Peter Cerutti

The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-κB

BACKGROUND The inducible, higher eukaryotic transcription factor NF-kappa B is activated by a variety of stimuli. Several lines of evidence have suggested that reactive oxygen intermediates (ROIs) serve as messengers for most if not all of these stimuli. To identify the relevant ROI species and to gain more direct evidence for an involvement of ROIs as messengers, we investigated whether changes in the levels of enzymes that control intracellular ROI levels affect the activation of NF-kappa B. RESULTS Cell lines stably overexpressing the H2O2-degrading enzyme catalase were deficient in activating NF-kappa B in response to tumor necrosis factor alpha (TNF) or okadaic acid. The catalase inhibitor aminotriazol restored NF-kappa B induction. In contrast, stable overexpression of cytoplasmic Cu/Zn-dependent superoxide dismutase (SOD), which enhances the production of H2O2 from superoxide, potentiated NF-kappa B activation. The level of cytoplasmic NF-kappa B-I kappa B complex was unchanged, indicating that synthesis of NF-kappa B was not affected. CONCLUSIONS Our data show that one ROI species, H2O2 acts as a messenger in the TNF- and okadaic acid-induced post-translational activation of NF-kappa B. Superoxide is only indirectly involved, as a source for H2O2. These data explain the inhibitory effects of many antioxidative compounds on the activation of NF-kappa B and its target genes. H2O2 is overproduced in response to various stimuli, and normal levels of catalase appear insufficient to remove it completely. H2O2 can therefore accumulate and act as an intracellular messenger molecule in the response to pathogens.

Formation and repair of γ-ray induced thymine damage in Micrococcus radiodurans

A reductive assay for the identification and determination of the major radiolysis products of thymine and its derivatives has been developed. Radiolysis products of the 6-(hydroxy or hydroperoxy)-5,6-dihydrothymine type ( t † ) are reductively cleaved with NaBH 4 and a four-carbon alcohol is released containing the thyminemethyl substituent. If thymine derivatives carrying a radioactive label in the methyl group are used as starting materials for the radiolysis, the amount of the four-carbon fragments produced by the treatment with NaBH 4 (mostly 2-methylglycerol after irradiation in air) can be determined by radioactivity measurements and is a measure of the extent of thymine radiolysis. The formation and repair of thymine base damage was investigated in Micrococcus radiodurans after exposure to γ -rays with the help of the reductive assay. The radiolysis products of thymine were determined in the cytoplasm and the culture medium after their release from the DNA during post-irradiation incubation. It is estimated that t † is formed in the DNA of M. radiodurans with an efficiency of about 1.2 × 10 −6 /rad, 10 6 daltons for irradiation in air and 10 −6 /rad, 10 6 daltons for irradiation in nitrogen. Damaged residues are released from the DNA of irradiated M. radiodurans into cytoplasm and medium during post-irradiation incubation in a biphasic, selective process. On the average, about 300 undamaged thymine residues are removed from the DNA per damaged residue during 240 minutes post-irradiation repair after irradiation in air. The first phase of removal (0 to 30 min at 37°C) and post-irradiation degradation may be part of the same process. Selectivity of product removal may be due to product clustering. Degradation is assumed to start at nearby radiation induced breaks. After a temporary halt, a second phase of product removal is observed (60 to 200 min).

Effects of ionizing radiation on mammalian cells

Soon after the discovery of ionizing radiation its beneficial and damaging effects on human health were noticed and scientists became interested in the physical interactions of radiation energy with living material and the resulting biological effects. Since that time environmental and accidental exposure to ionizing radiation has become a threat to the general population. In this article recent progress in the radiobiology of rapidly dividing mammalian cells in culture is discussed. Special emphasis is placed on the lethal, mutagenic and carcinogenic effects of ionizing radiation. These detrimental effects of ionizing radiation result, for the most part, from damage induced in the nuclear deoxyribonucleic acid (DNA). The formation and repair of radiationinduced breakage of one or both strands of the double stranded DNA helix and of damage to the heterocyclic nucleic acid bases are actively being investigated in mammalian cells. While important progress has recently been made, most of the DNA lesions produced in situ in the cell have not been chemically identified. The relation of the DNA damage to the impairment by ionizing radiation of the cellular functions leading to cell death, mutation and malignant transformation remains unknown.

Treatment of lymphocyte cultures with a hypoxanthine-xanthine oxidase system induces the formation of transferable clastogenic material

Culture medium of lymphocyte cultures that have been exposed to the superoxide generating system hypoxanthine plus xanthine oxidase (X-XO) contains substances with chromosome damaging properties. This is demonstrated by the ability of ultrafiltrates of such culture media to induce chromosomal aberrations and sister chromatid exchanges in the lymphocytes of blood test cultures. Culture medium becomes active about 15 hours after the addition of X-XO and stimulation by phytohemagglutinin. Concomitant with the accumulation of clastogenic material, assays for conjugated dienes and thiobarbituric acid-reactive material which measure lipid-peroxidation become positive in the culture media. When cells are pretreated with superoxide dismutase or glutathione peroxidase before the addition of X-XO neither clastogenic substances nor lipid peroxidation products are detected. Catalase is a less efficient protector.

Identification of Hydrogen Peroxide as the Relevant Messenger in the Activation Pathway of Transcription Factor NF-κB

The inducible higher eukaryotic transcription factor NF-κB is activated by a large variety of distinct simuli [1–3]. In unstimulated cells, this factor resides in a latent form in the cytoplasm [4]. Latency is achieved by association of the DNA-binding NF-κB dimer with an inhibitory subunit, called IκB [5]. IΚB suppresses DNA-binding and nuclear transport of NF-κB. Upon stimulation of cells, IκB is phosphorylated and proteolytically degraded [6–9]. Both reactions are required for activation [10]. The released NF-κB is then translocated to the nucleus where it initiates transcription of target genes. Among the numerous proteins which are induced by a concerted action of NF-kB with other transcription factors are cytokines, chemokines, cell adhesion molecules, hematopoetic growth factors and receptors, histocompatibility antigens and acute phase proteins [1–3]. While NF-κB may be indispensable as inducer of many immediate-early inflammatory and immune reactions, the transcription factor is likely to play a fatal role in certain diseases and syndromes that involve an abberrant expression of inflammatory cytokines [22–24].

Effect of the methylation of the 2′-hydroxyl groups in polyadenylic acid on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with polyuridylic acid☆

Abstract The influence of the methylation of the 2′-hydroxyl groups in poly A on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with poly U has been investigated by ultraviolet and circular dichroism spectroscopy. A major effect of the methylation of the backbone is to increase the thermal stability of the double-stranded, acid structure of poly A, while no significant effect on the single-stranded conformation was observed. The dissymmetric structures, as reflected in the circular dichroic spectra, were virtually identical for poly A and poly Am § for the tightly packed, double-stranded, acid structures (pH 4.6, 0.15 m -KCl) on one hand and for the single-stranded conformations (pH 7, 0.15 m -KCl, 25 °C) on the other. Circular dichroic spectra with characteristics intermediate between those of the tightly packed, double-stranded and the single-stranded structures of poly A and poly Am are obtained in the pH range from 5 to 7 at moderate to high ionic strength (for poly Am e.g. at pH 5.7, 0.15 m -NaCl, 4 to 44 °C). From the circular dichroic spectra and absorbance melting profiles it is tentatively concluded that poly A and poly Am form less tightly packed, less ordered double-stranded structures under these conditions. Methylation of the 2′-hydroxyl groups in poly A has virtually no effect on the thermal stability of its double-stranded complex with poly U, but considerably weakens the attachment of the third strand of poly U in poly(Am + 2U). It is concluded that the presence of an unsubstituted 2′-hydroxyl group in poly A is not essential for the stabilization of the double-stranded conformations of poly A in acidic solutions, for the partially-ordered single-stranded conformation of poly A at neutrality or for the double-stranded complex of poly A with poly U.

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

Abstract 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.

Oxidant Carcinogenesis and Antioxidant Defensea

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases. causes DNA breakage, and induces the growth competence‐related protooncogenes c‐fos and c‐myc.

Selective reduction of yeast transfer ribonucleic acid with sodium borohydride

A method for the selective reduction of dihydrouridine in transfer RNA from yeast has been developed. The chemistry of the reaction, which uses sodium borohydride as a reducing agent, was studied with dihydrouracil, dihydrouridine and dihydrouridine-2′(3′)-phosphate as starting materials. 3-Ureidopropane-1-ol, N-ribosyl-3-ureidopropane-1-ol and N-(2′(3′)-phosphorylribosyl)-3-ureidopropane-1-ol, respectively, were proved to be the major reduction products by spectral and chemical evidence. 3-Ureidopropane-1-ol was also synthesized by an independent route. In nucleotide mixtures and in yeast transfer RNA, dihydrouridine was reduced with sodium borohydride with high selectivity. The major nucleotides, pseudouridine and glucosyl-2-thiouridine, were found to be inert towards the reducing agent. N4-Acetyl-cytidine was reduced to N1-ribosyl-N4acetyl-3,4,5,6-tetrahydrocytidine and 4-thiouridine to N-ribosyl-2-oxohexahydropyrimidine under the conditions used for the reduction of RNA.

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.