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.

Synthesis and photoirradiation of isomeric ethylchrysenes by UVA light leading to lipid peroxidation.

Polycyclic aromatic hydrocarbons (PAHs) are widespread genotoxic environmental pollutants. We have recently demonstrated that photoirradiation of PAHs leads to cytotoxicity, DNA damage, and induction of lipid peroxidation. In this paper we report the synthesis of all the six isomeric ethylchrysenes and the study of light-induced lipid peroxidation by these ethylchrysenes. 5-Ethylchrysene was synthesized by reaction of 5-keto-5,6,6a,7,8,9,10,10a-octahydrochrysene with CH3CH2MgBr followed by dehydration catalyzed by p-toluenesulfonic acid and dehydrogenation with DDQ in benzene. 1- and 4-Ethylchrysenes were similarly prepared by reaction of 1-keto-1,2,3,4,5,6-hexahydrochrysene and 4-keto-1,2,3,4-tetrahydrochrysenes, respectively with CH3CH2MgBr followed by dehydration and dehydrogenation. Direct acetylation of chrysene followed by Wolff-Kishner or Clemmensen reduction resulted in the formation of 2-, 3-, and 6-ethylchrysenes in 4%, 16%, and 43% yields, respectively. Photoirradiation of these compounds with 7 and 21 J/cm2 UVA light in the presence of methyl linoleate all resulted in lipid peroxidation. For comparison, photoirradiation of 4-methylchrysene and 5-methylchrysene was similarly conducted. For irradiation at a UVA light dose of 21 J/cm2, the level of induced lipid peroxidation is in the order 4-methylchrysene = 5-methylchrysene = 5-ethylchrysene = 4-ethylchrysene = chrysene > 1-ethylchrysene = 2-ethylchrysene > 3-ethylchrysene > 6-ethylchrysene. Compared with chrysene, these results indicate that the ethyl group at C4 or C5 position either slightly enhances or has no effect on the light-induced lipid peroxidation, while at C1-, C2-, C3-, or C6 position reduces light-induced lipid peroxidation.

Photodecomposition of vitamin A and photobiological implications for the skin.

Vitamin A (retinol), an essential human nutrient, plays an important role in cellular differentiation, regulation of epidermal cell growth and normal cell maintenance. In addition to these physiological roles, vitamin A has a rich photochemistry. Photoisomerization of vitamin A, involved in signal transduction for vision, has been extensively investigated. The biological effects of light-induced degradation of vitamin A and formation of reactive species are less understood and may be important for light-exposed tissues, such as the skin. Photochemical studies have demonstrated that excitation of retinol or its esters with UV light generates a number of reactive species including singlet oxygen and superoxide radical anion. These reactive oxygen species have been shown to damage a number of cellular targets, including lipids and DNA. Consistent with the potential for damaging DNA, retinyl palmitate has been shown to be photomutagenic in an in vitro test system. The results of mechanistic studies were consistent with mutagenesis through oxidative damage. Vitamin A in the skin resides in a complex environment that in many ways is very different from the chemical environment in solution and in in vitro test systems. Relevant clinical studies or studies in animal models are therefore needed to establish whether the pro-oxidant activity of photoexcited vitamin A is observed in vivo, and to assess the related risks.

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 retinyl palmitate--formation of singlet oxygen and superoxide, and their role in induction of lipid peroxidation.

We have previously reported that photoirradiation of retinyl palmitate (RP) in ethanol with UVA light results in the formation of photodecomposition products, including 5,6-epoxy-RP and anhydroretinol (AR). Photoirradiation in the presence of a lipid, methyl linoleate, induced lipid peroxidation, suggesting that reactive oxygen species (ROS) are formed. In the present study, we employ an electron spin resonance (ESR) spin trap technique to provide direct evidence as to whether or not photoirradiation of RP by UVA light produces ROS. Photoirradiation of RP by UVA 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. Both 5,5-dimethyl N-oxide pyrroline (DMPO) and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) are specific probes for superoxide. When photoirradiation of RP was conducted in the presence of the DMPO or BMPO, ESR signals for DMPO-*OOH or BMPO-*OOH were obtained. These results unambiguously confirmed the formation of superoxide radical anion. Consistent with a free radical mechanism, there was a near complete and time-dependent photodecomposition of RP and its photodecomposition products. ESR studies on the photoirradiation of 5,6-epoxy-RP and AR indicate that these compounds exhibit similar photosensitizing activities as RP under UVA light.

Photo-induced DNA damage and photocytotoxicity of retinyl palmitate and its photodecomposition products.

Retinyl palmitate (RP) is an ester of retinol (vitamin A) and the predominant form of retinol found endogenously in the skin. We have previously reported that photoirradiation of RP with UVA light resulted in the formation of anhydroretinol (AR), 5,6-epoxyretinyl palmitate (5,6-epoxy-RP) and other photodecomposition products. While AR was formed through an ionic photodissociation mechanism, 5,6-epoxy-RP was formed through a light-mediated, free radical-initiated chain reaction. In the current study, the phototoxicity of RP, AR and 5,6-epoxy-RP in human skin Jurkat T-cells with and without light irradiation was determined using a fluorescein diacetate assay. Under similar conditions, the Comet assay was used to assess damage to cellular DNA. Nuclear DNA was not significantly damaged when the cells were irradiated by UVA plus visible light in the absence of a retinoid; however, when the cells were illuminated with UVA plus visible light in the presence of either RP, 5,6-epoxy-RP or AR (50, 100, 150 and 200 microM), DNA fragmentation was observed. Cell death was observed for retinoid concentrations of 100 microM or higher. When treated with 150 microM of RP, 5,6-epoxy-RP or AR, cell death was 52, 33 and 52%, respectively. These results suggest that RP and its two photodecomposition products, AR and 5,6-epoxy-RP, induce DNA damage and cytotoxicity when irradiated with UVA plus visible light. We also determined that photoirradiation of RP, AR and 5,6-epoxy-RP causes single strand breaks in supercoiled phi chi 174 plasmid DNA. Using a constant dose of UVA light (50 J/cm2), the level of DNA cleavage was highest in the presence of AR, followed by 5,6-epoxy-RP, then RP. The induced DNA strand cleavage was inhibited by NaN3. These results suggest that photoirradiation of RP, 5,6-epoxy-RP and AR with UVA light generates free radicals that initiate DNA strand cleavage.

Photodecomposition of retinyl palmitate in ethanol by UVA light-formation of photodecomposition products, reactive oxygen species, and lipid peroxides.

Photodecomposition of retinyl palmitate (RP), an ester and the storage form of vitamin A (retinol), in ethanol under UVA light irradiation was studied. The resulting photodecomposition products were separated by reversed-phase HPLC and identified by spectral analysis and comparison with the chromatographic and spectral properties of synthetically prepared standards. The identified products include 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), palmitic acid, ethyl palmitate, and four tentatively assigned cis and trans isomeric 15-ethoxy-ARs. AR was formed as a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Z-cis-AR with all-trans-AR predominating. 5,6-Epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP were also formed from reaction of RP with alkylperoxy radicals generated by thermal decomposition of 2,2'-azobis(2,4-dimethylvaleronitrile). Formation of these photodecomposition products was inhibited in the presence of sodium azide (NaN3), a free radical inhibitor. These results suggest that formation of 5,6-epoxy-RP, 4-keto-RP, 11-ethoxy-12-hydroxy-RP, and 13-ethoxy-14-hydroxy-RP from photoirradiation of RP is mediated by a light-initiated free radical chain reaction. AR and the isomeric 11-ethoxy-ARs were not formed from reaction of RP with alkylperoxy radicals generated from 2,2'-azobis(2,4-dimethylvaleronitrile), and their formation was not inhibited when NaN3 was present during the photoirradiation of RP. We propose that these products were formed through an ionic photodissociation mechanism, which is similar to the reported formation of AR through ionic photodissociation of retinyl acetate. RP and all its identified photodecomposition products described above (i) were not mutagenic in Salmonella typhimurium tester strains TA98, TA100, TA102, and TA104 in the presence and absence of S9 activation enzymes, (ii) were not photomutagenic in Salmonella typhimurium TA102 upon UVA irradiation, and (iii) did not bind with calf thymus DNA in the presence of microsomal metabolizing enzymes. These results suggest that RP and its decomposition products are not genotoxic; however, photoirradiation of RP, 5,6-epoxy-RP, and AR with UVA light in the presence of methyl linoleate resulted in lipid peroxide (methyl linoleate hydroperoxides) formation. The lipid peroxide formation was inhibited by dithiothreitol (DTT) (free radical scavenger), NaN3 (singlet oxygen and free radical scavenger), and superoxide dismutase (SOD) (superoxide scavenger) but was enhanced by the presence of deuterium oxide (D2O) (enhancement of singlet oxygen lifetime). These results suggest that photoirradiation of RP, 5,6-epoxy-RP, and AR by UVA light generated reactive oxygen species resulting in lipid (methyl linoleate) peroxidation.