4-monochlorobiphenyl (pcb3) induces mutations in the livers of transgenic fisher 344 rats

4-Monochlorobiphenyl (PCB3) induces mutations

in the livers of transgenic Fisher 344 rats

Leane Lehmanna, Harald L. Eschb, Patricia A. Kirbyc, Larry W. Robertsonb,

Gabriele Ludewigb*

aUniversity of Karlsruhe (TH), Institute of Applied Biosciences, Section of Food Chemistry and

Toxicology, Kaiserstraße 12, D-76131 Karlsruhe, Germany

bThe University of Iowa, College of Public Health, Dept. of Occupational and Environmental Health

100 Oakdale Campus, Iowa City, IA 52242-5000, USA

cThe University of Iowa, Department of Pathology, Iowa City, IA 52242, USA

*Correspondence:

Dr. Gabriele Ludewig, The University of Iowa,

Dept. of Occupational and Environmental Health

100 Oakdale Campus #234 IREH,

Iowa City, IA 52242-5000

Tel.: +001-319-335 4650; fax +001-319-335 4290

E-mail address: [email protected]

Key Words: 4-monochlorobiphenyl, PCB, BigBlue®, LacI, mutation, Fischer 344 rats

The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]

Carcinogenesis Advance Access published August 31, 2006 by guest on A

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ABBREVIATIONS: A, adenine; ATP, adenosine triphosphate; bp, base pairs; BW, body

weight; EDTA, ethylenediamine tetraacetic acid; C, cytosine; CTP, cytidine triphosphate; G,

guanine; GMP, guanosine monophosphate; HPLC ESI MS/MS, high performance liquid

chromatography electron spray injector/mass spectrometry; lacI, lactose inhibitor; 3-MC, 3-

methylcholanthrene; OD, optical density; PCB, polychlorinated biphenyl, PCB3, 4-

monochlorobiphenyl; pfu, plaque forming unit; PCR, polymerase chain reaction; PND,

postnatal day; T, thymine; TE, Tris EDTA; X-gal, 5-Bromo-4-chloro-3-indolyl β-D-

galactopyranoside.

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ABSTRACT

4-Monochlorobiphenyl (PCB3) is found in small amounts in commercial PCB mixtures,

indoor and outdoor air, and in food. In contrast to highly chlorinated congeners that are more

resistant to metabolic attack, PCB3 is more readily converted by xenobiotic-metabolizing

enzymes to monohydroxy-PCBs and further to dihydroxy-metabolites, which can be oxidized

to quinones. Our recent studies demonstrated the initiating action of PCB3 in the livers of

male rats. Therefore we hypothesized that PCB3 and/or its metabolite(s) are mutagenic in rat

livers in vivo. To investigate the mutagenicity and the types of mutations generated by

PCB3, male Fischer 344 BigBlue® rats, transgenic for the lacI gene, were injected

intraperitoneally with PCB3 (600 µmol/kg), 4-hydroxy-PCB3 (4-HO-PCB3, 400 µmol/kg),

3-methylcholanthrene (3-MC, 300 µmol/kg, positive control), or corn oil (negative control)

once per week, for 4 weeks. Animals were killed 10 days after the last injection and the

mutant frequency of the liver lacI gene determined. 3-MC induced a 4-fold increase of the

mutant frequency of the lacI gene in the liver. The mutant frequency in PCB3-treated

animals was also significantly elevated. In contrast, 4-HO-PCB3 induced a non-significant

doubling of the mutant frequency. The mutation spectrum of solvent control mutants was

characterized by transitions, whereas in 3-MC-animals, transversion and frameshift mutations

predominated. The PCB3-induced mutation spectrum was similar to that of the 3-MC-

induced mutants. In contrast, the mutation spectrum of the 4-HO-PCB3 group hardly

differed from that of the control animals. This study demonstrates for the first time the

mutagenicity of a PCB in vivo.



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INTRODUCTION

Polychlorinated biphenyls (PCBs) were produced world wide from 1929 to the early

1980s. During this period, an estimated 2 million tons of commercial PCB mixtures were

produced, of which about 0.2 million tons remain in mobile environmental reservoirs [1].

Despite the production ban and heavily restricted use, PCBs can still be found even in the

most remote areas like polar regions and mountain lakes due to atmospheric transport and

precipitation [2-5]. Vaporization of PCBs from landfills [6,7], contaminated surface water [8-

10] and construction material for public buildings [11-15] are main sources for outdoor and

indoor air contamination. Human exposure to PCBs occurs to 90% via food, the inhalation

contributes the remaining 10% [1]. In some populations, however, inhalation exposure was

found to be the major source for daily PCB uptake [16].

Commercial PCBs are mixtures of the 209 possible congeners, which differ in the

number and position of chlorines bound to the biphenyl core. PCB congeners that accumulate

in the food chain are mainly tetra- to hepta-chlorinated, are stored preferably in fatty tissues,

and are only slowly metabolized. In contrast, airborne PCBs are lower chlorinated, and are

susceptible to metabolic attack. They occur in commercial PCB mixtures [17,18], in the

atmosphere [18], especially in cities, in buildings, and near waste sites [19]. They can also be

found in food, however in a lower proportion and more likely in/on food items like

vegetables, possibly through air deposition, in vegetable oils, and in dairy products [20,21].

Congener-specific analysis of indoor air in houses built on soil contaminated with Aroclor

1260, one of the highest chlorinated commercial PCB mixtures, showed that the congeners

measured in indoor air were mostly 4-monochlorobiphenyl (PCB3, Fig. 1) and 2-

monochlorobiphenyl (PCB1) [22].

PCBs have repeatedly been shown to be complete carcinogens in rodents [23,24]. In

addition, commercial PCB mixtures as well as individual congeners have tumor promoting



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activity in two stage hepatocarcinogenesis assays [25-27]. Generally, those PCB congeners

that induce cytochrome P-450 dependent monooxygenases (CYPs) in the liver, e.g. PCB77,

PCB126 and PCB153, are efficacious as hepatic tumor promoters [28-30]. PCBs were

reported to have no or only little genotoxic activity in most in vitro genotoxicity tests [24,31]

and their potential as cancer initiators was therefore questioned. Recently, however, evidence

for tumor initiating activity of PCB3 and other lower chlorinated PCBs that can be

metabolically activated (PCB15, PCB52, PCB77) was provided using a modified Solt-Farber

protocol in male Fischer 344 rats [32,33]. Thus PCB mixtures could be complete carcinogens

due to the initiating activity of their lower chlorinated congeners and the promoting activity

of the higher chlorinated congeners.

PCB3 is a substrate for hepatic CYPs and can be activated to electrophiles, namely

arene oxides and quinones [34]. These reactive metabolites can bind to cellular nucleophiles

like glutathione and macromolecules like DNA, RNA, protein and hemoglobin [35-38]. The

generation of reactive oxygen species during oxidative metabolism as well as the resulting

formation of 8-oxo-deoxyguanosine and DNA strand breaks have been demonstrated in vitro

[39-41]. However, the question whether PCB3 or one of its metabolites is mutagenic in vivo

was still unanswered. Therefore, we investigated the mutagenicity of PCB3 in the livers of

transgenic male Fischer 344 (BigBlue®) rats which contain 30-40 copies of the bacterial lacI

gene in every cell as a mutable target sequence. Animals were treated with corn oil (negative

control), the mutagen 3-methylcholanthrene (3-MC, positive control), PCB3, or its main

metabolite 4-hydroxy-PCB3 (4-HO-PCB3) [34]. Mutated lacI sequences were detected after

transfection into E. coli on the basis of a blue plaque phenotype using 5-bromo-4-chloro-3-

indolyl-β-D-galactopyranoside (X-gal), and the type of mutational event was determined by

sequence analysis of the lacI gene. PCB3 as well as the mutagen 3-MC induced a significant



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increase in mutant frequency in the liver of BigBlue® rats, thus demonstrating for the first

time the mutagenicity of PCB3 in vivo.

MATERIALS AND METHODS

Chemical Substances

PCB3 and its hydroxylated metabolite 4-OH-PCB3 [42] were synthesized, purified and

characterized as described [32]. 3-Methylcholanthrene was purchased from Sigma (Cat # M-

6501; St Louis, MO) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) from

Research Products International (Prospect, IL). DNA isolation and transpackaging kits were

obtained from Stratagene (La Jolla, CA). All other reagents, media and media supplements

were from Fischer Scientific (Pittsburgh, PA), if not stated otherwise.

Animals and Treatment Protocol

Sixteen male Fisher 344 BigBlue® rats, homozygous for lacI transgene, were purchased from

Stratagene (La Jolla, CA) and obtained from the breeder, Taconic Laboratories, Germantown,

NY) at postnatal day (PND) 30. The animals were kept on a 12-hr light/dark cycle and were

provided with a standard 7013-NIH-13 Modified Open Formula Rat diet and water ad

libitum. After one week of acclimatization animals were weighed and distributed at random

into 4 groups with 4 animals each. Each animal received one weekly i.p. injection on PND

37, 44, 51 and 58 of either 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-HO-PCB3 in corn oil or 5

ml/kg BW corn oil alone (negative control). The doses of the PCB and metabolites were

selected based on previous studies [32,33]. Throughout the treatment period animals were

monitored daily for well being and weighed twice per week. Seventeen days after the last

injection (PND 75), all animals were euthanized by CO2 asphyxiation followed by cervical



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dislocation. The livers were immediately removed and weighed. Small tissue samples were

quickly excised from the central part of the large lobe of the liver and these samples and the

remaining liver were snap frozen in liquid nitrogen and stored at -80°C until use for analysis

of lacI mutation frequencies and mutant analysis as described below. Parts of the remaining

liver were used to prepare slides for histological analysis. Four micron sections were

prepared, placed on glass slides and stained with conventional hematoxylin and eosin stains.

Slides were then examined using an Olympus BX40 light microscope. All experiments were

conducted with the approval of the University of Iowa Institutional Animal Care and Use

Committee.

DNA Extraction and Packaging

DNA extraction, packaging, and plating for lacI mutant plaque detection were carried out in a

blocked manner (i.e. 1 animal from each group per cycle) to avoid bias from day-to-day

variations following the BigBlue® assay procedure according to the manufacturer�s

instructions (Stratagene). Genomic DNA was extracted from the livers using the BigBlue®

RecoverEase DNA isolation kit according to the manufacturer�s instruction manual. Briefly,

50 mg of liver tissue were homogenized with a Dounce homogenizer at 4°C, and nuclei were

pelleted by centrifugation for 10 min at 1100 g. RNA and proteins in the pellet were digested

with RNase and Proteinase K, and the genomic DNA was purified by dialysis in TE buffer

for 48 h.

For recovery of the lambda transgenic shuttle vector containing the lacI target, i.e.

excision of the lambda vector and packaging into a lambda head, the Lambda Phage

Transpack® packaging kit (Stratagene) was used with 10 µl of genomic DNA per reaction as

described in the manual. The mixture, now containing the lacI transgene packaged into

infectious phages (packaged lacI), was diluted to 1 ml with SM buffer (100 mM NaCl, 8 mM

MgSO4, 50 mM Tris-HCl, 0.004% (w/v) gelatin), and stabilized by addition of 50 µl



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chloroform. Packaging efficiency was checked by infecting E. coli bacteria, strain SCS-8 (2

ml SCS-8 suspension, OD = 0.5) with 1 µl packaged lacI extract and plating the suspension

with 3 ml top agarose onto 100 mm NZY-agar plates. Infected bacteria result in plaques in

the bacterial lawn.

Determination of lacI Mutant Frequency

For determination of the lacI mutant frequency, a volume of phage extract equivalent

to approximately 12,500 plaque forming units (pfu) was mixed with 2 ml SCS-8 suspension

(OD = 0.5) and incubated at 37°C for 15 min. Fifty µl of each 2 ml SCS-8/phages suspension

were added to 2 ml fresh SCS-8 suspension and mixed with 35 ml top agarose (titer trays).

The rest of the 2 ml SCS-8/phage suspension was mixed with 35 ml top agarose containing

1.5 mg/ml of X-gal. Top agarose containing infected and non-infected SCS-8 was poured

onto 500 mm2 assay trays (Corning, Acton, MA) containing NZY-bottom agar (assay trays).

The trays were vented for 30 min before incubation at 37°C for 20 h and scoring. Packaging

and plating were repeated until 95,000�339,000 pfu were scored for each DNA sample.

Titer trays were scored for pfu and assay trays were inspected for blue plaques. To

confirm the mutant phenotype, and for use in DNA sequence analysis, all putative mutants

from the 500 mm2 assay trays were picked, diluted 1:100 with SM buffer and replated on 100

mm diameter plates with 3.5 ml of top agarose containing 1.5 mg/ml of X-gal. The lacI

mutant frequency was calculated by dividing the number of verified mutant plaques by the

total number of plaques analyzed and presented as number of mutants ± standard error per

106 pfu.

Sequence Analysis of lacI Mutants

The lac I genes of verified mutants were PCR amplified using the following primers

(named according to the manufacturer�s instruction manual) and amplification conditions:

5�-GTATTACCGCCATGCATACTAG-3� (forward PCR primer),



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5�-CGTAATCATGGTCATAGCTG-3� (reverse PCR primer), 30 amplification cycles at

94°C for 30 sec, 53°C for 50 sec, and 72°C for 60 sec. Aliquots of the PCR products were

characterized by agarose gel electrophoresis (1% in TAE buffer, 5 volt/cm). Then, PCR

products were purified using QiaQuick columns (Qiagen, Valencia, CA) and quantified by

their OD at 260 nm. Sixty ng of this purified DNA were used per reaction to sequence the

entire lacI gene (1,083 bp) in both directions using the forward PCR primer, reverse PCR

primer, primer #5 (5�-TCTGGTCGCATTGGGTC-3�) and primer #12 (5�-

AGAACTTAATGGGCCCG-3�) and a dye terminator cycle sequencing kit (Applied

Biosystems, Foster City, CA) according to the manufacturer�s instructions. The sequencing

products were analyzed on an Applied Biosystems automated capillary DNA sequencer

Model 3700 following the procedure outlined by Applied Biosystems. All alterations in DNA

sequence were verified at least once. The same mutation at the same locus in DNA extracted

from the same animal may be due to the same event and was therefore counted only once for

the calculation of the mutation frequency, which is the number of independent mutations

(different locus and/or different type as verified by sequence analysis) per total number of

plaques analyzed and presented as number of mutations ± standard error per 106 pfu.

Statistical Analysis

Gains of body weight were analyzed by Student�s t-test. The lacI mutant frequencies and the

frequencies of recovered mutations were analyzed by one-way ANOVA and Chi-square.

Data are presented as means ± standard error (p< 0.05). Chi-square test and the statistical test

described by [43] using the classes �transition�, �transversion�, and �frameshift� were used to

analyze the lacI mutation profiles of the 4 treatment groups.



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RESULTS

The objective of the present study was to clarify whether PCB3 and/or its

monohydroxylated metabolite 4-HO-PCB3 are mutagenic at the lacI gene locus in the liver of

transgenic male Fischer 344 BigBlue® rats. The established mutagen 3-MC (Fig. 1) was used

as a positive control throughout this study.

Development of animal and liver weight

The bodyweights of male BigBlue® rats administered corn oil (negative control

group), 3-MC, PCB3, or 4-HO-PCB3 were monitored from two days before the first injection

(PND 35) until euthanization at PND 75. AT PND 35, bodyweights of all treatment groups

were similar (average 71.5 ± 4.32 g). Bodyweights as well as gains of bodyweight did not

differ significantly during the first 30 experimental days (Figure 2). However, at PND 65 and

75, the gain of bodyweight of animals treated with 3-MC were significantly reduced

compared to the other treatment groups (Figure 2). In contrast, liver weights of all treatment

groups (corn oil, 3-MC, PCB3, and 4-HO-PCB3) did not differ significantly at PND 75 (4.3 ±

0.37 g/100 g bodyweight, 4.7 ± 0.64 g/100 g bodyweight, 4.3 ± 0.26 g/100 g bodyweight, and

4.5 ± 0.33 g/100 g bodyweight, respectively.

Liver Histology

Liver samples were analyzed microscopically. Control animals had normal liver architecture,

one with rare lymphoid aggregates in the parenchyma. PCB3-treated animals had only very

minimal changes with or without small clusters of parenchymal lymphocytes and rare

acidophil bodies. Animals that had received 4-HO-PCB3 showed dilated sinusoid and rare



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acidophil bodies. Three of four animals had signs of minimal portal tract inflammation. 3-

MC-treated animals had mild to severe centrilobular necrosis with various degrees of

lymphocytes and neutrophiles in the necrotic areas. The portal tracts were unaffected.

LacI mutant frequency in the livers of BigBlue® rats

Liver lacI mutant frequencies for BigBlue® rats exposed to corn oil (negative

control), 3-MC (positive control), PCB3, and 4-HO-PCB3 were measured 17 days following

the last injection (PND 75). For control animals, the mutant frequency was 17 ± 4 x 10-6 pfu

(Figure 3 and Supplementary Table 1). Treatment with 3-MC induced a mutant frequency of

88 ± 15 x 10-6 pfu, approximately a fivefold increase over the untreated control (p = 0.004).

The mutant frequency in the liver of rats treated with PCB3 was 48 ± 4 x 10-6 pfu (p = 0.001).

Although elevated more than twofold, the mutant frequency for rats administered 4-HO-

PCB3 (40 ± 12 x 10-6 pfu) was not significantly different from that of the control rats (p =

0.115) using ANOVA for analysis due to the very high variation within this group. When the

Chi-square test was applied, the significance of 3-MC and PCB3 was confirmed and 4-HO-

PCB3 was significant as well (p<0.05).

Sequence analysis of lacI mutants

Sequence analysis of mutants provides information about the kind of mutation

induced spontaneously or by treatment with test compounds. Sixteen of the 17 mutants (95%)

from the vehicle-treated rats (Supplementary Table II), 47 of 56 (85%) from the 3-MC-

treated rats (Supplementary Table III), 24 of 29 (84%) of the mutants from rats treated with

PCB3 (Supplementary Tables IV), and 29 of 34 (83%) of those from rats treated with 4-HO-

PCB3 (Supplementary Table V) provided lacI sequences that were complete, readable, and



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contained a mutation. In some instances the same mutation (location in the sequence and type

of mutation) was identified in several plaques from the same animal. In this case a

conservative approach was used by assuming that these mutations were siblings and

represent a single in vivo mutational event. As a result a total number of 16, 44, 23, and 27

independent mutations from the corn-oil treated control, 3-MC-, PCB3-, and 4-HO-PCB3-

treated groups, respectively, were identified, as listed in Supplementary Tables II�V and

summarized in Table I. A calculation of the frequency for these recovered, sequenced,

independent mutations gave 16 ± 4, 67 ± 13, 38 ± 7, and 30 ± 9 per 106 pfu for animals

treated with corn oil, 3-MC, PCB3, and 4-HO-PCB3, respectively. The significance level

compared to corn oil control for 3-MC, PCB3 and 4-HO-PCB3 was p = 0.01, p = 0.03, and p

= 0.18 using ANOVA and p<0.001, p<0.05, and not significant, respectively, when the Chi-

square test was used. It should be noted that this is a very conservative approach, but even

so, the frequencies of recovered mutations of the 3-MC and PCB3 group were significantly

elevated compared to the control group thus confirming the results obtained with the less

stringent mutant frequencies.

Simple bp substitutions comprised most of the mutations recovered: 13/16 (81%)

from untreated animals, 32/44 (73%) from 3-MC-treated, 17/23 (74%) from PCB3-treated,

and 21/27 (78%) from 4-HO-PCB3-treated rats (Table I). Most of these mutations in

untreated (69%), 3-MC-treated (59%), PCB3-treated (53%) and 4-HO-PCB3-treated (62%)

rats were found within the first 400 bp. Substitutions at G:C accounted for 75% of the

control, 64% of the 3-MC-induced mutations, 61% of he PCB3, and 67% of the 4-HO-PCB3-

induced mutations (Table I and supplementary Tables II-V).

Characterists of lac I mutations in the different treatment groups



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The most frequent type of mutation in the negative control group was G:C→A:T

transition (9/16; 56%), with 22% of these occurring at CpG sites. Also 2 G:C→C:G

transversion (13%), 1 A:T→G:C transition (6%), and 1 G:C→A:T transversion (6%)

mutations were recovered from this group. No A:T→C:G or A:T→T:A transversions were

detected in this group. In addition to bp substitutions, 1 and 2 of the 16 total mutations from

the control group were deletions or insertions, respectively, leading to frameshift mutations

(19% of the total mutations; Table 1 and Supplementary Table II).

Forty-four different mutation sites were discovered in mutants from 3-MC-treated

animals. One mutation was recovered 3 times and another one twice from the same animal,

leaving 44 independent mutations. The predominant mutation types were G:C→T:A

transversions and G:C→A:T transition mutations (27% each). Seven of the 12 transition

mutations (58%) occurred at a CpG site. In addition, there were 7 single-base frameshifts,

three 2-5 bp insertions, and a 7 and a 67 bp deletion, leading to a frameshift mutation rate of

27% of the total mutations in this group (Table 1 and Supplementary Table 3). The total

number of transitions was significantly reduced in the 3-MC-treated animals compared to

solvent controls (Chi-square test, Table I). However, using the test described by Cariello et al.

[43], the 3-MC-induced mutation spectrum in total barely missed being significantly different

from the lacI spectrum of untreated rats (p=0.058).

Of the 17 PCB3-induced single bp substitutions, 3 mutations (18%) occurred at A:T

bp while 14 (82%) were at G:C bp (Table I and Supplementary Table 4). Like the 3-MC

spectrum, the predominant mutations in this group were G:C→A:T transition (30%; 7/23),

with 43% of them occurring at CpG sites, and G:C→T:A transversion (30%; 7/23), followed

by A:T→C:G transversion (9%; 2/23). Also, there was one (4%) A:T→T:A transversion

mutation recovered from this group. Additionally, there were 4 single-base frameshifts, a 4

bp insertion, and a 13 bp deletions, leading to frameshift mutations (26% of the total



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mutations, Table I). The proportion of transitions among PCB3-induced mutations was

significantly different from that of the control group (p < 0.05) and similar to the 3-MC-

induced mutation spectrum. Despite the apparent differences in the proportions of transitions

and transversions recovered from total mutants of the control and the PCB3-treated rats

(Table 1), the PCB3-induced mutation spectrum was not significantly different from the

spectrum of the control animals using the previously described statistical test method [43]

(p=0.117).

Of the 21 4-HO-PCB3-induced single bp substitutions, only 3 mutations (14%)

occurred at A:T bp while 18 (86%) were at G:C bp (Table I). Like the control spectrum, the

predominant mutations in this group were G:C→A:T transition (48%; 13/27), with 23% of

them occurring at CpG sites, followed by G:C→T:A transversions (15%; 4/27), and

A:T→G:C transition (7%; 2/27). There was only one (4%) AT→C:G transversion recovered

from this group and no A:T→T:A transversion. Additionally, there were 2 single-base

frameshifts, three insertions (4-8 bp long), and a 21 bp deletion, leading to frameshift

mutations (22% of the total mutations, Table I). Therefore, the 4-HO-PCB3-induced mutation

spectrum was not significantly different from the spectrum of the control group.

Discussion

The present study demonstrates for the first time that PCB3 is mutagenic in vivo.

Mutant frequencies and mutation spectra of the negative control

In our experiments, the mutant frequency as well as the mutation frequency of the lacI

gene in the liver of untreated BigBlue® rats was 17 ± 4 x 10-6 pfu which is in concordance

with the finding derived from the analysis of 35 independent experiments by Lambert et al.

[44] which yielded an average spontaneous mutation frequency of the lacI gene in the liver of

BigBlue® rats of 35 ± 3 x 10-6 pfu with a range of 10 - 85 x 10-6 pfu.



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The dominating kind of mutation in all four groups was bp substitutions (73 � 81%,

compared to 19 - 27% frameshift mutations). Most of these bp substitution mutations in

untreated (69%), 3-MC-treated (59%), PCB3-treated (53%) and 4-HO-PCB3-treated (62%)

rats were found within the first 360 bp of the lac I gene, which is in accordance with

observations of other laboratories [45]. This region contains the DNA binding domain (or

negative complementing region) of the lacI gene [46].

The most frequent type of mutation in the control group was G:C→A:T transition

(56%) which is in agreement with the findings of deBoer and Glickman [45]. Sixtysix

percent (4/6) of the G:C→A:T transitions recovered from our control animals occurred in the

first 400 bp and 50% of these (2/4) occurred at CpG sites (Supplementary Table II).

G:C→A:T transitions are known to be particularly concentrated in the first 400 bp, where

65�85% of them are located at 5�-CpG-3 sites due to spontaneous deamination of methylated

cytosines [47] and the unusual malleability of CpG steps [48,49].

In contrast, deletion events are threefold more common after the first 400 bp [45]. The

3 frameshift mutations recovered from the 16 mutants of the control animals occurred within

this region (Supplementary Table II). Deletion of DNA is frequently mediated by repeated

sequences and both insertions and deletions can be explained by a slipped-mispairing

mechanism [50]. For additional information, we added the location of the frameshift mutation

in Supplementary Tables II-V.

Mutations in 3-MC-treated animals The mutant frequency in the liver of 3-MC-treated rats in our experiments was 88 ±

15 x 10-6 pfu. Sequence analysis revealed that most of them, 93%, were true independent

mutations. If we subtract 7% from those 88 mutants to account for possible siblings we

obtain an estimated mutation frequency for the 3-MC group of about 82 x 10-6 pfu, giving a



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5-fold induction of mutations above background levels. Rihn et al. [51] analyzed the

mutagenicity of 3-MC in the liver of male BigBlue® mice. Fourteen days after a single

injection of 80 mg 3-MC/kg BW, a mutation frequency of 203 ± 29 x 10-6 pfu was observed.

This seems to be higher than in our experiment with BigBlue® rats using 4 once a week

injection of 80 mg 3-MC/kg BW, however, the mutation frequency in the liver of the control

animals (76 ± 27 x 10-6 pfu) was also much higher than in our study, resulting in a less than

3-fold induction of mutations by 3-MC. These differences between this mouse and our rat

study may be due to the differing species and treatment protocols.

The predominant mutations among 3-MC-induced total mutations were G:C→T:A

transversion and G:C→A:T transition mutations. Compared to the negative control, the 3-

MC-induced mutation spectrum showed a dramatic increase in G:C→T:A transerversions

(Table 1) and -1 frameshift mutations (Table 1). G:C→T:A transversions were also the

predominant mutations recovered from the 3-MC-induced lambda lacI mutant plaques

sequenced by Rihn et al. [51]. Metabolically activated polycyclic aromatic hydrocarbons are

known to react with the exocyclic amino group of G, A, and - to a lesser extend - C [52],

resulting in G:C→T:A and A:T→T:A transversions and G:C→A:T transition mutations.

Mutant frequencies and mutation spectra of PCB-induced mutants

The purpose of this study was primarily to analyze whether PCB3 and one of its

metabolites, 4-HO-PCB3, induce gene mutations in the livers of male Fischer 344 rats. The

basis for this question was the observation that both compounds and a downstream

metabolite, the 3,4-quinone (see Fig 4) induced γ−glutamyltranspeptidase-positive foci,

which are considered to be pre-neoplastic stages, and adenoma (only PCB3) in the livers of

male Fischer 344 when applied as initiating agent in a modified Solt-Farber protocol [32,33].



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Similarly to the Solt-Farber experiment a slightly lower dose of the 4-HO-PCB was used (4

injections of 400 µmol/kg BW) than of the PCB3 (4 injections of 600 µmol/kg BW) to

account for the fact that 4-HO-PCB3 is expected to be one metabolic step closer to the

proposed ultimate mutagen, the 3,4-quinone of PCB3, and the expected higher toxicity of the

hydroxylated metabolite compared to the parent compound.

In this present study, PCB3 indeed significantly induced mutations in the liver of

transgenic male BigBlue® rats. We did not observe any signs of liver toxicity due to PCB3-

treatment, neither morphologically nor histologically, which argues against indirect effects as

cause for this increase in mutations. Moreover, the mutation spectrum was significantly

different compared to that of control animals, with transitions, which are typical for

spontaneous mutations, contributing significantly less to the PCB3-induced mutation

spectrum than in controls. This characteristic was shared with 3-MC, our positive control and

a known mutagen and carcinogen in rodents. In the 4-HO-PCB3 group on the other hand, the

mutant frequency was elevated twofold, which was not statistically significant. This lack of

significance may in part be due to the large variability in mutation frequency between

animals within this group and the use of only 4 animals per treatment group However, the

mutation spectrum of mutants isolated from animals treated with 4-HO-PCB3, was also not

significantly different from that of the control animals. A slight shift towards more

transversion and frameshift mutations may be present, but it is nowhere near the one seen

with 3-MC or PCB3. Even though the mutant frequency may be underestimated by

inadvertently omitting light colored mutant plaques, the mutational spectra are unlikely to be

biased [45]. Of course, certain deletion or insertion mutations may not be detected in phage

based transgenic rodent systems because of packaging constraints. Thus, agents whose

genotoxicity arises primarily through clastogenic events are less likely to be detected in

transgenic rodent systems [44]. For these compounds existing clastogenicity assays such as



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the in vivo bone marrow micronucleus test may still be more sensitive, although organ

specificity could hamper the detection even in these systems [44]. The results in our

experiments are therefore not sufficient to clearly answer the questions whether 4-HO-PCB3

is mutagenic in the liver of male rats, but they show that the parent compound, PCB3, is a

significantly stronger mutagen with the concentrations and exposure protocol used in this

study.

What is the mechanism of PCB3 activation to a mutagen?

The second goal of this study was to analyze whether the mutation spectra of PCB3

and its metabolite 4-HO-PCB3 could give us any information about the ultimate mutagenic

species. This raises the question of the mechanism of mutation induction involved in the

PCB3-induced mutagenesis. PCB3 is efficiently metabolized by CYPs in the liver to

monohydroxy-PCB3, probably with an epoxide as intermediate, and further to dihydroxylated

metabolites (Figure 4) [34,40,53]. Dihydroxy-PCBs can be further oxidized to the

corresponding semiquinone and quinone. Several reports show metabolic activation of

radiolabeled PCBs to result in protein, RNA, and DNA binding of radioactivity in cell free

systems [54], in cultured cells [55] and in vivo [37]. The formation of such adducts by

monochlorinated biphenyls has been explained by activation through different metabolic

pathways [34,39,56,57].

32P-postlabeling studies with PCB3 and a metabolizing system showed that similar but

also unique adducts were formed depending on whether the incubation environment was

oxidizing or reducing [36]. It was suggested that some DNA adducts of lower chlorinated

biphenyls may be derived from arene oxides (reducing environment), but others may be

formed from the oxidized products of catechol and p-hydroquinone, i,e, semiquinones and



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quinones (oxidizing environment). PCB-quinones form adducts with nitrogen and sulfur

nucleophiles [40,56] as well as with guanosine or isolated DNA in vitro [36,40,53]. Recently,

the structure of the reaction product of PCB3-2,5-quinone with N2 of guanine has been

identified [53]. In addition to the cell-free experiments mentioned above, the induction of

DNA adducts in cultured human hepatocytes by PCB3 has been demonstrated by the 32P-

postlabeling assay [35] and quinonoid protein adducts of 2,5,2�,5�-tetrachlorobiphenyl

(PCB52) were discovered in the liver and brain of treated rats [58].

Besides the reactivity of arene oxide and quinoid PCB metabolites towards DNA, the

induction of oxidative stress might be another possible mechanism for the initiation potential

of PCB3 and some of its metabolites: incubation of isolated DNA with dihalogenated PCB

catechols in the presence of lactoperoxidase and metal ions induced 8-oxo-deoxyguanosine

formation [39]. Several metabolites of PCB3 induce DNA strand breaks in vitro and reactive

oxygen species in vitro as well as in cultured cells [41].

The G:C→T:A transversions recovered from PCB3-induced mutants in the present

study are in concordance with both possible mechanisms: arylalkylating agents such as

activated PCB3 metabolites (e.g. quinones, epoxides) react preferably with the exocyclic

aminogroup of G, A or C [52], which would generate a non-coding DNA lesion. When trying

to interpret such a lesion during transcription, DNA polymerases usually use ATP instead of

CTP resulting in G:C→T:A and A:T→T:A transversion, as well as G:C→A:T transition.

However, G:C→T:A transversion is also the predominant mutation induced by 8-oxo-

deoxyguanosin [59]. Thus both, oxidative stress and/or adduct formation, could have caused

the observed increase in mutations. The fact that -1 frameshift mutations and A:T→C:G and

A:T→T:A transversions also seem to be increased in PCB3-treated animals, similar to 3-MC-

treated animals, may be additional support for arylalkylation as mechanism. Also, the strong

mutagenicity of PCB3 compared to 4-HO-PCB3 seems to argue in favor of an arenoxide as



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mutagenic intermediate. However, it could also be argued that the 4-HO-PCB3 metabolite

was already too reactive to reach the target organ, the liver, whereas the non-reactive PCB3

was easily transported to the liver and there bioactivated to a mutagenic species with or

without the production of oxidative stress. Thus these in vivo experiments proved for the first

time that PCB3 is indeed mutagenic in the organ where it produced preneoplastic lesions, but

the ultimate mutagenic species and activation mechanism is still elusive.

PCB3 is found in the air of contaminated sites and in urban areas [18]. It can not be

detected in human blood samples [60], indicating its susceptibility to metabolism which

might involve the generation of mutagenic metabolites. In addition, many other lower

chlorinated PCBs which are ubiquitous in our environment may have a similar potential to be

activated to mutagenic and carcinogenic species as our model compound PCB3, as indicated

by the observation that several of them cause preneoplastic foci in rat liver [32,33].

Therefore, the here described mutagenicity of PCB3 in vivo might have strong implication

concerning the potential carcinogenicity of this whole group of ubiquitous environmental

pollutants.



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ACKNOWLEDGEMENTS

The authors would like to thank James A. Jacobus for injecting the animals and his help

in the laboratory, Dr. Lehmler for synthesizing and characterizing the study chemicals, and

Dr. Barbara Shane for useful suggestions concerning the assay procedure. This work was

financially supported by NIEHS (ES 07380 and ES13661), DOD (DAMD17-02-1-0241),

EPA (R-82902102-0) and a Hollaender Trainee award to L.L. from the EMS. The content of

this publication is the sole responsibility of the authors and does not necessarily represent the

official views of the funding agencies.

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Table I

Summary of sequenced, independent lacI mutations in the livers of BigBlue® rats treated

with corn oil, 3-MC, PCB3, or 4-HO-PCB3. Asterisks indicate significant differences of a

specific type of mutation (*, Chi-square test) or the total spectrum (#, test described by [43])

compared to the corn oil control group. Levels of significance: p < 0.05.

Types of mutations Number of mutations (% total mutations)

Corn oil 3-MC PCB3 4-HO-PCB3

Transitions (total) 10 (63) 13 (30)* 7 (30)* 15 (56)

G:C → A:T 9 (56) 12 (27) 7 (30) 13 (48)

CpG sites 2 (22) 7 (58) 3 (43) 3 (23)

A:T → G:C 1 (6) 1 (2) 2 (7)

Transversions (total) 3 (19) 19 (43) 10 (43) 6 (22)

G:C → T:A 1 (6) 12 (27) 7 (30) 4 (15)

G:C → C:G 2 (13) 4 (9) 1 (4)

A:T → C:G 2 (5) 2 (9) 1 (4)

A:T → T:A 1 (2) 1 (4)

Frameshifts (total) 3 (19) 12 (27) 6 (26) 6 (22)

-1 frameshift 6 (14) 3 (13) 1 (4)

+1 frameshift 1 (6) 1 (2) 1 (4) 1 (4)

Insertions 1 (6) 3 (7) 1 (4) 3 (11)

Deletions 1 (6) 2 (5) 1 (4) 1 (4)

Total 16 (100) 44 (100) 23 (100) 27 (100)

Significance versus control# P = 0.058

(0.047-0.069)

P = 0.117

(0.102-0.132)

P = 1.000



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FIGURE LEGENTS

Figure 1. Chemical structures of PCB3, 4-HO-PCB3, and 3-MC.

Figure 2. Weight gain of male BigBlue® rats treated with corn oil (negative control), 3-MC

(positive control), PCB3, and 4-HO-PCB3. Asterisks indicate significant differences to the

untreated control group. Levels of significance: *, p < 0.05; **, p < 0.01; (Student�s unpaired

t-test). Arrows, injection, PND, post natal day.

Figure 3. Mutant frequencies in the liver after treatment of male transgenic BigBlue® rats

with 5 ml/kg BW corn oil (negative control), 298 µmol/kg BW 3-MC (positive control), 600

µmol/kg BW PCB3, or 400 µmol/kg BW 4-HO-PCB3. Data represent mean of 4 animals ±

standard error (p<0.5). Asterisks indicate significant differences to the control group. Levels

of significance: **, p < 0.01 (One-way ANOVA).

Figure 4. Metabolic activation of PCB3 to quinones (Q), modified from [32].



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4-monochlorobiphenyl (pcb3) induces mutations in the livers of transgenic fisher 344 rats

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