Abstract
Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction–single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.
Keywords
Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of chemicals with widespread occurrence in the environment. They are a family of lipophilic nonpolar chemicals comprising two or more benzene rings, and are formed mainly as a result of pyrolytic processes, in particular the incomplete combustions of organic materials. Also, PAHs are formed naturally and are present in crude oil. Humans are exposed to PAHs by inhalation, ingestion, and skin contact. Tobacco smoke and urban air are the two major sources of nonoccupational respiratory exposure. Epidemiologic studies have associated PAHs exposure with an increase in the incidence of lung, skin, larynx, kidney, and bladder cancers, whereas lungs are the major target organs (IARC 1983; Boffetta, Jourenkova, and Gustavsson 1997).
Several hundred PAHs have been characterized; benzo[a]-pyrene (BP) and dibenzopyrenes (DBPs) are two of the most widely distributed and studied compounds. BP is a classical complete carcinogen; it can act both as an initiator and promoter in the carcinogenic process (IARC 1973). It has been thoroughly studied and requires metabolic activation for carcinogenicity. Active metabolites, of which benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is probably the ultimate carcinogen, which binds covalently to DNA, causing DNA, damage (Levin et al. 1978).
DBP is one of the most genotoxic PAH of environmental significance ever tested, because of its high in vivo and in vitro genotoxicity and also because of its wide distribution and stability in the environment (Cavalieri et al. 1989; Busby et al. 1995; Arif, Smith, and Gupta 1999; Durant et al. 1999). As well as BP, DBP requires metabolic activation for carcinogenicity (Shaw and Connell 1994; Shou et al. 1996).
The single-cell gel electrophoresis method (SCGE), also called comet assay, is a rapid and sensitive tool to demonstrate the damaging effects of different compounds or physical treatments on DNA at individual cell level. Cells with damaged DNA display increased migration of DNA fragments from the nucleus, generating a “comet” shape (Singh et al. 1988). Among the various versions of the assay, the alkaline method permits the detection of the broadest spectrum of DNA damage (DNA single- and/or double-strand breaks, DNA-DNA or DNA-protein adducts, apurinic/apyrimidic sites, oxidative base damages, and apoptosis) under different conditions (Tice et al. 2000; Hartmann et al. 2003). There are several parameters that enable the measurement of the DNA damage by this technique, such as the proportion of cells with tail, length of migration, percentage of DNA in the tail, tail moment, and comet moment (Tice et al. 2000).
This study was undertaken to investigate several potential mechanisms by which these PAHs might produce their genotoxic effects in a diploid human lung fibroblast cell line at noncytotoxic concentrations. The diploid human lung cell line MRC-5 was chosen for this study considering that inhalation is the main cause of human exposure and also because lung cancer is tightly associated to BP and DBP exposure (Boffetta, Jourenkova, and Gustavsson 1997). In this sense, the ability of BP and DBP to induce point mutations at codon 12 of the K-ras protooncogene was also evaluated. The effects observed in the comet assay were compared to another cytotoxic and genotoxic indicator such as sister-chromatid exchange (SCE) induction.
MATERIALS AND METHODS
Cell Culture and Treatments
The human lung fibroblast cell line MRC-5 (ATCC no. CCL171) was used for the experiments. Cells were grown as monolayers in minimal essential medium (GIBCO BRL, Los Angeles, USA), supplemented with 10% inactivated fetal calf serum, 50 IU/ml of penicillin, and 50 μg/ml of streptomycin sulphate at 37°C in a 5% CO2 atmosphere. Benzo[a]pyrene (BP) (Sigma, St Louis, Missouri, USA) (5 mM stock solution in DMSO) and dibenzo[a,l]pyrene (DBP) (Sigma) (5 mM stock solution in DMSO) were added directly to cell medium (1:100 in relation to the final volume of cell culture) in order to have the following final concentrations: 3.9 μM, 19.8 μM, and 39 μM of BP and 1.65 μM, 8.27 μM, and 16.53 μM of DBP. The highest dose for each compound was established according to the highest tolerable dose criteria, determined in pilot experiments (data not shown). The time of incubation depended on the assay and was described in each particular technique. Two control groups were included for each assay: one of untreated cells and the other group comprising DMSO exposed cells. All experiments were performed twice in independent trials to assess reproducibility.
Sister-Chromatid Exchange (SCE) Assay
Before each experiment, cells were grown until confluence in order to obtain cells in the G0/G1 phase due to the cessation of growth by contact inhibition. Then, cells were grown in culture medium added with 10 μg/ml of 5′-bromo-2′-deoxyuridine (BrdU) (Sigma) in complete darkness for 38 h. Twelve hours after the initiation of the culture, the cells were treated with PAHs and left for the final 26 h. Colchicine (0.1 μg/ml final concentration) was added for the last 2 h. prior to harvest the cultures. The SCE assay was performed according to Perry and Wolff (1974) with some modifications, as previously described (Mourón et al. 2004). SCEs were scored in 50 cells per treatment and experience, bringing a final number of 100 cells. Proliferation of cells was evaluated in more than 100 cells, determining the proportion of first (M1), second (M2), and third or more (M3) mitotic divisions. The proliferation index (PI) was calculated according to the formula PI = (M1 + 2M2 + 3M3)/total cells.
Comet Assay
Subcultures for experiments were set up the day before treatment. Approximately, 2 × 105 cells at logarithmic growth phase were treated with BP and DBP for 2 h. Cell viability was determined using the trypan blue stain exclusion method immediately after treatment.
The comet assay was performed according to the method of Singh and coworkers (1988) with some small modification as described previously (Mourón, Golijow, and Dulout 2001). Briefly, after agarose solidification, the slides were immersed overnight at 4°C in freshly lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10) containing 1% Triton X-100 and 10% dimethylsulfoxide, added just before use. Two slides were prepared from each control and treatment group under dimmed light conditions. After lysis, the slides were placed on an horizontal gel electrophoresis unit filled with fresh electrophoretic buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13), left for 20 min for DNA unwinding and then electrophoresed for 30 min at 1.25 V/cm (300 mA). These procedures were always performed at 4°C under dim light. After electrophoresis, the slides were washed with neutralizing buffer (0.4 M Tris, pH 7.5) and the cells were stained with SYBR Green I (Molecular Probes, Eugene, Oregon, USA) at the recommended dilution.
Observations were made at 400× magnification using a fluorescent microscope (Olympus BX40, equipped with a 515 to 560-nm excitation filter) connected through a Sony 3CCD-IRIS color video camera. The image for each individual cell was acquired immediately after opening the microscope shutter to the computer monitor, employing the Image Pro Plus 3.0 program (Media Cybernetics, Madison, USA). Pictures of 100 randomly selected cells (50 cells from each of the two replicate slides) were analyzed. The comet moment (Wang et al. 2001) was calculated using the Image Pro Plus 3.0 software. This parameter calculates a moment of the whole comet image, so it is considered a more sensitive estimator for individual fragments of DNA within different types of tails (Kent et al. 1995).
DNA Extraction
Approximately, 6000 confluent cells grown in 24-well dishes were treated with BP and DBP for 24 h at 37°C in a 5% CO2 atmosphere. Three repetitions for each treatment (controls and doses) were done for each experiment. After the culture lapse, cells were washed twice with phosphate-buffered saline (PBS) and then incubated for 3 h at 56°C in 300 μl of extraction buffer (50 mM Tris-HCl pH 8.5; 1 mM EDTA; 1% Triton X-100; and 0.5% Tween 20) and 15 μl of proteinase K (stock solution 10 mg/ml). The samples were boiled for 10 min to inactivate proteinase K and maintained at –80°C until their use. Ten microliters of the cell extract were directly used for PCR amplification.
Amplification of K-ras Protooncogene by Nested PCR
The amplification of a DNA fragment of K-ras gene was performed according to the method previously described (Golijow et al. 1999) with some small modifications (Mourón et al. 2004), in a Techne Progene PCR reactor (Techne, Cambridge, England). The primers used for PCR reaction were 5BKIM and 3KiE for the first round of PCR amplification and 5BKIM and 3AKIL for the second round (Ward et al. 1995).
DNA from the cellular line K562 was used as a negative control for codon 12 mutations and DNA from the HeLa cell line as a positive control for this point mutation. Detection of the amplification products were made by electrophoresis onto 6% polyacrylamide:bis-acrylamide (19:1) minigel in a vertical electrophoresis minisystem (17 × 11 cm; Aladin Enterprises, California, USA), at 170 volts for 45 min and then stained with ethidium bromide and exposed to ultraviolet (UV) 320 nm.
Analysis of Band Patterns of Exon 1 from K-ras Protooncogene by LIS-SSCP
The analysis of band patterns by LIS-SSCPs (low ionic strength–single-strand conformation polymorphisms) was done according to the method previously described (Abba and Golijow 2004; Mourón et al. 2004). Briefly, 5 to 8 μl of the PCR product was added to 10 μl of LIS (low ionic strength) solution containing 10% sucrose, 0.01% bromophenol blue, and 0.01% xylene cyanol FF (Sigma). Electrophoresis was performed on 10% polyacrylamide:bis-acrylamide (39:1) gel (In-vitrogene, California, USA) at 4°C for 4 h at 120 volts in TBE 1 × buffer (45 mM Tris-borate/1 mM EDTA), using the electrophoresis system mentioned above. Finally, the gels were stained with silver nitrate to visualize the bands. Positive and negative controls were included in all the electrophoretic assays.
Determination of K-ras Codon 12 Mutations by RFLP-Enriched PCR
The samples showing mobility shifts in the SSCP screening were further evaluated for point mutations in the first and second base of codon 12 of K-ras gene using a specific enriched PCR method (Ward et al. 1995) with some small modifications (Golijow et al. 1999; Mourón et al. 2004) in a Techne Progene PCR Reactor (Techne, Cambridge, England).
An aliquot of 12 μl from the first PCR reaction was incubated at 60°C for 2 h with 5 U of BstNI (New England Bio-Labs, Maryland, USA) and 100 μg/ml of bovine serum albumin (BSA), in a final volume of 17 μl. After the incubation time, the tubes were boiled for 10 min to inactivate the restriction enzyme.
The second round of amplification was performed using the entire digested product in the reaction mixture. An aliquot of this second reaction was then removed and incubated with the same restriction enzyme in the conditions mentioned above.
Detection of the amplification products were made by electrophoresis onto an 8% polyacrylamide:bis-acrylamide (19:1) gel, at 170 volts for 45 min and then stained with ethidium bromide and exposed to UV 320 nm. After digestion with BstNI the control DNA and those samples without K-ras codon 12 mutations demonstrated the 28- and 63-bp normal fragments. The samples with codon 12 K-ras mutations also showed the undigested 91-bp fragment as a consequence of the loss of the restriction site.
Statistical Analysis
Statistical evaluation was done using the SPSS 11.0.1 software (SPSS Inc., Illinois, USA, LEAD Technologies, Illinois, USA). In the SCE test a total of 100 metaphases were analyzed for each treatment and the mean and error standard were calculated. The effect of chemical treatment on the frequency of SCEs was analyzed using the nonparametric Kruskal-Wallis one-way analysis of variance (ANOVA) and the Mann-Whitney U test. Kruskal-Wallis one-way ANOVA test is defined as a distribution-free method, because it is not dependent on a given distribution. H values are distributed approximately as χ2 for large and small samples considering an α = 0.05. When the comparison was only between two samples, the nonparametric Mann-Whitney U test was used. This method is a semigraphical test and will be especially convenient when there are a few items in each sample (Sokal and Rohlf 1980).
For comet assay, a total of 200 cells were evaluated for each treatment. The mean and standard error were calculated for the comet parameter for each treatment. Also, the Kruskal-Wallis test was used in order to analyze total differences. Mann-Whitney test was also employed in order to evaluate differences between each sample pair.
Comparison between the frequencies of K-ras mutations in the cells exposed to different concentrations of PAHs was made using the χ2 test.
RESULTS
SCE Assay
Table 1 exhibits the SCE frequencies detected in MRC-5 cells treated with different doses of benzo[a]pyrene and dibenzo[a,l]pyrene and their controls (untreated cells and DMSO-treated cells). The proliferation index was slightly diminished in cells exposed with BP and with the highest concentration of 16.5 μM of DBP in relation to controls, as a consequence of an increased of M1 cells and a relative drop of M2 cells (Table 1).
SCE frequencies were significantly increased in cells exposed with benzo[a]pyrene in relation to controls (H = 136.7, p < .001). The Mann-Whitney test demonstrated significant differences between (i) untreated cells and DMSO treatment and each dose (p < .001); (ii) DMSO treatment and doses of 3.9 μM (p < .05), 19.8 μM (p < .01), and 39 μM (p < .001); (iii) doses 3.9 μM and 39 μM (p < .001); (iv) doses 19.8 μM and 39 μM (p < .01).
Also, SCE frequencies were significantly increased in the treatment with dibenzo[a,l]pyrene (H = 118.8, p < .001). The Mann-Whitney test showed significant differences (i) between untreated cells and DMSO treatment and each dose (p < .001); (ii) DMSO exposed cells with respect to doses of 8.3 μM and 16.5 μM (p < .001); (iii) between dose of 1.6 μM and doses of 8.3 μM and 16.5 μM (p < .05 and p < .001, respectively).
Comet Assay
The treatment of MRC-5 cells with both PAHs produced an increment in DNA migration in the standard alkaline comet assay (Table 2). The percentage of viable cells observed indicates the lack of cytotoxic effects with the employed doses.
When cells were treated with BP, the Kruskal-Wallis test showed highly significant differences between groups (p < .001). Mann-Whitney test revealed significant differences between (i) untreated cells and each treatment (p < .001) except for 3.9 μM BP; (ii) DMSO control and each dose (p < .001); (iii) dose 3.9 μM and doses 19.8 μM and 39 μM (p < .001).
Also, in the treatment with DBP the Kruskal-Wallis test revealed significant differences between groups (p < .001). Mann-Whitney test revealed significant differences between (i) untreated cells and those treated with DMSO (p < .05) and every dose of DBP employed (p < .001); (ii) DMSO-treated cells with respect to doses 1.65 μM (p < .005) and 8.27 μM (p < .005); (iii) dose 16.53 μM and doses of 1.65 μM and 8.27 μM (p < .005).
Detection of K-ras Codon 12 Mutations
In the treatment with BP, abnormal band mobilities were detected by PCR-LIS-SSCP screening in treated cells (4 of 17) as well as in the untreated group (2 of 6) and in the DMSO-exposed group (1 of 5). Nevertheless these differences were not significant (p > .05). The distribution of abnormal band patterns was 2 of 6 assays treated with 19.8 μM and 39 μM. However, mutations in codon 12 assessed by RFLP-enriched PCR were found in only one of the two untreated samples presenting mobility shifts in the SSCP analysis.
On the other hand, in the treatment with DBP, a total of 14 samples showed abnormal band patterns. The prevalence of samples presenting abnormal band patterns was 4 of 6 untreated assays, 2 of 6 DMSO group, 2 of 5 treated with 1.65 μM DBP, and 3 of 6 treated with 8.27 μM and 16.53 μM DBP. No significant differences were found between groups (p > .05). Mutations in codon 12 assessed by RFLP-enriched PCR were found in two of the untreated samples and in one of those treated with 8.27 μM presenting mobility shifts in the SSCP analysis.
DISCUSSION
The single-cell gel electrophoresis, also called comet assay, is a short-term genotoxicity test widely used to reveal a broad spectrum of DNA-damaging agents capable of inducing strand breakage, cross-links, and alkali-labile sites. It has been employed to investigate diverse areas such as DNA repair, radiation biology, environmental biomonitoring, genetic toxicology, and human epidemiology (Anderson et al. 1998; Rojas, Lopez, and Valverde 1999; Tice et al. 2000).
In this study the comet assay was applied to detect DNA damage induced in vitro, both by BP and DBP. The data showed a significant increase in comet moment when cells were exposed to PAHs in relation to controls (p < .001). In the SCGE, the extent of DNA migration is directly related to the frequency of strand breaks in the DNA as a consequence of a number of different type of reactions such as base and nucleotide excision repair; direct scission of the DNA backbone by chemical or radical attack; alkali-labile DNA adducts; and endonuclease or topoisomerase action.
Furthermore, DNA migration could be increased as a result of DNA fragmentation in the apoptosis and/or necrosis process. Cells undergoing active cell death, or apoptosis, could be clearly distinguished from viable cells exhibiting DNA strand breaks. These cells exhibit only a small percentage of the DNA associated with the comet head and most of the DNA remains in the typical apoptotic tail (Olive, Frazer, and Bańath 1993). However, in this study, there was not evidence of cytotoxicity analyzed by the percentage of viable and apoptotic cells. Survival was generally above 90% in all treatments (Table 2). In BP treatment, the proportion of apoptotic cells was 2/200 in untreated cells, 1/200 in DMSO group, 1/200 in dose of 3.9 μM, and 2/200 in dose of 39 μM. In DBP treatment, the proportion of apoptotic cells was 1/200 in dose of 1.65 μM, 4/200 in dose of 8.27 μM, and 3/200 in dose of 16.53 μM.
These results were concordant with other in vivo and in vitro studies in relation to BP exposure (Monteith and Vanstone 1995; Speit et al. 1996; Hanelt et al. 1997; Sasaki et al. 1997; Yusuf et al. 2000; van Delft et al. 2001; Garry et al. 2003; Valentin-Severin et al. 2003), but this work was the first that analyzed DNA damage in vitro by the comet assay after DBP exposure. In this sense, the highest dose of DBP used in the present study showed a drop in the values for comet moment (CM) in relation to the other two lower doses of the compund (Table 2). This situation could reflect the induction of DNA adducts interfering with DNA mobility and producing a small tail in the comet image. The induction of DNA adducts by DBP in vitro and in vivo was previously reported by other studies (Smith, Freeman, and Gupta 2001; Dreij, Seidel, and Jernstrom 2005; Mahadevan et al. 2005).
On the other hand, we found that BP and DBP induced SCEs without an exogenous metabolic activation system, indicating that these cells contain the activation mechanisms required to produce the DNA damage that elicits the SCE response (Rudiger et al. 1976; Wiencke, McDowell, and Bodell 1990). Other cytogenetic end points after BP exposure revealed a statistically significant increase in the number of micronuclei (Valentin-Severin et al. 2003) and in relation to aneuploidy induction in different cell lines (Matsuoka et al. 1997). However, they did not find a significant increase in SCE frequency at the dose range of 1.25 to 10 μg/ml BP.
Some epidemiologic studies revealed the induction of SCEs in workers exposed to PAHs (Forni et al. 1996; Kalina et al. 1998). Contrarily, other results did not found significant differences with respect to control groups (van Hummelen et al. 1993; van Delft et al. 2001) except for those workers who smoked (van Delft et al. 2001). This association between smokers and SCE induction was previously described by other authors (Kelsey, Christiani, and Little 1986; Perera et al. 1987).
It is well established that carcinogenic polycyclic aromatic hydrocarbons covalently bind to DNA in vivo with the development of DNA adducts in characteristic profiles. If these adducts are not repaired, or if repair introduces errors into the DNA sequence, gene mutations can be introduced upon replication of the damaged DNA. Certain mutations, particularly those resulting in activation of protooncogenes or inactivation of tumor suppressor genes, are thought to play a critical role in the carcinogenic process. In the present work, we could not find a positive response in the treatments with BP and DBP in relation to codon 12 K-ras point mutation induction. In a similar work, BP treatment caused a strong genotoxic effect in the comet assay but had only a marginal effect on the frequency of mutations at HPRT gene (Speit et al. 1996). Chakravarti and coworkers (1995; 2000; 2001) demonstrated the induction of point mutations at H-ras by DBP in vivo as a consequence of the deficient reparation of adducts between DBP and N7 or N3 of adenines. Likewise, another study revealed the induction of G → T transversions in the first base of codon 12, A → G transitions in the second base of codon 12 and A → T transversions in the second or third base of codon 61 of K-ras protooncogene in DBP-induced lung tumors of strain A/J mice following a single intraperitoneal (i.p.) injection (Prahalad et al. 1997).
Epidemiologic studies associated the occupational PAH exposure or smokers with point mutations at K-ras protooncogene (Vainio et al. 1993; Sarkar, Li, and Vallyathan 2001; DeMarini et al. 2001). Similar results were described for p53 tumor suppressor gene after BP exposure (Denissenko et al. 1996; Smith et al. 2000; DeMarini et al. 2001; Pfeifer and Denissenko 1998; Pfeifer et al. 2002).
The data presented here indicated that, under these experimental conditions, BP and DBP induced both DNA strand breaks and SCEs but not point mutations at codon 12 of K-ras protooncogene in MRC-5 cell line. The comet assay and SCE test seem to be a useful and sensitive tool for studying the indirect genotoxic effects of PAHs. Nevertheless, future studies are needed to investigate other conditions related to the induction of point mutations at particular and essential genes, such as protooncogenes and tumor suppressor genes, to reach a better understanding of the carcinogenic potential of these compounds.
Footnotes
Tables
This study was supported by the Agencia Nacional de Promoción Científica grant PICT99 01-5304 and Universidad Nacional de La Plata grant 11/V138. Dra Silvana Mourón, Fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.