Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Solubilization of Sodium Channel from Human Brain

[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5.     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

The penetration of sodium into the brain following a cisternal injection of sodium chloride with particular emphasis on the area postrema

Summary Following an intracisternal injection of sodium chloride, sodium has been localized in paraventricular and subpial tissues of the posterior fossa by means of the pyroantimonate histochemical technique, with the use of a buffered pyroantimonate medium. The electron dense deposit is present in these tissues within 4 minutes after injection and is found only extracellularly except in the area postrema. This finding supports the contention that sodium is chiefly an extracellular ion and that the cerebrospinal fluid and the extracellular fluid are in equilibrium at these sites. In the area postrema, an intracellular precipitate is noted in the vesicular structures of the atypical astrocytes of this structure and in pinocytic vesicles of the large blood vessels. The intraglial localization of sodium in the area postrema is discussed in relation to a possible function of this structure as a regulator of cerebrospinal fluid ionic content.This work was supported by grant number NB-08549-02 from the National Institute of Neurological Disease and Stroke, Bethesda, Maryland.     

The effect of sodium balance on sweat sodium secretion and plasma aldosterone concentration

The effect of manipulating sodium intake upon sweat sodium secretion was investigated during heat acclimation. Twenty-five male subjects were confined to an environmental chamber at a temperature of 25°C for 3 days, and then acclimated to heat by a further 5 days at 40°C. The subjects' daily sodium intake was controlled throughout as follows: high (HNa), 348.4 (0.8) mmol · day⁻¹, n = 7; moderate (MNa), 174.1 (0.6) mmol · day⁻¹, n = 9; or low (LNa), 66.3 mmol · day⁻¹, n = 9. Sodium losses were estimated from urinary, faecal and sweat collections using a whole-body washdown method. Plasma aldosterone concentration was also measured from venous blood sampled each morning. Measurements of body temperature and heart rate during the heat exposure phase indicated a degree of heat acclimation. During this heat phase there was a reduction (P < 0.01) in sweat sodium secretion for all three conditions which was greatest for the LNa condition, although this finding was not significant (P < 0.1). In the LNa condition, plasma aldosterone concentration increased (P < 0.05) prior to heat exposure, and the secretion of aldosterone was potentiated (P < 0.01) during the heat exposure in comparison with the MNa condition. In contrast, the HNa diet produced a fall (P < 0.05) in plasma aldosterone concentration prior to heat exposure and an attenuation of aldosterone secretion thereafter. These findings are inconsistent with the hypothesis that retention of sweat sodium is dependent upon a net body sodium deficit, but demonstrate that aldosterone secretion is potentiated under such conditions. Accepted: 22 May 1988     

Under-sulfation by PAPS synthetase inhibition modulates the expression of ECM molecules during chondrogenesis

Sulfation of proteoglycans is an important post-translational modification in chondrocytes. We previously found that 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2 levels increased more than 10-fold during mesenchymal cell chondrogenesis. Given that PAPS is the sole sulfur donor, and is produced only by PAPS synthetase in all cells, increased expression of PAPS synthetase-2 should be a prerequisite for increased sulfation activity of chondrocytes. We found that sodium chlorate, a specific inhibitor of PAPS synthetase, inhibited proteoglycan sulfation during chondrogenesis. In contrast, sodium chlorate unexpectedly induced early expression of type II collagen and increased the number of cartilage nodules during chondrogenesis. Inhibition of sulfation also accelerated the down-regulation of N-cadherin and fibronectin during chondrogenesis. These findings suggest that sulfation has an important regulatory role in coordinating the timely expression of extracellular matrix molecules during chondrogenesis, and that under-sulfation may cause the breakdown of this coordination, leading to premature chondrogenesis.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

Role of sodium ions for sulfate transport and energy metabolism in Desulfovibrio salexigens

The sodium ion gradient and the membrane potential were found to be the driving forces of sulfate accumulation in the marine sulfate reducer Desulfovibrio salexigens. The protonmotive force of –158 mV, determined by means of radiolabelled membrane-permeant probes, consisted of a membrane potential of –140 mV and a pH gradient (inside alkaline) of 0.3 at neutral pH_out. The sodium ion gradient, as measured with silicone oil centrifugation and atomic absorption spectroscopy, was eightfold ([Na⁺]_out/[Na⁺]_in) at an external Na⁺ concentration of 320 mM. The resulting sodium ionmotive force was –194 mV and enabled D. salexigens to accumulate sulfate 20000-fold at low external sulfate concentrations (<0.1 M). Under these conditions high sulfate accumulation occurred electrogenically in symport with three sodium ions (assuming equilibrium with the sodium ion-motive force). With increasing external sulfate concentrations sulfate accumulation decreased sharply, and a second, low-accumulating system symported sulfate electroneutrally with two sodium ions. The sodium-ion gradient was built up by electrogenic Na⁺/H⁺ antiport. This was demonstrated by (i) measuring proton translocation upon sodium ion pulses, (ii) studying uptake of sodium salts in the presence or absence of the electrical membrane potential, and (iii) the inhibitory effect of the Na⁺/H⁺ antiport inhibitor propylbenzilylcholin-mustard HCl (PrBCM). With resting cells ATP synthesis was found after proton pulses (changing the pH by three units), but neither after pulses of 500 mM sodium ions, nor in the presence of the uncoupler tetrachorosalicylanilide (TCS). It is concluded that the energy metabolism of the marine strain D. salexigens is based primarily on the protonmotive force and a protontranslocating ATPase.Abbreviations MOPS morpholinopropanesulfonic acid - TCS tetrachlorosalicylanilide - PrBCM propylbenzilylcholin-mustard HCl - Tris tris(hydroxymethyl)aminomethane - TPP⁺ bromide tetraphenylphosphonium bromide