CbE Activity Research Articles

Microplastics and plastic additives as contaminants of emerging concern: A multi-biomarker approach using Rhinella arenarum tadpoles

Polyethylene microplastics (PE-MPs), a whitish thermoplastic polymer with numerous applications, is one of the materials most widely used in the industrial sector, whereas tetrabromobisphenol A (TBBPA) is a brominated flame retardant. The aim of this study was to analyze the effects of PE-MPs and TBBPA on Rhinella arenarum tadpoles at the laboratory scale. Tadpoles were chronically exposed (30 days) to four treatments: PE-MPs (60 mg L−1), TBBPA (10 µg L−1), their mixture (PE-MPs + TBBPA), and dechlorinated water as negative control (CO). Biomarkers of enzymatic activity (acetylcholinesterase, AChE; carboxylesterase, CbE; glutathione reductase, GR; and glutathione-S-transferase, GST), hepatic physiological alteration (alkaline phosphatase; ALP activity, and cholesterol; CHOL level), and endocrine disruption through thyroid hormone (T4) levels were assessed. In addition, intestine and liver were histomorphologically evaluated. AChE activity in tadpoles was significantly inhibited after exposure to PE-MPs and TBBPA with respect to CO. In addition, CbE, GR, and ALP activities showed higher values in the mixture of PE-MPs+ TBBPA treatment than in CO, whereas CHOL level was higher in TBBPA and PE-MPs+ TBBPA treatments than in CO. GST activity did not show significant differences between treatments and CO. T4 levels increased significantly in all treatments with respect to CO. The intestinal structure of tadpoles exposed to PE-MPs and PE-MPs+ TBBPA showed signs of mechanical damage. The intestinal wall of tadpoles under PE-MPs, TBBPA and PE-MPs+ TBBPA treatments was thicker than that of CO individuals. The analysis of liver histology demonstrated the hepatotoxicity caused by PE-MPs+ TBBPA. This study provides quantitative evidence of the harmful effects of PE-MPs, TBBPA and their mixture on enzymatic and hormonal activities, and histological evidence of intestinal wall hypertrophy and liver damage of R. arenarum tadpoles.

Effect of microplastics on the activity of carboxylesterase and phosphatase enzymes in Scinax squalirostris tadpoles.

Microplastics (MPs) are critical emerging pollutants around the world. There is a growing interest in the effects of MP ingestion, non-digestion, and toxicity on aquatic organisms. Amphibian tadpoles are the vertebrate group that has received the least attention regarding this issue. The aim of the present study was to determine the ingestion of polyethylene MPs by Scinax squalirostris tadpoles by atomic force microscopy (AFM) and to evaluate the activities of carboxylesterase (CbE, using 4-naphthyl butyrate-NB-, and 1-naphthyl acetate -NA- as substrates) and alkaline phosphatase (ALP) under MP exposure. Enzyme activities were analyzed spectrophotometrically at 2 and 10days of exposure. Tadpoles were exposed to two different treatments during 10days: a negative control (CO, dechlorinated water) and MP (60mg L-1). AFM images of the digestive contents of tadpoles revealed the presence of MPs. After 10days of MP exposure, CbE (NB) activity was significantly higher and CbE (NA) activity was significantly lower in MP treatments than in controls. ALP activity decreased in MP treatments after 2 and 10days of exposure. The detection of MP particles in the intestinal contents and the effects on metabolic enzymes in a common frog species evidenced the potential health risk of MP to aquatic vertebrates. Thus, the differential response in enzymes and substrates demonstrate the need for considering the complex effects of contaminants and nutrients on ecosystems for ecotoxicological risk characterization.

Development of a universal antibiotic resistance screening reporter for improving efficiency of cytosine and adenine base editing

Base editing has emerged as a revolutionary technology for single nucleotide modifications. The cytosine and adenine base editors (CBEs and ABEs) have demonstrated great potential in clinical and fundamental research. However, screening and isolating target-edited cells remains challenging. In the current study, we developed a universal Adenine and Cytosine Base-Editing Antibiotic Resistance Screening Reporter (ACBE-ARSR) for improving the editing efficiency. To develop the reporter, the CBE-ARSR was first constructed and shown to be capable of enriching cells for those that had undergone CBE editing activity. Then, the ACBE-ARSR was constructed and was further validated in the editing assays by four different CBEs and two versions of ABE at several different genomic loci. Our results demonstrated that ACBE-ARSR, compared to the reporter of transfection (RoT) screening strategy, improved the editing efficiency of CBE and ABE by 4.6- and 1.9-fold on average, respectively. We found the highest CBE and ABE editing efficiencies as enriched by ACBE-ARSR reached 90% and 88.7%. Moreover, we also demonstrated ACBE-ARSR could be employed for enhancing simultaneous multiplexed genome editing. In conclusion, both CBE and ABE activity can be improved significantly using our novel ACBE-ARSR screening strategy, which we believe will facilitate the development of base editors and their application in biomedical and fundamental research studies.

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH.

In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl-/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.

Interspecific differences in biochemical and behavioral biomarkers in endogeic earthworms exposed to ethyl-parathion

Earthworms are common organisms in the soil toxicity-testing framework, and the epigeic Eisenia andrei and E. fetida are the recommended species. However, Eisenia species are rarely found in agricultural soils and recent studies have pointed out endogeic species are more sensitive to pesticides than Eisenia. Allolobophora chlorotica and Aporrectodea caliginosa are two endogeic soil-dwelling species that are abundant in the agroecosystem. However, knowledge on pesticide impact on this ecological group of earthworms is still incipient. Herein, we compared the biochemical (acetylcholinesterase [AChE] and carboxylesterase [CbE] activities) and behavioral (burrowing, casting and feeding) biomarker responses of these two endogeic earthworm species exposed for 7 days to soils contaminated with 0.1, 1 and 10 mg kg−1 ethyl-parathion. The results showed marked species-specific differences in both groups of biomarkers, suggesting A. caliginosa the most sensitive species to this organophosphorus pesticide under the exposure conditions in this study. Moreover, an in vitro inhibition trial with ethyl-paraoxon evidenced a higher sensitivity of A. caliginosa AChE activity compared with that of A. chlorotica. This finding suggested that this molecular target endpoint could contribute to the interspecific differences of behavioral responses rather than CbE activity; this latter considered a potent mechanism of OP removal. Our results suggest the inclusion of more than one endogeic earthworm species to assess toxicity from organophosphorus insecticides. However, the use of A. caliginosa in the environmental risk assessment framework of organophosphorus contamination is highly recommended because of its higher sensibility to this class of pesticides, in addition to its abundance in the agroecosystem.

Estrés oxidativo, metabolismo lipídico y neurotransmisión en el caracol dulceacuícola (Pomacea patula) expuesto a la fracción hidrosoluble de petróleo crudo

Background. Crude oil is a super mixture of chemical compounds and is commonly found in aquatic environments. The tegogolo ( Pomacea patula Baker, 1922) is a Mexican freshwater snail endemic to Lake Catemaco in Veracruz; currently, however, its distribution has expanded to many freshwater ecosystems that suffer the impact of crude oil spills and oil byproducts like fuels. Goals . To assess a series of biomarkers involved in oxidative stress, neurotransmission, and fatty acid metabolism in tegogolos exposed to the water-accommodated fraction (WAF) of Maya crude oil (MCO). Methods . Tegogolo specimens were exposed to WAF of MCO obtained from loads of 0.1, 1, 10 and 100 mg/L. We evaluated ROS ( O2 -* and H2O2), oxidative stress (TBARS and RC=O), enzymes involved in antioxidant defense (SOD, CAT, and GPx), some enzymes involved in neurotransmission (AChE, GDA, and CbE activities), and biomarkers of fatty acids metabolism (fatty acids levels and AOX activity). Results. Clear biomarkers responses were observed only in some tissues. ROS were clearly higher than controls in the foot, head, and kidney; however, others biomarkers of oxidative stress remain statistically unchanged. SOD response was irregular with respect to controls and treatments. In contrast, CAT (foot) and GPx (foot and intestine) were the more active enzymes and their activities were higher than in controls. The responses of some enzymes involved in neurotransmission suggest that compensation mechanisms exist between AChE and GDA in the foot and head. Fatty acids metabolism increased with exposure to WAF; however, these types of biomarkers seem unsuitable for monitoring the toxic effects produced by WAF at low environmental concentrations. Conclusions. We can conclude that under the exposure conditions discussed herein, the tegogolos showed acclimation to WAF of Maya crude oil by complex mechanisms.

Two Phase Modulation of [Formula: see text] Entry and Cl-/[Formula: see text] Exchanger in Submandibular Glands Cells by Dexmedetomidine.

Dexmedetomidine (Dex), a highly selective α2-adrenoceptor agonist, attenuates inflammatory responses induced by lipopolysaccharide (LPS) and induces sedative and analgesic effects. Administration of Dex also reduces salivary secretion in human subjects and inhibits osmotic water permeability in rat cortical collecting ducts. However, little is known about the mechanisms underlying the effects of Dex on salivary glands fluid secretion. We demonstrated the α2-adrenoceptor expression in the basolateral membrane of mouse submandibular glands (SMG). To investigate fluid secretion upon treatment with Dex, we studied the effects of Dex on the activity of Na+-K+-2Cl− cotransporter1 (NKCC1) and Cl−/ exchange (CBE), and on downstream pro-inflammatory cytokine expression in isolated primary mouse SMG cells. Dex acutely increased CBE activity and NKCC1-mediated and independent entry in SMG duct cells, and enhanced ductal fluid secretion in a sealed duct system. Dex showed differential effects on cholinergic/adrenergic stimulations and inflammatory mediators, histamine, and LPS, stimulations-induced Ca2+ in mouse SMG cells. Both, histamine- and LPS-induced intracellular Ca2+ increases were inhibited by Dex, whereas carbachol-stimulated Ca2+ signals were not. Long-lasting (2 h) treatment with Dex reduced CBE activity in SMG and in human submandibular glands (HSG) cells. Moreover, when isolated SMG cells were stimulated with Dex for 2 h, phosphodiesterase 4D (PDE4D) expression was enhanced. These results confirm the anti-inflammatory properties of Dex on LPS-mediated signaling. Further, Dex also inhibited mRNA expression of interleukin-6 and NADPH oxidase 4. The present study also showed that α2-adrenoceptor activation by Dex reduces salivary glands fluid secretion by increasing PDE4D expression, and subsequently reducing the concentration of cAMP. These findings reveal an interaction between the α2-adrenoceptor and PDE4D, which should be considered when using α2-adrenoceptor agonists as sedative or analgesics.

Earthworm-induced carboxylesterase activity in soil: Assessing the potential for detoxification and monitoring organophosphorus pesticides

Soil enzyme activities are attracting widespread interest due to its potential use in contaminant breakdown, and as indicators of soil deterioration. However, given the multiple environmental and methodological factors affecting their activity levels, assessment of soil pollution using these biochemical endpoints is still complex. Taking advantage of the well-known stimulatory effect of earthworms on soil microbes, and their associated enzyme activities, we explored some toxicological features of carboxylesterases (CbEs) in soils inoculated with Lumbricus terrestris. A microplate-scale spectrophotometric assay using soil–water suspensions was first optimized, in which kinetic assay parameters (Km, Vmax, dilution of soil homogenate, and duration of soil homogenization) were established for further CbE determinations. Optimal conditions included a soil-to-water ratio of 1:50 (w/v), 30-min of shaking, and 2.5mM of substrate concentration. As expected, CbE activity increased significantly in soils treated with L. terrestris. This bioturbed soil was used for exploring the role of CbE activity as a bioscavenger for organophosphorus (OP) pesticides. Soil treated with two formulations of chlorpyrifos revealed that CbE activity was a significant molecular sink for this pesticide, reducing its impact on soil microbial activity as shown by the unchanged dehydrogenase activity. Dose-dependent curves were adjusted to an exponential kinetic model, and the median ecological dose (ED50) for both pesticide formulations was calculated. ED50 values decreased as the time of pesticide exposure increased (14d-ED50s=20.4–26.7mgkg−1, and 28d-ED50s=1.8–2.3mgkg−1), which suggested that chlorpyrifos was progressively transformed into its highly toxic metabolite chlorpyrifos-oxon, but simultaneously was inactivated by CbEs. These results were confirmed by in vitro assays that showed chlorpyrifos-oxon was a more potent CbE inhibitor (IC50=35.5–4.67nM) than chlorpyrifos (0.41–0.84μM). The results showed that earthworm-induced CbE activity is an efficient bioscavengers for OP pesticides, acting as a soil safeguarding system. Moreover, the simple dose-response curves against OP exposure suggest that this enzyme – combined with other enzyme activities (e.g., dehydrogenase) – may be a suitable biomarker of pesticide exposure.

Hepatic biotransformation and antioxidant enzyme activities in Mediterranean fish from different habitat depths

Marine fish are threatened by anthropogenic chemical discharges. However, knowledge on adverse effects on deep-sea fish or their detoxification capabilities is limited. Herein, we compared the basal activities of selected hepatic detoxification enzymes in several species (Solea solea, Dicentrarchus labrax, Trachyrhynchus scabrus, Mora moro, Cataetix laticeps and Alepocehalus rostratus) collected from the coast, middle and lower slopes of the Blanes Canyon region (Catalan continental margin, NW Mediterranean Sea). The xenobiotic-detoxifying enzymes analysed were the phase-I carboxylesterases (CbEs), and the phase-II conjugation activities uridine diphosphate glucuronyltransferase (UDPGT) and glutathione S-transferase (GST). Moreover, some antioxidant enzyme activities, i.e., catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR), were also included in this comparative study. Because CbE activity is represented by multiple isoforms, the substrates α-naphthyl acetate (αNA) and ρ-nitrophenyl acetate (ρNPA) were used in the enzyme assays, and in vitro inhibition kinetics with dichlorvos were performed to compare interspecific CbE sensitivity.Activity of xenobiotic detoxification enzymes varied among the species, following a trend with habitat depth and body size. Thus, UDPGT and some antioxidant enzyme activities decreased in fish inhabiting lower slopes of deep-sea, whereas UDPGT and αNA-CbE activities were negatively related to fish size. A trend between CbE activities and the IC50 values for dichlorvos suggested S. solea and M. moro as potentially more sensitive to anticholinesterasic pesticides, and T. scabrus as the most resistant one. A principal component analysis considering all enzyme activities clearly identified the species but this grouping was not related to habitat depth or phylogeny. Although these results can be taken as baseline levels of the main xenobiotic detoxification enzymes in Mediterranean fish, further research is needed to evaluate their response to environmental contaminant exposure.

Impact of insecticide exposure on the predation activity of the European earwig Forficula auricularia.

The European earwig Forficula auricularia is an effective predator in apple orchards. It is therefore crucial to study whether insecticides affect this natural pest control agent. Predation activity, i.e., the number of aphids eaten in 24 h, was determined under laboratory conditions after exposure of fourth-instar nymphs and adult earwigs to widely used insecticides (acetamiprid, chlorpyrifos-ethyl, deltamethrin, and spinosad), which were applied at the normal application rates. Inhibition of acetylcholinesterase and carboxylesterase activities were also measured as indicators of pesticide exposure. Predation activity decreased significantly in nymphs exposed to spinosad (62%) and chlorpyrifos-ethyl (98%) compared with controls. A similar response was found for both esterase activities. Spinosad had a stronger effect on AChE (-33%) whereas chlorpyrifos-ethyl affected CbE activity preferentially (-59%). Spinosad (20% of controls), acetamiprid (28%), and chlorpyrifos-ethyl (66%) also significantly decreased the predation behavior of adult male but not female (5 to 40%) earwigs. Adult AChE and CbE activities were also significantly reduced (28 to 67% of controls) in pesticide-exposed earwigs. Our results suggest that earwigs should be included in the environmental risk assessment framework for authorization of newly marketed plant protection products. Their predation behavior appears to be a sensitive and complementary biomarker.

Esterase profile changes in ladybeetle, <i>Hippodamia variegata</i> Goeze (Coleoptera: Coccinellidae) and its prey, <i>Aphis fabae</i> Scop. (Homoptera: Aphididae) affected by two insecticides, thiamethoxam and pirimicarb

Black bean aphid, Aphis fabae Scopoli (Homoptera: Aphididae), is a cosmopolitan and serious pest of diverse plant species. In addition to inflicting direct damage, it is an important vector of viruses producing indirect damages. Hippodamia variegata , is considered as aphids predator both in their larval and adult stages. Thus, the aim of the current study was to investigate toxicological, biochemical and physiological effects of two widespread used pesticides against aphids including pirimicarb and thiamethoxam on third instar larvae of the ladybeetle and the adult female of the aphid in laboratory condition. Bioassay showed that LC 50 value in thiamethoxam treatment against A. fabae and H. variegata was 113.85 and 788.55 mg (ai)L -1 , respectively and LC 50 value for pirimicarb treatment against two insects was 2.94 and 2740.07 mg (ai)L -1 , respectively. Enzyme assays showed that AChE and CbE (evaluated by a -NA) of A. fabae inhibited significantly by pirimicarbtreatments(P>0.05), whilst thiamethoxam treatment did not affect both enzymes. Both insecticides did not affect H. variegata AChE and CbE activities, significantly. Polyacrylamide gel electrophoresisof general esterases of A. fabae by a solution mixture of both substrates ( a - and b -NA) showed that only one band present in the control and in the treated insects. However, in H. variegata larvae, there were four esterase isoforms in the control and the isoforms showed changes after insect treatment with the insecticides. Thus it is concluded that susceptibility of the two insects toward the two insecticides are different and these differences were obvious in their enzyme activity and their isoforms as well.

Biochemical changes in certain enzymes of Lysapsus limellium (Anura: Hylidae) exposed to chlorpyrifos

Different enzyme biomarkers (AChE: acetylcholinesterase, CbE: carboxylesterase, GST: glutathione-S-transferase, CAT: catalase) were measured in digestive tissues of Lysapsus limellum frogs collected from a rice field (RF: chlorpyriphos sprayed by aircraft) and a non-contaminated area (RS: reference site), immediately (24h) and 168h after aerial spraying with chlorpyrifos (CPF). CPF degradation was also searched in water samples collected from RF and RS, and found that insecticide concentration was reduced to≈6.78% of the original concentration in RF at 168h. A significant reduction of AChE and CbE activities was detected in L. limellum from RF in stomach and liver at 24 and 168h, and in intestine only at 24h, with respect to RS individuals. CAT activity decreased in intestine of L. limellum from RF 24h and 168h after exposure to CPF, whereas GST decreased in that tissue only at 24h. In stomach and liver, a decrease was observed only at 168h in both CAT and GST. The use of biomarkers (AChE, CbE, GST, and CAT) provides different lines of evidences for ecotoxicological risk assessment of wild frog populations at sites contaminated with pesticides.

Extracellular pesticide detoxification in the gastrointestinal tract of the earthworm Aporrectodea caliginosa

We provide evidences that indicate Aporrectodea caliginosa detoxifies extracelullarly organophosphorus (OP) pesticides through its digestive system. A high carboxylesterase activity (a group of esterases able to bind OP pesticides) was found in the luminal microenvironment of its gastrointestinal tract compared to casts (faeces) or bulk soil. In vitro inhibition kinetics and microcosm trials showed that this luminal CbE activity was highly sensitive to inhibition by chlorpyrifos-oxon. Our data suggest that CbE activity was an efficient bioscavenger for OP pesticides during gastrointestinal transit of soil, which increases our understanding of how earthworms are able to tolerate OP-contaminated soils.

Bile acids inhibit Na⁺/H⁺ exchanger and Cl⁻/HCO₃⁻ exchanger activities via cellular energy breakdown and Ca²⁺ overload in human colonic crypts.

Bile acids play important physiological role in the solubilisation and absorption of dietary lipids. However, under pathophysiological conditions, such as short bowel syndrome, they can reach the colon in high concentrations inducing diarrhoea. In this study, our aim was to characterise the cellular pathomechanism of bile-induced diarrhoea using human samples. Colonic crypts were isolated from biopsies of patients (controls with negative colonoscopic findings) and of cholecystectomised/ileum-resected patients with or without diarrhoea. In vitro measurement of the transporter activities revealed impaired Na⁺/H⁺ exchanger (NHE) and Cl⁻/HCO₃⁻ exchanger (CBE) activities in cholecystectomised/ileum-resected patients suffering from diarrhoea, compared to control patients. Acute treatment of colonic crypts with 0.3 mM chenodeoxycholate caused dose-dependent intracellular acidosis; moreover, the activities of acid/base transporters (NHE and CBE) were strongly impaired. This concentration of chenodeoxycholate did not cause morphological changes in colonic epithelial cells, although significantly reduced the intracellular ATP level, decreased mitochondrial transmembrane potential and caused sustained intracellular Ca²⁺ elevation. We also showed that chenodeoxycholate induced Ca²⁺ release from the endoplasmic reticulum and extracellular Ca²⁺ influx contributing to the Ca²⁺ elevation. Importantly, our results suggest that the chenodeoxycholate-induced inhibition of NHE activities was ATP-dependent, whereas the inhibition of CBE activity was mediated by the sustained Ca²⁺ elevation. We suggest that bile acids inhibit the function of ion transporters via cellular energy breakdown and Ca²⁺ overload in human colonic epithelial cells, which can reduce fluid and electrolyte absorption in the colon and promote the development of diarrhea.

Earthworm activity increases pesticide-sensitive esterases in soil

Carboxylesterases (CbEs) are serine hydrolases involved in the detoxification of anticholinesterase (organophosphorus and methylcarbamate) pesticides. Past studies have documented the occurrence of these esterases in soil, but little is known about their origin, function, and particularly, their reactivity against agrochemicals. In this study, it was compared the potential CbE activity in earthworm-treated and control (earthworm-free) soils by enzyme kinetics with multiple carboxylic esters and native polyacrylamide gel electrophoresis. After 12 weeks of inoculation, CbE activity was between two- and four-fold higher in the earthworm-treated soils (α-naphthyl acetate = 4.85 ± 1.58 μmol h−1 g−1 dry soil, α-naphthyl butyrate = 2.93 ± 1.60, α-naphthyl valerate = 2.64 ± 1.27, 4-nitrophenyl acetate = 1.41 ± 0.37, 4-nitrophenyl butyrate = 0.87 ± 0.15 and 4-nitrophenyl valerate = 0.89 ± 0.11; values are presented as mean ± standard deviation) than in controls. Although this enhanced esterase activity remained unchanged for 1 month following earthworm removal, it decreased under soil desiccation (31%–60%) or thermal denaturing (43%–82%). The potential sources for enhanced soil CbE activity were also examined through plate-count of microorganisms and zymographic techniques. The earthworm gut microenvironment was a significant source of soil CbE activity, and the casts were found to be the main contributors to the esterase activity analyzed. Soil CbE activity was strongly inhibited by organophosphorus (chlorpyrifos-oxon, paraoxon-ethyl and paraoxon-methyl) and, at less extent, by methylcarbamates (carbaryl and carbofuran). In vitro inhibition kinetics showed a biphasic curve that revealed at least two sensitive esterases and a resistant fraction; the latter varied widely depending on the enzyme substrate (7–68% of control activity). Likewise, spiking of earthworm-treated soils with 4 mg kg–1 (wet weight) of chlorpyrifos-oxon led to a significant inhibition of CbE activity 2 (40–72% inhibition) and 6 days (37–53%) after its application. Current results suggest that the soil-dwelling earthworm Lumbricus terrestris may be used as a promoter of soil enzyme activities with a direct benefit for pesticide bioremediation.

Tissue-Specific Variations of Esterase Activities in the Tadpoles and Adults of Pseudis paradoxa (Anura: Hylidae)

We determined basal levels of cholinesterase (ChE) and carboxylesterase (CbEs; two substrates: α-naphthyl acetate and 4-nitrophenylvalerate) in different tissues of tadpoles and adults of the frog Pseudis paradoxa and evaluated their use as complementary biomarkers of anti-cholinesterase pesticide exposure. ChE and CbEs sensitivity to malaoxon was also evaluated. Adults and tadpoles were collected with sweep net from temporary ponds located in natural riparian forests along the Parana River (Garay Department, Santa Fe province, Argentina). We found significant differences in B-esterase activities between adults and tadpoles and among different tissues. The in vitro inhibition tests indicated that ChE is more sensitive to inhibition than CbEs. Our results suggest that basal ChE and CbE (α-NA and 4-NPV) activities in different tissues of adult and tadpoles P. paradoxa would be suitable biomarkers of pesticide exposure, and this amphibian species could be used as sentinel in field monitoring.

What primary care physician teachers need to sustain community based education in Japan.

BackgroundCommunity based education (CBE), defined as “a means of achieving educational relevance to community needs and, consequently, of implementing a community oriented educational program,” is reported to be useful for producing rural physicians in Western countries. However, why some physicians withdraw from their teaching roles is not well known, especially in Asian countries. The aim of this study was to clarify the requisites and obstacles for taking part in CBE.MethodsWe combined two steps: preliminary semi-structured interviews followed by workshop discussions. First of all, we interviewed four designated physicians (all male, mean age 48 years) working in one rural area of Japan, with less than 10,000 residents. Secondly, we held a workshop at the academic conference of the Japan Primary Care Association. Fourteen participants attending the workshop (seven male physicians, mean age 45 years, and seven medical students (one female and six male), mean age 24 years) were divided into two groups and their opinions were summarized.ResultsIn the first stage, we extracted three common needs from interviewees; 1. Sustained significant human relationships; 2. Intrinsic motivation; and 3. Tangible rewards. In the second stage, we summarized three major problems from three different standpoints; A. Preceptors’ issues: more educational knowledge or skills, B. Learner issues: role models in rural areas, and C. System issues: supportive educational system for raising rural physicians.ConclusionsOur research findings revealed that community physicians require non-monetary support or intrinsic motivation for their CBE activities, which is in accordance with previous Western studies. In addition, we found that system support, as well as personal support, is required. Complementary questionnaire surveys in other Asian countries will be needed to validate our results.

Plasma B-esterase and Glutathione S-transferase Activities in the South American Reptiles Caiman latirostris (Crocodylia, Alligatoridae) and Phrynops hilarii (Testudines, Chelidae)

We determined normal plasma butyrylcholinesterase (BChE), carboxylesterase (CbE using α-NA substrate), and glutathione S-transferase (GST) activities in Caiman latirostris and Phrynops hilarii to obtain reference values for organophosphorus (OP) pesticide monitoring. BChE and CbE sensitivity to malaoxon was also evaluated. C. latirostris (N = 12; six males and six females) and P. hilarii (N = 12; seven males and five females) were obtained from the programs Yacare (Entre Rios Province, Argentina) and Zoo of Cordoba (Cordoba Province, Argentina). Mean total (female and male) plasma BChE activity was significantly different between reptile species, ranging between 0.337 ± 0.085 μmol min−1 ml−1 of plasma for C. latirostris and 0.251 ± 0.070 μmol min−1 ml−1 of plasma for P. hilarii. However, plasma CbE (α-NA) and GST activities were significantly higher in P. hilarii (4.81 ± 1.00 and 0.145 ± 0.045 μmol min−1 ml−1 of plasma, respectively) than in C. latirostris (0.57 ± 0.20 and 0.059 ± 0.013 μmol min−1 ml−1 of plasma, respectively). No significant differences in B-esterase and GST activities were detected between sexes, except CbE (α-NA) for C. latirostris. IC50 values for BChE and CbE (α-NA) suggested different sensitivity levels between species and between sexes. The results demonstrate that plasma esterase activity varied between species, but not between sexes (except CbE for C. latirostris). The in vitro inhibition tests indicated that CbE (α-NA) is more sensitive to inhibition than BChE. C. latirostris may be the reptile species most vulnerable to field pesticide exposure because this reptile presents the lowest CbE activity levels and its B-esterase levels seem more sensitive to OP.

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1 mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (∼50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the use of organophosphorus pesticides in the natural habitat of the axolotl.

Activity levels of B-esterases in the tadpoles of 11 species of frogs in the middle Paraná River floodplain: Implication for ecological risk assessment of soybean crops

Soybean fields provide habitats for many species of amphibians. However, the persistence and health of amphibian populations may be at risk from the increasing use of pesticides and other agricultural chemicals. We examined the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterases (CbEs) in 11 syntopic species of larval anurans. In vitro effects of malaoxon causing 50% BChE inhibition (IC 50) were also studied. In addition, we calculated a relative risk index (RI) based on the geographic distributions of the anurans, the phenology of soybean cultivation, and basal enzymatic values related to potential pesticide detoxification. Among the 11 species, AChE activity varied from 17.5±1.6 to 68.2±4.7 nmol min −1 mg −1 protein (PT). BChE activity also varied significantly, ranging from 3.3±0.4 to 7.5±0.4 nmol min −1 mg −1 PT. Both measures of CbE activities varied widely (CbE α-NA: 2.1±0.5–12.4±1.1 nmol min −1 mg −1 PT; CbE-4NPV: 21.8±1.8–102.6±7.9 nmol min −1 mg −1 PT). We also corroborate that lower BChE activity levels for the tadpoles were associated at minor IC 50 values. The results of this study demonstrate significant variation in enzymatic levels among several tadpole species and intermediate to high RI values for 7 species. Based on these results, it appears that a conversion of native ecosystems to soybean crops may lead to increased ecological risk for anuran amphibians.