Two zones of the prostate, the peripheral zone (PZ) and the transition zone (TZ), which differ in cancer susceptibility, were examined for the expression of Phase I/Phase II genes and polycyclic aromatic hydrocarbon (PAH)-DNA adduct formation. Clinically, there is a higher proportion of prostate tumors arising in the PZ, with fewer tumors in the TZ. Matched paraffin-embedded tissue sets (n=28) obtained from radical retropubic prostatectomies, were examined for the expression of cytochrome (CYP) P450s (1A1 and 1B1), N-acetyl transferase 1 and 2 (NAT1 and NAT2) and catechol-O-methyl transferase (COMT), using quantitative real-time reverse transcriptase (RT)-PCR. Levels of CYP1B1 and NAT1 were significantly higher in the PZ compared to the TZ (p<0.001 and <0.01, respectively). Immunohistochemical (IHC) staining of PZ and TZ sections with antiserum elicited against r7, t8-dihydroxy-t-9, 10-oxy-7, 8, 9, 10-tetrahydro-benzo[a]pyrene (BPDE)-DNA was followed by quantitation of nuclear color intensity by Automated Cellular Imaging System II (ACIS II). PAH-DNA adducts were concentrated in the prostate glandular epithelial cells, and levels were very high compared to other human tissues. However, there was no significant difference in PAH-DNA adduct levels for the PZ, and TZ samples. Therefore, though expression levels of CYP1B1 and NAT1 were higher in the PZ than in the TZ, PAH-DNA adduct levels were similar in both zones suggesting that localization of metabolite formation is independent of DNA adduct localization. Parental exposure to tobacco smoke carcinogens has been implicated in the development of childhood cancers. Cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots that line the placental chorionic villi are known to metabolize xenobiotics that subsequently enter fetal circulation. We examined human placenta for PAH-DNA adduct formation using semi-quantitative IHC/ACIS II. A standard curve was prepared using cultured human cervical keratinocytes exposed for 1 hr to increasing doses of BPDE, where cells were evaluated both by IHC/ACIS II, and by BPDE-DNA chemiluminescence immunoassay (CIA) of extracted DNA. The two approaches were highly correlated (R2 = 0.99). Anti-BPDE-DNA IHC staining of full-term human placenta samples showed positive nuclear color localized in CT cells and ST knots. Comparison of placental IHC values with the keratinocyte standard curve revealed statistically indistinguishable PAH-DNA adduct values of 223 27/108 nucleotides (mean SE) for 7 smokers and 187 34/108 nucleotides for 7 non-smokers. The data suggest that smoking is not the only exposure contributing to PAH-DNA adduct formation in human placenta. Normal human mammary epithelial cell (NHMEC) strains, cultured from human breast tissue obtained at reduction mammoplasty, provide a relevant model for investigation of human interindividual differences in carcinogen metabolism and DNA-damage response. In this study we hypothesized that the chemopreventive agent chlorophyllin (CHLN) might reduce benzo[a]pyrene (BP) genotoxicity. Using NHMECs exposed to 4M BP for 24 hr in the presence or absence of 5M CHLN, we measured r7, t8, t9-trihydroxy-c-10-(N2deoxyguanosyl)-7, 8, 9, 10-tetrahydro-benzo[a]pyrene (BPdG) by BPDE-DNA CIA. The protocol included the following experimental groups: BP alone;BP given simultaneously with CHLN for 24 hr;CHLN given for 24 hr followed by BP for 24 hr;and CHLN given for 48 hr with BP added for the last 24 hr. Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the group with 48 hr exposure to CHLN. By Affymetrix microarray we found BP-induced up-regulation of CYP1A1 and CYP1B1, as well as up-regulation of groups of interferon-inducible, inflammation signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. By reverse transcriptase real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 (p<0.01) and >70% for CYP1B1 (p<0.01), was observed in the group with 48 hr CHLN exposure. To explore the relationship between gene expression and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, compared to the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls (p = 0.066). Overall, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but the reduction in EROD activity was not statistically significant. The data suggest that CHLN may act not only by altering CYP1A1 and CYP1B1 expression levels but also by lowering the effective dose of carcinogen. Hypothesizing that tamoxifen (TAM)-induced gene expression changes may provide insight into breast tissue response, TAM-exposed (10 M TAM, 48 hr) NHMEC strains, derived from reduction mammoplasty tissue taken from 3 individuals, were subjected to microarray. Cells with expression changes of >3-fold were as follows: 13 genes up-regulated and 1 down-related for NHMEC strain 16;17 genes up-regulated for NHMEC strain 5;and 11 genes up-regulated for NHMEC strain 40. Five interferon-inducible genes (IFITM1, IFIT1, IFNA1, MXI and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all 3 strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. RT-PCR revealed up-regulation of interferon α(IFNA1) and confirmed the TAM-induced up-regulation of the 5 other interferon-inducible genes identified by microarray. The data suggest that, in addition to hormonal effects, TAM exposure may enhance immune response in normal breast tissue.
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