Biomedical subjects
Publications and source records attributed to R Santus.
Rhodamines 110 and 123, and rhodamine 110 linked via peptide bonds to Arg, Cbz-Arg and Cbz-Ile-Pr-Arg interact with free base porphyrins or cytochrome C. Rhodamines 110 and 123 essentially form 1:1 complexes while the other derivatives form 2:1 complexes. The possible biological implications of these results are discussed.
Fluoranthene, a non-carcinogenic polycyclic aromatic hydrocarbon, inactivates Escherichia coli cells in the presence of near-ultraviolet light (NUV; 300-400 nm). E coli cells carrying defects in the uvrA6 or katF genes are sensitized to inactivation by the simultaneous treatment with fluoranthene and NUV, suggesting that DNA is a target and that hydrogen peroxide is generated. Haemophilus influenzae transforming DNA can be inactivated by the simultaneous treatment with fluoranthene and NUV confirming DNA as a target. Using the photooxidation of imidazole and histidine as probes, fluoranthene was found to generate singlet oxygen in organic and aqueous media. In water, it participated in electron transfer reactions, reducing nitro blue tetrazolium as well as ferricytochrome C. This reduction took place both in the presence of air, where superoxide anion was formed, and under argon. Simultaneous treatment with fluoranthene and NUV was incapable of inducing histidine-independent mutations. Simultaneous treatment with fluoranthene and NUV was incapable of inducing the uvrA gene product as evidenced by the absence of the induction of beta-galactosidase in an E coli operon fusion strain [uvrA215::Mud(Ap,lac)].
DNA synthesis, as well as respiration, has been studied in CV-1 cells incubated with 5 or 25 micrograms/cm3 haematoporphyrin derivative Photofrin II (PF II) for 1, 24 or 48 h and then irradiated with various doses of UVA light (365 nm). The impairments of DNA synthesis increased with the duration of incubation with the porphyrin, its concentration and the dose of irradiation. The cellular consumption of oxygen is also inhibited by the treatment, but less severely. In the case of the higher PF II concentration (25 micrograms/cm3), the impairment of DNA synthesis after illumination seems to be mainly due to 3HTdR transport inhibition. This effect can be related to plasma membrane damage as shown by lactate dehydrogenase leakage. At 5 micrograms/cm3 PF II, DNA synthesis inhibition is observed even after short exposure to PF II and light without 3HTdR transport impairment. In that case, DNA and/or mitochondrial photodamage may explain the inhibition.
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Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL-P2, HDL-P2 or A-P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10-20 micrograms/ml) by LDL-P2 than by HDL-P2 or A-P2. Moreover, P2 delivery to cells by LDL-P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL-P2 or A-P2 is a linear process. Finally, reduction of the LDL-receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL-P2. These results suggest a special role of the LDL-receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.
Resonance energy transfer from tyrosine to tryptophan residues was detected in the phage fd. The magnitude of the transfer efficiency was estimated by both a traditional and an alternative method. The latter involved comparison of the indole acceptor excitation spectrum full-width half-maximum with a set of standard values differing in the amount of absorbance contributed by tyrosine donor. Both methods lead to the same conclusion: essentially all the tyrosine residues of the viral coat are within 0.9 nm of a tryptophan residue. Also, fluorescence lifetime measurements provide additional support for the hypothesis that there are at least two different environments for the coat protein's sole indole side-chain. Little if any DNA phosphorescence was seen, consistent with the nucleic acid bases being stacked in the DNA core.
Charge transfer has been observed between oxidised tryptophan-26 units and the tyrosine-21 or -24 of the coat protein of fd phage. The transfer is likely to be intramolecular. The rates suggest that the aromatic units are in a rigid region and that they may have at least two different environments. No apparent interaction occurs with the DNA, consistent with tryptophan and tyrosine units not being in contact with the bases.
The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.e. antiproliferative effect of anthralin, has been attributed to its action at the level of mitochondria or at the level of glucose-6-phosphate dehydrogenase which initiates the pentose phosphate shunt (cf. its prominent role in nucleic acid synthesis). Upon addition of 2.3 to 23 microM anthralin to the L cell culture, the characteristic structure of the anthralin anion fluorescence spectrum is recognized almost immediately in the cytoplasm (much weaker in the nucleus) but disappears within minutes. The vital mitochondrial fluorescence probe dimethylaminostyryl-pyridinium-methyl-iodine reveals striking structural alterations of the mitochondria within 15 min after addition of the drug. At the same time, there is a stimulation of the transient NAD(P)+ reduction observed upon microinjection into the L cell of the Krebs' cycle substrate malate, or the pentose cycle substrate 6-phosphogluconate. Specially, the injection of the latter to anthralin-treated cells suggests that upon release of the mitochondrial control, there is a tremendous disruption of metabolic activity which could have profound consequences on the proliferative activity of the cell. These findings, while they open new possibilities for the intracellular evaluation of therapeutic agents, create also a challenge in understanding the complex and dynamic interrelationships between intracellular organelles and bioenergetic or biosynthetic pathways.
The chemical mechanisms of the photosensitized peroxidation of unsaturated fatty acids and cholesterol are reviewed and the subsequent reactions of peroxide decomposition products with biological targets such as DNA and amino acids are analyzed. The importance of protein photooxidation and cross-linking in membrane function impairment is discussed.
A first attempt to impair a biological function by the synergistic effects of HpD and nitroimidazoles (metronidazole or misonidazole) under anaerobic conditions is reported. A function tightly linked to a membrane has been chosen as model and the Ca2+ movements through the inner mitochondrial membrane have been studied. When isolated mitochondria (1 mg/cm3) are irradiated in oxic condition with 25 micrograms/cm3 HpD and 1 mmol dm-3 nitroimidazole in the medium, Ca2+ uptake is stopped after 15 s of irradiation. When irradiated in anoxic conditions with 25 micrograms/cm3 HpD alone, addition of ATP triggers a normal Ca2+ uptake even after 10 min of irradiation. In contrast Ca2+ uptake is stopped by 1 min irradiation after addition of 1 mmol dm-3 misonidazole to HpD and by 2 min irradiation after addition of 1 mmol dm-3 metronidazole. Thus, under these experimental conditions, the enhancement ratio of the HpD action (defined as the ratio of the durations of irradiation necessary to obtain the cessation of Ca2+ uptake before and after addition of a component other than HpD) is of the order of 40, 10 and 5 for oxygen, misonidazole and metronidazole, respectively. Misonidazole is still efficient at a concentration of 0.1 mmol dm-3.
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We have studied the photosensitized oxidation, via singlet oxygen production, of histidine (His) and tryptophan (Trp) in the serum of porphyria patients and in the serum of healthy volunteers before or after addition of hematoporphyrin. It was observed that free plasma His and Trp are good probes of singlet oxygen production in the blood under visible light irradiation. However, Trp, which is mostly bound to serum albumin, is much less susceptible to photooxidation than His, which remains free in the plasma. These results must not be ignored in systemic effects of porphyrias and in photochemotherapy with hematoporphyrin derivatives.
The short-term effect of topical application of anthralin (dithranol) on normal human skin was investigated by electron microscopy. Mitochondria appeared markedly damaged. By contrast other cellular structures, particularly the nuclear and cytoplasmic membranes were unchanged. In vitro experiments were therefore performed on isolated rat liver mitochondria and it was shown that anthralin acts as an uncoupler of oxidative phosphorylation. These results suggest that anthralin can inhibit the adenosine triphosphate supply in epidermal cells. This loss of energy supply in keratinocytes could explain, at least in part, the therapeutic efficiency of anthralin in psoriasis.
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Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.