Oltipraz chemoprevention trial in Qidong, People's Republic of China: unaltered oxidative biomarkers

Aflatoxin, which leads to formation of carcinogen-DNA adducts as well as oxidized DNA, is a well-known risk factor for development of hepatocellular carcinoma. The aim of the present study was to investigate if the chemopreventive agent oltipraz had an effect on DNA oxidation measured as oxidized guanine derivatives in urine among healthy individuals living in a region of China at high risk of exposure to aflatoxin and development of hepatocellular carcinoma. Two hundred thirty-three healthy residents of Qidong, PRC, were randomized to 8 weeks treatment with placebo, oltipraz 125 mg daily, or oltipraz 500 mg weekly, with a subsequent 8-week follow-up period. Urine samples were collected as overnight voids. Samples collected 4 weeks into the treatment period and 6 weeks into the follow-up period were analyzed for oxidized guanine derivatives with a HPLC-MS/MS method. A repeated-measures analysis of variance showed no significant differences between the randomization groups regarding changes in oxidized guanine derivatives. In the present double-blind, randomized, placebo-controlled trial performed among healthy individuals, oltipraz had no major effect on oxidative DNA damage. Mechanisms other than prevention of oxidative DNA damage may be of higher importance when oltipraz is used as a chemopreventive agent in humans.     

UPTAKE OF GUANINE BY THE DIATOM,PHAEODACTYLUM TRICORNUTUM1

Nitrate-cultured cells of Phaeodactylum tricornutum Bohlin lack the ability to take up guanine but can do so after a period of nitrogen deprivation, i.e. photosynthesis in nitrogen-free medium. Maximum rate of uptake occurred after 24 h of nitrogen deprivation. The development of ability to take up guanine required CO₂ fixation and was prevented by cycloheximide, ammonium or nitrate. The guanine taken up accummulated in the cells almost entirely as a compound which is probably methylated hypoxanthine. Guanine uptake was dependent upon metabolism and exhibited Michaelis-Menten like kinetics with a half-saturation value of 0.48 ± 0.05 μM guanine and a maximum uptake rate for guanine of ca. 200 nmol · 10^?8 cells · h^?1. Rate of uptake increased hyperbolically with Na⁺ concentration, with 8.25 mM Na⁺ supporting half-maximal rate, and it was inhibited by K⁺ ions.     

Postradiation volatile secretion and development of immunosupression effectes by laboratory mice with various genotype

The appearance of lines CBA and C57Bl/6 in the urine of mice irradiation volatile excretions with immunosupression property was associated with the violation at mice of ability to immunogenesis. It was established, that immunosupression activity of excretions irradiated mice CBA and C57Bl/6 did not depend on genotip of mice. However, by irradiated mice C57Bl/6 immunosupression components, depressing antibody formation at intact mice, appeared earlier, than at CBA. Immunosypression, limited postradiation volatile secretion in view of depression humoral immune response at intact mice kept for not a long time and mostly expressed on the course 2-3 days after exposition with postradiation secretion.     

Communicative multiplication of secondary disorders in blood formula and immunity in groups of intact mice caused by volatile compounds excreted by irradiated animals

It was shown that immunosupressive substances excreted by the irradiated mice are highly volatile. Even the excretion from one irradiated mouse could induce disorders in the immune system and blood formula of intact recipients exposed to the volatile substances. In turn, a dominate mouse from a group of the exposed recipients could cause the disorders in the next intact group. Samples of urine from irradiated or exposed to the volatile substances could cause the same effect. The experiments showed a mechanism of secondary post-radiation disorders in animal groups through volatile chemical signals.     

Normal and modified nucleobases excreted in urine of patients with chronic nyeloproliferative disorders (author's transl)]

The qualitative and quantitative analysis by high performance liquid chromatography of the normal and modiefied nucleobases excreted in urine represents a new and versatile tool, especially in oncology. The excretion of 2 normal (adenine, guanine) and 4 modified nucleobases (methylated guanine derivatives) in urine was measured by cation exchange LC. All chronic myeloproliferative syndromes showed highly elevated total excretion values of all determined nucleobases, the "pattern" being characteristic with N2, N2-dimethylguanine most prominent (up to 29.8 S.D. above the pertinent normal value). A follow-up study of a case of CML with two episodes of extreme leukocytosis showed a correlation of the nucleobases excretion with the number of leukocytes. Thus, a method has been established which permits the assessment of myeloproliferation and probably therapy effects.     

Double base lesions in DNA X-irradiated in the presence or absence of oxygen

Previously, double lesions in which two adjacent bases are modified were identified in DNA oligomers exposed in solution to ionizing radiation. However, the formation of such lesions in polymer DNA had not been demonstrated. Using reference oligomer containing a specific double lesion and employing liquid chromatography-mass spectrometry (LC-MS), it was possible to show directly that double lesions are formed in irradiated calf thymus DNA. The double lesion in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized to 8-oxoguanine was detected in DNA X-irradiated in oxygenated aqueous solution. The double lesion in which the methyl carbon atom of a thymine base is covalently linked to carbon at the 8-position of an adjacent guanine base was detected in DNA irradiated in a deoxygenated environment.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Differentiation, Multiplication and Control of Bloodstream Form Trypanosoma (Duttonella) vivax in Mice

.The growth and differentiation of Trypanosoma vivax was studied in intact and irradiated C3H/He and C57B1/6 mice. In irradiated (800 R) or intact C3H/He and irradiated (800 R) C57B1/6 mice, T. vivax parasitaemia increased rapidly then entered a plateau phase and thereafter declined in an antibody-independent remission phase. Throughout the infection, variations were observed in parasite morphology, density, DNA content, number of organisms with 2 nuclei and 2 kinetoplasts and infectivity of parasites for mice. Parasites in exponential phase had the highest number of members in the S, G₂ and M phases of the cell cycle as determined by staining with the interchalating dye Chromomycin A, and analysis on a flow cytometer. During this phase there were numerous parasites with 2 nuclei and 2 kinetoplasts and infectivity was high for mice. As the parasitaemia approached and entered the plateau phase, the proportion of organisms in the S, G₂ and M phases of the cell cycle as well as the number of those with 2 kinetoplasts decreased slightly; the number of organisms with 2 nuclei decreased rapidly; and parasites had a considerably reduced capacity to infect mice. Organisms from the remission phase contained only 1 nucleus and 1 kinetoplast and were not infective for mice. The study suggests that T. vivax organisms transit from dividing to committed non-dividing forms and that some non-dividing, non-infective T. vivax organisms remain trapped in the S, G₂ and M stages of the cell cycle and die without completing binary fission. In contrast to the above, parasite wave remission occurred in T.vivax-infected intact C57B1/6 mice during exponential growth when there were large numbers of dividing form organisms present in the bloodstream as determined by both DNA content and the proportion of parasites with 2 kinetoplasts and 2 nuclei. Clearance of T. vivax from the bloodstream of infected intact C57B1/6 mice coincided with the production of a parasite-specific antibody response. The studies are discussed with reference to the mode of induction of host protective antibody responses to exponentially growing T. vivax.     

Plasmodium berghei: The spleen in sporozoite-induced immunity to mouse malaria

A/J mice were splenectomized (Splx) or sham-splenectomized (SSplx) prior to administration of a single injection of irradiated sporozoites. Following challenge 7 days after immunization, it was found that none of the splenectomized mice were protected whereas 50% of the sham-splenectomized and intact animals were resistant to challenge. In another series of experiments similar groups, along with mice splenectomized just prior to challenge, were injected with 1.5 × 10⁵ irradiated sporozoites over a 5 week period. This resulted in protection of (1) 60–100% of the animals splenectomized before immunization, and (2) 90–100% protection of the animals splenectomized prior to challenge, as well as the intact and sham-splenectomized mice. Serum levels of antisporozoite antibodies (CSP and SNA) increased during immunization of the intact animals. Only 15–20% of the animals splenectomized prior to immunization presented positive CSP reactions and little if any sporozoite neutralizing activity (SNA) was detected. Serum from intact animals immunized and found resistant to sporozoite challenge was used for passive transfer studies. Immune serum recipients were challenged with small numbers of sporozoites. Only one out of 18 recipients was protected against sporozoite challenge.     

Photoirradiation products of flavin derivatives,and the effects of photooxidation on guanine

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.     

Oxidation and turnover of renal metallothioneins after an injection of ferric nitrilotriacetate

Metallothioneins (MTs) have demonstrated strong antioxidant properties, however the biological significance of their effect against hydroxyl radical toxicity remains unclear. We investigated the oxidation and turnover of renal MTs in MT-preinduced mice after an injection of ferric nitrilotriacetate (Fe-NTA). Incubation of MTs with Fe-NTA and H(2)O(2) resulted in a loss of their metal-binding properties and a decrease in their thiol concentration independent of binding potential and isoforms. Moreover, in vitro reduction of renal oxidized MT with dithiothreitol (DTT) reversed these oxidative changes. An injection of Fe-NTA oxidized renal preinduced MT in Zn- and Cd-pretreated mice. The metal-binding properties of renal MTs were lost when the Fe-NTA dose was increased. However, analysis of renal MTs using an immunoassay showed that its protein concentration did not decrease 4h after the injection with various Fe-NTA doses. Furthermore, in vitro reduction of renal oxidized MTs with DTT resulted in an increase in the concentration of metals in the MT fraction. These data indicate that radicals produced by Fe-NTA may oxidize MTs in vitro and in vivo. When we investigated the turnover of oxidized MTs in Fe-NTA-treated mice, effects on the concentration of renal (35)S-labeled MTs were opposite to those observed in Cd-pretreated mice. The concentration of preinduced (35)S-labeled MTs in the kidneys of Cd-pretreated mice showed a significant decrease (p<0.05), whereas that of newly synthesized (35)S-labeled MTs showed a considerable increase. These data suggest that degradation of oxidized MTs may be faster than intact MTs. Therefore, the radical scavenging system of MTs may include their induction and degradation during oxidative stress conditions.     

Epidermal growth factor concentrations in submandibular salivary gland, plasma, liver, bile, kidneys and urine of male mice: dynamics after phenylephrine injection

To elucidate the dynamics of epidermal growth factor (EGF) in mice, I studied the effects of excision of the submandibular salivary gland (SMG) and intravenous injection of phenylephrine in adult male mice on the concentration of EGF in plasma, bile, urine, the SMG, the liver and the kidneys. After excision of SMG (sx), the mean concentration of EGF in the liver decreased. Phenylephrine increased the concentrations of EGF in the plasma and liver in both intact and sx mice. In the intact mice, the EGF concentrations also increased in the bile and kidneys but not in the urine. The expected dramatic decrease occurred in the SMG EGF. I conclude that alpha-adrenergic stimulation causes 1) in intact mice release of EGF from SMG into blood, from which it is excreted into bile but not into urine, and 2) release of EGF even from tissues other than SMG.     

Accumulation of 5′ guanine nucleotides by mutants of Brevibacterium ammoniagenes

5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture.     

Postradiation volatile secretion of mice attractive for intact individuals

In early period after exposure to a sub-lethal dose (4 Gy) the exposed mice secreted volatile components (in their urine), which were attractive for intact individuals independently of genotype. The postradiation components were more attractive for allogene individuals than for singene ones. This reaction was different in exposed intact mice, which showed singene attractiveness of volatile secretion. Attractiveness of postradiation volatile components differed from the attractiveness of ones secreted after stress.     

The influence of postradiation chemical signals of mice on the humoral immune response in recipients in different time relative to antigenic stimulus

The influence of volatile urine chemosignals of irradiated (4 Gy) mice on the primary humoral immune response to sheep red blood cells in intact recipients was investigated. It was demonstrated that the direction of immunomodulatory effect is dependent upon the time at which the postradiation chemosignals was initially applied. The antibody response to antigen was markedly suppressed in mice that were exposed before antigen injection. When chemosignals applied immediately following inoculation of antigen the antibody response was unaffected. The immune response was increased when chemosignals was loadeded for 1-10 days after immunization. The possible mechanisms of immunomodulation are considered.     

The Use of the Uromodulin Promoter to Target Production of Recombinant Proteins into Urine of Transgenic Animals

A uromodulin promoter has been isolated, sequenced, and used to generate two sets of transgenic mice for expression of the lacZ marker gene and for production of the human recombinant erythropoietin (rhEPO) in urine. We demonstrated that the 5.6-kb fragment of the uromodulin gene containing the 3.7-kb promoter area and, both the first exon and part of the second exon, were sufficient to provide kidney-specific expression of the lacZ gene. Histological analysis of the lacZ expression pattern revealed -galactosidase activity specifically in the thick limb of Henle's loop. However, due to random integration of the transgene, ectopic expression was detected in some transgenic lines. Analysis of the EPO-transgenic mice showed that rhEPO was secreted into the urine of founder mice (up to 6 ng/ml). We were able to breed and analyze only two sublines with a very low expression level of rhEPO (up to 260 pg/ml). All of our transgenic mice expressing rhEPO in urine developed disease symptoms similar to polycythemia in humans. These included a considerable increase in red blood cell counts, hemoglobin concentration, and hematocrit concomitant with severe thrombocytopenia, all of which were detected in the rhEPO-expressing mice. Although our model did not prove to be beneficial for commercial production of rhEPO, we concluded that the uromodulin promoter could be useful for expression of other important therapeutic proteins into the urine of transgenic animals.     

A new model for how O6-methylguanine-DNA methyltransferase binds DNA

Human methyltransferase (hAT) catalyzes the transfer of an alkyl group from the 6-position of guanine to an active site Cys residue. The physiological role of hAT is the repair of alkylated guanine residues in DNA. However, the repair of methylated or chloroethylated guanine bases negates the effects of certain chemotherapeutic agents. A model of how hAT binds DNA might be useful in the design of compounds that could inactivate hAT. We have used computer modeling studies to generate such a model. The model utilizes a helix-loop-wing DNA binding motif found in Mu transposase. The model incorporates a flipped out guanine base in order to bring the methylated oxygen atom close to the active site Cys residue. The model is consistent with a variety of chemical and biochemical data. Proteins 32:3–6, 1998. © 1998 Wiley-Liss, Inc.     

Cultured cells of Arachis hypogaea susceptible to induction of stilbene synthase (resveratrol-forming)

Callus cultures were established from cotyledons of Arachis hypogaea. The cultured cells were shown to produce stilbene synthase (resveratrol-forming) if the cells were irradiated with 254 nm UV light. The lag period after which the stilbene synthase activity appeared was similar for cultured cells and intact cotyledons.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.