Near-ultraviolet photolysis of beta-phenylpyruvic acid generates free radicals and results in DNA damage.

Ultraviolet A (UVA) light (315-400 nm) is ubiquitously found in our environment and constitutes about 95% of the total solar UV; all UVC and most UVB being absorbed by the stratospheric ozone layer. Compared with UVB and C, UVA does not show any direct effect on biological systems. Indirect effects of UVA, however, have been recognised overwhelmingly and this includes photosensitization of biological and non-biological compounds and production of free radicals many of which include oxygen and are hence known as reactive oxygen species or ROS. Several types of free radicals have been identified although their impacts on various macro- and micro-biomolecules are yet to be fully elucidated. beta-Phenylpyruvic acid is ubiquitously found in eukaryotic cells as a metabolite of phenylalanine, which is subsequently converted to phenyllactate and/or to 2-hydroxyphenylacetate and mandelate. In patients suffering from phenylketonuria the hydroxylation of phenylalanine to tyrosine is defective due to lack of phenylalanine hydroxylase. These result in accumulation and excretion of this compound in the urine. Here we present evidence that photolysis of beta-phenylpyruvic acid by a skin tanning lamp, emitting 99% UVA (315-400 nm) and 1% UVB (290-315 nm) generates carboxyl radicals (CO(2)(*)) and also possibly causes direct electron transfer (or type 1) reactions. Electron paramagnetic resonance was used to detect the free radicals. To determine the biological effects of this photolytic reaction, T7 was exposed to these photolytic reactive agents and found to lead to high levels of phage inactivation. Damage to DNA and/or components such as tail fibre proteins may be involved in T7 inactivation. In addition, our unpublished data suggest that certain phenylketonuria cell lines are more sensitive to PPA+NUV, lending importance to photolytic studies of this agent.

Near-ultraviolet photolysis of L-mandelate, formation of reactive oxygen species, inactivation of phage T7 and implications on human health.

Compared with ultraviolet B and C, UVA is considered to have little direct effects on biological systems. However, damaging effects of UVA on biological systems are often synergistically enhanced in the presence of sensitizers. Production of reactive oxygen species (ROS) has been implicated in the process. Several ROS have been identified but their involvement in inducing cellular damage is yet to be fully evaluated. Although membranes and proteins are affected, DNA is an important target and a variety of types of damage have been reported. Here, we present evidence that L-mandelate can act as a near UV (NUV) sensitizer, when activated by a lamp emitting 99% UVA and 1% UVB. Although evidence is available that H(2)O(2) and a small amount of *OH are produced, an alternative effect of the sensitization reaction may involve direct electron transfer. Studies have shown that NUV photolysis of mandelate can inactivate phage T7. Employment of tetrazolium blue test to detect superoxide anion may not be sufficient evidence as this agent may be reduced by alternative routes.

Localization of monoamine oxidase mRNA in human placenta.

Monoamine oxidase (MAO) oxidatively deaminates vasoactive and biogenic amines and exists in two distinct forms (A and B), coded for by separate genes, which exhibit distinct substrate specificities and inhibitor sensitivities. Using specific primers for MAO-A and MAO-B mRNA in a reverse transcription-polymerase chain reaction (RT-PCR) on RNA from human liver, the predicted products for both enzymes were detected. Furthermore, RT-PCR on RNA from human placenta, believed to contain predominantly (or only) MAO-A protein, also indicated the presence of both A and B gene transcripts. The cellular distribution of MAO mRNA in placental tissue was analyzed by in situ hybridization of MAO-A and MAO-B mRNA-specific cRNA probes on paraffin sections. MAO-A mRNA was mainly evident in the syncytiotrophoblastic layer. None was detected in the vascular endothelium/smooth muscles. Significantly, MAO-B mRNA signal was also evident in the placental villi, notably in the syncytiotrophoblasts, intermediate trophoblasts, cytotrophoblasts, and the vascular endothelium. To our knowledge, this is the first demonstration of the cellular distribution of MAO mRNA in human placenta via in situ hybridization. The expression of MAO-B in placental tissue rather than in blood elements within placenta is also unequivocally demonstrated. These highly specific cRNA probes can now be used to study the distribution of MAO-A and MAO-B expression in other tissues.

Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system.

Near-ultraviolet (NUV) light (280-400 nm) has a variety of effects on biological systems; these effects are increased, often synergistically, in the presence of sensitizers. A variety of both man-made and naturally occurring sensitizers have been identified, but their precise roles and relative contributions to cellular damage are not yet fully established. DNA seems to be a major target and a variety of types of damage have been observed. In this report we present evidence that histidine can also act as a sensitizer of NUV. Upon NUV photolysis a variety of reactive oxygen species, including superoxide anions, hydroxyl radicals and hydrogen peroxide, are produced as determined by the effects of various scavengers. pH influences the reaction, alkaline media being most effective, as has previously been reported for the photolysis of H2O2, tyrosine, phenylalanine and tryptophan. Exposure of phage T7 to a combination of histidine and NUV leads to synergistic inactivation and scavengers of O2.-, .OH and H2O2 reduce this effect. These results point to a possible involvement of sunlight-induced histidine photolysis in cellular damage. The fact that photolysis is maximal at high pH indicates that biological effects are likely to be highly localized, e.g., at enzyme active sites.

Thymine metabolism and thymineless death in prokaryotes and eukaryotes.

For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation [thymineless death (TLD)]. This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect. Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects. The direct effects involve both single- and double-strand DNA breaks. The former may be repaired effectively, but the latter lead to cell death. DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair. Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process. This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites. Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction. Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features. The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD. There are similarities between TLD of bacteria and death of eukaryotic cells. Also, bacteria have "survival" genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two. A model is presented for a molecular basis of TLD.

Synergistic action of near-UV and phenylalanine, tyrosine or tryptophan on the inactivation of phage T7: role of superoxide radicals and hydrogen peroxide.

Near ultraviolet (NUV) light can cause a variety of damage to biological systems. The effects of NUV are significantly enhanced in the presence of sensitizers. One of the most important targets of such synergistic effects is DNA. Cellular DNA exposed to NUV plus sensitizers is damaged in a variety of ways, DNA strand breaks and interstrand cross-links being the most common effects. In this study, phenylalanine, tyrosine and tryptophan are shown to act as sensitizers for NUV action of phage T7; superoxide anions are produced. The reactive species probably interacts with phage DNA causing damage responsible for phage inactivation. Superoxide dismutase reverses the synergistic activities of phenylalanine and tyrosine on NUV-induced phage inactivation, but catalase is additionally required to reverse the effect of tryptophan. Therefore, it is probable that NUV photolysis of tryptophan causes the production of superoxide ions and hydrogen peroxide, both of which contribute to phage inactivation. The ubiquitous nature of NUV in our environment and the presence of amino acids in skin cells suggests that an important mechanism for the induction of skin cancer in humans by solar exposure is amino acid photolysis by NUV.