Acaricidal efficacy evaluation of amitraz and diazinon against Amblyomma variegatum tick species in Waghimra zone, northern Ethiopia.

Ticks pose a substantial economic burden associated with production loss and treatment costs globally. Ethiopia has tremendous livestock resources, but its productivity is hindered by various animal health challenges in which ticks are the top priority, with a limited response to acaricidal treatments. Hence, we designed an acaricidal efficacy trial for the top commercially available Chemicals (amitraz and diazinon) to examine their efficacy against the widely distributed tick species (Amblyoma variegatum). Ticks were collected from animals admitted to veterinary clinics with no history of treatment with acaricides. Adult Immersion Technique (AIT) was employed to detect acaricidal resistance, and the mean percent control and antiparasitic efficacy were used to estimate tick susceptibility with a completely randomized laboratory-based trial (CRT). The mean weights of eggs laid by ticks subjected to amitraz and diazinon indicated that amitraz had a better egg-laying-inhibition effect than diazinon. The mean control percentages of amitraz and diazinon were 92.8 ± 5.6% and 69.7 ± 3.1%, respectively, with a highly significant difference (P-value = 0.00). The antiparasitic efficacy of the two drugs was 57.5 ± 0.96 and 37.5% ± 0.96 for amitraz and diazinon, respectively, which revealed that amitraz was statistically better than diazinon in killing adult ticks (P-value =0.026). In general, ticks treated with diazinon showed evidence of resistance development, and amitraz is relatively the most effective acaricide; we recommend its use in the study area and other locations with similar settings.

Status of Acaricide Resistance and Detecting the Knockdown Resistance Mutation T2134A in the Cattle Tick Rhipicephalus microplus (Acari: Ixodidae) from Northeastern Mexico.

Rhipicephalus microplus is the most important tick in veterinary medicine, given its repercussions on animal production. The principal strategy to avoid adverse effects associated with R. microplus is the chemical control of tick populations through organosynthetic acaricides. Therefore, monitoring susceptibility to acaricides is paramount in any control program. This study aimed to analyze the resistance status of 2 populations of R. microplus from northeastern Mexico to the organochlorine (OC) lindane, organophosphates (OP) coumaphos, chlorfenvinphos, diazinon, and chlorpyrifos, and the synthetic pyrethroids (SPs) flumethrin, deltamethrin, and cypermethrin. Discriminating doses (DD) of each acaricide were used in the larval packet bioassay (LPT). Additionally, the presence of the knockdown resistance (kdr) mutation T2134A associated with pyrethroid resistance was evaluated using allele-specific polymerase chain reaction (PCR). The populations of R. microplus showed a high frequency of resistance to SP, with mortality rates of less than 5%; they also showed resistance to the OPs (diazinon and chlorpyrifos) with mortality rates ranging from 1.29% to 34.62%; meanwhile, they were susceptible to coumaphos and chlorfenvinphos. Mortality rates higher than 66% were observed for lindane, indicating susceptibility. The mutant allele of the kdr mutation T2134A was detected in 75% and 100% of the pools analyzed. The populations studied presented a highly resistant profile to pyrethroids, with the presence of the kdr mutant allele A2134. The susceptibility to the organophosphates such as coumaphos and chlorfenvinphos of R. microplus from northeastern Mexico should be noted.

Prevalence of cattle ixodid ticks and acaricides efficacy evaluation in Amibara district of Afar Region, Ethiopia.

A cross sectional and randomized controlled trial study was conducted starting from July 2021 to July 2022 to determine the prevalence of ixodid ticks, and evaluate the efficacy of commonly used acaricides (amitraz 12.5% and diazinon 60%) in vitro and in vivo on cattle ticks in Amibara district. A total of 372 cattle were visited randomly from six kebeles of the district which were selected based on their livestock population. A total of 4330 adult ticks were collected and identified to the species level with stereomicroscope. For the invitro acaricide efficacy test, 90 engorged female ticks were collected from infested herds and grouped into three, each group having ten engorged ticks; two groups for the two test acaricides and the third groups were control to evaluate these acaricides in the laboratory. All the groups were replicated three times and incubated for seven days with similar temperature and humidity. Thirty-six infested cattle were selected and again grouped into three randomly, 12 each for the invivo test. The first groups of animals were sprayed with amitraz, the second with diazinon and the third groups were left untreated. Ticks were counted and recorded before, day 3, day 7, day 14 and day 21 of spraying. A total of 360 (96.7%) of the visited cattle were infested with ticks. Amblyomma. Rhipicephallus, Hyalomma and Bophillus were the genera of ticks identified with a prevalence of 44.9%, 41.4%, 7.5% and 6.2% respectively. A total of 15 species of ixodid ticks were identified from which Rhipicephallus pulchellus (39.86%) was dominant followed by Amblyomma lepidum (26.65%) and Amblyomma gemma (14.36%). Diazinon 60% EC and amitraz 12.5% were significantly inhibited the egg laying ability of engorged ticks' in vitro with percent inhibition of 100% and 99.1% respectively. On live animal, both acaricides showed statistically significant difference from the control group (p- value <0.05) with maximum 97.37% (amitraz) and 93.33% (diazinon) at day 7 and minimum 73.3% (amitraz) and 73.7% (diazinon) efficacy. However, there was no statistically significant difference on egg laying inhibition and tick count reduction between the two acaricides (p-value >0.05). Therefore, awareness should be created about the high prevalence as well as the application, dosage and dilution of the acaricides to pastoralists, community animal health workers and para veterinarians in the study area. Cattle owners, veterinarians and concerned government officials should address infested herds with manufacturer recommended dosage of these acaricides to control the tick infestation.

The protective effect of resveratrol on diazinon-induced oxidative stress and glucose hemostasis disorder in rats' liver.

This study was intended to assess the possible protective effect of resveratrol (Res) against oxidative stress and glucose hemostasis disorder in rats exposed to diazinon (DZN) for 4 weeks. Totally 25 Sprague-Dawley rats were divided randomly into five groups: Control (orally received corn oil), DZN group (orally received 70 mg/kg/day), and Res groups (received DZN 70 mg/kg/day plus Res doses of 5, 10, and 20 mg/kg bodyweight/-day), respectively. DZN significantly inhibited serum acetylcholinesterase enzyme (Ach E), serum and liver catalase, glutathione peroxidase activities, also total antioxidant capacities. On the other hand, DZN increased serum and liver malondialdehyde. DZN significantly increased Forkhead box protein O1 (Foxo1) expression and decreased phosphatase and tensin homolog (PTEN) and sirtuin 1 (Sirt-1) expression. DZN impaired glucose hemostasis. Instead, Res treatment significantly reversed status of oxidative stress and antioxidant enzymes activities induced by DZN. Also, Res improved glucose hemostasis. Res increased PTEN and Sirt-1 expression and reduced Foxo1 expression. Res administration ameliorated liver histopathological changes induced by DZN. These data confirmed that DZN significantly enhances oxidative stress and impairs glucose hemostasis. While Res showed a protective effect against the toxicity induced by DZN in rats.

Evaluation of Alterations in DNA Methylation of CYP3A4 Gene Upstream Regulatory Elements in Gastric Cancer and in Response to Diazinon Treatment.

BACKGROUND: Little is known about cytochrome P450 3A4 (CYP3A4) DNA methylation and transcription alterations in gastric cancer. OBJECTIVE: In this paper, we initially aimed to address the effect of diazinon pesticide on DNA methylation and transcription changes of the CYP3A4 gene in a human gastric cell line. In the next step, we studied the methylation differences of CpG sites within the upstream regulatory regions of the CYP3A4 gene among human gastric cancerous and healthy tissues. METHODS: For the in vitro assay, the methylation changes of the C/EBP response element and transcript level of the CYP3A4 gene were studied following treatment of the AGS cell line with various concentrations of diazinon pesticide. In the next phase, the methylation percentages of 24 CpG sites within or around the upstream regulatory elements, including near promoter, C/EBP binding site, XREM, and CLEM4, in 11 specimens of human gastric cancer tissue were compared to their adjacent healthy tissues. RESULTS: Treatment with 10 µM Diazinon significantly increased the CYP3A4 gene transcription by approximately 27-fold, which was correlated with the hypermethylation of 3 CpGs in C/EBP binding sites, including -5998, -5731 and -5725 (p<0.001 for all comparisons). Results of bisulfite sequencing revealed that the CpG sites which are located in -1521 (p=0.003), -1569 (p=0.027), -10813 (p=0.003), -10851 (p=0.001) and -10895 (p=0.0) bp from transcription start site, were significantly hypermethylated in cancerous tissues comparing to their healthy cohort. CONCLUSION: Hypermethylation of CLEM4 and a region near the core promoter may have a significant association with gastric cancer incidence.

Insight into natural medium remediation through ecotoxicity correlation with clay catalyst selectivity in organic molecule ozonation.

The oxidative degradation of diazinon (DAZ) and diclofenac sodium (DCF) in aqueous media was comparatively investigated and correlated with the mortality of Artemia salina in the presence of clay catalysts. For this purpose, montmorillonites (Mt) exchanged with Na+ and Fe2+ cations (NaMt and Fe(II)Mt), acid activated bentonites and hydrotalcite were used as clay catalysts. Surface interaction and adsorption on the clay surface were found to govern the catalyst dispersion in aqueous media and both activity and selectivity in ozonation. These catalysts' features were correlated with the ecotoxicity of ozonised reaction mixtures as expressed in terms of mortality rates of Artemia salina. DAZ and DCF display specific intrinsic ecotoxicity that evolves differently during ozonation according to the catalyst. The ecotoxicity was found to strongly depend on the distribution of the ozonation intermediates, which, in turn, was narrowly correlated with the acid-base properties of the catalyst surface. These valuable findings allow the prediction of the behaviour of the clay-containing media in natural remediation.

Variation of diazinon and amitraz susceptibility of Hyalomma marginatum (Acari: Ixodidae) in the Rabat-Sale-Kenitra region of Morocco.

In the present study, the acaricide susceptibility status of Hyalomma marginatum in Morocco was investigated in the Rabat-Sale-Kenitra region using the Larval Packet Test. The overall LC50 value for diazinon and amitraz was 115 ppm (95% CI: [104; 125]) and 22 ppm (95% CI: [21; 23]), respectively. The LC50 values varied significantly between the nine sampled locations (P<0.001) ranging from 75 ppm (95% CI: [65; 84]) in Ouelmes to 179 ppm (95% CI: [139; 201]) in Jorf El Melha for diazinon and from 18 ppm (95% CI: [15; 21]) in Skhirat to 28 ppm (95% CI: [24; 31]) in Ouelmes for amitraz. Sequencing of the target-site of diazinon, acetylcholinesterase 1 (AChE1), indicated that previously reported resistance mutations in AChE1 were absent in ticks from Jorf El Melha surviving 500 ppm diazinon. This study is the first report on the H. marginatum susceptibilty status to the most frequently used acaricides in Morocco and indicates that acaricide tick resistance is emerging.

Investigation of pesticides on honey bee carbonic anhydrase inhibition.

Carbonic anhydrase (CA, EC 4.2.1.1) plays crucial physiological roles in many different organisms, such as in pH regulation, ion transport, and metabolic processes. CA was isolated from the European bee Apis mellifera (AmCA) spermatheca and inhibitory effects of pesticides belonging to various classes, such as carbamates, thiophosphates, and pyrethroids, were investigated herein. The inhibitory effects of methomyl, oxamyl, deltamethrin, cypermethrin, dichlorodiphenyltrichloroethane (DDT) and diazinon on AmCA were analysed. These pesticides showed effective in vitro inhibition of the enzyme, at sub-micromolar levels. The IC50 values for these pesticides ranged between of 0.0023 and 0.0385 µM. The CA inhibition mechanism with these compounds is unknown at the moment, but most of them contain ester functionalities which may be hydrolysed by the enzyme with the formation of intermediates that can either react with amino acid residues or bid to the zinc ion from the active site.

The effects of diazinon on the cell types and gene expression of the olfactory epithelium and whole-body hormone concentrations in the Persian sturgeon (Acipenser persicus).

The olfactory function and imprinting of odorant information of the native stream play a critical role during the homing migration in fish. Pesticides may impair olfactory imprinting by altering olfaction and hormone functions. The present study aimed to determine how diazinon impacts olfactory epithelium morphology and cell composition, as well as hormone concentrations in Persian sturgeon (Acipenser persicus) during their lifetime in freshwater and, also during diazinon-free saltwater acclimation. Fingerlings were exposed to 0, 150, 300, and 450 µg·L-1 of diazinon in freshwater for 7 days and then were transferred to diazinon-free saltwater by gradually increasing salinity up to 12 ppt. After diazinon exposure, the number of olfactory receptor cells (ORCs) and goblet cells (GCs) decreased and increased, respectively, and the expression of G-protein αolf (GPαolf) and calmodulin-dependent kinase II delta (CAMKIId) was down-regulated and up-regulated, respectively. Transferring the fish to diazinon-free saltwater (8 and 12 ppt) raised the number of ORCs, supporting cells (SCs), GCs, and GPαolf expression, and down-regulated CAMKIId without any significant differences among treatments. Exposure to diazinon increased whole-body cortisol at the high concentration, while decreased whole-body thyroxin (T4) and triiodothyronine (T3) in a dose-dependent manner. Although whole-body T4 and T3 increased in all the treatments after saltwater acclimation (8 and 12 ppt), the level of these hormones was lower in fish that had been exposed to diazinon than in the control. These results showed that diazinon can disrupt olfactory epithelium morphology and cell composition as well as hormone concentrations, which in turn may affect the olfactory imprinting in Persian sturgeon fingerlings.

Sensitive and reversible perylene derivative-based fluorescent probe for acetylcholinesterase activity monitoring and its inhibitor.

A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water soluble perylene derivative, N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aqueous is quenched after combining with copper ions (Cu2+). Acetylcholinesterase (AChE) is well known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissociation between PASP and Cu2+. We proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL-1. Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC50 value of tacrine was calculated to be 0.43 µM, and the detection limit of diazinon was 0.22 µM. Both of these performances were much better than previous results, revealing our probe is sensitive and reversible for screening applications.

Overcoming insecticide resistance through computational inhibitor design.

Insecticides allow control of agricultural pests and disease vectors and are vital for global food security and health. The evolution of resistance to insecticides, such as organophosphates (OPs), is a serious and growing concern. OP resistance often involves sequestration or hydrolysis of OPs by carboxylesterases. Inhibiting carboxylesterases could, therefore, restore the effectiveness of OPs for which resistance has evolved. Here, we use covalent virtual screening to produce nano-/picomolar boronic acid inhibitors of the carboxylesterase αE7 from the agricultural pest Lucilia cuprina as well as a common Gly137Asp αE7 mutant that confers OP resistance. These inhibitors, with high selectivity against human acetylcholinesterase and low to no toxicity in human cells and in mice, act synergistically with the OPs diazinon and malathion to reduce the amount of OP required to kill L. cuprina by up to 16-fold and abolish resistance. The compounds exhibit broad utility in significantly potentiating another OP, chlorpyrifos, against the common pest, the peach-potato aphid (Myzus persicae). These compounds represent a solution to OP resistance as well as to environmental concerns regarding overuse of OPs, allowing significant reduction of use without compromising efficacy.

The role of ghrelin and tumor necrosis factor alpha in diazinon-induced dyslipidemia: insights into energy balance regulation.

The evidence shows that organophosphate compounds (OPCs), as toxic agents that stimulate the cholinergic system, can increase the incidence of metabolic disorders such as dyslipidemia. In the present study, we focused on the role of tumor necrosis factor alpha (TNF-α) and serum leptin and ghrelin in Diazinon (DZN)-induced dyslipidemia. The rats were randomly divided into five groups comprising eight animals, and all were treated via oral gavage for 28 consecutive days as follows: group one received only corn oil daily, while groups two through five received different doses of DZN dissolved in corn oil equal to 1/40, 1/20, 1/10 and 1/5 of the LD50 daily, respectively. The alteration of the serum lipid profile, such as triglycerides, high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL), was confirmed the occurrence of dyslipidemia in the range of doses 1/20-1/5 LD50 of DZN. Although no changes were found in the serum leptin levels, a significant increase was observed in the size of adipocytes, as well as in the TNF-α and ghrelin serum levels, and in the accumulation of epididymal fat, especially at a dose of 1/5 LD50 of DZN. It seems that interactions among the inflammatory reaction, cholinergic pathways and ghrelin secretion may be effective causes of DZN-induced dyslipidemia.

Age-Related Diazinon Toxicity Impact on Blood Glucose, Lipid Profile and Selected Biochemical Indices in Male Rats.

BACKGROUND: Diabetes and its complications are age-related diseases. Low-grade inflammation plays the main role in the aging processes. Diazinon (DZN), an organophosphate pesticide, has been found to induce metabolic disturbances. OBJECTIVE: The present study was designed to investigate the impact of DZN on age-related changes on inflammatory cells, blood glucose concentration, lipid profile, and liver and kidney function indices in adult and aged rats. METHODS: Male rats (2 and 16 month old) were orally administrated with DZN (15 mg/kg) for 4 weeks. Then the blood was obtained for measuring inflammatory cells, lipid profile, glucose and serum biochemical indices such as liver enzymes, albumin, total protein, creatinine (Cr), urea, and uric acid in the serum of adult and aged male rats. RESULTS: DZN increased the blood levels of glucose and the percentage of lymphocytes and also serum levels of TChol, TG, LDL-c, AST, ALT, ALP, LDH, Cr, urea, and uric acid in the adult and aged rats versus the aged matched control rats (p< 0.001). A marked reduction in HDL-c levels, total protein, albumin, and in the percentage of neutrophils were seen in the adult and aged animals exposed to DZN versus the aged matched control rats. DZN also increased the levels of LDL-c and ALT in the aged rats versus adult animals. CONCLUSION: The present study indicated that DZN can cause metabolic disturbance. However, the age-dependent effects of DZN on metabolic indices were not be confirmed by the present data.

Organophosphorus Pesticides Induce Cytokine Release from Differentiated Human THP1 Cells.

Epidemiologic studies link organophosphorus pesticides (OPs) to increased incidence of asthma. In guinea pigs, OP-induced airway hyperreactivity requires macrophages and TNF-α. Here, we determined whether OPs interact directly with macrophages to alter cytokine expression or release. Human THP1 cells were differentiated into macrophages and then exposed to parathion, chlorpyrifos, or diazinon, or their oxon, phosphate, or phosphorothioate metabolites for 24 hours in the absence or presence of reagents that block cholinergic receptors. TNF-α, IL-1ß, platelet-derived growth factor, and transforming growth factor-ß mRNA and protein were quantified by qPCR and ELISA, respectively. The effects of OPs on NF-κB, acetylcholinesterase, and intracellular calcium were also measured. Parent OPs and their oxon metabolites upregulated cytokine mRNA and stimulated cytokine release. TNF-α release, which was the most robust response, was triggered by parent, but not oxon, compounds. Cytokine expression was also increased by diethyl dithiophosphate but not diethyl thiophosphate or diethyl phosphate metabolites. Parent OPs, but not oxon metabolites, activated NF-κB. Parent and oxon metabolites decreased acetylcholinesterase activity, but comparable acetylcholinesterase inhibition by eserine did not mimic OP effects on cytokines. Consistent with the noncholinergic mechanisms of OP effects on macrophages, pharmacologic antagonism of muscarinic or nicotinic receptors did not prevent OP-induced cytokine expression or release. These data indicate that phosphorothioate OP compounds directly stimulate macrophages to release TNF-α, potentially via activation of NF-κB, and suggest that therapies that target NF-κB may prevent OP-induced airway hyperreactivity.

Diazinon exposure activated transcriptional factors CCAAT-enhancer-binding proteins α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) and induced adipogenesis in 3T3-L1 preadipocytes.

Environmental chemical exposure could be a contributor to the increasing obesity epidemic. Diazinon, an organophosphate insecticide, has been widely used in the agriculture, and exposure of the general population to diazinon has been reported. Diazinon has been known to induce neurotoxic effects mainly through the inhibition of acetylcholinesterase (AChE). However, its association with dysregulation of adipogenesis has been poorly investigated. The current study aimed to examine the mechanism of diazinon's effect on adipogenesis using the 3T3-L1 preadipocytes combined with a single-cell-based high-content analysis. The results showed that diazinon induced lipid droplet accumulation in a dose-dependent manner. The dynamic changes of adipogenic regulatory proteins and genes were examined at the three stages of adipogenesis (induction, differentiation, and maturation) in 3T3-L1 cells treated with various doses of diazinon (0, 1, 10, 100 µM) using real-time quantitative RT-PCR and Western Blot respectively. Diazinon significantly induced protein expression of transcriptional factors CCAAT-enhancer-binding proteins α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ), their downstream proteins, fatty acid synthase (FASN), acetyl CoA carboxylase (ACC), fatty acid-binding protein 4 (FABP4), lipoprotein lipase (LPL), adiponectin and perilipin in dose and time-dependent manners. Similarly, the adipogenic genes were significantly induced in a dose and time-dependent manner compared to the relative controls. The current study demonstrates that diazinon promotes lipid accumulation and activates the adipogenic signaling pathway in the in vitro model.

Establishment of a multi-acaricide resistant reference tick strain (IVRI-V) of Rhipicephalus microplus.

Tick-borne diseases is a global threat and tick resistance to commonly used acaricides is a growing problem, thus calling for improved resistance monitoring tools. To aid in monitoring of resistance in field tick populations, a resistant colony of Rhipicephalus microplus was characterized with the aim to establish a reference multi-acaricide resistant tick strain. Using a standardized adult immersion test, the Lethal Concentration(LC)50 values for deltamethrin, cypermethrin, fenvalerate and diazinon against the laboratory selected resistant tick (LSRT) strain were determined as 306.7 ppm, 2776.9 ppm, 30262.1 ppm and 9458.7 ppm. Relative to the susceptible IVRI-I tick strain, the LSRT strain showed 4.78- and 5.84-fold increases in activity of esterases, a 6-fold increase for monooxygenases and a 2.24 fold increase for glutathione S-transferase. In the acetylcholinesterase 2 gene, 22 single nucleotide polymorphisms (SNPs) were identified in the LSRT strain. Four of these SNPs lead to amino acid substitutions and were consistently found in resistant field populations in India. A C190A mutation in the domain II S4-5 linker region of sodium channel gene resulting in a L64I amino acid substitution was recorded in the LSRT strain. Monitorable indicators for the maintenance of the strain, designated as the reference IVRI-V tick strain and representing the first established multi-acaricide resistant tick strain in India, were identified.

Anti-Osteoarthritic and Anti-Inflammatory Activities of Diazine: In Vitro and In Vivo Studies.

BACKGROUND The present study evaluated the effects of diazine (DZN) on collagenase-induced osteoarthritis (OA) in rats. MATERIAL AND METHODS OA was produced via intra-articular injections of collagenase type II into the knee joint. The rats were then treated with DZN (25, 50, or 100 mg/kg, p.o.) for three weeks. At the end of the protocol, all rats were evaluated for paw latency, paw edema, and knee swelling. Additionally, serum concentrations of glycosaminoglycan (GAG), alkaline phosphatase (ALP), and C-reactive protein (CRP) were determined. X-rays were performed to estimate radiological and histopathological changes in the knee joint. The expressions of antioxidant enzymes, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were estimated in the synovial tissues. RESULTS DZN treatment attenuated inflammation in osteoarthritic rats, as evidenced by decreases in paw edema and knee swelling and enhanced paw latency compared to the negative control group. Additionally, there were significant decreases in the serum levels of CRP and GAG and increases in ALP in the DZN-treated groups compared to the negative control group. The radiological and histopathological results showed that DZN protected against cartilage damage in the knee joint. Additionally, MMP levels decreased and there were significant reductions in the expressions of antioxidant enzymes and TIPMs in the DZN-treated groups compared to the negative control group. CONCLUSIONS The present findings demonstrated the chondroprotective effects of DZN via its modulation of the expressions of TIMP-1 and MMPs in the synovial tissues of osteoarthritic rats.

Different response of acetylcholinesterases in salt- and detergent-soluble fractions of honeybee haemolymph, head and thorax after exposure to diazinon.

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study.

Planarian cholinesterase: molecular and functional characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity.

The asexual freshwater planarian Dugesia japonica has emerged as a medium-throughput alternative animal model for neurotoxicology. We have previously shown that D. japonica are sensitive to organophosphorus pesticides (OPs) and characterized the in vitro inhibition profile of planarian cholinesterase (DjChE) activity using irreversible and reversible inhibitors. We found that DjChE has intermediate features of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Here, we identify two candidate genes (Djche1 and Djche2) responsible for DjChE activity. Sequence alignment and structural homology modeling with representative vertebrate AChE and BChE sequences confirmed our structural predictions, and show that both DjChE enzymes have intermediate sized catalytic gorges and disrupted peripheral binding sites. Djche1 and Djche2 were both expressed in the planarian nervous system, as anticipated from previous activity staining, but with distinct expression profiles. To dissect how DjChE inhibition affects planarian behavior, we acutely inhibited DjChE activity by exposing animals to either an OP (diazinon) or carbamate (physostigmine) at 1 µM for 4 days. Both inhibitors delayed the reaction of planarians to heat stress. Simultaneous knockdown of both Djche genes by RNAi similarly resulted in a delayed heat stress response. Furthermore, chemical inhibition of DjChE activity increased the worms' ability to adhere to a substrate. However, increased substrate adhesion was not observed in Djche1/Djche2 (RNAi) animals or in inhibitor-treated day 11 regenerates, suggesting this phenotype may be modulated by other mechanisms besides ChE inhibition. Together, our study characterizes DjChE expression and function, providing the basis for future studies in this system to dissect alternative mechanisms of OP toxicity.

Comparative study of esterases in deltamethrin and diazinon resistant Rhipicephalus microplus and Hyalomma anatolicum ticks collected from the Trans-Gangetic plains of India.

A comparative analysis of esterases in susceptible and resistant ticks revealed six types of esterases (EST-1b, EST-2b, EST-3b, EST-4b, EST-5b and EST-6b) in Rhipicephalus microplus and four types (EST-1h, EST-2h, EST-3h, EST-4h) in Hyalomma anatolicum using α-naphthyl acetate substrate. Inhibition studies with eserine sulfate, p-chloromercuribenzoate, copper sulphate and phenylmethylsulfonyl fluoride revealed a marked variation in band intensity between susceptible and resistant ticks, with the latter being more intense. Qualitative expression of EST-4b along with an extra band of EST-5b and EST-6b were indicative of deltamethrin and diazinon resistance in R. microplus, whereas qualitative expression of EST-4h was probably responsible for diazinon resistance in H. anatolicum. The data suggest that increased esterase activity may represent a detoxification strategy leading to the development of resistance in these tick populations.