Skip Navigation


ToxSci Advance Access originally published online on August 8, 2007
Toxicological Sciences 2007 100(1):88-98; doi:10.1093/toxsci/kfm204
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
100/1/88    most recent
kfm204v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zettner, M. A.
Right arrow Articles by Lehmann, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zettner, M. A.
Right arrow Articles by Lehmann, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Quinoid Metabolites of 4-Monochlorobiphenyl Induce Gene Mutations in Cultured Chinese Hamster V79 Cells

Markus Alexander Zettner*, Susanne Flor*, Gabriele Ludewig{dagger}, Jörg Wagner*, Larry W. Robertson{dagger} and Leane Lehmann*,1

* Institute of Applied Biosciences, Section of Food Chemistry and Toxicology, University of Karlsruhe (TH), Kaiserstraße 12, D-76131 Karlsruhe, Germany {dagger} Department of Occupational and Environmental Health, College of Public Health, University of Iowa, 100 Oakdale Campus, Iowa City, Iowa 52242-5000

1 To whom correspondence should be addressed. Fax: +49-721-608-7255. E-mail: leane.lehmann{at}lmc.uni-karlsruhe.de.

Received May 22, 2007; accepted July 31, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
4-Monochlorobiphenyl (PCB3) is a component of commercial polychlorinated biphenyl (PCB) products and is an airborne environmental pollutant. Our recent study with transgenic Fischer 344 rats revealed the mutagenic potential of PCB3 in the livers of male rats. PCB3 is converted in vitro to hydroxylated metabolites, to hydroquinones (HQs, e.g., 2',5'-HQ and 3',4'-HQ), and can be further oxidized to quinones (Qs, e.g., 2',5'-Q and 3',4'-Q). This raises the question whether the mutagenic potential of PCB3 is due to the mutagenicity of PCB3 itself or of one of the metabolites. In this study, we investigated the mutagenicity of PCB3, of the monohydroxylated metabolites 2'-hydroxy (HO)-, 3'-HO-, and 4'-HO, of the HQs 3',4'-HQ and 2',5'-HQ and of the Qs 3',4'-Q and 2',5'-Q in cultured Chinese hamster V79 cells. The induction of gene mutations was determined at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene locus by selection with 6-thioguanine. The induction of chromosome and genome mutations was assessed using the micronucleus assay and immunochemical differentiation of micronuclei containing whole chromosomes (kinetochore positive) and DNA fragments (kinetochore negative). The induction of chromosome and genome mutations, detected as micronuclei, was only observed at higher, cytotoxic concentrations of monohydroxylated, catecholic, and quinoid metabolites of PCB3. However, both PCB3-Qs induced a significant increase in the mutant frequency of the hprt gene and did so at submicromolar concentrations. Thus, the present study demonstrates for the first time the mutagenicity of PCB3 metabolites in mammalian cells and identifies quinoid metabolites of PCB3 as potential ultimate mutagens.

Key Words: 4-monochlorobiphenyl; PCB; HPRT mutation; V79 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Polychlorinated biphenyls (PCBs) were produced world wide from 1929 to the early 1980's. 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 (WHO, 2003Go). 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 or condensation (Grimalt et al., 2004Go; Gustafsson et al., 2005Go). Vaporization of PCBs from landfills (Hansen and O'Keefe, 1996Go; Persson et al., 2005Go), contaminated surface water (Hermanson and Hites, 1989Go; Hornbuckle and Green, 2003Go), and construction materials for public buildings (Digernes and Astrup, 1982Go; Gabrio et al., 2000Go; Kohler et al., 2005Go) are the main sources of outdoor and indoor air contamination. Human exposure to PCBs, however, occurs 90% via food. PCB congeners that accumulate in the food chain are mainly tetra- to heptachlorinated, are stored preferably in fatty tissues, and are only slowly metabolized. In contrast, inhalation exposure contributes the remaining 10% (WHO, 2003Go). Airborne PCBs are lower chlorinated and are susceptible to metabolic attack. In some populations, however, inhalation exposure was found to be the major source for daily PCB uptake (Wilson et al., 2001Go), which raises the question about the toxicity of these congeners and their metabolites.

Airborne PCBs originate in commercial PCB mixtures (Hansen, 1999Go; Uraki et al., 2004Go) but volatilize into the atmosphere, especially in cities, buildings, and near waste sites (Imsilp et al., 2005Go). 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 (Duarte-Davidson and Jones, 1994Go; Gallani et al., 2004Go). 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 (Davis et al., 2002Go).


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Metabolism of PCB3 in vitro (McLean et al., 1996aGo), modified.

 
PCBs have repeatedly been shown to be complete carcinogens in rodents (Mayes et al., 1998Go; Silberhorn et al., 1990Go). In addition, commercial PCB mixtures as well as individual congeners have tumor-promoting activity in two-stage hepatocarcinogenesis assays (Dean et al., 2002Go; Haag-Gronlund et al., 1997Go). Generally, those PCB congeners that induce cytochrome P450 (CYP)–dependent monooxygenases in the liver, e.g., PCB77, PCB126, and PCB153, are efficacious as hepatic tumor promoters (Buchmann et al., 1991Go; Glauert et al., 2001Go). PCBs were reported to have no or only little genotoxic activity in most in vitro genotoxicity tests (summarized in Ludewig 2001) 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, and PCB77) was provided using a modified Solt-Farber protocol in male Fischer 344 rats (Espandiari et al., 2003Go, 2004Go). Moreover, in a recent study, we demonstrated the induction of predominately transversion mutations in the livers of male transgenic Fischer 344 rats by PCB3 (Lehmann et al., 2007Go). 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 (Qs) (McLean et al., 1996aGo, Fig. 1). These reactive metabolites can bind to cellular nucleophiles, like glutathione, and macromolecules, like DNA, RNA, protein, and hemoglobin (Borlak et al., 2003Go; McLean et al., 1996bGo; Pereg et al., 2001Go; Tampal et al., 2003Go). The generation of reactive oxygen species during oxidative metabolism as well as the resulting formation of 8-oxo-deoxyguanosine and DNA strand breaks has been demonstrated in vitro (Oakley et al., 1996aGo,bGo; Srinivasan et al., 2001Go). However, the question whether PCB3 or one of its metabolites is the mutagenic agent was still unanswered. Therefore, we investigated the mutagenicity of PCB3 and its most important oxidative metabolites 2'-hydroxy-, 3'-hydroxy-, 4'-hydroxy- PCB3 (2'-HO, 3'-HO, and 4'-HO, respectively), PCB3-2',5' hydroquinone (2',5'-HQ), 3',4'-HQ, PCB3-2',5' quinone (2',5'-Q), and 3',4'-Q at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in cultured Chinese hamster V79 cells. Furthermore, the induction of genome and chromosome mutations was determined by the micronucleus assay. Sufficient cell proliferation for the expression of mutations was assured by determination of the cell cycle distribution after compound treatment and the number of cells during the complete HPRT experiments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Chemical substances.
PCB3 and its mono- and dihydroxylated metabolites were synthesized, purified, and characterized as described (Espandiari et al., 2004Go). Purity was > 99%, unless otherwise stated. 4-nitroquinoline-N-oxide (NQO) and all other reagents, media, and media supplements were from Sigma-Aldrich (Taufkirchen, Germany) or Roth (Karlsruhe, Germany), if not stated otherwise.

Cell culture.
Male Chinese hamster V79 lung fibroblasts were kindly provided by H. R. Glatt (German Institute of Human Nutrition, Nuthetal, Germany). They were grown in Dulbecco's modified Eagle’s medium (DMEM) with 10% bovine fetal calf serum (FCS; Invitrogen, Karlsruhe, Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a water-saturated atmosphere containing 5% CO2. Test compounds were dissolved in dimethyl sulfoxide (DMSO) immediately before use and added to the medium to yield a final DMSO concentration of 1% (vol/vol). Negative controls received 1% DMSO without test compound.

HPRT assay and measurement of cytotoxicity.
The HPRT assay was performed as described previously (Schumacher et al., 2005Go). Briefly, 1.5 x 106 V79 cells were seeded in cell culture flasks (182 cm2, Greiner bio-one, Frickenhausen, Germany) containing 20 ml DMEM with FCS and penicillin/streptomycin as described above. After 24 h, the medium was changed (day 0) and cells were treated with different concentrations of PCB3, its metabolites, or NQO for another 24 h. A total of 1 x 106 treated cells were subcultured in fresh medium directly after treatment (day 1) and again on day 3 or 4. On days 1, 3 (4), and 6, the number of viable cells was counted with an electronic cell counter (CASY, Schärfe Systeme, Reutlingen, Germany) as a measure for cytotoxicity and proliferation. On day 6, cells with mutations at the hprt gene locus were selected by growing cells in medium containing 5% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 7 µg/ml 6-thioguanine (6-TG) using three tissue culture dishes (145 mm, Greiner bio-one) with 1 x 106 cells per dish. To determine the plating efficiency (PE) on days 1 and 6, cells were grown in the absence of 6-TG (500 cells per 94-mm dish, three dishes). After 1 week, cells were fixed with ethanol and stained with methylene blue. Colonies were counted and the PE, i.e., the number of colonies per number of seeded cells, and the mutant frequency (MF), i.e., the number of colonies/(number of seeded cells x the PE at day 6), were calculated. For relative PE1 and PE2 (%), the PE of cells treated with solvent was set 100%. Selection of mutants and determination of the PE were done in triplicate (three independent experiments with three plates each).

Determination of cell cycle distribution.
After treatment with test compounds for 24 h, aliquots of the suspended V79 cells from the HPRT experiments were fixed and stained with CyStain DNA Protein 2 staining solution (Partec, Muenster, Germany) following the manufacturer's instructions. Briefly, 2 x 105 suspended cells were pelleted by centrifugation at 310 x g for 5 min, resuspended in 200 µl cold calcium- and magnesium-free phosphate-buffered saline (PBS-CMF), and mixed with 600 µl of cold ethanol. Cell suspensions were stored at – 20°C for at least 12 h, then pelleted, and resuspended in CyStain DNA Protein 2 staining solution. Measurement of blue (4,4'-diamidino-2-phenylindole [DAPI]-stained nuclei) and red (sulforhodamin-stained protein) fluorescence intensities was performed with a PA II flow cytometer equipped with the following optical filter set: KG/BG38/UG1/TK420/TK560/GG435 (blue fluorescence) and GG590 (red fluorescence), respectively (Partec) as described previously (Schumacher et al., 2005Go). Cell cycle distribution was derived by analysis of the resulting histograms and of the dot plot with Flowmax software (Partec) assigning the first peak of the DAPI fluorescence to G1/G0 cells, the second peak with a twofold fluorescence intensity of that of the first peak to G2/M cells and intermediate fluorescence intensities to cells in S phase.

Micronucleus assay.
For micronucleus analysis, slides were prepared in two different ways:

  • Immediately after the treatment with compounds for 24 h and after the next cell passage (day 3, postincubation time = 48 h), aliquots of the V79 cell suspensions from the HPRT assay were spun onto glass slides pretreated with polylysine (10 min at 115 x g) and were subsequently fixed with Canoy's fixans (3 vol methanol + 1 vol acetic acid) at – 18°C for at least 12 h.
  • Parallel to the HPRT assay, 3 x 103 cells were seeded in each chamber of an eight-chamber slide (Nunc, Wiesbaden, Germany) and were treated in the same way and with the same incubation medium as the cells of the HPRT assay. Either immediately after compound treatment for 24 h or after a compound-free postincubation period of 6, 24, or 48 h, cells were fixed with methanol at – 18°C.

Slides prepared by either method were stained with CREST (calcinosis, Reynaud's phenomenon, esophageal mobility abnormalities, sclerodactyly and telangiectasia) antibodies for the detection of kinetochores and with antifade solution containing DAPI for the scoring of nuclei and micronuclei. Chamber slides were also stained with anti-{alpha}-tubulin antibodies for the evaluation of cell morphology as described previously (Lehmann and Metzler, 2004Go). Briefly, nonspecific binding was blocked by incubation with goat serum for 1 h at 37°C. Slides were then incubated with a 1% solution of bovine serum albumin (BSA) in PBS-CMF containing monoclonal mouse anti-{alpha}-tubulin antibody (Sigma-Aldrich, diluted 1:500) and purified CREST antibodies (Acris, Hiddenhausen, Germany, diluted 1:100) for 1 h at 37°C, followed by incubation with a solution of BSA in PBS-CMF containing the secondary antibodies goat anti-mouse antibody (CY3 conjugated, Jackson Immune Research Laboratories, Inc., West Grove, PA, diluted 1:250) and goat polyvalent anti-human antibody (FITC conjugated, Sigma-Aldrich, diluted 1:200) for 1 h at 37°C. Finally, slides were mounted in antifade solution containing 1 µg/ml DAPI. On each slide, cell nuclei and micronuclei were visualized using ultraviolet excitation and the microtubules (forming the cytoplasmic microtubule complex [CMTC], the midbody and the mitotic spindle) and kinetochore signals were analyzed under green and blue illumination, respectively. All signals could be visualized simultaneously by means of a triple band-pass filter. At least 2000 total cells or 100 cells with micronuclei were scored per slide with respect to micronuclei and signs of cytotoxicity, such as karyorrhectic cells, disrupted CMTC, and apoptotic bodies. Published recommendations concerning the scoring of micronuclei (Kirsch-Volders et al., 2003Go) were followed.

Statistics.
Data are given as means ± SDs of the means of three independent experiments (n = 3). When relative data are shown (PE1 and PE2), the value of solvent-treated cells was set 100%, and the percentages of the treatment groups were calculated prior to calculate the means. Statistical analysis of differences was performed with the untransformed data set. Cell numbers, PEs, cell cycle distributions, and MFs were statistically analyzed with Student's paired t-test of significance using the Origin program (Microcal Software, Northampton, MA). Statistical differences in the micronucleus test were also determined using Fisher's exact test and chi-square test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Acute Cytotoxicity of PCB3 and Metabolites of PCB3 in V79 Cells
In order to assess the concentration range for the HPRT assay, V79 cells were treated with NQO, PCB3, or its metabolites for 24 h, and the number of viable cells and the PE was determined. Treatment with the known mutagen NQO (0.5 and 1.0µM) for 24 h had no effect on cell number or PE in V79 cells (Fig. 2). A concentration-dependent reduction of viable cells and PE was observed after exposure to 200–300µM PCB3; 50–100µM 2-, 3-, and 4'-HO; > 5µM 3',4'-HQ and 2',5'-HQ; 2.5–15µM 3',4'-Q; and 2.0–2.5µM 2',5'-Q. PEs were reduced to 10–20% of solvent controls after exposure to the highest concentration of PCB3 (300µM), 2-HO- and 4'-HO (100µM), 3',4'-HQ (25µM), and 2',5'-HQ (7.5µM), indicating that the highest possible concentration for the HPRT assay was reached. Treatment with 100µM 3'-HO reduced the PE merely to 40 ± 5.0%, but the next highest concentration tested (150µM) decreased the number of cells below the number needed for the HPRT assay (data not shown); therefore, 100µM was considered the highest test concentration possible.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Number of viable cells (columns) and PE (PE1, symbols) of V79 cells immediately after 24-h treatment with solvent alone (1% DMSO), PCB3, metabolites of PCB3, or NQO. The PE1 of solvent-treated cells was between 0.80 and 0.91. Data represent mean of three independent experiments ± SDs. Asterisks indicate significant differences to the corresponding solvent control. Levels of significance (Student's paired t-test): *p < 0.05; **p < 0.01; ***p < 0.001.

 
Proliferation of V79 Cells during the HPRT Assay
Proliferation during PCB treatment.
Intracellular stress reactions caused by clastogenic compounds are likely to activate cell cycle checkpoints resulting in arrests or delays in the progression of the cell cycle, which might interfere with the conversion of DNA damage into mutations. To assess the impact of exposure to PCB3 or its metabolites on cell cycle progression, the cell cycle distribution was determined immediately after the 24-h treatment period by flow cytometry, and the number of cells was determined electronically during the HPRT study. In all experiments, cells treated with DMSO alone showed a proportion of cells in G1/G0 between 55.6 ± 0.9% and 60.1 ± 2.6%. Exposure to 0.5 or 1.0µM NQO for 24 h did not significantly affect the cell cycle distribution of V79 cells (data not shown). For PCB3 and its metabolites, a substance-related and statistically significant decrease in the proportion of cells in G1/G0 to 49.5 ± 3.3% (PCB3), 30.6 ± 1.9% (2'-HO), 53.0 ± 1.1% (3'-HO), 17.5 ± 1.8% (4'-HO), 23.8 ± 15.8% (3',4'-HQ), 41.6 ± 4.2% (2',5'-HQ), and 51 ± 1.3% (2',5'-Q) was observed at the highest concentrations tested (Supplementary Fig. 1). A slight, but statistically nonsignificant, reduction of the proportion of cells in G1/G0 (55.8 ± 5.2) was also observed after treatment with 3',4'-Q. The observed reductions in G1/G0 were due to a statistically significant increase in the proportion of cells in G2/M-phase only (PCB3, 3'-HO, 3',4'-HQ, and 3',4'-Q), in S-phase only (2',5'-HQ and 2',5'-Q), or in both phases of the cell cycle (2'-HO, 4'-HO [Supplementary Fig. 1]).

In order to determine whether the observed increase in the G2/M peak after treatment with highest concentrations of PCB3, HO-PCB3, 3',4'-HQ, and 3',4'-Q were due to cells in G2 or M phase of the cell cycle, V79 cells of the same experiments were centrifuged onto microscopic slides, or cells were grown on microscopic slides under the same conditions as in the HPRT assay, and the mitotic index was determined by fluorescence microscopy. The average mitotic index of V79 cells treated with solvent only was 2.8 ± 0.4% (data not shown). Of all PCB3 metabolites which induced an increase in the proportion of cells in G2/M phase of the cell cycle (Supplementary Fig. 1), only 10–25µM 3',4'-HQ induced both a concentration-dependent and statistically significant increase in the mitotic index (up to 5.1 ± 0.3% at 25µM, data not shown) indicating that the cell populations treated with PCB3, monohydroxylated PCB3, and 3',4'-Q accumulated in the G2 phase of the cell cycle. In order to analyze the influence of certain PCB3 metabolites on mitosis in more detail, the number of cells in pre-anaphase and ana/telophase was determined separately and expressed as percentage of total cells similar to the mitotic index. When V79 cells were treated with PCB3 metabolites which induced predominately kinetochore-positive micronuclei (HO-PCB3s and 2',5'-HQ, see Fig. 4), a reduction of the frequency of cells in ana/telophase from 1.3 ± 0.1% (average of all solvent controls) to 0.7 ± 0.2% (2'-HO), 0.4 ± 0.3% (3'-HO), 0.3 ± 0.2% (4'-HO), and 0.8 ± 0.1% (2',5'-HQ) was observed. This decrease caused an increase in the pre-anaphase:ana/telophase ratio from 1.2 (average of all solvent controls) to 3.0, 6.0, 8.3, and 2.5, respectively, suggesting an impaired transition into anaphase.


Figure 4
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. Micronucleated cells immediately after treatment of V79 cells with various concentrations of PCB3 or metabolites of PCB3 for 24 h. Data represent mean of three independent experiments ± SDs. Asterisks indicate significant differences to the corresponding vehicle control (1% DMSO). Levels of significance (Fisher's exact test): *p < 0.05; **p < 0.01; ***p < 0.001. Parenthesis, mathematical significance which is considered of no toxicological relevance.

 
Proliferation of V79 cells until the time point of selection.
Proliferation of cells is essential for the conversion of compound-induced DNA damage into mutations during the HPRT assay. Therefore, the number of cells was determined at every cell passage during the expression phase until the time point of selection. The slopes of the semilogarithmic growth curves were used as a measure for cell proliferation, assuming that equal rates of proliferation would be reflected in slopes of treated cells that parallel the slope of the solvent control. V79 cells treated with up to 100µM PCB3, 25–50µM of 2'-, 3'-, or 4'-HO, 5µM 2',5'-HQ, 10µM 3',4'-HQ, 2.5µM 2',5'-Q, or 15µM 3',4'-Q resumed growth and attained the same proliferation rate as the cells of the solvent control immediately after removal of the compounds. In contrast, V79 cells treated with the highest concentrations of PCB3, HO-PCB3s, or PCB3-HQs did not proliferate in the presence of the compounds (or even died) and needed a compound-free expression period of 3–4 days to resume a normal proliferation rate, confirming that these were the highest concentrations suitable for the HPRT assay (Supplementary Fig. 2).

Mutagenicity of PCB3 Metabolites in V79 Cells
Induction of mutations at the hprt locus.
MFs represent the number of 6-TG–resistant mutants per 106 viable cells (see "Materials and Methods" section for the equation). The frequency of spontaneous HPRT mutants in our V79 cell populations ranged from 4 ± 2 to 12 ± 2 TG-resistant mutants per 106 colony-forming cells (Fig. 3). Compound-induced MFs that were statistically significantly different from their respective solvent control, but lay within this normal range of spontaneous mutations, were considered of no toxicologic relevance. The mutagen NQO served as positive control: incubation with 0.5 or 1.0µM NQO for 24 h, which was not cytotoxic (Fig. 2), caused a significant increase in the MF that ranged from 43 ± 15 to 47 ± 11 and 105 ± 25 to 133 ± 30, respectively (Fig. 3). The reduction of the NQO concentration was due to the lack of or the comparatively small increases in the number of TG-resistant colonies observed with the PCB metabolites compared to the solvent control. A significant increase in the MF was also induced by treatment with 0.6–1.3µM 3',4'-Q (maximum 37 ± 6) and 0.5–1.5µM 2',5'-Q (maximum 63 ± 10). These PCB3-Q concentrations were noncytotoxic as indicated by cell count (Fig. 2) and PEs of nearly 100% immediately after PCB3-Q treatment (98 ± 5% and 90 ± 3%, respectively; Fig. 2). The induction of a maximum MF which declines with higher concentrations of the PCB3 Qs seems unusual. It is noticeable that the PE1 as the most sensitive marker for cytotoxicity was reduced in a concentration-dependent way after treatment with ≥ 2.5µM 3',4'-Q although significance was not reached before 10µM (Fig. 2). Likewise, a small reduction in PE1 was observed after treatment with 2.0 and 2.5µM 2',5'-Q, even if statistical significance was not reached due to large variations (Fig. 2). This seems to be in accordance with the finding that despite the very strong induction of mutations at the hprt locus by 1µM NQO, the PE1 remained unaffected (Fig. 2). In addition, cell growth was slightly but visibly affected by the higher concentrations of PCB3 Qs (Supplementary Fig. 2).


Figure 3
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. MFs (columns) and PEs at the time point of selection (PE2, symbols) after treatment of V79 cells with solvent alone (1% DMSO), PCB3, metabolites of PCB3, or either 0.5 or 1.0µM NQO for 24 h. Data represent mean of three independent experiments ± SDs. Asterisks indicate significant differences to the corresponding solvent control. Levels of significance (Student's paired t-test): *p < 0.05; **p < 0.01; ***p < 0.001. Parenthesis, mathematical significance which is considered of no toxicological relevance (as explained in the main text).

 
Induction of micronuclei.
Subsequently, we were interested to know whether the induction of gene mutations at the hprt locus correlated with the induction of chromosome and genome mutations (i.e., micronuclei containing DNA fragments and micronuclei containing whole chromosomes). Therefore, using fluorescence microscopy, aliquots of the cell suspensions obtained during the HPRT experiments, or cells treated under the same conditions as in the HPRT assay were analyzed with respect to micronuclei induction. The occurrence of micronucleated cells was expressed as number of micronuclei per 1000 cells. Micronuclei containing whole chromosomes were identified by immunochemical staining of kinetochore proteins. The spontaneous frequency of total micronucleated cells in our V79 cell line was between 9 ± 1 and 12 ± 2 after treatment with solvent alone for 24 h (Fig. 4). Treatment with noncytotoxic 0.5µM NQO for 24 h (Fig. 2) significantly induced mutations at the hprt locus of V79 cells (Fig. 3) and also significantly increased the frequency of micronucleated cells (21 ± 3, data not shown). About 79% of the NQO-induced micronuclei were kinetochore negative, i.e., chromosome fragments (data not shown).

Likewise, an increase in total micronuclei frequency was observed after treatment of V79 cells for 24 h with 75–100µM 2'-HO (maximum 42 ± 3), 75–100µM 4'-HO (maximum 99 ± 2), 25µM 3',4'-HQ (28 ± 9), 7.5µM 2',5'-HQ (28 ± 3), 10–15µM 3',4'-Q (maximum 36 ± 5), or 1.5µM 2',5'-Q (36 ± 1). In contrast, 3'-HO (100µM) induced merely a slight, yet statistically significant increase in micronuclei and treatment with up to 300µM PCB3 had no effect on the micronucleus frequency of V79 cells (Fig. 4). After a PCB3 (metabolite)-free postincubation period for 6, 24, or 48 h, no further increase in micronuclei frequencies was observed (data not shown).

Immunochemical staining to differentiate micronuclei containing whole chromosomes or DNA fragments revealed an increased incidence in both kinetochore-negative and kinetochore-positive micronuclei with PCB3 Qs and 3',4'-HQ (Fig. 4), indicating a clastogenic as well as an aneuploidogenic potential of these metabolites of PCB3. In contrast, monohydroxy-PCB3s preferably induced kinetochore-positive micronuclei (Fig. 4), suggesting a primarily aneuploidogenic potential of these compounds.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
The present study addresses the question which PCB3 metabolite may be responsible for the reactivity of PCB3 toward cellular macromolecules observed in vivo (Espandiari et al., 2003Go; Lehmann et al., 2007Go; Pereg et al., 2001Go). Here, we report that PCB3 itself did not induce any kind of mutations in V79 cells which are not capable of PCB3 hydroxylation due to lack of CYP activity (McGregor et al., 1991Go), whereas all tested mono- and dihydroxylated PCB3 metabolites (2'-HO, 3'-HO, 4'-HO, 3',4'-HQ, 2',5'-HQ) induced genome mutations (i.e., chromosome loss) and some of them (4'-HO, 3',4'-HQ) also weakly induced chromosome mutations at cytotoxic concentrations. In contrast, both PCB3 Qs (3',4'-Q and 2',5'-Q) induced gene mutations at lower and noncytotoxic concentrations, whereas chromosome as well as genome mutations were observed at slightly higher, but still noncytotoxic concentrations. The concentrations of quinoid PCB3 metabolites needed for the induction of gene mutations were in the sub- to low micromolar range, and an unusual nonlinear kinetics was observed (Fig. 3). Unusual concentration-response curves of gene mutations have been observed before: several compounds including benzo[a]pyrene, epichlorohydrin, Natulan, 2,4,7-trinitrofluorenone, and methyl iodide induced mutations with nonlinear kinetics at the thymidin kinase locus in L5178Y mouse lymphoma cells (Moore et al., 1985Go). The anomalous shape of the Natulan curve was characterized by a high MF at low concentrations followed by a decrease and subsequent second increase of the MF at concentrations that strongly reduced the survival and has been observed before with thioxanthenes (Clive, 1974Go). Moreover, the induction of mutations in the hprt locus of spleen lymphocytes after administration of 1,6-dinitropyrene to rats followed an inverse U-shaped kinetics (Beland et al., 1994Go). An unusual dose-response curve might have several reasons.

  • Although PE1 was only slightly reduced at concentrations of PCB3 Qs which were higher than those inducing maximum MF, cells might have died rather than convert the DNA damage into mutations. Likewise, we observed a slight reduction in cell growth (Supplementary Fig. 2). This possibility is supported by the finding that the positive control NQO did not affect PE1 and PE2 at all despite the very strong induction of mutations.
  • PCB3-derived Qs were shown to react with glutathione in vitro (Amaro et al., 1996Go), and quinoid electrophiles are known to be inactivated by glutathione-S-transferase (GST)–mediated conjugation with glutathione. The V79 cell line synthesizes glutathione (Schumacher et al., 2005Go) and expresses GST (activity of about 100 nmol chlorodinitrobenzene/min/mg protein, Lehmann, unpublished data) and therefore might be able to detoxify PCB3 Qs. We observed a statistically significant increase in intracellular glutathione of 140 and 195% of that of solvent-treated cells immediately after treatment with 10 and 15µM 3',4'-Q for 24 h (data not shown). Likewise, the intracellular glutathione concentration was slightly but statistically significantly increased to 106% after treatment with 2.5µM 2',5'-Q, suggesting the initiation of a cellular stress response. Since the GST activity was not affected (data not shown), we assume that the expression of {gamma}-glutamylcystein synthetase which catalyzes the speed-limiting step of glutathione synthesis and is under the control of transcription factor Nrf binding to antioxidant response elements (Rushmore and Kong, 2002Go) was stimulated by the PCB3 Qs. This or other pathways might have also stimulated other intracellular defense mechanisms. Moreover, the increase in glutathione levels might have been more pronounced at earlier time points.
  • The repair of some kinds of DNA damage at higher concentrations has been reported to cause nonlinear kinetics in the HPRT test (Jenssen, 1982Go, 1986Go) and might also apply for Q-derived DNA adducts.

Recent studies demonstrated a tumor-initiating (Espandiari et al., 2003Go) and mutagenic (Lehmann et al., 2007Go) potential of PCB3 in the livers of male F344 rats. This raises the question of the mechanism of mutation induction involved in the PCB3-induced mutagenesis. Since the major type of mutations induced by PCB3 in transgenic Fischer 344 rats (BigBlue) was base substitutions (predominately G:C->T:A transversions, Lehmann et al., 2007Go), an in vitro assay sensitive for gene mutations in mammalian cells was chosen in the present study to identify the most likely metabolite responsible for these base substitution mutations. In contrast, large deletions are only limitedly detected (e.g., Oberly et al., 1993Go) and aneuploidogens are not detected (e.g., Tsutsui et al., 2000Go) in the HPRT assay.

Based on the results of the V79 HPRT assay, the gene mutations observed in vivo were presumably caused by quinoid metabolites of PCB3.

Despite the conjugation of hydroxylated metabolites of PCBs with glucuronosyl and sulfonyl groups (James, 2001Go) which prevents further metabolic activation of PCBs and conjugation with glutathione which provides protection against electrophiles, several reports show that the administration of radiolabeled PCBs results in protein, RNA, and DNA binding of radioactivity in cultured cells (Wong et al., 1979Go) and in vivo (Pereg et al., 2001Go). The identity of the reactive intermediates remains elusive. Different metabolic pathways and/or end products were suspected to be involved in the adduct formation by monochlorinated biphenyls (Amaro et al., 1996Go; McLean et al., 1996aGo; Oakley et al., 1996aGo). 32P-postlabeling studies with metabolically activated PCB3 showed that similar, but also unique DNA adducts were formed depending on whether the incubation environment was oxidizing or reducing (McLean et al., 1996bGo). It was suggested that some DNA adducts of lower chlorinated biphenyls may be derived from CYP-generated arene oxides. Unfortunately, these arene oxides were unavailable and could therefore not be tested in the present study. V79 cells do not express CYPs (McGregor et al., 1991Go), and thus the negative result in the mutation assay with PCB3 is not surprising and does not completely exclude a possible role of arene oxides in mutagenesis under other conditions. However, there were reports that other adducts may be formed from the oxidized products of catechols and HQs, i.e., semiquinones and Qs. Qs form adducts with nitrogen and sulfur nucleophiles in vitro (Amaro et al., 1996Go; Oakley et al., 1996bGo), and quinoid-derived protein adducts have been detected in the brains and livers of rats administered 2,2',5,5'-tetrachlorobiphenyl (Lin et al., 2000Go). Adducts with guanosine or isolated DNA have been detected in vitro (McLean et al., 1996bGo; Oakley et al., 1996bGo; Zhao et al., 2004Go), and the chemical structure of the reaction product of PCB3-2,5-quinone with N2 of guanine has been identified (Zhao et al., 2004Go). Moreover, DNA adducts have been observed after treatment of cultured human hepatocytes with PCB3 (Borlak et al., 2003Go). All these studies are in accordance with the findings of the present study which identifies PCB3 Qs as mutagenic in the HPRT gene. Likewise, the aneuploidogenic potential of PCB3 Qs observed in this study might be explained by the reactivity of Qs toward microtubular proteins which are highly susceptible to electrophiles due to their high content of sulfhydryl groups (Little et al., 1981Go).

Besides adduct formation by PCB Qs with DNA and microtubules, the induction of oxidative stress might be another possible mechanism for the initiating potential of PCB3 and some of its metabolites. The V79 cell line used in the HPRT assay expresses peroxidase activity (McGregor et al., 1991Go) and could therefore be able to generate reactive oxygen species by redox cycling. The initial product of redox cycling is superoxide. Secondary products are hydrogen peroxide and finally hydroxyl radicals. All three reactive oxygen species cause oxidative DNA base modifications as well as DNA strand breaks (summarized in Epe, 1996Go). Yet, no gene mutagenic potential of PCB3-HQs was observed in the HPRT assay, and the 3',4'-HQ–induced micronuclei containing DNA fragments were only observed at highly cytotoxic concentrations.

In contrast to the HPRT assay, the micronucleus assay represents a sensitive tool for the detection of both clastogens and aneuploidogens. All mono-and dihydroxylated PCB3 metabolites tested induced micronuclei containing whole chromosomes, but only at cytotoxic concentrations. Micronuclei containing DNA fragments were observed as well after treatment with 4'-HO and 3',4'-HQ, but again only at concentrations that induced cytotoxicity. The high concentrations necessary to cause those aneuploidogenic and clastogenic effects might indicate in vitro artifacts, yet, no micronuclei were observed after treatment with PCB3 at concentrations causing equal or even higher cytotoxicity. One indication of aneuploidogenic events is the activation of the anaphase checkpoint which controls the transition from metaphase to anaphase and is often reflected by an increase in the mitotic index due to accumulation of cells in metaphase. Surprisingly, only 3',4'-HQ induced an increase in the mitotic index. However, pre-anaphase:ana/telophase ratio was decreased, suggesting impairment with the transition into anaphase. The lack of an increase in the mitotic index by most of the putative aneuploidogenic metabolites might be due to the long exposure time, 24 h, equivalent to two population doublings. After such a long time period, the cell cycle distribution and the microscopic determination of the mitotic index do not necessarily reflect the initial cell cycle arrest (Schumacher et al., 2005Go). Furthermore, a second cell cycle arrest can prevent an accumulation of cells in mitosis, even if the transition of cells into anaphase is delayed or inhibited. In this case, a mitotic arrest can only be detected by differentiation between early mitotic cells prior to anaphase and cells in ana- and telophase (Lehmann and Metzler, 2004Go).

Further investigations are needed in order to clarify the relevance of the putative aneuploidogenic potential the PCB3 metabolites. However, the predominant finding of toxicologic relevance is the direct DNA reactivity of PCB3 Qs resulting in gene mutations. PCB3 is found in the air of contaminated sites and in urban areas (Uraki et al., 2004Go) and can be detected in human blood samples (DeCaprio et al., 2005Go). It is susceptible to metabolic attack and metabolic activation, and as we have shown here, this process generates metabolic species that are potent mutagens. Like our model compound PCB3, other lower chlorinated PCBs such as tetrachlorobiphenyls which are ubiquitous in our environment form reactive intermediates in vivo and may thus be of so far unknown and unexpected toxicological importance.


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
FUNDING
 REFERENCES
 
Supplementary data are available online at http://toxsci.oxfordjournals.org/.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
REFERENCES
 
National Institutes of Health (P42ES013661 to L.W.R.); Department of Defence (DAMD17-02-1-0241 to G.L.).


   ACKNOWLEDGMENTS
 
The authors would like to thank Heike Newill, Carolin Müller, and Dr Harald L. Esch for their help in the laboratory and Dr Hans Lehmler for synthesizing and characterizing the study chemicals.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Amaro AR, Oakley GG, Bauer U, Spielmann HP, Robertson LW. Metabolic activation of PCBs to quinones: Reactivity toward nitrogen and sulfur nucleophiles and influence of superoxide dismutase. Chem. Res. Toxicol. (1996) 9:623–629.[CrossRef][Web of Science][Medline]

Beland FA, Fullerton NF, Smith BA, Heflich RH. Formation of DNA adducts and induction of mutations in rats treated with tumorigenic doses of 1,6-dinitropyrene. Environ. Health Perspect. (1994) 102(Suppl. 6):185–189.

Borlak J, Hock A, Hansen T, Richter E. DNA adducts in cultures of polychlorinated biphenyl-treated human hepatocytes. Toxicol. Appl. Pharmacol. (2003) 188:81–91.[CrossRef][Web of Science][Medline]

Buchmann A, Ziegler S, Wolf A, Robertson LW, Durham SK, Schwarz M. Effects of polychlorinated biphenyls in rat liver: Correlation between primary subcellular effects and promoting activity. Toxicol. Appl. Pharmacol. (1991) 111:454–468.[CrossRef][Web of Science][Medline]

Clive D. Mutagenicity of thioxanthenes (hycanthone, lucanthone and four indazole derivatives) at the TK locus in cultured mammalian cells. Mutat. Res. (1974) 26:307–318.[Web of Science][Medline]

Davis B, Beach JM, Wade M, Klein AK, Hoch K. Risk assessment of polychlorinated biphenyls (PCBs) in indoor air. Toxicologist Suppl. Toxicol. Sci. (2002) 66:516.

Dean CE Jr, Benjamin SA, Chubb LS, Tessari JD, Keefe TJ. Nonadditive hepatic tumor promoting effects by a mixture of two structurally different polychlorinated biphenyls in female rat livers. Toxicol. Sci. (2002) 66:54–61.[Abstract/Free Full Text]

DeCaprio AP, Johnson GW, Tarbell AM, Carpenter DO, Chiarenzelli JR, Morse GS, Santiago-Rivera AL, Schymura MJ. Polychlorinated biphenyl (PCB) exposure assessment by multivariate statistical analysis of serum congener profiles in an adult Native American population. Environ. Res. (2005) 98:284–302.[Medline]

Digernes V, Astrup EG. Are datascreen terminals a source of increased PCB-concentrations in the working atmosphere? Int. Arch. Occup. Environ. Health (1982) 49:193–197.[CrossRef][Web of Science][Medline]

Duarte-Davidson R, Jones KC. Polychlorinated biphenyls (PCBs) in the UK population: Estimated intake, exposure and body burden. Sci. Total Environ. (1994) 151:131–152.[CrossRef][Medline]

Epe B. DNA damage profiles induced by oxidizing agents. Rev. Physiol. Biochem. Pharmacol. (1996) 127:223–249.[Web of Science][Medline]

Espandiari P, Glauert HP, Lehmler HJ, Lee EY, Srinivasan C, Robertson LW. Polychlorinated biphenyls as initiators in liver carcinogenesis: Resistant hepatocyte model. Toxicol. Appl. Pharmacol. (2003) 186:55–62.[CrossRef][Web of Science][Medline]

Espandiari P, Glauert HP, Lehmler HJ, Lee EY, Srinivasan C, Robertson LW. Initiating activity of 4-chlorobiphenyl metabolites in the resistant hepatocyte model. Toxicol. Sci. (2004) 79:41–46.[Abstract/Free Full Text]

Gabrio T, Piechotowski I, Wallenhorst T, Klett M, Cott L, Friebel P, Link B, Schwenk M. PCB-blood levels in teachers, working in PCB-contaminated schools. Chemosphere (2000) 40:1055–1062.[Medline]

Gallani B, Boix A, Di Domenico A, Fanelli R. Occurrence of NDL-PCB in food and feed in Europe. Organohal. Comp. (2004) 66:3561–3569.

Glauert HP, Robertson LW, Silberhorn EM. PCBs and tumor promotion. In: PCBs, Recent Advances in Environmental Toxicology and Health Effects—Robertson LW, Hansen LG, eds. (2001) Lexington, KY: The University Press of Kentucky. 355–371.

Grimalt JO, van Drooge BL, Ribes A, Vilanova RM, Fernandez P, Appleby P. Persistent organochlorine compounds in soils and sediments of European high altitude mountain lakes. Chemosphere (2004) 54:1549–1561.[Medline]

Gustafsson O, Andersson P, Axelman J, Bucheli TD, Komp P, McLachlan MS, Sobek A, Thorngren JO. Observations of the PCB distribution within and in-between ice, snow, ice-rafted debris, ice-interstitial water, and seawater in the Barents Sea marginal ice zone and the North Pole area. Sci. Total Environ. (2005) 342:261–279.[CrossRef][Medline]

Haag-Gronlund M, Kato Y, Fransson-Steen R, Scheu G, Warngard L. Promotion of enzyme altered foci in female rat 2,3,3',4,4',5-hexachlorobiphenyl. Toxicol. Appl. Pharmacol. (1997) 147:46–55.[CrossRef][Web of Science][Medline]

Hansen LG. The Ortho Side of PCBs: Occurence and Disposition (1999) Boston, MA: Kluwer Academic Publishers.

Hansen LG, O'Keefe PW. Polychlorinated dibenzofurans and dibenzo-p-dioxins in subsurface soil, superficial dust, and air extracts from a contaminated landfill. Arch. Environ. Contam. Toxicol. (1996) 31:271–276.[Web of Science]

Hermanson ME, Hites RA. Long-term measurements of atmospheric polychlorinated biphenyls in the vicinity of Superfund dumps. Environ. Sci. Technol. (1989) 23:1253–1258.

Hornbuckle KC, Green ML. The impact of an urban-industrial region on the magnitude and variability of persistent organic pollutant deposition to Lake Michigan. Ambio (2003) 32:406–411.[Medline]

Imsilp K, Schaeffer DJ, Hansen LG. PCB disposition and different biological effects in rats following direct soil exposure vs. PCBs off-gassed from the soil. Toxicol. Environ. Chem. (2005) 87:267–285.[CrossRef]

James MO. Polychlolinated biphenyls: Metabolism and metabolites. In: PCBs, Recent Advances in Environmental Toxicology and Health Effects—Robertson LW, Hansen LG, eds. (2001) Lexington, KY: The University Press of Kentucky. 35–46.

Jenssen D. Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells. Mutat. Res. (1982) 106:291–296.[Web of Science][Medline]

Jenssen D. The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells. Mutat. Res. (1986) 165:185–190.[Web of Science][Medline]

Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate M Jr, Kirchner S, Lorge E, Morita T, et al. Report from the in vitro micronucleus assay working group. Mutat. Res. (2003) 540:153–163.[Web of Science][Medline]

Kohler M, Tremp J, Zennegg M, Seiler C, Minder-Kohler S, Beck M, Lienemann P, Wegmann L, Schmid P. Joint sealants: An overlooked diffuse source of polychlorinated biphenyls in buildings. Environ. Sci. Technol. (2005) 39:1967–1973.[Medline]

Lehmann L, Esch HL, Kirby PA, Robertson LW, Ludewig G. 4-monochlorobiphenyl (PCB3) induces mutations in the livers of transgenic Fischer 344 rats. Carcinogenesis (2007) 28:471–478.[Abstract/Free Full Text]

Lehmann L, Metzler M. Bisphenol A and its methylated congeners inhibit growth and interfere with microtubules in human fibroblasts in vitro. Chem. Biol. Interact. (2004) 147:273–285.[CrossRef][Web of Science][Medline]

Lin PH, Sangaiah R, Ranasinghe A, Upton PB, La DK, Gold A, Swenberg JA. Formation of quinonoid-derived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2, 2', 5, 5'-tetrachlorobiphenyl. Chem. Res. Toxicol. (2000) 13:710–718.[CrossRef][Web of Science][Medline]

Little M, Krauhs E, Ponstingl H. Tubulin sequence conservation. Biosystems (1981) 14:239–246.[CrossRef][Web of Science][Medline]

Ludewig, G. (2001). Cancer Initiation by PCBs. In PCBs, Recent Advances in Environmental Toxicology and Health Effects (L. W. Robertson and L. G. Hansen, Eds.), pp. 337–354. The University Press of Kentucky, Lexington, KY.

Mayes BA, McConnell EE, Neal BH, Brunner MJ, Hamilton SB, Sullivan TM, Peters AC, Ryan MJ, Toft JD, Singer AW, et al. Comparative carcinogenicity in Sprague-Dawley rats of the polychlorinated biphenyl mixtures Aroclors 1016, 1242, 1254, and 1260. Toxicol. Sci. (1998) 41:62–76.[Abstract/Free Full Text]

McGregor DB, Edwards I, Wolf CR, Forrester LM, Caspary WJ. Endogenous xenobiotic enzyme levels in mammalian cells. Mutat. Res. (1991) 261:29–39.[CrossRef][Web of Science][Medline]

McLean MR, Bauer U, Amaro AR, Robertson LW. Identification of catechol and hydroquinone metabolites of 4-monochlorobiphenyl. Chem. Res. Toxicol. (1996a) 9:158–164.[CrossRef][Web of Science][Medline]

McLean MR, Robertson LW, Gupta RC. Detection of PCB adducts by the 32P-postlabeling technique. Chem. Res. Toxicol. (1996b) 9:165–171.[CrossRef][Web of Science][Medline]

Moore MM, Clive D, Howard BE, Batson AG, Turner NT. In situ analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/- mouse lymphoma cells. Mutat. Res. (1985) 151:147–159.[CrossRef][Web of Science][Medline]

Oakley GG, Devanaboyina U, Robertson LW, Gupta RC. Oxidative DNA damage induced by activation of polychlorinated biphenyls (PCBs): Implications for PCB-induced oxidative stress in breast cancer. Chem. Res. Toxicol. (1996a) 9:1285–1292.[CrossRef][Web of Science][Medline]

Oakley GG, Robertson LW, Gupta RC. Analysis of polychlorinated biphenyl-DNA adducts by 32P-postlabeling. Carcinogenesis (1996b) 17:109–114.[Abstract/Free Full Text]

Oberly TJ, Michaelis KC, Rexroat MA, Bewsey BJ, Garriott ML. A comparison of the CHO/HGPRT+ and the L5178Y/TK+/- mutation assays using suspension treatment and soft agar cloning: Results for 10 chemicals. Cell. Biol. Toxicol. (1993) 9:243–257.[CrossRef][Web of Science][Medline]

Pereg D, Tampal N, Espandiari P, Robertson LW. Distribution and macromolecular binding of benzo[a]pyrene and two polychlorinated biphenyl congeners in female mice. Chem. Biol. Interact. (2001) 137:243–258.[CrossRef][Web of Science][Medline]

Persson NJ, Pettersen H, Ishaq R, Axelman J, Bandh C, Broman D, Zebuhr Y, Hammar T. Polychlorinated biphenyls in polysulfide sealants–occurrence and emission from a landfill station. Environ. Pollut. (2005) 138:18–27.[CrossRef][Medline]

Rushmore TH, Kong AN. Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr. Drug Metab. (2002) 3:481–490.[CrossRef][Web of Science][Medline]

Schumacher DM, Metzler M, Lehmann L. Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione. Arch. Toxicol. (2005) 79:110–121.[CrossRef][Web of Science][Medline]

Silberhorn EM, Glauert HP, Robertson LW. Carcinogenicity of polyhalogenated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol. (1990) 20:440–496.[Medline]

Srinivasan A, Lehmler HJ, Robertson LW, Ludewig G. Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites. Toxicol. Sci. (2001) 60:92–102.[Abstract/Free Full Text]

Tampal N, Myers S, Robertson LW. Binding of polychlorinated biphenyls/metabolites to hemoglobin. Toxicol. Lett. (2003) 142:53–60.[CrossRef][Web of Science][Medline]

Tsutsui T, Tamura Y, Suzuki A, Hirose Y, Kobayashi M, Nishimura H, Metzler M, Barrett JC. Mammalian cell transformation and aneuploidy induced by five bisphenols. Int. J. Cancer (2000) 86:151–154.[CrossRef][Web of Science][Medline]

Uraki Y, Suzuki S, Yasuhara A, Shibamoto T. Determining sources of atmospheric polychlorinated biphenyls based on their fracturing concentrations and congener compositions. J. Environ. Sci. Health (2004) A39:2755–2772.

WHO. (2003) Polychlorinated Biphenyls: Human Health Aspects. Concise International Chemical Assessment Document. World Health Organization, Geneva, Switzerland.

Wilson NK, Chuang JC, Lyu C. Levels of persistent organic pollutants in several child day care centers. J. Expo. Anal. Environ. Epidemiol. (2001) 11:449–458.[CrossRef][Web of Science][Medline]

Wong A, Basrur P, Safe S. The metabolically mediated DNA damage and subsequent DNA repair by 4-chlorobiphenyl in Chinese hamster ovary cells. Res. Commun. Chem. Pathol. Pharmacol. (1979) 24:543–550.[Web of Science][Medline]

Zhao S, Narang A, Ding X, Eadon G. Characterization and quantitative analysis of DNA adducts formed from lower chlorinated PCB-derived quinones. Chem. Res. Toxicol. (2004) 17:502–511.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
100/1/88    most recent
kfm204v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (2)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zettner, M. A.
Right arrow Articles by Lehmann, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zettner, M. A.
Right arrow Articles by Lehmann, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?