Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues.

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.

Lower induction of p53 and decreased apoptosis in NQO1-null mice lead to increased sensitivity to chemical-induced skin carcinogenesis.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic protein that catalyzes metabolic detoxification of quinones and protects cells against redox cycling and oxidative stress. NQO1-null mice deficient in NQO1 protein showed increased sensitivity to 7,12-dimethylbenz(a)anthracene- and benzo(a)pyrene-induced skin carcinogenesis. In the present studies, we show that benzo(a)pyrene metabolite benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide and not benzo(a)pyrene quinones contributed to increased benzo(a) pyrene-induced skin tumors in NQO1-null mice. An analysis of untreated skin revealed an altered intracellular redox state due to accumulation of NADH and reduced levels of NAD/NADH in NQO1-null mice as compared with wild-type mice. Treatment with benzo(a)pyrene failed to significantly increase p53 and apoptosis in the skin of NQO1-null mice when compared with wild-type mice. These results led to the conclusion that altered intracellular redox state along with lack of induction of p53 and decreased apoptosis plays a significant role in increased sensitivity of NQO1-null mice to benzo(a)pyrene-induced skin cancer.

Presence and induction of the enzyme NAD(P)H: quinone oxidoreductase 1 in the central nervous system.

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes a reductive detoxification that is thought to protect cells against the adverse effects of quinones and related compounds. NQO1 activity is present in all tissues. Absence of the enzyme produces abnormalities in the redox state and seizures, suggesting an important role of the protein in the central nervous system. Immunohistochemical analysis showed that the protein was found throughout the brain of the adult rat and mouse, with complete absence of the protein in brains from NQO1-/- mice. NQO1 was not seen in any neuronal population, but was localized to Bergmann glial in the cerebellum and a subset of the oligodendrocytes throughout the brain. Prolonged seizures induced in adult rats with kainic acid resulted in an increase in activity of the enzyme throughout the brain, most prominently in the cerebellum, but immunoreactivity did not appear in neurons. Comparison of the axons in the corpus callosum from a wild-type mouse to a knockout mouse showed that myelin is produced in the absence of NQO1, but there appears to be more small-diameter axons in the knockout animal. These results suggest that NQO1 has a role in myelination in the central nervous system or in the insulating/wrapping function of glial cells.

Disruption of dihydronicotinamide riboside:quinone oxidoreductase 2 (NQO2) leads to myeloid hyperplasia of bone marrow and decreased sensitivity to menadione toxicity.

Dihydronicotinamide riboside (NRH):quinone oxidoreductase 2 (NQO2) is a flavoenzyme that catalyzes the reductive metabolism of quinones. To examine the in vivo role of NQO2, NQO2-null (NQO2-/-) mice were generated using targeted gene disruption. Mice lacking NQO2 gene expression showed no detectable developmental abnormalities and were indistinguishable from wild-type (NQO2+/+) mice. However, NQO2-null mice exhibited myeloid hyperplasia of the bone marrow and increased neutrophils, basophils, eosinophils, and platelets in the peripheral blood. Decreased apoptosis of bone marrow cells and circulating granulocytes contributed to myeloid hyperplasia and hyperactivity of bone marrow in NQO2-null mice. The hematological changes in NQO2-/- mice were specifically associated with loss of the NQO2 gene because histological analysis of various tissues including spleen, thymus, blood cultures, and urine analysis demonstrated no sign of infection. NQO2-null mice also demonstrated decreased toxicity when exposed to menadione or menadione with NRH. These results establish a role for NQO2 in protection against myelogenous hyperplasia and in metabolic activation of menadione, leading to hepatic toxicity. The NQO2-null mice are a model for NQO2 deficiency in humans and can be used to determine the role of this enzyme in sensitivities to toxicity and carcinogenesis.

Disruption of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene in mice causes myelogenous hyperplasia.

NAD(P)H:quinone oxidoreductase1 (NQO1) is a cytosolic protein that reduces and detoxifies quinones and their derivatives, thus protecting cells against redox cycling and oxidative stress. Disruption of the NQO1 gene in mice caused myeloid hyperplasia of bone marrow and highly significant increases in blood neutrophils, eosinophils, and basophils. NQO1-null mice also showed a decrease in lymphocytes and WBCs as compared with wild-type mice. Various techniques also demonstrated an increase in megakaryocytes without an increase in blood platelets. Histological analysis of liver, kidney, spleen, and thymus did not demonstrate a difference between wild-type and NQO1-null mice or a sign of infection. Blood cultures and urine analysis also did not demonstrate any sign of infection in NQO1-null and wild-type mice. Additional analysis of the bone marrow from NQO1-null mice revealed that loss of NQO1 alters the intracellular redox status because of accumulation of NAD(P)H, cofactors for NQO1. This causes a reduction in the levels of pyridine nucleotides and tumor suppressor proteins p53 and p73, and a decrease in apoptosis. The decrease in apoptosis causes myelogenous hyperplasia in NQO1-null mice. These results demonstrate that NQO1 acts as an endogenous factor in the protection against myelogenous hyperplasia. This is significant because 2-4% of human individuals without known abnormalities, and >25% of individuals with benzene poisoning and acute myelogenic leukemia are homozygous for a mutant allele (P187S) of NQO1 and lack NQO1 protein/activity.