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.
click on figure to enlarge |
PCR products were derived from reverse-transcribed mRNA from
benign prostatic hyperplasiatissues (n=4).
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Figure 1. RT-PCR analysis of CYP1A1, CYP1A2 and
CYP1B1 expression in human prostatic tissue.
click on figure to enlarge
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. |
click on figure to enlarge
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click on figure to enlarge
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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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