Human cytochrome P450 reductase can act as a source of endogenous oxidative DNA damage and genetic instability

Studies with repair-deficient mice and other experiments suggest that oxidative DNA modifications are generated in all types of cells even under physiological conditions and that this type of endogenous DNA damage contributes to spontaneous cancer incidence. However, the cellular sources of reactive oxygen species that are relevant for nuclear oxidative DNA damage are largely unknown. Here, we report that expression of human NADPH-cytochrome P450 reductase (hOR) in cultured V79 Chinese hamster cells gives rise to elevated basal levels of oxidative purine modifications after depletion of glutathione. Also, the basal levels of micronuclei are increased in the hOR-expressing cells, and again the effect is enhanced when the antioxidant defense system of the cells is diminished by depletion of glutathione. The oxidative DNA damage is increased when duroquinone, a substrate of hOR, is added, both in the presence and absence of glutathione. In contrast, hOR-expressing cells are similarly sensitive as the parental cells when oxidative DNA damage and micronuclei are induced by a mechanism independent of hOR, i.e., exposure to bromate. The results identify hOR as a potential source of endogenous oxidative DNA damage and subsequent genetic instability in mammalian cells.     

Three unique mutants of Arabidopsis identify eds loci required for limiting growth of a biotrophic fungal pathogen

To identify components of the defense response that limit growth of a biotrophic fungal pathogen, we isolated Arabidopsis mutants with enhanced disease susceptibility to Erysiphe orontii. Our initial characterization focused on three mutants, eds14, eds15, and eds16. None of these is considerably more susceptible to a virulent strain of the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). All three mutants develop a hypersensitive response when infiltrated with Psm expressing the avirulence gene avrRpt2, which activates resistance via the LZ-NBS/LRR resistance protein encoded by RPS2. The growth of Psm(avrRpt2), while somewhat greater in the mutants than in the wild type, is less than growth of the isogenic virulent strain. These results indicate that resistance mediated via LZ-NBS/LRR R genes is functional. Analysis of the growth of avirulent Peronospora parasitica strains showed that the resistance pathway utilized by TIR-NBS/LRR R genes is also operative in all three mutants. Surprisingly, only eds14 and eds16 were more susceptible to Erysiphe cichoracearum. Analysis of the expression profiles of PR-1, BGL2, PR-5 and PDF1.2 in eds14, eds15, and eds16 revealed differences from the wild type for all the lines. In contrast, these mutants were not significantly different from wild type in the deposition of callose at sites of E. orontii penetration. All three mutants have reduced levels of salicylic acid after infection. eds16 was mapped to the lower arm of chromosome I and found by complementation tests to be allelic to the salicylic acid-deficient mutant sid2.     

How to avoid pharmaceuticals in the aquatic environment

Pharmaceuticals and other micropollutants in wastewater pose a new challenge to wastewater professionals as well as to the pharmaceutical industry. Although there is a great deal of uncertainty concerning the possible detrimental effects on the aquatic ecosystems, the precautionary principle--or possibly new scientific evidence--may give rise to more stringent demands on wastewater treatment in the future. In conventional wastewater treatment plants, a combination of biological treatment with high sludge residence times and ozonation of the effluent seems to be the most promising technology. Ozonation, however, is an energy-intensive technology. Moreover, in conventional end-of-pipe systems a large part of the pollutants will always be lost to the environment due to leaking, primarily during rain. In the long term, source separation offers the more sustainable solution to the entire wastewater problem, including organic micropollutants. Urine source separation is an elegant solution to the problems of nutrients and pharmaceuticals alike and losses of untreated pollutants to the environment can be minimized. Although few technologies for the separate treatment of urine have been developed to date, the 100-500 times higher concentrations of micropollutants promise more efficient conditions for all removal technologies known from conventional wastewater treatment.     

Colonial differentiation in Streptomyces coelicolor depends on translation of a specific codon within the adpA gene

We identified adpA as an araC-like regulatory gene needed for colonial morphogenesis in Streptomyces coelicolor and showed that its activity depended on a unique TTA triplet corresponding to the leucyl-tRNA gene (bldA). These findings partially explained the dependence of aerial mycelium formation on a rare tRNA that is postulated to have developmental control functions.     

Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants

A genetically tractable model plant pathosystem, Pseudomonas syringae pv. tomato DC3000 on tomato and Arabidopsis thaliana hosts, was used to investigate the role of salicylic acid (SA) and iron acquisition via siderophores in bacterial virulence. Pathogen-induced SA accumulation mediates defense in these plants, and DC3000 contains the genes required for the synthesis of SA, the SA-incorporated siderophore yersiniabactin (Ybt), and the fluorescent siderophore pyoverdin (Pvd). We found that DC3000 synthesizes SA, Ybt, and Pvd under iron-limiting conditions in culture. Synthesis of SA and Ybt by DC3000 requires pchA, an isochorismate synthase gene in the Ybt genomic cluster, and exogenous SA can restore Ybt production by the pchA mutant. Ybt was also produced by DC3000 in planta, suggesting that Ybt plays a role in DC3000 pathogenesis. However, the pchA mutant did not exhibit any growth defect or altered virulence in plants. This lack of phenotype was not attributable to plant-produced SA restoring Ybt production, as the pchA mutant grew similarly to DC3000 in an Arabidopsis SA biosynthetic mutant, and in planta Ybt was not detected in pchA-infected wild-type plants. In culture, no growth defect was observed for the pchA mutant versus DC3000 for any condition tested. Instead, enhanced growth of the pchA mutant was observed under stringent iron limitation and additional stresses. This suggests that SA and Ybt production by DC3000 is costly and that Pvd is sufficient for iron acquisition. Further exploration of the comparative synthesis and utility of Ybt versus Pvd production by DC3000 found siderophore-dependent amplification of ybt gene expression to be absent, suggesting that Ybt may play a yet unknown role in DC3000 pathogenesis.     

Strategy for genotoxicity testing--metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.     

Environmental and developmental controls over the seasonal pattern of isoprene emission from aspen leaves

Isoprene emission from plants represents one of the principal biospheric controls over the oxidative capacity of the continental troposphere. In the study reported here, the seasonal pattern of isoprene emission, and its underlying determinants, were studied for aspen trees growing in the Rocky Mountains of Colorado. The springtime onset of isoprene emission was delayed for up to 4 weeks following leaf emergence, despite the presence of positive net photosynthesis rates. Maximum isoprene emission rates were reached approximately 6 weeks following leaf emergence. During this initial developmental phase, isoprene emission rates were negatively correlated with leaf nitrogen concentrations. During the autumnal decline in isoprene emission, rates were positively correlated with leaf nitrogen concentration. Given past studies that demonstrate a correlation between leaf nitrogen concentration and isoprene emission rate, we conclude that factors other than the amount of leaf nitrogen determine the early-season initiation of isoprene emission. The late-season decline in isoprene emission rate is interpreted as due to the autumnal breakdown of metabolic machinery and loss of leaf nitrogen. In potted aspen trees, leaves that emerged in February and developed under cool, springtime temperatures did not emit isoprene until 23 days after leaf emergence. Leaves that emrged in July and developed in hot, midsummer temperatures emitted isoprene within 6 days. Leaves that had emerged during the cool spring, and had grown for several weeks without emitting isoprene, could be induced to emit isoprene within 2 h of exposure to 32°C. Continued exposure to warm temperatures resulted in a progressive increase in the isoprene emission rate. Thus, temperature appears to be an important determinant of the early season induction of isoprene emission. The seasonal pattern of isoprene emission was examined in trees growing along an elevational gradient in the Colorado Front Range (1829–2896 m). Trees at different elevations exhibited staggered patterns of bud-break and initiation of photosynthesis and isoprene emission in concert with the staggered onset of warm, springtime temperatures. The springtime induction of isoprene emission could be predicted at each of the three sites as the time after bud break required for cumulative temperatures above 0°C to reach approximately 400 degree days. Seasonal temperature acclimation of isoprene emission rate and photosynthesis rate was not observed. The temperature dependence of isoprene emission rate between 20 and 35°C could be accurately predicted during spring and summer using a single algorithm that describes the Arrhenius relationship of enzyme activity. From these results, it is concluded that the early season pattern of isoprene emission is controlled by prevailing temperature and its interaction with developmental processes. The late-season pattern is determined by controls over leaf nitrogen concentration, especially the depletion of leaf nitrogen during senescence. Following early-season induction, isoprene emission rates correlate with photosynthesis rates. During the season there is little acclimation to temperature, so that seasonal modeling simplifies to a single temperature-response algorithm.     

Bacterial mutagenicity investigation of epoxides: drugs,drug metabolites,steroids and pesticides

Although it has been observed that many epoxides are ultimate mutagens, surprisingly little is known about epoxides to which man may be extensively exposed, e.g., physiological compounds, drugs, drug metabolites and pesticides. We have now investigated 35 such and related epoxides for mutagenicity, using reversion of his^?Salmonella typhimurium TA98 and TA100 as biological end-point. None of the tested steroids (12 compounds), vitamin K epoxides (3 compounds) and pesticides (dieldrin, endrin, HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a5,6,7,8,8a-octahydro-1,4-methanonaphthalene), heptachlor epoxide) showed any mutagenic activity. Negative results were also obtained with the antibiotics oleandomycin, anti-capsin and asperlin, the cardiotonic drug resibufogenin, the widely used parasympatholytic drugs butylscopolamine and scopolamine, the sedatives valtratum, didovaltratum and acevaltratum, the tranquilizer oxanamide as well as with the drug metabolites carbamazepine 10,11-oxide and diethylstilbestrol α,β-oxide. Three barbiturate epoxides, formed by metabolism of allobarbital, alphenal and secobarbital, caused weak but reproducible mutagenic effects at high concentrations. The cytostatic agent ethoglucide was the only drug having substantial mutagenic activity. Its mutagenic potency was similar to those of the control epoxides styrene 7,8-oxide, p-bromostyrene 7,8-oxide and m-bromostyrene 7,8-oxide, but much lower than those of benzo[a]pyrene 4,5-oxide, benzo[e]pyrene 4,5-oxide and 7,12-dimethylbenz[a]-anthracene 5,6-oxide.Some epoxides were also tested in other Salmonella typhimurium strains or in the presence of rat-liver S9 mix. Positive results were only obtained with compounds that had already been detected as mutagens in the direct test with strain TA100.