High glucose induces DNA damage in cultured human endothelial cells.

Morphologic and functional abnormalities of vascular endothelium are well recognized in diabetes. In view of our previous finding that high glucose concentrations accelerate death and hamper replication of cultured human endothelial cells, we have investigated in the same model the possibility that exposure to high glucose may result in DNA damage. DNA from human endothelial cells--but not from fibroblasts--exposed to 30 mM glucose for 9-14 d manifested an accelerated rate of unwinding in alkali indicative of an increased number of single strand breaks (P less than 0.001 vs. control). Endothelial cells exposed to high glucose also manifested an increased amount of hydroxy-urea-resistant thymidine incorporation (333 +/- 153 cpm/10(5) cells vs. 88 +/- 42 in control cells, mean +/- SD, P = 0.04), which is indicative of increased DNA repair synthesis. Neither DNA damage nor repair synthesis were increased by medium hypertonicity achieved with 30 mM mannitol. These findings suggest the possibility that, under conditions of high ambient glucose, excess glucose entry in cells that are insulin independent for glucose transport may, directly or indirectly, perturb DNA function. Further, they suggest the possibility that different individual capabilities to repair DNA damage--a process that is under genetic control--may represent a mechanism for different individual susceptibilities to development of diabetic vascular complication.

Increased single strand breaks in DNA of lymphocytes from diabetic subjects.

Certain aspects of the chronic complications of diabetes suggest that, with time, the abnormal metabolic milieu leads to irreversible changes in some cell populations. Since we have previously observed that high glucose concentrations induce an increase in single strand breaks in the DNA of cultured human endothelial cells, we have investigated whether the same abnormality occurs in cells derived from the in vivo diabetic environment. Peripheral blood lymphocytes obtained from 21 type I diabetic patients and age- and sex-matched controls were tested for rate of unwinding in alkali (a measure of DNA single strand breaks). The patients were subdivided into two groups on the basis of glycohemoglobin values above or below 9%. The group with glycohemoglobin values of 12.9 +/- 2.4% (mean +/- SD), but not the group with glycohemoglobin values of 7.4 +/- 1.5%, showed accelerated unwinding of lymphocyte DNA when compared to controls (P less than 0.01). These studies suggest that poorly controlled diabetes may result in DNA lesions, whose impact on long-term complications deserves to be investigated.

Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

Use of tannic acid to study cytoplasmic vesicles and membrane invaginations in epididymal fat pads of ageing mice.

A staining technique based upon the known ability of tannic acid to selectively stain the outer layer of the triple-layered plasma membrane was used, along with electron microscopic examination of stained, serial sections, to differentiate between surface invaginations, clusters of invaginations, free vesicles and tubular channels in epididymal fat pads from young, fed and fasted mice and from old, fed mice. A preliminary semiquantitative evaluation of the average number of each type of structure per cell was attempted. There were no significant numbers of free cytoplasmic vesicles of approximately 50 nm diameter (the dimension of most surface invaginations) under any conditions studied. Most apparent vesicles were stained by tannic acid and were, therefore, actually invaginations of the plasma membrane. There were no tubular channels of this size seen in any of the electron micrographs examined in serial sections. We estimated that there are about 50 single invaginations per micron2 of plasma membrane surface in both young and old fed mice. In addition there were about 20 invaginations/micron2 grouped as clusters of 2-15 per group (mean, 4 per group) in the fed mice. There was a tendency for the number of invaginations in clusters to increase during fasting; about 40% of the surface invaginations were grouped in clusters in adipocytes of fasted mice. Although there was no effect of ageing on the concentrations of surface invaginations or in their groupings as clusters, the total number of invaginations per cell must have increased almost 3-fold as the cells enlarged. The function of these surface invaginations and deeply penetrating groups of invaginations remains to be elucidated.

H2O2 increases expression of pulmonary artery endothelial cell platelet-derived growth factor mRNA.

Endothelial cells subjected to cell injury are capable of producing platelet-derived growth factor (PDGF), a mitogen for the stimulation of fibroblast and smooth muscle cell proliferation. Cultured bovine pulmonary artery endothelial cells were exposed to low concentrations of H2O2 for 30 min. Total cell RNA was isolated and subjected to Northern analysis with use of a v-sis PDGF cDNA probe. Results demonstrate a fourfold increase in cell PDGF mRNA immediately after exposure of bovine pulmonary artery endothelial cells to 50 microM H2O2. Evidence of expression of PDGF was sought in samples of cell supernatant collected 48 h after exposure. No evidence of PDGF activity or PDGF antigen could be demonstrated in those supernatants. Although the biologic activities of PDGF suggest that PDGF production by endothelial cells may contribute to the pulmonary pathology associated with acute lung injury, our results suggest that posttranscriptional events may prevent expression of PDGF under the experimental conditions of this investigation.

Association between mitochondria and rough endoplasmic reticulum in rat liver.

Mitochondria were isolated from rat liver homogenate by both zonal and sedimentation equilibrium centrifugation. From electron microscopic examination of thin sections it was observed that 81% of the isolated mitochondria were in contact with rough endoplasmic reticulum (RER). Intact, non-sectioned mitochondria subjected to negative staining procedures appeared to show points of connection between RER and the outer mitochondrial membrane. After treatment of mitochondria with digitonin to remove outer membranes, some of the resulting mitoplasts (intact inner membranes) remained closely associated with RER. Serial section analysis of intact rat liver indicated that RER saccules fit over mitochondria like caps providing broad areas of contact between the two organelles. The RER saccules were also observed communicating with more than one mitochondrion.

The polyol pathway and glucose 6-phosphate in human endothelial cells cultured in high glucose concentrations.

In an attempt to identify the mechanisms underlying the ill effects of high glucose previously described in cultured human endothelial cells, we have investigated in these cells the activity of the polyol pathway and accumulation of glucose 6-phosphate, a powerful agent of non-enzymatic glycosylation. Sorbitol accumulation varied among different batches of cells (primary cultures). In presence of 5 mmol/l glucose the cellular sorbitol content ranged from 0.04 to 0.12 nmol/10(6) cells. When cells were exposed to 20 mmol/l glucose the sorbitol content increased by 2- to 3-fold to concentrations of 0.08-0.38 nmol/10(6) cells (p less than 0.01). Addition to the culture medium of 100 mumol/l Sorbinil, an inhibitor of aldose reductase, resulted in a substantial inhibition of sorbitol accumulation throughout the 14 days in culture, but the degree of inhibition varied inversely with the duration of cell exposure to high glucose (70% inhibition in cells exposed to high glucose and Sorbinil for 1-3 days versus 14% inhibition in cells exposed for 14 days, p less than 0.01). Sorbinil treatment failed to improve even slightly the abnormalities in cellular replication induced by high glucose. The cellular content of glucose 6-phosphate was augmented 3-fold by exposure to 20 mmol/l glucose (p less than 0.001). In conjunction with other studies these results indicate that in this model the polyol pathway is not an osmotically or metabolically important mechanism of glucotoxicity, and that the inhibitory activity of Sorbinil on the polyol pathway of human tissues may be a function of their length of exposure to hyperglycaemia. The consequences of intracellular accumulation of glucose 6-phosphate await investigations.

DNA-mediated restoration of aryl hydrocarbon hydroxylase induction in a mouse hepatoma mutant defective in nuclear translocation of the Ah receptor.

In order to study the induction mechanism of aryl hydrocarbon hydroxylase (AHH), non-inducible mutants have previously been isolated from the mouse hepatoma cell line, Hepa-1. With the ultimate goal of isolating the corresponding genes, restoration of AHH inducibility to representative mutants by means of DNA-mediated gene transfer has been set out. The successful transfection of a C- mutant, which is defective in nuclear translocation of the Ah receptor-inducer complex, is described here, using rat genomic DNA as donor material. Primary and secondary rat transfectants were obtained, and they were assayed for hydroxylase activity, receptor translocation, and homology with a rat repetitive DNA sequence.