Gene expression profiling of di-n-butyl phthalate in normal human mammary epithelial cells.

Studies show that female workers in the personal-care industry have an increased risk of developing cancer believed to be the result of increased exposure to toxic and/or carcinogenic chemicals found in cosmetics, hair dyes, and nail polish. One chemical found in multiple personal-care products, di-n-butyl phthalate (DBP), is a known endocrine disruptor and has been found in increased levels in women of childbearing age. The goal of this study was to elucidate mechanisms of phthalate toxicity in normal human cells to provide information concerning interindividual variation and gene-environment interactions. Normal human mammary epithelial cell strains were obtained from discarded tissues following reduction mammoplasty [Cooperative Human Tissue Network (sponsors: NCI/NDRI)]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (U133A, Affymetrix) and changes in the expression of selected genes were verified by real-time polymerase chain reaction (PCR) (ABI). DNA microarrays were hybridized with total RNA that was collected after DBP treatment for 5 hr and 10 hr. RNA was harvested from the vehicle control (acetone) at 10 hr. Data Mining Tool software (Affymetrix) was used to separate genes in clusters based on their expression patterns over time. Only 57 genes were found to be altered in all four cell strains following exposure to DBP. These included genes involved in fertility (inhibin, placental growth factor), immune response (tumor necrosis factor induced protein), and antioxidant status (glutathione peroxidase). Data from this study will help clarify the role of DBP in reproductive toxicity, and yield biomarkers of exposure for future epidemiology studies.

Differential gene expression in normal human mammary epithelial cells treated with malathion monitored by DNA microarrays.

Organophosphate pesticides are a major source of occupational exposure in the United States. Moreover, malathion has been sprayed over major urban populations in an effort to control mosquitoes carrying West Nile virus. Previous research, reviewed by the U.S. Environmental Protection Agency, on the genotoxicity and carcinogenicity of malathion has been inconclusive, although malathion is a known endocrine disruptor. Here, interindividual variations and commonality of gene expression signatures have been studied in normal human mammary epithelial cells from four women undergoing reduction mammoplasty. The cell strains were obtained from the discarded tissues through the Cooperative Human Tissue Network (sponsors: National Cancer Institute and National Disease Research Interchange). Interindividual variation of gene expression patterns in response to malathion was observed in various clustering patterns for the four cell strains. Further clustering identified three genes with increased expression after treatment in all four cell strains. These genes were two aldo-keto reductases (AKR1C1 and AKR1C2) and an estrogen-responsive gene (EBBP). Decreased expression of six RNA species was seen at various time points in all cell strains analyzed: plasminogen activator (PLAT), centromere protein F (CPF), replication factor C (RFC3), thymidylate synthetase (TYMS), a putative mitotic checkpoint kinase (BUB1), and a gene of unknown function (GenBank accession no. AI859865). Expression changes in all these genes, detected by DNA microarrays, have been verified by real-time polymerase chain reaction. Differential changes in expression of these genes may yield biomarkers that provide insight into interindividual variation in malathion toxicity.

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.

The effect of oxythioquinox exposure on normal human mammary epithelial cell gene expression: a microarray analysis study.

BACKGROUND: Inter-individual variation in normal human mammary epithelial cells in response to oxythioquinox (OTQ) is reported. Gene expression signatures resulting from chemical exposures are generally created from analysis of exposures in rat, mouse or other genetically similar animal models, limiting information about inter-individual variations. This study focused on the effect of inter-individual variation in gene expression signatures. METHODS: Gene expression was studied in primary normal human mammary epithelial cells (NHMECs) derived from four women undergoing reduction mammoplasty [Cooperative Human Tissue Network (National Cancer Institute and National Disease Research Interchange)]. Gene transcription in each cell strain was analyzed using high-density oligonucleotide DNA microarrays (HuGeneFL, Affymetrix) and changes in the expression of selected genes were verified by real-time polymerase chain reaction at extended time points (ABI). DNA microarrays were hybridized to materials prepared from total RNA that was collected after OTQ treatment for 15, 60 and 120 min. RNA was harvested from the vehicle control (DMSO) at 120 min. The gene expression profile included all genes altered by at least a signal log ratio (SLR) of +/- 0.6 and p value < or = 0.05 in three of four cell strains analyzed. RESULTS: RNA species were clustered in various patterns of expression highlighting genes with altered expression in one or more of the cell strains, including metabolic enzymes and transcription factors. Of the clustered RNA species, only 36 were found to be altered at one time point in three or more of the cell strains analyzed (13 up-regulated, 23 down-regulated). Cluster analysis examined the effects of OTQ on the cells with specific p53 polymorphisms. The two strains expressing the major variant of p53 had 83 common genes altered (35 increased, 48 decreased) at one or more time point by at least a 0.6 signal log ratio (SLR). The intermediate variant strains showed 105 common genes altered (80 increased, 25 decreased) in both strains. CONCLUSION: Differential changes in expression of these genes may yield biomarkers that provide insight into inter-individual variation in cancer risk. Further, specific individual patterns of gene expression may help to determine more susceptible populations.