IRREVERSIBLE BINDING OF CHLORDIAZEPOXIDE TO HUMAN PLASMA PROTEIN INDUCED BY UV-A RADIATION

Abstract— In view of the phototoxicity of chlordiazepoxide (Librium^®) the kinetics of the reaction in the presence of plasma proteins was studied for chlordiazepoxide upon UV-A irradiation and for its oxaziridine in the dark. Two different methods were used to determine the extent of irreversible binding to protein (up to ∼ 50% was found for both situations). Kinetic data support the conclusion that the formation of oxaziridine from photoexcited chlordiazepoxide is the basic event making chlordiazepoxide phototoxic. The half life of oxaziridine is about 30 min even in the presence of a high concentration of plasma proteins, is in agreement with previous in vivo results, showing covalent binding not only to biomolecules of the UV-A irradiated skin but also to those of inner organs, e.g. liver and kidneys.     

Irreversible photobinding of nitrofurantoin and of nitrofurfural to plasma proteins in vitro

Allergic reactions form a serious problem in therapy with the urinary antiseptic nitrofurantoin (NFT). The formation of conjugates between NFT and plasma proteins is considered to be a first step in the development of such reactions. We investigated the possibility that UV-A irradiation of NFT in the presence of plasma proteins results in covalent binding. Binding to blood cell proteins was +/- 100X less. Efficient photobinding to albumin was demonstrated for NFT (up to 50 nmol mg-1 protein) and its photoproduct 5-nitrofurfural. Incubation of photodecomposition products from these two nitrofurans with plasma proteins also resulted in irreversible binding. Protein amino and, to a lesser extent, thiol groups proved to be targets for binding. Furthermore, upon photolysis both compounds induced a significant decrease in the iso-electric point of albumin. Photobinding, along with such alterations in the structure of plasma proteins, may well be able to trigger immunological responses when taking place in vivo. In this manner activation of nitrofurantoin by light may be a cause of allergic reactions by nitrofurantoin.     

In vivo PHOTODEGRADATION OF CHLORPROMAZINE

The in vivo photodegradation of chlorpromazine (CPZ) in the skin was investigated after systemic administration of 3H-CPZ to shaven Wistar rats and exposure to UV-A. Promazine (PZ) and 2-hydroxy-promazine (2-OH-PZ) appeared to be formed in irradiated rats, but not in the skin of rats kept in the dark. This indicates that upon irradiation with UV-A the PZ-radical is formed which can be held responsible for the photobinding to eye and skin constituents as observed earlier [Schoonderwoerd and Beijersbergen von Henegouwen (1987) Photochem. Photobiol. 46, 501-505]. Chlorpromazine-sulfoxide (CPZSO) is a major metabolite of CPZ. Less CPZSO was found in the skin of irradiated rats compared to those kept in the dark. As this appeared not to be caused by photobinding or photodegradation of CPZSO it can be concluded that CPZSO is not a photoproduct of CPZ under these experimental conditions. This study shows that the in vivo photodegradation of CPZ proceeds via the promazinyl radical rather than via the radical cation.     

PHOTOPHARMACOLOGY OF THE TRANQUILIZER CHLORDIAZEPOXIDE IN RELATION TO ITS PHOTOTOXICITY

Abstract— This study demonstrates that photobiological effects by exogenous molecules are not per se restricted to the skin. As an example the photopharmacology of chlordiazepoxide (e.g. Librium^R) was studied. Rats being exposed to UV-A after administration of ¹⁴C₂ labeled chlordiazepoxide (CDZ) showed a marked change in the pattern of urinary metabolites; the quantity of metabolites without N₄-oxide function increased, whereas that of compounds with N₄-oxide function decreased. Covalent binding to organ tissue of especially liver and skin of the irradiated rats was found. This would mean that in the irradiated rats oxaziridines which may also react with tissue of the inner organs are formed from CDZ and its N₄-oxymetabolites.
Further, the combination of light and CDZ leads to a 30% decrease in weight of the liver, indicating liver damage. This is also supported by the percentage of conjugated urinary metabolites being 1.5–2.5 times lower than in the non-irradiated CDZ-treated rats, possibly caused by a decrease of enzymatic activity.
The administration of CDZ-oxaziridine to non-irradiated rats provoked the same effects, such as a decrease of the weight of the liver comparable to that of irradiated CDZ-treated rats. On the other hand the combination of light and treatment with N₄-desoxy CDZ did not have any effect, as expected.     

Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.     

IRREVERSIBLE PHOTOBINDING TO SKIN CONSTITUENTS AFTER SYSTEMIC ADMINISTRATION OF CHLORPROMAZINE TO WISTAR RATS

From in vitro experiments it is known that chlorpromazine binds to protein and DNA/ RNA upon UV-irradiation. In the present study the possible photobinding of chlorpromazine (or its metabolites) in vivo was examined. Tritium labeled drug was administered intraperitoneally to female albino Wistar rats after which they were irradiated with light with maximum intensity at 310, 370 or 420 nm. After homogenization, unbound radioactivity in tissue of several organs was removed by dialysis. In the ears, eyes and skin of the back irreversibly bound radioactivity could be detected after irradiation with 310- and 370- but not with 420 nm light. Binding in the skin of the back after UVA irradiation was examined in more detail by separating epidermal lipids, DNA/RNA and proteins by a selective extraction/precipitation method. Radioactivity appeared to be bound to lipids and proteins but not to DNA/RNA.     

Photochemical decomposition of lomefloxacin in vitro and in vivo

To obtain an idea of the photostability of Lomefloxacin (Lom) under in vivo conditions the compound was exposed to UV-A (310-360 nm) in PBS buffer pH 7.4. Exposure of 10 microg/ml of Lom in PBS pH 7.4 led to more than 50% decomposition within 10 min. Loss of the fluorine atom at C-8 and partial breakdown of the piperazine ring occurred. The only two photoproducts formed under these conditions were AEA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminoethyl-amino)-4-oxo-3-quinolinecarboxylic acid, and APA, 1-ethyl-6-fluoro-1,4-dihydro-7-(2-aminopropyl-amino)-4-oxo-3-quinolinecarboxylic acid. When Lom was exposed in whole blood in vitro, the same photochemical decomposition was observed in the plasma as in PBS buffer: APA and AEA were the only products. During UV-A exposure, Lom was shown to be taken up by the leukocytes. This process appeared to be less rapid during UV-A exposure than in the dark. As soon as UV-A exposure commenced, AEA and APA were found. As in the plasma, the total amount of Lom and the two photoproducts in the leukocytes was not significantly different from the amount of Lom found in unexposed cells at the same time point. The erythrocytes did not take up Lom, but exposure of whole blood to Lom and UV-A under the above conditions led to more than 7% haemolysis. Treatment of rats with a combination of Lom and UV-A demonstrated photodecomposition of Lom in vivo. In urine produced during exposure and by the irradiated rats during the twilight period after exposure, a considerable amount of AEA and APA was found. The blood plasma from rats exposed simultaneously to UV-A and Lom proved to contain AEA and APA and, in the leukocytes, APA. This was not the case with animals kept in twilight.     

EFFECTS OF UV IRRADIATION ON A LIVING SKIN EQUIVALENT

Abstract— The Living Skin Equivalent (LSE™) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280–400 nm) or UV-A (320–400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R₅₀ or UV-A₅₀) in different lots of LSE were 0.053 ± 0.021 J/cm² (n = 29) and 11.6 ± 4.9 J/cm² (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-α, tumor necrosis factor-α and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of U V radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.     

PHOTOREAaIVITY OF CHLORAMPHENICOL in vitro AND in vivo

Abstract The negative side effects of chlorarnphenicol (CAP) mostly involve blood dyscrasias (e.g. irreversible nondose-dependent aplastic anemia), allergic skin reactions and eye damage. To learn the cause of these side effects, most research focuses on metabolically formed nitroso- and hydroxylamino derivatives in the predisposed patient. In previous investigations it was demonstrated that photochemical decomposition of CAP in vitro by UV-A leads to formation of p-nitrobenzaldehyde (pNB), p-nitrobenzoic acid (pNBA) and p-nitrosobenzoic acid (pNOBA); the latter comprises up to 45 mol% of the starting amount of CAP. Incubation of these photoproducts in rat blood showed that pNB and pNOBA rapidly react and that PNBA is stable under these conditions. Reaction products from pNB (half-life 1.7 min) proved to be pNBA and p-nitrobenzyl alcohol (pNBOH) while pNOBA (half-life 3.7 min) was converted into p-aminobenzoic acid (pABA). Exposure of CAP in rat blood to UV-A yielded the same end products: pNBA, PABA and pNBOH. To estimate the amount of oxidative stress generated in vivo by these compounds, the ability to form methemoglobin (MetHb) in erythrocytes was tested; only pNOBA and p-hydroxylaminobenzoic acid (pHABA), a possible intermediate in the decomposition of pNOBA, proved to be reactive. Ultraviolet-A exposure of rats, after intraperitoneal injection of CAP, led to 3.6 times the basic level of MetHb. In addition, covalent binding of ³H-labeled CAP photoproducts to the skin of the back and to the ears was found, which was 9.1 and 3.2 times higher, respectively, than the dark values. Toxicity toward bone marrow cells of all photoproducts was established in vitro. p-Nitrobenzaldehyde, pNOBA andpHABA were 20, 6 and 6 times more toxic than CAP, respectively. These results show that photodecomposition of CAP in vivo does occur. Its reactive photoproducts are able to cause damage that may lead to (systemic) side effects. The latter is supported by the fact that the nature of the reactive products, nitroso- and hydroxylamino derivatives, is the same as the expected metabolites.     

二维材料“遇见”生物大分子:机遇与挑战

二维材料的超薄原子层结构使其具有独特的力学性能、导热导电性以及巨大的比表面积,在能源存储、催化、传感和生物医学等领域引起了国内外学者的广泛关注。将二维材料与具有生物活性的生物大分子相结合可以为开发具有优异电学、力学和生物学功能的特种功能材料提供新的方法和途径。近年来,科研工作者针对这一方向展开了广泛的研究,取得了一系列重要的成果,使二维材料与生物大分子的结合与应用成为了新的研究热点。本文综述了近年来二维材料和生物大分子之间的相互作用及应用的研究进展,重点介绍了二维材料与生物大分子在分子水平上的相互作用机理,还总结了基于二维材料与生物大分子之间的相互作用在工程、疾病治疗和抗菌中的应用,并对其未来的研究趋势提出了展望。     

In vitro PHOTODEGRADATION OF CHLORPROMAZINE

The in vitro photodecomposition of chlorpromazine (CPZ) was investigated with the aim to evaluate possible reactive determinants that could play a role in the occurrence of the in vivo -observed photosensitivity. In view of the in vivo situation, CPZ was dissolved in low concentration in buffered aqueous solution (pH 7.4) or in dilute human serum and irradiated with low intensity (5–7 W m^-2) UV-A and UV-B. No distinct difference was found between UV-A or UV-B irradiation as far as photoproduct formation is concerned. This suggests the same degradation mechanism at both wavelength ranges. In buffered aqueous solution, irradiation of CPZ resulted in 65 and 90% 2-hydroxypromazine (PZOH), 5 and 7% promazine (PZH) and 2 and 0% chlorpromazinesulfoxide (CPZSO) under aerobic and anaerobic conditions, respectively. In dilute human serum, there was only a shift in the PZH/PZOH ratio, probably as a result of H-atom or electron donation by sulfur containing groups present in proteins. The results demonstrate that photodegradation of CPZ in vitro , under conditions relevant to the in vivo situation, proceeds almost entirely by dechlorination rather than by radical cation formation (the essential pathway of CPZSO production). Thus we conclude that the thiyl radical cation probably does not play a major role in the in vivo -observed phototoxic reactions.     

Different susceptibility of cells of porcine skin and internal organs to ultraviolet A-induced breaking of nuclear DNA

There is a continuously growing interest in medical applications of ultraviolet radiation (UV-A and long-wavelength UV-B) especially for laser surgery, phototherapy and photodiagnostics of human internal organs. UV-B and UV-A radiation is potentially mutagenic, however, there has been very little information published to date concerning the significance of possible deleterious action of such photons on cells of internal tissues. The aim of this study is to compare the sensitivities of skin cells to those of internal organs upon exposure to UV-A. To assess this sensitivity we have determined the UV-A dose-dependent frequency of nuclear DNA breaks detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) technique. The materials for the study were macroscopic samples of porcine skin, colon and esophagus. The UV-A dose ranged from 0.1 to 1000 mJ/cm2, which is similar to doses received by cells in regions examined with laser-induced fluorescence or by cells surrounding areas subject to a laser ablation. To reduce the influence of DNA repair processes the tissue samples were kept at a low temperature during the irradiation and were deep frozen immediately after completing the irradiation procedure. The cells of the internal organs are much more susceptible to UV-A-induced breaking of DNA than the skin cells. The percentage fractions and the spatial distributions of the damaged cells and the characteristics of the UV-A dose dependence seem to vary by type of internal organ.     

CAROTENOIDS QUENCH EVOLUTION OF EXCITED SPECIES IN EPIDERMIS EXPOSED TO UV-B (290-320 nm) LIGHT

Abstract— Reactions involving singlet oxygen and other free radicals have been identified in epidermis containing either exogenous or endogenous photosensitizers. soaked in a singlet oxygen/free radical trap, and then exposed to visible or UV-A (320-400 nm) light. Such reactions can be quenched by the presence of the carotenoid pigments β-carotene and canthaxanthin which accumulate in epidermis after oral administration. We report here that the carotenoid pigments β-carotene. canthaxanthin and phytoene accumulating in epidermis can also quench to some degree those photochemical reactions involving singlet oxygen and free radicals that occur when epidermis is exposed to the sunburn spectrum of light (UV-B. 290–320 nm).     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

POTENTIATION OF OXIDATIVE DAMAGE TO PROTEINS BY ULTRAVIOLET-A AND PROTECTION BY ANTIOXIDANTS

We have studied the damage of alcohol dehydrogenase (ADH) and glyceraldehyde 3-phosphate dehydrogenase (GAPD) induced by Fe++/EDTA + H2O2 in combination with UV-A (main output at 365 nm). Enzyme inactivation, formation of hydroxyl radicals (measured in the absence of enzymes), increase in protein carbonyls, oxidation of sulfhydryl (SH) groups, loss of native protein fluorescence, and enhanced protease degradation were used to determine protein damage. Hydroxyl radical production was greatly enhanced by the combination of UV-A with Fe++/EDTA + H2O2. The combined treatment increased protein carbonyls but decreased native protein fluorescence and SH groups. The combined treatment caused turbidity in GAPD but not in ADH, whereas trypsin susceptibility was increased more in ADH than in GAPD. These measurements of protein oxidation correlated well with enzyme activities. Glyceraldehyde 3-phosphate dehydrogenase and dithiothreitol were most protective against such damage, while hydroxyl radical and singlet oxygen scavengers were partially effective. Superoxide dismutase had no effect. Thus, UV-A potentiation of protein damage induced by FE++/EDTA + H2O2 appeared to involve hydroxyl radicals and perhaps singlet oxygen but not superoxide radicals. The damage to proteins induced by combination of UV-A with physiological oxidants, iron ions and H2O2 may be relevant to UV-A-induced skin and tissue damage.     

Method for measuring endogenous 3-O-methyldopa in urine and plasma.

The present report describes a method using column liquid chromatography with electrochemical detection for assaying concentrations of 3-O-methyldopa in urine and plasma. The technique combines a one-step sample preparation scheme with post-column flow-through electrodes in series, allowing adequate chromatographic separation of 3-O-methyldopa from other endogenous substances in urine. The validity of the method was confirmed by markedly decreased urinary 3-O-methyldopa levels after administration of an inhibitor of catechol-O-methyltransferase to rats, radioactivity in chromatographic fractions corresponding to 3-O-methyldopa in urine of rats undergoing infusion of [3H]-L-DOPA, and correlations between excretion rates of 3-O-methyldopa and catechols in humans. In healthy humans, urinary excretion of 3-O-methyldopa averaged 974 +/- 707 (S.D.) nmol per day, and plasma levels of 3-O-methyldopa averaged 89 +/- 32 nmol/l. The method should be useful in studies about the metabolism of endogenous and exogenous DOPA.     

Phototesting in lupus erythematosus tumidus--review of 60 patients

Photosensitivity is an important characteristic feature of several forms of lupus erythematosus (LE), and induction of skin lesions by UV-A and UV-B irradiation has been proved to be an optimal model for evaluating light sensitivity in patients with this disease. Because lupus erythematosus tumidus (LET) has rarely been documented in the literature and is often difficult to differentiate from other photodermatoses such as polymorphous light eruption, we performed photoprovocation tests in 60 patients with LET according to a standardized protocol. Areas of uninvolved skin on the upper back were irradiated with single doses of UV-A (100 J/cm2) and/or UV-B (1.5 minimal erythema dose) daily for three consecutive days. Interestingly, patients with LET are more photosensitive than those with subacute cutaneous lupus erythematosus, and in our study experimental phototesting revealed characteristic skin lesions in 43 patients (72%). Because of the latency period in developing positive phototest reactions, it might be difficult for these patients to link sun exposure with their skin lesions. Furthermore, our data revealed a positive correlation of antinuclear antibodies and positive provocative phototest reactions in these patients as seen for other forms of LE. In conclusion, the high incidence of positive phototest reactions in correlation with the clinical findings, history of photosensitivity and antinuclear antibodies enable the classification of LET as the most photosensitive type of LE.     

SUNSCREENS WITH BROAD-SPECTRUM ABSORPTION DECREASE THE trans TO cis PHOTOISOMERIZATION OF UROCANIC ACID IN THE HUMAN stratum corneum AFTER MULTIPLE UV LIGHT EXPOSURES

Abstract The trans to cis photoisomerization of urocanic acid (UCA) in skin is considered to play an important role in the mechanism of immunosuppression. We have investigated the effects of skin type and various sunscreens with low sun protection factor (SPF) on the UV-induced cis -UCA formation in human skin after exposure to artificial IJV light. The rate of cis -UCA formation depends little on the skin type and is reduced by topical application of sunscreens. The rate of cis -UCA formation decreases with increasing SPF and only broad-spectrum, highly protective sunscreens offer protection against the UV-induced formation of cis -UCA, which accumulates in the stratum corneum after multiple UV exposures. A theoretical approach to estimate the distribution of cis -UCA after irradiation indicates that this compound may diffuse into the deeper layers of the epidermis with D ∼ 10⁻¹⁷ m²/s, and that its elimination from the stratum corneum is mainly due to desquamation.     

BIOLOGICAL PROPERTIES OF SOME BENZOPSORALEN DERIVATIVES

Abstract— The biological activity of some benzopsoralen derivatives, prepared with the aim of obtaining new drugs for photochemotherapy, has been studied. The more interesting compounds are 4-hydroxy-methyl-4',5'-benzopsoralen and 4-hydroxymethyl-4',5'-tetrahydro-benzopsoralen, which were found to be active in the dark also: DNA and RNA synthesis were both inhibited in Ehrlich cells, even if in a partially reversible fashion, while protein synthesis remained unaffected. In Chinese hamster ovary cells cultured in vitro , the clonal growth was strongly inhibited by incubation in the dark with both drugs, while a number of chromosomal aberrations was observed in the fraction of growing cells. Using alkaline elution, DNA strand breaks were detected. In addition, in the presence of aphidicolin, a specific inhibitor of DNA polymerase, the clonal growing capacity was completely restored; in contrast, the number of DNA strand breaks remained unchanged. All these results suggest that DNA topoisomerases are probably the target of these two benzopsoralens. These compounds are also good sensitizers; by UV-A irradiation they have a good capacity to produce singlet oxygen, but they appeared to be unable to induce erythemas on guinea-pig skin. Under UV-A light, they induced a strong inhibition of DNA synthesis in Ehrlich cells. Thus, benzopsoralens appear to be capable of inducing strong antiproliferative effects by two different mechanisms, by UV-A irradiation and in the dark.