Induction of CYP1A1 and CYP1B1 and formation of carcinogen-DNA adducts in normal human mammary epithelial cells treated with benzo[a]pyrene.
Inter-individual variation in formation of carcinogen-DNA adducts and induction of cytochrome P450 genes was measured in 23 cultured normal human mammary epithelial cell (NHMEC) strains established from reduction mammoplasty tissue. Semi-confluent cells were exposed to 4 microM benzo[a]pyrene (BP) for 12 h and BP-DNA adduct levels were measured by chemiluminescence immunoassay using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). BP-DNA adduct levels for 22 of 23 different cell strains ranged from non-detectable (three samples) to about 15 adducts/10(8) nucleotides. Increases in levels of CYP1A1 and CYP1B1 were detected using both oligonucleotide arrays and reverse transcription/quantitative real-time polymerase chain reactions (RT-PCRs). For CYP1A1 and CYP1B1, the oligonucleotide array data and RT-PCR data were highly correlated (r=0.73 and 0.70, respectively), suggesting that oligonucleotide arrays are a suitable gene discovery tool, and demonstrating that the complementary and efficient RT-PCR may be used to confirm microarray data for a specific gene in a large number of samples. As measured by RT-PCR, inter-individual variation in CYP1A1 induction was 100-fold, while the variation in CYP1B1 induction was almost 40-fold. On a per-person basis, CYP1A1 and CYP1B1 induction were well-correlated (r=0.88, P<0.001), which is to be expected as they are under the control of a common transcriptional regulation mechanism in response to BP exposure. Inter-individual variation in carcinogen-DNA adduct formation could not be explained only by variation in levels of CYP1A1 or CYP1B1 induction, as neither was well-correlated with BPDE-DNA adduct level (r=0.40 and 0.50 for CYP1A1 and CYP1B1, respectively). Evaluation of glutathione-S-transferase M1 genotype (GSTM1 positive or null) revealed an apparent correlation between positive GSTM1 genotype and BPDE-DNA adduct levels (r=0.84 and 0.77 for CYP1A1 and CYP1B1, respectively); however, after removal of the single outlier this relationship was not significant. Overall the data suggest that BPDE-DNA adduct levels in normal human breast tissue may be modulated by multiple factors that include, but are not exclusive to, CYP1A1 and CYP1B1 inducibility and the presence or absence of GSTM1.
Keshava
Channa
C
Toxicology and Molecular Biology Branch, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Road, Morgantown, WV 26505-2888, USA.
Divi
Rao L
RL
Whipkey
Diana L
DL
Frye
Bonnie L
BL
McCanlies
Erin
E
Kuo
Maryanne
M
Poirier
Miriam C
MC
Weston
Ainsley
A
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D002273:Carcinogens / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D065633:Cytochrome P-450 CYP1B1; D018736:DNA Adducts / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D005838:Genotype; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D042361:Mammary Glands, Human / Q000187:drug effects* / Q000378:metabolism; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D011110:Polymorphism, Genetic; D014158:Transcription, Genetic / Q000187:drug effects
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D001189:Aryl Hydrocarbon Hydroxylases; D002273:Carcinogens; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; D018736:DNA Adducts; D005982:Glutathione Transferase; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C585496:CYP1B1 protein, human