Induction of anchorage-independent growth in primary human cells exposed to protons or HZE ions separately or in dual exposures.

Travelers on space missions will be exposed to a complex radiation environment that includes protons and heavy charged particles. Since protons are present at much higher levels than are heavy ions, the most likely scenario for cellular radiation exposure will be proton exposure followed by a hit by a heavy ion. Although the effects of individual ion species on human cells are being investigated extensively, little is known about the effects of exposure to both radiation types. One useful measure of mammalian cell damage is induction of the ability to grow in a semi-solid agar medium highly inhibitory to the growth of normal human cells, termed neoplastic transformation. Using primary human cells, we evaluated induction of soft-agar growth and survival of cells exposed to protons only or to heavy charged particles (600 MeV/nucleon silicon) only as well as of cells exposed to protons followed after a 4-day interval by silicon ions. Both ions alone efficiently transformed the human cells to anchorage-independent growth. Initial experiments indicate that the dose responses for neoplastic transformation of cells exposed to protons and then after 4 days to silicon ions appear similar to that of cells exposed to silicon ions alone.

Frequencies and relative levels of clustered damages in DNA exposed to gamma rays in radioquenching vs. nonradioquenching conditions.

Clustered damage induced by ionizing radiation--two or more oxidized bases, abasic sites, or strand breaks within a few DNA helical turns--have been postulated to be major lethal and/or mutagenic sites. Although they have recently been shown to be induced in genomic DNAs by ionizing photons and particles, little is known of the factors that affect their yields or the relative levels of the classes of clusters. Toward this aim we have investigated the effect of DNA milieu, specifically, a nonradioquenching (phosphate) or radioquenching (Tris) solution, upon the generation of clustered lesions in a well-defined molecule, T7 bacteriophage DNA. Irradiation of DNA in Tris reduces the yields of all clustered damages to 1-3% of the levels formed in phosphate. Further, although the percentage of the total clusters in oxidized purine clusters is largely unchanged, and the level of abasic clusters decreases, the frequencies of double-strand breaks and oxidized pyrimidine clusters increase in the radioquenching solution. The ratio of the level of oxidized pyrimidine clusters to double-strand breaks in a DNA in radioquenching solution is similar to that obtained in DNA in human cells, also a radioquenching environment.

Quantifying DNA damage by gel electrophoresis, electronic imaging and number-average length analysis.

DNA damages that can be converted to single- or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge-coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled DNA.

Clustered DNA damages induced by high and low LET radiation, including heavy ions.

Clustered DNA damages--here defined as two or more lesions (strand breaks, oxidized purines, oxidized pyrimidines or abasic sites) within a few helical turns--have been postulated as difficult to repair accurately, and thus highly significant biological lesions. Further, attempted repair of clusters may produce double strand breaks (DSBs). However, until recently, there was no way to measure ionizing radiation-induced clustered damages, except DSB. We recently described an approach for measuring classes of clustered damages (oxidized purine clusters, oxidized pyrimidine clusters, abasic clusters, along with DSB). We showed that ionizing radiation (gamma rays and Fe ions, 1 GeV/amu) does induce such clusters in genomic DNA in solution and in human cells. These studies also showed that each damage cluster results from one radiation hit (and its track), thus indicating that they can be induced by very low doses of radiation, i.e. two independent hits are not required for cluster induction. Further, among all complex damages, double strand breaks comprise--at most-- ~20%, with the other clustered damages being at least 80%.

Clustered DNA damages as dosemeters for ionising radiation exposure and biological responses.

Clustered DNA damages--two or more lesions (oxidised bases. abasic sites, or strand breaks) within a few DNA helical turns on opposing strands--are induced in DNA in solution and in vivo in human cells by ionising radiation. They have been postulated to be difficult to repair, and thus of potentially high biological significance. Since the total of clustered damages produced by ionising radiation is at about 3 to 4 times higher levels than double-strand breaks and are apparently absent in unirradiated cells, levels of clustered damages present immediately alter radiation exposure could serve as sensitive dosemeters of radiation exposure. Since some clusters may not be repairable and may accumulate in cells, they might also be useful as integrating dosemeters of biological effects of radiation damage.

UV radiation-sensitive norin 1 rice contains defective cyclobutane pyrimidine dimer photolyase.

Norin 1, a progenitor of many economically important Japanese rice strains, is highly sensitive to the damaging effects of UVB radiation (wavelengths 290 to 320 nm). Norin 1 seedlings are deficient in photorepair of cyclobutane pyrimidine dimers. However, the molecular origin of this deficiency was not known and, because rice photolyase genes have not been cloned and sequenced, could not be determined by examining photolyase structural genes or upstream regulatory elements for mutations. We therefore used a photoflash approach, which showed that the deficiency in photorepair in vivo resulted from a functionally altered photolyase. These results were confirmed by studies with extracts, which showed that the Norin 1 photolyase-dimer complex was highly thermolabile relative to the wild-type Sasanishiki photolyase. This deficiency results from a structure/function alteration of photolyase rather than of nonspecific repair, photolytic, or regulatory elements. Thus, the molecular origin of this plant DNA repair deficiency, resulting from a spontaneously occurring mutation to UV radiation sensitivity, is defective photolyase.

Clustered damages and total lesions induced in DNA by ionizing radiation: oxidized bases and strand breaks.

Ionizing radiation induces both isolated DNA lesions and clustered damages-multiple closely spaced lesions (strand breaks, oxidized purines, oxidized pyrimidines, or abasic sites within a few helical turns). Such clusters are postulated to be difficult to repair and thus potentially lethal or mutagenic lesions. Using highly purified enzymes that cleave DNA at specific classes of damage and electrophoretic assays developed for quantifying isolated and clustered damages in high molecular length genomic DNAs, we determined the relative frequencies of total lesions and of clustered damages involving both strands, and the composition and origin of such clusters. The relative frequency of isolated vs clustered damages depends on the identity of the lesion, with approximately 15-18% of oxidized purines, pyrimidines, or abasic sites in clusters recognized by Fpg, Nth, or Nfo proteins, respectively, but only about half that level of frank single strand breaks in double strand breaks. Oxidized base clusters and abasic site clusters constitute about 80% of complex damages, while double strand breaks comprise only approximately 20% of the total. The data also show that each cluster results from a single radiation (track) event, and thus clusters will be formed at low as well as high radiation doses.

Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.

Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha.

Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells. We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.

A robust, inexpensive filter for blocking UVC radiation in broad-spectrum 'UVB' lamps.

Accurate studies of the biological effects of UBV radiation require suitable laboratory sources. Lamps labeled as UVB sources often emit UVC radiation that contributes significantly to the levels of DNA damage. The UVC from an unfiltered UVB source produced more pyrimidine dimers in soybean DNA than a lamp filtered by a Pyrex dish that removes wavelengths of < 280 nm. Calculations based on action spectra and on the emission spectra of unfiltered lamps indicate that UVC contributes approximately 13%, 4% and approximately 1% of the total dimers induced in unshielded cells or DNA, alfalfa cotyledons, and human skin, respectively. Further, relative to a Pyrex dish-filtered lamp, an unfiltered lamp would produce approximately 7-, 2.4- or 2.8-fold more dimers in these three biological systems. We report here that a Pyrex dish provides an effective, stable, robust and inexpensive filter for reducing or excluding the contribution of UVC to damage induced by broad-spectrum 'UVB' lamps.

UV effects in "the real world": problems of UV dosimetry in complex organisms.

Understanding the effects of UV radiation on higher plants and animals is essential for evaluating the consequences of environmental changes which could alter the UV levels in the biosphere. Although analytical approaches for quantifying UV exposures and resulting cellular damage levels in optically simple systems (DNA in dilute solution, viruses or microorganisms in dilute suspension and monolayers of cultured cells) are well known, these methods may not be applicable to optically complex systems, such as multilayered plants and animals, or microorganisms in absorbing milieu such as natural lakes or oceans. Understanding the problems, pitfalls and solutions to the quantification of UV exposures and cellular damage is critical for evaluating the effects of environmental UV on complex organisms.

Computer network for data acquisition, storage and analysis.

Modern scientific instruments can produce huge quantities of data, usually in digital form. However, data acquisition is only one of three important functions. To be useful, data must also be stored and analyzed. Fortunately, the same computer-based technologies that facilitate the generation of large data-sets provide tools to accomplish these tasks. We describe a data system based on computers connected to a network, developed for this purpose.

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair.

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.

Isolation of high-molecular-length DNA from human skin.

An agarose plug method for isolating high-molecular-length DNA from mammalian tissues has been developed, including from those that are difficult, such as skin. It gives high yields of DNA that contain a minimum of single-strand breaks and is readily digested by restriction and other nucleases. The method requires only simple equipment and is readily adaptable to field or clinical studies.

Double strand breaks induced by low doses of gamma rays or heavy ions: quantitation in nonradioactive human DNA.

We have developed a method of quantitating low frequencies (0-30 sites/10(9) base pairs) of double strand breaks in approximately 1 microgram of nonradioactive human DNA. Unirradiated or irradiated DNA is digested with the restriction endonuclease NotI, producing cleavage fragments that include a major group centered at approximately 1.2-1.3 Mbp. The DNA molecules are separated as a function of size by transverse alternating field electrophoresis. The frequency of double strand breaks is computed directly from the decrease in number average molecular length induced in the 1.2- to 1. 3-Mbp cleavage fragment group by 137Cs gamma or Fe26+ (1.1 GeV/nucleon) irradiation vs the corresponding unirradiated DNA samples. The double strand break frequency can be quantitated easily in the dose range of 0-10 cGy of gamma rays. The frequency of breaks per unit dose calculated for gamma irradiation of DNA in human cells (approximately 4.6 double strand breaks/10(9) bp/Gy) is within the range of values obtained by others (2-8 sites/10(9) bp/Gy) who used methods requiring higher doses.

Action spectroscopy in complex organisms: potentials and pitfalls in predicting the impact of increased environmental UVB.

Evaluating the effects of broad spectrum sources, including sunlight, on humans and their environment requires knowledge both of the effects of various wavelengths of radiation in those sources on biological systems and of the kinds and magnitude of changes in those sources. Action spectroscopy-the determination of the effect of radiation on an organism as a function of wavelength-provides a major method for evaluating the effects of a source, including that of changes in its intensity and wavelength distribution. Although action spectra for UV radiation damage to higher organisms can give useful information, inappropriate analyses can lead to misleading or even incorrect results. It is thus essential to consider carefully methods of analyzing action spectra and their consequences for evaluating the effects of changes in the solar spectrum.

Nursing practice: the regulatory arena.

State nursing laws and boards of nursing significantly impact nurses, nursing education, and practice. As healthcare delivery systems continue to change, roles of boards of nursing and the value of licensure will be questioned. In this article, the purpose of nursing laws and licensure, and the mission, authority, and functions of boards of nursing are reported. A decision-making model to guide nurses in determining scope of practice matters is provided. The delegation of nursing tasks and use of unlicensed assistive persons under nurse supervision is addressed.

Human white blood cells contain cyclobutyl pyrimidine dimer photolyase.

Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (supercoiled pET-2, nonsupercoiled bacteriophage lambda, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells.