Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks.

Triketones, derived chemically from a natural phytotoxin (leptospermone), are a good example of allelochemicals as lead molecules for the development of new herbicides. Targeting a new and key enzyme involved in carotenoid biosynthesis, these latest-generation herbicides (sulcotrione, mesotrione and tembotrione) were designed to be eco-friendly and commercialized fifteen-twenty years ago. The mechanisms controlling their fate in different ecological niches as well as their toxicity and impact on different organisms or ecosystems are still under investigation. This review combines an overview of the results published in the literature on ß-triketones and more specifically, on the commercially-available herbicides and includes new results obtained in our interdisciplinary study aiming to understand all the processes involved (i) in their transfer from the soil to the connected aquatic compartments, (ii) in their transformation by photochemical and biological mechanisms but also to evaluate (iii) the impacts of the parent molecules and their transformation products on various target and non-target organisms (aquatic microorganisms, plants, soil microbial communities). Analysis of all the data on the fate and impact of these molecules, used pure, as formulation or in cocktails, give an overall guide for the assessment of their environmental risks.

Grape marc extract causes early perception events, defence reactions and hypersensitive response in cultured tobacco cells.

Grape marc extract (GME) showed elicitor activity on suspension-cultured cells of tobacco. The BY-2 cells reacted to GME (0.25% and 0.125%) with a long-sustained pH rise in their growth medium. Using EGTA or LaCl3, we showed that extracellular alkalinization depended on Ca(2+) mobilization. The tobacco BY-2 cells challenged with GME promoted cell death and the upregulation of defence-related genes such as PR3, PAL and CCoAOMT. Cell death rate was quantified using an experimental calibrated Evans Blue assay. The GME-induced cell death was dose-dependent and occurred in 24 h. Longer exposure increased the extent of tobacco cell death. To investigate a potential hypersensitive reaction, we tested the effect of various inhibitors of protein synthesis (cycloheximide) and proteases (aprotinin, pepstatin and E-64) on GME-induced cell death. All these chemicals reduced GME-induced cell death rate in 30 min. Overall, our findings indicate that GME elicits early perception events, defence reactions and cell death requiring protein synthesis and proteases.

Exposure of Vicia faba and Pisum sativum to copper-induced genotoxicity.

The potential genotoxicity of Cu(2+) was investigated in Vicia faba and Pisum sativum seedlings in hydroponic culture conditions. Cu(2+) caused a dose-dependent increase in micronuclei frequencies in both plant models. Cytological analysis of root tips cells showed clastogenic and aneugenic effects of this heavy metal on V. faba root meristems. Cu(2+) induced chromosomal alterations at the lowest concentration used (2.5 mM) when incubated for 42 h, indicating the potent mutagenic effect of this ion. A spectrum of chromosomal abnormalities was observed in V. faba root meristems, illustrating the genotoxic events leading to micronuclei formation.

Sucrose assimilation during early developmental stages of chicory (Cichorium intybus L.) plants.

The activities of enzymes of both sucrose and fructan metabolism were measured in chicory (Cichorium intybus L. cv. Turbo) plants during early vegetative growth. From 21 to 42 d after sowing (phase I), carbohydrates were used for structural growth and sucrose was predominantly cleaved by acid invertase whereas neutral invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13) activities were low. From 49 to 63 d after sowing (phase II) a cambium formed producing secondary tissues, concomitant with induced sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) and fructan:fructan-1-fructosyl transferase (EC 2.4.1.100) activities, and fructan synthesis in the roots. Accumulation of 1-SST mRNA occurred at the onset of thickening, indicating that 1-SST is controlled at a transcriptional level. Acid invertase activity gradually increased during phase I and remained high during early phase II. It subsequently decreased. The pattern of invertase mRNA accumulation correlated with the enzyme activities, indicating that acid invertase is controlled at the transcriptional level. Both acid invertase and 1-SST probably contributed to the sink strength in the root at the beginning of phase II.

Nitrate assimilation in chicory roots (Cichorium intybus L.) which acquire radial growth.

Nitrate assimilation was analysed in chicory plants (Cichorium intybus L. cv. Turbo) during the early vegetative growth. Nitrate reductase (NR, EC 1.6.6.1) activity (NRA) was measured in roots and leaves at different developmental stages. During phase I, which corresponds to the structural growth (21-42 DAS), nitrate reduction mainly occurred in the roots. At the onset of the tuber formation (phase II), which is characterized by the formation of a cambium inducing a radial growth (42-63 DAS), NRA rapidly decreased in roots and developed in leaves. A tight correlation was found between the nitrate content, the amino acid level and NRA in roots and leaves. Northern blot and ELISA analysis showed that both levels of NR mRNA and NR protein were not modified during the time-course of the experiment suggesting that modification of nitrate assimilation was not controlled at a transcriptional level. In vitro NRA assayed in presence of either Mg2+ ions or EDTA showed that NR was influenced at least in part by a reversible phosphorylation/dephosphorylation reaction. Okadaic acid, a serine-threonine protein phosphatases inhibitor, strongly decreased NRA. Conversely, staurosporine, a serine-threonine protein kinases inhibitor, did not significantly change NRA in roots or leaves. Therefore, NRA was regulated at a post-translational level during the early vegetative growth by modifying the phosphorylation balance of the NR protein in chicory.

Expression of DC8 is associated with, but not dependent on embryogenesis.

DC8 is a late embryogenesis-abundant (LEA) protein gene isolated from carrot (Daucus carota). Deletion analysis of the DC8 promoter was performed to determine the sequences required for ABA and seed-specific regulation of DC8 transcription. To investigate the mechanism of DC8 expression during seed development, chimeric gene constructs containing DC8 promoter fragments fused to a promoterless beta-glucuronidase gene (DC8:GUS) were introduced into carrot, tobacco (Nicotiana tobacum) and Arabidopsis thaliana plants. Seed-specific DC8 expression patterns was conserved among the three plant species. However, differences among the species in the patterns of DC8 expression in the embryo and endosperm that correlated with differences in the rates of embryo and endosperm growth were found. Lack of correspondence between DC8 activation and embryo development among the seeds of the three species suggests that DC8 expression, which is associated with seed maturation, is not coupled to the embryo development program. The presence of DC8 activity in carrot callus and endosperm is consistent with the notion that DC8 expression is independent of embryo morphogenesis. A similar DC8 activity time-course during callus induction and seed development suggests that explantation and 2,4-D treatment initiates a course of events similar to that in the carrot ovule. After fertilization, two pathways one leading to embryo development and another to seed maturation are initiated, but they are not closely linked. As a result we find DC8, part of the maturation program, being activated at different embryonic stages in different plant species.

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots.

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 nM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.

Phase II trial of intraperitoneal carboplatin in ovarian carcinoma patients with macroscopic residual disease at second-look laparotomy. A multicentre study of the French Fédération Nationale des Centres de Lutte Contre le Cancer.

BACKGROUND: Because of its antitumour activity and its pharmacological advantage when administered by the intraperitoneal route, carboplatin was studied in a phase II multicentric trial. The aim of the study was to determine the response rate and the toxicity of carboplatin administered intraperitoneally and to determine if pathological complete response could be attained in women with macroscopic residual ovarian cancer at second-look laparotomy after intravenous cisplatin chemotherapy. PATIENTS AND METHODS: Twenty-nine patients with macroscopical residual disease after intravenous cisplatin-based chemotherapy at second-look laparotomy, were treated at that time with 300 mg/m2 of carboplatin administered in the abdominal cavity every four weeks for six cycles. In instances of negative findings at physical and CT scan examination, laparotomy evaluation was performed and the catheter was removed. The dose of carboplatin was increased or decreased according to hematological toxicity. RESULTS: Efficacy is evaluable in 25 pts: 2 pts had pathological complete responses and 1 pt had microscopic disease (12% response rate of evaluable patients). Toxicity is evaluable for 135 cycles in 29 patients. No grade 4 hematological toxicity was observed, 2 pts had grade 3 leukopenia and 3 pts had grade 3 thrombocytopenia; grade 3 vomiting was observed in 11% of cycles. No peritoneal complication was observed; catheter dysfunction occurred after the first cycle in one patient who refused a surgical procedure to remove the catheter and to pursue treatment. CONCLUSION: Intraperitoneal carboplatin demonstrates efficacy in patients with macroscopical residual disease at second-look laparotomy after first-line cisplatin chemotherapy. The recommended dose for further studies is 300 mg/m2 administered every 4 weeks. A low response rate does not favour a randomised study.

Molecular cloning and physiological analysis of an invertase isoenzyme in Helianthus tissues.

A soluble acid invertase activity isolated from Helianthus tuberosus (Jerusalem artichoke) shoots and analyzed by immunochromatography using polyclonal yeast antibodies, represents around 5% of the total invertase activity. This invertase isoenzyme was also isolated from dormant tuber parenchyma. In these partially dormant tissues, the specific activity of this isoenzyme is low suggesting a partial inactivation of the invertase molecules. Polyacrylamide gel electrophoresis of immunopurified fractions yields similar levels of the 58 kDa polypeptide both in shoots and dormant tubers, but with much lower activity of the enzyme in the tubers. A cDNA library was constructed in pUEX 1 from poly (A)+ RNA extracted from Jerusalem artichoke tubers. This library was screened for invertase using (i) a Bacillus subtilis invertase DNA probe and (ii) anti-yeast invertase antibodies. A recombinant clone of approximately 1.8 kb size was selected by these two methods. Using Northern blots, a temporal sequence in the expression of invertase gene was observed during the breaking of dormancy with the main level after 8 weeks of cold treatment at 4 degrees C. A 2.5 kb transcript was detected, translation of which would yield a 97 kDa polypeptide representing the precursor of Jerusalem artichoke invertase.

Transcriptional regulation of a seed-specific carrot gene, DC8.

Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to beta-glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from -170 to -51 contained ABA-responsive sequences that required a 5' upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5' upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.