Our Science – Poirier Website

Miriam C. Poirier, Ph.D.

Laboratory of Cancer Biology and Genetics Head, Carcinogen-DNA Interactions Section Senior Investigator Building 37, Room 4032C 37 Convent Drive Bethesda, MD 20892 Phone:   301-402-1835 Fax:   301-402-8230 E-Mail:   poirierm@exchange.nih.gov

Biography

Dr. Poirier received a B.Sc. magna cum laude in chemistry. She obtained an M.Sc. in experimental oncology from the McArdle Laboratories at the University of Wisconsin under Drs. James A. and Elizabeth C. Miller. She received her Ph.D. from Catholic University, Washington, D.C. Since 1997, she has been head of the Carcinogen-DNA Interactions Section of the Laboratory of Cellular Carcinogenesis and Tumor Promotion.

Research

Carcinogen-DNA Interactions: Their Extent, Mechanisms, and Biological Consequences

This research effort has 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. We are particularly interested in searching out themes that are common to both animal models and human subjects, with the intention of applying the knowledge gained to either enhance or reduce a specific effect in humans. The compounds of intensive investigation include the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs), as well as the chemotherapeutic agents cisplatin and tamoxifen (TAM), and the polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants.

Drug combinations that include one or two nucleoside analogs have become the standard of care for individuals infected with the Human Immunodeficiency Virus (HIV), and are given to both adults and to HIV-infected pregnant women to prevent vertical viral transmission. However these drugs are known to cause mitochondrial dysfunction in HIV-1-infected adults and in the current study we demonstrated that there is some mitochondrial compromise in infants exposed in utero. Two indicators of mitochondrial toxicity, mitochondrial morphology by EM and mtDNA quantity, were examined in umbilical cord and cord blood leukocytes taken from HIV-1-uninfected infants born to either HIV-1-infected women receiving Combivir (AZT plus 3TC) therapy (n=10), or HIV-1-uninfected women (n=9). Moderate to severe mitochondrial morphological damage was observed by EM in umbilical cords from 6 Combivir-exposed infants and none of the unexposed infants. In addition, mtDNA depletion of at least 35% was found in umbilical cord and cord blood of Combivir-exposed infants, compared to unexposed controls. The data suggest that, in the face of an unremarkable clinical presentation, some Combivir-exposed children sustain molecular and cellular mitochondrial damage at birth.

A high level of variability was previously observed for AZT-DNA incorporation in human leukocytes from exposed mothers and infants. To explore this normal human mammary epithelial cell (NHMEC) strains were used to investigate variability in human AZT metabolism. NHMEC strains (n=19) were exposed for 24 hours to 0 or 200M AZT. AZT-DNA incorporation ranged from 6 to >100 AZT molecules/106 nucleotides in 12 strains, with no detectable AZT-DNA in 7 strains. Because the active form of the enzyme thymidine kinase (TK1) performs the initial AZT phosphorylation on the pathway to AZT-DNA incorporation, we examined TK1 levels by Western blot and showed that NHMEC strains with no AZT-DNA incorporation had little or no TK1, while the majority of those with measurable AZT-DNA showed evidence of constitutive or inducible TK1 activity. Therefore, the high variability for AZT-DNA incorporation levels in human samples may be at least partially due to variability in levels of active TK1.

Aneuploidy and chromosomal instability are important events in malignant transformation, and here we have shown significant AZT-induced abnormalities in kinetochore and spindle proteins, that may lead to aneuploidy in CHO cells and in NHMECs. Centrosomal amplification and fragmentation occurred in CHO cells exposed to 200, 400 or 800 μM AZT and in NHMECs exposed to 200 μM AZT. Because aberrations in centrosome morphology are associated with chromosomal mis-segregation, micronuclei bearing intact chromosomes (identified by CREST antibody) were scored revealing increased positive signal for centromeric kinetochores within micronuclei of cells exposed to AZT. Therefore, AZT-induced genomic instability that is associated with alterations in proteins involved in centrosomal activation may be related to the carcinogenic potency of this compound.

In ongoing studies we are investigating mitochondrial toxicity in fetal Erythrocebus patas monkeys taken at birth and at 1 year of age from dams exposed to human-equivalent protocols containing 3TC, AZT, AZT/3TC, AZT/Didanosine (ddI), 3TC/Stavudine (d4T), AZT/3TC/abacavir (ABC) or AZT/3TC/nevirapine. The antiretroviral drugs were given to pregnant patas dams (n=3-4/group) during the last half of gestation and to their neonates for the first 6 weeks after birth. Mitochondrial morphology, examined by EM, revealed mild to moderate changes in heart and skeletal muscle in fetuses exposed to 3TC or AZT alone, while the 2- and 3-drug combinations showed severe mitochondrial EM damage in these organs at birth. There was little improvement in the overall mitochondrial morphology at 1 year of age. mtDNA quantity, determined by hybrid capture-chemiluminescence immunoassay (HC-CIA), was substantially (>50%) depleted at birth in heart and skeletal muscle from infants exposed in utero to the 2-NRTI combinations. At 1 year of age NRTI-exposed heart mtDNA levels were higher than in the unexposed controls, and skeletal muscle mtDNA levels were depleted. Alterations in OXPHOS enzyme specific activities at birth and 1 year age were not extensive in heart and skeletal muscle from NRTI-exposed monkeys. The order of increasing toxicity was as follows: 3TC < AZT < AZT/3TC < AZT/d4T < AZT/ddI = 3TC/d4T

An epidemiological association between ingestion of well-cooked meat and colon adenoma incidence, and the observation that PAH=s are produced during high-temperature cooking of meats, have lead to the hypothesis that PAH-DNA adduct formation may be associated with colon adenoma incidence. Samples of human leukocyte DNA were obtained from 82 individuals with rectal adenoma and 111 controls, all of whom were non-smokers. PAH-DNA adducts, measured by BPDE-DNA Chemiluminescence Immunoassay (CIA) were found to be higher among colorectal adenoma cases (1.4 adducts/108 nucleotides) than polyp-free controls (1.2 adducts/108 nucleotides) (p=0.05). Compared to individuals in the lowest quartile of PAH-DNA adduct level, those in the highest quartile had an odds ratio (OR) of 2.8 for risk of colorectal adenoma formation (p=0.048). The data support a link between PAH-exposure and colorectal neoplasia

Epidemiological studies have shown that, among women with cancer associated HPV infection, smoking may increase the cervical cancer risk an additional 2 4 fold. In an effort to elucidate the contribution of smoking we examined human cervical paraffin-embedded sections for the presence of PAH-DNA adducts using immunohistochemical staining and the ACIS. The study, involving 142 coded samples of human cervix, has shown a broad range - between 25 and 191 adducts/108 nucleotides - for PAH-DNA adduct formation. There was, however, no association between smoking and PAH-DNA adduct levels or between degree of cervical neoplasia and PAH-DNA adduct levels. The data suggest that tobacco-associated cervical cancer in HPV-infected women has multifactorial origins.

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. To elucidate the formation of BP-DNA adducts and understand the underlying metabolism, 15 NHMEC strains and MCF-7 cells were exposed to 4 µM BP. The NHMECs contained 0.2-10.6 BP-DNA adducts/108 nucleotides while the MCF-7 cells had 790 BP-DNA adducts/108 nucleotides. CYP1B1 and NQO1 gene abundance (transcripts/ng RNA [tpn]) were determined by quantitative real-time PCR (qRT-PCR). In unexposed cells, CYP1B1 abundance was 569-3,452 tpn in NHMECs, and 12,897 tpn in MCF-7 cells. After 12h of BP exposure, CYP1B1 abundance increased 7- to 8-fold in NHMECs and 2.8-fold in MCF-7 cells. In unexposed cells, NQO1 gene abundance was 9,004-22,530 tpn in NHMECs, and 4,962 tpn in MCF-7 cells. After 12h of BP exposure, NQO1 abundance increased 1.1- to 1.4-fold in NHMECs, and 2.7-fold in MCF-7 cells. To compare gene expression with enzyme activity, CYP1A1 and 1B1 enzyme activities (by EROD assay), and dicumorol sensitive NQO1 enzyme activity, were measured in NHMEC strain M98016 and in MCF-7 cells. In unexposed MCF-7 cells, EROD activity was 4 fold higher than that found in the NHMECs. In BP-exposed NHMECs and MCF-7 cells EROD activity was increased 1.6- to 5-fold. NQO1 enzyme activity decreased by 45% and 13% in BP-exposed MCF-7 cells and NHMECs, respectively. Therefore, the high BP-DNA adduct level observed in MCF-7 cells appears to be driven by high levels of CYP1B1 expression and enzyme activity, and low levels of NQO1 enzyme activity. Overall, these results suggest that normal human breast cells are protected from BP-DNA damage, and possibly mutagenesis, by their high capacity for detoxification and comparatively low levels of activation.

Collaborators on this research include Sanford Dawsey, Philip Taylor, Mark Roth, Nathaniel Rothman, Gene Shearer, and Rashmi Sinha, NIH; Bernadette Schoket, National Institute of Public Health, Budapest, Hungary; Vernon Walker, Lovelace Respiratory Research Institute, Inhalation Toxicology Laboratory, Albuquerque, NM; and Ainsley Weston, NIOSH-CDC, Morgantown, WV.

This page was last updated on 6/12/2008.