Photoreduction of methemoglobin by irradiation in the near-ultraviolet region

Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O(2) binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi transitions, whereas the photoreduction did not occur in Ar, N(2), or O(2). The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O(2) bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.     

Nucleosomal distribution of thymine photodimers following far- and near-ultraviolet irradiation

The nucleosomal distribution of cis-syn cyclobutyl-type thymine photodimers was determined in normal human skin fibroblasts following irradiation with low doses of far-ultraviolet light at 254 nm and nearultraviolet light at 313 nm. The thymine photodimer concentrations were determined by high pressure liquid chromatography in acid hydrolysates of total cellular DNA and of nucleosomal core- and chromatosomal-DNA. The lesion concentrations in linker-DNA were calculated from these data. While thymine photodimers were distributed uniformely following 254 nm irradiation they were enriched by a factor of 2.4 – 4.2 in nucleosomal linker DNA after exposure to 313 nm light.     

Method for the isolation of Escherichia coli relaxed mutants, utilizing near-ultraviolet irradiation.

Near-ultraviolet radiation (300 to 380 nm) induces a transient growth inhibition in "stringent" (rel+) strains of Escherichia coli, whereas "relaxed" (rel-) strains are only mildly affected. This difference permits rapid isolation of large numbers of relaxed mutants of E. coli.     

Changes in polyphosphate composition and localization in Propionibacterium acnes after near-ultraviolet irradiation.

Electron microscopy showed that electron-dense granules accumulated in Propionibacterium acnes in larger amounts when the bacteria were grown on a phosphate-rich medium. X-ray microanalysis demonstrated that the granules contained mostly phosphorus and potassium, indicating that the cells contained polyphosphate granules. When cells were grown on a complex Bacto-agar medium, the amount and the size of the polyphosphate granules were reduced. Polyphosphate was also detected with 31P nuclear magnetic resonance (31P-NMR). Of the polyphosphates observed with 31P-NMR, 20% seemed to be located outside the cell membrane. Broad-band near-ultraviolet irradiation (emission maximum 366 nm) corresponding to doses that killed 37% of the cells increased the amount of polyphosphate in cells grown on the phosphate-rich medium. The fluorescent chromophore 4',6-diamidino-2-phenylindole (DAPI) shifted the fluorescence emission from 478 to 538 nm when bound to polyphosphate and excited at 340 nm. DAPI was used to detect polyphosphates generated after near-ultraviolet irradiation of the cells. Nonirradiated cells showed no increased fluorescence at 538 nm, indicating no polyphosphate is presented in the cells. We conclude that DAPI did not have "access" to the intracellular polyphosphate as long as the cells were not light damaged. This observation is important for the interpretation of near-UV damage to cells.     

A 31P-NMR study of Propionibacterium acnes, including effects caused by near-ultraviolet irradiation

161.8 MHz 31P-NMR spectra were recorded from the light sensitive skin bacterium Propionibacterium acnes. The cells were grown anaerobically on synthetic phosphate-buffered Eagle's medium or on a complex yeast extract medium. The spectra showed a large accumulation of polyphosphates when grown on Eagles medium. A splitting of the inorganic phosphate peak indicated a difference between internal and external pH of the cells. Addition of glucose to the cell suspension gave rise to a change in the pH gradient across the cell membrane, as reported for other Gram-positive bacteria. A decrease in the polyphosphate peak was observed after addition of glucose. A lethal dose of broad-band near-ultraviolet light (corresponding to a 10% survival in a survival test), increased the amount of polyphosphates visible in the NMR-spectra. The addition of glucose to irradiated cells decreased the pH in the external solution, but no splitting of the inorganic phosphate peak could however be observed. 31P-NMR can, therefore, be used to study immediate near-ultraviolet-induced effects at the cellular level, at least in the case of P. acnes.     

Formation of directly mutagenic alpha-hydroxy-N-nitrosopiperidine phosphate ester by near-ultraviolet irradiation of N-nitrosopiperidine in phosphate buffer

Previously we found that direct-acting mutagens can be formed from N-nitrosodialkylamines on exposure to near-ultraviolet light in the presence of phosphates. We have now isolated the mutagenic photoproduct formed from N-nitrosopiperidine and inorganic phosphate and identified its structure as the phosphate ester of alpha-hydroxy-N-nitrosopiperidine. This reaction represents a new, non-enzymatic activation of promutagenic N-nitrosodialkylamines.     

Repair in E. coli of transforming plasmid DNA damaged by psoralen plus near-ultraviolet irradiation

Treatment of DNA with psoralen plus near-ultraviolet irradiation gives rise to both monoadducts and cross-links. We have examined the repair of plasmid NTP16 DNA treated in this way in vitro and then used to transform E. coli. Monoadducts are found to be potentially lethal, and can be repaired by uvr-dependent and recA-dependent pathways. The presence of a related resident plasmid in the transformed cells can enhance the survival of the incoming damaged NTP16 DNA. This effect is not recA-dependent, and a similar effect (designated resident enhanced repair) has been observed previously with UV-irradiated plasmids of this particular incompatibility group. Removal of unbound psoralen from the plasmid DNA and exposure to further NUV is known to increase the ratio of cross-links to monoadducts, and we demonstrate that such cross-linked plasmid DNA is not readily repaired following transformation. However in the presence of homologous DNA (related resident plasmid) there is evidence for the repair, and hence uptake by the cell, of cross-linked DNA.     

Mutagenicity of particulates from the laboratory combustion of plastics

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (nitro-PAHs) have been identified in airborne particulate organic matter extracts. The pollutant sources were generally contributed by motor vehicles and industrial activity. Massive quantities of urban solid wastes, containing plastic materials such as PVC, PET, PS, and PE, burnt in the open air in local garbage dumps are frequently found in developing countries. In this study, the smog particulates from the combustion of these synthetic polymers were produced in a laboratory combustion chamber. The mutagenicity of acetone extracts from the smog particulates was evaluated with Salmonella typhimurium TA98 and TA100 in the presence and absence of S9 mix. Four samples in TA98 exhibited higher mutagenicity than those in TA100. The greatest mutagenicity was observed from the extracts of particulates from combustion of PVC followed by that of PS, PET, and PE. To determine the major mutagenic compounds in these samples, mutagens were partially purified through TLC and their mutagenicity was monitored with TA98. 1-NP and DNPs in the above samples were also determined by HPLC. The amounts of 1-NP and DNPs generally corresponded with their mutagenicity. Higher levels of 1-NP and DNPS from the combustion of PVC, PET, and PS. the combustion of synthetic polymer wastes might be responsible for the presence of high levels of 1-NP and DNPs in Taiwan urban air.     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

Mutagenicity of extracts from typewriter ribbons and related items

Extracts of typewriter ribbons and carbon papers were found to be mutagenic in the Salmonella/microsome assay with strain TA98. Fractionation of ribbon extracts indicates that at least 2-3 different classes of mutagenic component are present in these extracts. Nitro-containing compounds may be responsible for the high mutagenicity observed for some of the ribbon extracts in the absence of S9. The results indicate that impurities in the products may be causing part of the mutagenic effect.     

Mutagenicity of emissions from a natural gas fueled truck

Concern about the potential health risks of particulate exhaust emissions from diesel-fueled vehicles has led regulatory agencies to foster the use of natural gas fueled heavy duty vehicles. However, the potential health risks of particulate exhaust emissions from natural gas fueled vehicles have not been well-studied. The present study investigated the mutagenicity of particulate exhaust emissions from a natural gas fueled refuse truck currently in-service. Organic solvent extracts of exhaust particulate emissions from the natural gas fueled truck were positive in both Salmonella tester strains TA98 and TA100 in the presence and absence of S-9. The maximum mutagenic responses ranged from 7-fold in the TA100 strain to 87-fold in the TA98 strain when compared to negative controls. Our results show that current in-service natural gas fueled heavy duty trucks have particulate exhaust emissions that possess mutagenic activity. This finding requires follow-up studies to develop a database on natural gas fueled vehicles for comparison with data on diesel-fueled vehicles to aid in making decisions on use of alternative fuels to reduce air pollution health risks.     

Mutagenicity of pan residues and gravy from fried meat

Lean pork meat was fried with or without the addition of frying-fat at 200 or 250 degrees C. The pan residues were collected by washing the hot pan with boiling water. When producing thickened gravy the water was substituted by a mixture of water and flour, milk and flour or cream and flour. The basic extracts were tested for mutagenicity in Ames' Salmonella test on strain TA98 with the addition of S9 mix. High amounts of mutagenicity were found in all samples. The amounts of mutagenicity in the pan residues were at a comparable level of the amounts found in the meat crusts. Thickening of the gravy caused only small changes in the mutagenicity.