Toxicity of engineered nanomaterials mediated by nano-bio-eco interactions.

Engineered nanomaterials may adversely impact human health and environmental safety by nano-bio-eco interactions not fully understood. Their interaction with biotic and abiotic environments are varied and complicated, ranging from individual species to entire ecosystems. Their behavior, transport, fate, and toxicological profiles in these interactions, addressed in a pioneering study, are subsequently seldom reported. Biological, chemical, and physical dimension properties, the so-called multidimensional characterization, determine interactions. Intermediate species generated in the dynamic process of nanomaterial transformation increase the complexity of assessing nanotoxicity. We review recent progress in understanding these interactions, discuss the challenges of the study, and suggest future research directions.

Assessing the effect of different natural dissolved organic matters on the cytotoxicity of titanium dioxide nanoparticles with bacteria.

Titanium dioxide nanoparticles (TiO2 NPs) are among the most widely manufactured nanomaterials on a global scale. However, prudent and vigilant surveillance, incumbent upon the scientific community with the advent of new technologies, has revealed potentially undesirable effects of TiO2 NPs on biological systems and the natural environment during their application and discharge. Such effects are likely best evaluated by first assessing the fate of the TiO2 NPs in natural environments. In this study, the effects of terrestrial humic acid (HA) and tannic acid (TA), two major members of the collective: dissolved organic matter (DOM), on the cytotoxicity of TiO2 NPs to Escherichia coli were investigated in the presence and absence of natural sunlight. Qualitative (transmission electron microscopy (TEM)) and quantitative (LC50) analyses were employed in this study. In addition, the production of reactive oxygen species (ROS) in the form of OH was further assessed-as HA or TA increased the production of ROS decreased. The inhibition of bacterial viability in the light treatment groups, with respective treatment organics at concentrations of 10 ppm, was less in TA than in terrestrial HA. SAS was used to analyze the treatment effect of individual factors of light irradiation, DOM, and concentration of TiO2 NPs.

Assessing the effects of surface-bound humic acid on the phototoxicity of anatase and rutile TiO2 nanoparticles in vitro.

In this study, the cytotoxicity of two different crystal phases of TiO2 nanoparticles, with surface modification by humic acid (HA), to Escherichia coli, was assessed. The physicochemical properties of TiO2 nanoparticles were thoroughly characterized. Three different initial concentrations, namely 50, 100, and 200 ppm, of HA were used for synthesis of HA coated TiO2 nanoparticles (denoted as A/RHA50, A/RHA100, and A/RHA200, respectively). Results indicate that rutile (LC50 (concentration that causes 50% mortality compared the control group)=6.5) was more toxic than anatase (LC50=278.8) under simulated sunlight (SSL) irradiation, possibly due to an extremely narrow band gap. It is noted that HA coating increased the toxicity of anatase, but decreased that of rutile. Additionally, AHA50 and RHA50 had the biggest differences compared to uncoated anatase and rutile with LC50 of 201.9 and 21.6, respectively. We then investigated the formation of reactive oxygen species (ROS) by TiO2 nanoparticles in terms of hydroxyl radicals (·OH) and superoxide anions (O2(·-)). Data suggested that O2(·-) was the main ROS that accounted for the higher toxicity of rutile upon SSL irradiation. We also observed that HA coating decreased the generation of ·OH and O2(·-) on rutile, but increased O2(·-) formation on anatase. Results from TEM analysis also indicated that HA coated rutile tended to be attached to the surface of E. coli more than anatase.

Photoinactivation of Escherichia coli by sulfur-doped and nitrogen-fluorine-codoped TiO2 nanoparticles under solar simulated light and visible light irradiation.

Titanium dioxide (TiO2) is one of the most widely used photocatalysts for the degradation of organic contaminants in water and air. Visible light (VL) activated sulfur-doped TiO2 (S-TiO2) and nitrogen-fluorine-codoped TiO2 (N-F-TiO2) were synthesized by sol-gel methods and characterized. Their photoinactivation performance was tested against Escherichia coli under solar simulated light (SSL) and VL irradiation with comparison to commercially available TiO2. Undoped Degussa-Evonik P-25 (P-25) and Sigma-TiO2 showed the highest photocatalytic activity toward E. coli inactivation under SSL irradiation, while S-TiO2 showed a moderate toxicity. After VL irradiation, Sigma-TiO2 showed higher photoinactivation, whereas S-TiO2 and P-25 showed moderate toxicity. Oxidative stress to E. coli occurred via formation of hydroxyl radicals leading to lipid peroxidation as the primary mechanism of bacterial inactivation. Various other biological models, including human keratinocytes (HaCaT), zebrafish liver cells (ZFL), and zebrafish embryos were also used to study the toxicity of TiO2 NPs. In conclusion, N-F-TiO2 did not show any toxicity based on the assay results from all the biological models used in this study, whereas S-TiO2 was toxic to zebrafish embryos under all the test conditions. These findings also demonstrate that the tested TiO2 nanoparticles do not show any adverse effects in HaCaT and ZFL cells.

Effect of humic acids and sunlight on the cytotoxicity of engineered zinc oxide and titanium dioxide nanoparticles to a river bacterial assemblage.

The effect of a terrestrial humic acid (HA) and Suwannee River HA on the cytotoxicity of engineered zinc oxide nanoparticles (ZnONPs) and titanium dioxide nanoparticles (TiO2NPs) to natural aquatic bacterial assemblages was measured with spread plate counting. The effect of HA (10 and 40 ppm) on the cytotoxicity of ZnONPs and TiO2NPs was tested factorially in the presence and absence of natural sunlight (light irradiation (LI)). The experiment was of full factorial, completely randomized design and the results were analyzed using the General Linear Model in SAS analytical software. The method of least squares means was used to separate the means or combinations of means. We determined the mechanism of toxicity via measurements of oxidative stress and metal ions. The toxicity of ZnONPs and TiO2NPs to natural aquatic bacterial assemblages appears to be concentration dependent. Moreover, the cytotoxicity of ZnONPs and TiO2NPs appeared to be affected by HA concentration, the presence of sunlight irradiation, and the dynamic multiple interactions among these factors. With respect to light versus darkness in the control group, the data indicate that bacterial viability was inhibited more in the light exposure than in the darkness exposure. The same was true in the HA treatment groups. With respect to terrestrial versus Suwanee River HA for a given nanoparticle, in light versus darkness, bacterial viability was more inhibited in the light treatment groups containing the terrestrial HA than in those containing Suwanee River HA. Differences in the extent of reactive oxygen species formation, adsorption/binding of ZnONPs/TiO2NPs by HA, and the levels of free metal ions were speculated to account for the observed cytotoxicity. TEM images indicate the attachment and binding of the tested nanoparticles to natural bacterial assemblages. Besides the individual parameter, significant effects on bacterial viability count were also observed in the following combined treatments: HA-ZnONPs, HA-LI, ZnONPs-LI, and HA-ZnONPs-LI. The main effects of all independent variables, plus interaction effects in all cases were significant with TiO2NPs.

Determination of the mechanism of photoinduced toxicity of selected metal oxide nanoparticles (ZnO, CuO, Co3O4 and TiO2) to E. coli bacteria.

Cytotoxicity of selected metal oxide nanoparticles (MNPs) (ZnO, CuO, Co3O4 and TiO2) was investigated in Escherichia coli both under light and dark conditions. Cytotoxicity experiments were conducted with spread plate counting and the LC50 values were calculated. We determined the mechanism of toxicity via measurements of oxidative stress, reduced glutathione, lipid peroxidation, and metal ions. The overall ranking of the LC50 values was in the order of ZnO < CuO < Co3O4 < TiO2 under dark condition and ZnO < CuO < TiO2 < Co3O4 under light condition. ZnO MNPs were the most toxic among the tested nanoparticles. Our results indicate depletion of reduced glutathione level and elevation of malondialdehyde level correlated with the increase in oxidative stress. Released metal ions were found to have partial effect on the toxicity of MNPs to E. coli. In summary, the dynamic interactions of multiple mechanisms lead to the toxicity of the tested MNPs to E. coli.

In vitro cytotoxicity of CdSe/ZnS quantum dots with different surface coatings to human keratinocytes HaCaT cells.

Quantum dots (QD) nanoparticles have been widely used in biomedical and electronics fields, because of their novel optical properties. Consequently it confers enormous potential for human exposure and environmental release. To increase the biocompatibility of QDs, a variety of surface coatings or functional groups are added to increase their bioactivity and water solubility. Human adult low calcium high temperature (HaCaT) cells are the epithelial cells derived from adult human skin that exhibits normal differentiation capacity and a DNA fingerprint pattern that is unaffected by long-term cultivation, transformation, or the presence of multiple chromosomal alternations. Human keratinocytes, HaCaT cells were used to systematically evaluate the cytotoxicity of biocompatible QD made of CdSe metal core and ZnS shell with three different coatings and at three different wavelengths (530, 580 and 620 nm). In terms of half-maximal inhibitory concentration, QSA-QDs with amine-polyethyleneglycol coating and QSH-QDs with amphiphilic polymer coating were not cytotoxic, while QEI-QDs with polyethylenimine coating were highly toxic to the HaCaT cells in comparison to a reference CuInS2/ZnS. QEI-QDs led to significant increase in reactive oxygen species, decrease in mitochondrial membrane potential and DNA damage in HaCaT cells. The mechanisms of toxicity of QEI-530 and QEI-580 can be attributed to the combination of intracellular reactive oxygen species production and loss of MMP. The QDs toxicity can be attributed to the polyethylemimine surface coating which was highly toxic to cells in comparison with amine-polyethyleneglycol, but not due to the release of cadmium ions.

In vitro evaluation of cytotoxicity of engineered metal oxide nanoparticles.

The recent advances in nanotechnology and the corresponding popular usage of nanomaterials have resulted in uncertainties regarding their environmental impacts. In this study, we used a systematic approach to study and compare the in vitro cytotoxicity of selected engineered metal oxide nanoparticles to the test organisms--E. coli. Among the seven test nano-sized metal oxides, ZnO, CuO, Al2O3, La2O3, Fe2O3, SnO2 and TiO2, ZnO showed the lowest LD(50) of 21.1 mg/L and TiO2 had the highest LD(50) of 1104.8 mg/L. Data of 14C-glucose mineralization test paralleled the results of bacteria viability test. After regression calculation, the cytotoxicity was found to be correlated with cation charges (R(2) = 0.9785). The higher the cation charge is, the lower the cytotoxicity of the nano-sized metal oxide becomes. To the best of our knowledge, this finding is the first report in nanotoxicology.

A study of the degradation of organophosphorus pesticides in river waters and the identification of their degradation products by chromatography coupled with mass spectrometry.

The degradation of selected organophosphorus pesticides (OPs), i.e., malathion and parathion, in river water, has been studied with solar simulator irradiation. The degradation of OPs and formation of degradation products were determined by chromatography coupled with mass spectrometry analysis. The effect of a photosensitizer, i.e., riboflavin, on the photolysis of OPs in a river-water environment was examined. There was no significant increase in the degradation rate in the presence of the photosensitizer. Degradation products of the OPs were identified with gas chromatography coupled with mass spectrometry (GC-MS) after derivatization by pentafluorobenzyl bromide (PFBB) and with high-performance liquid chromatography-mass spectrometry (HPLC-MS) with electrospray (ESI) or atomospheric pressure chemical ionization (APCI). Malaoxon, paraoxon, 4-nitrophenol, aminoparathion, O,O-dimethylthiophosphoric acid, and O,O-dimethyldithiophosphoric acid, have been separated and identified as the degradation products of malathion and parathion after photolysis in river water. Based on the identified transformation products, a rational degradation pathway in river water for both OPs is proposed. The identities of these products can be used to evaluate the toxic effects of the OPs and their transformation products on natural environments.

Effect of Size and Crystalline Phase of TiO2 Nanoparticles on Photocatalytic Inactivation of Escherichia coli.

We studied the size dependent toxicity of TiO2 nanoparticles (TiO2 NPs; 5-50 nm) of the anatase and rutile crystalline phases (including the mixture of anatase and rutile) against the model organism Escherichia coli. All the TiO2 NPs were characterized and their photocatalytic inactivation of E. coli was studied under the solar simulated light irradiation and dark conditions. In addition, the mechanism of toxicity was studied by measurement of reactive oxygen species (ROS), an indicator of oxidative stress. Rutile TiO2 NPs (TiO2-R-30 nm) of 30 nm showed the highest photocatalytic activity against E. coli (LC50 of 14.11 mg/L), followed by rutile TiO2 NPs (TiO2-R-50 nm) (LC50 of 35.96 mg/L). The anatase and rutile mixture of 20 nm size produced LC50 of 17.12 and 27.26 mg/L for A80%-R20% and A20%-R80% respectively, whereas none of the anatase TiO2 NPs with various sizes (5 nm, 15 nm and 30 nm) showed any toxicity against E. coli. The results indicate that the rutile had higher photocatalytic activity than anatase and the toxicity is size dependent, while the mixture of anatase and rutile had the median toxicity. Hydroxyl radical formation is the major ROS causing oxidative stress in E. coli, the primary mechanism of toxicity.

The current application of nanotechnology in food and agriculture.

The rapid development of nanotechnology has been facilitating the transformations of traditional food and agriculture sectors, particularly the invention of smart and active packaging, nanosensors, nanopesticides and nanofertilizers. Numerous novel nanomaterials have been developed for improving food quality and safety, crop growth, and monitoring environmental conditions. In this review the most recent trends in nanotechnology are discussed and the most challenging tasks and promising opportunities in the food and agriculture sectors from selected recent studies are addressed. The toxicological fundamentals and risk assessment of nanomaterials in these new food and agriculture products are also discussed. We highlighted the potential application of bio-synthesized and bio-inspired nanomaterial for sustainable development. However, fundamental questions with regard to high performance, low toxic nanomaterials need to be addressed to fuel active development and application of nanotechnology. Regulation and legislation are also paramount to regulating the manufacturing, processing, application, as well as disposal of nanomaterials. Efforts are still needed to strengthen public awareness and acceptance of the novel nano-enabled food and agriculture products. We conclude that nanotechnology offers a plethora of opportunities, by providing a novel and sustainable alternative in the food and agriculture sectors.

Comparing the relative toxicity of malathion and malaoxon in blue catfish Ictalurus furcatus.

Malathion inhibits the critical body enzyme, acetylcholinesterase (AChE). This capability requires that malathion should first be converted to malaoxon to become an active anticholinesterase agent. Conversion can be caused by oxidation in mammals, insects, plants, and in sunlight. In this study, the effects of malathion and malaoxon on catfish Ictalurus furcatus were evaluated. After 96-h exposures, the LC(50) (concentration that causes 50% mortality) and IC(50) (concentration that causes 50% enzyme inhibition) for malaoxon were lower than corresponding values for malathion. The overall mean 96-h LC(50) is 17.0 ppm for malathion and 3.1 ppm for malaoxon. IC(50) values for malathion are 8.5 ppm for brain, 10.3 ppm for liver, and 16.6 ppm for muscle. Corresponding values for malaoxon are 2.3, 3.7, and 6.8 ppm, respectively. All the AChE activities in malathion- and malaoxon-exposed catfish brain showed significant inhibition. The oxidation product malaoxon demonstrated higher inhibition on AChE activity than did malathion. Moreover, malaoxon showed significant inhibition on butyrylcholinesterase (BChE) in the liver if the concentrations were increased to more than 1 ppm. Malathion showed no difference between treatment group and control group. Compared with malathion, malaoxon showed higher inhibition on monoamine activity than that of malathion. The results indicated that the oxidative product malaoxon is more toxic than the parent compound malathion. AChE, BChE, and monoamine activities are confirmed as bioindicators of malathion exposure in blue catfish, I. furcatus.

Enhancing ethanol fermentability of an artificial acid hydrolyzate with anion exchange resin treatment.

To assess the effectiveness of anion exchange resins (Dowex M43 and Dowex monosphere 66) in neutralization and detoxification of an acid hydrolyzate solution, a fermentation medium containing inhibitors was inoculated with Saccharomyces cerevisiae. When treated with resins at a 1:1 ratio (vol:wt) for up to 20 min, 55-67% of furan and more than 95% of phenolic compounds were removed. Ethanol fermentation activity in resin-treated fermentation medium was the same as the control. There was 21-43% of the total sugar loss after one resin treatment, depending on the sugar concentration. Additional treatments increased sugar retention rate to 95%.

Biodegradation of benzo[a]pyrene with immobilized laccase: genotoxicity of the products in HaCat and A3 cells.

Benzo[a]pyrene (BaP) is listed as a priority pollutant by the U.S. Environmental Protection Agency because it is one of the most potent carcinogens of all known polycyclic aromatic hydrocarbons (PAHs). The biodegradation of BaP is of interest as a means for mitigating its effects in polluted ecosystems. In the present study, BaP was oxidized with laccase from Trametes versicolor, which was immobilized on functionalized kaolinite particles, and the cytotoxicity and genotoxicity of BaP and its degradation intermediates were measured in human HaCaT keratinocytes and A3 T lymphocytes. Cytotoxicity was assessed by fluorescein diacetate (FDA) uptake, while the alkaline Comet assay measured genotoxicity, using tail moment, tail DNA content, and tail length as metrics for DNA damage. On the basis of first-order reaction kinetics, the half life (t(1/) (2)) for the oxidization of BaP by immobilized laccase was 58.5 hr. After 87 hr of oxidation, 20 muM of BaP had decreased to 9.6 muM. HPLC analysis identified 1,6-benzo[a]pyrene quinone (1,6-BaQ), 3,6-benzo[a]pyrene quinone (3,6-BaQ), and 6,12-benzo[a]pyrene quinone (6,12-BaQ) among the oxidation products. Most treatments of HaCaT cells and A3 lymphocytes with BaP or its quinone intermediates resulted in significant decreases in viability (P < 0.05); dose-dependent decreases in cell viability were detected at concentrations of 0.1, 1, and 5 muM, but none of these treatments resulted in decreases of >30%. While treatment of HaCaT cells with as little as 0.1 muM 6,12-BaQ caused significant DNA damage, DNA damage was detected in HaCaT cells only with 1 and 5 muM 1,6-BaQ and 3,6-BaQ, and 5 muM BaP. In Comet assays conducted with A3 lymphocytes, all three quinone intermediates caused significant increase in tail DNA content at 1 and 5 muM. The results indicate that immobilized laccase is capable of degrading BaP, but several of those biodegradation products produce significant levels of DNA damage in human cells.

Study of biodegradation products from azo dyes in fungal degradation by capillary electrophoresis/electrospray mass spectrometry.

Biodegradation products from four model sulfonated azo dyes Orange II, Acid Orange 8, Food Yellow 3, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonic acid, sodium salt (4HABA), during fungal degradation were determined by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) with electrospray ionization and a coaxial sheath flow interface. The development and optimization of this analytical method including the sheath liquid composition and flow rate, nebulizing gas flow rate, carrier electrolyte, and MS voltage are described herein. Detection of unknown biodegradation products was carried out under negative ion mode with base peak electrophorogram (BPE) or extractive ion electrophorogram (EIE) monitoring. A volatile ammonium acetate buffer (10 mM) without organic modifier and a shealth liquid made from 2-propanol and water (80:20, v/v) were suited for the separation and ESI interface. The sulfonated ion was the base peak for model azo dyes and their metabolites containing sulfonic group. Results showed that the tested azo dyes were degraded quickly in the culture of white rot fungus, Pleurotus ostreatus in 3 days with the major biodegradation products being 4-hydroxy-benzenesulfonic acid, 3-methyl-4-hydroxy-benzenesulfonic acid, benzenesulfonic acid, 1,2-naphthoquinone-6-sulfonic acid and 3-methyl-benzenesulfonic acid.

Comparative study of immobilized Trametes versicolor laccase on nanoparticles and kaolinite.

Laccase from Trametes versicolor was immobilized on nanoparticles and kaolinite by physical adsorption or chemical covalence in which the supporters were activated by cross-linked with glutaraldehyde. Thermal and pH stabilities of immobilized laccase on these different supporters were compared. The degradation efficiencies of these immobilized laccases on oxidation of benzo[a]pyrene (BaP) were also compared. The results showed that the immobilized laccases on nanoparticles were more stable in resisting pH and thermal changes. After 48h oxidation, laccase immobilized on kaolinite using the covalent coupling method showed a higher efficiency of oxidation with the BaP residue of 23% in the presence of 1mM HBT and with BaP residue of 37% in 1mM ABTS as the mediator. The results also exhibited a significant inhibition by 1% surfactant Tween 80. According to the HPLC analysis, the oxidation products including 1,6-benzo[a]pyrene quinone, 3,6-benzo[a]pyrene quinone and 6,12-benzo[a]pyrene quinone were identified.

Phototransformation of 2,4,6-trinitrotoluene: sensitized by riboflavin under different irradiation spectral range.

Riboflavin-sensitized phototransformation of 2,4,6-trinitrotoluene (TNT) under natural sunlight was investigated with reverse-phase high performance liquid chromatography/mass spectrometry (HPLC/MS) and gas chromatography/mass spectrometry (GC/MS). The effect of different spectral region of sunlight on TNT phototransformation in the absence or presence of riboflavin was also investigated by using optical filters with cut-off at 400 or 455 nm. The concentration of riboflavin in the phototransformation of TNT was optimized. Concentration of riboflavin and TNT was 1.0 and 50 microM, respectively. The rates of phototransformation of TNT under natural sunlight in the presence or absence of riboflavin were conformed to initial pseudo-first-order rate equation. The photolysis half life of TNT in the presence of riboflavin was 21.87 min, compared to 39 min in the absence of riboflavin under natural sunlight. Two major phototransformation products of TNT, 3,5-dinitroaniline (3,5-DNA) and 1,3,5-trinitrobenzene (1,3,5-TNB), were detected in the samples in the presence of riboflavin receiving irradiation at full wavelength or wavelength >400 nm. The results indicate that riboflavin mediates TNT sensitized-phototransfomation under natural sunlight or near-UV-vis light.

Nanostructures: Current uses and future applications in food science.

Recent developments in nanoscience and nanotechnology intend novel and innovative applications in the food sector, which is rather recent compared with their use in biomedical and pharmaceutical applications. Nanostructured materials are having applications in various sectors of the food science comprising nanosensors, new packaging materials, and encapsulated food components. Nanostructured systems in food include polymeric nanoparticles, liposomes, nanoemulsions, and microemulsions. These materials enhance solubility, improve bioavailability, facilitate controlled release, and protect bioactive components during manufacture and storage. This review highlights the applications of nanostructured materials for their antimicrobial activity and possible mechanism of action against bacteria, including reactive oxygen species, membrane damage, and release of metal ions. In addition, an overview of nanostructured materials, and their current applications and future perspectives in food science are also presented.

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.

Comparing cytotoxicity and genotoxicity in HaCaT cells caused by 6-aminochrysene and 5,6-chrysenequinone under ultraviolet A irradiation.

Chrysene is one of the basic polycyclic aromatic hydrocarbons that are toxic environmental pollutants. The photoproducts of 6-aminochrysene (6AC) include 5,6-chrysenequinone (5,6-CQ) along with some minor products. In this study, cytotoxicity and genotoxicity of 6AC and 5,6-CQ to a human skin cell line, HaCaT, were measured with the fluorescein diacetate uptake (FDA) test and comet assay, respectively, in the presence or absence of ultraviolet A (UVA) irradiation. The FDA test result showed that HaCaT cell viability decreased dose dependently after exposure to UVA irradiation in both 6AC (0, 0.1, 0.5, 1, 5, 10, 50 microM) and 5,6-CQ (0, 0.05, 0.25, 0.5, 2.5, 5, 25 microM) groups, with the 6AC group having lower cell viability at the same substrate concentrations; therefore, 6AC was more cytotoxic. Results of the comet assay showed that the extent of DNA damage was also dose dependent after the combined UVA and 6AC treatment (0, 0.05, 0.1, 0.5, 1 microM), although no DNA damage was detectable in the 6AC group without UVA irradiation. In addition, no DNA damage was found in the 5,6-CQ group with or without UVA irradiation. Our study indicated that 5,6-CQ, the major photoproduct of 6AC, was less photocytotoxic than the parent compound and was not photogenotoxic to HaCaT cells under the experimental conditions.