Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Two 2-year research grants from NIEHS/NIH supported the pathways research: (1) "Biochemistry of mismatch-repair responses to DNA lesions" and (2) "MMR-coupled translesion DNA synthesis during suppression of PAH-induced mutation. A supplemental NIEHS grant covered undergraduate research opportunities. The federal grants totaled over $410,000, addressing the hypothesis that active MMR suppresses PAH-induced DNA mutations and carcinogenesis, and that PAH exposure would represent cancer risk for MMR-deficient individuals. We investigated biochemical and cellular responses dependent upon MMR proteins to a model PAH. Preliminary data suggested that MMR plays a role in signaling the DNA replication arrest seen following BPDE-exposure, perhaps by helping to resolve the signal for arrest. A second research focus looked at the effectiveness of natural substances in the human diet for cancer protection. Red raspberries contain anthocyanins which may reduce the risk of various diseases, and have been shown to reduce the incidence of certain cancers. Currently we lack information on anthocyanin dietary chemoprevention in transplacental exposure to carcinogens. We conducted a dose response study examining the chemo preventative activity of raspberry extract and C3OG against DBC carcinogenesis in a mouse model and determined if these neutraceuticals inhibit Nf-κB levels induced in the lung by DBC. Results determined pretreatment and dose concentration in a mouse tumor study to monitor offspring survival and incidence of lymphoma over a 10-mon period and measure expression of NfκB with western immunodetection. Animals succumbed to T-ALL during the 10- mon period were euthanized and tissues fixed in buffered formalin. Ten-month survivors were necropsied and analyzed. Early results using standard HPLC with MS/MS show a reduction of lung adducts from mothers fed C3OG diet compared to control fed animals, but the method was not sensitive enough to determine differences in fetal treatment groups. We optimized a method using ultra pressure liquid chromatography (UPLC) to increase sensitivity. Preliminary results showed decrease levels in neonate thymus and liver compared to controls but no difference in adduct levels in lung. A 10-mon tumor study using the same dietary intervention through gestation and lactation was completed using the carcinogen DBC 15 mg/kg. Dietary intervention had no effect on the pathway in thymus cytosol and nuclear fraction. The remaining progeny were monitored for an aggressive T-cell lymphoblastic lymphoma (T-ALL), the typical outcome with this treatment. Overall 10 mon survival in the control diet group was 14%, compared to 19% in the raspberry diet and 17% in the C3OG diet groups. Kaplan-Meier survival curves showed no significant difference among the treatment groups (p=0.23). Superfund support allowed testing of PAHs from Superfund site at Portland Harbor in mouse models. Resulting skin tumors were confirmed; the first study demonstrated transplacental lung carcinogenesis with environmental PAH mixture. This would be the first study to document the tissue distribution of this potent carcinogen in a pregnant animal model. PARTICIPANTS: Dave Williams, Linus Pauling Institute, OSU Ian Blair, Center for Cancer Pharmacology, University of Pennsylvania TARGET AUDIENCES: The general public will ultimately benefit from risk assessment and preventative strategies based on individual generic profiles and the threats posed by particular classes of PAHs. Information and insights generated by the research proposed will guide formulation of these strategies by the medical research community, as well as state and federal regulatory agencies. PROJECT MODIFICATIONS: The original project laid out a plan for measuring DNA adducts by 33P-post labeling. This method has been the standard procedure for detecting low amounts of cancer inducing DNA interaction products (adducts) for over 17 years and we did not anticipate any difficulty in carrying out these analyses. We were unable to carry through with this plan as an important enzyme, prostatic acid phosphatase, used for removing a phosphate from the DNA was no longer available. We therefore put a significant amount of time and effort into developing an LC/MS/MS method for measuring adducts from lung, liver, and thymus.
Impacts
Understanding the biochemical mechanisms and the interactions of different cellular pathways will help to identify individuals and populations at risk of cancer from exposure to PAHs, allowing for a reduction in societal costs through targeted educational and preventative measures. Our data demonstrate for the first time that MMR-proficiency does impact the mutagenicity of a model PAH, consistent with our hypothesis that MMR status would similarly influence risk of PAH-induced carcinogenesis. On-going analyses will expand the analysis of the mutational spectra in MMR-proficient versus -deficient cells treated in parallel at different doses, and address the biochemical mechanism of the MMR-dependent responses through analysis of PAH-lesion-provoked excision and processing. So far, survival curves for mouse models using raspberry extracts showed no significant difference among the treatment groups. The research supports the recruitment, retention, training, graduation, and placement of the next generation of research scientists. Students conducted research aimed at preparation of DNA plasmid substrates for use in biochemical assays of the DNA mismatch repair pathway, to quantify the abundance of DNA replication enzymes and to measure cell cycle reponses of cultured human cells following exposure to BPDE. The principal objective of the proposed research was to improve the scientific knowledge base concerning the interaction between genes and diet, and how genetic diversity affects the relationship between whole diet as a risk factor for chronic disease. The general public will ultimately benefit from risk assessment and preventative strategies based on individual generic profiles and the threats posed by particular classes of PAHs. Information and insights generated by the research proposed will guide formulation of these strategies by the medical research community, as well as state and federal regulatory agencies.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Our work in this project period focused on determining the effect of inactivation of DNA mismatch repair (MMR) on polycyclic aromatic hydrocarbon (PAH) -induced mutagenesis and carcinogenesis. Objectives were: (1) To identify classes of PAH-induced mutations specifically suppressed by MMR, and (2) To evaluate the general utility of genetic models in mice to investigate the gastrointestinal carcinogenicity of PAHs. Transgenic mice, MMR-deficient or -proficient, were exposed to diolepoxide racemic mixtures of benzo[a]pyrene (B[a]P) or benzo[c]phenanthrene (B[c]Ph), and the frequency and spectra of PAH-induced mutations in the intestines and other internal organs will be determined using reporter genes. Specifically, the project is testing the hypothesis that loss of MMR function increases PAH-induced carcinogenicity, the incidence, multiplicity and size of intestinal tumors and preneoplastic lesions in the intestine of MMR-deficient vs. - proficient mice. This will be determined following neonatal exposure to (B[a]P) or (B[c]Ph) diolepoxides. This effort will directly evaluate risk of gastrointestinal tumorigenesis due to exposure to representative examples of relevant human carcinogens (PAHs) in a genetically susceptible population (animals with MMR-deficiency) and will address mechanism(s) that may increase risk. The information gained will help identify human populations at potentially high risk from environmental PAH exposure. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
MMR is an important component of apoptotic signaling pathways in intestinal stem cells triggered by several different types of DNA damage. In addition, we evaluated the effect of MMR status on the carcinogenic potential of PhIP in mice [71], and found that induction of aberrant crypt foci (ACF; monoclonal preneoplastic lesions thought to represent an early step in colorectal carcinogenesis [72, 73]) in Mlh1-/- mice was four-times greater than in wild-type littermates, indicating that loss of MMR increases the likelihood of PhIP-induced ACF. Together, our data are consistent with the hypothesis that MMR suppresses cancer risk due to dietary carcinogens via suppression of damage-induced mutation and via apoptotic killing of damaged stem cells, together reducing the chance of propagating mutated genomes prone to carcinogenic transformation. Based on our experience in these studies, we anticipate no difficulty with similar experiments evaluating other effects of PAH exposure in MMR-deficient mice
Publications
- No publications reported this period
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