DNA strand breaks and DNA repair response in lymphocytes after chronic in vivo exposure to very low doses of ionizing radiation in mice

In contrast to the well-documented negative effects of high-dose oxidant exposure, accumulating evidence supports a positive, perhaps essential physiologic role for very low-level oxidant stress. For example, low-level oxidant exposure, within or below the physiologic range, has been reported to stimulate membrane signal transduction, proliferation, antioxidant defense and DNA repair. In the present study, we have examined whether whole-body exposure to low-dose radiation (LDR) results in an alteration in constitutive (steady state) levels of DNA-strand breaks and whether an adaptive increase in DNA-repair response is induced. C57B1/6J mice were exposed to 0.04 Gy (4 cGy) of gamma-radiation as a model of low level oxidant stress. End points measured after chronic in vivo LDR included: (1) constitutive expression of DNA-strand breaks in quiescent spleen cells; (2) sensitivity to DNA damage after high-dose radiation exposure in vitro; (3) repair of constitutive and radiation-induced DNA strand breaks after mitogen stimulation: (4) activity of the DNA-repair associated enzyme, poly(ADP-ribose)transferase (ADPRT) and its substrate, NAD. The results indicated that the constitutive expression of DNA-strand breaks is significantly decreased after chronic LDR; however, DNA-repair capacity after high-dose radiation exposure is not increased above that observed in sham-irradiated mice. Associated with the reduction in constitutive DNA-strand break accumulation was a decrease in resting levels of the DNA-repair-associated enzyme poly(ADP-ribose) transferase (ADPRT). These results are consistent with the interpretation that cumulative DNA damage and associated DNA-repair activity in unstimulated cells are both reduced after chronic LDR exposure.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

Determination of acid α-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa)

We examined gazelle peripheral blood leucocytes using the α-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1–2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes.     

Alpha1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.