Our Science – Poirier Website
Miriam C. Poirier, Ph.D.
Carcinogen-DNA Interactions: Their Extent, Mechanisms, and Biological Consequences
Interactions between chemical carcinogens and DNA, which result in damage to DNA, are considered a necessary, but not sufficient, early 'initiating' step in the process of chemical carcinogenesis. Our studies have concentrated on mechanisms of interaction between chemical carcinogens, some of which are commonly used drugs, and DNA. Topics under study include both the extent of DNA adduct formation and persistence, and the biological consequences of DNA damage in cultured cells, animal models and human tissues. Information on DNA adduct processing in nuclear and mitochondrial DNA are correlated with specific effects of exposure, including tumorigenesis, clinical response, specific toxicities and functional alterations in target organs and organelles. Having pioneered one of the earliest methodologies for measuring DNA damage in humans, we are interested in searching out themes that are common to both animal models and human subjects. We wish to apply the knowledge gained to enhance or reduce a specific effect in humans, and to lower cancer risk through the use of chemoprevention. The carcinogens of intensive investigation include: polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants; tamoxifen (TAM), used for adjuvant chemotherapy; and the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) used for therapy of human immunodeficiency virus type 1 (HIV-1).
Our long-term studies in the area of PAH carcinogenesis have demonstrated that DNA damage, caused by a family of carcinogenic PAHs, can be measured in a variety of human tissues by immunoassay, and localized in particular cell types by immunohistochemistry (IHC). We have shown that human blood cell PAH-DNA adducts are formed as a result of dietary ingestion of PAHs, and increase or decrease as a function of the level of ingested PAHs. In addition, we found that PAH-DNA adducts from dietary exposure have a half-life of about a week in human blood cell DNA. When PAH levels in the diet are low, inhalation is a significant source of PAH exposure, and we reported that moving the same cohort from an area with a clean ambient environment to one that was much more polluted resulted in significant increases in blood cell PAH-DNA adduct levels. Pollution may vary by season, and we found a significantly higher level of blood cell PAH-DNA adducts in Mexico City in the dry season, when ambient PAH levels were the highest, compared to the rainy season. More recently, we have shown localization of PAH-DNA adducts in all human tissues investigated, including placenta, cervix, vulva, prostate and esophagus, suggesting that PAH-DNA adducts have widespread distribution in human organs. With respect to cancer risk, we demonstrated a 3-fold increased risk of colorectal adenoma in individuals with the highest leukocyte PAH-DNA adducts, compared to polyp-free controls. These were, coincidentally, the individuals who consistently ate the most heavily cooked beef. Taken together, our PAH studies suggest that PAH-DNA levels vary with PAH exposure, and decreasing PAH-DNA levels may decrease human cancer risk.
Cell culture studies using normal human mammary epithelial cells (NHMECs) cultured from 20 different individuals and exposed to benzo[a]pyrene (BP, a PAH and known human carcinogen) have revealed broad interindividual variability in BP-DNA adduct formation, induction of CYP1A1 and 1B1 expression, and induction of their combined enzyme (EROD) activity. However, the addition of chlorophyllin (CHL), a chemopreventive agent, in the presence of BP, caused a marked reduction (20-80%) in BP-DNA adduct formation in all the strains tested, suggesting that CHL may be a useful chemopreventive agent in the human population. The CHL studies are currently being extended to DNA repair-deficient, p53 haploinsufficient mice.
The formation of TAM-DNA adducts in human endometrium is a controversial topic of interest, as TAM-exposed women are at risk for endometrial cancer. Our current efforts are focused on searching for evidence of a genotoxic mechanism in tissues from women and female monkeys exposed to TAM. We have recently demonstrated the presence of TAM-DNA adducts in human endometrial tumor and normal surrounding tissue from cancer patients receiving TAM therapy, but not in corresponding endometrial tissues from cancer patients receiving no TAM. These studies suggest that TAM induces endometrial cancer at least partially through genotoxicity.
Drug combinations that include two nucleoside reverse transcriptase inhibitors (NRTIs) are used as therapy for individuals infected with the human HIV-1. These drugs are incorporated into DNA acting as replication chain terminators. However, mitochondrial toxicities limit NRTI use, and genotoxicity studies suggest that exposure may confer a long-term cancer risk. We have hypothesized that NRTI incorporation and sequelae are fundamental to both the nuclear and mitochondrial manifestations of toxicity. We are particularly interested in HIV-1-uninfected infants born to HIV-1-infected mothers receiving antiretroviral (ARV) therapy because these children, though born with no HIV-1 infection, are particularly vulnerable as a result of their in utero exposure.
We have reported novel manifestations of NRTI-induced genotoxicity in cultured cells, including: arrest of cells in S-phase, centrosomal amplification, and chromosomal loss. In addition, our studies using DNA repair-deficient cells suggest that nucleotide excision repair (NER) may play a role in the processing of azidothymidine (AZT)-modified DNA. We showed that the variable AZT-DNA incorporation found in 19 different NHMEC strains correlated with induction of thymidine kinase-1 (TK-1), the first step in activation of AZT. In addition, using tissues from neonatal HIV-1-uninfected infants born to HIV-1-infected mothers receiving NRTI therapy we demonstrated AZT-DNA incorporation, as well as DNA fragmentation by Comet assay, and mutagenesis by glycophorin A (GPA) assay. The mutagenesis persisted in ARV drug-exposed infants at 1 year of age.
Manifestations of mitochondrial toxicity in children born to HIV-1-infected mothers are considered due to both maternal HIV-1 status and maternal ARV-drug use. Using Erythrocebus patas (patas) monkey dams exposed to human-equivalent ARV-drug protocols in the absence of virus, we demonstrated that mitochondrial compromise can be induced by the drugs alone. We showed persistent mitochondrial toxicity in skeletal muscle, and progressive mitochondrial toxicity in heart, liver and brain of 1 year-old patas offspring exposed in utero and for 6 weeks after birth. These studies suggest the potential usefulness of chemoprotective agents, and we are testing amifostine, dexrazoxane and tempol for protective effects in cultured cardiomyocytes exposed to NRTIs. Evaluation of mitochondrial integrity is by Seahorse XF24. Studies to date suggest that tempol may be a promising protective agent.
Collaborators on this research include several NCI investigators: Ettore Appella, Qing Lan, James Mitchell, Nathaniel Rothman, Gene Shearer and Rashmi Sinha. Collaborators outside of the NIH include: Frances L. Martin, Lancaster University, Lancaster, UK; Radim Sram, Institute of Experimental Medicine, Prague, Czech Republic; David Manchester, Denver Children's Hospital, Denver, CO; Maria Eugenia Gonsebatt, UNAM, Mexico City, Mexico; Vernon E. Walker, University of Vermont, Burlington, VT; Eugene Herman, FDA/CDER, White Oak, MD; and Igor Pogribny and Frederick A. Beland, both of NCTR/FDA, Jefferson, AR.
This page was last updated on 2/21/2013.