Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

The study of colon cancer has taken advantage of the development of a model in animals in which tumors in the colon are easily induced by chemical treatment. When 1,2-dimethylhydrazine (DMH) is injected into rats tumor growth is observed in colon in preference to other tissues. This observation led us to investigate the Cytochrome P450 system in colon and its participation in the particular “colon sensitivity” to DMH. It has been established that the Cytochrome P450 system participates in the metabolism of DMH and the methyl carbonium product of Cytochrome P450 activation of DMH is responsible for DNA damage which is considered an initial step to carcinogenesis. The Cytochrome P450 system is a reasonable place to search for an explanation of this organotropic effect of DMH and we feel that the knowledge obtained from this study can take us closer to understanding the development of colonic malignancy. In our study we used a human colon cell line (LS174T) treated with DMH. The Cytochrome P450 system in the cells was manipulated with inducers of different isoforms of Cytochrome P450. The effect of DMH on colon cells was measured by determination of O-6-methylguanine which is a DNA adduct derived from the metabolism of this chemical and is associated with development of tumors. Our results support the hypothesis that Cytochrome P450 plays an important role in the damage to cellular DNA by DMH. This damage is increased after induction of Cytochromes P450 1A1 and 2E1. The effect of inhibition of the methyltransferase and glutathione systems on protection against DMH damage in colon demonstrated the importance of the protective role of the former and the lack of effective protection of the latter system. ^

CHARACTERIZATION AND TREATMENT OF A NOVEL MOUSE MODEL OF TSC-ASSOCIATED AUTISM

Tuberous sclerosis complex (TSC) is a dominant tumor suppressor disorder caused by mutations in either TSC1 or TSC2. The proteins of these genes form a complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1), which controls protein translation and cell growth. TSC causes substantial neuropathology, often leading to autism spectrum disorders (ASDs) in up to 60% of patients. The anatomic and neurophysiologic links between these two disorders are not well understood. However, both disorders share cerebellar abnormalities. Therefore, we have characterized a novel mouse model in which the Tsc2 gene was selectively deleted from cerebellar Purkinje cells (Tsc2f/-;Cre). These mice exhibit progressive Purkinje cell degeneration. Since loss of Purkinje cells is a well-reported postmortem finding in patients with ASD, we conducted a series of behavior tests to assess if Tsc2f/-;Cre mice displayed autistic-like deficits. Using the three chambered social choice assay, we found that Tsc2f/-;Cre mice showed behavioral deficits, exhibiting no preference between a stranger mouse and an inanimate object, or between a novel and a familiar mouse. Tsc2f/-;Cre mice also demonstrated increased repetitive behavior as assessed with marble burying activity. Altogether, these results demonstrate that loss of Tsc2 in Purkinje cells in a haploinsufficient background lead to behavioral deficits that are characteristic of human autism. Therefore, Purkinje cells loss and/or dysfunction may be an important link between TSC and ASD. Additionally, we have examined some of the cellular mechanisms resulting from mutations in Tsc2 leading to Purkinje cell death. Loss of Tsc2 led to upregulation of mTORC1 and increased cell size. As a consequence of increased protein synthesis, several cellular stress pathways were upregulated. Principally, these included altered calcium signaling, oxidative stress, and ER stress. Likely as a consequence of ER stress, there was also upregulation of ubiquitin and autophagy. Excitingly, treatment with an mTORC1 inhibitor, rapamycin attenuated mTORC1 activity and prevented Purkinje cell death by reducing of calcium signaling, the ER stress response, and ubiquitin. Remarkably, rapamycin treatment also reversed the social behavior deficits, thus providing a promising potential therapy for TSC-associated ASD.