Cataract formation in a strain of rats selected for high oxidative stress.

The primary purpose of this study was to define the clinical and morphological features of cataractogenesis in the OXYS strain of rats that generate excess reactive oxygen species. Rats were sequentially examined from birth to the development of mature cataracts with slit lamp biomicroscopy. Morphology of selected stages of cataract development was studied using light and transmission electron microscopy (TEM), immunohistochemical localization of the lipid peroxidation product 4-hydroxynonenal (HNE) and fluorescent antibody labeling for DNA oxidation products. Lenses from age-matched normal rats were used as controls. OXYS rats developed cataracts as young as two weeks of age with progression to maturity by 1 year. Clinically, cataracts appeared initially either as nuclear or sub-capsular cortical changes and progressed to pronounced nuclear cataracts within months. TEM confirmed the light microscopic impression of region-specific alterations in both fiber cell cytoplasmic protein matrix and membrane structure. The outer adult nuclear region showed extensive cellular damage similar to osmotic cataracts, which is consistent with the postulated high uptake of glucose in the OXYS strain. The adult and outer fetal nuclear cells displayed several types of focal damage. The inner fetal and embryonic nuclear cells demonstrated textured cytoplasm, suggesting protein degradation or redistribution. Staining for HNE was increased in epithelium, cortex and nucleus compared to control lenses. Fluorescent antibody probes demonstrated increased levels of DNA oxidation products in OXYS rat lenses compared to age-matched controls. Fourier analysis of nuclear cytoplasm revealed significant components with corresponding sizes greater than 100 nm and, using a new theoretical approach, the texturing of the cytoplasm was shown to be sufficient to cause opacification of the nucleus. The OXYS rat appears to be an ideal model for oxidative stress cataractogenesis. The potential oxidative damage observed is extensive and characteristic of the developmental region. The source of oxidative damage may in part be a response to elevated levels of glucose. Because oxidative stress is thought to be a major factor in cataract formation in both diabetic and non-diabetic aging humans, this animal model may be a useful tool in assessing efficacy of antioxidant treatments that may slow or prevent cataract formation.

Dietary depletion of vitamin E and vitamin A inhibits mammary tumor growth and metastasis in transgenic mice.

We showed previously that dietary antioxidant depletion enhances tumor reactive oxygen species (ROS) and apoptosis, resulting in a reduction in brain tumor size in the TgT(121) transgenic mouse model, a nonmetastatic tumor model. Here, in a transgenic mouse model of mammary tumorigenesis with defined rates of tumor growth and lung-targeted metastasis, we determined the ability of dietary antioxidant depletion to inhibit tumor growth and metastasis. Compared with control mice fed a standard diet, antioxidant-depleted mice exhibited tumor-targeted generation of ROS manifested by increased levels of oxidatively modified DNA/RNA (8- hydroxy-2'-deoxyguanine, 8-hydroxyguanine) and lipid peroxidation (4-hydroxy-2-nonenal) in primary and metastatic tumor foci. In addition to increased tumor-targeted ROS, the number of apoptotic cells was increased approximately 500% (P < 0.01) and terminal dUTP nucleotide DNA end-labeling-positive cells 200% (P < 0.01) in mice fed the antioxidant-depleted diet, whereas the percentage of tumor cells undergoing mitosis was >50% lower than in controls (P < 0.01). The proportional distribution of small (<1.5 cm) and large (> or = 1.5 cm) primary mammary tumors differed. The mice fed the antioxidant-depleted diet had more small primary tumors (P <0.05) and fewer large primary tumors (P < 0.05). Importantly, they also had fewer lung metastatic tumor foci compared with mice fed the control diet (4.5 +/- 1.3 vs. 15.8 +/- 8.5 foci/lung, P < 0.01). These findings may be important in understanding the role of dietary antioxidant vitamins in tumor growth and metastasis.

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.