Characterization of cyclin D1 expression in a rat global model of cerebral ischemia.

During normal development of the central nervous system there is expression of cyclins that regulate the progression of cells through various stages of mitosis. Cyclins have also been implicated in neuronal degeneration and apoptosis in adult brain, especially cyclin D1 as it is permissive for the transition from growth phase to synthesis phase in mitotic cell division. There is controversy as to whether cyclin D1 expression increases in both in vitro and in vivo models of cerebral ischemia. In this study we use immunohistochemistry and Western blot analysis to characterize cyclin D1 expression in an in vivo rat global model of cerebral ischemia to address the hypothesis that cyclin D1 alterations are involved in ischemic neuronal death. Although there was no change in cyclin D1 expression in either the vulnerable CA1 or resistant CA3 regions of the hippocampus prior to neuronal cell death (<3 days reperfusion), concomitant with the death of CA1 neurons and the loss of cyclin D1 in these cells, there was an increase in non-neuronal cyclin D1 positive cells. Some of the non-neuronal cyclin D1 expressing cells were identified to be activated microglia. In contrast to the cytoplasmic expression of cyclin D1 in neurons, the cyclin D1 expression in the microglia and other non-neuronal cells in CA1 was both nuclear and cytosolic. This study suggests that cyclin D1 does not play a role in the death of vulnerable CA1 neurons in global ischemia.

Dietary ortho phenols that induce glutathione S-transferase and increase the resistance of cells to hydrogen peroxide are potential cancer chemopreventives that act by two mechanisms: the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics.

Oxidative stress is implicated in the etiology of cancer, hence compounds that alleviate oxidative stress by inducing enzymes that defend against free radical damage might be useful as cancer chemopreventives. Glutathione S-transferase (GST) has been suggested to be a candidate for a critical enzyme in protecting cells against free radical damage, in part, because its level of induction correlates with protection of the cell line IMR-32 against hydrogen peroxide-induced oxidative stress. The present study identified dietary ortho phenols that both induce GST and protect the cell line IMR-32 against hydrogen peroxide-caused oxidative stress. The ortho phenol (o-phenol) inducers were better protectors against oxidative stress than a number of GST inducers that did not bear phenolic groups, possibly because the phenol residues of the ortho phenols allowed their action as antioxidants as well as inducers of GST. GST has previously been thought to protect cells against cancer by detoxifying mutagenic xenobiotics. The present results suggest that ortho phenol inducers of GST might be useful as cancer chemopreventives that act by two independent mechanisms, the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics.

Compounds that induce isoforms of glutathione S-transferase with properties of a critical enzyme in defense against oxidative stress.

Compounds that upregulate enzymes that play critical roles in protection against free radical damage might be useful in treating diseases in which free radicals are pathological. To identify critical enzymes and their upregulators, compounds that were not free radical scavengers were screened for the ability to increase the IC(50) of the human neuronal cell line IMR-32 for hydrogen peroxide. Subsequently, enzymes upregulated by compounds that increased the IC(50) were identified. All of the compounds identified that increased the IC(50) also increased the specific activity of glutathione S-transferase (GST). In addition, compound-caused increases in the specific activity of GST correlated with compound-caused increases in the IC(50), the expected behaviour if GST was a critical enzyme. The GST isoform composition changed on upregulation, suggesting the upregulation of isoforms with anti-free radical activities. Structural features of compounds concurrently increasing the IC(50) and upregulating GST were identified.

Bile acid inhibition of xenobiotic-metabolizing enzymes is a factor in the mechanism of colon carcinogenesis: tests of aspects of the concept with glucuronosyltransferase.

A factor in colon carcinogenesis might be the partial defeat in colon epithelial cells of the protective enzymic barrier against xenobiotics, via bile acid inhibition of enzymes that detoxify mutagens. The applicability of aspects of this concept to glucuronosyltransferase, a phenol detoxification enzyme, was tested in a colon cancer cell line. Inhibition of glucuronidation of the test substrate, 4-methylumbelliferone, occurred at bile acid concentrations found in faecal water, and depended on pH for some bile acids. Lithocholate was the most inhibitory: the concentration causing 50% inhibition of the initial rate of glucuronidation (IC50) was about 3 microM at pH 7.4 and at pH 6.2. The inhibitory potency of deoxycholate and chenodeoxycholate increased when pH decreased, but still remained less than that of lithocholate: the IC50 for deoxycholate was 88.5 microM at pH 7.4, and 14.8 microM at pH 6.2, and for chenodeoxycholate the IC50 was 67.4 microM at pH 7.4, and 21.7 microM at pH 6.2. Cholate did not cause appreciable inhibition. The inhibitory effects were additive when lithocholate was present together with either deoxycholate or chenodeoxycholate. The results provide a mechanism for the comutagenicity of bile acids, a feature of which is the inter-relation of bile acid comutagenicity specifically with mutagens that are inactivated by a bile acid-inhibitable enzyme. The results are also in accord with the view that high concentrations of bile acids in solution in faecal water, especially lithocholate, are a risk factor for colon cancer.

Toxicity of bile acids to colon cancer cell lines.

Quantitative aspects of bile acid cytotoxicity to colon cancer cell lines were investigated because of the etiological role in colon carcinogenesis attributed to the toxic effects of bile acids on colon mucosal cells. The cytotoxicity of major colonic bile acids differed. Lithocholate was the most toxic, followed by chenodeoxycholate and deoxycholate, with cholate being non-toxic over the concentration range studied. Cytotoxicity increased with time of exposure. Values for IC50 for some of the acids were determined to be in the physiological range, as estimated from their concentrations in fecal water. The results suggest dietary factors that contribute to bile acid mucosal damage. They also identify factors of possible importance in the association of high concentrations of bile acids in fecal water with risk for colon cancer.