LET Dependence of 8-Hydroxy-2'-deoxyguanosine (8-OHdG) Generation in Mammalian Cells under Air-Saturated and Hypoxic Conditions: A Possible Experimental Approach to the Mechanism of the Decreasing Oxygen Effect in the High-LET Region.

One of the most distinguished features in biological effects of heavy ions would be the decrease of oxygen effect in the high-LET region. This feature has been referred to as the radiobiological basis for the control of hypoxic fraction in cancer radiotherapy. However, mechanisms to explain this phenomenon have not been fully understood. One of the explanations was given by the oxygen in the track hypothesis, which proposes that oxygen is produced along ion tracks even in the hypoxic irradiation condition. In the present study, we designed an experimental approach to support this hypothesis by using 8-hydroxy-2'-deoxyguanosine (8-OHdG) as DNA damage requiring oxygen to produce. The LET dependence of 8-OHdG under hypoxic condition revealed that with increasing LET 8-OHdG yield seems to increase, despite that the yield of OH radical, which is also required for the production of 8-OHdG, decreases in the high-LET region. This result is consistent with the explanation that the local generation of oxygen along ion tracks contributes to the increase of 8-OHdG yield.

Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

Deletion of 12 carboxyl-terminal residues from pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase affects steroid metabolism.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase (3alpha/beta,20beta-HSD) is 80-85% identical to human, rat, and mouse carbonyl reductases. However, pig 3alpha/beta,20beta-HSD contains an extra 12 amino acids at its COOH-terminus that these other mammalian carbonyl reductases lack. We constructed a pig 3alpha/beta,20beta-HSD mutant, G278opal, which lacks these amino acids and found that compared to wild-type 3alpha/beta,20beta-HSD, G278opal has a 10-fold lower catalytic efficiency for testosterone and progesterone. G278opal also has lower 3alpha- and 20beta-reductase and increased 3beta-reductase activity compared to wild-type 3alpha/beta,20beta-HSD. Binding of NADPH to G278opal was similar to that of wild-type 3alpha/beta,20beta-HSD. The recently determined three-dimensional structure of 3alpha/beta,20beta-HSD, without a steroid substrate, shows the 12 COOH-terminal amino acids in a random configuration. Our data indicate that the 12 COOH-terminal amino acids have a role in steroid metabolism suggesting that binding of steroid to wild-type 3alpha/beta,20beta-HSD induces a conformational change in which the 12 COOH-terminal amino acids interact with the steroid substrate.

Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase.

Pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3alpha/beta,20beta-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20beta-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3alpha/beta,20beta-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4-1% and the mutant with arginine-198 has a catalytic efficiency of 4-23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3alpha/beta,20beta-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.

Carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase.

The carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) was examined using a recombinant enzyme. Kinetic parameters were obtained for 48 carbonyl group-containing substrates, including aromatic aldehydes, aromatic ketones, cycloketones, quinones, aliphatic aldehydes and aliphatic ketones. 20 beta-HSD showed a high affinity towards quinones, such as 9,10-phenanthrenequinone, alpha-naphthoquinone and menadione (Km values of 4, 2 and 5 microM, respectively), and the substrate utilization efficiency (Vmax/Km) of the enzyme against these quinones was very high. Cyclohexanone and 2-methylcyclohexanone were also reduced with a high Vmax/Km value, but not cyclopentanone or 2-methylcyclopentanone. Various aromatic aldehydes and ketones including benzaldehyde- and acetophenone-derivatives were reduced by 20 beta-HSD. Especially, 4-nitrobenzaldehyde and 4-nitroacetophenone were reduced with high Vmax/Km values in the related compounds. The enzyme also reduced the pyridine-derivatives, 2-, 3-, and 4-benzoylpyridine, with the Vmax/Km value for 2-benzoylpyridine being the highest. 20 beta-HSD reduced aliphatic aldehydes and aliphatic ketones, but was more effective on the former. The correlation between the structure of carbonyl compounds and their substrate Vmax/Km is discussed.

Monoclonal antibody directed against neuroendocrine properties of both normal and malignant cells.

A monoclonal antibody, 6H7, was produced by the immunization of small cell carcinoma of the lung (SCCL). Immunohistochemical examination indicated that 6H7 reacted not only with SCCL but also various neuronal and/or endocrine tumors such as neuroblastoma, pheochromocytoma, carcinoid and adrenal cortical tumors. 6H7 was also reactive with normal neuroendocrine tissues including brain, spinal cord, thyroid follicular cells, pancreatic islet cells and adrenal cells. 6H7 did not react with squamous cell carcinomas, one large cell carcinoma or most adenocarcinomas of the lung, or carcinomas of the stomach, colon, pancreas, breast and esophagus. The antigen recognized by 6H7 was analyzed on gel filtration after purification of the antigen by liquid chromatography which indicated the molecular weight of the antigen to be 270,000-300,000. From SDS-PAGE analysis the antigen reactive with 6H7 appeared to consist of polypeptide dimers of 128,000.