Long‐term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes

Photoprotection is essential to prevent the long‐term deleterious effects of ultraviolet (UV ), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE ) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA ‐induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA ‐irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE ‐treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.     

N-acetyl-L-cysteine prevents DNA damage induced by UVA, UVB and visible radiation in human fibroblasts

The thiol N-acetyl-L-cysteine (NAC) is a source of cysteine for the synthesis of the endogenous antioxidant glutathione (GSH) which is depleted by ultraviolet radiation. It is also associated with the scavenging of reactive oxygen species (ROS). In this study the effects of NAC were examined in cultured human fibroblasts during prolonged exposure to ultraviolet B (UVB), ultraviolet A (UVA) and visible irradiation (280-700 nm), delivered by a 150 W xenon-arc lamp. The alkaline comet assay was used to assess the DNA damage in individual cells. It was found that incubating skin and lung fibroblasts at 37 degrees C for 1 h with an optimal 6 mM NAC supplement prior to light exposure, significantly reduced the level of DNA damage in both cell types, however, the skin fibroblasts were less sensitive to xenon-arc lamp irradiation than lung fibroblasts. NAC incubation resulted in an initial delay in DNA damage when the cells were irradiated. There was also a significant reduction in the overall levels of DNA damage observed with continued irradiation. NAC significantly reduced the DNA damage produced in lung fibroblasts depleted of normal GSH protection by the glutamylcysteinyl synthetase inhibitor, L-buthionine-[S,R]-sulfoximine. Although the specific mechanism of NAC protection has not yet been elucidated, these results support the hypothesis that NAC may protect the cells directly, by scavenging ROS induced by UVA and visible radiation, and indirectly by donating cysteine for GSH synthesis.     

Kinetics and mechanism of the addition of nucleophiles to alpha,beta-unsaturated thiol esters

Compounds containing the UV-absorbing chromophores p-methoxycinnamate, p-methoxycinnamide, or anthranilate and an alpha,beta- or alpha,beta,gamma,delta-unsaturated thiol ester (crotonyl or sorboyl) have been prepared. These compounds are subject to nucleophilic attack at the C=C conjugated to the thiol ester carbonyl group. The kinetics of the reactions of these thiol esters with N-acetyl-l-cysteine (NAC), N-acetylcysteamine, and N(2)-acetyl-L-lysine (NAL) have been studied, and the thiol addition products have been identified. The reaction rates increased at higher pH, and the reaction of NAC thiolate with a crotonyl thiol ester in 1:1 (v/v) acetonitrile/aqueous HEPES exhibited buffer catalysis as a result of protonation of the enolate intermediate. At the same concentration, NAC underwent approximately 300-fold more reaction than NAL with a crotonyl thiol ester at pH 9.8. Additionally, a crotonyl thiol ester was found to be 7.9 times more reactive than a sorboyl thiol ester toward NAC addition. These unsaturated thiol esters may serve as a means of covalently binding UVA and UVB sunscreens to the outer layer of skin to provide long-lasting protection.     

Quantification of Thiopurine/UVA-Induced Singlet Oxygen Production

Thiopurines were examined for their ability to produce singlet oxygen ((1)O(2)) with UVA light. The target compounds were three thiopurine prodrugs, azathioprine (Aza), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), and their S-methylated derivatives of 6-methylmercaptopurine (me6-MP) and 6-methylthioguanine (me6-TG). Our results showed that these thiopurines were efficient (1)O(2) sensitizers under UVA irradiation but rapidly lost their photoactivities for (1)O(2) production over time by a self-sensitized photooxidation of sulfur atoms in the presence of oxygen and UVA light. The initial quantum yields of (1)O(2) production were determined to be in the range of 0.30-0.6 in aqueous solutions. Substitution of a hydrogen atom with a nitroimidazole or methyl group at S decreased the efficacy of photosensitized (1)O(2) production as found for Aza, me6-MP and me6-TG. (1)O(2)-induced formation of 8-oxo-7,8-dihydro-2'-dexyguanosine (8-oxodGuo) was assessed by incubation of 6-methylthiopurine/UVA-treated calf thymus DNA with human repair enzyme 8-oxodGuo DNA glycosylase (hOGG1), followed by apurinic (AP) site determination. Because more 8-oxodGuo was formed in Tris D(2)O than in Tris H(2)O, (1)O(2) is implicated as a key species in the reaction. These findings provided quantitative information on the photosensitization efficacy of thiopurines and to some extent revealed the correlations between photoactivity and phototoxicity.     

Multiple forms of DNA damage caused by UVA photoactivation of DNA 6-thioguanine

Thiopurines are prescribed frequently as medication for cancer and for inflammatory disorders. One of them, azathioprine, has been the immunosuppressant of choice for organ transplant recipients for many years. Thiopurine use is associated with elevated sun sensitivity and skin cancer risk. Skin sensitization is selective for UVA. 6‐TG integrates into DNA and unlike the canonical DNA bases, it is a strong UVA chromophore with an absorbance maximum at 342 nm. DNA 6‐TG is a photosensitizer and a source of reactive oxygen species. Reactive oxygen that is generated from the photochemical activation of DNA 6‐TG causes extensive damage to DNA and proteins. This damage is mutagenic and extremely toxic to cultured human cells. Here we describe some of the lesions that are known to be generated from UVA irradiation of DNA 6‐TG. We discuss how this photochemical damage might contribute to the toxic effect of thiopurine/UVA treatment on cultured cells and to the high risk of skin cancer in thiopurine‐treated patients.     

The Endogenous Tryptophan‐derived Photoproduct 6‐formylindolo[3,2‐b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo

UV‐chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6‐formylindolo[3,2‐b]carbazole (FICZ), a tryptophan‐derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol^?1 cm^?1), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC‐25, HaCaT‐ras II‐4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm^?2) and FICZ (≥10 nm ), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg‐sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 “high‐risk” mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV‐induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.     

The Copper(II)‐Catalyzed Oxidation of Glutathione

The kinetics and mechanisms of the copper(II)‐catalyzed GSH (glutathione) oxidation are examined in the light of its biological importance and in the use of blood and/or saliva samples for GSH monitoring. The rates of the free thiol consumption were measured spectrophotometrically by reaction with DTNB (5,5′‐dithiobis‐(2‐nitrobenzoic acid)), showing that GSH is not auto‐oxidized by oxygen in the absence of a catalyst. In the presence of Cu²⁺, reactions with two timescales were observed. The first step (short timescale) involves the fast formation of a copper–glutathione complex by the cysteine thiol. The second step (longer timescale) is the overall oxidation of GSH to GSSG (glutathione disulfide) catalyzed by copper(II). When the initial concentrations of GSH are at least threefold in excess of Cu²⁺, the rate law is deduced to be ?d[thiol]/dt=k[copper–glutathione complex][O₂]^0.5[H₂O₂]^?0.5. The 0.5^th reaction order with respect to O₂ reveals a pre‐equilibrium prior to the rate‐determining step of the GSSG formation. In contrast to [Cu²⁺] and [O₂], the rate of the reactions decreases with increasing concentrations of GSH. This inverse relationship is proposed to be a result of the competing formation of an inactive form of the copper–glutathione complex (binding to glutamic and/or glycine moieties).     

Characterization of non-enzymatic acylation of amino or thiol groups of bionucleophiles by the acyl-adenylate or acyl-CoA thioester of cholic acid

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are highly electrophilic acyl-linked metabolites which can undergo transacylation reactions with amino and thiol groups of nucleophilic groups on acceptor molecules such as amino acids, peptides, and proteins. Here, non-enzymatic acylation at pH 7.4 of glycine, taurine, glutathione (GSH), and N-acetylcysteine (NAC) by cholyl-adenylate (CA-AMP) was compared with that mediated by cholyl-CoA thioester (CA-CoA) using a 1:1 mixture of stable isotopically labeled CA-AMP and unlabeled CA-CoA. The transacylation products of these substrates were analyzed by liquid chromatography/electrospray ionization linear ion-trap mass spectrometry in negative-ion detection mode. CA-AMP was more reactive than CA-CoA with the amino group of glycine or taurine than with the thiol group of GSH or NAC. In contrast, CA-CoA was more reactive than CA-AMP with the thiol group of GSH or NAC and was far less reactive with the amino group of glycine or taurine. These differences in the reactivity of CA-AMP as compared with that of CA-CoA towards amino and thiol groups may be attributed to the electrophilicity of the carbonyl carbon of these acyl-linked cholic acid metabolites and the nucleophilicity of the amino and thiol group in the bionucleophiles that were studied.     

A Mechanistic Study of the Effects of Antioxidants on the Formation of Malondialdehyde‐Like Products in the Reaction of Hydroxyl Radicals with Deoxyribose

The reactions of ^.OH radicals with deoxyribose, DR, form five different DR^. radicals, only one of which is transformed into malondialdehyde (MDA)‐like products. The radiolytic yield of the MDA‐like products increases with the increase in the DR concentration indicating that some of the initially formed “unproductive” radicals react with DR to form the “productive” radicals. The yield of the MDA‐like products also increases with the dose rate delivered to the solution suggesting that the formation of the MDA‐like products involves the reaction of the “productive” radicals with a radical. The addition of ascorbate, AH^?, to the solution decreases the yield of the MDA‐like products as expected from the relative rates of the reaction of DR and AH^? with ^.OH radicals. On the other hand the addition of the exogenous thiol, N‐acetylcysteine (NAC), to the solutions decreases the yield of the MDA‐like products considerably more than expected from the rate constants of the reaction with ^.OH radicals. The addition of the endogenous thiol, glutathione (GSH), to the solutions affects the yield of the MDA‐like products at low concentration less than expected and at “high” concentrations more than expected from the rate constant of the reaction. Addition of low concentration of AH^? to solutions containing GSH increases considerably its antioxidant activity whereas addition of small concentrations of AH^? to solutions containing NAC has no effect on its antioxidant activity. The results point out that the DR^. radicals react differently with NAC and GSH and that the GS^. and NAC^. radicals react differently with DR, the GS^. radical being considerably more active than the NAC^. radical. Thus it has to be concluded that the relative activity of antioxidants depends also on the rate constants of many secondary reactions and on the concentrations of all the solutes present in the system.     

Synthesis and Structure–Activity Correlation Studies of Secondary‐ and Tertiary‐Amine‐Based Glutathione Peroxidase Mimics

In this study, a series of secondary‐ and tertiary‐amino‐substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H₂O₂, cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert‐amino groups in benzylamine‐based diselenides by sec‐amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N‐propyl‐ and N‐isopropylamino‐substituted diselenides are 8–18 times more active than the corresponding N,N‐dipropyl‐ and N,N‐diisopropylamine‐based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec‐amino‐substituted diselenides is similar to that of the tert‐amine‐based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx‐like activities. It is observed that the sec‐amino groups are better than the tert‐amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol‐exchange reactions in the selenenyl sulfides derived from sec‐amine‐based diselenides. Furthermore, the seleninic acids (RSeO₂H) derived from the sec‐amine‐based compounds are more stable toward further reactions with peroxides than their tert‐amine‐based analogues.     

The B6‐vitamer Pyridoxal is a Sensitizer of UVA‐induced Genotoxic Stress in Human Primary Keratinocytes and Reconstructed Epidermis

UVA‐driven photooxidative stress in human skin may originate from excitation of specific endogenous chromophores acting as photosensitizers. Previously, we have demonstrated that 3‐hydroxypyridine‐derived chromophores including B₆‐vitamers (pyridoxine, pyridoxamine and pyridoxal) are endogenous photosensitizers that enhance UVA‐induced photooxidative stress in human skin cells. Here, we report that the B₆‐vitamer pyridoxal is a sensitizer of genotoxic stress in human adult primary keratinocytes (HEKa) and reconstructed epidermis. Comparative array analysis indicated that exposure to the combined action of pyridoxal and UVA caused upregulation of heat shock (HSPA6, HSPA1A, HSPA1L, HSPA2), redox (GSTM3, EGR1, MT2A, HMOX1, SOD1) and genotoxic (GADD45A, DDIT3, CDKN1A) stress response gene expression. Together with potentiation of UVA‐induced photooxidative stress and glutathione depletion, induction of HEKa cell death occurred only in response to the combined action of pyridoxal and UVA. In addition to activational phosphorylation indicative of genotoxic stress [p53 (Ser15) and γ‐H2AX (Ser139)], comet analysis indicated the formation of Fpg‐sensitive oxidative DNA lesions, observable only after combined exposure to pyridoxal and UVA. In human reconstructed epidermis, pyridoxal preincubation followed by UVA exposure caused genomic oxidative base damage, procaspase 3 cleavage and TUNEL positivity, consistent with UVA‐driven photooxidative damage that may be relevant to human skin exposed to high concentrations of B₆‐vitamers.     

UVA II Exposure of Human Skin Results in Decreased Immunization Capacity, Increased Induction of Tolerance and a Unique Pattern of Epidermal Antigen-Presenting Cell Alteration

Abstract— The risks incurred from increased exposure to UVA II (320-340 nm) (i.e. during sunscreen use and extended outdoor exposure, tanning parlors) are not well understood. Therefore, we explored the effects of UVA II on skin immune responses in humans. After a single local exposure (4 minimum erythemal dose [MED]) using a xenon arc lamp filtered with a narrow bandpass filter (335 ± 5 nm full width at half maximum), individuals were contact-sensitized with dinitrochlorobenzene (DNCB) through a UVA II exposure site or through normal skin. UVA II induced a marked decrease in the magnitude of skin immune responses (P < 0.0001). The UVA II group had only 29% successful sensitizations, as compared to 83% in the control group. The percentage of individuals who remained tolerant to DNCB after two sensitizations was 23.6% for the UVA II-exposed group, as compared to 3.8% in the controls (P= 0.006). UVA II also uniquely altered the type of antigen-presenting cells present in the epidermis. Human leukocyte antigen (HLA)-DR+ cells in control epidermal cell suspensions (C-EC) comprised a single, homogeneous population of Langerhans cells (LC) with the phenotype: CD1a^hi DR^mid CD11b^? CD36^? (1.5 ± 0.3% of EC). UVA II irradiation reduced the number of such LC to 0.6 ± 0.2% of EC. Although cells expressing the macrophage phenotype: CD1a DR^hi CD11b+ CD36+ were increased in UVA II skin, relative to C-EC, these comprised only 10.1 ± 6.1% of the DR+ cells, which is less than that after UVB exposure. Also distinct from UVB, a third population was found in UVA II-EC, which exhibited a novel phenotype: CD1a+ DR+ CD36+ CDllb+; these comprised 11.1 ± 6.9% of the DR+ UVA II-EC. In conclusion, despite the above differences in infiltrating DR cells, both UVB and UVA II reduce the skin's ability to support contact sensitization, induce active suppression (tolerance) and induce a reduction in LC.     

Antiviral properties of photosensitizers

Abstract— We have studied the antiviral properties of three different groups of photo-sensitizers, viz. (i) various furyl compounds; (ii) β-carboline alkaloids; (iii) thiophenes and their acetylene derivatives. In general the antiviral potency of the furyl compounds correlated with their ability to produce DNA photoadducts. Among the naturally occurring β-carboline alkaloids, harmine was considerably more potent (in the presence of long wavelength UV radiation, UVA) than several other harmane-related compounds. Slight alterations in chemical structure had profound effects on their antiviral activities. Harmine was shown to inactivate the DNA-virus murine cytomegalovirus (MCMV) by inhibiting viral gene expression, although other targets may also exist. Several eudistomins, carboline derivatives isolated from a tunicate, were also photoactive against viruses. Various plant thiophenes and polyacetylenes were studied in detail. These compounds also required UVA for antiviral activity, and some of them were extremely potent against viruses with membranes, e.g. α-terthienyl, which showed significant activity at only 10^-5μg/ml. When MCMV had been treated with α-terthienyl plus UVA, the virus retained its integrity and penetrated cells normally; but the virus did not replicate. More than 30 additional thiophenes have recently been evaluated, including many synthetic ones, and some of these are even more potent than a-terthienyl. We believe that certain thiophenes possess potential therapeutic value and should be tested against model virus infections in animals.     

Diazido Mixed‐Amine Platinum(IV) Anticancer Complexes Activatable by Visible‐Light Form Novel DNA Adducts

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side‐effects. Photoactivatable Pt^IV prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X‐ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Py)] ( 1 ; MA=methylamine, Py=pyridine) and trans,trans,trans‐[Pt(N₃)₂(OH)₂(MA)(Tz)] ( 2 ; Tz=thiazole), and interpret their photophysical properties by TD‐DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1 p and 1 q . Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin‐resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf‐thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono‐ and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross‐links, with evidence for DNA strand cross‐linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin‐type lesions. The photo‐induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt^II compounds trans‐[PtCl₂(MA)(Py)] ( 5 ) and trans‐[PtCl₂(MA)(Tz)] ( 6 ). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1 , whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.     

Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells against UVA Light

This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L‐buthionine‐(S,R)‐sulfoximine (BSO) or 1‐chloro‐2,4‐dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O₂, 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm^?2 of UVA radiation (338–400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O₂ compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA‐induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA‐induced cell damage.     

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells

The crucial role of DNA polymerase eta in protecting against sunlight‐induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP‐V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XP‐V cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP‐V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP‐V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and γH2AX signaling. Corroborating ATR participation in UVA‐DDR, a significant increase in Chk1 protein phosphorylation, as well as S‐phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N‐acetylcysteine (NAC), which significantly protects XP‐V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA‐induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells.     

UVA-Potentiated Damage to Calf Thymus DNA by Fenton Reaction System and Protection by para-Aminobenzoic Acid

Abstract— Calf thymus DNA was irradiated with low-intensity UVA (main output at 365 nm, 2 mW cm^?2 or 36 kj m ² for 30 min), and the role of metal ions, hydrogen peroxide and reactive oxygen species (ROS) was examined. DNA damage was measured as thiobarbituric acid-reactive substances (possibly from degradation of deoxyribose) and as changes in ethidium bromide-DNA fluorescence due to unwinding from strand breaks. Under the present experimental conditions, UVA alone or in the presence of H₂0₂ had no effect on DNA but slightly enhanced the damage by iron/EDTA. Ultraviolet A strongly enhanced DNA damage (ca four- to five-fold) by the Fenton reaction system (50 μM Fe²⁺/100 μM EDTA + 0.5 mM H₂0₂). The results suggest that the Fenton reaction system was “photosensitized” to damage DNA by low-intensity UVA radiation. The enhanced damage by UVA was attributed in part to the reduction of Fe³⁺ to Fe²⁺. Ultraviolet A had no effect when iron (ferric or ferrous) ions were replaced by Cu²⁺, Zn²⁺, Mn²⁺ or Cd²⁺. The ROS involved in the UVA-enhanced damage to DNA by the Fenton reagents were OH and, to a lesser extent, superoxide anions. The UVA-potentiated DNA damage by the Fenton reaction system was then used to examine the protective effect of para-aminobenzoate (PABA), a UVB-absorbing sunscreen that protects against photocarcinogenesis in hairless mice. The results show that PABA and mannitol dose-dependently inhibited the damage with concentrations required for 50% inhibition at 0.1 mM and 3 mM, respectively. The protection by PABA was attributed to its radical-scavenging ability because PABA does not absorb light in the UVA region. These findings may be relevant to the biological damage by UVA and suggest that PABA is useful in protection against photocarcinogenesis by wide-range UV radiation.     

Synthesis of Novel 3‐Aryl‐5‐dichloromethyl‐Δ2‐1,2,4‐oxadiazolines

The first synthetic approach to hitherto unknown 3‐aryl‐5‐dichloromethyl‐Δ²‐1,2,4‐oxadiazolines, of synthetic and biological interest, has been developed involving high‐yield reactions between N‐(2,2‐dichlorovinyl)benzimidoyl chlorides and hydroxylamine. The molecular structure of one member of this new family of compounds—5‐dichloromethyl‐3‐(4‐fluorophenyl)‐1,2,4‐oxadiazoline—has been determined by X‐ray crystallography. Density functional theory calculations supporting the proposed reaction pathway for the formation of these products have been carried out.