Blue light induced A2E oxidation in rat eyes--experimental animal model of dry AMD.

Previous studies have shown that short-wavelength blue visible light induces retinal injury and may be a risk factor for age related macular degeneration. A2E is a blue light absorbing retinal chromophore that accumulates with age. Our previous in vitro studies have determined that, although A2E itself has a low phototoxic efficiency, the oxidation products of A2E that are formed in the presence of visible light can contribute to observed retinal pigment epithelial photodamage. The purpose of this study was to investigate the effects of blue light on retinal phototoxicity and its relationship to A2E, oxidized A2E and its isomers. Sprague-Dawley albino rats were dark adapted for 24 h. Control rats remained in the dark while experimental rats were exposed to blue light (λ = 450 nm, 3.1 mW cm(-2)) for 6 h. Isolated retinas were homogenized in Folch extraction mixture and then in chloroform. The dried extracts were reconstituted and divided for determination of organic soluble compound. Esters of fatty acids were determined with GC-MS, A2E and other chromophores using HPLC, and A2E oxidation products with LC-MS. Exposure of rat eyes to blue light did not significantly change the fatty acid composition of the retina. The A2E concentration (normalized to fatty acid content) in blue light exposed animals was found to be lower than the A2E concentration in control rats. The concentrations of all-trans-retinal-ethanolamine adduct and iso-A2E a precursor and an isomer of A2E respectively, were also lower after blue-light exposure than in the retinas of rats housed in the dark. On the other hand, the amount of oxidized forms of A2E was higher in the animals exposed to blue light. We conclude that in the rat eye, blue-light exposure promotes oxidation of A2E and iso-A2E to the products that are toxic to retinal tissue. Although high concentrations of A2E may be cytotoxic to the retina, the phototoxicity associated with blue light damage to the retina is in part a result of the formation of toxic A2E oxides. This effect may partially explain the association between blue light induced retinal injury and macular degeneration.

Photochemical reactions involved in the phototoxicity of the anticonvulsant and antidepressant drug lamotrigine (Lamictal).

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (1O2) with a quantum yield of 0.22 in CH2Cl2, 0.11 in MeCN and 0.01 in D2O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak 1O2 quencher (k(q) = 3.2 x 10(5) M(-1) s(-1) in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched 1O2 very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated 1O2 twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of 1O2 prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.

Oxidation of flame retardant tetrabromobisphenol A by singlet oxygen.

Wide use of flame retardants can pose an environmental hazard, and it is of interest to investigate how they may degrade. We report here that 3,3',5,5'-tetrabromobisphenol A (TBBPA) is subject to photosensitized oxidation involving singlet molecular oxygen ((1)O2). By using visible light and rose bengal or methylene blue as 102 photosensitizers, we have found that TBBPA is a 102 quencher. The quenching rate constant, k(q), depends on TBBPA ionization (pK = 7.4). In acetonitrile, where TBBPA is undissociated, the kq value is 6.1 x 10(5) M(-1) s(-1) for a TBBPA monomer and decreases to 2.9 x 10(4) M(-1) s(-1) for TBBPA dimers and/or aggregates. TBBPA dissociates in aqueous solutions, and its kq value is 1.44 x 10(9) M(-1) s(-1) in alkaline solution, decreasing to 3.9 x 10(8) M(-1) s(-1) at pH 7.2. The strong 102 quenching by TBBPA anion initiates an efficient oxidation of TBBPA, which results in oxygen consumption in aqueous micellar (e.g., Triton X-100) solutions containing photosensitizer. This oxygen consumption is mediated by transient radical species, which we detected by using EPR spectroscopy. We observed two major radicals and one minor radical generated from TBBPA by reaction with 102 at pH 10. One was identified as the 2,6-dibromo-p-benzosemiquinone radical (a2H = 2.36 G, g = 2.0056). A second radical (aH = 2.10 G, g = 2.0055) could not be identified butwas probably a 2,6-dibromo-p-benzosemiquinone radical containing an EPR-silent substituent at the 3-position. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DPMO) showed that other minor radicals (hydroxyl, carbon-centered) are also generated during the reaction of TBBPA with (1)O2. The photosensitized production of radicals and oxygen consumption were completely inhibited by the azide anion, an efficient physical (1)O2 quencher. Because TBBPA is a stable compound that at neutral pH does not absorb much of the atmosphere-filtered solar radiation, its photosensitized oxidation by (1)O2 may be the key reaction initiating or mediating TBBPA degradation in the natural environment.

EPR studies of in vivo radical production by 3,3',5,5'-tetrabromobisphenol A (TBBPA) in the Sprague-Dawley rat.

Brominated flame retardants (BFRs) are present in many consumer products ranging from fabrics to plastics and electronics. Wide use of flame retardants can pose an environmental hazard and it is of interest to determine the mechanism of their toxicity. Of all the BFRs, 3,3',5,5'-tetrabromobisphenol A (TBBPA) is produced in the largest volume. Previous studies by Szymanska et al. (2000) have shown that TBBPA is hepatotoxic in rats. We report here that when TBBPA (100 or 600 mg/kg) dissolved in DMSO and alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) was administered ip to male Sprague-Dawley rats the POBN/CH(3) spin adduct was detected by electron paramagnetic resonance (EPR) in the bile. When (13)C-DMSO was employed the POBN/C(13)H(3) adduct was observed. Also present in the bile was the 2,6-dibromobenzosemiquinone radical derived from 2,6-dibromohydroquinone, a known metabolite of TBBPA. Reaction of the 2,6-dibromobenzosemiquinone radical with oxygen would generate superoxide from which hydrogen peroxide can form by dismutation. The hydroxyl radical generated via the Fenton reaction from hydrogen peroxide reacts in vivo with DMSO to give the methyl radical which is trapped by POBN. These observations suggest that the hepatotoxicity of TBBPA in rats may be due to the in vivo generation of the hydroxyl radical as a result of redox reactions involving the TBBPA metabolite 2,6-dibromohydroquinone and its corresponding semiquinone radical.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.). 2. Palmatine, hydrastine, canadine, and hydrastinine.

Goldenseal is an herb that is widely used in dietary supplements, eye washes, and skin lotions. The presence of Goldenseal root powder in dietary supplements and the topical application of Goldenseal preparations raise the possibility that an adverse phototoxic reaction may result from an interaction between its constituent alkaloids and light in exposed tissues. We have previously shown that berberine, the major alkaloid in Goldenseal powder, in combination with UVA causes DNA damage and cell death in HaCaT keratinocytes [(2001) Chem. Res. Toxicol. 14, 1529]. We have studied the photochemical and photobiological properties of four minor alkaloids found in Goldenseal, namely, hydrastine, palmatine, canadine, and hydrastinine. UVA radiation of palmatine in aqueous solutions generated no (1)O(2), but in CH(2)Cl(2), copious amounts of (1)O(2) were detected (Phi = 0.2). Palmatine also photogenerated oxygen-centered radicals, (*)OH and O(2)(*)(-) in aerated aqueous buffer and acetonitrile, respectively, as detected by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In nitrogen-sparged acetonitrile containing DMPO, we observed the neutral palmatine radical formed by one-electron reduction. UVA irradiation (4 J/cm(2)) of HaCaT keratinocytes in the presence of palmatine (50 microM) resulted in a 50% decrease in cell viability but no DNA damage as measured by the comet assay. UVA irradiation of hydrastine, hydrastinine, or canadine (50 microM) did not cause DNA damage or cell death in keratinocytes. Although palmatine is photoactive, it is present in such small amounts in Goldenseal root powder that the phototoxicity of the herb is most likely due to berberine, the major constituent alkaloid.

Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance.

Ultraviolet A (UVA, 315-400 nm), constituting about 95% of ultraviolet irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. It has proven difficult to show direct actions of UVA as a carcinogen in human cells. Here, we demonstrate that chronic UVA exposures at environmentally relevant doses in vitro can induce malignant transformation of human keratinocytes associated with acquired apoptotic resistance. As evidence of carcinogenic transformation, UVA-long-treated (24 J/cm(2) once/week for 18 weeks) HaCaT (ULTH) cells showed increased secretion of matrix metalloproteinase (MMP-9), overexpression of keratin 13, altered morphology and anchorage-independent growth. Malignant transformation was established by the production of aggressive squamous cell carcinomas after inoculation of ULTH cells into nude mice (NC(r)-nu). ULTH cells were resistant to apoptosis induced not only by UVA but also by UVB and arsenite, two other human skin carcinogens. ULTH cells also became resistant to apoptosis induced by etoposide, staurosporine and doxorubicin hydrochloride. Elevated phosphorylation of protein kinase B (PKB, also called AKT) and reduced expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were detected in ULTH cells. The resistance of ULTH cells to UVA-induced apoptosis was reversed by either inhibition of phosphatidylinositol 3-kinase (PI-3K) or adenovirus expression of PTEN or dominant negative AKT. These data indicate that UVA has carcinogenic potential in human keratinocytes and that the increased AKT signaling and decreased PTEN expression may contribute to this malignant transformation. Further comparisons between the transformed ULTH and control cells should lead to a better understanding of the mechanism of UVA carcinogenesis and may help identify biomarkers for UVA-induced skin malignancies.

Cleavage of epidermal growth factor receptor by caspase during apoptosis is independent of its internalization.

Epidermal growth factor receptor (EGFR) plays a critical role in cell proliferation, differentiation, and transformation. EGFR downregulation attenuates its signaling intensity and duration to maintain cellular homeostasis. Here, we report that during apoptosis EGFR is cleaved by activated caspase-3 or related proteases at its C-terminus domain. EGFR downregulation by activation of caspases is neither stimulus- nor cell type-specific. EGFR internalization during apoptosis required dynamin and cholesterol since dominant-negative dynamin (K44A) or cholesterol depletion by methyl-beta-cyclodextrin prevented EGFR internalization. However, EGFR downregulation did not require its internalization. The EGFR cleavage fragment was detected in the membrane blebs in addition to the cell pellets. Mutations at the consensus sequence (DXXD) at the C-terminus domain revealed that DVVD1012 and to a lesser extent DNPD1172 may be target sites for active recombinant caspase-3 in vitro and activated caspase-3 or related proteases in vivo. We have detected the N-terminus and C-terminus fragments in vitro and in vivo. A cleavage-deficient EGFR mutant delayed apoptosis process. We conclude that the evolutionarily conserved C-terminus domain of EGFR is the target of caspases and subjected to degradation during apoptosis to shut down its signaling.

Interaction of singlet molecular oxygen with melatonin and related indoles.

Singlet molecular oxygen (1O2) is one of the major agents responsible for (photo)oxidative damage in biological systems including human skin and eyes. It has been reported that the neural hormone melatonin (MLT) can abrogate 1O2-mediated cytotoxicity through its purported high antioxidant activity. We studied the interaction of MLT with 1O2 in deuterium oxide (D2O), acetonitrile and methanol by measuring the phosphorescence lifetime of 1O2 in the presence of MLT and related indoles for comparison. Rose bengal (RB) was used as the main 1O2 photosensitizer. The rate constant (kq) for the total (physical and chemical) quenching of 1O2 by MLT was determined to be 4.0 x 10(7) M(-1) s(-1) in D2O (pD 7), 6.0 x 10(7) M(-1) s(-1) in acetonitrile, and 6.1 x 10(7) M(-1) s(-1) in methanol-d1. The related indoles, tryptophan, 5-hydroxyindole, 5-methoxytryptamine, 5-hydroxytryptamine (5-OH-T, serotonin), 6-hydroxymelatonin (6-OH-MLT) and 6-chloromelatonin quenched 1O2 phosphorescence with similar kq values. We also compared the photosensitized photobleaching rate of MLT with that of other indoles, which revealed that MLT is the most sensitive to 1O2 bleaching. Hydroxylation of the indole moiety in 5-OH-T and 6-OH-MLT makes them more sensitive to photodegradation. In the absence of exogenous photosensitizers MLT itself can generate 1O2 with low quantum yield (0.1 in CH3CN) upon UV excitation. Thus, the processes we investigated may occur in the skin and eyes during physiological circadian rhythm (photo)signaling involving MLT and other indoles. Our results indicate that all the indoles studied, including MLT, are quite efficient yet very similar 1O2 quenchers. This directly shows that the exceptional antioxidant ability proposed for MLT is unsubstantiated when merely chemical mechanism(s) are considered in vivo, and it must predominantly involve humoral regulation that mobilizes other antioxidant defenses in living organisms.

Photochemical characterization of water samples from Minnesota and Vermont sites with malformed frogs: potential influence of photosensitization by singlet molecular oxygen (1O(2)) and free radicals on aquatic toxicity.

Environmental pollutants activated by UV sunlight may have contributed to the recent decline in frog populations and the concomitant increase in malformations in the USA and abroad. UV radiation is able to mutate DNA and to initiate photosensitization processes that generate mutagenic and biologically disruptive oxygen transients. We have examined water from selected sites in Minnesota and Vermont using singlet molecular oxygen (1O(2)), detected by its phosphorescence and free radicals detected by spin trapping, as markers for photosensitization. Water from a pond in Minnesota with malformed frogs, which also causes malformations in the laboratory, photosensitized more 1O(2), even though it absorbed less UV light compared to water from a site that did not cause malformations. This suggested that unknown natural or pollutant agents were present, and that photosensitization may be involved. Although UV irradiation of the two Minnesota water samples in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of the DMPO/*OH, DMPO/*H(e(aq)-) and DMPO/*C(unknown) adducts there were no qualitative or quantitative differences between them. We also examined water samples from several sites in Vermont, and compared them by measuring the quantum yield of 1O(2) photosensitization. While all the Vermont samples produced a small amount of 1O(2), there was no clear correlation with the incidence of frog malformations. However, the samples differed strongly in absorption spectra and the ability to quench 1O(2). These factors may determine how much UV light is absorbed and converted into chemical reactions. Our results show that photochemical characterization of 1O(2) photosensitization is possible in untreated natural water samples. Photosensitization falls into the category of global factors that may be closely associated with the effects of UV irradiation of the Earth's environments. Thus, photosensitization might be an important component in global amphibian malformation and decline. The observation of 1O(2) emission directly from natural water may also provide new opportunities to investigate the involvement of 1O(2) in other complex environmental processes.

Photocarcinogenesis in the Tg.AC mouse: lomefloxacin and 8-methoxypsoralen.

The Tg.AC mouse is a good predictor of carcinogenic potential when the test article is administered by dorsal painting (Tennant et al. (1995) Environ. Health Perspect. 103, 942). We have used lomefloxacin (LOME) and 8-methoxypsoralen (8-MOP) in combination with UVA to determine whether the Tg.AC transgenic mouse also responds to parenterally administered photocarcinogens. Female Tg.AC mice were given LOME (25 mg/kg intraperitoneal in normal saline) followed by UVA (25 J/cm2) 1-2 h later, five times every 2 weeks on a repetitive schedule. Other groups received LOME, UVA or vehicle alone. After 16 weeks, the mean numbers of papillomas/mouse +/- SD (% responding) were: saline, 0.3 +/- 0.5 (33%); UVA + saline, 1.3 +/- 0.6 (100%); LOME, 1.9 +/- 1.6 (86%) and LOME-UVA, 1.5 +/- 1.9 (64%). Only the 100% incidence of tumors in the UVA group and the maximum tumor yields in the LOME and UVA groups are significant (P < 0.05) when compared with the control. In a second study, Tg.AC mice were administered the classical photocarcinogen 8-MOP (8 mg/kg intragastric in corn oil) followed by 2 J/cm2 UVA 1-2 h later, five times every 2 weeks on a repetitive schedule. The second group received 8-MOP, whereas the third was exposed to UVA alone. Papillomas began to appear at 2 weeks in the 8-MOP-UVA group, and after 17 weeks the mean numbers of papillomas/mouse +/- SD (% responding) were: 8-MOP-UVA, 6.9 +/- 8.6 (93%); UVA + corn oil, 1.1 +/- 1.2 (69%) and 8-MOP, 1.1 +/- 1.6 (50%). The maximum tumor yield in the 8-MOP-UVA group was significantly higher (P < 0.01) than that in the other two groups. Our findings suggest that more studies need to be done before the Tg.AC mouse can be used with confidence to identify parenterally administered photocarcinogens.

Interaction of singlet molecular oxygen with double fluorescent and spin sensors.

Double fluorescent and spin sensors were recently used to detect transient oxidants via simultaneous fluorescence change and production of the nitroxide radical detected by electron paramagnetic resonance. One such oxidant, singlet molecular oxygen ((1)O(2)), was detected in thylakoid membrane using these probes. In the present study, we investigated the total (physical and chemical) quenching of (1)O(2) phosphorescence by sensors composed of the 2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole moiety attached to xanthene or dansyl fluorophores. We found that the quenching rate constants were in the range (2-7) x 10(7) M(-1)s(-1) in acetonitrile and D(2)O. Quenching of (1)O(2) is usually an additive process in which different functional groups may contribute. We estimated that the (1)O(2) quenching by the amine fragments was ca. one to two orders of magnitude lower than that for the complete molecules. Our data suggest that the incorporation of a fluorescent chromophore results in additional strong quenching of (1)O(2), which may in turn decrease the nitroxide yield via the (1)O(2) chemical path, possibly having an effect on quantitative interpretations. We have also found that probes with the dansyl fluorophore photosensitized (1)O(2) upon UV excitation with the quantum yield of 0.087 in acetonitrile at 366 nm. This result shows that care must be taken when the dansyl-based sensors are used in experiments requiring UV irradiation. We hope that our results will contribute to a better characterization and wider use of these novel double sensors.

Photosensitized oxidation of 2',7'-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2',7'-dichlorofluorescein.

2',7'-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 microM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 microM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D(2)O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O(2) in DCF formation, even though DCFH is an efficient (physical) quencher of 1O(2) (k(q) = 1.4 x 10(8) M(-1)s(-1) in methanol). DCF is also a moderate photosensitizer of 1O(2) with a quantum yield of circa phi = 0.06 in D(2)O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 1. Berberine.

Goldenseal is an herb which is widely used for many medical applications such as in eyewashes and skin lotions and which is currently undergoing testing by the National Toxicology Program. The main alkaloid constituent of Goldenseal is berberine. The topical application of Goldenseal or berberine to the skin or eyes raises the possibility that an adverse phototoxic reaction may result from an interaction between the alkaloid and light. We have therefore studied the photochemistry of berberine in different solvents and its phototoxicty to HaCaT keratinocytes. Irradiation of berberine in aqueous solutions does not generate (1)O(2), but in CH(2)Cl(2), (1)O(2) is produced with a quantum yield phi = 0.34. With the aid of the electron paramagnetic resonance (EPR) spin trapping technique and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we have detected oxygen-centered radicals photogenerated by berberine in water and acetonitrile. In the latter solvent and in the absence of oxygen, the neutral berberine radical formed by one electron reduction was observed. Methanol radicals were detected by EPR in water/alcohol low-temperature glasses irradiated in the berberine long-wavelength absorption band. In such alcoholic glasses, we have also detected an EPR signal from the berberine triplet at 77 K, in contrast to aqueous glasses where neither triplet nor radicals were detectable. Our data show that, although a weak photosensitizer in water, berberine is able to produce both (1)O(2) and radical species in a nonpolar environment. UVA irradiation of HaCaT keratinocytes in the presence of 50 microM berberine resulted in an 80% decrease in cell viability and a 3-fold increase in DNA damage as measured by the Comet assay. These findings suggest that exposure to sunlight or artificial light sources emitting UVA should be avoided when topical preparations derived from Goldenseal or containing berberine are used.

The effect of short intermittent light exposures on the melatonin circadian rhythm and NMU-induced breast cancer in female F344/N rats.

We investigated the effects of altered endogenous nighttime melatonin concentrations on mammary tumor production in an N-nitroso-N-methylurea (NMU)-induced breast cancer model in female Fischer 344 (F344)/N rats. Experiments were designed 1) to evaluate whether short-duration intermittent exposures to light at night would affect the nocturnal rise of melatonin, resulting in a decrease in nighttime serum melatonin concentrations, 2) to evaluate whether any suppression of nighttime serum melatonin concentrations could be maintained for a period of weeks, and 3) to determine the effects of suppressed serum melatonin concentrations on the incidence and progression of NMU-induced breast cancer. In vivo studies were used to assess serum melatonin concentrations after 1 day and 2 and 10 weeks of nightly administration of short-duration intermittent light exposure at night and incidence of NMU-induced tumors. Five 1-minute exposures to incandescent light every 2 hours after the start of the dark phase of the light: dark cycle decreased the magnitude of the nocturnal rise of serum melatonin concentrations in rats by approximately 65%. After 2 weeks of nightly intermittent light exposures, an average decrease of the peak nighttime serum melatonin concentrations of approximately 35% occurred. The amelioration continued and, at 10 weeks, peak nighttime serum melatonin concentrations were still decreased, by approximately 25%. Because peak endogenous nighttime serum melatonin values could be moderately suppressed for at least 10 weeks, a 26-week NMU mammary tumor study was conducted. Serum melatonin concentrations and incidence, multiplicity, and weight of NMU-induced mammary tumors were assessed. A group of pinealectomized (Px) animals was also included in the tumor study. No effect on the development of mammary tumors in an NMU-induced tumor model in rats occurred when endogenous nighttime serum melatonin concentrations were moderately suppressed by short-duration intermittent light exposures at night. At necropsy, there were no alterations in mammary tumor incidence (28/40 NMU controls, 28/40 NMU + light, 31/40 NMU + Px), multiplicity (2.18 tumors/tumor-bearing NMU control, 1.89 NMU + light, 2.39 NMU + Px), or average tumor weight (1.20 g NMU control, 1.19 g NMU + light, 0.74 g NMU + Px). Tumor burden had no effect on the serum melatonin cycle. At 26 weeks, however, animals exposed to intermittent light at night exhibited approximately 3-fold higher serum melatonin concentrations as compared with controls. Additionally, rats that had been pinealectomized at 4 weeks of age had serum melatonin concentrations that were markedly higher than the expected baseline concentrations for pinealectomized rats (<15 pg/ml), suggesting the reestablishment of a melatonin cycle. This finding was unexpected and suggests that melatonin can be produced by an organ or tissue other than the pineal gland.

Quenching of singlet molecular oxygen (1O2) by azide anion in solvent mixtures.

The azide ion is a strong physical quencher of singlet molecular oxygen (1O2) and is frequently employed to show involvement of 1O2 in oxidation processes. Rate constants (k(q)) for the quenching of 1O2 by azide are routinely used as standards to calculate k(q) values for quenching by other substrates. We have measured k(q) for azide in solvent mixtures containing deuterium oxide (D2O), acetonitrile (MeCN), 1,4-dioxane, ethanol (EtOH), propylene carbonate (PC), or ethylene carbonate (EC), mixtures commonly used for many experimental studies. The rate constants were calculated directly from 1O2 phosphorescence lifetimes observed after laser pulse excitation of rose bengal (RB), used to generate 1O2. In aqueous mixtures with MeCN and carbonates, the rate constant increased nonlinearly with increasing volume of organic solvent in the mixtures. k(q) was 4.78 x 10(8) M(-1) s(-1) in D2O and increased to 26.7 x 10(8) and 27.7 x 10(8) M(-1) s(-1) in 96% MeCN and 97.7% EC/PC, respectively. However, in EtOH/D2O mixtures, k(q) decreased with increasing alcohol concentration. This shows that a higher solvent polarity increases the quenching efficiency, which is unexpectedly decreased by the proticity of aqueous and alcohol solvent mixtures. The rate constant values increased with increasing temperature, yielding a quenching activation energy of 11.3 kJ mol(-1) in D2O. Our results show that rate constants in most solvent mixtures cannot be derived reliably from k(q) values measured in pure solvents by using a simple additivity rule. We have measured the rate constants with high accuracy, and they may serve as a reliable reference to calculate unknown k(q) values.

Photochemical studies on xanthurenic acid .

The tryptophan metabolite xanthurenic acid (Xan) has been isolated from aged human cataractous lenses. The photophysical properties of Xan were examined to determine if it is a potential chromophore for age-related cataractogenesis. We found that Xan produces singlet oxygen (psi delta = 0.17 in CD3OD) with the same efficiency as the lenticular chromophore N-formyl kynurenine and quenches singlet oxygen at a rate similar (2.1 x 10(7); CD3OD) to other tryptophan metabolites found in the eye. As the mechanisms of induction of cataracts may also involve redox reactions, the interactions of hydrated electrons (e(aq)-), the azide radical (N3*) and hydroxyl radical (OH*) with Xan were studied using the technique of pulse radiolysis. The reaction rate constants of e(aq)-, N3* and OH* with Xan were found to be of the same order of magnitude as other tryptophan metabolites. The rate constant for reaction of Xan with e(aq)- solvated electrons was found to be diffusion controlled (k = 1.43 x 10(10) M(-1) s(-1); the reaction with N3* was very fast (k = 4.0 x 10(9) M(-1) s(-1)); and with OH* was also near diffusion controlled (k = 1.0 x 10(10) M(-1) s(-1)). Superoxide O2*- production by irradiated Xan in methanol was detected by electron paramagnetic resonance and substantiated by determining that the enhanced rate of oxygen consumption of Xan irradiated in the presence of furfuryl alcohol was lowered by superoxide dismutase.

Photophysical studies on melatonin and its receptor agonists.

Previous work has demonstrated that melatonin inhibits the growth of both dermal and uveal melanoma cells. Recent clinical trials have found that melatonin is an efficacious treatment for metastatic dermal melanoma. The goal of this study was to provide further insight into the oncostatic mechanism(s) of melatonin. The inhibition of the growth of uveal melanoma cells is dose-dependent (0.1-10 nM) within the range of endogenous melatonin concentrations (2 nM) found in the human aqueous humor. We know that this inhibition of growth is receptor-mediated, at least in part, because uveal melanoma cell growth was also blocked by the agonists of melatonin receptors. There are two known membrane receptors for melatonin (Mel(1a) and Mel(1b)) and one known nuclear receptor (Mel2). To determine if singlet oxygen production and/or quenching contributed to the growth inhibition of melatonin, we examined the photophysical properties of melatonin and its agonists. Using flash photolysis, we determined that melatonin and its membrane receptor agonist 6-chloromelatonin (Mel(1a-b), Lilly, Indianapolis, IN) produced very little singlet oxygen (psidelta = 0.073 and psidelta = 0.01, respectively). There was no detectable singlet oxygen phosphorescence at 1,270 nm for the nuclear receptor agonist CG-52608 (Mel2, Novartis, Basel, Switzerland). In contrast, the agonist of the Mel(1b) receptor, S-20098 (Servier, Paris, France), produced singlet oxygen with a quantum efficiency of psidelta = 0.34. Singlet oxygen was quenched by melatonin and 6-chloromelatonin at approximately the same rate (6.1 x 10(7) M(-1)s(-1) and 6.0 x 10(7) M(-1)s(-1) in CD3OD), while the rate of quenching for the nuclear receptor agonist CG-52608 and membrane receptor agonist S-20098 was less (2.2 x 10(7) M(-1)s(-1) and 1.5 x 10(7) M(-1) s(-1), respectively). It appears that the production of singlet oxygen by melatonin would not be sufficient to directly block the proliferation of melanoma cells, but may activate gene products that could contribute to the oncostatic effect.

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort).

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.

Investigation of the mechanism of action of microperoxidase-11, (MP11), a potential anti-cataract agent, with hydrogen peroxide and ascorbate.

The interaction of hydrogen peroxide, ascorbate and microperoxidase-11 (MP11), a ferriheme undecapeptide derived from cytochrome c, has been investigated using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR), and mass spectroscopy techniques. It is shown that in 50 m M phosphate pH 7. 0-7.4 in the absence of other reactants H(2)O(2)induces a concentration-dependent decrease in absorption at the Soret band (399 nm) of the microperoxidase, with concomitant H(2)O(2)decomposition and oxygen evolution. The reaction causes irreversible heme degradation, concomitant with loss of enzymatic activity. Ascorbate effectively protects MP11 from degradation and inhibits oxygen evolution. At ascorbate concentrations greater than that of H(2)O(2), microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H(2)O(2)oxidizes the microperoxidase to a monooxygenated product, which did not form if ascorbate was included in the reaction system. There appears to be a 1:1 relationship between H(2)O(2)degradation and ascorbate oxidation. EPR experiments revealed that an ascorbate radical was formed during the reaction. These reactions may be described by a scheme where a putative 'compound I' of the microperoxidase is reduced by ascorbate back to the original redox state (ferric) of the peroxidase in two one-electron steps, concomitantly with oxidation of the ascorbate to an ascorbate radical or in one two-electron transfer step forming dehydroascorbate. In the absence of ascorbate, the 'compound I' reacts further with the peroxide causing microperoxidase degradation and partial oxygen evolution. These observations are relevant to the interaction of ferrihemes with H(2)O(2)and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.

Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group.

Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light-absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co-Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose-effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity.