Toxicological potential of 2-alkylcyclobutanones--specific radiolytic products in irradiated fat-containing food--in bacteria and human cell lines.

Food irradiation has been considered as a safe processing technology to improve food safety and preservation, eliminating efficiently bacterial pathogens, parasites and insects. This study aims to characterize the toxicological potential of 2-alkylcyclobutanones (2-ACBs), radiolytic derivatives of triglycerides, formed uniquely upon irradiation of fat-containing food. In irradiated food they are generated proportionally to fat content and absorbed radiation dose. The cyto- and genotoxic potentials of various highly pure synthetic 2-ACBs were studied in bacteria and human cell lines. While pronounced cytotoxicity was evident in bacteria, no mutagenic activity has been revealed by the Ames test in Salmonella strains TA 97, TA 98 and TA 100. In mammalian cells genotoxicity was demonstrated mainly by the induction of DNA base lesions recognized by the Fpg protein as determined by both the Comet Assay and the Alkaline Unwinding procedure. Formation of DNA strand breaks was observed by the Alkaline Unwinding procedure but not by the Comet Assay. The extent of cytotoxicity and genotoxicity were dependent on chain length and degree of unsaturation of the fatty acid chain. Further studies will have to clarify mechanisms of action and potential relevance for human exposure situation.

Activation of JNK and p38 but not ERK MAP kinases in human skin cells by 5-aminolevulinate-photodynamic therapy.

5-Aminolevulinate (ALA) photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. ALA is applied as a precursor of porphyrins serving as endogenous photosensitizers. Irradiation of HaCaT cells preincubated with 1 mM ALA for 24 h with red light of 570-750 nm at a dose of 4.5 J/cm2 leads to a 6-fold elevation of cellular c-Jun N-terminal kinase activity; phosphorylation of p38 mitogen-activated protein kinase (MAPK) is enhanced to a similar extent. In contrast, neither activation nor increased phosphorylation of the extracellular stimulus-regulated kinase MAPKs is detected. p38 is also phosphorylated by ALA-PDT in the human melanoma cell lines Bro and SkMel-23, applying doses that lead to 80-95% cell death after 24 h. Hence, the effects of ALA-PDT on MAPKs are similar to stresses like UV irradiation or exposure to hydrogen peroxide with respect to activation of JNK and p38 MAPKs. They are different, however, in that extracellular stimulus-regulated kinase activity is not raised by ALA-PDT. Of the 830 pmol porphyrins/mg protein that were present at 24 h in HaCaT cells, 99 pmol/mg were intracellular. When extracellular porphyrins had been removed by washing, p38 responses were retained. Thus, intracellular porphyrins synthesized from ALA are sufficient to elicit activation of p38 on photosensitization.

Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.

Reactive oxygen species (ROS) play a pivotal role in UVA-induced cell damage. As expression of the inducible nitric oxide synthase (iNOS) is a normal response of human skin to UV radiation we examined the role of nitric oxide (NO) as a protective agent during or even after UVA1- or ROS-exposure against apoptosis or necrosis of rat endothelial cells. When added during or up to 2 h subsequent to UVA1 or ROS exposure the NO-donor S-nitroso-cysteine (SNOC) at concentrations from 100-1000 microM significantly protects from both apoptosis as well as necrosis. The NO-mediated protection strongly correlates with complete inhibition of lipid peroxidation (sixfold increase of malonedialdehyde formation in untreated versus 1.2-fold with 1 mM SNOC). NO-mediated protection of membrane function was also shown by the inhibition of cytochrome c leakage in UVA1 treated cells, a process not accompanied by alterations in Bax and Bcl-2 protein levels. Thus, the experiments presented demonstrate that NO exposure during or even after a ROS-mediated toxic insult fully protects from apoptosis or necrosis by maintaining membrane integrity and function.

Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo.

The stratum corneum is located at the interface between body and environment and thus is constantly exposed to a pro-oxidative environment. Previously, we have demonstrated that stratum corneum lipids are targets of oxidative stress induced by ozone and by ultraviolet A and B exposure. Here, we employed an immunoblotting technique to detect protein oxidation in human stratum corneum obtained by tape stripping. After lysis, protein carbonyl groups were measured by derivatization with dinitrophenylhydrazine, separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against dinitrophenyl groups. Keratin 10, identified by use of specific antibodies and by microsequencing, was demonstrated in vitro to be oxidizable by ultraviolet A irradiation, hypochlorite, and benzoyl peroxide. In vivo, a keratin 10 oxidation gradient with low levels in the lower stratum corneum layers, and about 3-fold higher contents of carbonyl groups towards the outer layers was demonstrated in forehead stratum corneum of healthy volunteers (n = 6). As protein oxidation can be associated with an increased susceptibility to proteases, this finding may be important for better understanding the process of desquamation.

Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase.

Singlet oxygen has been postulated to be generated by Ultraviolet (UV) A irradiation of mammalian cells. We studied the role of singlet oxygen in the downstream signaling of the complex UV response leading to the induction of matrix-metalloproteinase-1 (interstitial collagenase/MMP-1). Exposure of cultured human fibroblasts to singlet oxygen, generated in a dark reaction by thermodissociation of the endoperoxide of the disodium salt of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) induced collagenase mRNA steady state levels in a dose dependent manner. The increase in collagenase expression after singlet-oxygen exposure generated with 3 mM NDPO2 was equivalent to that observed with UVA at a dose rate of 200-300 kJ/m2 and developed in a similar time course. In contrast, mRNA levels of TIMP-1, the specific tissue inhibitor of metalloproteinases, remained unchanged. Indirect evidence for the role of singlet oxygen in the UVA induction of collagenase comes from studies using singlet oxygen enhancer or quencher. Accordingly, incubation in deuterium oxide, an enhancer of singlet-oxygen lifetime, led to an additional increase in steady-state levels of collagenase mRNA after exposure to NDPO2 or to UVA irradiation. In contrast, sodium azide, a potent quencher of singlet oxygen, almost totally abrogated the induction of collagenase after exposure of fibroblasts to NDPO2 or to UVA irradiation. Similar results were obtained in studies of the proteins by radioimmunoprecipitation of MMP-1 and TIMP-1 using specific antibodies. Collectively, our data provide circumstantial evidence that singlet oxygen mediates the UVA induction of collagenase in vitro, whereas it does not exert any effect on TIMP-1 synthesis. The unbalanced synthesis of interstitial collagenase may contribute to the connective tissue damage in vivo related to photoaging and other photocutaneous disorders.

Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation.

In response to the attack of reactive oxygen species, the skin has developed a complex antioxidant defense system including among others the manganese-superoxide dismutase (MnSOD). MnSOD dismutates the superoxide anion (O2*-) derived from the reduction of molecular oxygen to hydrogen peroxide (H2O2), which is detoxified by glutathione peroxidase to water and molecular oxygen. We have addressed the question whether MnSOD is inducible upon UVA irradiation and whether repetitive UV exposure, as practiced for the light-hardening during phototherapy of various photodermatoses, can even enhance the adaptive antioxidant response. Single exposure of four different strains of fibroblasts to UVA irradiation resulted in a dose- and time-dependent increase in specific MnSOD mRNA levels. Interestingly, repetitive UVA exposure at days 1, 2, and 3 at a dose rate of 200 kJ per m2 resulted in a 5-fold induction of specific MnSOD mRNA levels following the third UVA exposure. Similar results were obtained for MnSOD activity. This adaptive response in terms of upregulation of the antioxidant enzyme MnSOD correlates with the protection against high UV doses, if cells were preexposed to sublethal UV doses. Importantly, MnSOD substantially differed between the tested individuals in both mRNA and activity levels. Taken together, we here provide evidence for the increasing induction of MnSOD upon repetitive UVA irradiation that may contribute to the effective adaptive UVA response of the skin during light hardening in phototherapy. Interindividual differences in the inducibility of MnSOD might account for differences in the susceptibility to develop photodermatologic disorders related to photosensitivity, photoaging, and skin cancer. The molecular basis for interindividual differences in the inducibility of antioxidant enzymes remains to be elucidated.

Singlet oxygen mediates the activation of JNK by UVA radiation in human skin fibroblasts.

Ultraviolet A (UVA: 320-400 nm) radiation activates c-Jun-N-terminal kinase (JNK 2) in human skin fibroblasts. Exposure of cells to UVA (300 kJ/m2) led to a 5-fold induction of JNK-activity which was significantly increased in the presence of D2O, an enhancer of the lifetime of singlet oxygen. Sodium azide, a quencher of singlet oxygen, abolished the activation of JNK. A hydroxyl radical scavenger, mannitol, had no effect. Furthermore, photochemically produced singlet oxygen (Rose Bengal plus white light) was found to induce JNK activity. This was enhanced by D2O and inhibited by azide. Thus, singlet oxygen activates and mediates the UVA-induced activation of JNK.

Assessment of the C-525 laser dye as a chemiluminescence sensitizer for lipid peroxidation in biological membranes: a comparison with chlorophyll-a.

The C-525 laser dye at micromolar concentration range is shown to enhance up to two to three orders of magnitude the chemiluminescence (CL) accompanying tert-butyl hydroperoxide (t-BHP)-induced rat liver microsome oxidation and Fe(2+)-induced lipid peroxidation (LPO) in liposomes. C-525 is shown to be a more efficient sensitizer of CL accompanying LPO in membrane systems than the known low-energy excited triplet carbonyl sensitizer, chlorophyll-alpha, (Cl-a). Regarding the sensitization mechanism, C-525 and Cl-a were compared in (a) a peroxyl radical-producing system (2,2'-azobis(2-dimethylvaleronitrile) (AMVN); (b) excited carbonyl-producing systems (3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD) thermal decomposition and horseradish peroxidase (HRP)-catalyzed isobutanal oxidation); and (c) excited singlet oxygen-producing system [endoperoxide of 3,3-(1,4-naphthylidene)-dipropionate (NDPO2)]. C-525 sensitized CL only in the systems where peroxyl radical and/or triplet excited carbonyls are produced, the mechanism of CL sensitization apparently is energy transfer from the excited triplet carbonyls formed in the peroxyl radical self-reaction via Russell's mechanism or by dioxetane decomposition. Cl-a was found to considerably sensitize CL related to NDPO2 thermal decomposition, a source of singlet oxygen, in addition to acting as a sensitizer of triplet carbonyl CL. The chemical stability of the C-525 laser dye in excited state-generating systems was shown to be appropriate for its application as a sensitizer of CL related to LPO reactions in membranes, but not in the HRP-catalyzed peroxidation system.

Protection against peroxynitrite.

Peroxynitrite formed in vivo from superoxide and nitric oxide can mediate oxidation, nitration, or nitrosation reactions, leading to impaired function, toxicity, and alterations in signaling pathways. Protection against peroxynitrite is important for defense of normal tissue, especially during inflammation. Biological protection against peroxynitrite is organized in three categories: prevention, interception, and repair. Prevention is the control of the formation of peroxynitrite precursors, nitric oxide and superoxide. Interception is by direct reaction with peroxynitrite, leading to non-toxic products. In this regard, organoselenium compounds, metalloporphyrin derivatives, and peroxidases (e.g. glutathione peroxidase and myeloperoxidase) exhibit high second-order rate constants with peroxynitrite. Ebselen, like glutathione peroxidase, protects in a catalytic fashion utilizing glutathione as reductant in the peroxynitrite reductase reaction. Protection by metalloporphyrins can be maintained through glutathione or ascorbate. Repair processes remove damaged products and restitute intact biomolecules.

Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts.

Reactive oxygen species (ROS) have been shown to be important messenger molecules in the induction of several genes. In human dermal fibroblasts the herbicide paraquat (PQ2+) was used to induce intracellular oxidative stress that was modulated by the inhibition of copper, zinc superoxide dismutase (Cu,ZnSOD), glutathione peroxidase (GSHPx), catalase, and blocking of the Fenton reaction. Interstitial collagenase (MMP-1) mRNA increased time dependently for up to 72 h following paraquat treatment. A correlation with the translation of MMP-1 could, however, only be detected up to 24 h, indicating an uncoupling of transcription and translation. Interleukin-1 alpha and beta mRNA showed two peaks at 6 h and 72 h. The inhibition of catalase by aminotriazol (ATZ), inhibition of GSHPx by buthionine sulfoximine (BSO), and blocking the Fenton reaction by the iron chelator desferrioxamine (DFO) in concert led to an increase in steady-state MMP-1 mRNA levels, possibly dependent on intracellular H2O2 increase. This combined treatment potentiated MMP-1 mRNA induction up to 6.5-fold compared to paraquat treated controls. Furthermore, exogenously added H2O2 caused an increase in MMP-1 mRNA levels. In contrast, inhibition of Cu,ZnSOD by diethyldithiocarbamate (DDC), leading to diminished H2O2 production from O2.-, decreased MMP-1 mRNA induction. Collectively, our data provide evidence that H2O2 is an important intermediate in the downstream signalling pathway finally leading to the induction of increased steady state MMP-1 mRNA levels. The synthesis of MMPs may contribute to connective tissue damage in vivo related to photoaging, inflammatory diseases, and tumor invasion.

Singlet oxygen induces collagenase expression in human skin fibroblasts.

Singlet oxygen generated in a dark reaction by thermodissociation of an endoperoxide (NDPO2) elicits an increase in mRNA of interstitial collagenase (MMP-1) in cultured human fibroblasts. The effect is enhanced in deuterium oxide-based medium and is abolished in the presence of non-toxic doses of sodium azide. In contrast, the mRNA level of the tissue inhibitor of metalloproteinases (TIMP-1) remains unaltered under these experimental conditions. These observations support the suggestion that an unbalanced synthesis of collagenase and TIMP reported to occur following UV-A irradiation or during inflammatory conditions may be mediated by singlet oxygen.

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation.

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro.

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL-1alpha, IL-1beta, and IL-6. These effects are enhanced in deuterium oxide-based medium and are diminished in the presence of non-toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal-coated resin particles with visible light (lambda > 450 nm) results in the induction of interstitial collagenase, IL-1 and IL-6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL-1 and IL-6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

Carotenoid mixtures protect multilamellar liposomes against oxidative damage: synergistic effects of lycopene and lutein.

Antioxidant activity of carotenoids in multilamellar liposomes assayed by inhibition of formation of thiobarbituric acid-reactive substances was in the ranking: lycopene> alpha-tocopherol > alpha-carotene > beta-cryptoxanthin > zeaxanthin = beta-carotene > lutein. Mixtures of carotenoids were more effective than the single compounds. This synergistic effect was most pronounced when lycopene or lutein was present. The superior protection of mixtures may be related to specific positioning of different carotenoids in membranes.

Singlet molecular oxygen production in the reaction of peroxynitrite with hydrogen peroxide.

Peroxynitrite and hydrogen peroxide are mediators of cytotoxicity. This study shows that the peroxynitrite anion reacts with hydrogen peroxide to release oxygen accompanied by emission of chemiluminescence (CL). Direct characterization of this light emission attributes it to the transition of singlet molecular oxygen to the triplet ground state. Chemiluminescence was monitored: (i) by dimol light emission in the red spectral region (> 610 nm) using a red-sensitive photomultiplier; and (ii) by monomol light emission in the infrared (1270 nm) with a liquid nitrogen-cooled germanium diode. These properties of photoemission and the enhancing effect of deuterium oxide on CL intensity as well as the quenching effect of sodium azide are diagnostic of molecular oxygen in the excited singlet state. For comparison, singlet molecular oxygen arising from the thermolysis of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate or from the hypochlorite/H2O2 system was also monitored. These novel observations identify a potential singlet oxygen-dependent mechanism contributing to cytotoxicity mediated by peroxynitrite and hydrogen peroxide.

Protection by organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions.

Diaryl tellurides effectively protect against peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR), hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetate (HPA). Bis(4-aminophenyl) telluride offered the most efficient protection against oxidation of DHR induced by peroxynitrite. Protection by this compound was approximately 3 times more effective than that afforded by its selenium analog, bis(4-aminophenyl) selenide, and 11 times more effective than selenomethionine. When peroxynitrite was infused to maintain a steady-state concentration, bis(4-aminophenyl) telluride in the presence of GSH, but neither bis(4-aminophenyl) telluride nor GSH alone, effectively inhibited the peroxynitrite-mediated hydroxylation of benzoate. The inhibition of nitration was most pronounced using bis(4-hydroxyphenyl) telluride, and this compound was ca. 3 times more effective than selenomethionine. Bis(4-aminophenyl) telluride also protected proteins in lysates from human skin fibroblasts from peroxynitrite-mediated nitration of tyrosine residues more effectively than selenomethionine. These data establish a potential biological or pharmacological role of organotellurium compounds in the defense against peroxynitrite.

One-electron reduction of selenomethionine oxide.

Experimental evidence is provided that selenomethionine oxide (MetSeO) is more readily reducible than its sulfur analogue, methionine sulfoxide (MetSO). Pulse radiolysis experiments reveal an efficient reaction of MetSeO with one-electron reductants, such as e(aq)-, (k = 1.2x10(10) M(-1) s(-1)), CO2*- (k = 5.9x10(8) M(-1) s(-1)) and (CH3)2 C*OH (k = 3.5x10(7) M(-1) s(-1)), forming an intermediate selenium-nitrogen coupled zwitterionic radical with the positive charge at an intramolecularly formed Se(three-electron bond)N 2sigma/1sigma* three-electron bond, which is characterized by an optical absorption with lambda(max) at 375 nm, and a half-life of about 70 micros. The same transient is generated upon HO* radical-induced one-electron oxidation of selenomethionine (MetSe). This radical thus constitutes the redox intermediate between the two oxidation states, MetSeO and MetSe. Time-resolved optical data further indicate sulfur-selenium interactions between the Se(three-electron bond)N transient and GSH. The Se(three-electron bond)N transient appears to play a key role in the reduction of selenomethionine oxide by glutathione.

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Ultraviolet B wavelength dependence for the regulation of two major matrix-metalloproteinases and their inhibitor TIMP-1 in human dermal fibroblasts.

The wavelength dependence for the regulation of two major matrix-metalloproteinases, interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP-1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP-1 and MMP-3 mRNA steady-state levels and the secretion of the corresponding proteins up to 4.4-fold, whereas almost no increase was observed at wavelengths < 290 nm. In contrast, the synthesis of TIMP-1 increased only marginally. This imbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP-1 and MMP-3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earth's surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Antioxidant activity of the pyridoindole stobadine in liposomal and microsomal lipid peroxidation.

Stobadine, a pyridoindole derivative, is an efficient inhibitor of lipid peroxidation in phosphatidylcholine liposomes and in rat liver microsomes treated with iron/ADP/NADPH as pro-oxidant. Accumulation of thiobarbituric acid-reactive substances (TBARS) or low-level chemiluminescence were taken as a measure of lipid peroxidation and 5 microM stobadine doubled the duration of the lag phase preceding the onset of rapidly increasing chemiluminescence. Inhibition of lipid peroxidation was not observed with tocopherol-deficient microsomes, suggesting that the antioxidant effect of stobadine depends on vitamin E in the membrane. The cis(-) isomer was most effective, with the cis(+) and trans(rac) as well as dehydro- or acetyl derivatives being less active. In liposomes, the presence of reductant (NADPH or ascorbate) protects from the loss of stobadine.