THE CYTOTOXIC AND MUTAGENIC EFFECTS OF UVA RADIATION ON L5178Y MOUSE LYMPHOMA CELLS

Abstract— A broad-band UVA source that emits primarily350–400 nm radiation and no measurable radiation below 340 nm was used to test toxicity and mutagenicity at the thymidine kinase locus in L5178Y, subclone 3.7.2C (TK⁺/^-) mouse lymphoma cells. Cells were exposed to a fluence of 0 to 80 × 10⁴ J/m². The relationship between UVA fluence and survival was found to have a shoulder region followed by an exponential decrease in survival at higher fluence levels. An exposure-dependent increase in mutation was observed with increasing fluences from 0 to about 60 × 10⁴ J/m². An approximately 3- to 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells was seen at a fluence that resulted in 90% cell killing. We conclude that UVA radiation is a mutagen in the L5178Y mouse lymphoma cells used in this study.     

MUTATION AND SISTER CHROMATID EXCHANGE INDUCTION IN CHINESE HAMSTER OVARY (CHO) CELLS BY PULSED EXCIMER LASER RADIATION AT 93 nm AND 308 nm AND CONTINUOUS UV RADIATION AT 254 nm

We compared mutagenesis and sister chromatid exchange (SCE) induction by 193 nm and 308 nm pulsed excimer laser radiation with 254 nm low intensity continuous wave UV light in Chinese hamster ovary (CHO) cells in culture. The 254 nm radiation was most mutagenic of the radiations, in accordance with expectation, and also was most effective in increasing the level of SCEs. The 193 nm radiation was mutagenic at the ouabain resistance locus, but not at the HGPRT locus. However, 193 nm radiation was also strongly cytotoxic at energies producing measurable mutations. This radiation also caused a dose-related increase in SCEs. Pulsed excimer radiation at 308 nm was mutagenic at both loci, and also increased the incidence of SCEs. Comparison of the ratio of mutants/surviving cells at the D37 after radiation showed similar values for 254 nm and 308 nm at the HGPRT locus, but at the ouabain resistance locus, the ratio for the 308 nm radiation was about 5 times that for 254 nm radiation. These results indicate that some risk for mutagenesis may accompany the use of excimer radiation in the UVA region in therapeutic applications.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.     

CYTOTOXICITY AND GENOTOXICITY OF UVA IRRADIATION IN CHINESE HAMSTER OVARY CELLS MEASURED BY SPECIFIC LOCUS MUTATIONS, SISTER CHROMATID EXCHANGES AND CHROMOSOME ABERRATIONS

Abstract— The increasing use of artificial UVA (320-400 nm) suntanning devices has brought attention to possible hazardous effects of UVA. In contrast with earlier studies, several groups recently have described that UVA possibly is mutagenic. In this paper we evaluate the genotoxic properties of broad band UVA using CHO cells and three different assays: specific locus (HGPRT) mutations, chromosome aberrations, and sister chromatid exchanges (SCEs). The UVA-source was an UVASUN 2000 S (Mutzhas), emitting UVA above 340 nm. The survival curve of the cells exhibited a shoulder up to 200 kJ/m², that was followed by exponential killing at higher fluences. Mutations were induced linearly in the fluence range from 0-200 kJ/m² ( P < 0.001) to a level seven fold higher than the spontaneous, followed by a decrease at fluences above 300 kJ/m². Over the total range of tested fluences (0-300 kJ/m²) a linear dose-response relationship was observed for UVA-induced SCEs ( P < 0.001). A significantly higher percentage of the cells showed chromosomes with aberrations at the higher levels of exposure (200, 300 and 400 kJ/m²), but no dose response was demonstrated. Our results confirm recent findings showing that UVA is mutagenic in mammalian cells and suggest that UVA exposure may contribute to the total burden of genetic damage caused by exposure to ultraviolet light.     

Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp. (Cryptophyta).

The single-cell gel/comet assay is an electrophoretic technique used to detect single-strand breaks in DNA. Damage is assessed examining individual cells under an epifluorescent microscope. UV-induced DNA damage consists mostly of the formation of pyrimidine dimers; therefore, most of the damage cannot be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers. The main objective of this work is to evaluate the comet assay to detect UV-induced damage in DNA after an initial treatment of cells with T4 endonuclease V. This work was conducted on Rhodomonas sp. (Cryptophyta), a marine unicellular flagellate. Cells of Rhodomonas sp. were exposed to 12 h visible + ultraviolet-A + ultraviolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclease V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damage than control if these were treated with T4 endonuclease V. Rhodomonas sp. were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB. Damage induced by VIS + UVA + UVB as detected by the comet assay increased along with exposure time. However, damage caused by VIS and VIS + UVA remained relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunction with T4 endonuclease V. This modification of the comet assay can be used as an alternative technique to detect DNA damage in single cells caused by UV radiation.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

LONG WAVE ULTRAVIOLET RADIATION STIMULATES ARACHIDONIC ACID RELEASE AND CYCLOOXYGENASE ACTIVITY IN MAMMALIAN CELLS IN CULTURE

Human fibroblasts and mouse C3H 10T1/2 cells in culture were prelabeled with [3H]arachidonic acid and exposed to UVA radiation. Cells released labeled arachidonate metabolites into medium in a dose-dependent fashion (5-20 J cm-2). The time course of release appeared biphasic with peak responses occurring immediately and at 2 h post irradiation. Release of radiolabel was oxygen and calcium ion dependent and was inhibited by the addition of phenylglyoxal, indomethacin, and dibucaine to the medium. High performance liquid chromatographic examination of medium extracts revealed UVA stimulation of cyclooxygenase metabolism of [3H]arachidonic acid and specifically, prostaglandin E2 production by cells in culture. Furthermore, UVA stimulated a dose-dependent release of membrane incorporated [3H]choline from cells in culture. Paper chromatographic analysis of the medium provided evidence that choline release from the membrane was predominantly accompanied by release of phosphorylcholine with some glycerophosphorylcholine suggesting indirectly that the major pathway for UVA-stimulated arachidonic acid release was via phospholipase C and diacylglycerol lipase enzyme systems.     

LONG-WAVELENGTH UVA RADIATION INDUCES OXIDATIVE STRESS,CYTOSKELETAL DAMAGE and HEMOLYSIS*

Abstract— We investigated the ability of the different wavelength regions of UV radiation, UVA(320–400 nm), UVB(290–320 nm) and UVC(200–290 nm), to induce hemolysis. Sheep erythrocytes were exposed to radiation from either a UVA1 (>340 nm) sunlamp, a UVB sunlamp, or a UVC germicidal lamp. The doses used for the three wavelength regions were approximately equilethal to the survival of L5178Y murine lymphoma cells. Following exposure, negligible hemolysis was observed in the UVB- and UVC-irradiated erythrocytes, whereas a decrease in the relative cell number (RCN), indicative of hemolysis, was observed in the UVA 1-exposed samples. The decrease in RCN was dependent on dose(0–1625 kj/m²), time(0–78 h postirradiation) and cell density (10⁶-10⁷ cells/mL). Hemolysis decreased with increasing concentration of glutathione, hemoglobin or cell number, while the presence of pyruvate drastically enhanced it. Because scanning spectroscopy(200–700 nm) showed that hemoproteins and nicotinamide adenine dinucleotides were oxidized, cytoplasmic oxidative stress was implicated in the lytic mechanism. Further evidence of oxidation was obtained from electron micrographs, which revealed the formation of Heinz bodies near the plasma membrane. The data demonstrate that exposure of erythrocytes to UVA1, but not UVB or UVC, radiation causes oxidation of cytoplasmic components, which results in cytoskeletal damage and hemolysis.     

REASSESSMENT OF THE DIFFERENTIAL EFFECTS OF ULTRAVIOLET AND IONIZING RADIATION ON HIV PROMOTER: THE USE OF CELL SURVIVAL AS THE BASIS FOR COMPARISONS

Abstract: Effects of different radiation treatments on the human immunodeficiency virus-1 (HIV) promoter were reassessed for exposures comparable to those encountered in clinical or cosmetic practice, using survival of the host cell as a basis for comparisons. The exposures were performed with two ultraviolet radiation sources commonly used as medical or cosmetic devices (UVASUN 2000 and FS20 lamps), a germicidal (G15T8) lamp and an X-ray machine. The UVC component of the FS20 lamp was filtered out. The emission spectra of the lamps were determined. The characteristics of these sources allowed us to discriminate among effects of UVA1 (340–400 nm), UVB + UVA2 (280–340 nm) and UVC (254 nm) radiations. Effects of irradiation were ascertained using cultures of HeLa cells stably transfected with the HIV promoter linked to a reporter—chloramphenicol acetyl transferase—gene. The exposures used caused at least two logs of cell killing. In this cytotoxicity range, UVA1 or X radiations had no effect on the HIV promoter, whereas UVB + UVA2 or UVC radiations activated the HIV promoter in a fluence-dependent manner. Survivals following exposure to UVB + UVA2 or UVC radiation were (1) at the lowest measurable HIV promoter activation, 30 and 20%, respectively, (2) at one-half maximal activation, 6 and 3%, respectively and (3) at the maximal activation, 0.5 and 0.2%, respectively. The results suggest that, among the radiations studied, UVB is the most important modality from the viewpoint of its potential effects on HIV-infected individuals, since (1) UVA1 or X radiations have no effects on the HIV promoter, (2) human exposure to UVC radiation is infrequent and (3) human UVB exposure is very common.     

ISOLATION OF V79 FIBROBLAST CELL LINES CONTAINING ELEVATED METALLOTHIONEIN LEVELS THAT HAVE INCREASED RESISTANCE TO THE CYTOTOXIC EFFECTS OF ULTRAVIOLET-A RADIATION

Abstract Isolated clones of V79 Chinese hamster lung fibroblasts, selected for resistance against cadmium toxicity, were exposed to monochromatic 365 nm ultraviolet-A (UVA, 320 nm to visible light) radiation and examined for cell survival. All three of the Cd-resistant V79 clones (V79Cd) tested exhibited significant increases in survival after irradiation compared with control cultures similar to the increased survival observed in Zn acetate-induced V79 cells. Dose-modifying factors calculated for these survival experiments were all approximately 1.5. When characterized for steady-state levels of metallothionein (MT) mRNA and associated Cd-binding activity, all of the Cd-resistant V79Cd clones demonstrated elevated constitutive levels of both, implicating MT as the mechanism responsible for the observed cellular resistance to Cd and also to 365 nm UVA radiation. However, whereas levels of intracellular MT protein correlated with differences in survival against Cd, MT intracellular levels did not correlate well with protection against 365 nm UVA. Increased cell survival after exposure to 365 nm UVA radiation mediated by MT appeared to reach a threshold level and MT only provided a limited degree of protection. Since UVA radiation is known to cause cell death mediated through the intracellular generation of reactive oxygen species (ROS), these results suggest that the role of MT in ameliorating cellular photooxidative damage produced by UVA is by reducing intracellular ROS.     

PHOTOFRIN II PHOTOSENSITIZATION IS MUTAGENIC AT THE tk LOCUS IN MOUSE L5178Y CELLS

Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AlPcCl). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AlPcCl. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AlPcCl may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.     

An animal model for extracorporeal photochemotherapy based on contact hypersensitivity.

Recently, photopheresis was introduced as a specific immune suppressor in several T cell mediated disorders. In order to study photopheresis, animal models are indispensable. This report describes an easy to handle model for this purpose. It concerns the Wistar-derived rat with contact hypersensitivity (CHS), also a T cell mediated disorder that has already been studied extensively in several other fields of research. After subsequent exposure to 8-methoxypsoralen (8-MOP) and ultraviolet A radiation (UVA), white blood cells from CHS rats were intravenously injected into other syngeneic rats suffering from the same disorder. This treatment appears to be very efficacious in suppressing the immunological response against the applied contact allergen, 2,4-dinitrofluorobenzene (DNFB). Cells subsequently exposed to UVA and 8-MOP did not have any effect.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET LIGHT—INDUCED CELL KILLING AND MUTAGENESIS IN L5178Y MOUSE LYMPHOMA CELLS

Abstract The wavelength dependence of ultraviolet radiation-induced cell killing and mutagenicity in L5178Y mouse lymphoma cells has been determined from 235 nm to 313 nm. Cells were irradiated in phosphate buffered saline at 20°C. The amount of cell killing was determined by cloning in soft agar medium immediately after irradiation. Mutation frequency was determined, after a 3-day expression time, by cloning in soft agar medium in the presence and the absence of 5-bromo-2'-deoxyuridine (BrdUrd). The endpoint used to quantitate lethal effects was the exposure necessary to reduce the surviving fraction to 10%, while the endpoint for mutagenesis was the exposure necessary to increase the frequency of BrdUrd-resistant colonies ten-fold over the background level. Data were corrected for quantum energy and the action spectra for cell killing and mutagenesis were plotted as relative biological effectiveness per quantum vs wavelength, relative to the effect at 265.2 nm. Both action spectra show broad maxima at 270 nm, and are very similar to the action spectra determined by Rothman and Setlow (1979) for pyrimidine dimer formation and cell killing in V-79 cells.     

SPECTRAL DEPENDENCE OF UV-INDUCED IMMEDIATE AND DELAYED APOPTOSIS: THE ROLE OF MEMBRANE AND DNA DAMAGE

Abstract— The phototoxicity of each waveband region of UV radiation (UVR), i.e., UVA (32CM100 nm), UVB (290–320 nm) and UVC (200–290 nm), was correlated with an apoptotic mechanism using equilethal doses (10% survival) on murine lymphoma L5178Y-R cells. Apoptosis was qualitatively monitored for DNA "ladder" formation (multiples of 200 base pair units) using agarose gel electrophoresis, while the percentages of apoptotic and membrane-permeabilized cells were quantified over a postexposure time course using flow cytometry. The UVA1 radiation (340–400 nm) induced both an immediate (<4 h) and a delayed (>20 h) apoptotic mechanism, while UVB or UVC radiation induced only the delayed mechanism. The role of membrane damage was examined using a lipophilic free-radical scavenger, vitamin E. Immediate apoptosis and membrane permeability increased in a UVA1 dose-dependent manner, both of which were reduced by vitamin E. However, vitamin E had little effect on UVR-induced delayed apoptosis. In contrast, the DNA damaging agents 2,4- and 2,6-diaminotoluene exclusively induced delayed apoptosis. Thus, immediate apoptosis can be initiated by UVA 1-induced membrane damage, while delayed apoptosis can be initiated by DNA damage. Moreover, the results suggest that immediate and delayed apoptosis are two independent mechanisms that exist beyond the realm of photobiology.     

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm⁻² UVA, 0.07 and 0.9 J cm⁻² UVB, 20 n m β-PDH (pigment dispersing hormone) or 10 μ m SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo . Cultured cells were exposed to 250 μ m L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or β-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and β-PDH. The same responses to UVA and UVB were observed in vivo . SIN-1 did not induce pigment dispersion in the cell cultures. l-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by β-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.     

Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract— The fluoroquinolone(FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fle-roxacin, BAYy3118 (a recently developed monofluori-nated quinolone) and nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kj/m₂ UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4A_oC in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 norfloxacin ciprofloxacin lomefloxacin fleroxacin nalidixic acid. If cells were incubated after treatment for 1 h at 37_oC, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase, inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but that FQ-pho-tosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.     

Histopathological Changes in Liver and Heart Tissue Associated with Experimental Ultraviolet Radiation A and B Exposure on Wistar Albino Rats

This study aims to evaluate the influences of ultraviolet radiation A and B ( UVA + B) exposure on the liver and heart organs of albino rats. Female Wistar Albino rats, whose hair of the dorsal skin was shaved, were exposed to a combined UVA + B radiation for 2 h/day, for 4 weeks in order to be compared with the control group. Histopathological findings in vital organs (liver and heart) were evaluated. Tissues were fixed in 10% buffered formalin (pH = 7.2) and embedded in paraffin. The histopathological findings were examined on the H&E stained sections with light microscopy. The results show that the liver and the heart were injured in the UVA + B group. Liver tissue in the UVA + B group showed minimal vacuolation, enlargement of hepatocytes and bile duct proliferation, and the heart tissue showed hibernomas; uniform large cells resembling brown fat with coarsely granular to multivacuolated cytoplasm that is eosinophilic or pale with a small central nucleus. The number of hibernoma cases was significantly higher in the UVA + B group compared with the control group (P = 0.021). The control group showed normal liver and heart histology with normal adipose tissue in the pericardium. As a result, UVA + B exposure has toxic effects, especially on the liver and the heart of Wistar albino rats. UV radiation may cause such adverse effects in humans. Therefore, protection against the harmful effects of UV radiation is of significant importance for skin and organs.     

Early induction of binucleated cells by ultraviolet A (UVA) radiation: a possible role of microfilaments.

The effects of UVA (365 nm) radiation on the cellular distribution of F-actin and formation of binucleated cells have been studied using 3T3 Swiss albino mouse fibroblasts and V79 Chinese hamster fibroblasts. Ultraviolet A at biologically relevant fluences was found to disintegrate the actin filaments in the cells shortly (5 min) after irradiation, concomitant with the formation of cells with two nuclei. In 76-100% of the bi- and multinucleated cells the distribution of F-actin was clearly altered. Cells in GI phase of the cell cycle were most probably involved in the formation of binucleated cells. The disintegration of F-actin was presumably not due to depolymerization of F-actin to G-actin, as the amount of F-actin in the cells was unaltered after UVA exposure but rather due to direct breakage of the actin filaments. Ultraviolet B (297/302 nm) had no effect on the cellular distribution of microfilaments, not even at highly lethal fluences.     

Electroanalysis of Benzalkonium Chloride in Ophthalmic Formulation by Boron-doped Diamond Electrode

Benzalkonium chloride (BAK) is a mixture of alkyl benzyl dimethyl ammonium chlorides, which is used primarily as a biocide, surfactant, preservative, and antimicrobial agent in the pharmaceutical industry, in particular in ophthalmologic and nasal solutions. However, BAK may cause harmful consequences on the eye structures of the anterior segment. Control of BAK identity and content is necessary by applying a sensitive detection method. This study unravels the use of a glassy carbon (GC) electrode and a pristine boron-doped diamond electrode (BDD) for the detection of four BAK homologs in a non-aqueous medium using square wave voltammetry (SWV). The BDD provided more reproducibility of the oxidation potential than GC with a correlation coefficient of 0.999. The irreversible oxidation peak was very broad and deconvoluted into 3 peaks corresponding to C₁₂, C₁₄, and combined C₁₆–C₁₈ to reflect their concentration ratio in the mixture. The method was then extended to the detection of the C₁₂ homolog in the ophthalmic formulations with a limit of detection (LOD) of 0.4 μg/mL. The estimated BAK levels in three  ophthalmic formulations were in agreement with the specified values by the manufacturers. The results were validated by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, confirming the presence of a single homolog (C₁₂) in the eye drops.     

The Effect of Chronic Exposure to Artificial UVB Radiation on the Survival and Reproduction of Daphnia magna Across Two Generations

We examined the effects of daily (chronic) exposure to artificial UVB radiation on the survival and reproduction of Daphnia   magna over two generations. Control and experimental animals in each generation (parental and F1) were exposed to 16 h of UVA radiation and photosynthetically active radiation daily. In addition, experimental animals were exposed to 6 h of UVB during the middle of the light period. Survival and reproduction were followed for 12 days for each individual. Survival and production of F1 were significantly lower in the UVB exposed parental generation Daphnia than in controls. F1 exposure to UVB significantly decreased F1 survival and reproduction. Reproduction was lowest in UVB exposed F1 animals whose parents were also exposed to UVB. Adverse effects of UVB on offspring production may be magnified in successive generations suggesting that short-term experiments could underestimate the impact of increased UVB exposure on populations.