Fritz Pliquett

PUVA-induced erythema and changes in mechanoelectrical properties of skin. Inhibition by tocopherols

SummaryInfluence of antioxidants on two phototoxic effects of 8-methoxypsoralen (8-MOP) was studied: erythema and changes in mechanoelectrical properties of skin. α-Tocopherol and its analogs with shortened lateral hydrocarbon chains at C2-atoms of chromane groups (chromanols) were used as antioxidants. α-Tocopherol and its analogs inhibited both phototoxic effects of 8-MOP. Inhibition was observed only if antioxidants were present in skin during irradiation. When applied after irradiation these antioxidants produce no inhibitory effect. The antioxidant antierythemal action depends greatly on their concentration. The protective effect is maximal at antioxidant concentrations 2.5·10-10 – 5·10-9mol·cm-2 of skin, at concentrations higher than 5·10-9mol·cm-2 the protective action is decreased. The protective effect of antioxidants depends on the irradiation dose.

Reticulocyte lipoxygenase changes the passive electrical properties of bovine heart submitochondrial particles.

Purified reticulocyte lipoxygenase oxygenates the polyunsaturated phospholipids of sonified submitochondrial particles from bovine heart as measured by a burst of oxygen uptake. Over the frequency range of 0.5 to 100 MHz, the complex impedance of the submitochondrial particles as a function of the frequency before and after lipoxygenase attack was measured. From these data, the membrane capacity, the conductivity of the membrane and the conductivity inside the particles were calculated. Lipoxygenase action causes a 4-fold increase in the membrane capacity and a 2-fold increase in the membrane conductivity. Using the method of deformation of electric pulses, kinetic measurements were performed. In parallel to the changes of the passive electric properties, a partial inhibition of NADH oxidase and succinate oxidase was caused by the lipoxygenase attack. Oxygen uptake, changes of the passive electric properties and the inhibition of respiratory enzymes were prevented by lipoxygenase inhibitors. Owing to the high oxygen consumption produced by the lipoxygenase reaction, anaerobiosis was reached within the first 30 s in the closed chamber. Therefore, it must be concluded that the changes in passive electric properties and the inhibition of the respiratory enzymes are due to secondary anaerobic processes such as the hydroperoxidase reaction catalyzed by the lipoxygenase or a slow redistribution of peroxidized membrane lipids. The results are discussed in relation to the breakdown of mitochondria during the maturation process of red cells.

Passive electrical properties of RBC suspensions: changes due to distribution of relaxation times in dependence on the cell volume fraction and medium conductivity

The intra- and extracellular conductivity, κa and κi as well as the cell membrane capacitance Cm of RBC suspensions are often calculated using the Pauly and Schwan equations which neglected the parameter α of the empirical Cole–Cole function. However, in complex materials such as RBC suspensions with several overlapping relaxation processes the total electrical response is characterized by a broad distribution of relaxation times yielding an α>0. The locus diagram in the conductivity-plane (κ-plane) is a depressed semicircle with the center below the real axis. It is suggested, that α characterizes both the distribution in relaxation times and the interaction between the relaxing units. Here we focus on the influence of interaction on α. Therefore complex conductivity measurements were done on RBC suspensions with the hematocrit p and the conductivity κ of the medium as parameter. The hematocrit was varied from 0.04 to 0.95 while three different media (κ=1.447 S/m, 0.571 S/m, and 0.064 S/m) were chosen. From this we could evaluate the dependence of α on the interactions of the cells. The electrical parameters of the suspension were calculated by the Pauly and Schwan equations and correlated with the hematocrit. The correct value is obtained at low p when α is small (p and α<0.1), because the Pauly and Schwan equations are derived under the presumption of low cell concentration. We compared our values with findings from cell rotation experiments. We propose an algorithm to estimate the correct passive electrical values from measurements carried out at higher p values.

Passive electrical properties of human stratum corneum in vitro depending on time after separation.

The passive electrical properties of human skin after separation from the body are predominated by the stratum corneum. Skin within a bath medium (150 mM phosphate buffered saline) at constant temperature (37 degrees C) exhibits a characteristic change of the passive electrical properties with time. Independent of the time the locus in the Z-plane is a depressed semicircular arc. The angle between the lines from the center of the arc to the points where the locus reaches the real axis remains unchanged. The difference between the high and low frequency resistivity (R0-Rx) increases over 10 h, reaches a plateau and decays after 20 h exponentially with a time constant of about 40 h. As model for the impedance we used a 5 element electrical circuit (R0, R1, R2, C1, C2), describing 3 pathways, (0) the dc path (appendages; R0), (1) tortuous pathways around the cell structures (R1, C1) and (2) direct pathways involving the corneocytes (R2, C2). There are characteristic changes with time in the elements of the equivalent circuit up to about 200 h after excision. Dramatic changes in C1 and R2 at about this time after separation strongly suggests destruction of the lipid structures. It will be suggested that the use of separated human stratum corneum as model for in vivo yields unreliable results after this time.

Inhibition of erythema of the skin photosensitized with 8-methoxypsoralene by α-tocopherol

8-Methoxypsoralene (8-MOP) and other psoralene derivatives are effective photosensitizers of the animal and human skin to near ultraviolet radiation (320-400 nm), and under these circumstances erythema (PUVA erythema) [5] and pigmentation of the skin [8] develop. In clinical practice the psoralenes have found widespread application for the phototherapy of vitiligo, psoriasis [5], and certain other skin diseases. The photochemical reaction of the psoralenes which has received the most study is their covalent photoaddition to DNA with the formation of cyclobutane adducts [3]. Many workers consider that this photochemical reaction of the psoralenes lies at the basis of induction of photobiological effects observed in the skin, for the ability of different psoralene derivatives to photosensitize the skin correlates closely with their ability to undergo photoaddition to DNA [4, 14]. However, the psoralenes can also take part in other photochemical reactions. For example, in the presence of oxygen 8-MOP photosensitizes inactivation of lysozyme [13] and for ribosomes from Escherichia coli [15], uncoupling of oxidative phosphorylation in mitochondria [ii], and accumulation of oxidation products of lipids in suspensions of mitochondria [ii] and liposomes [7]. It is considered that these processes take place through the participation of singlet oxygen generated by 8-MOP under the influence of near UV radiation [2]. The psoralene can thus take part in a wide variety of photochemical reactions. As yet no attempt has been made to evaluate the effect of photo-oxidative reactions involving psoralenes on the induction of erythema of the skin. One way of studying this problem is to use antioxidants. In the investigation described below the role of the antioxidant a-tocopherol in the induction and development of erythema of the human and rabbit skin, photosensitized with 8-MOP, was investigated.

Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.

Stress action on biological tissue and tissue models detected by the Py value.

Abstract: The Py value, a fast measurable combination of the conductivity at the corner frequencies of the β‐dispersion, is a measure of the relative cell volume concentration in tissue. In many cases, if the biological object is stressed, for instance, by mechanical deformation, shortage of oxygen, electric field strength, temperature rise, or ischemia, Py increases. Depending on the object and the kind of stress, Py plateaus for minutes up to hours and then it decreases continuously. Values of passive electrical parameters of biological tissues are often given without information about the time following a stimulation or stress situation, for example, death, surgery, field application, etc. However, since the passive electrical properties change with time, information about their history, for example, time after death, should be given.

Pre-irradiated photo-oxidized psoralen induces hemolysis and changes in electrical parameters of erythrocyte membranes

Abstract The effects of pre-irradiated photo-oxidized psoralen (POP) on human erythrocytes (RBCs) were investigated. Psoralen in ethanol solutions was photo-oxidized and then added to a suspension of erythrocytes. The hemolysis and the electric impedance of the suspension were measured in the frequency range 5–20 MHz. The membrane capacity CM, and the extracellular and intracellular conductivities ka and ki were calculated from these data. There appeared to be a threshold concentration of POP for hemolysis. When the concentration of POP slightly exceeded this threshold, the hemolytic effect rapidly increased to a maximum, but a further increase in POP concentration slowed down the rate of hemolysis. POP induced a decrease in extracellular conductivity and an increase in membrane capacity, but did not affect intracellular conductivity. The changes in extracellular conductivity correlated well with hemolysis.

Influence of the antitumour antibiotics daunomycin and violamycin B I on membranes of human erythrocytes: Passive electrical investigations

Abstract The interaction of several anthracycline antitumour drugs with human erythrocytes was investigated by passive electrical measurements. The results show that mainly the cell membrane capacity is changed after addition of violamycin B I, while daunomycin used in chemotherapy affects the conductivity inside the red blood cells significantly. An attempt was made to find a correlation between the observed changes of the electrical parameters and the chemical structures of these drugs.

Disaggregation of HeLa-Cx43- and HeLa-spheroids induced by PUVA and photo-oxidized psoralen (POP)

To investigate the effects of PUVA (psoralen + UVA-irradiation) and photooxidized psoralen (POP) on cell-cell junctions, two kinds of multicellular spheroids, which were grown from HeLa cells of epithelioid human cervix carcinoma, were used as a model systems: i) defective in intercellular communication through gap junctions (HeLa-spheroids) and ii) transfected with coding sequences of murine connexin Cx43 with restored gap-junction coupling (HeLa-Cx43-spheroids). It was been found that both PUVA and POP induced disaggregation of HeLa-spheroids as well as HeLa-Cx43-spheroids. It implies that gap-junction plaques are not, apparently, critical targets in psoralen-photosensitized disaggregation. The rate of disaggregation was estimated as inverse time of disaggregation of 50% or 100% spheroids in suspensions (1/t50 or 1/t100, respectively). The rate of PUVA-induced disaggregation was found to increase with the increase of UVA-fluence up to 85 kJ/m2. Photosensitization coefficient was highest at low UVA-fluences (4-6 kJ/m2) and significantly decreased with increase in UVA-fluence. The viability of cells in spheroids was estimated with the use of trypan blue stain. At low UVA-fluences, the process of disaggregation was found to occur without the formation of trypan positive cells in spheroids. Results obtained evidence that PUVA-induced disaggregation of spheroids may occur, at least partially, through the action of POP-products.