Hydrocaffeic and p-coumaric acids,natural phenolic compounds,inhibit UV-B damage in WKD human conjunctival cells in vitro and rabbit eye in vivo

This paper studied the effect on UV-B ocular damage of 10µm hydrocaffeic acid (HCAF) alone and as a mixture (MIX) (5µm HCAF+5µm p-coumaric acid). Since ocular UV-B damage is mediated by reactive oxygen species, the aim was to test if HCAF and MIX could reduce oxidation damage in human conjunctival cells (WKD) in vitro and in cornea and sclera of rabbits in vivo. After UVB irradiation (44 J/m²) of WKD cells, 8-oxodG levels in DNA were markedly increased and this effect was attenuated by HCAF and MIX. Rabbit eyes were treated by application of HCAF and MIX drops before UV-B exposure (79 J/m²). Corneal and scleral DNA oxidation damage, xanthine-oxidase (XO) activity and malondialdehyde levels (MDA) in corneal tissue and prostaglandin E₂ (PGE₂) in the aqueous humour were reduced by HCAF alone and in combination with p-coumaric acid, showing their potential as a topical treatment against UV-B damage.     

In vitro fertilization and expression of transgenes in gametes and zygotes

The in vitro fertilization system of maize is the well characterized model system for the fertilization process and early zygotic embryogenesis of higher plants. Application of molecular methods to the in vitro fertilization system led to the isolation of new genes and uncovered specific expression patterns of cell cycle regulators. Recent studies showed that expression of transgenes is possible in gametes and zygotes, thus transgenic approaches might offer an opportunity to unravel the roles of genes during fertilization and early development. The competence of gametes and zygotes to express transgenes will also enable the expression of GFP based reporter genes for the visualization of subcellular components in these cells in vivo. This review focuses on the data concerning the expression of transgenes in gametes and zygotes and describes some examples of recent developments in transgenic technology illustrating the emerging possibilities in experimental design by combining this technology with in vitro fertilization. Received: 20 December 2001 / Revision accepted: 6 June 2001     

Effects of vesicular-arbuscular mycorrhizal inoculation at different soil P availabilities on growth and nutrient uptake of in vitro propagated coffee (Coffea arabica L.) plants

 In a pot experiment, the growth and the nutrient status of in vitro propagated coffee (Coffea arabica L.) microcuttings were investigated for 5 months following vesicular-arbuscular mycorrhizal (VAM) inoculation with either Acaulospora melleae or Glomus clarum at four soil P availabilities. Control plants remained P-deficient even at the highest soil P availability while mycorrhizal plants were P-sufficient at all soil P availabilities. Growth of control plants was only improved at the highest soil P availability. In P-deficient soil, neither of the two VAM species improved plant growth. Plant growth increased by 50% following inoculation with either A. melleae or G. clarum when P availability went from deficient to low. No further plant growth improvement was induced by either VAM species at intermediate and high soil P levels. Nevertheless, growth of plants inoculated with G. clarum was still significantly greater than that of non-mycorrhizal plants at the highest soil P availability. Root colonization by G. clarum increased with increasing soil P availability while root colonization by A. mellea decreased with soil P level increasing above low P availability. Soil P availability also affected Zn nutrition through its influence on VAM symbiosis. With increasing soil P availability, foliar Zn status increased with G. clarum or decreased with A. mellea in parallel to root colonization by VAM. This study demonstrates the beneficial effects of VAM inoculation on in vitro propagated Arabica coffee microcuttings, as shown previously for seedlings. This study also demonstrates differences in tolerance to soil P availability between VAM species, most likely resulting from their differing abilities to enhance coffee foliar P status. Accepted: 14 November 1996     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

In vitro pollen tube growth and penetration of female gametophyte in Douglas fir (Pseudotsuga menziesii)

 Pollen tube and female gametophyte interactions in Douglas fir (Pseudotsuga menziesii) were examined in vitro. Formation of pollen tubes in Douglas fir occurred on a modified Murashige and Skoog medium in which concentrations of H₃BO₃ and Ca(NO₃)₂ were altered and supplemented with sucrose and polyethylene glycol. Addition of 100 μg/ml H₃BO₃ and 300 μg/ml Ca(NO₃)₂ resulted in optimum pollen viability. Lack of H₃BO₃ inhibited pollen tube formation. Addition of H₃BO₃ and Ca(NO₃)₂ significantly increased pollen tube formation within one week in culture. Using a medium supplemented with mannitol, viability of Douglas fir pollen can be sustained for 7 weeks in culture, about the same length of time as in vivo. However, pollen tubes are not formed. This suggests that the factors responsible for tube formation reside in the external environment of the pollen. Culture of female gametophytes to examine egg viability and longevity had not been done previously. We found that egg viability in culture is short-lived, and therefore the window to study and manipulate events of fertilization in Douglas fir is very limited. In spite of this, about 7% of the female gametophytes that were co-cultured became penetrated by pollen tubes. In vitro archegonial penetration has been repeatedly achieved, but pollen tubes also penetrated other parts of the female gametophytes. Pollen tubes also penetrated non-viable eggs. Most female gametophytes were not penetrated because of pollen tube branching and swelling, failure of tubes to orient towards the female gametophytes, or premature pollen tube death due to plasmolysis. This report outlines the first attempt towards in vitro fertilization in conifers. Received: 13 March 1997 / Revision accepted: 6 June 1997     

In vitro morphogenesis of pearl millet [Pennisetum glaucum (L.) R.Br.]: efficient production of multiple shoots and inflorescences from shoot apices

 This report presents a procedure for high-frequency multiple shoot production from cultured shoot apical meristems of pearl millet [Pennisetum glaucum (L.) R. Br.]. Shoot apices from 1-week-old aseptically germinated seedlings were cultured in vitro on MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) with biweekly subculture. A low concentration of 2,4-D coupled with four different concentrations of BA induced the production of adventitious shoots from the enlarged shoot apical meristems. Somatic embryogenesis was also observed at higher concentrations of BA. The use of higher levels of 2,4-D resulted in callusing of shoot apical meristems, while the shoot tips produced many leaves and in vitro flowering in 2,4-D-free media containing BA. All four pearl millet genotypes produced similar results. Fertile pearl millet plants were produced from in vitro-produced multiple shoots. Received: 1 April 1999 / Revision received: 8 July 1999 / Accepted: 17 August 1999     

Inhibition by singlet oxygen quenchers of oxidative damage to DNA produced in cultured cells by exposure to a quinolone antibiotic and ultraviolet A irradiation

The photogenotoxicity mechanism of quinolone antibiotics was investigated by measuring oxidative DNA damage in lomefloxacin- and UVA-exposed cultured liver-derived cells. The combination of lomefloxacin and UVA irradiation produced a dose-dependent increase in 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG) in cell DNA. This DNA damage was substantially inhibited by co-incubation with sodium azide (NaN₃) or 2,2,6,6-tetramethyl-4-piperadone (TMP), chemicals that specifically quench singlet oxygen. No significant reduction of 8-oxo-dG formation was produced by N-t-butyl--phenylnitrone (TBP) or -tocopherol, which primarily scavenge hydroxyl radicals. We conclude that the photodynamic generation of 8-oxo-dG by quinolones is mediated, at least in part, by singlet oxgen.     

Protective effects of melatonin against oxidation of guanine bases in DNA and decreased microsomal membrane fluidity in rat liver induced by whole body ionizing radiation

The aim of the study was to examine the potential protective effect of melatonin against whole body ionizing radiation (800 cGy). Changes in 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver, 12 h after their exposure to radiation. To test the potential protective effects of melatonin, the indole was injected (i.p. 50 mg/kg b.w.) at 120, 90, 60 and 30 min prior to radiation exposure. Both 8-OH-dG levels and microsomal membrane rigidity increased significantly 12 h after radiation exposure. Melatonin completely counteracted the effects of ionizing radiation. Changes in 8-OH-dG levels and membrane fluidity are early sensitive parameters of DNA and microsomal membrane damage, respectively, induced by ionizing radiation and our findings document the protective effects of melatonin against ionizing radiation.     

Differential effect of creatine on oxidatively-injured mitochondrial and nuclear DNA

Creatine is a naturally occurring compound obtained in humans from endogenous production and consumption through the diet. It is used as an ergogenic aid to improve exercise performance and increase fat-free mass. Lately, creatine’s positive therapeutic benefits in various oxidative stress-associated diseases have been reported in literature and, more recently, creatine has also been shown to exert direct antioxidant effects. Oxidatively-challenged DNA was analysed to show possible protective effects of creatine. Acellular and cellular studies were carried out. Acellular assays, performed using molecular approaches, showed that creatine protects circular and linear DNA from oxidative attacks.     

Photo-irradiated titanium dioxide catalyzes site specific DNA damage via generation of hydrogen peroxide

Titanium dioxide (TiO₂) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO₂ was examined using [₃₂P]-5'-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO₂ (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2' -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO₂ particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO₂.     

DNA polymerase gamma and mitochondrial disease: Understanding the consequence of POLG mutations

DNA polymerase γ is the only known DNA polymerase in human mitochondria and is essential for mitochondrial DNA replication and repair. It is well established that defects in mtDNA replication lead to mitochondrial dysfunction and disease. Over 160 coding variations in the gene encoding the catalytic subunit of DNA polymerase γ (POLG) have been identified. Our group and others have characterized a number of the more common and interesting mutations, as well as those disease mutations in the DNA polymerase γ accessory subunit. We review the results of these studies, which provide clues to the mechanisms leading to the disease state.     

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N = 7) seeded singly yielded clonal cultures with 253 ± 167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5 ± 4 cells after 21 days.     

Oxidative damage to proteins and DNA in rats exposed to cadmium and/or ethanol

The study was aimed to estimate whether rat's exposure to cadmium (Cd; 50 mg/l in drinking water for 12 weeks) and/or ethanol (EtOH; 5 g/kg b.wt./24 h p.o. for 12 weeks), noted by us to induce oxidative stress and stimulate lipid peroxidation, can cause oxidative damage to proteins and DNA, and whether and to what extent the effects of co-exposure differ from those observed under the treatment with each substance alone. Protein carbonyl groups (PC) and protein thiol groups (PSH) in the serum, liver and kidney, as markers of oxidative protein damage, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the serum, as a marker of DNA oxidation, were determined. The exposure to Cd or/and EtOH led to oxidative protein damage (increased PC and decreased PSH concentrations in the serum and/or liver), and to DNA oxidation (increased 8-OHdG concentration in the serum). The effects were more advanced at the co-exposure than at the treatment with each substance alone. The more serious damage to proteins and DNA at the co-exposure to Cd and EtOH seems to be the effect of independent action of both xenobiotics. The results of the present paper together with our recent findings in the same rats seem to indicate that at co-exposure to Cd and EtOH proteins and DNA may be more vulnerable to oxidation than lipids. The paper is the first report suggesting that excessive EtOH consumption during exposure to Cd may increase the risk of health damage via enhancing protein and DNA oxidation.     

Evaluation of urinary biomarkers of oxidative/nitrosative stress in adolescents and adults with Down syndrome

Urinary biomarkers of oxidative stress have been little studied in adults with Down syndrome (DS), usually no more than two biomarkers have been measured in the population studied and controversial results are reported in literature. Thus, we aimed to assess a set of oxidative and nitrosative stress biomarkers in urine samples of adolescents and adults with DS, with and without hypothyroidism, which comprise: 8-hydroxy-2′-deoxyguanosine (8-OHdG), isoprostane 15-F_2t-IsoP, thiobarbituric acid-reacting substances (TBARS), advanced glycation end products (AGEs), dityrosine (diTyr), hydrogen peroxide (H₂O₂) and nitrite/nitrate (NOx). Fluorimetric and spectrophotometric assays were performed in DS (n = 78), some of them taking levothyroxine for hypothyroidism (n = 24), and in their healthy age-matched controls (n = 65). We found that levels of AGEs, diTyr, H₂O₂ and NOx are increased in DS patients in any or in all age groups, whereas Cr levels were lower in DS than in controls in all age groups. Besides, correlations with age in DS were positive for diTyr and negative for Cr, TBARS, 15-F_2t-IsoP and NOx. We also found lower levels of Cr from 15 to 19 years, higher levels of TBARS and AGEs from 20 to 40 years and higher levels of diTyr from 15 to 40 years in DS patients receiving levothyroxine than in DS without hypothyroidism diagnosed. We conclude that AGEs, diTyr, H₂O₂ and NOx could be used as oxidative stress biomarkers in DS in contrast to 8-OHdG, 15-F_2t-IsoP and TBARS, at least with the methods used. However, renal impairment could occur in DS and Cr adjustment may bias the results, particularly in hypothyroid patients.     

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant ‘Quickstart II’ and salt-sensitive ‘DP1′ were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4 d after treatment (DAT), but a lower level of enzyme activity at 8 and 12 d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.     

Inhibition of NADPH oxidase alleviates germ cell apoptosis and ER stress during testicular ischemia reperfusion injury

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.