Parental exposure to methyl methane sulfonate of three-spined stickleback: contribution of DNA damage in male and female germ cells to further development impairment in progeny.

Data regarding the link between DNA integrity of germ cells and the quality of progeny in fish exposed to genotoxicant are scarce although such information is of value to understand genotoxic effects of contaminants in aquatic fauna. This work aimed at studying the consequences of a parental exposure during the breeding season on offspring quality in three-spined stickleback. After in vivo exposure of adult fish to methyl methane sulfonate, a model alkylating compound, a clear increase in DNA damage was observed in erythrocytes of both genders, here used as a biomarker of exposure. MMS exposure significantly affected sperm DNA integrity but neither female fecundity nor fertilization success. In order to understand the contribution of each sex to potential deleterious effects in progeny due to parental exposure, mating of males and females exposed or not to MMS, was carried out. Exposure of both males and females or of males alone led to a significant increase in both mortality during embryo-larval stages and abnormality rate at hatching that appeared to be sensitive stages. Thus, in accordance with recent studies carried out in other freshwater fish species, such development defects in progeny were clearly driven by male genome, known to be devoid of DNA repair capacity in spermatozoa. The next step will be to investigate the link between DNA damage in stickleback sperm and reproductive impairment in natural populations exposed to complex mixture of genotoxicants.

The goby fish Sicydium spp. as valuable sentinel species towards the chemical stress in freshwater bodies of West Indies.

Implementation of the European Water Framework Directive in tropical areas such as the French West Indies (FWI) requires to select relevant aquatic sentinel species for investigating the ecological status of surface waters. The present work aimed to study the biological response of the widespread fish Sicydium spp. towards river chemical quality in Guadeloupe island through a set of proper biomarkers. During a 2-year survey, the hepatic EROD activity, the micronucleus formation and the level of primary DNA strand breaks in erythrocytes were measured respectively as an enzymatic biomarker of exposure and genotoxicity endpoints in fish living upstream and downstream of two chemically-contrasted rivers. Hepatic EROD activity was shown to be variable along the time but always significantly higher in fish from the most contaminated river (Rivière aux Herbes) compared to the low contaminated one (Grande Rivière de Vieux-Habitants). Fish size did not influence EROD activity. Female fish exhibited a lower EROD activity compared to males depending on the catching period. We observed significant temporal variation in micronucleus frequency and primary DNA damage level measured in fish erythrocytes that did not depend on the fish size. Micronucleus frequency and to a lesser extent DNA damage were significantly higher in fish from the Rivière aux Herbes compared to the Grande Rivière de Vieux-Habitants. Our results argue for the interest of using Sicydium spp. as sentinel species to assess river quality and chemical pressures in FWI.

Genotoxic pressure of vineyard pesticides in fish: field and mesocosm surveys.

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2 microg L(-1) for diuron and 0.5-1 microg L(-1) for axoxystrobin). A single 6h pulse of pesticide (14 microg L(-1) of diuron and 7 microg L(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.

Genotoxic potential associated with low levels of the Fusarium mycotoxins nivalenol and fusarenon X in a human intestinal cell line.

This study aims to assess the genotoxic potential of nivalenol (NIV) and fusarenon X (FusX), produced by various Fusarium on cereals. Toxins were applied in time and dose-dependent experiments to the human enterocyte-like Caco-2 cell-line, both in dividing (undifferentiated) and in 10-12 days post-confluent cells (differentiated). Genotoxicity was evaluated through the alkaline Comet assay in a concentration range defined for each toxin as below the cytotoxicity threshold IC(10), determined by the MTS and the neutral red assays, to prevent false positive results because of DNA damage stemming from necrosis. Thus, genotoxicity was explored in the sub-cytotoxic 0-0.5 microM and 0-0.05 microM ranges respectively for NIV and FusX as the latter was found about 10-fold more cytotoxic than NIV. For both toxins, a 3h exposure did not cause any DNA damage, unlike after 24 and 72 h exposure in post confluent Caco-2 cells where DNA damage was significantly observed with a dose-dependent relationship. In dividing cells, only FusX increases DNA strand breaks in the 0.01-0.05 microM range after 72 h. These results demonstrated the existence of a genotoxic potential for NIV and FusX at low exposure levels and could contribute to the risk assessment process of these toxins that are of growing concern.

Evaluation of HIV serial and parallel serologic testing algorithms in Abidjan, Côte d'Ivoire.

OBJECTIVE: To evaluate HIV serologic testing algorithms based on a combination of three enzyme linked immunosorbent assays (ELISA) for the confirmation of HIV infection in Abidjan, Côte d'Ivoire, where HIV-2 and HIV-1 non-B subtypes are prevalent. METHODS: A total of 1069 human sera with known serologic status, in addition to a seroconversion and low titer antibody panel were initially tested by six ELISA to determine the sensitivity, specificity and delta values of the assays. On the basis of the performance of the assays, three ELISA (Enzygnost, ICE 1.0.2, and Vironostika) were selected for use in a parallel and serial testing algorithm in analyzing 8283 consecutively collected sera. In the parallel testing algorithm, sera concordantly reactive or non-reactive by Enzygnost and ICE 1.0.2 were considered as true positive or true negative, respectively. In the serial algorithm, sera reactive by Enzygnost were retested by ICE 1.0.2. Sera with discordant results were tested by Vironostika, and the results was considered definitive. All reactive sera, plus a random sample of negative sera were tested for confirmation by Peptilav. In addition, a random sample of reactive sera was tested by Western blot. RESULTS: All ELISA had 100% sensitivity; specificities ranged from 96.8 to 100%. Positive and negative delta values of the ELISA were high (range, 6.89 to 46.07 and -2.05 to -5.75, respectively). Of the 8283 sera, 2054 were considered true positives and were correctly classified by the parallel testing algorithm (sensitivity, 100%). Of the 6229 true negative sera, 6226 were negative by the parallel testing algorithm (specificity, 99.95%). The sensitivity of the serial algorithm was 99.96%, and specificity was 99.95%. None of the 250 concordant ELISA-negative sera in the algorithm that were randomly tested in Peptilav was positive; similarly, all of the 103 concordant ELISA-positive sera were confirmed by Western blot. The three-ELISA algorithm resulted in reagent cost-savings of at least 50% compared with the Peptilav-based algorithm. CONCLUSION: These results suggest that a combination of ELISA using different principles or antigens in a serial or parallel algorithm is an efficient and cost-effective alternative to the standard algorithm in areas where HIV-1 and HIV-2 are prevalent.

Rapid analysis of ergovaline in ovine plasma using high-performance liquid chromatography with fluorimetric detection.

A rapid high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in ovine plasma is described here. Ergotamine was used as an internal standard. A simple extraction procedure with diethyloxide was carried out, before chromatography on a C8 column, with the excitation and emission wavelengths fixed at 250 and 420 nm respectively, on a fluorimetric detector. The method, which was found to be linear between 3.5 and 15 ng/ml, had good specificity, precision and accuracy. The limit of quantification and the limit of detection were 3.5 and 1.2 ng/ml, respectively. A preliminary application of the described assay to a plasma kinetic study, after intravenous administration of a single dose of ergovaline (17 micrograms/kg body mass) to four sheep, showed a very rapid decrease of the plasma ergovaline levels. The terminal half-life and the total clearance of the mycotoxin were found to be 23.6 min and 0.020 l/min kg-1 body mass, respectively.

Influence of consumption of endophyte-infested tall fescue hay on performance of heifers and lambs.

Two experiments were conducted to evaluate performance and physiological responses of heifers and lambs to Neotyphodium coenophialum-infested tall fescue hay fed under European rearing conditions. Endophyte-free (E-) or 100% endophyte-infested (E+) hay was derived from the same cultivar (cv. Clarine) so that the effect of the endophytic fungus could be clearly separated from a possible cultivar effect. In Exp. 1, starting in June 1996, 20 age- and body weight-paired Holstein dairy heifers were assigned for 97 d to one of two treatments consisting of ad libitum access to either E- or E+ hay, corresponding to 0 and .41 mg/kg ergovaline, respectively. During the experimental period, no significant difference (P>.20) in forage consumption, rectal temperature, or behavioral status of the animals was observed between the two treatments. The E+ diet induced a 10% apparent decrease in ADG and a clear reduction in prolactin (PRL) plasma concentration compared to the E- diet. When animals were all reassigned to a common endophyte-free diet, the E+ group recovered body weight and PRL to levels similar to those in animals fed E- after 7 wk. In Exp. 2, 30 Texel ram lambs were assigned to two treatments consisting of dietary E- or E+ tall fescue hay. The E- and E+ hays were harvested from the same plots as used in Exp. 1 and contained 0 and .96 mg/kg ergovaline, respectively. No effect of the endophyte was found on intake or carcass or testicle weight (P>.20) after the 95-d feeding period. The E+ treatment resulted in a slight reduction in BW at slaughter, mainly explained by a lower ruminal fill (P<.01). In E+ treated animals, prolactin concentrations dropped significantly (P<.001) from d 27. Hay assessment in both experiments showed no difference in chemical composition and IVDMD. The endophytic fungus strongly lowered the palatability of the E+ hay, although there was no effect on intake with heifers (Exp. 1) or with lambs (Exp. 2). The potential of severe heat stress, as expressed by the temperature humidity index, was not high in our experimental conditions, although they were considered rather unusually stressful for the western part of northern Europe. Yet, no economic effect on cattle was observed, in disagreement with results obtained in many previous U.S. studies.

Identification of the Fungal Endophyte Epichloe festucae in the Fine Fescue Festuca ampla.

Festuca ampla is native to the Iberian Peninsula (4). Endophytic mycelium was observed by microscopy (2) in stem pith samples of two of eight asymptomatic plants of F. ampla collected in one population from a natural grassland in Salamanca, Spain. The fungus could be isolated (2) only from these two plants, and conidiophores and reniform conidia typical of Epichloe species (3) were observed in pure cultures. The two infected plants maintained in pots outside, developed ectostromata in some reproductive stems (choke disease) the year after the field sampling. Six seeds collected from an infected plant were germinated, and all six seedlings were found to be infected based on microscopy (2), implying seed transmission of the endophyte. These observations suggest that this is a pleiotropic symbiont, having both mutualistic and pathogenic states in its host. An ergovaline concentration of 120 ng/g dry weight was detected in a sample of leaves and leaf sheaths of an infected plant. All of the above characteristics are typical of the genus Epichloe, and in particular of the fine fescue endophyte E. festucae (1,3). To determine the species, internal transcribed spacer and 5.8-rDNA sequences as well as a partial sequence of the ß-tubulin gene were obtained. These two sequences (EMBL Accession Nos. AJ488497 and AJ488498) showed 100% sequence homology to the corresponding sequences in E. festucae. To our knowledge, this is the first report of this endophyte species in the grass F. ampla. References: (1) L. P. Bush et al. Plant Physiol. 114:1, 1997. (2) E. M. Clark et al. J. Microbiol. Methods 1:149, 1983. (3) A. Leuchtmann et al. Mycologia 86:802, 1994. (4) I. Markgraff-Dannenberg. Festuca. Pages 125-153 in: Flora Europaea, Vol 5. Cambridge University Press, Cambridge, 1980.

Genotoxic potential of several naphthenic acids and a synthetic oil sands process-affected water in rainbow trout (Oncorhynchus mykiss).

The exploitation of oil sands has raised major environmental concerns, particularly regarding the presence of high concentration in contaminants such as polycyclic aromatic hydrocarbons (PAHs) and naphthenic acids (NAs) in oil sands process-affected water (OSPW). The purpose of this study was, first to evaluate the genotoxic impact of OSPW-related compounds such as NAs and PAHs in a salmonid species and secondly to assess if OSPW exposure leads to genotoxicity. For this purpose, rainbow trout hepatocytes were exposed in vitro to environmentally relevant concentrations of synthetic NAs, naphtalene, benzo(a)pyrene, and extracts of synthetic OSPW (generated by a laboratory bitumen extraction) and of oil sands leaching water (OSLW, mimicking leaching of oil sands in river water). Primary DNA damage was assessed by the formamidopyrimidine-DNA glycolyase (Fpg)-modified comet assay. Genotoxicity was observed in hepatocytes exposed to several NAs, mixture of them, OSPW and OSLW extracts. The chemical structure of NAs influences the genotoxicity potential: among the NAs tested, the most cyclic NA was the most genotoxic. It also appears that genotoxicity was more marked for OSPW than for OSLW. Because exposure to OSPW led to oxidative DNA damage, while after exposure to several NAs, these types of DNA damage were limited, the NAs tested in this study could not be qualified as the only major contaminants responsible for OSPW genotoxicity. Notwithstanding, it should be noteworthy that exposure to NAs resulted in genotoxic impact at concentrations lower than those documented by literature for fresh OSPW. Further research is needed to explore the relationships between the chemical structure of NAs and their genotoxicity in the light of the distribution of NAs in fresh OSPW samples as well as in surface waters.

Relationship between DNA damage in sperm after ex vivo exposure and abnormal embryo development in the progeny of the three-spined stickleback.

Many xenobiotics released in the aquatic environment exhibit a genotoxic potential toward organisms. Long term exposure to such compounds is expected to lead to multigenerational reproductive defects, further influencing the recruitment rate and hence, the population dynamics. Paternal exposure to genotoxicants was previously shown to increase abnormal development in the progeny of mammalian or aquatic species. The aim of this study was to evaluate the relationship between DNA damage in sperm of the fish three-spined stickleback and progeny developmental defects. Spermatozoa were exposed ex vivo to an alkylating agent (methyl methanesulfonate) before in vitro fertilization and DNA damage was assessed by the alkaline comet assay. A significant relationship between abnormal development and sperm DNA damage was underlined. This study illustrates the interest to use germ cell DNA damage after ex vivo exposure to evaluate the impact of genotoxic compounds on progeny fitness in aquatic organisms.

Toxicokinetics of ergovaline in the horse after an intravenous administration.

The toxicokinetics of ergovaline (an ergopeptine mycotoxin present in some grasses infected with endophytic fungus of the genus Neotyphodium) were studied after intravenous administration of a single dose of 15 microg/kg bwt in four gelding horses. Plasma ergovaline concentrations were measured by high performance liquid chromatography, and the kinetic data were described by a three-compartment model. The elimination half-life and the total clearance of ergovaline were found to be 56.83 +/- 13.48 min and 0.020 +/- 0.004 L/min x kg, respectively. According to the toxicological data previously reported in the horse, and in spite of the very low dose administered, clinical signs were observed, including excessive coolness of the ears and the nose, excessive sweating and prostration.

Analysis of ergovaline in milk using high-performance liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic method for the determination of the mycotoxin ergovaline in goat's milk is described here. Ergotamine was used as an internal standard. For a sample size of 5.0 ml, the cleanup method included precipitation of milk protein with acetone. Then, ergovaline was extracted twice with chloroform and purified by elution on an Ergosil column. HPLC separation of the extract was accomplished on a C18 column: an isocratic elution, using acetonitrile-ammonium carbonate, was performed, and the analyte was detected by fluorimetry. The method was found to be linear between 0.7 and 8 ng ml(-1), a mean recovery rate of 99.8% was obtained, and the described assay appeared both repeatable and reproducible. The limit of detection and the limit of quantitation of ergovaline in milk were 0.2 ng ml(-1) and 0.7 ng ml(-1), respectively. In order to apply the proposed method, four lactating goats were administered the toxin intravenously at a dose of 32 mg kg(-1) body weight. The concentrations of the drug in plasma and milk were then determined at standardized intervals. Ergovaline (unequivocally identified by LC-MS-MS) could not be detected in the milk beyond eight hours post-dosing. Therefore, in goats, milk does not appear to be a major excretion route for the unmetabolized toxin.

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes.

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na. In contrast, the attachment was affected by the removal of divalent cations (Mg and Ca), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100 degrees C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg and Ca), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca and Mg. Hydrophobic bonds and enzymes may also be involved.

[Schwannoma of the parotid region. Apropos of a case report]. / Schwannome de la région parotidienne. A propos d'un cas clinique.

Schwannomas are rarely located in the parotid regions. We report a case in a 6-year old child and recall the epidemiology of this type of tumours together with the clinical presentations and therapeutic approaches.

Microbial thiamin metabolism in the rumen simulating fermenter (RUSITEC): the effect of acidogenic conditions, a high sulfur level and added thiamin.

The effects of acidogenic conditions, a high S level and the addition of thiamin on the rumen microbial metabolism of thiamin were investigated in vitro in a semi-continuous fermenter (RUSITEC), using a factorial design. Acidogenic conditions were obtained by simultaneously increasing the starch: cellulose ratio and the amount of solid substrate fed, and by decreasing the buffering capacity of the liquid phase of the fermenter. S in the form of sulfate was supplied at two levels, one corresponding to a control amount of S (2 g/kg dietary DM), the second to an excess (5 g/kg DM) which is sufficient to trigger cerebrocortical necrosis (CCN) when used in vivo. Acidogenic conditions decreased the pH of the fermenters, CH4 production and cellulose digestibility, increased the short-chain fatty acid production, but had no effect on thiamin production. The high S level enhanced the production of sulfide considerably, had no effect ont he microbial metabolism of energy and N, and decreased thiamin production (326 v. 266 nmol/d). The added thiamin was rapidly converted into phosphorylated compounds which largely decreased the apparent synthesis of this vitamin by the rumen microflora. The total thiamin flow was increased by added thiamin. In no case was thiaminase activity in the fermenter liquid phase significantly modified. The high level of S induced only a limited decrease of total thiamin flow. Consequently, it is unlikely that the investigated factors could be considered to be high risk factors for the thiamin-dependent CCN.

Use of a semi-continuous culture system (RUSITEC) to study the effect of pH on microbial metabolism of thiamin (Vitamin B1).

Polioencephalomalacia (P.E.M.) in ruminants is often associated with high concentrate diets and rumen acidosis; this syndrome is classically related to a disturbance of the rumen metabolism of thiamin. An in vitro model using a semicontinuous system (RUSITEC) was used to investigate the effect of pH on microbial metabolism and on production of thiamin in the rumen. These effects were tested using either a natural diet (hay/wheat) or a semi-synthetic one. Lowering the pH decreased total volatile fatty acids, methane and microbial nitrogen production. Molar proportions of VFA were modified by an increase in butyric, valeric and caproic acids. Microbial production of thiamin was comparable to in vivo synthesis but decreased when the diet was enriched with thiamin. The diet of the donors of inoculum had no effect on this metabolism. For all diets, lowering of pH did not reduce microbial production of thiamin. Thiaminase activity in the liquid phase of fermentors was very low and was not modified by pH. Thus lowering of pH in vitro, had no deleterious effect on microbial production of thiamin. Therefore, lowering of the rumen pH in acidotic conditions may not be a factor which promotes P.E.M.

Effect of a high sulfur diet on rumen microbial activity and rumen thiamine status in sheep receiving a semi-synthetic, thiamine-free diet.

A semi-synthetic thiamine-free diet was used on weaned lambs to test the effect of a high sulfur level on the rumen, microbial activity and on the microbial production of thiamine. In vivo and in vitro kinetic studies, as well as the determination of the thiamine concentrations and thiaminase activity in the rumen, were performed during the 16 week experiment. A high sulfur level (0.6%) in the diet, in comparison with a normal sulfur level (0.2%), did not modify the microbial activity of the rumen with the exception of a slightly retarded decrease in the volatile fatty acid (VFA) rumen concentration. The rumen thiamine level and the thiaminase activity were not modified by the dietary sulfur level. In contrast, the rate of sulfate reduction into sulfide in the rumen increased progressively with the 0.6% sulfur diet. In conclusion, a high sulfur level (0.6%) in the diet of sheep did not modify the thiamine status of the rumen. It strongly increased the production of sulfides but an adaptation period of several weeks was required by the rumen microflora to reduce sulfate at a maximal rate.