Effects of Salinity and Ultraviolet Radiation on the Bioaccumulation of Mycosporine‐like Amino Acids in Artemia from Lake Urmia (Iran)

We investigated the effects of salinity and artificial UV radiation on the accumulation of mycosporine‐like amino acids (MAAs) in sexual and parthenogenetic Artemia from Lake Urmia. The nauplii hatched from the cysts were cultured until adulthood under two salinities (150 and 250 g L^?1) and two light treatments (PAR and PAR+UVR) in the laboratory. Finally, the Artemia were analyzed for their concentration of MAAs. In most of the cases, the higher salinity level applied was found to increase the MAA concentrations in both Artemia populations significantly. The acquisition efficiency of MAAs in both Artemia populations increased under exposure to UVR‐supplemented photosynthetically active radiation (PAR) compared to those raised under PAR, except for Porphyra‐334. It was observed that combination of UV radiation and elevated salinity significantly increased the bioaccumulation of MAAs. Thus, the presence of these compounds in these populations of Artemia may increase their adaptability for living in high‐UV and high‐salinity conditions prevailing in Lake Urmia. Higher concentrations of MAAs in the parthenogenetic population of Artemia could be probably attributed to its mono sex nature and higher adaptation capacities to extreme environmental conditions.     

Mycosporine-like Amino Acids in Freshwater Copepods: Potential Sources and Some Factors That Affect Their Bioaccumulation

Mycosporine-like amino acids (MAAs) are ubiquitous photoprotective compounds in aquatic environments. MAAs are synthesized by a wide variety of organisms (i.e. bacteria, fungi and algae) and their production is photoinducible by ultraviolet radiation (UVR) (280–400 nm) and/or photosynthetically active radiation (400–750 nm). Most animals however, are unable to synthesize MAAs and must acquire these compounds through their diet or from symbiotic organisms. In this paper, we investigate the possible sources of MAAs and factors (temperature and initial MAA concentration) that may affect their bioaccumulation in freshwater copepods. We found that MAA accumulation may occur even if the copepods are cultured on a MAA-free diet. In addition, we found that the bacteriostatic antibiotic, chloramphenicol, inhibits the bioaccumulation of MAAs. These two pieces of evidence suggest that the source of MAAs in these copepods may be prokaryotic organisms in close association with the animals. The two factors investigated in this study, temperature and initial MAA concentrations, were found to affect the rates at which MAAs are accumulated. Temperature had positive effects on both uptake and elimination rates. On the other hand, the rate of uptake decreased at the highest assayed initial MAA concentration, probably because the concentration of MAAs was already close to saturation.     

Cellular Changes Associated with the Acclimation of the Intertidal Sea Anemone Actinia tenebrosa to Ultraviolet Radiation

To assess the relative importance of long‐ and short‐term cellular defense mechanisms in seasonally UV‐R‐acclimated Actinia tenebrosa (Anthozoa, Actiniidae), individuals were exposed to summer doses of PAR, UV‐A, UV‐B and enhanced UV‐B (20%) for a period of 4 days. Mycosporine‐like amino acids (MAAs) and cyclobutane pyrimidine dimer (CPD) concentrations were quantified, while oxidative damage to lipids and proteins, and the activities or levels of the antioxidant enzymes SOD, CAT, GR, GPOX and total glutathione were determined. Our results show that summer UV‐R‐acclimated individuals had a higher UV‐R tolerance, with no significant increases in CPDs levels, than winter‐acclimated sea anemones possibly due to higher MAA concentrations. Summer‐acclimated individuals showed increased lipid and protein oxidation and GPOX activity only when they were exposed to UV‐B at 20% above ambient UV‐R levels. In contrast, winter‐acclimated sea anemones showed elevated levels of oxidative damage, GPOX and SOD activities after exposure to UV‐A or UV‐B at ambient and elevated levels. Thus, this study indicates that long‐term UV‐R acclimation mechanisms such as the accumulation of MAAs could be more important than short‐term increases in antioxidant defenses with respect to reducing indirect UV‐R damage in intertidal sea anemones.     

Mycosporine-like amino acids in the marine dinoflagellate Gyrodinium dorsum: induction by ultraviolet irradiation

Cultures of the marine dinoflagellate Gyrodinium dorsum have been exposed to polychromatic radiation (photosynthetically active radiation and UV) from a solar simulator for up to 72 h. Different irradiance spectra in the ultraviolet are produced by inserting cut-off filters between lamp and samples. The mycosporine-like amino acid (MAA) content and composition are investigated by spectroscopic and chromatographic analysis. The study reveals that G. dorsum contains a complex mixture of several aminocyclohexenimine-MAAs and one aminocyclohexenone-MAA. UV irradiation around 320 nm induces an increase in the concentration of all MAAs in the samples. In contrast, exposure to short-wavelength UV-B radiation results in decreased overall MAA production. Furthermore, there is a spectral shift in the absorption of the MAA mixture towards shorter wavelengths, indicating that short-wavelength UV-B induces an altered MAA composition. The amount of MAAs is normalized to the chlorophyll a concentration.     

Can Mycosporine‐Like Amino Acids Act as Multifunctional Compounds in Gymnodinium catenatum (Dinophyceae)?

MAAs originating from Gymnodinium catenatum were subjected to H₂O₂ oxidation, light and heat. Shinorine and porphyra‐334 were the more resistant to all treatments, mycosporine‐glycine (MYGL) was the least resistant to oxidation and heat, whereas palythene and M‐370 were the least resistant to light. MYGL and M‐311 were similarly resistant to photodegradation and oxidation in the dark and low temperature, but M‐311 was more resistant to oxidation under light or heat. The ratio M‐370/M‐365 changed from 29:1 to 6:1 ratio after 240 h of exposure to fluorescent light, indicating that M‐365 could represent the M‐370 cis‐isomer. The role of MAAs as antioxidants and/or osmolytes was evaluated by studying effects of abrupt salinity reduction. Both increases or decreases in concentrations were observed and were dependent on the MAA initial concentration and its chemical structure. The relative increase in MAAs with a known antioxidant capacity (MYGL, palythene) followed an exponential decay trend related to initial concentration. The relative decrease in highly polar MAAs (shinorine, porphyra‐334, M‐332) with a suspected osmolyte role followed a rise to a maximum with the increase in initial concentration. Whether or not MAAs play a significant role in osmoregulation, their loss can occur upon hypoosmotic shock.     

Novel glycosylated mycosporine-like amino acids with radical scavenging activity from the cyanobacterium Nostoc commune

Mycosporine-like amino acids (MAAs) are UV absorbing pigments, and structurally distinct MAAs have been identified in taxonomically diverse organisms. Two novel MAAs were purified from the cyanobacterium Nostoc commune, and their chemical structures were characterized. An MAA with an absorption maximum at 335 nm was identified as a pentose-bound porphyra-334 derivative with a molecular mass of 478 Da. Another identified MAA had double absorption maxima at 312 and 340 nm and a molecular mass of 1,050 Da. Its unique structure consisted of two distinct chromophores of 3-aminocyclohexen-1-one and 1,3-diaminocyclohexen and two pentose and hexose sugars. These MAAs had radical scavenging activity in vitro; the 1050-Da MAA contributed approximately 27% of the total radical scavenging activities in a water extract of N. commune. These results suggest that these glycosylated MAAs have multiple roles as a UV protectant and an antioxidant relevant to anhydrobiosis in N. commune.     

An unusual case of photoreactivation observed in an insect egg (Smittia Spec., chironomidae, diptera).

Abstract— An action spectrum for inactivation by ultraviolet (UV) radiation of Smittia eggs during intravitelline cleavage was established, taking into account wavelength-dependent shielding of the effective targets. Under the assumption of a random distribution of the effective targets in the egg, the action spectrum displayed only one very distinct peak at 295 nm. The eggs were photoreactivable with an action spectrum similar but not identical to that found for direct photoreactivation (PR) in E. coli Indirect PR seems not involved because light was effective only after but not before UV. Temperature dependence and dose rate saturation could not be observed. The photoreactivable sector (PRS) was 0.75 after UV inactivation at 295 nm but only 0.32 after UV inactivation at 265 nm. Initial PR rates were highest after 295 nm and lowest after 265 nm. During migration of cleavage nuclei into the periplasm, when the shielding of nuclei by yolk material decreases by an order of magnitude, no corresponding increase in the sensitivity of the eggs to UV was observed. After inactivation at the blastoderm stage, when the nuclei are no longer shielded by yolk material, the PRS was also high (0.79) after UV of 295 nm but again lower (0.59) after 265 nm. These data are difficult to understand within the conceptual framework of light-dependent enzymatic splitting of UV-induced pyrimidine dimers in nucleic acids. Yet this type of PR seems to play a vital role in the survival of Smittia eggs under sunlight without need for pigmentation or shading.     

Effects of abiotic stressors on synthesis of the mycosporine-like amino acid shinorine in the Cyanobacterium Anabaena variabilis PCC 7937

In the present investigation we show that the cyanobacterium Anabaena variabilis PCC 7937 produces a single mycosporine-like amino acid (MAA), shinorine (retention time = 2.3 min and absorption maximum at 334 nm) when isolated and purified by HPLC. Although there was significant induction of MAA synthesis from its initial value under 395 or 320 nm cutoff filters, MAA induction was significantly more pronounced in samples covered with 295 nm cutoff filters after 72 h of exposure. Heat as a stress factor had no effect on MAA induction with or without UV radiation. In contrast, salt and ammonium treatment had synergistic effects with UV stress. MAA synthesis was also induced by salt and ammonium in a concentration-dependent manner without UV stress in samples covered with 395 nm cutoff filters. The results indicate that MAAs may have other functions in addition to photoprotection in this organism.     

Isolation and Structural Elucidation of Novel Mycosporine‐Like Amino Acids as Alarm Cues in the Defensive Ink Secretion of the Sea Hare Aplysia californica

Three new mycosporine‐like amino acids (MAAs), aplysiapalythines A, B, and C ( 1, 2 , and 3 , resp.) were isolated from opaline, a glandular component of the defensive ink secretion of sea hares (Aplysia californica) collected from waters off southern California. Here, we report the structure of these MAAs determined by mass spectrometry and NMR data. These new MAAs are structurally related to two known MAAs that are also present in sea hare opaline, i.e., asterina 330 ( 4 ) and palythine ( 5 ), and for which we also provide detailed data here for comparison. The fact that three of the five MAAs that we identified from sea hare opaline are novel molecules is interesting given that this represents a relatively large addition to the current list of known MAAs. This is likely because most researchers have identified MAAs through HPLC with UV detection, which is imprecise given similarities in UV spectra for different MAAs. Our findings suggest that there is much greater diversity of MAAs than is currently known. Results published elsewhere show that 1, 2 , and 4 are alarm cues for conspecific sea hares at natural concentrations, but 3 and 5 are not.     

Effects of Light and Salinity Stresses in Production of Mycosporine‐Like Amino Acids by Gymnodinium catenatum (Dinophyceae)

Mycosporine‐like amino acids (MAAs) were analyzed in a Portuguese Gymnodinium catenatum strain when transferred to high salinity and high light conditions. Total MAA concentrations increased progressively between 30 and 36 psu, attaining at 36 psu 2.9‐fold the 30 psu treatment. When abruptly transferred to solar light in an outdoor shadowed location, MAA concentration increased steadily along the day for most compounds. After 8 h, mycosporine–glycine, palythene and M‐319 attained or surpassed 25‐fold their initial concentration, while M‐370 only attained 4‐fold concentration. When transferred from halogen to fluorescent light, polar MAAs such as shinorine and porphyra‐334, increased until day two and then declined, while M‐370 increase slowly, becoming the dominant compound from the profile after 1 week. These experiments put into evidence the relation of palythene with M‐319, which was further identified as its acid degradation product, palythine. Acid degradation of M‐370 originated M‐324, while M‐311 seems to be the precursor of M‐370. Under high salinity and high light conditions chain formation was altered toward shorter chains or solitary cells. This alteration can represent a morphological stress sign, which in the natural environment could affect average population speed during daily vertical migrations.     

Gas chromatographic-mass spectrometric assay for 2-methoxyethanol and 2-methoxyacetic acid in human plasma and its application to pharmacokinetic studies

Summary A new assay has been developed for simultaneous measurement of 2-methoxyethanol (ME) and its major toxic metabolite, 2-methoxyacetic acid (MAA) in human blood by gas chromatography-mass spectrometry. Over the concentration range 0.3 to 200 μgml⁻¹, ME and MAA could be determined with a pooled coefficient of variation (eleven concentrations, six replicate samples) of 6.5% and 8.7%, respectively. The limits of quantification of ME and MAA were 0.19 and 0.05μg mL⁻¹, respectively. The respective mean (SD) recoveries of ME and MAA were 101.3 (2.6)% and 98.2 (4.9)% (n=3). A study of the effects of human contact with ME liquid showed that this new method is a specific, sensitive, simple, and reliable method both for the pharmacokinetic studies and for the biological monitoring of occupational exposure to ME.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Sulfur Deficiency Changes Mycosporine-like Amino Acid (MAA) Composition of Anabaena variabilis PCC 7937: A Possible Role of Sulfur in MAA Bioconversion

In the present investigation we show for the first time that bioconversion of a primary mycosporine-like amino acid (MAA) into a secondary MAA is regulated by sulfur deficiency in the cyanobacterium Anabaena variabilis PCC 7937. This cyanobacterium synthesizes the primary MAA shinorine (RT = 2.2 min, λ_max = 334 nm) under normal conditions (PAR + UV-A + UV-B); however, under sulfur deficiency, a secondary MAA palythine-serine (RT = 3.9 min, λ_max = 320 nm) appears. Addition of methionine to sulfur-deficient cultures resulted in the disappearance of palythine-serine, suggesting the role of primary MAAs under sulfur deficiency in recycling of methionine by donating the methyl group from the glycine subunit of shinorine to tetrahydrofolate to regenerate the methionine from homocysteine. This is also the first report for the synthesis of palythine-serine by cyanobacteria which has so far been reported only from corals. Addition of methionine also affected the conversion of mycosporine-glycine into shinorine, consequently, resulted in the appearance of mycosporine-glycine (RT = 3.6 min, λ_max = 310 nm). Our results also suggest that palythine-serine is synthesized from shinorine. Based on these results we propose that glycine decarboxylase is the potential enzyme that catalyzes the bioconversion of shinorine to palythine-serine by decarboxylation and demethylation of the glycine unit of shinorine.     

Photodegradation and photosensitization of mycosporine-like amino acids

The photodegradation and photosensitization of several mycosporine-like amino acids (MAAs) were investigated. The photodegradation of the MAA, palythine, was tested with three photosensitizers: riboflavin, rose bengal and natural seawater. For comparison of degradation rates, the riboflavin-mediated photosensitization of six other MAAs was also examined. When riboflavin was used as a photosensitizer in distilled water, MAAs were undetectable after 1.5h. Palythine showed little photodegradation when rose bengal was added as the photosensitizer (k=0.12x10(-3)m(2)kJ(-1)). Palythine dissolved in natural seawater containing high nitrate concentrations also showed slow photodegradation rate constants (k=0.26x10(-3)m(2)kJ(-1)) over a 24-h period of constant irradiation. Similar experiments in deep seawater with porphyra-334 and shinorine resulted in 75% of the initial MAA remaining after 4h of irradiation and rates of 0.018 and 0.026x10(-3) m(2) kJ(-1), respectively. Experiments conducted in deep seawater with riboflavin additions resulted in photodegradation rate constants between 0.77x10(-3) and 1.19x10(-3)m(2)kJ(-1) for shinorine and porphyra-334, respectively. Photoproduct formation appeared to be minimal with the presence of a dehydration product of the cycloheximine ring structure indicated as well as the presence of amino acids. Evidence continues to build for the role of MAAs as potent and stable UV absorbers. This study further highlights the photostability of several MAAs in both distilled and seawater in the presence of photosensitizers.     

Photoprotection and long-term acclimation to UV radiation in the marine diatom Thalassiosira weissflogii.

Unialgal cultures of the marine diatom Thalassiosira weissflogii (Grunow) were exposed for 40 days to artificial UV-B radiation in the presence of PAR and UV-A to examine long-term acclimation to UV, PAR and UV-A were supplied 14 h daily, while UV-B (two levels: 0.16 and 0.30 W m(-2) unweighted) was supplied for 4 h/day. Growth rates and photochemical capacity (CFC ratio) both decreased over the first 10-15 days, then recovered. No obvious differences were noted between the responses to the two UV-B treatments. The concentration of the major pigments (chlorophyll a and c(1+2), fucoxanthin and beta,beta-carotene) changed very little with time, except for diatoxanthin. which increased over the first 16 days, decreased over the next 13 days, then increased again from day 29 to the end of the experiment. The concentration of total mycosporine-like amino acids (MAAs) was initially undetectable, then increased from day 16 in the high UV-B treatment and after day 22 in the low UV-B treatment, reaching a maximum on day 29 for both treatments and decreasing afterwards. The synthesis of MAAs proceeded only once photochemical capacity had recovered from the initial UV stress and this recovery likely involved the xanthophyll cycle (diatoxanthin increase). The concentration of MAAs decreased when the cells showed signs of photoinhibition (decrease in CFC ratio). It also showed an inverse trend with diatoxanthin. UV-B alone had little regulatory effect over these responses, except possibly for an earlier synthesis of MAAs under HUV-B conditions. This suggests that the observed changes were due to UV-A rather than to UV-B exposure. The overall response of this coastal diatom to prolonged UV exposure indicates that T. weissflogii is a relatively UV-tolerant species and that its long-term response to UV exposure involves an activation of the xanthophyll cycle followed by the synthesis of MAAs, which may proceed only when photoinhibition is relieved.     

Ultraviolet (280-400 nm)-induced DNA damage in the eggs and larvae of Calanus finmarchicus G. (Copepoda) and Atlantic cod (Gadus morhua)

In previous work, we evaluated the effects of ultraviolet (UV = 280-400 nm) radiation on the early life stages of a planktonic Calanoid copepod (Calanus finmarchicus Gunnerus) and of Atlantic cod (Gadus morhua). Both are key species in North Atlantic food webs. To further describe the potential impacts of UV exposure on the early life stages of these two species, we measured the wavelength-specific DNA damage (cyclobutane pyrimidine dimer [CPD] formation per megabase of DNA) induced under controlled experimental exposure to UV radiation. UV-induced DNA damage in C. finmarchicus and cod eggs was highest in the UV-B exposure treatments. Under the same spectral exposures, CPD loads in C. finmarchicus eggs were higher than those in cod eggs, and for both C. finmarchicus and cod embryos, CPD loads were generally lower in eggs than in larvae. Biological weighting functions (BWF) and exposure response curves that explain most of the variability in CPD production were derived from these data. Comparison of the BWF revealed significant differences in sensitivity to UV-B: C. finmarchicus is more sensitive than cod, and larvae are more sensitive than eggs. This is consistent with the raw CPD values. Shapes of the BWF were similar to each other and to a quantitative action spectrum for damage to T7 bacteriophage DNA that is unshielded by cellular material. The strong similarities in the shapes of the weighting functions are not consistent with photoprotection by UV-absorbing compounds, which would generate features in BWF corresponding to absorption bands. The BWF reported in this study were applied to assess the mortality that would result from accumulation of a given CPD load: for both C. finmarchicus and cod eggs, an increased load of 10 CPD Mb(-1) of DNA due to UV exposure would result in approximately 10% mortality.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) I. Photosensitized production and light-dependent disappearance of pyrimidine dimers

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength-dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV-irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono-merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light-dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.     

PHOTODYNAMIC INHIBITION OF ESCHERICHIA COLI DNA POLYMERASE I BY 8-METHOXYPSORALEN PLUS NEAR ULTRAVIOLET IRRADIATION

Abstract— Morphological abnormality due to the UV irradiation of sperm and its modification by photoreactivation (PR) were studied in the sea urchin, Hemicentrotus pukherrimus. When sperm was UV-irradiated and allowed to fertilize unirradiated eggs, the effect of the UV was manifested as an abnormal morphology of embryos in the gastrula or later stages. The UV-induced morphological abnormality was prevented by photoreactivation when the fertilized eggs were illuminated with visible light. In the experiments on a stage-dependent change of PR effectiveness, it was found that an illumination sufficed to effect a nearly complete PR when applied up to the onset of the first DNA synthetic phase, while the PR effectiveness declined thereafter. Illumination after the completion of DNA synthesis had little effect for PR.     

Synthesis and application of carbohydrate-derived morpholine amino acids

The synthesis of series of diversely functionalized epsilon-morpholine amino acids (MAAs, 5a-h) starting from an epsilon-sugar amino acid and following a two-step oxidative glycol cleavage/reductive amination strategy, is described. In an alternative synthetic scheme, diastereoisomerically pure delta-MAAs (12a,b) were obtained. Oligopeptides containing MAAs were prepared either by direct incorporation of a MAA building block or by subjecting a fully assembled SAA-containing peptide to the two-step glycol cleavage/reductive amination procedure.     

Sunscreens Protect from UV-Promoted Squamous Cell Carcinoma in Mice Chronically Irradiated with Doses of UV Radiation Insufficient to Cause Edema

Previously we reported that the broad-spectrum sunscreen microfine titanium dioxide (MTD) could completely protect C3H/HeJ mice from UV radiation-induced immunosuppression to a contact sensitizer. In contrast, 2-ethylhexyl p-methoxycinnamate (2-EHMC), a UVB-absorbing sunscreen, only partially protected the skin immune system. In this study we investigated further this differential protection of the skin immune system by comparing the ability of 2-EHMC and MTD to protect these mice from the promotion phase of tumorigenesis. The mice were initiated using a single subcarcinogenic dose of 7,12-dimethylbenz(a)anthracene (DMBA) followed by promotion with chronic low-dose solar-simulated UV radiation for 32 weeks. We used doses of UV insufficient to cause edema in order to simulate daily human exposure to solar UV radiation. Mice were observed for the appearance of squamous cell carcinomas for 48 weeks. The DMBA-initiation alone and DMBA-initiated, sunscreen-treated groups did not develop tumors. Ultraviolet alone induced the appearance of tumors in 46% of mice at week 48 and therefore some tumors were initiated by UV. Initiation with DMBA prior to UV irradiation enhanced tumorigenesis such that 87% of mice at week 48 had tumors. Both 2-EHMC and MTD completely protected these mice from UV-induced promotion as well as from complete carcinogenesis despite the different UV-absorption spectra of the sunscreens and their differential abilities to protect from UV-induced immunosuppression. Furthermore, we have shown that, if UV exposure is not increased to compensate for tolerance to edema, protection from tumorigenesis is afforded by sunscreens.