Disruption of oogenesis and molting by methoprene and glyphosate in Gammarus fossarum: involvement of retinoic acid?

In the last decade, the freshwater amphipod Gammarus fossarum proved to be a promising sentinel species in active biomonitoring programs to assess the effects of environmental contamination on non-target organisms. Given that the highly conserved retinoid (RETs) metabolism supports many biological functions and is perturbed by xenobiotics and used as biomarker for vertebrates, we explored the RETs functions in the crustacean model Gammarus fossarum. More specifically, we studied the implication of all -trans retinoic acid (atRA) in the reproduction (embryo, oocyte, and juvenile production) and development (success and delay of molting) by exposing G. fossarum females to atRA and citral (CIT), a known inhibitor of RA synthesis. In parallel, we exposed gammarids to methoprene (MET) and glyphosate (GLY), two pesticides suspected to interfere with atRA metabolism and signaling and frequently found in water systems. After 14 days of exposure, atRA, CIT, and MET reduced the number of oocytes, whereas only MET caused a reduced number of embryos. After 44 days, MET and GLY showed a tendency to decrease juvenile production. The duration of the molting cycle increased following the exposures to atRA and MET, while the treatment with CIT caused a typical endocrine disruptive inverted U-shaped curve. The exposure to GLY led to increased duration of the molting cycle at the lowest concentrations and lowered molting success at the highest concentration tested. This study highlights for the first time the implication of RA in the oogenesis and molting of G. fossarum and suggests that it may be a potential mediator of MET-induced effects on these processes. This study adds to the comprehension of the reproductive and developmental control in G. fossarum and opens new research avenues to study the effects of xenobiotics on the RET system in this sentinel species. Ultimately, our study will drive the development of RET-based biomarkers for non-target aquatic invertebrates exposed to xenobiotics.

The retinoid metabolism of Gammarus fossarum is disrupted by exogenous all-trans retinoic acid, citral, and methoprene but not by the technical formulation of glyphosate.

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status.

Pesticides Inhibit Retinoic Acid Catabolism in PLHC-1 and ZFL Fish Hepatic Cell Lines.

The population of yellow perch (Perca flavescens) in lake Saint-Pierre (QC, Canada) has been dramatically declining since 1995 without any sign of recovery. Previous studies have shown disrupted retinoid (vitamin A) metabolic pathways in these fish, possibly due to the influence of pesticides. Our study aimed to evaluate the impact of some herbicides and neonicotinoids on retinoic acid catabolism in the fish hepatic cell lines PLHC-1 and ZFL. We hypothesized that pesticides accelerate the catabolism of retinoic acid through oxidative stress that exacerbates the oxidation of retinoic acid. Results obtained with talarozole, a specific CYP26A1 inhibitor, and ketoconazole, a generalist inhibitor of cytochrome-P450 enzymes, revealed that CYP26A1 is mainly responsible for retinoic acid catabolism in ZFL but not PLHC-1 cells. The impacts of pesticides on retinoic acid catabolism were evaluated by incubating the cells with all-trans-retinoic acid and two herbicides, atrazine and glyphosate, or three neonicotinoids, clothianidin, imidacloprid, and thiamethoxam. Intracellular thiols and lipid peroxidation were measured following pesticide exposure. The possible causal relation between oxidative stress and the perturbation of retinoic acid catabolism was investigated using the antioxidant N-acetylcysteine. The data revealed that pesticides inhibit retinoic acid catabolism, with the involvement of oxidative stress in the case of atrazine, imidacloprid, and thiamethoxam but not with clothianidin and glyphosate. Pesticides also affected the isomerization of all-trans-retinoic acid over time, leading to an increased proportion of active isomers. These results hint at a possible perturbation of retinoic acid catabolism in fish living in pesticide-contaminated waters, as suggested by several in vivo studies. Such a disruption of retinoid metabolism is worrying, given the numerous physiological pathways driven by retinoids.

Interactive effects of neonicotinoids and natural ultraviolet radiation on yellow perch (Perca flavescens) larvae.

Neonicotinoids (NEOCs) are insecticides that are widely used worldwide in the culture of maize and soya. Whereas they specifically target terrestrial insects by acting as agonists of the neurotransmitter acetylcholine in their nervous system, their effects on the cholinergic system of vertebrates is still unclear. Moreover, there is an increasing concern about their effects on aquatic biota because of their high leaching potential. In the agricultural watershed of Lake St. Pierre (LSP) (St. Lawrence River System, Québec, Canada), for example, NEOC concentrations considered toxic for aquatic biota (>8.3 ng L-1) have frequently been detected. These conditions may affect the yellow perch (Perca flavescens) population in LSP, which collapsed in the mid 1990s and is now experiencing poor recruitment. Moreover, because their larvae are found in shallow waters (<80 cm) near agricultural land, they are also exposed to ultraviolet radiation (UVR), with unknown potential interactions with NEOCs. The objective of this study was to test the synergistic effects of two commonly used NEOCs (imidacloprid and thiamethoxam) with natural UVR on yellow perch larvae using survival analysis and biomarkers to better quantify lethal and sublethal effects. Three common garden experiments were conducted with thiamethoxam and/or imidacloprid and natural UVR following a factorial design. Our results showed an interaction between UVR and thiamethoxam in terms of larval mortality. At the sublethal level, imidacloprid was associated with increased protein content and, in the presence of UVR, with increased acetylcholinesterase activity, thus indicating a cholinergic perturbation like that found in insects. Finally, we also found unexpected reduced lipid peroxidation associated with imidacloprid. A reduction in the overall lipid accumulation is suspected to be behind this puzzling result. These results will open new research avenues related to the effects of NEOCs on proteins and lipid accumulation.

Chronic exposure to imidacloprid or thiamethoxam neonicotinoid causes oxidative damages and alters carotenoid-retinoid levels in caged honey bees (Apis mellifera).

Over the last decade, the persistent dwindling of the populations of honey bees has become a growing concern. While this phenomenon is partly attributed to neonicotinoids (NEOCs), chronic exposures to these insecticides at environmentally-relevant concentrations are needed to fully estimate their implications. In this study, honey bees were orally exposed for 10 days to low field-realistic concentrations of NEOCs known for their effects on the cholinergic system (imidacloprid - IMI or thiamethoxam - THM). Selected biomarkers were measured such as acetylcholinesterase (AChE) activity, lipid peroxidation (LPO), α-tocopherol as well as several forms of vitamin A (retinoids) and carotenoids. Bees exposed to IMI showed lower levels of two carotenoids (α-carotene and α-cryptoxanthin) and α-tocopherol. The THM exposure increased the oxidized vitamin A metabolites in bees conjointly with the LPO. These results could be the consequence of a pro-oxidant effect of NEOCs and were observed at levels where no effects were recorded for AChE activity. This study reveals that exposure to low levels of NEOCs alters the carotenoid-retinoid system in honey bees. This would merit further investigation as these compounds are important in various aspects of bees' health. Overall, this study contributes to the development of biomonitoring tools for the health of bees and other pollinators.

RNA-sequencing to assess the health of wild yellow perch (Perca flavescens) populations from the St. Lawrence River, Canada.

This study aimed to better understand in situ cumulative effects of anthropogenic stressors on the health of St. Lawrence River (QC, Canada) yellow perch populations using high-throughput transcriptomics and a multi-biological level approach. Fish were collected in the upstream fluvial Lake Saint-François (LSF) with low degree of environmental perturbations; Lake Saint-Louis (LSL) considered having a moderate degree of anthropogenic stressors, and Lake Saint-Pierre (LSP) a sector where the perch population has been severely declining. Morphometric results indicated that fish from the downstream LSP showed lower body condition compared to LSF and LSL. Liver transcriptomic responses were assessed by RNA-sequencing. Two hundred and eighty genes were over-transcribed in LSP perch while 200 genes were under-transcribed compared to LSF and LSL. In LSP fish, genes transcripts related to reproduction, retinol, iron, thyroid hormones, oxidative stress, lipid metabolism and immune functions were among the most abundant suggesting that multiple metabolic and physiological pathways were impacted by environmental stressors at this site. Inhibition of liver superoxide dismutase, catalase and glutathione S-transferase activities were also observed at the cellular level. Overall, identified impacted biological pathways in perch from LSP may help understand the precarious state of this population and identify the factors inhibiting its recovery.

Temporal trends of mercury and organohalogen contaminants in great blue heron eggs from the St. Lawrence River, Québec, Canada, 1991-2011, and relationships with tracers of feeding ecology.

Since 1991, great blue heron (Ardea herodias) eggs have been collected and analyzed for mercury (Hg), persistent organic contaminants (OCs), brominated and non-brominated flame retardants (FRs) as well as stable isotopes δ13C and δ15N. In the present study, temporal trends of contaminants were analyzed in eggs sampled in four regions along the St. Lawrence River (Quebec, Canada) and inland sites using new and previously published data. Most contaminants declined significantly over time in most regions. Globally, the highest annual change, -17.5%, was found for pp'-DDD, while the smallest annual decline, -0.54%, was observed for Hg. Concentrations of ΣDDT and ΣFR8 (sum of 8 congeners) decreased by -11.6% and -7.3%, respectively. Declines in ΣPCBs differed among regions, from -5.6% in the fluvial section to -14.7% in the inland region. The highest concentration of ΣFR8 was measured in eggs from Grande Ile in the fluvial section of the river in 1996 (2.39µg/g). Stable isotope ratios also showed temporal trends in some regions: δ13C decreased in the fluvial section and increased in Gulf region, while δ15N decreased in the fluvial section and increased in the upper estuary. Significant positive relationships were found between ΣDDT, ΣPCBs and ΣFRs and δ15N and δ13C in freshwater colonies, but not in estuarine or marine colonies. These results suggest that changes in trophic level and foraging areas over time were influential factors with respect to contaminant burden in great blue heron eggs in the fluvial section, but not in the other regions.

Foraging in maize field areas: A risky business?

In Quebec, Canada, the cultivation of maize dominates the agricultural territory. This crop requires a sustained supply of fertilizers from different sources: chemical, natural or from residual materials (sludge). These amendments contain metallic trace elements, which may lead to metal-contaminated maize pollen, a possible source of prooxidants for the foraging bees. Our objective was to determine whether maize fields environment influences the oxidation processes and the accumulation of metals in bees. A few days prior to pollen shedding, beehives were installed in maize fields: one organically grown (site A) and three conventionally grown (sites B, C and D). Soil, maize pollen and bees were analyzed for metal content. Every 15days, bees were collected and analyzed for peroxidation of lipids, metallothionein-like proteins (MTLPs), proteins, retinoids and lipophilic antioxidants (carotenoids and α-tocopherol). The compound ß-carotene was the most abundant in bees from all sites, followed by α-carotene, ß-cryptoxanthin, α-cryptoxanthin, zeaxanthin and lutein. Retinaldehyde and retinol varied according to times and sites without demonstrating clear trends. However, significant differences between sites were noted in 13-cis-retinoic acid and two retinoic acid metabolites measured in bees, suggesting alteration in the reduction-oxidation processes. In line with these results, the level of lipid peroxidation was globally higher in sites B, C and D compared with the organic site. Higher concentrations of metals were observed in soil and pollen from the field A, but bees metal contents were equal or less than those measured in bees from other sites. Higher bee MTLP levels were measured in sites B, C and D. For most sampling times, the discriminant analysis revealed that the conditions were distinguished by the oxidation processes in bees. Our data suggest that bees foraging in conventionally grown maize fields are at risk of increased oxidative damages which can alter the fine regulation of retinoids.

Lipophilic antioxidants and lipid peroxidation in yellow perch subjected to various anthropogenic influences along the St. Lawrence River (QC, Canada).

In Lake Saint-Pierre (LSP), the last great widening of the St. Lawrence River (province of Québec, Canada), the yellow perch has been experiencing a significant decline since the mid-1990s. The combined effect of several stressors (deterioration of habitats appropriate for reproduction and growth, invasive species and poor water quality) seems to exert considerable influence on the yellow perch population in LSP, characterized by low recruitment. To better understand possible stressor effects at the biochemical level, LSP yellow perch were compared with other sites along a gradient of increasing human influences from upstream to downstream along the St. Lawrence River. Morphometry (size, weight, circumference and Fulton's condition factor) and biomarkers associated to the peroxidation of lipids, lipophilic antioxidants (α-tocopherol and carotenoids), along with retinoids (vitamins A1and A2) and proteins were compared between sites at the larval, juvenile and adult stages. Fulton's condition factor was similar between sites for juveniles but was significantly lower in LSP adults, suggesting a weakened physiological condition. In most contaminated sites as LSP, lipid peroxidation tended to be higher in juveniles and adults whereas the lipophilic antioxidant lycopene and proteins content were lower. Retinyl esters were significantly lower for LSP fish compared to other sites, not only in larvae but also in the livers of juveniles and adults. These results are consistent with possible altered metabolism in the retinoid system of LSP yellow perch. The overall results reflect the "pressure" gradient tested, where the yellow perch from the most affected sites located downstream had impaired physiological and biochemical conditions compared to the upstream sectors.

Thyroid Hormones, Retinol and Clinical Parameters in Relation to Mercury and Organohalogen Contaminants in Great Blue Heron (Ardea herodias) Nestlings from the St. Lawrence River, Québec, Canada.

The exposure and effects of persistent environmental contaminants were investigated in great blue heron (Ardea herodias) nestlings sampled in 2001, 2002, 2006, and 2007 in freshwater and estuarine heronries along the St. Lawrence River, Québec (Canada). Biomarkers (retinoids, thyroid hormones, and clinical parameters) and contaminants (organochlorine contaminants, polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs), and mercury (Hg)) were analyzed in blood, and Hg was analyzed in feathers (generally 9 nestlings per colony and 4 colonies per year). Feather Hg and most contaminants detected in blood were found in higher concentrations in birds from freshwater than estuarine colonies more distant from the pollution sources. Among freshwater colonies, Ile aux Hérons showed the highest levels of contaminants, with mean Hg concentrations of 8.4 and 0.55 mg/kg in feathers and plasma, respectively, and plasma ΣBFRs of 19.6 ng/g ww. The highest mean ΣPCBs, 56.5 ng/g ww, was measured at Grande Ile in 2001. The levels of contaminants in heron nestlings were generally below critical thresholds for adverse effects observed on reproduction or survival. Retinol, dehydroretinol (DROH), and thyroid hormone concentrations differed significantly among colonies. Retinol concentrations were negatively related to ΣPCBs, whereas DROH concentrations were negatively related to Hg and total and free triiodothyronine (T3) concentrations were negatively related to ΣBFRs. These results indicate that contaminants from the St. Lawrence River could impair the development and fitness of great blue heron nestlings and emphasize the need for more research on the great blue heron population to assess their health and nutritional status.

Mixtures of herbicides and metals affect the redox system of honey bees.

The increasing loss of bee colonies in many countries has prompted a surge of studies on the factors affecting bee health. In North America, main crops such as maize and soybean are cultivated with extensive use of pesticides that may affect non-target organisms such as bees. Also, biosolids, used as a soil amendment, represent additional sources of metals in agroecosystems; however, there is no information about how these metals could affect the bees. In previous studies we investigated the effects of environmentally relevant doses of herbicides and metals, each individually, on caged honey bees. The present study aimed at investigating the effects of mixtures of herbicides (glyphosate and atrazine) and metals (cadmium and iron), as these mixtures represent more realistic exposure conditions. Levels of metal, vitamin E, carotenoids, retinaldehyde, at-retinol, retinoic acid isomers (9-cis RA, 13-cis RA, at-RA) and the metabolites 13-cis-4-oxo-RA and at-4-oxo-RA were measured in bees fed for 10 days with contaminated syrup. Mixtures of herbicides and cadmium that did not affect bee viability, lowered bee α- and ß-carotenoid contents and increased 9-cis-RA as well as 13-cis-4-oxo-RA without modifying the levels of at-retinol. Bee treatment with either glyphosate, a combination of atrazine and cadmium, or mixtures of herbicides promoted lipid peroxidation. Iron was bioconcentrated in bees and led to high levels of lipid peroxidation. Metals also decreased zeaxanthin bee contents. These results show that mixtures of atrazine, glyphosate, cadmium and iron may affect different reactions occurring in the metabolic pathway of vitamin A in the honey bee.

Integrated spatial health assessment of yellow perch (Perca flavescens) populations from the St. Lawrence River (QC, Canada), part B: cellular and transcriptomic effects.

Multi-biological level assessments have become great tools to evaluate the health of aquatic ecosystems. Using this approach, a complementary study was designed to evaluate the health of yellow perch (Perca flavescens) populations in the St. Lawrence River (Quebec, Canada). In the present study, stress responses were compared at the transcriptomic, cellular, and tissue levels in yellow perch collected at six sites along the river: Lake St. François, Lake St. Louis (north and south), Beauregard Island and Lake St. Pierre (north and south). These results complement the physiological and chemical parameters as well as pathogen infection investigated in a companion paper published in the present issue. Thiobarbituric acid reactive substance (TBARS) analyses indicated the presence of oxidative stress in fish collected in the southern part of Lake St. Louis and at the downstream sites of Lake St. Pierre. High lipid peroxidation levels were found in the muscle of yellow perch caught at Beauregard Island, located downstream of the Montreal's wastewater treatment plant, suggesting an impact of the municipal effluent on redox homeostasis. Transcriptomic results indicated the down-regulation of genes related to lipid, glucose, and retinoid in southern Lake St. Pierre as well as a decrease in retinoid storage. Overall, biochemical and molecular markers indicated that the health status of yellow perch followed a decreasing gradient from upstream to downstream of the St. Lawrence River. This gradient is representative of the cumulative negative impacts of human activities on water and habitat quality along the river.

Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera).

Several hypotheses have been proposed to explain the abnormally high mortality rate observed in bee populations in Europe and North America. While studies based on the effects of pesticides are paramount, the metals present in agroecosystems are often overlooked. Sources of metals are linked to the nature of soils and to agricultural practices, namely the use of natural or chemical nutrients as well as residual materials from waste-water treatment sludge. The aim of this study was to investigate the effects of metals on honey bees exposed for 10 days to environmentally realistic concentrations of Al, Pb and Cd (dissolved in syrup). The monitoring of syrup consumption combined with the quantification of metals in bees revealed the following order for metal bioconcentration ratios: Cd > Pb > Al. Alpha-tocopherol, metallothionein-like proteins (MTLPs) and lipid peroxidation were quantified. When bees were exposed to increasing amounts of Cd, a marked augmentation of MTLPs levels was found. Lead (Pb) and Cd caused an increase in α-tocopherol content, while alteration of lipid peroxidation was observed only with Al exposure. These findings raise concerns about the bioavailability and the additional threat posed by metals for pollinators in agricultural areas while providing new insights for potential use of the honey bee as a sentinel species for metal exposure.

Effects of realistic doses of atrazine, metolachlor, and glyphosate on lipid peroxidation and diet-derived antioxidants in caged honey bees (Apis mellifera).

The decline in the population of pollinators is a worrying phenomenon worldwide. In North America, the extensive use of herbicides in maize and soya crops may affect the health of nontarget organisms like the honey bee. In this study, caged honey bees were exposed to realistic doses of atrazine, metolachlor, and glyphosate for 10 days via contaminated syrup. Peroxidation of lipids was evaluated using the thiobarbituric acid reactive substance (TBARS) test, and diet-derived antioxidants-carotenoids, all-trans-retinol (at-ROH) and α-tocopherol-were detected and quantified using reversed-phase HPLC techniques. Significant increases in syrup consumption were observed in honey bees exposed to metolachlor, and a lower TBARS value was recorded for the highest dose. No relationship was observed between the peroxidation of lipids and the levels of antioxidants. However, ß-carotene, which was found to be the most abundant carotenoid, and at-ROH (derived from ß-carotene) both decreased with increasing doses of atrazine and glyphosate. In contrast, metolachlor increased levels of at-ROH without any effects on ß-carotene. These results show that the honey bee carotenoid-retinoid system may be altered by sublethal field-realistic doses of herbicides.

Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments.

In Québec, as observed globally, abnormally high honey bee mortality rates have been reported recently. Several potential contributing factors have been identified, and exposure to pesticides is of increasing concern. In maize fields, foraging bees are exposed to residual concentrations of insecticides such as neonicotinoids used for seed coating. Highly toxic to bees, neonicotinoids are also reported to increase AChE activity in other invertebrates exposed to sub-lethal doses. The purpose of this study was therefore to test if the honey bee's AChE activity could be altered by neonicotinoid compounds and to explore possible effects of other common products used in maize fields: atrazine and glyphosate. One week prior to pollen shedding, beehives were placed near three different field types: certified organically grown maize, conventionally grown maize or non-cultivated. At the same time, caged bees were exposed to increasing sub-lethal doses of neonicotinoid insecticides (imidacloprid and clothianidin) and herbicides (atrazine and glyphosate) under controlled conditions. While increased AChE activity was found in all fields after 2 weeks of exposure, bees close to conventional maize crops showed values higher than those in both organic maize fields and non-cultivated areas. In caged bees, AChE activity increased in response to neonicotinoids, and a slight decrease was observed by glyphosate. These results are discussed with regard to AChE activity as a potential biomarker of exposure for neonicotinoids.

Oxidation of retinoic acids in hepatic microsomes of wild bullfrogs Lithobates catesbeianus environmentally-exposed to a gradient of agricultural contamination.

Agricultural contaminants are suspected of contributing to the increased incidence of deformities and the decline of amphibians populations worldwide. Many authors have further suggested that a retinoid effect could be implicated in teratogenic mechanisms since the reported deformities resemble those caused by abnormal levels of retinoic acid (RA). We previously reported altered retinoid concentrations in male bullfrogs from the Yamaska River basin (Québec, Canada) associated with moderate-to-high agricultural activity, and the findings were consistent with a possible effect on hepatic RA oxidation. An in vitro assay was therefore optimized and hepatic microsomal RA oxidation in bullfrogs was found to be quite different from that of other vertebrates. With either all-transRA (atRA) or 13cisRA as the substrate, the major metabolite generated was at4-oxo-RA. The reaction with 13cisRA as substrate, markedly greater compared with atRA, was enhanced in the presence of a reducing agent and inhibited by cytochrome P450 inhibitors in a dose-dependent manner. Hepatic RA oxidation in male bullfrogs showed significant differences between sites with no clear relationship to a gradient of agricultural activity or 13cis-4-oxo-RA quantified in plasma. In contrast, the in vitro RA oxidation in females increased with the levels of contamination and coincided in vivo with higher plasma 13cis-4-oxo-RA concentration. The levels of circulating 4-oxo-derivatives could be influenced by hepatic RA oxidative metabolism as well as isomerization conditions or RA precursor levels.

P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity.

Atrazine and nitrogenous fertilizers are agrochemical contaminants frequently detected in water systems in North America. Several studies reported their ability to affect amphibian and mammalian development. Retinoids, supplied in the diet or synthesized by cells, are essential to embryogenesis. Disturbance of their homeostasis may lead to teratogenic effects. Retinoic acid (RA) is a major retinoid regulator of cell proliferation and differentiation. Previous studies reported alterations of retinoid stores in bullfrogs of Yamaska River subwatersheds (Québec, Canada), a region of intensive agricultural activities associated with atrazine, nitrate, and nitrite contaminants. These contaminants could affect RA metabolism and RA-mediated processes. Mouse P19 embryonic stem cells, which can differentiate to neurons in response to RA, were used to test this hypothesis. Cells were cultured in the absence or presence of contaminants during neuroinduction with RA and assayed by flow cytometry for expression of stage-specific embryonic antigen-1 (SSEA1) (embryonic marker) and betaIII-tubulin (neuronal marker). Cell cultures were also analyzed for RA metabolism by high performance liquid chromotagraphy (HPLC). Downregulation of SSEA1 paralleled betaIII-tubulin upregulation in an RA concentration-dependent manner. Atrazine, nitrate, and nitrite did not affect differentiation at environmentally encountered micromolar concentrations. However, low molar nitrite prevented RA-induced SSEA1 downregulation and decreased betaIII-tubulin appearance. Decreased cell viability/proliferation accompanied these differentiation effects. P19 cells metabolized RA to polar retinoids. RA metabolism was not affected at any concentration of atrazine, nitrate, or nitrite. Environmentally relevant levels of these contaminants, thus, had no gross effect on neurodifferentiation and RA catabolism of embryonic stem cells. P19 cell-based bioassays may provide valuable tools in monitoring developmental toxicity.

Retinoid metabolism (LRAT, REH) in the liver and plasma retinoids of bullfrog, Rana catesbeiana, in relation to agricultural contamination.

Retinoids have been extensively studied in birds, fish and mammals where their imbalances are associated with adverse effects on growth and reproduction along with decreased embryo survival and deformities. Organochlorine compounds may alter the retinoid system but little is known about the effects of agricultural contaminants on their metabolism. In the Yamaska River project, the retinoid system in bullfrogs is monitored to investigate the possible impact of agricultural contaminants on retinoid homeostasis. Retinoids were measured in liver and plasma of male bullfrogs collected from six locations subject to increasing agricultural activity in the Yamaska River watershed. Bullfrogs living in medium and high agricultural activity areas demonstrated lower hepatic retinyl palmitate and higher hepatic retinol levels when compared to frogs associated with low contaminated sites. Changes in the concentration of hepatic esters could be related to an altered activity of REH or LRAT, enzymes respectively linked to the hydrolysis of retinyl palmitate and the esterification of retinol. A partial characterization and the analysis of liver microsomial REH and LRAT showed significantly higher hydrolysis and lower esterification activities in highly contaminated sites. Enzymatic activities seemed to be influenced by plasma DROH but not by plasma retinol. Bullfrogs from the most contaminated sites showed altered retinoic metabolism that should increase concern for frogs living in intensive agricultural areas.

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Study design, water quality, morphometrics and age of the bullfrog, Rana catesbeiana, in sub-watersheds of the Yamaska River drainage basin, Québec, Canada.

The Yamaska River basin is renowned for its poor water quality, which has been attributed to intensive agriculture (corn, soya, high-density pork and poultry production). Six locations within the Yamaska watershed were selected to evaluate the impact of agriculture on water habitats and study the bullfrog as a sentinel species of potential exposure and effects. The selected sub-watersheds were chosen according to the percentage of surface area under cultivation and classified as low (0-19%), moderate (20-59%) or high (>60%). In 2004 and 2005, analysis of surface water samples demonstrated that pesticide concentrations and most water quality parameters increased with increasing agricultural activity. Sixteen adult bullfrogs were sampled from each site. Animals were weighed, measured for length (total, snout-vent, tibia, tympanum), sexed, and evaluated for the colour of the throat. Skeletochronology was used to estimate the growth and age of the frogs. Cross-sections of decalcified phalanges and femurs were treated in order to count LAGs (lines of arrest growth) and for the observations of other parameters related to bone growth. The bullfrogs from highly contaminated sites had the lowest mean age and the smallest snout-vent length compared to sites of low contamination.