA, 28.1 fold down) which negatively regulate cell growth and induces apoptosis, syvn1 (synovial apoptosis inhibitor 1, 26.7-fold up), pigt (phosphatidylinositol glycan 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, both treatments up-regulated phase II enzyme sult1b1 (sulfotransforase 1b1) to a similar extent (7-fold), indicating xenobiotic metabolism response after exposure to these organic compounds.
The goal of this study was to evaluate the mutagenicity of PCB3 and a hydroxylated metabolite 4′-OH-PCB3 in the livers of female rats using transgenic animals. This specific metabolite was chosen, since it is a major metabolite of PCB3 (McLean et al. 1996) and it was positive in the modified Solt-Farber Initiation Protocol (Espandiari et al. 2004). 3-MC was chosen as positive control since it is a known carcinogen and shown to be a mutagen in the male BigBlue rat model (Rihn et al. 2000). We did not expect our female BigBlue animals to be less affected than males from 3-MC- and PCB-induced genotoxicity, as female rats had been more prone to the development of hepatocellular carcinoma after chronic treatment with commercial PCB mixtures (Mayes et al. 1998). However, results from the current study ran contrary to our initial hypothesis, indicating the importance of three likely contributing factors: 1) Mayes and coworkers used Sprague-Dawley rats, while the current study's BigBlue rats are on a F344 background, 2) In the Mayes study rats were fed diets spiked with commercial Aroclor mixtures which were composed mostly of higher chlorinated congeners, not a single, monochlorinated PCB 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 of carcinogenesis, DNA mutations, while the Mayes study's endpoint was tumors. In fact, an NCI study 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 of hepatocellular proliferative lesions in both male and female rats was related to administration of the chemical” (NTP, 1978). Careful analysis of our data and the literature now indicate that gender differences as cause for differet susceptibility may be more common than expected.
In contrast to male BigBlue rodents, the present study indicates that neither PCB3 nor its hydroxylated metabolite significantly increased the mutant or mutation frequency of the Lac1 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 been seen (Lehmann et al. 2007). Nevertheless, the mutation frequency was approximately doubled in 3-MC exposed female rats.
Differential response to xenobiotic exposure based both on the strain, and on the gender within one strain, was noted over thirty years ago (Kato 1974). Often, these differences are based on 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 involved in detoxification of a phase I metabolite, can also differ greatly between the genders, and this has been shown in specifically for F344 rats (Ganem and Jefcoate 1998). As bioactivation of PCB3 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 versus female 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 many BigBlue studies use only male or female animals probably because of the costs and difficulty of this assay system. However, one recent study examining a carcinogen was performed simultaneously with both male and female BigBlue rats: Yang and coworkers studied the genotoxic potential of 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and found that male BigBlue rats had a higher mutation frequency in the kidneys than female rats (Yang et al. 2002). The data reported by Yang and coworkers are very similar to our data in terms of mutant frequency in control animals of both genders, intra-group variability, and gender-based chemical susceptibility, despite having assayed tissue from the kidney whereas our current study examined liver tissue (Yang et al. 2002). Another interesting study used intact and ovariectomized female BigBlue rats to study the effect of endogenous hormones, particularly estrogens, 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 in ovariectomized than in intact female livers, pointing towards an endogenous modulator, The authors 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 this sample size is quite normal and frequently used (for example in Rihn et al., 2000 and Chen et al., 2005) and was the same number of animals used in the Lehmann et al. (2007) study which resulted in statistically significantly elevated mutant frequencies with 3-MC and PCB3, it was not sufficient in this study to provide statistical significance in part because one female control animal had five verified mutations. This greatly increased not only the overall mean for the mutation frequency in the control group, but also magnified the standard deviation, a critical parameter in the statistical analysis, even though the overall mutation frequency for the female control group did not exceed the general range of mutation frequencies for normal BigBlue rats, which is 4-6 × 10-5. However, it has been stated earlier that mutational events should be evaluated at the DNA sequence level and not only by mutant number, because weak mutagens may only induce few mutations but of a different type (Heddle et al., 2000).
Mutation spectra data for our corn oil treatment were surprising. Most mutations isolated from control animals in in vivo mutagenicity studies in transgenic animal models have been reported to be transitions, i.e. a purine or pyrimidine DNA base is replaced by another nucleoside of the same class (Lambert et al. 2005). However, we found four transitions (36% of mutations) and five transversions out of eleven total mutants recovered from 496,967 pfu in the corn oil control animals. The remaining two mutations were an insertion and a deletion. Although this percent of transitions is still within the range described in the literature, Thornton and coworkers found 22.2% in males and 44% in females, it was lower than expected (Thornton et al. 2001. Lehmann and coworkers found mostly transitions after sequencing mutants from male solvent control animals done in parallel with the current study, particularly G:C → A:T transitions (Lehmann et al. 2007). This is in agreement with spontaneous deamination as a possible mechanism for these mutations in control animals (Ehrlich et al. 1990). Transversions are believed to be derived more by exogenous chemicals or reactive oxygen species (ROS) interacting with DNA nucleosides. Despite this ‘rule of thumb’, our current female BigBlue dataset displayed a high number of transversions and low number of transitions in female control animals. The exact mechanism and importance remains elusive.
Although no significant change in percentage of base mutations due to chemical treatment couldbe observed, there seem to be an increase in frameshift mutations (deletions and insertions) due to chemical exposure, since this number increased from only 18% in the solvent controls to 32% in 3-MC, 27% in PCB3, and 36% in 4′-OH-PCB3 animals. This indicates a possible shift in the mutation spectrum. Although the number of sequenced mutations is too small to make a definite statement these changes are in agreement with the finding in male livers, 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 in mutation spectrum could indicate that the test compounds are weak mutagens, as suggested by Heddle and coworkers (Heddle et al., 2000).
We attempted to gain better understanding of the effects of our test compounds on female livers by performing gene array studies.
Besides inducing genes in the AhR gene battery, exposure to the positive control 3MC caused the female rats to change their gene expression profile in order to maintain homeostasis in many 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-regulated including epoxide hydrolase 1, glutathione S transferase 2, prostaglandin endoperoxide synthase 1, glutaredoxin 1 and aldo-keto reductase7a3. Such response could render the female rats more resistant to 3MC-induced oxidative stress and/or endogenous stressors and hence protect them from 3-MC's or other compounds mutagenic effect. Moreover, the apoptosis cluster 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 contribute to 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 through different mechanisms from 3-MC, which is AhR-mediated. Microarray data from these treatments shared the functional cluster of apoptosis and cell proliferation, containing both anti-apoptotic and apoptotic genes, most of which were up-regulated. Although it is impossible to conclude at this point whether cells were going toward apoptosis or proliferation due to these changes, the presence of this cluster suggests adverse effects and disruption of homeostasis caused by these chemicals. In addition, both treatements caused expression changes in fatty acid metabolism and in circulatory system. Despite the similarities of responses to these two compounds, 4′OH-PCB3 appeared to have a more profound impact compared to the parent compound PCB3, at least at the messenger level, judging by the number of significantly affected genes in each treatment group. This could be explained by the fact that the metabolite is more bio-chemically active than the parent compound.
These findings from the microarray study await further verification since the rat RNA samples in each treatment group were pooled into one sample for microarray hybridization due to financial constraints. This makes it impossible to perform statistical tests on the data, although the pooling step was also designed to reduce data variation. Another point worth noticing is that the fixation time required for mutation induction makes this experimental design, where mutation frequency is the primary endpoint, less optimal for detecting changes in gene expression. PCB3 is rapidly metabolized and excreted (Kania-Korwel et al.; McLean et al. 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 return to baseline levels. 3-MC, on the other hand, has a longer half-life in the body and can be expected to persist longer and cause persistent changes in gene expression. It was reported that 3MC persists in the liver for 2 weeks (Li et al. 1997). Also, ip injected polycyclic aromatic hydrocarbons (PAHs) dissolved in corn oil were reported to sequester into the peritoneum from where they were constantly released (van Delft et al. 1998). This could very well be the mechanistic reason for the long-lasting effect of 3-MC on gene regulation.
Male 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 was only slightly decreased in comparison to controls. This was the first sign of considerable gender-differences in these studies. The largest effect, however, was seen in the change in liver weight (Figure 2). Transgenic Fischer 344 female rats greatly increased their liver weight in response to 3-MC, and a small increase was even seen after PCB3-exposure. Compared to male BigBlue 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 therefore there liver-to-body ratio increased even more dramatically, their livers nearly doubled in weight per grams of body weight compared to corn oil treated rats. The increased weight seen in the female liver and concomitant maintenance of growth through four 3-MC injections highlights the female gender's greater ability to defend against this xenobiotic insult compared to male BigBlue rats administered an identical dose.
While only correlative, these results suggest that an adaptive response in female animals could account for the lower mutation frequency compared to male F344 rats seen in our study. Even PCB3-treatment slightly increased liver weights in female rats, producing significantly larger livers in treated animals than control animals. The larger organs could result in a larger distribution volume for the xenobiotic compounds, thereby lowering their toxic impact as is clearly visible in the sustained growth curves of female 3-MC rats compared to males. The enourmosly enlarged liver would also decrease the number of mutants if the mutation was a late event. Not only livers were affected. 3-MC also increased spleen weights and decreased the thymus and adrenal gland weights relative to controls in females. No such changes were observed in male 3-MC animals. Clearly male and female F344 rats react very differently at both the macroscopic and microscopic level to the toxic insult of 3-MC.
The histological findings suggest that overt toxicity was not involved in any changes seen in the treatment groups. Nevertheless some interesting observations were made. The least pathology of all chemical treatment goups was seen in animals exposed to PCB3 in both genders, reflecting the relatively small changes in organ weight and gene transcription. 3-MC treatment induced some signs of acute toxicity, including lymphocyte infiltration, acute inflammation, and necrosis, and these changes were more pronounced in male rats than in females, which is in agreement with the resistance against toxicity of females in this study.
4′-OH-PCB3 treatment caused pathological changes including chronic inflammation and patchy necrosis in female rats. The gene expression changes seen with 4′-OH-PCB3 included transcripts involved in leukocyte differentiation (the cluster of differentiation, or CD proteins) and function (LAMP3), indicating the presence of these immune cells in response to treatment with hydroxylated PCB3. All four male livers of 4′-OH-PCB3 rats had dilated sinosoids, a change that cannot be explained at this time.
Female total lung weights in 3-MC-treated rats markedly increased resulting in a more than doubling of the relative lung weight. It could be speculated that the ARDS (acute respiratory distress syndrome), which was observed only in females, may be the cause of the increase in the weight of lungs. In 3-MC males the total lung weights decreased less than the body weight resulting in an increase in the relative lung weight. No histological abnormalities were seen in the 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 a reduced mantle of the white pulp was seen in both genders. The significance of this change is not 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 PCB3 may have unknown effects in the liver and spleen of animals.
The current study presents data indicating that female transgenic F344 rodents are less sensitive to increases in mutation frequency of the liver caused by i.p. administration of 3-MC, PCB3 and 4′-OH-PCB3. Differences in gene expression of female rodents compared to male rodents may be responsible for this difference in sensitivity. Organ weights also responded to PCB and 3-MC treatments in a dichotomous manner according to gender. Female F344 have previously been shown to be more resistant to various toxic agents than their male counterparts, and our current 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 were calculated by comparison to female corn oil controls only, and comparison to male gene expression was hampered due to the different microchips used. Basal and induced gene expression patterns in female versus male F344 rats might provide more insight into possibly mechanisms causing gender differences, including already observed gender differences in CYP regulation in F344 rats (Ikegwuonu et al. 1996; Larsen et al. 1994). It should be mentioned that recent gene expression studies with several PCB congeners found profound congener specific effects for males and femal mice, with by far more genes affected in female mice compared to males and only a very small overlap in affected genes between both genders (Heimer et al, oral presentation 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/ for more informationn about all PCB workshops).
Evidence is accumulating that some lower chlorinated biphenyls, especially PCB3, possess genotoxic 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 are not 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 hand and the pronounced gene regulation changes in females on the other may suggest that DNA damage may be a more important factor for hepatocarcinogenesis of these compounds in males whereas promotion should be considered in females. This possibility as well points towards a need 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 mimic environmental human exposures and the lung as possible target organ. Finally, it has been established that PCB3 exposure in animal and cell culture models results in gene mutations and preneoplastic foci, but the progression of these effects is poorly understood and needs further studies.
Our finding about different sensitivity of male and female rats to mutagenic effects of our test compounds highlight the importance of gender-influenced biotransformation and response in PCB-mediated genotoxicity and initiation processes and the need to understand and consider these factors if we want to perform accurate risk assessment.
The authors express their gratitude to Dr. Hans Lehmler for providing the PCBs and to the University of Iowa DNA facility for mutant sequencing. We also thank Dr. Chris Bradfield and the EDGE project at the University of Wisconsin for microarray analysis. This publication was made possible by grant number P42 ES 013661 from the National Institute of Environmental Health Sciences (NIEHS). Funds were also available from P30 ES 05605. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS.
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