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BACKGROUND. Caloric restriction (CR) retards aging and diseases in mice, rats, and other animals by unknown mechanisms. A popular hypothesis is that CR acts by opposing age-associated increases in oxidative stress. METHODS. Because influences of CR on antioxidant enzymes in the prostate have not been previously investigated, immunohistologic methods (light and electron microscopy) were used to determine the prostatic localization of catalase (CAT) in rats of diverse ages (3-32 months) fed either normally or subjected to CR from age 16 months. RESULTS. In 20-month-old rats fed either diet, CAT appeared as dense deposits at the apical poles of the epithelium in the lateral lobes, and within the ductular lumens, suggesting that CAT is secreted. Confirmation of both liver peroxisomal and prostatic apical cytoplasmic localization of CAT was provided by electron microscopic immunogold staining. The amount of CAT was reduced at 30 months in normally fed rats but not in those on CR. CONCLUSIONS. CAT appears to be a secretory product of the epithelial cells in the lateral lobes of the rat prostate. Further, CR from late-middle age opposed the age-associated loss of this intracellular enzyme activity.

Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathioneS-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and α class glutathioneS-transferase Ya subunit were detected in renal tubules before birth. μ class glutathioneS-transferase subunits Yb1 and Yb2 stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.

Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun N-terminal kinase activation and induction of apoptosis-necrosis require incremental amounts of the organic chemicals and increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mitochondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial membrane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N-acetylcysteine to cytoprotective glutathione.

Immunoperoxidase and immunogold techniques were used to localize the following antioxidant enzyme systems in the adult hamster kidney at the light and ultrastructural levels: superoxide dismutases, catalases, peroxidases and glutathione S-transferases. Each cell type in the kidney showed specific patterns of labelling of these enzymes. For example, proximal and distal tubular and transitional epithelial cells showed significant staining for all of these enzymes, while glomerular cells and cells of the thin loop of Henle did not show significant staining at the light microscope level. In addition, high levels of glutathione peroxidase were found in smooth muscle cells of renal arteries. At the ultrastructural level, each enzyme was found in a specific subcellular location. Manganese superoxide dismutase was found in mitochondria, catalase was localized in peroxisomes, while copper, zinc superoxide dismutase and glutathione S-transferase (liver and placental forms) were found in both the nucleus and cytoplasm. Glutathione peroxidase was found to have a broad intracellular distribution, with localization in mitochondria, peroxisomes, nucleus, and cytoplasm. Microvilli of tubular cells were labelled by antibodies to catalase, copper, zinc superoxide dismutase, glutathione peroxidase, and glutathione S-transferases. Cell types that were negative by light microscopy immunoperoxidase studies showed definite labelling with immunogold post-embedding ultrastructural techniques (glomerular cells and cells of the loop of Henle), demonstrating the greater sensitivity of the latter technique. These observations demonstrate that there are large variations in the levels of antioxidant enzymes in different cell types, and that even within a distinct cell type, the levels of these enzymes vary in different subcellular locations. Our results demonstrate for the first time the overall antioxidant enzyme status of individual kidney cell types, thereby explaining why different cell types have differing susceptibilities to oxidant stress. Possible physiological and pathological consequences of these findings are discussed.

Analysis of activities of the antioxidant enzyme manganese superoxide dismutase in human renal cell carcinomas often showed greatly altered enzyme levels (either elevated or depressed) compared to the cell of origin, the kidney proximal tubule. In order to better understand the variability observed, immunogold studies were performed on human renal cell carcinomas using a polyclonal antibody to human kidney manganese superoxide dismutase. For comparison, studies were also performed using antibodies to other antioxidant enzymes. For histologic studies, renal cell carcinomas were subclassified on the basis of light microscopy and ultrastructural analysis into clear cell, granular cell, or mixed clear and granular cell variants. In all three types of tumor, immunogold studies showed little staining using antibodies to copper, zinc superoxide dismutase or glutathione-dependent enzymes. However, intensity of labeling for manganese superoxide dismutase and catalase depended on the cell type(s) in the tumor. Clear cell variants demonstrated trace staining for manganese superoxide dismutase and catalase, while granular cell variants exhibited heavy staining for both of these enzymes. Mixed types of tumors showed clear cells with trace staining for all antioxidant enzymes examined, while granular cells again showed intense labeling for manganese superoxide dismutase and catalase. Using normal kidney proximal tubule as a comparison, immunogold ultrastructural analysis using antibody to manganese superoxide dismutase demonstrated infrequent small lightly labeled mitochondria in clear cell variants, while granular cell variants exhibited numerous medium-sized heavily labeled mitochondria. These data suggest that: 1) the variability in activity values for manganese superoxide dismutase may be due to heterogeneity of cell types in these tumors and 2) manganese superoxide dismutase immunoreactive protein was elevated in granular cells both because of an increase in number of mitochondria and because the labeling density in mitochondria was increased compared to mitochondria in clear cell types or in normal proximal tubular cells.