Photophysical properties and in vitro photocytotoxicity of disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX (Dimegine).

Photophysical and in vitro photocytotoxicity studies were performed for the photosensitizer Dimegine, a disodium salt 2.4-di(alpha-methoxyethyl)-deuteroporphyrin-IX with very low systemic toxicity. The singlet oxygen and luminescence quantum yield were ΦΔ = 0,65 ± 0,06, and Φƒ=0,11 ± 0,01 respectively, and were independent of the excitation wavelength. The photobleaching coefficients for Dimegine dissolved in phosphate buffer (pH 7.4), and DMEM medium at concentration 2 µM/l and in phosphate buffer (pH 7.0) at concentration 10 µM/l were 16·10-5, 9·10-5 and 2·10-5 respectively. In vitro cellular distribution and photocytotoxicity was studied in two human (U87 - primary glioblastoma and HT1376 - bladder cancer) and two rat cell lines (RG2 - glioma, and AY27 - bladder carcinoma). Fluorescence microscopy analysis shows primary Dimegine accumulation as small fluorescent inclusion bodies around the nuclei, suggesting an apoptotic over a necrotic cell death mechanism. The PDT efficacy was slightly higher for the rat cell lines over the human-derived cell lines, with LD50 values of 2,5 µM/l, 2.8 µM/l, 4.5 µM/l, 2.8 µM/l using 530 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively, and 1.8 µM/l, 2 µM/l, 5 µM/l, 2.4 µM/l using 625 nm excitation wavelength for AY27, RG2, HT1376 and U87 respectively. Comparison to literature data showed that Dimegine demonstrated improved phototherapeutic characteristics comparing to PpIX-mediated PDT.

Dynamics of micronuclei in rat skin fibroblasts after X irradiation.

In a previous study, we demonstrated DNA damage, expressed as micronuclei, in binucleate dermal fibroblasts obtained from human skin 2-9 weeks after fractionated radiotherapy. Here we assessed micronuclei in X-irradiated skin fibroblasts from 9-14-week-old female Lewis rats as a function of time after a single dose of radiation to determine the lifetime of such damage in the skin. After irradiation with 5, 10, 15 and 18 Gy, formation of micronuclei at 1 day or 2 months postirradiation increased up to about 10 Gy, with evidence for a plateau at higher doses. The time course of micronuclei present in the skin fibroblasts demonstrated a plateau region (approximately 20 days after 18 Gy and about 2 months after 10 Gy) before the number of micronuclei started to decline. Residual micronuclei were observed for more than 1 year after irradiation. Monomicronucleated cells predominated in fibroblasts from nonirradiated skin, whereas in fibroblasts from irradiated skin, multimicronucleated cells predominated and persisted (together with monomicronucleated cells) in the residual levels of damage at late times. The results suggest that DNA damage in dermal fibroblasts can be assayed by the micronucleus assay in samples from irradiated skin up to 1 month after irradiation for doses up to at least 10 Gy. Further studies are needed to define the dose-response relationship in detail.

Heat acclimation and heat stress have different effects on cholinergic-induced calcium mobilization.

There is evidence that the signal transduction array responsible for the secretion of water in evaporative cooling by the submaxillary gland of the rat is subject to heat acclimatory responses. The objectives of the present study were 1) to examine whether heat acclimation affects intracellular Ca(2+) mobilization and, in turn, submaxillary glandular responsiveness; 2) to assess whether the acclimatory responses differ from those evoked on heat stress (HS). Experiments were conducted on submaxillary glands of rats acclimated at 34 degrees C for 0, 2 [short-term heat acclimation (STHA)], and 30 [long-term heat acclimation (LTHA)] days. The resting cytosolic calcium concentration ([Ca(2+)](c)) and the carbamylcholine-evoked calcium signal ([Ca(2+)](s)) of dispersed glandular cells were measured using the fluorescent dye fura 2 AM. Inositol-1,4,5-trisphosphate (IP(3))-sensitive endoplasmic reticulum Ca(2+) stores were determined in permeabilized cells using fura 2 potassium salt. STHA resulted in a drop in both [Ca(2+)](s) and IP(3)-sensitive Ca(2+) stores. On LTHA, the [Ca(2+)](s) amplitude reverted to the preacclimation value, whereas the IP(3)-sensitive Ca(2+) stores remained low. The drop in [Ca(2+)](s) on STHA is in accord with the decreased glandular output (measured by (86)Rb efflux) observed during this acclimation phase. However, after LTHA the enhanced glandular output despite reduced [Ca(2+)](s) levels suggests an increased efficiency of cellular secretory mechanisms in that group. Collectively, the alterations in [Ca(2+)](s) support our biphasic acclimation model (Horowitz M, Kaspler P, Marmari Y, and Oron Y. J Appl Physiol 80: 77--85, 1996.). In nonacclimated glands, HS caused an elevation in [Ca(2+)](s) coincidentally with a decrease in the IP(3) Ca(2+) stores. In contrast, [Ca(2+)](s) in both STHA and LTHA glands was not affected by HS, despite a marked increase in the IP(3)-sensitive Ca(2+) stores in the LTHA glands. The opposing responses to HS and heat acclimation in calcium signaling and stores confirm the specificity of each process.

Effect of heat acclimation and heat shock on oscillations of carbamyl-choline-evoked Ca2+ signal in HSY cell line.

We studied effects of heat acclimation (HA) and acute heat stress (HS) on Ca2+ signal oscillations following supramaximal carbamyl-choline (CCh) stimulation, using HSY cell line as a model. In the control cells, oscillations decreased their amplitude with time. HS alone did not change either oscillation amplitude or frequency, although calcium release to the cytosol upon CCh stimulation was faster. HA increased maximal oscillation amplitude only. There was no change in basal cytosolic calcium level and peak evoked signal in all experimental conditions. Collectively, the data suggest that HA affects the oscillation profile. Changes in the oscillation profile did not correlate with changes in the resting and evoked Ca2+ signal, which suggests that the oscillations are a separate target for heat acclimation.

Effect of cadmium and zinc salts on energy metabolism and survival of Unio tumidus Philipsson.

Energy metabolism of aquatic invertebrates is one of the most important targets of environmental pollutants, in particular, heavy metals. In this study, we determined changes in survival, oxygen uptake and hepato-pancreas glycogen level of the bivalve mollusk Unio tumidus Philipsson following chronic exposure of Cd and Zn salts (chlorides and sulfates). The concentrations of Cd salts were equal to 0.001, 0.01 and 0.1 mg/l and concentrations of Zn salts were equal to 0.01, 0.1, 1.0 mg/l (maximal contamination level for Cd is 0.005 mg/l and for Zn is 0.01 mg/l). Survival was registered daily whereas oxygen consumption and glycogen level were determined on days 3, 7 and 14. Changes in survival of U. tumidus along the course of Cd and Zn salt concentrations was not monotonic; the sulfates were more inhibitory than the chlorides. No correlation was found between changes in survival and changes in the oxygen uptake and the glycogen level. By analysis of data in the literature, it was possible to construct a general scheme of adaptation of aquatic invertebrates' energy metabolism to heavy metals impact: (1) "exhaustive" or "economical" activation of aerobic metabolism (with or without depletion of energy resources); (2) change to "exhaustive" anaerobic mechanism (with depletion of glycogen); (3) change to "economical" anaerobic metabolism (involving mechanisms of glycogen expenditure economy); (4) late-term activation of aerobic metabolism due to need for binding and excretion of accumulated metals. Different severity of the pollutants' impact may lead to prevalence of different stages of this scheme. Our results revealed most of these stages in U. tumidus: "economical" activation of aerobic metabolism, change to "exhaustive" and "economical" anaerobic metabolism, and late-term activation of aerobic metabolism. It can be suggested that Cd is more toxic due to late-term decrease in oxygen consumption whereas Zn resulted only in transient early-term decrease. On the other hand, mechanisms of glycogen economy during anaerobic metabolism were involved in the effect of Cd and not involved in the effect of Zn. Concerning the effect of anions, chlorides promoted transient activation of aerobic metabolism, while sulfates promoted the passage to anaerobic metabolism. Thus, the effect of sulfates seems to be more inhibitory than the effect of chlorides.

Heat acclimation and hypohydration: involvement of central angiotensin II receptors in thermoregulation.

This investigation attempted to confirm the involvement of central ANG II-ergic signals in thermoregulation. Experiments were conducted on rats undergoing short (STHA)- and long (LTHA)-term heat acclimation, with and without superimposed hypohydration. Vasodilatation (VTsh) and salivation (STsh) temperature thresholds, tail blood flow, and heat endurance were measured in conscious rats during heat stress (40 degrees C) before and after losartan (Los), an ANG II AT(1)-selective receptor antagonist, administration either to the lateral ventricle or intravenously. Heat acclimation alone resulted in decreased VTsh. STsh decreased during STHA and resumed the preacclimation value, together with markedly increased heat endurance on LTHA. Hypohydration did not affect this biphasic response, although STsh was elevated in all groups. The enhanced heat endurance attained by LTHA was blunted. Neither Los treatment affected the nonacclimated rats. In the heat-acclimated, euhydrated rats, intracerebroventricular Los resulted in decreased VTsh, whereas intravenous Los resulted in elevated STsh. Both intracerebroventricular and intravenous Los led to markedly enhanced heat endurance of the LTHA hypohydrated rats. It is concluded that the LTHA group showed a loss of the benefits acquired by acclimation on hypohydration, whereas the STHA rats, which show an accelerated autonomic excitability in that phase, gained some benefit. It is suggested that ANG II modulates thermoregulation in conditions of chronic adjustments. Central ANG II signals may lead to VTsh upshift, whereas circumventricular structures, activated via circulating ANG II, decrease STsh. On hypohydration these responses seem to be desensitized.

Heat acclimation and heat stress have different effects on cholinergic muscarinic receptors.

In this brief report we have proved that membranal changes take place upon short-term heat acclimation. These changes may switch on a cascade of transient acclimatory compensatory responses as well as long-term processes. The changes observed in the MR profile are heat acclimation specific, and differ from those observed upon heat stress. These bring about functional changes in the signal transduction pathway for water secretion in the submaxillary salivary gland. A recovery period from heat stress, similar to STHA, leads to MR upregulation and decreased binding affinity. This response, however, is more pronounced than that observed upon STHA. The functional importance of the phenomenon is unclear. However, it could designate a novel component of the heat shock response.

Evidence for contribution of effector organ cellular responses to the biphasic dynamics of heat acclimation.

The involvement of cellular processes in the biphasic dynamics of heat acclimation was studied. Key steps in the cholinergic signal transduction pathway for water secretion were measured in the submaxillary gland of acclimating [2-day short-term heat acclimation (STHA) and 30-day long-term heat acclimation (LTHA) at 34 degrees C] or acute heat-stressed (2 h at 40 degrees C) rats in vitro. Both the carbamylcholine (CCh)-induced maximal fractional rate and the total 86Rb+ efflux, reflecting K+ efflux and water transport, transiently decreased in STHA (P < 0.001). In LTHA, the total K+ efflux increased (P < 0.001), whereas the maximal fractional rate of efflux increased only slightly. During STHA, the density of the high-affinity binding site of the muscarinic receptors (MRs) increased by 50% and their affinity for the muscarinic antagonist [3H]-N-methylscopolamine decreased transiently by 87%. Basal cytosolic Ca2+ concentration ([Ca2+]i) decreased (P < 0.05), but the peak CCh-induced [Ca2+]i increase resembled the control values. In LTHA, MR density continued to increase (100%; P < 0.05), whereas affinity resumed control values. Basal and CCh-induced [Ca2+]i increases returned to control levels. We conclude that glandular cellular processes follow a biphasic pattern with major apparent changes attributable to events distal to the [Ca2+]i rise. This was further validated by employing heat stress, which produced qualitatively different effects on the MR profile with a decrease in 86Rb+ efflux comparable to STHA. Hence, although heat-induced changes in the proximal components of the signal transduction pathway may contribute to altered regulatory span, the predominant apparent cellular effect is on the distal part of the pathway.