Marina Cecilia González

Effect of dithiocarbamate pesticide zineb and its commercial formulation, azzurro. IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells.

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 microg/ml) or azzurro (100.0 microg/ml) for 80min, washed and reincubated in pesticide-free medium for 0-12h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6h of incubation. After 12h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0-12h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5-12h repair time for both pesticides. At 12h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0-12h repair period was observed.

Effect of reconstituted discoidal high‐density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012.

Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11β-hydroxysteroid dehydrogenase type 1.

BACKGROUND AND AIMS Data about glucocorticoids role in the development of atherosclerosis are controversial showing different effects in human than in experimental animal models. Atherosclerosis is the result of a chronic inflammatory response to an injured endothelium where an uncontrolled uptake of OxLDL by macrophages triggers the development of foam cells, the main component of fatty streaks in atherosclerotic plaque. There are few data about the direct effect of glucocorticoids in macrophages of atherosclerotic plaque. The aim of the study was to elucidate the role of glucocorticoids in the development of foam cells in atherosclerosis initiation. METHODS For this purpose we used THP1 cells differentiated to macrophages with phorbol esters and incubated with OxLDL alone or with cortisol or cortisone. THP1 cells were also incubated with cortisone plus an inhibitor of 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) activity to determine the role of this enzyme on glucocorticoid action in this process. RESULTS Ours results showed that cortisol and cortisone decreased significantly the inflammation promoted by OxLDL, and also diminished the expression of genes involved in influx and efflux of cholesterol resulting in a reduced lipid accumulation. Likewise cortisol and cortisone decreased 11βHSD1 expression in THP1 cells. The presence of the inhibitor of 11βHSD1 abolished all the effects elicited by cortisone. CONCLUSION Our results indicate a direct effect of glucocorticoids on macrophages braking atherosclerosis initiation, reducing pro-inflammatory markers and OxLDL uptake and cholesterol re-esterification, but also inhibiting cholesterol output. These effects appear to be mediated, at least in part, by 11βHSD1 activity.

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.

Data related to inflammation and cholesterol deposition triggered by macrophages exposition to modified LDL

This article supports experimental evidence on the time-dependent effect on gene expression related to inflammation and cholesterol deposition in lipid-loaded cells. The cells employed were human monocytes THP1 line transformed into macrophages by treatment with phorbol esters. Macrophages were treated at different times with oxidized low density lipoprotein (Ox-LDL) and then gene expression was measured. We also include data about the different types of oxidized lipoprotein obtained (low, media or high oxidation) for differential exposure with Cu ions. These data include characterization to lipid and protein peroxidative damage and also quantification of cell viability by exposure to native and modified LDL. The present article complements data published in “Decreased OxLDL uptake and cholesterol efflux in THP1 cells elicited by cortisol and by cortisone through 11β-hydroxysteroid dehydrogenase type 1” Ledda et al. (in press) [1].