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Mutagenesis Advance Access originally published online on January 20, 2007
Mutagenesis 2007 22(2):111-116; doi:10.1093/mutage/gel060
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Growth kinetics in MCF-7 cells modulate benzo[a]pyrene-induced CYP1A1 up-regulation

Haiyan Jiao1,3, Sarah L. Allinson1, Michael J. Walsh1, Rebecca Hewitt1, Kathy J. Cole2, David H. Phillips2 and Francis L. Martin1,*

1Biomedical Sciences Unit, Department of Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK 2Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton SM2 5NG, UK

Pro-carcinogens, such as benzo[a]pyrene (B[a]P), that are exogenous ligands of the aromatic hydrocarbon receptor may influence the susceptibility of target-cell populations through the up-regulation of cytochrome P450 (CYP) mixed function oxidases. We examined whether the growth kinetics of MCF-7 cells might determine the level of up-regulation of CYP1A1, CYP1A2 or CYP1B1 by B[a]P, and whether this could then influence subsequent levels of DNA damage. Cell cultures manipulated to be G0/G1-phase concentrated, S-phase concentrated or G2/M-phase concentrated were treated with B[a]P and the expression levels of CYP1A1, CYP1A2, CYP1B1, cyclin-dependent kinase inhibitor 1A [CDKN1A (P21WAF1/CIP1)], B-cell leukaemia/lymphoma-2 (BCL-2), and Bcl-2-associated X levels were determined. Levels of DNA damage were measured as DNA single-strand breaks (SSBs) by the alkaline single-cell gel electrophoresis (comet) assay or as DNA adducts by 32P-postlabelling analysis. B[a]P-induced up-regulation of CYP1A1 was >100-fold in S-phase-concentrated cells, but in G0/G1-phase- or G2/M-phase-concentrated cultures up-regulation occurred to a significantly lower extent. Consistent with this, B[a]P-treated S-phase-concentrated cultures exhibited markedly up-regulated P21WAF1/CIP1, higher levels of dose-related increases in DNA SSBs, and increased DNA adduct levels presumably as a result of CYP1A1-mediated activation of B[a]P to B[a]P-diol-epoxide compared with the cultures enriched for the other cell cycle phases. Growth kinetics in vitro may be an important predeterminant of susceptibility to an exogenous pro-carcinogen in short-term test systems and these findings have important implications when extrapolating such results to a particular target-cell population in vivo.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Metabolic conversion of pro-carcinogens to genotoxic metabolites is often mediated by the cytochrome P450 (CYP) mixed function oxidase system. Thus, the expression levels of specific CYP isoforms may modulate the inherent susceptibility of target-cell populations to pro-carcinogens (1Go). The dynamics of tissue turnover may also be an important mediator of susceptibility to the initiating effects of DNA-damaging agents (2Go). For instance, the transformation zone of the cervix exhibits a cyclical cell turnover and it is this region that is most vulnerable to the development of carcinoma postinfection with oncogenic human papilloma virus (3Go). However, effects in an in vitro test system in which high levels of proliferation occur may not be representative of cells that are normally quiescent in vivo. For instance, primary prostate epithelial cells in vitro are sensitive to the genotoxic effects of benzo[a]pyrene (B[a]P) (4Go) although the activity of the required bioactivation enzymes appears to be comparatively low in vivo (5Go).

A stereoselective epoxidation and hydration of B[a]P can be generated through a reaction catalysed by CYP isoforms and epoxide hydrolase (6Go). This results in bioactivation to an electrophilic species, anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (7Go), that may then bind covalently to nucleophilic sites in cellular macromolecules such as the N2 position of guanine to form a B[a]P-DNA adduct (8Go). Other metabolic routes could include the conversion of B[a]P-7,8-diol to the reactive and redox-active o-quinone (B[a]P-7,8-dione) by aldo-keto reductase 1A1 (9Go); such pathways might give rise to the formation of B[a]P-DNA adducts of differing molecular weights (10Go).

Exposure of cells to non-cytotoxic B[a]P concentrations results in their accumulation in S-phase and has led to the hypothesis that evasion of G1-phase arrest contributes to the transforming activity of DNA-adduct-forming carcinogens (7Go,11Go). The planar B[a]P binds, as does dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin), to the ligand-activated transcription factor known as the aromatic hydrocarbon receptor (AhR) (12Go); the endogenous ligand is unknown (13Go). Upon binding, the ligand–receptor complex translocates into the nucleus where the AhR dissociates and then binds to a nuclear protein Arnt (AhR nuclear translocator) (14Go). This heterodimer binds to the dioxin responsive element (15Go) of various genes, including CYP1A1, CYP1A2 and CYP1B1; binding may result in elevated gene transcription (16Go,17Go). AhR appears to have important cell cycle regulatory roles both by ligand-activated and ligand-independent mechanisms (16Go). Up-regulated AhR-dependent activation of CYP1A1 may also be dependent on cell cycle phase (16Go,18Go).

AhR influences G1-phase progression through interaction with retinoblastoma protein (19Go,20Go). Agonist-activated AhR/Arnt heterodimer also associates with oestrogen receptor (ER){alpha} and ERß; through this mechanism, dioxin-type environmental contaminants may give rise to adverse oestrogen-related actions (21Go). Such hormone-induced effects mediated via ER{alpha} and ERß (e.g. AhR-ER crosstalk) play important roles in cell proliferation (22Go). Through cell culture manipulation, we set out to determine whether B[a]P-induced up-regulation of CYP1A1 is dependent on growth kinetics in ER-positive breast carcinoma MCF-7 cells and whether this influences subsequent genotoxic events. Such investigations might determine whether treatment in a particular cell cycle phase is an important modulator of susceptibility within a target-cell population.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals and media
Unless otherwise stated, chemicals were obtained from Sigma Chemical Co. (Poole, UK) and cell culture consumables from Invitrogen Life Technologies (Paisley, UK).

Cell culture
The MCF-7 cell line was grown in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin. MCF-7 cells were cultured routinely in 75-cm2 flasks at 5% CO2 in air and 37°C in a humidified atmosphere and subcultured (1:10, v/v) twice weekly. Prior to subculture or incorporation into experiments, cells were disaggregated with 0.05% trypsin/0.02% EDTA. B[a]P treatments, in dimethylsulphoxide, were at a concentration of 0.5% v/v.

Flow cytometry
Cells were re-suspended in aliquots of complete DMEM medium (10 ml, {approx}1 x 106 cells), seeded into 75-cm2 flasks and allowed to attach for 24 h prior to analysis, as indicated. For cell cycle analysis following disaggregation, cell aliquots were washed twice with phosphate-buffered saline (PBS) prior to fixation with ice-cold 70% ethanol (aqueous) and storage overnight at –20°C. Cell aliquots were again washed twice with PBS prior to incubation with RNase A (10 µg/ml) and propidium iodide (50 µg/ml) for 60 min at 37°C. DNA content of 10 000 events per tissue culture manipulation analysed using a Becton Dickinson FACSCaliber flow cytometer and the CELLQuest software version provided by the manufacturer (23). Cell cycle analysis was carried out using ModFitLT for Mac v2.0.

Culture conditions were manipulated in order to generate G0/G1-phase-concentrated (cells in a confluent state), S-phase-concentrated (cells in an exponential growth-phase 24 h postseeding) or G2/M-phase-concentrated (18-h exposure of exponentially growing cells to 0.4 µg/ml nocodozole) cultures (Table I). At this point, cell cycle distributions were verified using flow cytometry. Parallel cultures were then exposed to B[a]P, after which the end points outlined below were determined.


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  		Table I. Cell cycle distribution of MCF-7 cells prior to B[a]P treatment

 
Results are the means ± standard deviation (SD) of three separate experiments (an average of triplicate measurements per experiment contributed to each experimental value) (Table 1). The proportions of cells (untreated) in G0/G1-phase, S-phase or G2/M-phase did not markedly alter for at least another 24 h after each tissue culture manipulation (data not shown).

Quantitative real-time reverse transcriptase (RT)–PCR
Routinely cultured cells were disaggregated and re-suspended in complete medium (DMEM, 10% FCS) prior to seeding aliquots (5 ml, {approx}1 x 105 cells) into 60-mm petri dishes (24Go). Following cell cycle phase manipulation (Table I), MCF-7 cells were treated with B[a]P, as indicated. Cells were then washed twice with PBS prior to lysis and total RNA extraction using the Qiagen RNeasy® Kit in combination with the Qiagen RNase-free DNase kit (QIAGEN Ltd, Crawley, UK). DNase was incorporated into the extraction procedure in order to remove residual DNA, e.g. pseudogene. RNA quality was routinely assessed in a 1.2% formaldehyde agarose gel; yield and purity were checked using a spectrophotometer. RNA (0.4 µg) was reverse transcribed in a final volume of 20 µl containing Taqman® reverse transcription reagents (Applied Biosystems, Warrington, UK): 1 x Taqman reverse transcriptase (RT) buffer; MgCl2 (5.5 mM); oligo d(T)16 (2.5 µM); dNTP mix (dGTP, dCTP, dATP and dTTP; each at a concentration of 500 µM); RNase inhibitor (0.4 U/µl); RT (MultiScribeTM) (1.25 U/µl) and RNase-free water. Reaction mixtures were then incubated at 25°C (10 min), 48°C (30 min) and 95°C (5 min).

cDNA samples were stored at –20°C prior to use. Primers (Table II) for cyclin-dependent kinase inhibitor 1A [CDKN1A (P21WAF1/CIP1, GenBank accession no. NM_078467)], B-cell leukaemia/lymphoma-2 (BCL-2, GenBank accession no. NM_000633) and Bcl-2-associated X (BAX, GenBank accession no. AF007826), CYP1A1 (GenBank accession no. BC023019), CYP1A2 (GenBank accession no. NM_000761), CYP1B1 (GenBank accession no. NM_000104) and endogenous control ß-ACTIN (GenBank accession no. AK222925) were chosen using Primer Express software 2.0 (Applied Biosystems) and designed so that one primer spanned an exon boundary (24Go,25Go). Specificity was confirmed using the NCBI BLAST search tool. Quantitative real-time PCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Reaction mixtures contained 1 x SYBR® Green PCR master mix (Applied Biosystems); forward and reverse primers (Invitrogen Life Technologies) at a concentration of 300 nM (P21WAF1/CIP1, BCL-2, BAX, CYP1A1, CYP1A2, CYP1B1 or ß-ACTIN); for P21WAF1/CIP1, BCL-2, BAX, CYP1A1, CYP1A2 or CYP1B1 amplification 20 ng cDNA template or for ß-ACTIN amplification 5 ng cDNA template; made to a total volume of 25 µl with sterile H2O. Thermal cycling parameters included activation at 95°C (10 min) followed by 40 cycles each of denaturation at 95°C (15 sec) and annealing/extending at 60°C (1 min). Each reaction was performed in triplicate and ‘no-template’ controls were included in each experiment. Dissociation curves were run to eliminate non-specific amplification, including primer dimers. In control cell populations in all three culture conditions and at both the 3 and 24 h time points, averaged threshold cycle values of amplified cDNA were in the 25–30 range for CYP1A1 and CYP1B1 while they were >35 or undetectable (as indicated) for CYP1A2 analyses (Table III).


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  		Table II. Primers used for quantitative real-time RT–PCR analyses

 


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  		Table III. mRNA transcript levels in B[a]P-treated MCF-7 cells

 
The alkaline single-cell gel electrophoresis (comet) assay
Alkaline lysis followed by alkaline gel electrophoresis was employed in order to detect DNA single-strand breaks (SSBs) (24Go,26Go,27Go). Cell cultures in 75-cm2 flasks were manipulated to be G0/G1-phase concentrated, S-phase concentrated, or G2/M-phase concentrated (Table I) after which they were treated with B[a]P at 37°C for 24 h, as indicated. Treatments were conducted with or without hydroxyurea (HU, 1 mM) and cytosine arabinoside (ara-C, 120 µM) (28Go); these DNA-repair inhibitors were incorporated into the treatment 1 h prior to disaggregation of cells and their incorporation into the comet assay. Single-cell suspensions in low melting point agarose were then evenly applied to microscope slides pre-coated with normal melting point agarose and allowed to set on a cold surface for 5 min. The slides were subsequently submerged in cold lysis solution (2.5 M NaCl, 100 mM EDTA disodium salt, 10 mM Tris, 1% Triton X-100 and 10% DMSO), protected from light and stored at 4oC overnight. Then the slides were transferred to a light-tight container and covered in electrophoresis solution (0.3 M NaOH, 1 mM EDTA, freshly prepared, pH > 13), and stored for 40 min to allow DNA unwinding. Finally, slides were transferred to a horizontal electrophoresis tank and covered in fresh electrophoresis solution prior to electrophoresis at 0.8 V/cm and 300 mA for 24 min. After electrophoresis, slides were neutralized (Tris, 0.5 M, pH 7.5) and stained with ethidium bromide (20 ng/ml) after which comet tail length (CTL) (µm) was visualized by epifluorescence using a Leitz Dialux 20 EB microscope. A total of 100 digitized images per data point, 50 from each of duplicate slides, were measured in each experiment. Experiments were repeated independently on three separate occasions and all the results obtained were combined for the purposes of analysis. CTL measurements were compared using a Mann–Whitney test.

32P-postlabelling analysis
Following tissue culture manipulation (Table I), MCF-7 cells in 75-cm2 flasks were treated with B[a]P at 37°C for 24 h, as indicated. DNA subsequently isolated was subjected to 32P-postlabelling analysis (4 µg per sample) using the nuclease P1 digestion method of sensitivity enhancement (29Go,30Go). Solvents for chromatography of the labelled digests on polyethyleneimine-cellulose TLC plates were D1, 1.0 M phosphate, pH 6.0; D2, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D3, 0.8 M lithium chloride, 0.5 M Tris–HCl, 8.5 M urea, pH 8.0. Chromatograms were scanned for radioactivity using an InstantImager (Canberra Packard, Pangbourne, UK). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the postlabelling chromatograms and from the specific activity of the [{gamma}-32P]ATP used in the labelling procedure.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Gene expression in B[a]P-treated MCF-7 cells
In G0/G1-phase-concentrated or G2/M-phase-concentrated cultures, B[a]P-induced dose-related increases in CYP1A1 expression following 24-h exposure (up to a maximum of {approx}30-fold); however, these were markedly lower compared to {approx}120-fold elevations observed in S-phase-concentrated cultures (Table III). Higher levels of CYP1A1 induction (up to a maximum of some {approx}10-fold) occurred in G0/G1-phase-concentrated cultures after 3-h than after 24-h exposure to B[a]P, except with 1-µM treatment. A 3-h exposure to 0.01 µM B[a]P was sufficient to induce a 6-fold increase in CYP1A1 expression in S-phase-concentrated cells but levels of induction appeared to plateau at higher concentrations. Treatment for 24 h resulted in clear dose-related increases in CYP1A1 expression up to 0.5 µM B[a]P. In contrast, exposure for 3 h did not markedly alter CYP1A1 expression in G2/M-phase-concentrated cultures whereas after 24-h treatment, dose-related increases in CYP1A1 expression up to 0.5 µM were observed (Table III). Although not as marked, a similar profile of differential gene expression seemingly governed by growth kinetics was observed with B[a]P induction of CYP1A2 or CYP1B1. In fact, in some G0/G1-phase-concentrated cultures, B[a]P exposure was necessary to detect CYP1A2 expression; in the absence of B[a]P induction, levels of CYP1A2 expression were so low in some G0/G1-phase-concentrated cultures that mRNA transcripts were undetectable. Because of this, a mean ± SD for CYP1A2 expression levels in G0/G1-phase-concentrated cultures could not be estimated from triplicate experiments. Consistent with the notion that induction of CYP isoforms may determine bioactivation of B[a]P to BPDE that in turn results in the formation of elevated levels of bulky DNA adducts, S-phase-concentrated cultures exhibited markedly up-regulated P21WAF1/CIP1 ({approx}20-fold), down-regulated BCL-2 ({approx}5-fold) and up-regulated BAX ({approx}2-fold). In comparison, G0/G1-phase- and G2/M-phase-concentrated cultures appeared to be relatively resistant to fluctuations in the levels of P21WAF1/CIP1, BCL-2 or BAX expression (Table III).

DNA damage induction in B[a]P-treated MCF-7 cells
Table IV and Figure 1 show the comet-forming activity of B[a]P in the presence or absence of DNA-repair inhibitors, HU/ara-C, in G0/G1-phase-concentrated, S-phase-concentrated or G2/M-phase-concentrated cultures. In all the three culture conditions, clear dose-related increases in B[a]P-induced DNA SSBs were observed (Figure 1). In the absence of HU/ara-C, G2/M-phase-concentrated cultures appeared to be significantly (P < 0.001) more susceptible to the comet-forming activity of B[a]P compared to G0/G1-phase-concentrated or S-phase-concentrated cultures. Incorporation of HU/ara-C clearly elevated comet-forming activity under all the three culture conditions (Table IV). However, in the presence of DNA-repair inhibitors, S-phase-concentrated cultures were significantly (P < 0.001) more susceptible to the comet-forming activity of B[a]P at all concentrations tested compared to G0/G1-phase-concentrated or G2/M-phase-concentrated cultures (Table IV and Figure 1). Interestingly, control levels of DNA SSBs measurable in S-phase-concentrated cultures were significantly (P < 0.0001) higher than levels found in G0/G1-phase-concentrated cultures both in the presence or in the absence of HU/ara-C; when S-phase-concentrated cultures were compared to G2/M-phase-concentrated cultures, significantly (P < 0.01) higher levels were evident in the former only in the presence of DNA-repair inhibitors.


Figure 1
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  		Fig. 1. Comet-forming activity of B[a]P in G0/G1-phase-concentrated, S-phase-concentrated or G2/M-phase-concentrated cultures, as indicated. Cells were treated with B[a]P for 24 h in the absence (A) or presence (B) of incorporation of DNA-repair inhibitors, HU and ara-C, for 1 h prior to disaggregation. Following treatment, cells were disaggregated prior to incorporation into the comet assay as described in the Materials and Methods.

 


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  		Table IV. Comet-forming activity of B[a]P in MCF-7 cells

 
Following 32P-postlabelling analysis for B[a]P-DNA adduct levels, a clear association with growth kinetics of MCF-7 cells was noted (Table V). After 24-h incubation with 1.0 µM B[a]P, markedly higher levels of B[a]P-DNA adducts were observed compared to G2/M-phase-concentrated cultures ({approx}15-fold) and G0/G1-phase-concentrated cultures ({approx}40-fold); {approx}6-fold higher levels were observed in S-phase-concentrated cultures following 0.1 µM B[a]P 24-h exposure compared to either corresponding G0/G1-phase-concentrated cultures or G2/M-phase-concentrated cultures (Table V).


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  		Table V. B[a]P-DNA adduct formation in MCF-7 cells

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The CYP multigene family consists of constitutively expressed and inducible isoenzymes that are expressed mainly in the liver but also to varying degrees in extrahepatic sites (31Go). Expression of particular CYP isoforms may be associated with an elevated susceptibility to carcinoma (25Go) although whether this results in an increase in DNA conformational changes remains to be determined (32Go). The occurrence of sporadic cancers in the absence of germ-line mutations is mitigating evidence for an important role for xenobiotics in cancer causation (33Go). An important mediator in a mammalian cell's responsiveness to xenobiotics might be the AhR (15Go). There is increasing evidence to suggest that the transcriptional activation of CYP isoforms via exogenous ligands of AhR may be cell cycle dependent (16Go,34Go). However, it is unknown whether transcriptional activation of CYP expression may determine cell susceptibility to pro-carcinogens requiring metabolic activation to DNA-reactive species.

To explore this possibility, we investigated the role of MCF-7 cell growth kinetics on the effectiveness of B[a]P (an AhR agonist whose metabolite is DNA reactive) to induce CYP1A1, CYP1A2 and/or CYP1B1 (Table III) following manipulation of culture conditions so that G0/G1-phase-concentrated cultures, S-phase-concentrated cultures and G2/M-phase-concentrated cultures (Table I) could be independently assessed. Induction of CYP1A1 up-regulation, and to lesser degree that of CYP1A2 and CYP1B1, was most marked in S-phase-concentrated cultures compared to G2/M-phase-concentrated cultures and, especially, G0/G1-phase-concentrated cultures (Table III). This might suggest a role for CYP1A1 in G0/G1-phase to S-phase transition (18Go).

Compared to G0/G1-phase-concentrated cultures, MCF-7 cells concentrated in S-phase contained higher levels of CYP1A1 (along with CYP1A2 and CYP1B1) mRNA transcripts following exposure to B[a]P. AhR-mediated up-regulation of CYP1A1 induction was associated with elevated expression of P21WAF1/CIP1 and, presumably as a consequence of this DNA damage, there was a propensity towards apoptosis as reflected by a down-regulation of BCL-2 and an up-regulation of BAX. This points to an elevated susceptibility of exponentially-growing cell populations to B[a]P-induced DNA damage and was reflected in elevated levels of comet-forming activity (Table IV and Figure 1) and DNA adduct formation (Table V). Interestingly, cells in S-phase-concentrated cultures exhibited a significantly higher (P < 0.0001) level of DNA SSBs than those in G0/G1-phase-concentrated cultures or G2/M-phase-concentrated cultures. Increased susceptibility to DNA damage could be due to strand discontinuities occurring during normal DNA replication (35Go) or modulation of DNA ligase I (36Go) and may result in a persistent BPDE-induced S-phase arrest (37Go).

The inhibitor of microtubule formation, nocodazole, used here to synchronize MCF-7 cells in G2/M-phase, may itself generate intracellular-damaging mechanisms (38Go) but would be unlikely to interfere with B[a]P agonist effects on the AhR. B[a]P-induced CYP1A1 mRNA transcript levels were lower in G2/M-phase than in S-phase-concentrated cultures (Table III). The apparent lack of elevated P21WAF1/CIP1 expression following 24-h B[a]P exposure may point to an underlying toxic effect induced by this aneugen (39Go) (Table III). Suppressed CYP1A1 transcription in G2/M-phase-concentrated cultures [16Go] would account for the reduction in B[a]P-DNA adduct formation compared to S-phase-concentrated cells (Table V) but nocodazole toxicity might explain an increased susceptibility to DNA damage measurable in the comet assay (Table IV and Figure 1).

B[a]P, an exogenous ligand of AhR, elevated expression of CYP1A1, CYP1A2 or CYP1B1 in MCF-7 cells was most profound in S-phase-concentrated cultures. Although complete synchronization was not achieved, our results appear to indicate that CYP induction in S-phase-concentrated cultures exceeds that which could be numerically accounted for on the basis of an increased number of cells in this phase. Thus, CYP induction may also be dependent on the cycling properties of the whole-cell population as opposed to a subset of cells in a particular cell cycle phase.

Many substrates for CYP isoforms are also inducers of these metabolizing enzymes (40Go) and cell growth kinetics might be an important predictor of target-cell susceptibility. Our results suggest that regulation of xenobiotic-metabolizing genes is associated with MCF-7 cell growth kinetics and that this in turn influences the extent of genotoxic damage induced by a DNA-reactive pro-carcinogen.


   Acknowledgments
 
The authors acknowledge grants from the North West Cancer Research Fund (H.J., S.L.A., R.H., F.L.M.), Rosemere Cancer Foundation (M.J.W., F.L.M.) and Cancer Research UK (K.J.C., D.H.P).

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +44 1524 594505; Fax: +44 1524 593192; Email: f.martin{at}lancaster.ac.uk

3 Present address: Department of Medical Genetics and Cell Biology, Ningxia Medical College, 692 Shengli Street, Yinchuan, Ningxia 750004, People's Republic of China Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Received on July 29, 2006; revised on October 27, 2006; accepted on October 31, 2006.


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