ARNT PROTEIN, DEVELOPMENT AND CARCINOGENESIS

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The Ah receptor mediates all, or nearly all, of the toxicological effects of halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). These compounds act as tumor promoters in rodents. The Ah receptor is also directly involved in carcinogenesis by many initiating agents. The receptor may also play a role in embryonic development. The DNA-binding, transcriptionally active form of the Ah receptor in cultured liver cells is a heterodimer of the Ah receptor nuclear translocator (Arnt) protein and the ligand-binding subunit of the Ah receptor. However, it is not clear whether some activities of the ligand-binding subunit and of Arnt can be manifested independently of the other protein. This project proposes to use transgenic mouse technology to address the potential role of the Arnt protein in carcinogenesis by non-genotoxic and genotoxic carcinogens, and in developmental processes. One Arnt allele in an embryonal stem (ES) cell line will be inactivated by homologous recombination. The targeted ES cells will be used to generate mice homozygous for the disrupted Arnt allele. If they are inviable, the developmental abnormalities and defects in the reproductive system of homozygous fetuses or newborns will be determined, in order to provide insight into the role of Arnt in development. If Arnt deficient mice develop to a late enough stage, we will determine whether they are resistant to the toxic effects of TCDD on development. If Arnt-knockout mice are fully viable we will investigate whether the adult mice are resistant to acute toxic responses to TCDD, thus testing the hypothesis, proposed by others, that some toxic effects of TCDD are produced via an Arnt-independent pathway. If the Arnt- knockout mice are fully viable we will also generate derivatives which are homozygous for a concatemer of the lacI/q gene located on mouse chromosome 4. The lacI/q gene can be used to quantitate mutations in all cells of the body. We will use the lacI/q derivatives to test the hypothesis that TCDD (which is non-genotoxic in bacteria but a complete carcinogen to mouse liver), enhances spontaneous mutagenesis or mutagenicity of initiators in mouse fee, and if so, whether enhancement is dependent on Arnt. We will also ascertain whether the liver and colon carcinogen, 2- amino-3-methylimidazo[4,5-f]quinoxaline (IQ) increases the frequency of liver and colon mutations in mice fed with the compound, whether cofeeding with TCDD (and therefore stimulation of Ah receptor activity) affects mutagenicity, and if any effects of TCDD are observed, whether these are dependent on Arnt.