National Cancer Research Institute South of England
Prostate Cancer Collaborative
Research

Environmental factors in prostate cancer

Authors
Professor David H. Phillips davidp@icr.ac.uk
Institute of Cancer Research

Aim
To investigate carcinogen activation and gene expression in human prostate and to shed light on the causes of prostate cancer

Synopsis
Environmental and/or dietary factors appear to be important in the aetiology of prostate cancer, but the nature of these factors remains obscure. Very little is known about the capability of prostate tissue to metabolise and activate putative environmental carcinogens. We are carrying out a systematic study of the metabolic capabilities of the prostate. We are investigating (1) the expression of xenobiotic metabolising genes in human prostate using real-time RT-PCR and microarray analysis with arrays customised to contain Phase I and II genes; (2) the ability of the tissue to activate putative environmental carcinogens to DNA-binding species; (3) interindividual variations and whether any are due to genetic polymorphisms; (4) the effects of maintaining non-tumorous prostate cells in culture; (5) the presence of DNA adducts formed in prostate in vivo. These studies will shed light on the causes of prostate cancer and determine whether cultured prostate cells are suitable for metabolic studies.


Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue.
Epidemiological evidence suggests a link between meat consumption and prostate cancer. In this study, benign prostatic hyperplasia tissues, obtained by transurethral resection or radical retropubic prostatectomy from UK-resident individuals (n=18), were examined for CYP1 expression and for their ability, in short term organ culture, to metabolically activate carcinogens found in cooked meat. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of CYP1 expression detected CYP1A2 mRNA transcripts in the prostates of 4/4 individuals, as well as mRNA transcripts from CYP1A1 and CYP1B1 (figure 1). The compounds tested for metabolic activation were 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 500mM, n=9) and its metabolite N-hydroxy PhIP (20mM, n=8), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 500mM, n=6) and benzo[a]pyrene (B[a]P, 50mM, n=5). After incubation (PFMR medium, 22h, 37°C) DNA was isolated from tissue fragments and DNA adducts were detected and quantified by 32P-postlabelling analysis. DNA adduct formation was detected in all samples incubated with PhIP (mean, 3 adducts/108 nucleotides), N-hydroxy-PhIP (2736/108), or B[a]P (1/108). IQ-DNA adducts were detected in 5/6 tissues (mean, 1/108) (figure 2). The CYP1 inhibitor a-naphthoflavone (10mM) reduced B[a]P-DNA adduct formation in tissues from two individuals by 96% and 64%, respectively. This pilot study shows that human prostate tissue can metabolically activate 'cooked meat' carcinogens, a process that could contribute to prostate cancer development.


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PCR products were derived from reverse-transcribed mRNA from benign prostatic hyperplasiatissues (n=4).

Figure 1. RT-PCR analysis of CYP1A1, CYP1A2 and CYP1B1 expression in human prostatic tissue.

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Figure 2. 32P-postlabelling analysis of heterocyclic amine-DNA and B[a]P-DNA adducts in human prostatic tissues.

Heterocyclic amine-DNA adduct patterns are shown after exposure to the following compounds at 37°C for 22h (a) Vehicle only, (b) PhIP (500mM), (c) N-hydroxy-PhIP (20mM), (d) IQ (500mM) and postlabelling using the ATP-deficient method. The PhIP-DNA adducts shown in (b) and (c) were further enzymatically digested, giving rise to adduct spot 1 shown in (e) (88% of the total radioactivity) and (f), (94% of the total radioactivity) respectively. Using the nuclease P1 postlabelling technique (g) DMSO vehicle only, (h) B[a]P-DNA standard (adduct spot 2), (i) after incubation with B[a]P (adduct spot 2, 50mM). The origin of the radioactive spot in figure 2g, and to the right of adduct spot 2 is unknown.

Primary cultures of prostate cells and their ability to activate carcinogens.
In a further study, prostate tissues were obtained following transurethral resection of the prostate (TURP) or radical retropubic prostatectomy. Tumour-adjacent tissue fragments were minced in warm PFMR-4A medium (37oC). Medium containing tissue fragments was then pipetted into collagen-coated petri dishes. Non-adherent material was removed by washing with fresh medium after 12 h. Adhered cells subsequently reacted positively with monoclonal antibodies to prostate-serum antigen (PSA). PSA was also detected in the medium. The genotoxicities of the chemical carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), its N-hydroxy (N-OH) metabolite and benzo[a]pyrene (B[a]P) in adherent cell populations from different donors (n=8) were examined. Cells were treated in suspension for 30 min at 37oC in the presence of the DNA repair inhibitors, hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA single strand breaks were detected in cells by the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) (mm). All three carcinogens induced dose-related increases in CTLs (P <0.0001) in cells from four donors 24 h post-seeding (Figures 3 and 4). However, in cells from a further two donors the genotoxic effects of PhIP, N-OH-PhIP and B[a]P observed after 24 h in culture were much less apparent after 48 h. After 96 h in culture cells from these donors appeared to be resistant to the comet-forming activity of these compounds (Figure 5). However, B[a]P-DNA adducts were still measurable by 32P-postlabelling for up to 14 days following 24-h exposure to 50 mM B[a]P in adhered cells from another two donors. This study shows that primary cultures of cells derived from the prostate can activate members of two classes of chemical carcinogens. Future development(s) may provide a robust model system to investigate the aetiology of prostate cancer.

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Figure 3. DNA strand breaks, measured as CTLs, in prostate epithelial cells (PECs) obtained at radical retropubic prostatectomy. CTLs were determined, using the Comet assay after incubation for 30 min either in the absence (A) or presence (B-H) of HU/ara-C. (A and B) Control PECs. (C, E and G) PECs incubated in the presence of HU/ara-C and increasing concentrations of PhIP. (D, F and H) PECs incubated in the presence of HU/ara-C and increasing concentrations of B[a]P.

Figure 4. DNA strand breaks, measured as comet tail lengths (CTLs), in prostate epithelial cells (PECs) obtained from TURP. CTLs were determined after incubation for 30 min either in the absence (A) or presence (B-H) of HU/ara-C. (A and B) Control PECs. (C, E and G) PECs incubated in the presence of HU/ara-C and increasing concentrations of PhIP. (D, F and H) PECs incubated in the presence of HU/ara-C and increasing concentrations of N-OH-PhIP.

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Figure 5. The ability of adhered prostate epithelial cells (PECs) in culture to activate carcinogens over time (24 h, 48 h and 196 h) as measured using the Comet assay. PECs were incubated for 30 min at 37oC with PhIP (200 mM), N-OH-PhIP (1.0 mM) or B[a]P (90.0 mM).


Publications
Williams, J.A., Martin, F.L., Muir, G.H., Hewer, A., Grover, P.L. and Phillips, D.H. Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue. Carcinogenesis, 21, 1683-1689 (2000).
Kooiman, G., Martin, F.L., Williams, J.A., Grover, P.L., Phillips, D.H. and Muir, G.H. The influence of dietary and environmental factors on prostate cancer risk. Prostate Cancer and Prostatic Diseases, 3, 256-258 (2000).
Martin, F.L., Cole, K.J., Muir, G.H., Kooiman, G.G., Williams, J.A., Sherwood, R.A., Grover, P.L. and Phillips, D.H. Primary cultures of prostate epithelial cells and their ability to activate carcinogens. Prostate Cancer and Prostatic Dis., 5, 96-104 (2002).

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