A 90-day toxicity study of tripeptide, arginine-alanine-lysine.

There is a growing global interest in using peptides in the health industry for pharmaceuticals, cosmetics, and natural food products. Peptides contain two or more linked amino acids, whereas more than 50 amino acids are classified as polypeptides. Although there is a growing level of interest in the use of peptides in the health and wellness industry, there is a lack of literature pertaining to a specific tripeptide derived from arginine, alanine, and lysine (RAK) that is of interest for human dietary use. Therefore, a 90-day repeated-dose toxicity study was performed in rats to evaluate the subchronic oral toxicity of RAK. Eighty Han:WIST rats were administered RAK by gavage at doses of 0, 250, 500, or 1000 mg/kg bw/day. There were no mortalities or other treatment related effects, and no target organs were identified. A no-observed-adverse-effect-level (NOAEL) of 1000 mg/kg bw/day, the highest dose tested, was determined. This study will contribute to the body of research in regard to the safety of the use of RAK.

Strengthened rebamipide ocular nanoformulation to effectively treat corneal alkali burns in mice through the HMGB1 signaling pathway.

Corneal alkali burns are a major ophthalmic emergency, as current therapeutic treatments are limited. Novel treatment targets and new potential agents are required to combat this severe ocular injury. Glycyrrhizin and rebamipide (RBM) are both FDA-approved drugs with potential effects against corneal alkali burns, but RBM is limited by its low aqueous solubility and low bioavailability. This study aimed to utilize dipotassium glycyrrhizinate (DG, a dipotassium salt of glycyrrhizin) as a nanocarrier encapsulating RBM to formulate an ophthalmic solution (marked DG-RBM) with strengthened activities to treat corneal alkali burns. Results showed that an easy DG-RBM preparative process generated particles with high encapsulation efficacy and ultra-small micellar size. The solubility of RBM in DG-RBM in aqueous solution was 3.1 × 105-fold enhanced than its free solution. DG-RBM exhibited excellent storage stability. In vitro cytotoxicity, ex vivo conjunctival responses, and rabbit eye tolerance tests showed that DG-RBM possessed good ocular safety profiles. DG-RBM exhibited improved in vivo corneal permeation profiles and demonstrated a strong effect against H2O2-induced oxidative damage, with a significant effect on promoting epithelial wound healing in corneal cells in vitro. As expected, in a mouse model of corneal alkali burns, the topical administration of DG-RBM achieved a strengthened efficacy against alkali burn damages. The mechanism of this therapeutic effect involved regulating high-mobility group box 1 (HMGB1) signaling and its related angiogenic and proinflammatory cytokines. These findings demonstrate the ease of preparing DG-RBM and its great potential as a novel ocular topical formulation to treat corneal alkali burns by regulating HMGB1 signaling.

Transcriptome analysis to elucidate the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil on the hepatopancreas of Procambarus clarkii.

Most antibiotics, insecticides, and other chemicals used in agricultural and fishery production tend to persist in the environment. Fenvalerate, sulfide gatifloxacin, and ridomil are widely used in aquaculture as antibacterial, antifungal, and antiparasitic drugs; however, their toxicity mechanism remains unclear. Thus, we herein analyzed the effects of these three drugs on the hepatopancreas of Procambarus clarkii at the transcriptome level. Twelve normalized cDNA libraries were constructed using RNA extracted from P. clarkii after treatment with fenvalerate, sulfide gatifloxacin, or ridomil and from an untreated control group, followed by Kyoto Encyclopedia of Genes and Genomes pathway analysis. In the control vs fenvalerate and control vs sulfide gatifloxacin groups, 14 and seven pathways were significantly enriched, respectively. Further, the effects of fenvalerate and sulfide gatifloxacin were similar on the hepatopancreas of P. clarkii. We also found that the expression level of genes encoding senescence marker protein-30 and arylsulfatase A was downregulated in the sulfide gatifloxacin group, indicating that sulfide gatifloxacin accelerated the apoptosis of hepatopancreatocytes. The expression level of major facilitator superfamily domain containing 10 was downregulated, implying that it interferes with the ability of the hepatopancreas to metabolize drugs. Interestingly, we found that Niemann pick type C1 and glucosylceramidase-ß potentially interact with each other, consequently decreasing the antioxidant capacity of P. clarkii hepatopancreas. In the fenvalerate group, the downregulation of the expression level of xanthine dehydrogenase indicated that fenvalerate affected the immune system of P. clarkii; moreover, the upregulation of the expression level of pancreatitis-associated protein-2 and cathepsin C indicated that fenvalerate caused possible inflammatory pathological injury to P. clarkii hepatopancreas. In the ridomil group, no pathway was significantly enriched. In total, 21 genes showed significant differences in all three groups. To conclude, although there appears to be some overlap in the toxicity mechanisms of fenvalerate, sulfide gatifloxacin, and ridomil, further studies are warranted.

Cytotoxicity and genotoxicity of methomyl, carbaryl, metalaxyl, and pendimethalin in human umbilical vein endothelial cells.

Pesticides have adverse effects on the cellular functionality, which may trigger myriad of health consequences. However, pesticides-mediated toxicity in the endothelial cells (ECs) is still elusive. Hence, in this study, we have used human umbilical vein endothelial cells (HUVECs) as a model to quantify the cytotoxicity and genotoxicity of four pesticides (methomyl, carbaryl, metalaxyl, and pendimethalin). In the MTT assay, HUVECs exposed to methomyl, carbaryl, metalaxyl, and pendimethalin demonstrated significant proliferation inhibition only at higher concentrations (500 and 1000 µM). Likewise, neutral red uptake (NRU) assay also showed proliferation inhibition of HUVECs at 500 and 1000 µM by the four pesticides, confirming lysosomal fragility. HUVECs exposed to the four pesticides significantly increased the level of intracellular reactive oxygen species (ROS). Comet assay and flow cytometric data exhibited DNA damage and apoptotic cell death in HUVECs after 24 h of exposure with methomyl, metalaxyl, carbaryl, and pendimethalin. This is a first study on HUVECs signifying the cytotoxic-genotoxic and apoptotic potential of carbamate insecticides (methomyl and carbaryl), fungicide (metalaxyl), and herbicide (pendimethalin). Overall, these pesticides may affect ECs functions and angiogenesis; nonetheless, mechanistic studies are warranted from the perspective of vascular biology using in vivo test models.

The different toxic effects of metalaxyl and metalaxyl-M on Tubifex tubifex.

Metalaxyl and Metalaxyl-M are the fungicides that widely used in many countries. In this study, the environmental behaviors between metalaxyl and metalaxyl-M in Tubifex tubifex (T. tubifex) were quantitative analyzed by using a high performance liquid chromatography with photo-diode-array-detector (HPLC-DAD). Results demonstrated that there was no significant difference (p > 0.05) in the concentration of metalaxyl and metalaxyl-M in T. tubifex during the exposure process. However, the dissipation behaviors of metalaxyl and metalaxyl-M in T. tubifex were different (p < 0.05) during the non-exposure culture process. Meanwhile, the toxic effects were also evaluated by comparing the different influences of these two compounds on related physiological indicators, and functional enzyme activities. The survival rates of T. tubifex were 63.33 ± 15.28% (20 mg L-1), 63.33 ± 5.77% (200 mg L-1) treated with metalaxyl and were 50.00 ± 10.00% (20 mg L-1), 46.67 ± 11.55 (200 mg L-1) treated with metalaxyl-M at the non-exposure culture process. The autotomy rates were increased significantly compared with the initial in all treatments. Besides, the activities of CAT, SOD, and GST in T. tubifex were also inhibited by metalaxyl and metalaxyl-M treatments. Finally, the high-throughput transcriptome sequencing technology was applied to investigate the metabolic pathways of target analytes in T. tubifex, and results proved that the metabolic pathways associated with human diseases (such as viral myocarditis) were up-regulated expression for metalaxyl and metalaxyl-M treatments, and metalaxyl-M up-regulated more significantly. All the results demonstrated that metalaxyl-M had a higher toxicity than metalaxyl on T. tubifex.

Exposure to Metalaxyl Disturbs the Skeletal Development of Zebrafish Embryos.

Metalaxyl is broadly applied in agriculture to control peronosporales-caused diseases in plant. To investigate the toxic effects, zebrafish embryos were exposed to metalaxyl at 5, 50 and 500 ng/L for 72 h, the development of larvae were assessed. A significant decreased survival rate, body length, hatching rate (48 h post-fertilization), and a significant increased spinal curvature rate were observed in the 500 ng/L treatment. The lengths of lower jaw, upper jaw and hyomandibular were significantly decreased in the 5, 50 and 500 ng/L groups; while the lower jaw width was significantly increased in the 500 ng/L group. The lengths of palatoquadrate, ceratohyal and ethmoid plate were reduced. Though cyp26a1 mRNA levels showed no significant change, the transcription of bmp2b (in the 500 ng/L group), ihh (in the 50 and 500 ng/L groups), shh (in the 5, 50 and 500 ng/L groups) were significantly up-regulated, which may be related to the abnormal development of the skeleton.

L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions.

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a 'safety-valve' to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine.

Identifying Metabolic Perturbations and Toxic Effects of Rac-Metalaxyl and Metalaxyl-M in Mice Using Integrative NMR and UPLC-MS/MS Based Metabolomics.

Although metabolic perturbations are sensitive indicators for low-dose toxic effects, the metabolic mechanisms affected by rac-metalaxyl and metalaxyl-M in mammals from a metabolic profiling perspective remain unclear. In this study, the metabolic perturbations and toxic effects of rac-metalaxyl and metalaxyl-M in mice were carefully investigated using integrative nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based metabolomics. Histopathology, NMR-based untargeted urine profile, multivariate pattern recognition, metabolite identification, pathway analysis, UPLC-MS/MS based targeted serum amino acids, and tryptophan pathway analysis were determined after rac-metalaxyl and metalaxyl-M exposure, individually. Histopathology indicated that metalaxyl-M induced greater hepatocellular inflammatory, necrosis, and vacuolation in mice than rac-metalaxyl at the same exposure dosage. The metabolic perturbations induced by rac-metalaxyl and metalaxyl-M were directly separated using partial least-squares discriminant analysis (PLS-DA). Furthermore, metabolite identification and pathway analysis indicated that rac-metalaxyl mainly induced ten urine metabolite changes and four pathway fluctuations. However, metalaxyl-M induced 19 urine metabolite changes and six pathway fluctuations. Serum amino acids and tryptophan pathway metabolite changes induced by rac-metalaxyl and metalaxyl-M were also different even at the same exposure level. Such results may provide specific insight into the metabolic perturbations and toxic effects of rac-metalaxyl and metalaxyl-M, and contribute to providing available data for health risk assessments of rac-metalaxyl and metalaxyl-M at a metabolomics level.

Exposure to low-level metalaxyl impacts the cardiac development and function of zebrafish embryos.

Metalaxyl is an anilide pesticide that is widely used to control plant diseases caused by Peronosporales species. In order to study the toxic effects, zebrafish embryos were exposed to metalaxyl at nominal concentrations of 5, 50 and 500 ng/L for 72 hr, and the cardiac development and functioning of larvae were observed. The results showed that metalaxyl exposure resulted in increased rates of pericardial edema, heart hemorrhage and cardiac malformation. The distance between the sinus venosus and bulbus arteriosus, stroke volume, cardiac output and heart rate were significantly increased in larvae exposed to 50 and 500 ng/L metalaxyl compared to solvent control larvae. Significant upregulation in the transcription of tbx5, gata4 and myh6 was observed in the 50 and 500 ng/L treatments, and that of nkx2.5 and myl7 was observed in the 5, 50 and 500 ng/L groups. These disturbances may be related to cardiac developmental and functional defects in the larvae. The activity of Na+/K+-ATPase and Ca2+-ATPase was significantly increased in zebrafish embryos exposed to 500 ng/L metalaxyl, and the mRNA levels of genes related to ATPase (atp2a11, atp1b2b, and atp1a3b) (in the 50 and 500 ng/L groups) and calcium channels (cacna1ab) (in the 500 ng/L group) were significantly downregulated; these changes might be associated with heart arrhythmia and functional failure.

Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity.

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

CYP450 enzyme-specific enantioselective species-specific response for metalaxyl in in vitro hepatic cells.

Although enantioselective accumulation of chiral pesticide has been reported in organisms, the mechanisms remain unclear. In this study, the effects of chiral pesticide metalaxyl on CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP2E1 and CYP3A were investigated in human hepatoma HepG2, rat hepatic H4IIE, chicken hepatic LMH and grass carp hepatic L8824 cells. Moreover, the residual concentrations and enantiomeric ratios (ERs) of metalaxyl were also detected in the medium. The results showed the responses of these CYP450s to metalaxyl were enzyme-dependent and species-dependent in the four cells. CYP1A1, CYP1A2, and CYP2B1 were induced in HepG2 cells, CYP2A1 and CYP2B1 were induced in H4IIE cells, CYP1A1 and CYP2B1 were induced in LMH cells, and CYP2B1 was induced in L8824 cells. The enantioselective residual of metalaxyl was detected in the medium and found to be species-specific. HepG2, H4IIE and LMH cells were inclined to attenuate S-metalaxyl and lead to decrease of ER of metalaxyl, while L8824 cells were inclined to remove R-metalaxyl and resulted in an inverse shift of ER. These findings suggest an enantioselective metabolism of metalaxyl in various species which is not only related with CYP450s and CYP450 enzyme-specific, but also species-specific.

Placing arbuscular mycorrhizal fungi on the risk assessment test battery of plant protection products (PPPs).

Arbuscular mycorrhizal fungi (AMF) are mutualistic symbionts considered a key group in soil systems involved in the provision of several ecosystem services. Recently they have been listed by EFSA as organisms to be included in the test battery for the risk assessment of plant protection product (PPPs). This study aimed to contribute to improve the ISO Protocol (ISO 10832: 2009) by assessing the feasibility of using other AMF species under different test conditions. Overall, results showed that AMF species Gigaspora albida and Rhizophagus clarus (selected out of five AMF species) are suitable to be used in spore germination tests using the ISO protocol (14 days incubation with sand or artificial soil as substrate) to test PPPs. However, several modifications to the protocol were made in order to accommodate the use of the tested isolates, namely the incubation temperature (28 °C instead of 24 °C) and the change of reference substance (boric acid instead of cadmium nitrate). The need for these changes, plus the results obtained with the three fungicides tested (chlorothalonil, mancozeb and metalaxyl-M) and comparisons made with literature on the relevance of the origin of AMF isolates in dictating the adequate test conditions, emphasize the importance of adjusting test conditions (AMF species/isolates and test temperature) when assessing effects for prospective risk assessment targeting different climatic zones. So, further studies should be conducted with different AMF species and isolates from different climatic regions, in order to better define which species/isolate and test conditions should be used to assess effects of a particular PPP targeting a given climatic zone.

Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine.

The non-proteinogenic amino acid lanthionine is a byproduct of hydrogen sulfide biosynthesis: the third endogenous vasodilator gas, after nitric oxide and carbon monoxide. While hydrogen sulfide is decreased in uremic patients on hemodialysis, lanthionine is increased and has been proposed as a new uremic toxin, since it is able to impair hydrogen sulfide production in hepatoma cells. To characterize lanthionine as a uremic toxin, we explored its effects during the early development of the zebrafish (Danio rerio), a widely used model to study the organ and tissue alterations induced by xenobiotics. Lanthionine was employed at concentrations reproducing those previously detected in uremia. Light-induced visual motor response was also studied by means of the DanioVision system. Treatment of zebrafish embryos with lanthionine determined acute phenotypical alterations, on heart organogenesis (disproportion in cardiac chambers), increased heart beating, and arrhythmia. Lanthionine also induced locomotor alterations in zebrafish embryos. Some of these effects could be counteracted by glutathione. Lanthionine exerted acute effects on transsulfuration enzymes and the expression of genes involved in inflammation and metabolic regulation, and modified microRNA expression in a way comparable with some alterations detected in uremia. Lanthionine meets the criteria for classification as a uremic toxin. Zebrafish can be successfully used to explore uremic toxin effects.

Simultaneous determination and risk assessment of metalaxyl and azoxystrobin in potato by liquid chromatography with tandem mass spectrometry.

A liquid chromatography with tandem mass spectrometry method was developed and validated to simultaneously determine metalaxyl and azoxystrobin in soil, potato, and potato foliage samples. The samples were extracted by 20 mL of acetonitrile and purified with dispersive solid-phase extraction using octadecyl silane as sorbent. The method showed good linearity (determination coefficients ≥ 0.9926) for metalaxyl (2.5-500 ng/mL) and azoxystrobin (5-1000 ng/mL). The limits of detection and quantification for both fungicides were 1.5-20 µg/kg. The average recoveries in soil, potato, and potato foliage were 83.07-92.87% for metalaxyl and 82.71-98.53% for azoxystrobin. The intra- and inter-day relative standard deviations were all less than 9%. The method was successfully applied on the residual analysis of metalaxyl and azoxystrobin in field trial samples. The results showed that the concentrations of metalaxyl and azoxystrobin in potato samples collected from Guizhou and Hunan were below 50 and 100 µg/kg (maximum residue limit set by China), respectively, at 5 days after the last application. When following the recommended application manual, metalaxyl and azoxystrobin do not present health concerns to the population because the risk quotients are far below 100%. All the above data could help and promote the safe and proper use of metalaxyl and azoxystrobin in potato.

Identification of dietary alanine toxicity and trafficking dysfunction in a Drosophila model of hereditary sensory and autonomic neuropathy type 1.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is characterized by a loss of distal peripheral sensory and motorneuronal function, neuropathic pain and tissue necrosis. The most common cause of HSAN1 is due to dominant mutations in serine palmitoyl-transferase subunit 1 (SPT1). SPT catalyses the condensation of serine with palmitoyl-CoA, the initial step in sphingolipid biogenesis. Identified mutations in SPT1 are known to both reduce sphingolipid synthesis and generate catalytic promiscuity, incorporating alanine or glycine into the precursor sphingolipid to generate a deoxysphingoid base (DSB). Why either loss of function in SPT1, or generation of DSBs should generate deficits in distal sensory function remains unclear. To address these questions, we generated a Drosophila model of HSAN1. Expression of dSpt1 bearing a disease-related mutation induced morphological deficits in synapse growth at the larval neuromuscular junction consistent with a dominant-negative action. Expression of mutant dSpt1 globally was found to be mildly toxic, but was completely toxic when the diet was supplemented with alanine, when DSBs were observed in abundance. Expression of mutant dSpt1 in sensory neurons generated developmental deficits in dendritic arborization with concomitant sensory deficits. A membrane trafficking defect was observed in soma of sensory neurons expressing mutant dSpt1, consistent with endoplasmic reticulum (ER) to Golgi block. We found that we could rescue sensory function in neurons expressing mutant dSpt1 by co-expressing an effector of ER-Golgi function, Rab1 suggesting compromised ER function in HSAN1 affected dendritic neurons. Our Drosophila model identifies a novel strategy to explore the pathological mechanisms of HSAN1.

Capmul MCM/Solutol HS15-Based Microemulsion for Enhanced Oral Bioavailability of Rebamipide.

Rebamipide (RBP) is a potent anti-ulcer and anti-oxidative agent, which is a BCS class IV drug with a low oral bioavailability of less than 10%. Thus, the systemic absorption of RBP into the blood circulation is an essential prerequisite for exerting its pharmacological activities after oral dosing. Herein, we report on microemulsion (ME) systems for the enhancement of oral RBP bioavailability. In this study, MEs consisting of Capmul MCM (oil), Solutol HS15 (surfactant), and ethanol (co-surfactant) were prepared by the construction of pseudo-ternary phase diagram. The RBP-loaded MEs had spherical nano-sized droplets with narrow size distribution and neutral zeta potential. Moreover, the prepared MEs significantly enhanced the dissolution and oral bioavailability of RBP with no discernible intestinal toxicity. These results suggest that the present ME system could be further developed as an alternative oral formulation for RBP.

In vitro metabolism of [14C]-benalaxyl in hepatocytes of rats, dogs and humans.

The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [14C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [14C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

Enantioselectivity in Developmental Toxicity of rac-metalaxyl and R-metalaxyl in Zebrafish (Danio rerio) Embryo.

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac-metalaxyl and R-metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac-metalaxyl and R-metalaxyl, and the LC50 s at 96 h are 416.41 (353.91, 499.29) mg · L(-1) and 320.650 (279.80, 363.46) mg · L(-1) , respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic-pituitary-gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic-pituitary-thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac-metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R-metalaxyl. And likewise, rac-metalaxyl only caused the upregulation of dio2, while R-metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489-494, 2016. © 2016 Wiley Periodicals, Inc.

Neurotoxic Non-proteinogenic Amino Acid ß-N-Methylamino-L-alanine and Its Role in Biological Systems.

Secondary metabolites of photoautotrophic organisms have attracted considerable interest in recent years. In particular, molecules of non-proteinogenic amino acids participating in various physiological processes and capable of producing adverse ecological effects have been actively investigated. For example, the non-proteinogenic amino acid ß-N-methylamino-L-alanine (BMAA) is neurotoxic to animals including humans. It is known that BMAA accumulation via the food chain can lead to development of neurodegenerative diseases in humans such as Alzheimer's and Parkinson's diseases as well as amyotrophic lateral sclerosis. Moreover, BMAA can be mistakenly incorporated into a protein molecule instead of serine. Natural sources of BMAA and methods for its detection are discussed in this review, as well as the role of BMAA in metabolism of its producers and possible mechanisms of toxicity of this amino acid in different living organisms.

Combined toxicity of pesticide mixtures on green algae and photobacteria.

Different organisms have diverse responses to the same chemicals or mixtures. In this paper, we selected the green algae Chlorella pyrenoidosa (C. pyrenoidosa) and photobacteria Vibrio qinghaiensis sp.-Q67 (V. qinghaiensis) as target organisms and determined the toxicities of six pesticides, including three herbicides (simetryn, bromacil and hexazinone), two fungicides (dodine and metalaxyl) and one insecticide (propoxur), and their mixtures by using the microplate toxicity analysis. The toxicities of three herbicides to C. pyrenoidosa are much higher than those to V. qinghaiensis, and the toxicities of metalaxyl and propoxur to V. qinghaiensis are higher than those to C. pyrenoidosa, while the toxicity of dodine to C. pyrenoidosa is similar to those to V. qinghaiensis. Using the concentration addition as an additive reference model, the binary pesticide mixtures exhibited different toxicity interactions, i.e., displayed antagonism to C. pyrenoidosa but synergism to V. qinghaiensis. However, the toxicities of the multi-component mixtures of more than two components are additive and can be predicted by the concentration addition model.