The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Influences of genetic variants in interleukin-15 gene and serum interleukin-15 levels on coronary heart disease

Interleukin-15 (IL-15) is a potent proinflammatory cytokine that is now considered a key component of atherosclerosis. Proinflammatory gene polymorphisms lead to variations in the production and level of the proteins. In light of these findings, we hypothesized that variations in the gene coding for IL-15 influence the risk of coronary heart disease (CHD) by modulating the IL-15 levels. To test this hypothesis, we examined 5 single nucleotide polymorphisms (SNPs) in IL-15 gene and IL-15 levels in 102 patients with acute coronary syndrome (ACS), 102 patients with chronic ischemic stable CHD and 162 healthy control subjects. This study is the first report showing the influences of IL-15 gene variants and IL-15 levels on CHD. The five single nucleotide polymorphisms (SNPs) within the IL-15 gene, G367A, C267T, A14035T, C13687A, and A10504G were carried out by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Serum IL-15 levels were significantly higher in both acute and chronic patients than in controls. Genetic variants of IL-15 gene and IL-15 levels were associated with CHD. In conclusion, our study supports the hypothesis that genetic variation in IL-15 gene and IL-15 levels influence the risk of CHD. Further studies are needed to confirm our hypothesis.     

An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

A preliminary microspectrofluorometric study of NAD(P) reduction in diBenzo(a,e) Fluoranthene-treated single living cells

Summary The fluorescence increase, due to NAD(P) reduction, following microelectrophoretic injection of glucose 6-P (G6P) into EL2 and NCTC 8739 single living cells treated with diBenzo(ae)Fluoranthene (diB(ae)F) and non-treated, has been studied with a rapid microspectrofluorometer. This study shows the enhanced capacity of treated cells to utilize larger doses (6–10 times more) of G6P than control cells. The time course of the return to the initial fluorescence level is essentially related to the magnitude of the injection dose. There are alterations (e.g. red & blue shifts) in the fluorescence spectrum of diB(ae)F-treated cells before injection and in the increase spectrum after injection of G6P, as compared to the same spectra in the diB(ae)F-untreated cells. This is discussed in reference to the metabolization of diB(ae)F as an alternative pathway for the reoxidation of NAD(P)H.     

The effect of intracellular oxygen on the metabolization of Benzo(a)pyrene and Benzo(k)Fluoranthene. A microspectrofluorometric analysis in single living cells

Summary The fluorescence increase which accompanies the injection of glycolytic intermediates to Benzo(a)pyrene (BP) and Benzo(k) Fluoranthene-B(k)F treated EL2 ascites cancer cells, under aerobic and anaerobic conditions, has been studied in a microspectrofluorometer. In the carcinogen-treated cells the altered fluorescence increase pattern (in reference to control cells) which is observed at aerobiosis and attributed to BP or B(k)F metabolization, is not any more observable at anaerobiosis, in which case the fluorescence increase of the carcinogen-treated cells resembles that of the controls. This difference in behavior is discussed and a comparison is initiated between the response to injection in cells treated with BP (compound with K region) or B(k)F (compound without K region).     

Vitamin E supplementation modulates gingival crevicular fluid lipid peroxidation and antioxidant levels in patients with orthodontic tooth movement

The aim of this study was to investigate the levels of the oxidant and antioxidant changes in orthodontic tooth movement and the effects of vitamin E on these parameters. For this purpose, 50 orthodontic patients (aged 13-18 years) required non-extracted treatment were divided randomly into the following groups: Control and Vitamin E. Same pre-adjusted appliances were applied to all patients, and vitamin E (300 mg day(-1)) was given during 1 month in vitamin E group. Gingival crevicular fluid was collected and periodontal indexes were recorded at the baseline and after 1 month. Lipid peroxidation (LP) levels as malonyldialdehyde, reduced glutathione (GSH) and glutathione peroxidase (GSH-Px), vitamin C and E levels were measured in the anterior and posterior regions of the dentition. After 1 month, orthodontic treatment LP levels increased in control group in both anterior and posterior regions in vitamin E group. LP levels also increased in vitamin E group in only posterior region. The level of GSH and vitamin C did not change statistically in control and vitamin E groups. Periodontal indexes did not show any differences in comparison with the groups. In conclusion, we observed protective role of vitamin E on LP levels in anterior region of patients with orthodontic tooth movement.     

Rapid microspectrofluorometric studies in EL2 cells following intracellular accumulation of dibenzocarbazoles

Summary Microspectrofluorometric observations were carried out in EL2 ascites cancer cells and dibenzo(a,e)fluoranthene (diB(a,e)F)-grown EL2 cells, following treatment (5 min) with three dibenzocarbazoles (1,2,7,8; 1,2,5,6 and 3,4,5,6). After microinjection of glucose-6-P leading to reduction of NAD(P), a sequence of difference spectra (after substrate minus before) is recorded. In dibenzocarbazole-untreated cells, maximum NAD(P) reduction (emission maximum at 465–475 nm) is attained within 5 s, followed by a gradual return to initial fluorescence within 20 to 200 s (faster in the diB(a,e)F-grown). In dibenzocarbazole-treated cells there is a rather regular increase in the intensity of the difference spectrum up to 300–500 s. Initially the increase is more predominant in the region around 460–470 nm, but it gains later prominence in the shorter wavelength region (420–430 nm) characteristic of the hydrocarbon (higher and steadier increase in the 3,4,5,6, dibenzocarbazole-treated diB(a,e)F-grown). Subsequently there is a gradual decrease of fluorescence which may or may not return to initial level. The observed increase spectra require evaluation in terms of possible components (e.g. a mixture of NAD(P)H and hydrocarbon, binding changes, succession of fluorescent metabolites).     

A study of metabolic control and intracellular transport by multifunctional photon counting and two channel microfluorometry

A multifunctional photon counting method with operation on a ‘background subtract’ mode has made possible observations of small changes in the redox state of pyridine nucleotides (e.g. with microelectrophoretic additions of 6-phosphogluconate, mannose-6-phosphate). In EL 2 ascites cells, the utilization of glucose-6-phosphate along the Embden Meyerhof pathway can be described by a power formula (R = a [S]^b, for R, rate, [S], concentration, a and b, constants), that of 6-phosphogluconate by an exponential formula (R = ce^b[s], E = 2.7183), while that of mannose-6-phosphate exhibits a considerable lag, possibly at the level of phosphomannose isomerase. Considerable difficulty is associated with in situ observations of responses to Krebs cycle intermediates (e.g. malate), possibly due to intracellular steady states, endogenous substrate levels, as well as the complexity of redox changes in NAD (P)⁺ apparently not confined exclusively to the mitochondrion.The aerobic glycolysis which characterizes EL2 cells cannot be detected in Chang conjunctiva culture cells where a pronounced Pasteur effect, a highly active anaerobic glycolysis, and a strong lactate control are observed. Operation of the photon counter on a ‘chopping’ mode, with alternate sampling of two positions on the photocathode (e.g. 4 msec/position) of a deflectable photomultiplier tube makes possible parallel observations on two cellular sites. Microelectrophoretic additions of fluorescein have been used for determination of intracellular transport from the slopes of fluorescence rise at cellular sites 10–15 μm apart.