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[Cancer Research 62, 4605-4609, August 15, 2002]
© 2002 American Association for Cancer Research

Carcinogenesis

Neoplastic Transformation of Human Lung Fibroblast MRC-5 SV2 Cells Induced by Benzo[a]pyrene and Confluence Culture1

Huijun Zhu and Nigel Gooderham2

Molecular Toxicology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzo[a]pyrene (BaP) is potent rodent carcinogen and a reputed human carcinogen. Although much is known about its metabolic activation leading to DNA damage, the mechanisms of its actions are not as well understood at a cellular level. In addressing this, we have established an in vitro model that follows the progression toward neoplastic transformation induced by BaP. The model uses immortal nontumorigenic human lung fibroblast MRC-5 SV2 cells as effectors, cocultured with a metabolically competent human lymphoblastoid line h1A1v2 (activator cells). Treatment of the coculture with BaP for 48 h induced a dose-dependent decrease in cloning efficiency of the MRC-5 SV2 cells; nevertheless, cultures continued to progress to confluence. At prolonged confluence culture (day 11), an elevation in the proportion of G2-M phase cells was detected by flow cytometry. By day 15, the G2-M phase peak disappeared, accordant with the appearance of a population with DNA content greater than the cells in G2-M phase. These changes in DNA ploidy were coincident with changes in morphology, specifically the appearance of enlarged and irregular-shaped nuclei. Confluence culture of BaP-treated MRC-5 SV2 cells for more than 2 weeks resulted in cell death; however, a few colonies survived the crisis to reach confluence again after an additional 10–14 days. The number of death-resistant colonies was proportional to the dose of BaP, with the majority of the cells exhibiting abnormal morphology. The degree of morphological change progressively increased with successive rounds of confluence. Cells that survived three rounds of confluence adopted a vastly different morphology, becoming polygonal, spindle, or other irregular-shaped, and acquired the ability to form large dense clumps that grew in an anchorage-independent manner. In parallel experiments, treatment with the vehicle alone (DMSO) resulted in substantially less death resistance and lower numbers of high-density clumps. Our studies demonstrate that a single pulse treatment of human MRC-5 SV2 cells with metabolically activated BaP increased DNA ploidy, induced resistance to confluence-initiated cell death and morphological change, and was accompanied by substantial changes in growth pattern under prolonged confluence culture. The multiple-step nature of this process is characteristic of the development of neoplastic disease, and prolonged confluence seemed to play a pivotal role in selecting for carcinogen-induced transformants.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is thought that malignancy is a multistep process in which cells acquire multiple genetic alterations followed by selective clonal expansion leading to the neoplastic phenotype. Studies of spontaneous neoplastic transformation in rodent cells provide considerable insight on progression of the process. Mouse embryo fibroblasts passaged at high density gained capacity to induce sarcomas in syngeneic mice within 3 months, whereas those passaged at low density did not show such tumorigenic properties despite 9 months of observation (1) . Prolonged confluence, which is necessary for display of the foci, also appears to be obligatory to induce transformation in NIH 3T3 cells (2) , in which variants emerged that had undergone clonal expansion and acquired transformation-related genetic changes under high-density cell culture pressure.

Although epidemiology indicates that cancer results from exposure to carcinogens, primary human cells in vitro have never been transformed to malignancy by carcinogen treatment. This is because, unlike rodent cells, primary human cells in culture rarely spontaneously acquire infinite life span. After acquisition of immortalization, however, human cells can be transformed by carcinogen treatment and subsequent prolonged culture (3) . It seems that confluence culture selects cells with preexisting genetic abnormality to confer growth advantage leading to clonal expansion and neoplastic conversion. Confluence culture may also heritably damage cells, creating more diversity for selection.

The PAH,3 benzo[a]pyrene (BaP), is a ubiquitously distributed environmental pollutant generated by incomplete combustion of organic substances such as cigarettes and fossil fuels. It exerts a potent carcinogenic activity in a range of animal species (4 , 5) . BaP, like many other PAHs, is metabolized by cytochrome P450 enzymes (CYP1A family) to mutagenic derivatives that can form DNA adducts. It is this property that is generally believed to account for the tumorigenic activity of BaP (6 , 7) . By smoking or the consumption of contaminated food, air, and water, humans are daily exposed to these carcinogens. Evidence from numerous experimental studies suggests a positive link between exposure to PAHs containing BaP and cancer in animals and in humans (5 , 7, 8, 9, 10) .

Studies of the genetics of human cancer have emphasized the role of carcinogens; however, they provide little information on the dynamics of the process. Cancer-unique genetic changes in human cells have been poorly defined thus far. It would, therefore, be valuable to study the progress of neoplastic transformation of human cells in vitro, stepwise to the end-stage disease.

In the present study, we used immortalized human lung fibroblast MRC-5 SV2 cells as model to investigate the roles of BaP and confluence culture in neoplastic transformation in vitro. MRC-5 SV2 cells were derived from MRC-5 cells by transfection with SV40. Although the expression of SV40 leads to p53 sequestration and immortality, the cells are stable in terms of maintenance of chromosome number, uniformity in cell size and shape, and growth pattern. They do not cause tumors when injected into nude mice (11) . In the present study, we show that MRC-5 SV2 cells acquired changes in DNA ploidy and morphology after pulse exposure to activated BaP. Continued culture under prolonged confluence induced the cells to develop aggressive growth properties characteristic of the neoplastic phenotype. These results suggest that neoplastic transformation in vitro is a multiple-stage progressive process, in which genetic alterations are obligatory and subsequent selective pressure under growth inhibition at confluence, plays a significant role in progression to more advanced neoplastic stages.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents were purchased from Sigma Chemical Co. (Poole, United Kingdom) unless otherwise indicated.

Cell Lines.
MRC-5 SV2 cells, a human fetal lung fibroblast line transfected with SV40 (11) , were purchased from the European Collection of Cell Cultures. Cells were cultivated in MEM with Earle’s salts, supplemented with 10% FCS, 2 mM glutamine (Life Technologies, Inc., Paisley, United Kingdom), 100 units/ml penicillin, and 100 µg/ml streptomycin, in a humidified incubator at 37°C with 5% CO2.

A human B-lymphoblastoid cell line (h1A1v2) transfected with human CYP1A1 cDNA was purchased from Gentest (12) and cultured in RPMI 1640 (without L-histidine), supplemented with 2 mM 1-histidinol, penicillin (100 units/ml), streptomycin (100 µg/ml), and 9% horse serum. For coculture experiments with MRC-5 SV2 cells, the h1A1v2 cells were preirradiated (50 Gy) with a cobalt source, which effectively sterilized the cells, preventing their replication while maintaining their metabolic competency (13) .

Coculture System.
The coculture system comprised irradiated h1A1v2 cells as metabolic activator and MRC-5 SV2 cells (which express low basal levels of CPY 1A1 and CPY 1B1), as effectors. MRC-5 SV2 cells (106) were seeded into 75 cm2 flasks one day before experimental treatment. The irradiated (50 Gy) h1A1v2 cells (2 x 106/flask) were added to flasks containing MRC-5 SV2 cells just before commencement of BaP treatment. Cocultures were treated with BaP (5–25 µM, in DMSO) or with vehicle (DMSO) for up to 48 h. The final concentration of DMSO was <1%. At the end of treatment, the culture media containing h1A1v2 cells (suspension cells) were discarded and the remaining adherent MRC-5 SV2 cells were harvested by trypsinization or were, alternatively, maintained in the flasks for further culture.

Cloning Efficiency Assay.
After treatment, 1000 MRC-5 SV2 cells were seeded into 25-cm2 flasks and were cultured for 14 days after which cells were stained with methylene blue solution, and surviving colonies (containing more than 50 cells) were scored to assess cloning efficiency.

Flow Cytometry.
Cellular DNA content was determined by propidium-iodide-staining flow cytometry. Briefly, target MRC-5SV2 cells were collected and fixed with 70% ethanol at -20°C for 1 h. Cells were resuspended in 1 ml of PBS containing propidium iodide (5 µg/ml) and RNase A (0.1 mg/ml). The suspensions were incubated at 37°C for 30 min. The ploidy determination of nuclei was estimated by flow cytometry DNA content as described previously (14 , 15) . The DNA content per nucleus was evaluated in a FACScan flow cytometer (Becton-Dickinson, San, Jose, CA). This analysis was carried out using a double discriminator module, which distinguishes between signals coming from a single nucleus and those from two or more aggregated nuclei. Only signals from single nuclei were analyzed (104 nuclei/assay).

Selection of Death-resistant Colonies.
After treatment with DMSO or BaP, cells were allowed to grow to confluence. Medium was changed weekly. When the majority of the confluent cells had detached from the culture surface, normally between 15 and 20 days after confluence, the culture medium containing floating cells was discarded, and the surviving colonies were again allowed to grow to confluence in fresh media. This process was repeated once more, and at the end of the third confluence, surviving colonies (containing more than 50 cells) were scored by light microscopy.

Morphological Transformation and Growth Pattern Change.
Concurrent with analysis for DNA content by flow cytometry, cells were examined for morphological changes by fluorescence microscopy. Cell morphology together with growth pattern was also monitored after survival from confluence culture, by phase-contrast microscopy. The formation of dense clumps was observed and scored at the end of the third confluence. After scoring, cells were trypsinized and reseeded into 100-mm dishes at 105/dish. Cell morphology and growth pattern were reexamined 2 weeks later by staining with methylene blue solution (50% methanol containing 0.5% methylene blue) for 5 min.

Anchorage-independent Growth.
Ten thousand cells, suspended in 5 ml of 0.3% agarose, were loaded onto a 0.6% agarose base prepared with complete culture medium in 100-mm dishes. The culture dishes were examined 96 h later for signs of cell growth by phase-contrast microscopy. The size (positive growth, defined as colonies that were >0.1 mm in diameter) and number of clumps were determined two weeks later.

Digital Image.
All pictures were taken using a Nikon digital camera E950 and processed with Adobe Photoshop software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning Efficiency.
To assess cytotoxicity, MRC-5 SV2 cells were treated with DMSO or BaP (5–25 µM) for 48 h with/without metabolically competent irradiated h1A1v2 cells. After treatment, h1A1v2 cells (in suspension) were removed from adherent MRC-5 SV2 cells. The MRC-5 SV2 cells were trypsinized and seeded (1000 cells) into 25-cm2 flasks. Surviving colonies (determined as clones of more than 50 cells) were scored on day 14. Under these conditions, BaP caused a dose-dependent decrease in MRC-5 SV2 cell cloning efficiency (Fig. 1)Citation . The effect was more pronounced when irradiated h1A1v2 cells were included and was partially prevented by the addition of {alpha}-NF (25 µM), a CYP1A1 enzyme inhibitor, which indicated that CYP1A1 was involved in the BaP cytotoxicity. Therefore, all of the subsequent experiments were performed using coculture with irradiated h1A1v2 cells. It was noticed that {alpha}-NF appeared to enhance the effect of BaP on cloning efficiency in the absence of h1A1v2 cells. The mechanism of this effect has not been further investigated.



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  		Fig. 1. The effect of BaP on cloning efficiency in MRC-5 SV2 cells. MRC-5-SV2 cells were treated with BaP for 48 h in the presence or absence of h1A1v2 cells. After treatment, MRC-5 SV2 cells were seeded into 25-cm2 flasks at 1000 cells/flask. Surviving colonies (containing more than 50 cells) were counted after 14 days. Values are mean ± SD; n = 3. *, significantly different from vehicle control (P < 0.05); +, significantly different from value in the absence of {alpha}-NF (P < 0.05).

 
Change in Cell Cycle and DNA Ploidy.
Changes in cellular DNA ploidy are believed to be indicative of increased genetic instability (16 , 17) . To examine the effect of BaP treatment on cell cycle and the degree of ploidy, we measured cellular DNA content by flow cytometry (13 , 14) . MRC-5 SV2 cells, treated with DMSO for 48 h, displayed a well-distributed cell cycle profile with only a small M1 cell population (representing cells with DNA content less than those in G1 phase) and M5 cell population (representing cells with DNA content greater than those in G2-M phase, Fig. 2Citation ). The M1 cell population increased in a time-dependent manner, indicating cell death initiated by confluence culture. MRC-5 SV2 cells, treated with BaP, showed an abnormal cell cycle profile compared with vehicle-treated control. The G2-M phase peak was elevated after 48-h treatment and continued to increase over the next 11 days with a concomitant decrease in cells in G1 phase. By day 15, the majority of cells in G1 and G2-M phases had disappeared, which coincided with the appearance of an M5 cell population and an increase in the M1 cell population. These results suggest that the initial transient exposure of cells to metabolically activated BaP induced a remarkable degree of genetic instability. Such dramatic changes in cellular DNA content are probably the result of the induction of instability of karyotype by BaP along with an increased frequency of apoptotic cell death under confluence culture.



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  		Fig. 2. The effect of BaP on DNA ploidy in MRC-5 SV2 cells. MRC-5 SV2 cells were treated with DMSO or BaP (25 µM) and cocultured with irradiated h1A1v2 cells for 48 h. After treatment, the MRC-5 SV2 cells were cultured in fresh medium until the indicated time points when cells were collected for flow cytometry analysis of DNA content as described in "Materials and Methods." The gating represent the percentage of cells in the M1 population (M1), G0-G1 (M2), S phase (M3), and G2-M (M4) phases and cells in the M5 population (M5).

 
Resistance to Confluence-initiated Cell Death.
Confluence culture has been used to select and display spontaneous transformants in murine cells (2) . MRC-5 SV2 cells (106) were treated with BaP (5–25 µM) or with vehicle for 48 h. After treatment, cells grew to confluence in 3 days. They were then maintained in stationary growth for up to 20 days. By far, the majority of cells became detached from the culture surface within the 15–20 days postconfluence, indicating confluence-initiated cell death. Surviving colonies grew to confluence again in the next 2 weeks. After 3 subcultures at prolonged confluence, very few colonies survived from the vehicle (DMSO)-treated cell population. In contrast, the initial transient exposure to activated BaP (5–10 µM for 48 h) improved colony survival after three subcultures at prolonged confluence, which indicated the acquisition of a survival phenotype. The survival rate in BaP-treated cells was 30–100 x 10-6, more than 10-fold greater than that of DMSO-treated cells (~3 x 10-6). The ability of the BaP treatment to promote colony survival was not apparent when MRC-5 SV2 cells were treated with BaP in the absence of h1A1v2 cells. An identical result was obtained when MRC-5 SV2/h1A1v2 cocultures were pretreated with the CYP1A1 inhibitor {alpha}-NF (25 µM), supporting the proposal that this effect was mediated by CYP1A1 enzyme activity.

Morphological Changes.
Untreated MRC-5 SV2 cells exhibit epithelia-like morphology, with uniformity in cell size and shape (11) that was unaffected by treatment with vehicle (DMSO), even after two (Fig. 3c)Citation and three rounds of prolonged confluence culture (Fig. 3, e and g)Citation . However, in comparison, cells treated with activated BaP showed heterogeneous morphological changes by day 15 posttreatment (compare Fig. 3aCitation with Fig. 3bCitation ), when a M5 population was detected by flow cytometry (Fig. 2j)Citation . They were generally larger and most strikingly contained enlarged or multiple nuclei (Fig. 3b)Citation . These morphological changes progressed with each confluence culture and by the third round of confluence had assumed very irregular morphology, with a significant number of cells being polygonal and others more spindle-shaped or giant-sized (Fig. 3, f and h)Citation . These results imply that the transient initial CYP1A1-mediated BaP activation induced a heritable morphological change, which, we suggest, may be the consequence of the changes in ploidy.



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  		Fig. 3. The effect of BaP on morphology and growth pattern of MRC-5 SV2 cells. Morphology of MRC-5 SV2 cells was examined at different time points after a 48-h treatment with DMSO or BaP (25 µM) in the presence of irradiated h1A1v2 cells. Cells stained with propidium iodide for flow cytometry analysis on day 15 after treatment were also examined by fluorescence microscopy (a and b). DMSO-treated cells maintained normal morphology (a), whereas BaP-treated cells showed heterogeneous morphological changes (b). c and d, representative cells that survived a second round of confluence culture after treatment with DMSO (c) or BaP (d), respectively for 48 h. e–h, cells that survived the third round of confluence culture, in which e and g are two random views of cells treated with DMSO, whereas f and h are two representative views of cells treated with BaP (25 µM); x200. i and j, representative cells that were treated with DMSO or BaP (25 µM) for 48 h and were at second confluence. Cells treated with DMSO formed a normal monolayer (i), whereas cells treated with BaP displayed irregular multilayer growth pattern (j). k and l, representative cells that survived the third round of confluence culture. These cells were grown in 100-mm culture dishes at 105/dish for 2 weeks and were stained with methylene blue. DMSO-treated cells showed normal monolayer growth pattern (k), but BaP-treated cells formed dense clumps (l). m, the methylene blue staining pattern of culture dishes containing DMSO (left panel) or BaP (right panel) treated cells.

 
Growth Pattern Changes.
Normal MRC-5 SV2 cells grew to a confluent monolayer after 3–4 days seeded at 106cells/75-cm2 flask. Although cells treated with vehicle (DMSO) for 48 h in coculture with irradiated h1A1v2 cells exhibited gradually decreased growth rate with each round of confluence culture, they were able to form a monolayer after the first and second round of confluence (Fig. 3i)Citation . At the third time of prolonged confluence culture, very few colonies, or none in some experiments, survived, yet those that did retained a near normal morphology (Fig. 3k)Citation . The decrease of growth rate with each round of confluence indicates confluence-associated damage. MRC-5 SV2 cells cocultured with h1A1v2 cells and BaP for 48 h also showed decreased growth rate; yet in addition, they exhibited a changed growth pattern at the second time of confluence. Instead of growing as a monolayer, these cells started to grow on top of one another to form a multilayer composite (Fig. 3j)Citation . After survival of a third round of prolonged confluence, the MRC-5 SV2 cells, exposed to activated BaP, acquired the ability to form dense, large clumps (Fig. 3l)Citation . The frequency of clump formation was 30–50 x 10-6 in cells exposed to activated BaP and was ~3 x 10-6 in cells treated with DMSO.

To test whether the clump-forming cells had anchorage-independent growth activity, a frequently observed feature of neoplastic transformation, cells that survived 3 rounds of prolonged confluence culture were seeded into soft agar. MRC-5 SV2 cells exposed to activated BaP started growing within 3 days of seeding (Fig. 4)Citation . The rate of colony formation for cells treated with activated BaP was always more than 10-fold higher than that for cells treated with DMSO.



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  		Fig. 4. The effect of BaP on growth pattern in soft agar (anchorage-independent growth). Cells that survived the third round of confluent culture were seeded into 0.33% agar and examined after 3 days. Cells initially treated with BaP formed growing colonies, whereas DMSO-treated cells did not.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present work has established a cellular system to select confluence-initiated cell death resistance and to promote these variants to progress to fully neoplastic transformation by repeated confluence culture. We further show that transient exposure to activated BaP efficiently promotes these events. Such treatment induces changes in DNA content, followed by morphological and growth pattern changes. Although the M4 population could represent either G2 phase or tetraploid cells, the coexisting morphological changes found for the nuclei strongly favored the presence of a tetraploid cell population. Studies of human neoplastic and preneoplastic tissues by cytogenetic and flow cytometric analysis have demonstrated a linear relationship between DNA content and chromosome number (16, 17, 18) . Results from our study, using SV40-immortalized human cells, are in accordance with a study in a SV40 transgenic mouse model, in which progression to cancer occurs by a diploidy->tetraploidy->aneuploidy sequence that is associated with biological progression from phenotypically normal cells to hyperplasia and then to dysplasia and, finally, to carcinoma (19) . Studies in rodent cells also demonstrated that the induction of aneuploidy correlates with the ability to induce morphological transformation of Syrian hamster embryo cells (20) and Chinese hamster cells (21) . Our study parallels the development of human cancers because it has been reported that tetraploid or aneuploid cells are precursors of cancers in colon carcinoma (22) , bladder cancer (23) , and esophageal adenocarcinoma (15) . In addition, in Barrett’s esophagus, the transition from tetraploidy to aneuploidy is associated with progression from dysplasia to carcinoma (24) . However, the significance of early aneuploidy is questionable in view of the diploid nature of most early tumors like papillomas (25) and some intestinal polyps (26) .

The mechanisms of activated BaP-induced changes in ploidy in these cells were not investigated in the present work. However, DNA adduct formation was detected in MRC-5 SV2 cells after BaP treatment (data not shown). It is known that DNA-adduct-forming agents can induce chromosome breaks, which can result in chromosome gains via fusion of unrepaired breaks and subsequent nondisjunction or loss of defective chromosomes at mitosis, leading to a change in DNA content. MRC-5 SV2 cells were immortalized by transfection with SV40 resulting in sequestration of p53, a key protein in the control of cell cycle, DNA repair and apoptosis (27 , 28) . Therefore, cells with abnormal DNA ploidy would not be effectively excluded from cell cycle and could continue to divide. These cells will be subject to asymmetric chromosome segregation every time they divide (29) . In our studies, the progressive irregular morphological changes, including the number and the size of nuclei, reflect differences in DNA content, which suggests that changes in ploidy play a significant role in BaP-induced transformation. It has been demonstrated that the genotoxic metabolite of BaP, (±)-7ß,8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) induced neoplastic transformation in a human fibroblast cell strain MSU-1.1 (3) . Although the mutagenic effect of the compound was thought to be a driver of transformation, the diverse features of the transformants could be caused by the effects of DNA ploidy changes. In support of this, recent studies suggest that changes in ploidy, rather than gene mutations, determine the fate of cells toward neoplastic transformation (30) .

In mouse NIH 3T3 cells, repeated, prolonged confluence culture results in neoplastic transformation (31) . In contrast, in our experiments, immortal human lung fibroblast MRC-5 SV2 cells were resistant to confluence-induced transformation, probably because of an intrinsic mechanism that triggers cell death. Treatment of MRC-5 SV2 cells with activated BaP conferred the ability to survive confluence crisis and increased the chances for transformation. Under confluence culture, the spontaneous transformation rate of MRC-5 SV2 cells was very low (~3/106), but this was increased up to 30–50/106 when pulse treated with activated BaP. These data show that for these human cells, immortalization alone (untreated cells) is not sufficient for transformation to the neoplastic phenotype. It has been suggested that confluence culture induces the accumulation of metabolically related damage, leading to the destabilization of genetic material (32) , although in our experiments, this appears to have been significantly promoted by exposure to activated BaP. Thus the confluent state not only allows for the selective localized growth of the pretransformed cells, but also adds heritable damage to the cells, leading to progressive neoplastic development.

The relatively low incidence of neoplastic transformation induced by activated BaP compared with its effect on cloning efficiency indicates that the selection for rare variants that are susceptible to BaP transformation is a key for initiating the process. In agreement with our observations, Blagosklonny suggests that genetic instability provides a repertoire of mutants from which the environment selects favorable variants (33) . A central role for progressive selection of clones in tumor development has been frequently suggested (34, 35, 36) , and the particular nature of the cellular microenvironment determines the type of cells to be selected. For example, in a rat model the carcinogen N-nitroso-N-methylurea selectively induced mammary malignancy in patches with preexisting Ha-ras 1 mutation (37) .

In our model human cell culture system, three key events appear to be necessary for the development of chemical-induced neoplastic transformation: (a) preexisting potential for unlimited growth; (b) chemical-induced genetic modification; and (c) clonal expansion under confluence culture. Each of these events would appear to be required for full transformation. Studies of the progression of carcinogen-induced neoplastic transformation in vitro can provide significant insights for the development of chemopreventative and chemotherapeutic strategies for human disease. Furthermore, understanding the key cellular decisions in this stepwise progression toward the neoplastic phenotype, offers the prospect of selective intervention and prevention at multiple stages.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Food Standards Agency, United Kingdom. Back

2 To whom requests for reprints should be addressed, at Molecular Toxicology, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, United Kingdom. Phone: 44-0207-594-3188; Fax: 44-0207-594-3050; E-mail: n.gooderham{at}ic.ac.uk Back

3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbon; BaP, benzo[a]pyrene; CYP1A1, cytochrome P4501A1; {alpha}-NF, {alpha}-napthoflavone. Back

Received 2/19/02. Accepted 6/19/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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