Evaluation of the UV/H2O2 system for treating natural water with a mixture of anthracene and benzo[a]pyrene at ultra-trace levels.

The presence of polycyclic aromatic hydrocarbons, such as anthracene (AN) and benzo[a]pyrene (BaP), in water has become a problem of great concern due to the detrimental health effects caused to humans and living beings. In this work, the efficiency of the UV/H2O2 system for degrading the target compounds at ultra-trace levels in surface water has been evaluated. For this purpose, a previous optimization step using a face-centered central composite experimental design has been conducted, considering the effect of the UV-C irradiance and the initial concentration of H2O2. It was evidenced that under optimal operating conditions (11 mg L-1 H2O2 and 0.63 mW cm-2 irradiance), AN and BaP removal percentages were higher than 99.8%. Additionally, 69.3% of the organic matter, in terms of total organic carbon, was mineralized without the production of transformation by-products more harmful than the parent compounds. These findings demonstrate the oxidation capacity of the examined system in a natural matrix for degrading micropollutants that cannot be converted through conventional treatment processes. Consequently, new horizons are opened for the effective use of the UV/H2O2 system for drinking water production, providing the accomplishment of other regulated parameters related to water quality.

Solar simulated light exposure alters metabolization and genotoxicity induced by benzo[a]pyrene in human skin.

Skin is a major barrier against external insults and is exposed to combinations of chemical and/or physical toxic agents. Co-exposure to the carcinogenic benzo[a]pyrene (B[a]P) and solar UV radiation is highly relevant in human health, especially in occupational safety. In vitro studies have suggested that UVB enhances B[a]P genotoxicity by activating the AhR pathway and overexpressing the cytochrome P450 enzymes responsible for the conversion of B[a]P into DNA damaging metabolites. Our present work involved more realistic conditions, namely ex vivo human skin explants and simulated sunlight (SSL) as a UV source. We found that topically applied B[a]P strongly induced expression of cutaneous cytochrome P450 genes and formation of DNA adducts. However, gene induction was significantly reduced when B[a]P was combined with SSL. Consequently, formation of BPDE-adducts was also reduced when B[a]P was associated with SSL. Similar results were obtained with primary cultures of human keratinocytes. These results indicate that UV significantly impairs B[a]P metabolism, and decreases rather than increases immediate toxicity. However, it cannot be ruled out that decreased metabolism leads to accumulation of B[a]P and delayed genotoxicity.

Photocatalytic degradation of polycyclic aromatic hydrocarbon benzo[a]pyrene by iron oxides and identification of degradation products.

Photocatalytic decay profiles of polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) have been investigated on various synthesized iron oxides and on soil surfaces under a set of diverse conditions. Samples were analysed using the developed HPLC procedure. Results of the present study demonstrate fastest photodisintegration of B[a]P on goethite followed by haematite, magnetite, akaganeite and maghemite, respectively. The effect of soil pH, irradiation wavelength and iron oxide and oxalic acid dose on the degradation of B[a]P was evaluated. The studies revealed enhancement in photodegradation in the presence of oxalic acid due to the occurrence of fenton like reaction. The results showed faster B[a]P degradation under short wavelength UV radiation. Rate constants in acidic, neutral and alkaline soils under optimum dissipation conditions were 1.11×10(-2), 7.69×10(-3) and 9.97×10(-3) h(-1), respectively. The study indicates that iron oxides along with oxalic acid are effective photocatalyst for the remediation of benzo[a]pyrene contaminated soil surfaces. The degradation products of B[a]P in the soils of different pH in presence of goethite were identified and degradation pathways proposed. Peaks due to toxic metabolites such as diones, diols and epoxides disappear after 120 h in all the three soils.

UVA photoirradiation of benzo[a]pyrene metabolites: induction of cytotoxicity, reactive oxygen species, and lipid peroxidation.

Benzo[a]pyrene (BaP) is a prototype for studying carcinogenesis of polycyclic aromatic hydrocarbons (PAHs). We have long been interested in studying the phototoxicity of PAHs. In this study, we determined that metabolism of BaP by human skin HaCaT keratinocytes resulted in six identified phase I metabolites, for example, BaP trans-7,8-dihydrodiol (BaP t-7,8-diol), BaP t-4,5-diol, BaP t-9,10-diol, 3-hydroxybenzo[a]pyrene (3-OH-BaP), BaP (7,10/8,9)tetrol, and BaP (7/8,9,10)tetrol. The photocytotoxicity of BaP, 3-OH-BaP, BaP t-7,8-diol, BaP trans-7,8-diol-anti-9,10-epoxide (BPDE), and BaP (7,10/8,9)tetrol in the HaCaT keratinocytes was examined. When irradiated with 1.0 J/cm(2) UVA light, these compounds when tested at doses of 0.1, 0.2, and 0.5 µM, all induced photocytotoxicity in a dose-dependent manner. When photoirradiation was conducted in the presence of a lipid (methyl linoleate), BaP metabolites, BPDE, and three related PAHs, pyrene, 7,8,9,10-tetrahydro-BaP trans-7,8-diol, and 7,8,9,10-tetrahydro-BaP trans-9,10-diol, all induced lipid peroxidation. The formation of lipid peroxides by BaP t-7,8-diol was inhibited by NaN3 and enhanced by deuterated methanol, which suggests that singlet oxygen may be involved in the generation of lipid peroxides. The formation of lipid hydroperoxides was partially inhibited by superoxide dismutase (SOD). Electron spin resonance spin trapping experiments indicated that both singlet oxygen and superoxide radical anion were generated from UVA photoirradiation of BPDE in a light dose responding manner.

UVB in solar-simulated light causes formation of BaP-photoproducts capable of generating phosphorylated histone H2AX.

Polycyclic aromatic hydrocarbons (PAHs), wide-spread mutagenic and carcinogenic environmental pollutants, are consistently exposed to sunlight in the environment. Our previous paper showed that benzo[a]pyrene (BaP) exposed to solar-simulated light (SSL) induced phosphorylation of histone H2AX (γ-H2AX) [T. Toyooka, G. Ohnuki, Y. Ibuki, Solar-simulated light-exposed benzo[a]pyrene induces phosphorylation of histone H2AX, Mutat. Res. 650 (2008) 132-139], a marker of DNA double strand breaks. In this study, we found the ultraviolet B (UVB) region of SSL to produce photomodified BaP with high cytotoxicity and the ability to generate γ-H2AX. Degradation of BaP by SSL, resulting in an increase in cytotoxicity and the generation of γ-H2AX, was decreased by UVB-masking using a glass plate. Exposure to UVB itself increased the cytotoxicity of BaP and amount of γ-H2AX generated. Other PAHs, 1,2-benzoanthracene and 1,2:5,6-dibenzoanthracene, which absorb UVB, also showed enhanced cytotoxicity and the promoted the generation of γ-H2AX after exposure to SSL, whereas naphthalene and chrysene, which have low absorption in the UVB region, did not. These findings suggested that UVB is important for the degradation of PAHs having absorbance in this region, but that the production of genotoxic intermediates during the degradation process needs to be considered. UVB is a two-edged blade in environments, effectively degrading toxic chemicals but also producing genotoxic compounds as reactive intermediates.

Effects of UVA and visible light on the photogenotoxicity of benzo[a]pyrene and pyrene.

This study investigated the role of UVA/visible light (U, 320-800 nm) and visible light (V, 400-800 nm) in the phototoxicity and photogenotoxicity of two ubiquitous polycyclic aromatic hydrocarbons (PAH): benzo[a]pyrene (BaP) and Pyrene (Pyr). These mechanisms were evaluated by the WST-1 test and the comet assay on normal human keratinocytes (NHK) and by the micronucleus test on CHO cells. The production of reactive oxygen species (ROS) was assessed through the induction of 8-oxodeoxyguanine (8-oxodG) lesions by immunofluorescence staining in NHK. Results of the WST-1 test revealed the phototoxic properties of BaP and Pyr after irradiation with U and V lights. BaP presented the highest phototoxic properties. Results of the comet assay showed that U- and V-irradiated BaP and Pyr induced increasing rates of DNA single-strand breaks in NHK, in a dose dependent manner. The tested PAH could also induce increased levels of micronuclei in CHO cells after U and V irradiations. Increasing 8-oxodG levels were detected after U and V irradiations in BaP- and Pyr-treated keratinocytes and confirmed the involvement of ROS in the photogenotoxicity of PAH. Overall, this study highlighted the existence of an alternative pathway of PAH genotoxicity that is induced by UVA and/or visible light. Visible light is suggested to photoactivate PAH by a mechanism which is mainly based on oxidative reactions.

Enhanced bio-mineralization by riboflavin photosensitization and its significance to detoxification of Benzo[a]pyrene.

In this study, (14)C-benzo[a]pyrene (BaP) was chosen as a model compound to investigate if photosensitization by riboflavin enhances the subsequent microbial mineralization of polycyclic aromatic hydrocarbons (PAHs) in natural aquatic environments. After photolysis, BaP showed an increased toxicity to human epithelial cell and natural microbial assemblage. However, BaP mineralization rate in a river water sample containing riboflavin is roughly twice of that without riboflavin after the 2-day incubation. Thus, the results imply that microbial assemblage can mineralize BaP photoproducts to carbon dioxide and a combination of riboflavin photosensitization and microbial degradation could lead to complete detoxification of PAHs.

Effects of photodegradation of dissolved organic matter on the binding of benzo(a)pyrene.

Dissolved organic matter (DOM) in natural waters can bind various organic pollutants, and the affinity of this binding is strongly influenced by the chemical characteristics of the DOM and water pH. This study examined the effects of photochemically induced alteration of the DOM's chemical properties and water pH on the binding of benzo(a)pyrene (BaP). Time- and pH-series of solar-simulated irradiations were performed on a natural water sample and aqueous DOM solutions prepared from aquatic and soil humic substances. The binding affinity of BaP, expressed as a partition coefficient of a compound to DOM, decreased substantially after the DOM samples were irradiated over environmentally relevant radiation doses and pH ranges. The lowering of the pH due to the photoproduction of acidic products often partly offsets the reduction of the binding affinity caused by direct photoalteration of the DOM's chemical structure. The decrease of the binding affinity, after correction for the photoinduced pH change, was positively correlated with the decrease in the molecular weight and the aromaticity of the DOM in the course of irradiation. Increasing O(2) abundance accelerated the decrease of the binding affinity as a result of enhanced DOM photodegradation. Visible light played a more important role in reducing the molecular weight and aromaticity of the DOM than in reducing the content of dissolved organic carbon (DOC) via photoremineralization while the reverse was true for UV radiation, indicating that photochemical reduction of the binding affinity may occur in natural waters at depths greater than UV radiation can reach. A decrease of the affinity of DOM for binding BaP will increase the free dissolved fraction of BaP and thus its availability and toxicity to aquatic organisms. The results from this study may have similar implications for organic pollutants other than BaP.

Effects of riboflavin on the phototransformation of benzo[a]pyrene.

Riboflavin (Vitamin B2) is a natural dye-sensitizer habitually present in natural waters. Effects of riboflavin as photosensitizer on the transformation of benzo[a]pyrene (BaP) (10 microM) in the aqueous-organic solvent (water/acetonitrile/methanol 50/40/10) were investigated in this study. The photolysis half life of BaP in solution containing 50 microM riboflavin was 5 min, compared to 98 min in the absence of riboflavin. The rate of phototransformation of BaP increased as the concentration of riboflavin was raised from 10 microM to 100 microM under both natural sunlight and UVA irradiation. The half life of BaP in the presence of 50 microM riboflavin was 10.6 min and 43.1 min when exposed to visible range of natural sunlight and UVA irradiation respectively. Riboflavin decomposes under natural sunlight. Lumichrome, a principal photoproduct of riboflavin, was shown to photosensitize BaP under natural sunlight after photolysis of riboflavin. Our study indicated that other photoproducts from riboflavin, such as lumiflavin, were also involved in the phototransformation of BaP under sunlight when riboflavin diminished. The major photoproducts in the photolysis of BaP were identified as 1,6-benzo[a]pyrene-dione, 3,6-benzo[a]pyrene-dione, 6,12-benzo[a]pyrene-dione by using high performance liquid chromatography (HPLC). All these products were detected in the samples which were irradiated under different light sources and in the presence or absence of riboflavin. The possible phototransformation mechanism was discussed.

Increased polycyclic aromatic hydrocarbon toxicity following their photomodification in natural sunlight: impacts on the duckweed Lemna gibba L. G-3.

The authors previously demonstrated that simulated solar radiation (SSR), with a fluence rate of only 40 mumol m-2 sec-1, increased polycyclic aromatic hydrocarbon (PAH) toxicity to the duckweed Lemna gibba and that PAHs photomodified in SSR (generally oxygenation of the ring system) are more toxic than the parent compounds (Huang et al., Environ. Toxicol. Chem., 1993, 12, 1067-1077). It is not known, however, to what extent toxicity of PAHs can increase due to photomodification. Thus, natural sunlight, which has a high fluence rate (approximately 2000 mumol m-2 sec-1), was used to photomodify anthracene, benzo[a]pyrene, fluoranthene, phenanthrene, and pyrene. Toxicity was based on growth inhibition of L. gibba, measured as the rate of production of new leaves over an 8-day period. Initially, the toxicity of the PAHs applied in intact form was probed, with the compounds demonstrating greater toxicity in sunlight than in SSR. Next the PAHs were photomodified in sunlight prior to incubation with the plants. The half-lives of the PAHs in sunlight ranged from 12 min to 30 hr. Although most of the products of PAH photomodification are not yet identified, the degree that PAH toxicity increased following photomodification in sunlight could still be probed. The mixtures of photomodified chemicals that were derived from each PAH in sunlight were applied of L. gibba and growth inhibition under 100 mumol m-2 sec-1 of SSR was determined. The LC50s for the PAH photoproducts generated in sunlight were an order of magnitude lower than the LC50s for the PAHs applied in intact form.

The light-dependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA.

In vitro, the photodynamic compound benzo[a]pyrene (BAP) generates singlet oxygen efficiently when irradiated in organic solvents. It also photogenerates superoxide anion radical in water and can act as a photoreducing agent in the absence of oxygen. In vivo, the hemolysis of human erythrocytes, the inactivation of Escherichia coli cells representing a series of strains differing in excision repair and catalase proficiency, and the inactivation of Haemophilus influenzae transforming DNA activity were used to characterize the phototoxicity of BAP in the presence of near-UV light (290-400 nm). The results are consistent with BAP behaving as a photosensitizer that generates both superoxide and singlet oxygen, and that damages chiefly membranes. DNA does not seem to be a major target in the phototoxic reactions investigated.

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of photochemical reaction products of polycyclic aromatic hydrocarbons with nitrite.

Six kinds of polycyclic aromatic hydrocarbons (PAHs) were subjected to ultraviolet light irradiation with nitrite for 1, 4, 8 and 24 h, and the irradiated samples were tested for mutagenicity towards Salmonella typhimurium TA 98, TA 100 and TA 1538. Irradiated samples of pyrene, fluoranthene and benzo[a]pyrene showed marked mutagen responses towards TA 98 and TA 1538, especially in the absence of S9 mix. The direct-acting mutagenic activity of these samples, showing high activities at 1-8 h, decreased greatly with the development of irradiation. Further, these direct-acting mutagens were mostly present in the neutral fraction. On the other hand, the mutagenicity of the irradiated sample of 5,6-benzoquinoline was high both with and without S9 mix, and was mostly present in the basic fraction because of its authentic characteristic. There was no correlation between the yield of 1-nitropyrene and the mutagenic activity of the photochemical reaction product of pyrene with nitrite. Further studies by TLC separation suggested that a considerable number of direct-acting mutagens formed in this experiment were more polar than nitrated PAH such as 1-nitropyrene.