Massachusetts Institute of Technology

Global Responses to AflatoxinB1 & Alkylating Agents

Leona Samson, Ph.D.
lsamson@mit.edu

Project Description

We have proposed a highly integrated plan to explore the transcriptional responses of a variety of organisms, including humans, to a broad range of toxic agents found in our environment. Transcriptional profiling is expected to reveal a truly global view of how cells, tissues and animals attempt to recover, not always successfully, from environmental insults. We hope to learn what features of environmentally induced transcriptional responses are predictive of success or failure, and in addition to learn what features correlate with specific outcomes of toxic exposures, for instance, cell death, apoptosis, mutagenesis and carcinogenesis.

The model environmental agents to be studied are Aflatoxin B, and its metabolites, plus a range of SN1 an SN2 alkylating agents. Together these agents are representitive of toxic agents found in our external environment, our food supply, the endogenous cellular environment, and the cancer chemotherapy clinic. The model organisms and cell systems whose transcriptional responsiveness will be scrutinized include E. coli, S. cerevisiae, cultured rodent and human cell lines, engineered rat and mouse liver tissues, rats and mice. We have integrated an in vitro tissue engineering project into this proposal with the goal of developing an alternative to using animals to test for toxicity and carcinogenicity. It seems to us that appropriate transcriptional responsiveness in this system represents a rigorous test of its similarity to in vivo tissues.

Two platforms for mRNA profiling will be employed, namely Affymetrix GeneChip DNA oligonucleotide micorarrays, and cDNA arrays fabricated in the MIT BioMicro Center. The Microarray and Bioinformatics Core will have its center of gravity in the MIT BioMicro Center, and this Core will serve as the focal point for three Research Projects and the Toxicology Research Core Project (TRCP). The TRCP activities will be closely aligned with the activities of the other 4 or 5 members of the Toxicogenomics Research Consortium. Together the overarching goals will be to establish good working practices in transcriptional profiling as it relates to the area of Toxicogenomics, and to contribute to the development of an extensive relational database as a repository for high quality data emerging from the field of Toxicogenomics. We anticipate that this research will uncover a myriad of hitherto unknown ways in which which cells respond to environmental agents.

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Project 1: Differential Expression of Genes Following Exposure to Aflatoxin B1

Principal Investigator: John Essigmann
jessig@mit.edu

The goal of this project is to use genome microarrays to investigate the mechanisms by which the potent human liver carcinogen aflatoxin B1 (AFB1) exerts its toxic and carcinogenic effects. Yeast, Escherichia coli and rodent cells will be treated with the 8,9-epoxide of AFB, or with other chemically reactive forms of the toxin. The time and dose dependent effects of the toxin on the expression of approximately 103-104 genes will be monitored. A key model will be infant B6C3F1 mice. While mice generally are refractory to liver carcinogenesjs by aflatoxin, and largely immune to its toxic effects, this strain shows a transient high level of sensitivity to carcinogenesis if the animals are dosed 4-7 days after birth. After that window of sensitivity, the mouse becomes resistant to the toxin. We plan to monitor the gene expression profile in this animal model during the change from sensitivity to resistance. These studies will complement the work done by L. Griffith on the aflatoxin sensitivity of rat liver cells; rats are sensitive to the carcinogenic effects of aflatoxin regardless of their age at the time of toxin administration.

The AFB1 epoxide forms a host of different DNA lesions. The DNA lesions most workers view as the precursors to genotoxicity and/or carcinogenesis include AFB|-N7-Guanine (the major adduct formed shortly after dosing in vivo), AFB|-FAPY (a highly persistent adduct in vivo) and the abasic site. In a pilot experiment we shall introduce DNA enriched for each of the above lesions into mammalian cells to determine whether we can observe specific patterns of gene expression associated with specific DNA adducts. These preliminary studies will be done in HeLa cells. If successful, they will provide information on gene expression in cells that contain only damaged DNA - i.e., the electrophilic carcinogen will not have damaged the other constituents of the cell. The genes expressed following introduction of DNA enriched for each of the above lesions will eventually help define which DNA lesion(s) give rise to biologically important endpoints.

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Project 2: Transcriptional Responses of Mice to Alkylating Agent

Principal Investigator: Leona Samson
lsamson@mit.edu

The ultimate goal of this project is to paint an integrated picture of how mammalian cells in culture and in the intact organism, respond upon exposure to alkylating agents. The specific agents are chosen to represent environmental toxicants as well as those commonly used for chemotherapy. Certain alkylated bases interfere with replication (and probably transcription too) and it is now known that these DNA base lesions can be cytotoxic, mutagenic and ultimately carcinogenic. Two major DNA repair pathways deal with alkylated base lesions present in cellular DNA. (i) Base excision repair (BER), initiated when the damaged base is enzymatically removed by a 3-methyladenine (3MeA) DNA glycosylase. (ii) Direct reversal of base damage wherein the unwanted alkyl group on O6-methylguanine (O6-MeG) is transferred to a DNA repair methyltransferase (MTase), thus inactivating the MTase and restoring the normal DNA base. It has long been known that exposure of cells to alkylating agents causes the levels of certain mRNA transcripts to be altered in a responsive manner. This was first shown for E. coli and then for the yeast S cerevisiae and mammalian cells. More recently monitoring global transcriptional responses of S. cerevisiae to alkylating agents has revealed that these agents induce a very much broader response that previously imagined (Jelinsky and Samson, 1999; Jelinsky et al., 2000; Begeley et al 2001). Here we propose to paint a picture of the global transcriptional response of mammalian cells and tissues to carcinogenic alkylating agents, and in addition to examine the details of tmTglObal picture that represent the response elicited by the presence of specific alkylated bases in the genome (e.g., 3MeA and O6MeG). For this, we will exploit two knock-out mouse model systems that we recently generated. One is deficient in 3MeA DN glycosylase repair activity (Aag null mice), and the other is deficient in the O6-MeG DNA repair MTase activity (Mgmt null mice).

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Project 3: Liver MicroBioreactors: A New Tool for Toxicology

Principal Investigator: Linda Griffith
griff@mit.edu

The objective of this work is to demonstrate the effectiveness of a newly-developed microscale liver tissue bioreactor for studies of environmental toxicity, particularly for agents that exert effects through chronic exposure for which adequate in vitro models are currently lacking. The microscale bioreactor allows 3D perfusion culture of liver cells in organoid structures and promotes retention of high levels of liver-specific synthetic and metabolic functions. We will examine the acute and chronic response to two agents, aflatoxin BI (AFBi) and the alkylating agent N-methyl-N-nitrosourea (MNU), comparing the response of tissue formed in the bioreactor to cells in conventional culture and to the in vivo response. Mathematical modeling of transport and metabolism of agents will be used to estimate in vitro dosing regimens that will mimic exposures from in vivo regimens. Cells will be exposed to AFBi under single dose (acute) or multiple doses over 2 weeks (chronic), and the total level of DNA adducts will be used to optimize the dosing regimen to obtain similar levels in vitro and in vivo. Transcriptional responses to the agents will be examined under the optimal conditions, and used to evaluate the relative ability of the in vitro bioreactor to serve as a model for in vivo exposure. A similar protocol will be conducted with MNU, using acute responses.

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