Mutagenesis, Vol. 17, No. 4, 301-307,
July 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Mutation spectrum of 1,2-dibromo-3-chloropropane, an endocrine disruptor, in the lacI transgenic Big Blue Rat2 fibroblast cell line
1 Toxicology Laboratory, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Korea and 2 Department of Biochemistry and Molecular Biology, Hanyang University, Ansan, Kyunggi-do, Korea
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Abstract |
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1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant against nematodes, has been extensively studied for genotoxicity, carcinogenicity and damage to male reproduction-related organs, as a possible endocrine disruptor. However, the precise mechanisms involved in DBCP-induced mutagenesis and carcinogenesis are as yet unknown. Thus, in this study the mutagenicity and mutation spectrum of DBCP was determined using the lacI transgenic Big Blue Rat2 fibroblast cell line. In determining the optimal concentration of DBCP in Big Blue Rat2 fibroblast cells, the 50% inhibition concentration was calculated to be 0.75 mM. When cells were exposed to DBCP concentrations of 0.21, 0.39 and 0.75 mM, the respective relative survival rates were ~80, 70 and 50%. The mean mutant frequencies (MFs) (x 10-5, ± SEM) of the medium and 1% DMSO solvent controls were determined as 6.43 ± 0.616 and 5.28 ± 1.086, respectively. The MFs (x 10-5, ± SEM) of cells exposed to 0.21, 0.39 and 0.75 mM DBCP were 8.09 ± 1.02, 10.86 ± 2.17 and 12.26 ± 0.79, respectively, with a dose-dependent effect (ANOVA, P = 0.007). Moreover, MF values for the 0.75 and 0.39 mM DBCP-treated groups were statistically significant (ANOVA, P < 0.05). The majority of recovered mutations (31/40, 77.5%) were single base pair substitutions in the DBCP-induced groups. Among 31 single base pair substitutions, 25 (62.5%) occurred at G:C base pairs, while six (15%) were at A:T base pairs. The predominant mutation was G:C
A:T transitions (16/40, 40%), followed by G:CT:A transversions (9/40, 22.5%). We conclude that DBCP is a possible base substitution mutagen, especially at guanine bases. |
Introduction |
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1,2-Dibromo-3-chloropropane (DBCP), a soil fumigant against nematodes, is a halogenated alkane compound, which is widely used in agriculture. In addition, DBCP has been extensively studied as an endocrine disruptor (Sandifer et al., 1979
; Potashnik and Yanai-Inbar, 1987) for its potential to cause damage in male reproduction-related organs such as testis and sperm cells. This compound is also classified as a multi-organ animal carcinogen and a possible human carcinogen (IARC, 1986). Also, DBCP is known to be genotoxic in several in vitro and in vivo bacterial and mammalian test systems, i.e. reverse mutations in Salmonella typhimurium (Moriya et al., 1983; Teramoto and Shirasu, 1989) and recessive color spots in mouse (Sasaki et al., 1986), suggesting that it induces point mutations. It was also reported that DBCP induces chromosome aberrations and sister chromatid exchange in cultured Chinese hamster cells (Tezuka et al., 1980), non-disjuction of the Y-chromosome in workers exposed to DBCP (Kapp and Jacobson, 1979) and DNA breakage in multiple mouse organs with the alkaline single cell gel electrophoresis (Comet) assay (Sasaki et al., 1998). In addition, it has been reported that DBCP is metabolized to DNA-reactive metabolites formed by P450 oxidative metabolism and by conjugation with glutathione (Lag et al., 1994; Soderlund et al., 1995).
Mutagenic assays such as those stated above could be used to screen environmental mutagens, such as DBCP, for carcinogenic potential on the basis that mutagens often produce characteristic patterns of DNA sequence alterations (Dogliotti, 1996), called a mutation spectrum. Elucidation of the mutation spectrum of specific carcinogens provides a basis for understanding the cancer etiology and mechanisms of action behind chemical carcinogenesis (Gorelick, 1995).
Recently developed transgenic mutagenesis assay systems, including the Big BlueTM mutagenesis system, are useful and powerful tools for evaluating the genotoxicity of chemicals, providing insight into mechanisms of carcinogenesis and mutagenesis of chemicals based on such information as mutation pattern, frequency and location in the sequence context of the lacI target gene (Gorelick, 1995). The lacI transgenic Big Blue Rat2 fibroblast cell line carries over 60 copies of the 
shuttle vector (Dycaico et al., 1994) containing the lacI gene as a target (Summers et al., 1989; Lundberg et al., 1993; Heddle and Tao, 1995). The lacI gene is very useful, as a mutational target, for the study of the mutational characteristics of carcinogens for several reasons. First, the relatively small size (1080 bp coding region) of the lacI gene facilitates sequence analysis. Second, expression of the repressor protein permits a rapid colorimetric assay to screen for mutations. In addition, it is possible to make use of the large historical database in subsequent comparisons, allowing elucidation of the underlying mechanisms leading to mutations (Kohler et al., 1991). The mutations induced in the lacI gene can be easily quantified by mutant frequency (MF) and the precise mutation type and distribution can quickly be identified by direct sequencing. Moreover, mutations in the lacI gene induced by chemicals reflect the effects of mutagens on other endogenous genes, such as protooncogenes and tumor suppressor genes, and mutations occurring in these genes are the most common events in many types of human cancer (Gossen et al., 1989; Kohler et al., 1991; Tao et al., 1993). In our previous reports, we elucidated the mutation spectrum of 4-nitroquinoline N-oxide (Ryu et al., 1998, 1999). We also reported the mutant frequency of atrazine with cytogenetic analysis using Big Blue Rat2 cells (Ryu et al., 2000).
The goal of this study was to elucidate how the mutagenicity and/or carcinogenicity of DBCP affects the mechanism of action as a carcinogen through the analysis of mutant frequency (MF) and mutation spectrum in the lacI transgenic Big Blue Rat2 fibroblast cell line after exposure to DBCP.
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Materials and methods |
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Chemicals
1,2-Dibromo-3-chloropropane (97%, lot. no. CKM5599; Wako Pure Chemical Co., Japan), N-methyl-N-nitrosourea (MNU) (Sigma, St Louis, MO), dimethylsulfoxide (DMSO) (Sigma), 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), Big Blue top agarose, Big Blue bottom agar and Big Blue PCR primers (Stratagene, La Jolla, CA) were used in the experiments.
Cell culture
The lacI transgenic Big BlueTM Rat2 fibroblast cell line was purchased from Stratagene (La Jolla, CA). This cell line is derived from a Rat2 embryonic fibroblast cell line (CRL 1764; ATCC, Rockville, MD) transfected with the Big Blue shuttle vector ( lacI/
lacZ shuttle vector) and plasmid pSV2NEO, which provides an antibiotic selection marker. The shuttle vector, which has been integrated into the Rat2 genome at two sites (~60 copies/cell), is identical to that used to generate the Big BlueTM mouse. The lacI transgenic Big Blue Rat2 fibroblast cells were cultured as a monolayer in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, NY) containing 50 U/ml penicillin, 50 mg/ml streptomycin, 200 mg/ml geneticin (G418) and 10% heat-inactivated defined fetal bovine serum (Gibco BRL) at 37°C in a humidified incubator containing 5% CO2 (Ryu et al., 1999). Under these conditions the average cell cycle time was ~19 h. Low passage (<5) cells were used in each experiment. Cells were treated at 30–40% confluence (~3.5x105 cells in a 25 cm2 flask). A solution of 0.25% trypsin–EDTA (Gibco BRL) was used for subculture.
Cytotoxicity of DBCP
The survival rate of the lacI transgenic Big Blue Rat2 fibroblast cells following exposure to DBCP was assessed by exposing cells in 25 cm2 flasks (each containing ~3.5x105 cells) to each of 0, 0.2, 0.4, 0.8, 1.6 and 3.2 mM DBCP dissolved in DMSO for 24 h. Following the exposure for 24 h, cells from each flask were trypsinized and counted. Based on the cell count, replicate flasks were plated with 2.5x104 cells each in 75 cm2 flasks. The survival rate at each exposure concentration was assessed as the cumulative cell growth relative to the medium control at 120 h after plating with trypan blue dye exclusion (Saranko et al., 1998).
Mutagenesis assay Exposure to DBCP
The mutagenicity of DBCP in the lacI transgenic Big Blue Rat2 fibroblast cell line was assessed by exposing cells in two 25 cm2 flasks per exposure concentration (0.21, 0.39 or 0.75 mM DBCP) for 24 h. These concentrations correspond to ~80, 70 and 50% relative survival, respectively. This experiment was repeated in duplicate. Solvent (1% DMSO), medium and positive controls (100 µg/ml MNU) were included. After treatment, the cells from each exposed flask were trypsinized and counted. Then 4x105 cells were plated in a 75 cm2 flask. When they reached confluence, cells were trypsinized, pelleted and flash frozen in liquid nitrogen. Cell pellets were stored at –80°C.
Isolation of genomic DNA
Genomic DNA was isolated from lacI transgenic Big Blue Rat2 fibroblast cells using the RecoverEase protocol (Stratagene, La Jolla, CA). In essence, cell pellets were thawed on ice, resuspended in 8 ml of ice-cold lysis buffer (8.20 g/l NaCl, 0.22 g/l KCl, 120 g/l sucrose, 0.30 g/l EDTA, 10 ml Triton X-100, 1.58 g/l Tris–HCl, pH 8.3) and homogenized with a tissue grinder. The homogenate was filtered through a 100 µm nylon mesh filter into 50 ml tubes and centrifuged at 1100 g for 12 min at 4°C. The supernatant was discarded and residual supernatant was removed from the tubes using a cotton swab. The pellet was incubated at 50°C for 45 min in the presence of 70 µl of digestion buffer containing RNace-It RNase cocktail (20 µl/ml digestion buffer; Stratagene) and 70 µl of proteinase K solution. The contents of the tube were dialyzed for 48 h by placing the DNA extract on the wetted surface of a membrane floating on 10 mM Tris, 1 mM EDTA buffer, pH 7.4. The viscous DNA was collected and stored at 4°C until packaging.
Packaging and plating of DNA
After DNA concentrations were adjusted to 0.5 mg/ml, the genomic DNA was incubated with Transpack packaging extract (Stratagene) to excise the vector target and package it into a head according to the Stratagene Big Blue instruction manual. The titer of the rescued phage was estimated by plating the packaged phage in serial dilutions. For plating, the volume of the phage equivalent to 15 000 plaque forming units was added to 2 ml of Escherichia coli SCS-8 cells (Stratagene) in 10 mM MgSO4. Pre-warmed Big Blue top agarose containing 1.5 mg/ml X-gal (dissolved in dimethylformamide) was added and the contents were poured onto 25x25 cm assay trays containing 250 ml Big Blue bottom agar (agar, casein peptone, yeast extract, MgSO4 and NaCl). Trays were incubated for 18 h at 37°C and scored for blue mutant plaques. The total number of plaques in each plate was estimated by counting four sectors of 5x5 cm area, averaging these numbers and multiplying by a scaling factor. Blue mutant plaques were counted and picked into individual tubes containing 0.5 ml SM buffer (5.8 g/l NaCl, 2.0 g/l MgSO4·7H2O, 50 ml 1 M Tris–HCl, pH 7.5, and 5 ml of 2% w/v gelatin) for further characterization. To confirm the mutant phenotype and for future use in DNA sequence analysis, all recovered putative mutant phages were diluted to 1:200 and replated on 100 mm plates with 3.5 ml of top agarose containing 1.5 mg/ml X-gal. Observed sectored plaques were also verified for their phenotype as previously specified and confirmed sectored plaques were scored separately. The lacI MF value was calculated by dividing the number of verified mutant plaques by the total number of plaques analyzed.
Mutation spectrum
The lacI gene was sequenced from lacI mutant of solvent and medium control cells (collectively considered spontaneous mutants) and of cells exposed to 0.75 mM DBCP. Each mutant was purified by replating on a 100 mm plate at low density. From each plate, a single mutant plaque was isolated, cored, placed into 25 µl of deionized water and then boiled for 5 min. The lacI gene was amplified by PCR in 100 µl of PCR reaction mixture containing 15 µl of phage DNA, 2 µl each of forward (positions –53 to –37, 5'-CCCGACACCATCGAATG-3') and reverse (positions 1201 to 1185, 5'-ACCATTCCACACAACATAC-3') Big Blue PCR primers, 25 mM dNTPs and 5 U ExTaq polymerase (TaKaRa Shuzo Co., Japan). After the initial denaturation step at 95°C for 5 min and an extension step at 72°C for 5 min, 30 cycles of amplification were performed as follows: denaturation at 95°C for 90 s, annealing at 55°C for 90 s and elongation at 72°C for 150 s, with a final extension at 72°C for 10 min, using a thermocycler (Robocycler 40 temperature cycler; Stratagene). The resulting 1300 bp fragment was purified with a PCR purification kit (Inje Biotech, Seoul, Korea) according to the manufacturer's instructions and visualized on a 1% low melting point (LMP) agarose gel containing 1 mg/ml ethidium bromide. The PCR products were sequenced with an ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer, CA) on an Applied Biosystem 373 DNA sequencer (Perkin-Elmer). The primers used are shown in Table I. The purified DNA sample corresponding to ~200 ng was added to 3.2 pmol/ml primer and terminator dyes. The sequencing reactions were carried out for 25 cycles as follows: 96°C for 30 s; 50°C for 15 s; 60°C for 4 min.
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Statistical analysis
Analysis of variance (ANOVA) was performed to evaluate differences in lacI mutant frequency between treatment groups at the level of
= 0.05. A Dunnett's multiple comparison test was used to compare each treatment group with untreated controls. The 
2 test was used for statistical treatment of mutational spectra. |
Results |
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Cytotoxicity of DBCP
Relative survival of lacI transgenic Big Blue Rat2 fibroblast cells following exposure to a range of concentrations of DBCP was determined by recording their cumulative growth over a period of 180 h. The survival rate relative to the medium control was determined as a percentage of the number of cells surviving 120 h after plating. Exposure of lacI transgenic Big Blue Rat2 fibroblast cells to 0.1, 0.2, 0.4, 0.8 and 1.6 mM DBCP resulted in 82, 81, 68, 44 and 5% survival compared to the medium control, respectively (Figure 1
). The 50% inhibition concentration (IC50) of DBCP was calculated as 0.75 mM in lacI transgenic Big Blue Rat2 fibroblast cells by the equation y = –55.3686x + 91.4365 for relative survival. In addition, a dose-dependent relationship between DBCP and survival rate was observed with correlation efficiency r2 = 0.9875.
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Mutagenesis assay Mutant frequencies (MFs)
The mutagenicity of DBCP to the lacI transgene in lacI transgenic Big Blue Rat2 fibroblast cells was determined. The MFs obtained from each independent experiment were dose-dependent (ANOVA, P = 0.007). Cells exposed to medium only and to medium containing 1% DMSO (solvent control) showed mean MFs (x 10-5, ± SEM) of 6.43 ± 0.616 and 5.28 ± 1.086, respectively. The MFs (x 10-5, ± SEM) of cells exposed to 0.21, 0.39 and 0.75 mM DBCP were 8.09 ± 1.02, 10.86 ± 2.17 and 12.26 ± 0.79, respectively. Moreover, the 0.75 and 0.39 mM DBCP-treated groups showed a statistically significant difference (ANOVA, P < 0.05) compared to the medium control (Table II
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Analysis of mutational spectrum
The sequences of the lacI gene were determined in mutants recovered from the medium and solvent controls (mutation identified in 33 of 40 mutants, with 26 independent mutations) and 0.75 mM DBCP-induced mutants (mutation identified in 42 mutants, with 40 independent mutations). Mutants were considered to be siblings if the same mutation occurred more than once in independently exposed cell culture samples and were considered to represent a single in vitro mutational event by clonal expansion. One mutant was found to have two mutations in the DBCP-induced and spontaneous groups. For each mutant the entire lacI gene (1080 bp) was sequenced and the results are summarized in Tables III–VI
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Single base pair substitution mutations were prevalent in both the control (Table III
) and DBCP-induced groups (Table IV). Single base pair substitutions from recovered mutations were 19 of 26 (73%) in the control and 31 of 40 (77.5%) in the DBCP-induced group (Tables III, IV and VI). Of 40 independent DBCP-induced mutations, 31 were single base pair substitutions (Table IV) and nine (22.5%) were deletions or insertions (Table V). Among single base pair substitutions of the DBCP-induced group, it was observed that 25 (62.5%) occurred at G:C base pairs, while six (15%) were at A:T base pairs, as shown in Table VI. The predominant type of mutation after DBCP treatment was G:CA:T transitions (16/40, 40%), followed by G:CT:A transversions (9/40, 22.5%) (Table VI). In addition, two single base insertions, four single base deletions, one double base pair deletion and one quadruple base pair deletion, leading to a frameshift mutation and triple base pair deletion, were observed in the DBCP-induced group (Table V). In consequence, eight frameshift mutations and one amino acid deletion out of 40 total mutations (22.5%) after DBCP treatment are summarized in Table VI.
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The mutation spectrum in the DBCP-induced cells was significantly different (P = 0.05) when compared with controls (Table VI
). G:CA:T transitions were >2-fold more frequent in the DBCP-treated group (47.5%) when compared with the spontaneous groups (23.1%). The percentage of transitions at CpG sites was higher in the spontaneous (4/6, 67%) versus the treatment group (8/16, 50%). While G:CC:G transversions in the control group formed a large proportion (23.1%), few such mutations occurred in the DBCP treatment group. |
Discussion |
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Many synthetic chemicals are produced by industry to improve the quality of human life. The establishment of methods for detecting synthetic chemicals that may pose a genetic hazard in the environment is the subject of great concern at present (WHO, 1971
).
Several rapid and reliabile assay systems have been introduced for this purpose, including the bacterial gene mutation reversion test (Ames et al., 1973, 1975; Maron and Ames, 1983), the mammalian cell chromosome aberration assay (Ishidate and Odashima, 1977) and the rodent micronucleus assay (Schmid, 1975; Hayashi et al., 1992). These assay systems are now widely used to evaluate the genotoxicity of chemicals and also frequently adopted as the methods of choice in the preparation of an index of genotoxicity world-wide. Furthermore, they have applications in screening for the detection of possible carcinogenic substances in the environment. Since tens of thousands of man-made chemicals that have been introduced into the environment in the last few decades must also be tested for their damaging effects on DNA, agents that cause this damage should be identified. Some chemicals, such as endocrine-disrupting chemicals, cause severe problems for humans and the ecosystem because they have existed ubiquitously at very low concentrations for some time in nature, subjecting humans and the wildlife to long-term exposure. However, such toxicity evaluation tools cannot elucidate the mode and/or mechanisms of action of chemicals, especially carcinogens and mutagens. The focus is on recently developed transgenic mutagenesis assay systems to meet this requirement, as these assay systems can serve as powerful tools in more accurately predicting the mutation spectrum induced in cancer-related genes.
In this study, lacI transgenic Big Blue Rat2 fibroblasts were exposed to DBCP in an effort to understand how it may contribute to the mutational spectrum in the lacI transgene. It was observed that exposure of lacI transgenic Big Blue Rat2 fibroblasts to 0.39 and 0.75 mM DBCP for 24 h resulted in a 2-fold increase in MF compared with controls (P < 0.05). The MFs (x 10-5, ± SEM) of cells exposed to 0.21, 0.39 and 0.75 mM DBCP were 8.09 ± 1.02, 10.86 ± 2.17 and 12.26 ± 0.79, respectively, and the effect was dose-dependent (ANOVA, P = 0.007) (Table II). These genotoxic results are consistent with previously reported results in that treatment of cultured Chinese hamster cells with DBCP caused a significant dose-dependent increase in the frequency of sister chromatid exchanges and chromosomal aberrations at concentrations of 0.01–1 mM, without a metabolic activation system (Tezuka et al., 1980). In addition, DBCP inhibited DNA synthesis in HeLa cells at a concentration of 10 mM without a metabolic activation system (Painter and Howard, 1982). These results suggest that DBCP is mutagenic in mammalian cells in vitro, although there is some controversy as to whether metabolic activation is required for carcinogenic and mutagenic activity of DBCP (Lag et al., 1994; Brunborg et al., 1996). Because an impurity, epichlorohydrin, which is used as a stabilizer in commercial DBCP, is also a potent mutagen in a bacterial reversion assay (Teramoto and Shirasu, 1989), the genotoxicity of DBCP may be affected by the quantity of this chemical present.
From the results of the sequencing analysis, the majority of recovered mutations consisted of single base pair substitutions in both the control (Table III) and DBCP-induced groups (Table IV): 73 (19/26) and 77.5% (31/40), respectively (Table VI). Most of these mutations (56%) were found within the first 360 bp, which contains the negative complementing region of the lacI gene. A similar phenomenon in the lacI gene was reported by Adler et al. (1972) and in our previous results (Ryu et al., 1999). The predominance of single base substitution mutations in our experiment suggests that these mutations arose in the lacI transgenic Big Blue fibroblast cells, not in E.coli during the mutant recovery process, as only 11% of the spontaneous mutations recovered in bacteria were single base substitutions (Schaaper et al., 1986).
The background lacI mutational spectrum generated in these experiments was very similar to the background mutational spectra previously reported in lacI transgenic Big Blue Rat2 fibroblast cells (Saranko et al., 1998; Ryu et al., 1999; Sprung et al., 2001) and to those of untreated lacI transgenic rats (Bol et al., 2000) and mice (Shane et al., 2000). Among 19 spontaneous base substitution mutations, two nonsense mutations and one silent mutation with no change in amino acid sequence were observed (Table III). The remaining mutations were all missense mutations. G:CA:T transitions were the prevalent mutation in the background spectrum. Many of these (4/6, 67%) occurred at CpG dinucleotides. These transition mutations at CpG sites are thought to be the result of hypermethylation of the lacI transgene by mammalian DNA methylase and subsequent deamination of these 5-methylcytosine residues at CpG sites (Kapp and Jacobson, 1979; Sisk et al., 1994).
The DBCP-induced lacI mutation spectrum was significantly different from the spontaneous mutation spectrum. As shown in Table VI, the most common DBCP-induced mutations were G:CA:T transitions (40%), followed by G:CT:A transversions (22.5%). G:CA:T transitions in the DBCP-treated group (47.5%) were >2-fold more frequent when compared with the spontaneous groups (21.4%). The percentage of transitions at CpG sites was higher in the spontaneous (4/6, 67%) versus the treatment group (8/16, 50%). Among 31 single base pair substitutions, 25 (62.5%) occurred at G:C base pairs, while six (15%) occurred at A:T base pairs.
DBCP was reported to alkylate calf thymus DNA to form several N7-guanyl adducts. Among adducts that were detected in DBCP-exposed rat liver, a bis-guanyl adduct may be particularly important in the toxicity of DBCP, since it may lead to DNA crosslinking (Humphreys et al., 1991). These adducts are known to induce G:CA:T transitions.
Consequently, we conclude that DBCP is a possible base substitution mutagen, especially affecting guanine bases. Furthermore, this study is the first report of the mechanism of DBCP-induced mutagenesis in the lacI gene using the transgenic Big Blue mutagenesis system. The possibility of DBCP causing mutations in endogenous genes (protooncogenes, tumor suppressor genes, repair-related genes, etc.) suggests that the mutagenic properties of DBCP are involved in the initiation stage of carcinogenesis. To provide a better understanding of the mechanism of DBCP mutagenesis in vivo, further investigations of the mutagenic properties of DBCP and its metabolites in different tissues and in different species are needed.
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Acknowledgments |
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The authors thank Ms Wonhee Jang (Rice University, TX) for helpful assistance in manuscript preparation.
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Notes |
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3 To whom correspondence should be addressed. Tel: +82 2 958 5070; Fax: +82 2 958 5059; Email: ryujc{at}kist.re.kr
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Received on October 1, 2001; accepted on February 5, 2002.
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