Photoirradiation of dehydropyrrolizidine alkaloids--formation of reactive oxygen species and induction of lipid peroxidation.

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and human. PAs require metabolic activation to generate pyrrolic metabolites (dehydro-PAs) that bind cellular protein and DNA, leading to hepatotoxicity and genotoxicity, including tumorigenicity. In this study we report that UVA photoirradiation of a series of dehydro-PAs, e.g., dehydromonocrotaline, dehydroriddelliine, dehydroretrorsine, dehydrosenecionine, dehydroseneciphylline, dehydrolasiocarpine, dehydroheliotrine, and dehydroretronecine (DHR) at 0-70 J/cm2 in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose-responsive manner. When irradiated in the presence of sodium azide, the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a photo-induced product. When irradiated in the presence of superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. Electron spin resonance (ESR) spin trapping studies confirmed that both singlet oxygen and superoxide anion radical were formed during photoirradiation. These results indicate that UVA photoirradiation of dehydro-PAs generates reactive oxygen species (ROS) that mediated the initiation of lipid peroxidation. UVA irradiation of the parent PAs and other PA metabolites, including PA N-oxides, under similar experimental conditions did not produce lipid peroxidation. It is known that PAs induce skin cancer and are secondary (hepatogenous) photosensitization agents. Our results suggest that dehydro-PAs are the active metabolites responsible for skin cancer formation and PA-induced secondary photosensitization.

UVA photoirradiation of oxygenated benz[a]anthracene and 3-methylcholanthene--generation of singlet oxygen and induction of lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants and potentially pose a health risk to humans. Although the biological and toxicological activities, including metabolism, mutagenicity, and carcinogenicity, of PAHs have been thoroughly studied, their phototoxicity and photo-induced biological activity have not been well examined. We have long been interested in phototoxicity of PAHs and their derivatives induced by irradiation with UV light. In this paper we report the photoirradiation of a series of oxygenated benz[a]anthracene (BA) and 3-methylcholanthene (3-MC) by UVA light in the presence of a lipid, methyl linoleate. The studied PAHs include 2-hydroxy-BA (2-OH-BA), 3-hydroxy-BA (3-OH-BA), 5-hydroxymethyl-BA (5- CH2OH-BA), 7-hydroxymethyl-BA (7-CH2OH-BA), 12-hydroxymethyl-BA (12-CH2OH-BA), 7-hydroxymethyl-12- methyl-BA (7-CH2OH-12-MBA), 5-formyl-BA (5-CHO-BA), BA 5,6-cis-dihydrodiol (BA 5,6-cis-diol), 1-hydroxy-3- methylcholanthene (1-OH-3-MC), 1-keto-3-methylcholanthene (1-keto-3-MC), and 3-MC 1,2-diol. The results indicate that upon photoirradiation by UVA at 7 and 21 J/cm2, respectively all these compounds induced lipid peroxidation and exhibited a relationship between the dose of the light and the level of lipid peroxidation induced. To determine whether or not photoirradiation of these compounds by UVA light produces ROS, an ESR spin-trap technique was employed to provide direct evidence. Photoirradiation of 3-keto-3-MC by UVA (at 389 nm) in the presence of 2,2,6,6-tetramethylpiperidine (TEMP), a specific probe for singlet oxygen, resulted in the formation of TEMPO, indicating that singlet oxygen was generated. These overall results suggest that UVA photoirradiation of oxygenated BA and 3-methylcholanthrene generates singlet oxygen, one of the reactive oxygen species (ROS), which induce lipid peroxidation.

Photoirradiation of retinyl palmitate in ethanol with ultraviolet light--formation of photodecomposition products, reactive oxygen species, and lipid peroxides.

We have previously reported that photoirradiation of retinyl palmitate (RP), a storage and ester form of vitamin A (retinol), with UVA light resulted in the formation of photodecomposition products, generation of reactive oxygen species, and induction of lipid peroxidation. In this paper, we report our results following the photoirradiation of RP in ethanol by an UV lamp with approximately equal UVA and UVB light. The photodecomposition products were separated by reversed-phase HPLC and characterized spectroscopically by comparison with authentic standards. The identified products include: 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), and trans- and cis-15-ethoxy-AR. Photoirradiation of RP in the presence of a lipid, methyl linoleate, resulted in induction of lipid peroxidation. Lipid peroxidation was inhibited when sodium azide was present during photoirradiation which suggests free radicals were formed. Our results demonstrate that, similar to irradiation with UVA light, RP can act as a photosensitizer leading to free radical formation and induction of lipid peroxidation following irradiation with UVB light.

UVA photoirradiation of halogenated-polycyclic aromatic hydrocarbons leading to induction of lipid peroxidation.

Since the finding in the 1930s, a large number of polycyclic aromatic hydrocarbons (PAHs) of different structures have been tested for potential tumorigenicity. Structure-activity relationships of halo-PAHs have been investigated to determine the regions of a PAH that may be involved in cancer initiation. From these studies, a number of halo-PAHs were found to be tumorigenic in experimental animals. It was not until the 1980s that halo- PAHs were found to be present in the environment, including municipal incinerator fly ash, urban air, coal combustion, soil, snow, automobile exhausts, and tap water. Due to their widespread presence in the environment and their genotoxic activities, including carcinogenicity, many of these compounds may pose a health risk to humans. Although the biological activities, including metabolism, mutagenicity, and carcinogenicity, of halo- PAHs have been studied their phototoxicity and photo-induced biological activity have not been well examined. In this study, we study the photoirradiation of a series of structure-related halo-PAHs by UVA light in the presence of a lipid, methyl linoleate, and determine as to whether or not these compounds can induce lipid peroxidation. The halo-PAHs chosen for study include 2-bromonaphthalene, 1-chloroanthracene, 9,10- dibromoanthracene, 9-chlorophenanthrene, 9-bromophenanthrene, 7-chlorobenz[a]anthracene, 7- bromobenz[a]anthracene, 7-bromo-5-methylbenz[a]anthracene, 6-chlorobenzo[a]pyrene, and 6- bromobenzo[a]pyrene. The results indicate that upon photoirradiation by UVA all these compounds induced lipid peroxidation at different levels. These results suggest that halo-PAHs may be harmful to human health.

Effect of propofol on in vitro lipid peroxidation induced by different free radical generating systems: a comparison with vitamin E.

Propofol has been reported to have antioxidant properties and to inhibit lipid peroxidation. In this study, we examined the ability of propofol to inhibit lipid peroxidation induced by three free radical systems (hydroxyl, ferryl, and oxo-ferryl radicals), and we compared the effect of propofol with that of vitamin E, an endogenous antioxidant. Lipid peroxidation was induced by exposing a linoleic acid emulsion to either water gamma radiation, a ferrous iron-ascorbate solution, or human hemoglobin, generating the hydroxyl, ferryl, and oxo-ferryl radicals, respectively. Each experiment was performed in triplicate with and without propofol or vitamin E at concentrations between 10(-5) and 10(-4) M. Lipid peroxidation was quantified by gas chromatography measurement of the pentane released (nmoles) from lipid decomposition. In each condition, a significant dose-response relationship was found between the release of pentane and the concentration of either propofol or vitamin E. The antioxidant activities of both agents were similar but significantly higher against the hydroxyl than the ferryl and oxo-ferryl radicals. The study suggests that propofol could be beneficial as an anesthetic or sedative drug in patients presenting pathologies associated with free radical reactions.

UV-initiated autoxidation of methyl linoleate in micelles studied by optical absorption.

The UV-induced dissociation of lipid hydroperoxides, naturally present in methyl linoleate and methyl linoelaidate, was monitored by absorption spectroscopy. The decay of hydroperoxides in homogeneous solution, for a fluence rate of 0.82 mW.cm-2 at 240 nm, followed an exponential course with an average rate constant of k = (2.0 +/- 0.1) . 10(-3) s-1. Addition of alpha-tocopherol led to an increase in the magnitude of k until it reached a limiting value of (3.4 +/- 0.2) . 10(-3)s-1. When methyl linoleate (containing adventitous traces of hydroperoxides) was incorporated in SDS micelles, sustained UV irradiation caused initially an increase in the amounts of lipid hydroperoxides, due to linoleate autoxidation, followed by an exponential decrease due to photodissociation. From the initial rate of hydroperoxide formation (due to linoleate autoxidation) and the rate constant of the subsequent hydroperoxide decay, an oxidizibility of 0.61 . 10(-2) (Ms)-1/2 of methyl linoleate in micelles was deduced. It is suggested that UV irradiation and optical absorption afford an easy and reliable means for studying autoxidation of polyunsaturated fatty acids.

Lipid peroxidation in human milk and infant formula: effect of storage, tube feeding and exposure to phototherapy.

Preformed lipid peroxidation products present in the feed may contribute to the total reactive oxygen radical load infants have to deal with and may play a role in the pathogenesis of necrotizing enterocolitis and bronchopulmonary dysplasia. In this study, the occurrence of lipid peroxidation in human milk and feeding formulas for preterm babies was evaluated in vitro. Free linoleic acid (18:2) and its hydroperoxide (18:2OOH) were measured by gas chromatography-mass spectrometry and the concentration of 18:2OOH and the 18:2OOH/18:2 ratio were used as indices of peroxidation. In all feeds peroxidation products were present, but the proportion of peroxidized 18:2 was greater in infant formula. Storage of human milk (+4 degrees C for four days) increased lipid peroxidation. Exposure to light during tube feeding increased peroxidation in infant formula but not in human milk. Different procedures for preparation, storage and feeding may decrease the concentration of these potentially toxic peroxidized lipids in human milk and infant formula.

Gas chromatographic analysis of reactive carbonyl compounds formed from lipids upon UV-irradiation.

Peroxidation of lipids produces carbonyl compounds; some of these, e.g., malonaldehyde and 4-hydroxynonenal, are genotoxic because of their reactivity with biological nucleophiles. Analysis of the reactive carbonyl compounds is often difficult. The methylhydrazine method developed for malonaldehyde analysis was applied to simultaneously measure the products formed from linoleic acid, linolenic acid, arachidonic acid, and squalene upon ultraviolet-irradiation (UV-irradiation). The photoreaction products, saturated monocarbonyl, alpha,beta-unsaturated carbonyls, and beta-dicarbonyls, were derivatized with methylhydrazine to give hydrazones, pyrazolines, and pyrazoles, respectively. The derivatives were analyzed by gas chromatography and gas chromatography-mass spectrometry. Lipid peroxidation products identified included formaldehyde, acetaldehyde, acrolein, malonaldehyde, n-hexanal, and 4-hydroxy-2-nonenal. Malonaldehyde levels formed upon 4 hr of irradiation were 0.06 micrograms/mg from squalene, 2.4 micrograms/mg from linolenic acid, and 5.7 micrograms/mg from arachidonic acid. Significant levels of acrolein (2.5 micrograms/mg) and 4-hydroxy-2-nonenal (0.17 micrograms/mg) were also produced from arachidonic acid upon 4 hr irradiation.

[Oxidative degradation of linoleic acid methyl ester in suspensions of inorganic excipients. 1. Auto-oxidation in the presence of silicic acid products and aluminum oxide]. / Oxidativer Abbau von Linolsäuremethylester in Suspensionen anorganischer Hilfsstoffe. Teil 1: Autoxidation in Gegenwart von Kieselsäureprodukten und Aluminiumoxid.

Linoleic acid methyl ester (LME) was selected as a model to study the autoxidation of unsaturated compounds at the surfaces of inorganic excipients. From IR spectra of LME-silica adsorbates it was concluded that the interaction between LME and the silica surface is mainly due to hydrogen bonds, established between the ester carbonyl and the silica silanol groups. The autoxidation was continuously monitored by measuring the oxygen consumption of LME under UV light exposure and isobaric conditions, and, in parallel, by determining the carbonyl and peroxide values of LME. The oxidative degradation of LME is enhanced by porous and colloidal silicas as well as by colloidal aluminium oxide. From the oxygen consumption and the ratio of intermediates (carbonyls, peroxides) formed during the degradation of LME it was concluded that the prooxidative effect of the inorganic excipients can be attributed to an accelerated reaction of the peroxides of LME.

Identification of a diene conjugated component of human lipid as octadeca-9,11-dienoic acid.

The predominant diene conjugated acyl residue in triacylglycerols, cholesteryl esters and phospholipids in human serum was identified by high performance liquid chromatography and capillary gas chromatography-mass spectrometry. It is an octadeca -9,11-dienoic acid.

Comparison of the radiosensitivity of unsaturated fatty acids, structured as micelles or liposomes, under different experimental conditions.

A comparison has been made between the peroxidation rate as a result of ionizing radiation in liposomes prepared from phospholipids which were extracted from biological membranes, in single component micelles and in micelles of mixed composition. The ease of fatty acid oxidation in the different preparations was studied at a variety of pH values. The damage has been quantified spectrophotometrically in terms of diene conjugation (233 nm) and as the disappearance of fatty acids by gas chromatography. The ease of fatty acid oxidation was in the following order for the liposomal and mixed micelle preparations: 22:6 greater than 20:4 greater than 18:2. For single component micelles the order was reversed: 18:2 greater than 18:3 greater than 20:4 greater than 22:6. The micellar lipid preparations were pH-dependent in their response to radiation, which was demonstrated by a dip in the pH-response curve. Peroxidation of especially 22:6 was promoted when present in mixed micelles with 18:2.

[Free radicals formed during UV-irradiation of unsaturated fatty acids containing peroxidation products]. / Svobodnye radikaly, obrazuiushchiesia pri UF-obluchenii nenasyshchennykh zhirnykh kislot, soderzhashchikh produkty perekisnogo okisleniia.

On the irradiation of oxidized linolic acid an assymetric ESR signal with g-factor 2.0039 and the linewidth 30--33 Oe has been registered. The signal could be the result of photolysis of hydroperoxides. During prolonged irradiation of all the samples accumulation of alkyl radicals characterized by a symmetric signal with a well-defined ultrafine structure has been observed. An increase of the sample temperature up to 87 K in the presence of oxygen was preceded by transformation of alkyl radical R into RO2. All the radicals disappeared at the temperature of 160 K. All these processes seem to be the first phases of photoperoxidation of unsaturated fat acids in lipid-containing systems, including biological membranes.

Promotion of radiation peroxidation in models of lipid membranes by caesium and rubidium counter-ions: micellar linoleic and linolenic acids.

Caesium and rubidium counter-ions increase peroxidation in irradiated micelles of linoleic (18 : 2) and linolenic (18 : 3) acids. The effect is specific to Cs+ and Rb+ in the alkali metal series. The effect is independent of the salts used (Cl-, NO3-, ClO4-) and, therefore, independent of the chaotropic nature, and reactivity with hydroxyl radicals of Cl-, NO3- and ClO4-. The promotion of peroxidation by Cs+ and Rb+ is interpreted in terms of their effect on fatty acid micelle structure. The dependence of radiation peroxidation on lipid structure in the micelles may be significant for studies of peroxidation in highly structured cell membranes.

[Development of volatile compounds from radicals arising during intermediate steps of the lipoxygenase-reaction (author's transl)]. / Untersuchungen über die Entstehung flüchtiger Verbindungen aus radikalischen Reaktionszwischenstufen bei der Lipoxygenase-Reaktion.

The development of volatile compounds arising from radicals during intermediate reaction steps of the lipoxygenase-linoleic-acid-reaction (from soy) was investigated in model experiments with defined conditions. The results indicate as follows: Among the volatile compounds formed, hexanal has a special position. Its development is closely connected to the enzymatic formation of the 13-linoleic-acid-hydroperoxide because it is formed from the 13-linoleic-acid-peroxy-radical, which is a direct precursor of the 13-linoleic-acid-hydroperoxide. The other volatile products are apparently formed in the same way as by autoxidation. Their development is favoured by lack of oxygen, when the primarily formed linoleic-acid-radicals cannot react rapidly with the oxygen-radicals to form hydroperoxides. A small part of the volatile compounds is formed by autoxidation which always accompanies the enzymatic reaction.