Protective effect of vitamin C against thiamethoxam-induced toxicity in male rats.

Background: Thiamethoxam (THM) is a neonicotinoid insecticide used to control different insect pests on fruits, vegetables, and field crops. The misuse and continuous exposure to THM cause many harmful effects on health and the reproductive system. Aim: This work aims to investigate the efficiency of vitamin C (vit C) in reducing or eliminating the harmful effects of THM on the testes, liver, and kidney of male rats. Methods: Forty-eight sexually mature male Wister albino rats (weight: 170-190 g; age: 10-11 weeks) were randomly allocated into six groups (8 males/group). The control group was orally given distilled water, vit C group was orally treated with 200 mg/kg b.wt of vit C, group 1/10 of THM LD50 orally treated with 156.3 mg/kg b.wt of THM, group 1/20 of THM LD50 orally treated with 78.15 mg/kg b.wt of THM, group 1/10 of THM LD50 + vit C orally treated with 156.3 mg/kg b.wt of THM + 200 mg/kg b.wt of vit C, and group 1/20 of THM LD50 + vit C orally treated with 78.15 mg/kg b.wt of THM + 200 mg/kg b.wt of vit C. All groups were treated for five days per week for a whole period of 58 days. Blood samples were collected at the end of the experiment, and serum was extracted for liver and kidney functions and antioxidant measurements. Reproductive organs (testis, epididymis, and seminal vesicles) were collected and weighed at the end of the experiment. Results: The results showed that groups exposed to 1/10 and 1/20 of THM LD50 significantly (p < 0.05) decreased the body weight, the reproductive organ weights (testis, epididymis, and seminal vesicles), spermatid count, sperm (count and motility), and testosterone concentration with an increase in abnormalities. In addition, the groups exposed to THM showed a decrease in protein concentration, albumin, and globulin, and caused an increase in glucose concentration. The activities of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea, and malondialdehyde (MDA) were increased while caused decrease in total antioxidant capacity (TAC) due to exposure to THM. The co-administration of vit C with HM modulated the harmful effects of the insecticide on testicular, liver, and kidney parameters, which confirmed in histopathological examination of testis. Groups orally treated with vit C showed a significant increase in spermatogenesis, spermatid numbers, and the weight of seminal vesicles. Conclusion: This study showed the importance of vit C in reducing toxic effects from exposure to THM. Accordingly, the intake of vit C by individuals who regularly handle this insecticide will be beneficial in reducing the adverse effects that may occur in the liver and kidney.

Development of a sandwich ELISA for the specific quantitation of hemagglutinin (HA)-tagged proteins during their inducible expression in Escherichia coli.

Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical fields due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purification. In order to determine the optimal purification strategy for VHH to improve the yield, a method to monitor purification at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purification steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD·mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9-106.0% and 100.7-108.0%. The method was applied to a variety of real samples for the detection of different VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purification protocols especially for nanobodies.

Residue Analysis and Risk Exposure Assessment of Multiple Pesticides in Tomato and Strawberry and Their Products from Markets.

Pesticides are used on fruit and vegetable crops to obtain greater yield and quality. Residues can be detected in these crops or their products if applied pesticides do not degrade naturally. Therefore, this study aimed to estimate pesticide residues in some strawberry and tomato-based products available in the market for human consumption and associated dietary risks. Contamination with 3-15 pesticides in the tested samples was found. The total number of pesticides detected in the tested samples was 20, belonging to the group of insecticides (84%) and fungicides (16%). Pesticides of cypermethrin, thiamethoxam, chlorpyrifos, and lambda-cyhalothrin appeared at 100% in a number of samples, where the most detected was cypermethrin followed by thiamethoxam. The average values of pesticide residues detected in the tested samples ranged from 0.006 to 0.568 mg kg-1, where it was found that cypermethrin had the highest residue value and appeared in strawberry jam obtained from the market. The recovery rate of pesticides from fortified samples with pyrethroids ranged from 47.5% (fenvalerate) to 127% (lambda-cyhalothrin). Home processing of fortified tomato and strawberry samples had a significant effect on reducing residues in tomato sauce and strawberry jam, where the reduction reached 100%. The results of acute and chronic risk assessment showed that their values were much lower than 100%, indicating minimal risk of dietary intake.

Pesticide Residues in Vegetables and Fruits from Farmer Markets and Associated Dietary Risks.

The use of pesticides leads to an increase in agricultural production but also causes harmful effects on human health when excessively used. For safe consumption, pesticide residues should be below the maximum residual limits (MRLs). In this study, the residual levels of pesticides in vegetables and fruits collected from farmers' markets in Sharkia Governorate, Egypt were investigated using LC-MS/MS and GC-MS/MS. A total number of 40 pesticides were detected in the tested vegetable and fruit samples. Insecticides were the highest group in detection frequency with 85% and 69% appearance in vegetables and fruits, respectively. Cucumber and apple samples were found to have the highest number of pesticide residues. The mean residue levels ranged from 7 to 951 µg kg-1 (in vegetable samples) and from 8 to 775 µg kg-1 (in fruit samples). It was found that 35 (40.7%) out of 86 pesticide residues detected in vegetables and 35 (38.9%) out of 90 pesticide residues detected in fruits exceeded MRLs. Results for lambda-cyhalothrin, fipronil, dimothoate, and omethoate in spinach, zucchini, kaki, and strawberry, respectively, can cause acute or chronic risks when consumed at 0.1 and 0.2 kg day-1. Therefore, it is necessary for food safety and security to continuously monitor pesticide residues in fruits and vegetables in markets.

Olive Leaf Extract Attenuates Chlorpyrifos-Induced Neuro- and Reproductive Toxicity in Male Albino Rats.

Chlorpyrifos (CPF) is a common organophosphorus insecticide. It is associated with negative consequences such as neurotoxicity and reproductive injury. This study aimed to observe the ability of olive leaf extract to attenuate chlorpyrifos toxicity, which induced neuro- and reproductive toxicity in male albino rats. Olive leaf extract (OLE) exhibits potent antioxidant and antiapoptotic properties. Twenty-two mature male rats were divided into four groups: control (saline), CPF (9 mg/kg), OLE (150 mg/kg), and CPF + OLE. Treatment was administered orally for 80 days. The CPF significantly reduced serum sex hormones, sperm counts and motility, high oxidants (MDA), and depleted antioxidants (GSH, SOD, TAC) in the brain and testes homogenate; additionally, it decreased serum AChE and brain neurotransmitters, increased Bax, decreased Bcl-2, and boosted caspase-3 immune expression in neural and testicular cells. Immunological expression of Ki 67 in the cerebrum, cerebellum, choroid plexus, and hippocampus was reduced, and α-SMA in testicular tissue also decreased. Histopathological findings were consistent with the above impacts. OLE co-administration significantly normalized all these abnormalities. OLE showed significant protection against neural and reproductive damage caused by CPF.

Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil.

Occupational medical surveillance is highly desirable in manufacturing facilities where exposure to chemicals is significant. The insecticide fipronil is generally considered safe for humans but with increasing use, exposure to fipronil is of concern. Identification of urinary metabolites of fipronil may allow development of affordable, cheap and rapid procedures for human exposure evaluation. In this study we developed a fast and easy approach for synthesis of hydroxy-fipronil, a potential urinary metabolite of fipronil. This standard was used to develop a sensitive analytical LC-MS/MS method with a limit of quantification (LOQ) of 0.4ng/mL. Fipronil sulfone, a known metabolite, and hydroxy-fipronil were quantified in urine samples from rats treated with a fipronil containing diet. Fipronil sulfone concentration centered around 20ng/mL, while the concentration of hydroxy-fipronil was dose-dependent ranging in 10-10,000ng/mL and thus being a more sensitive marker of fipronil exposure. A fipronil immunoassay with cross-reactivity to hydroxy-fipronil showed a good correlation in signal intensity with LC-MS data. It was also used to demonstrate the applicability of the method for sample screening in the evaluation of exposure levels.

Effects of juvenile hormone (JH) analog insecticides on larval development and JH esterase activity in two spodopterans.

Juvenile hormone analog (JHA) insecticides are biological and structural mimics of JH, a key insect developmental hormone. Toxic and anti-developmental effects of the JHA insecticides methoprene, fenoxycarb, and pyriproxyfen were investigated on the larval and pupal stages of Spodoptera littoralis and Spodoptera frugiperda. Bioassays showed that fenoxycarb has the highest toxicity and fastest speed of kill in 2nd instar S. littoralis. All three JHAs affected the development of 6th instar (i.e., final instar) and pupal S. frugiperda. JH esterase (JHE) is a critical enzyme that helps to regulate JH levels during insect development. JHE activity in the last instar S. littoralis and S. frugiperda was 11 and 23 nmol min(-1) ml(-1) hemolymph, respectively. Methoprene and pyriproxyfen showed poor inhibition of JHE activity from these insects, whereas fenoxycarb showed stronger inhibition. The inhibitory activity of fenoxycarb, however, was more than 1000-fold lower than that of OTFP, a highly potent inhibitor of JHEs. Surprisingly, topical application of methoprene, fenoxycarb or pyriproxyfen on 6th instars of S. littoralis and S. frugiperda prevented the dramatic reduction in JHE activity that was found in control insects. Our findings suggest that JHAs may function as JH agonists that play a disruptive role or a hormonal replacement role in S. littoralis and S. frugiperda.

Toxic effects of sub-chronic exposure of male albino rats to emamectin benzoate and possible ameliorative role of Foeniculum vulgare essential oil.

Emamectin benzoate (EB) is an avermectin insecticide used extensively in pest control on vegetable and field crops. Few studies have been done for evaluating adverse effects of EB. In the current study, we evaluated the toxic effects of EB on male rats and the possible ameliorative role of fennel essential oil (FEO). Thirty two male rats were randomly divided into 4 equal groups. All groups were treated orally with distilled water (control group), 0.5mlFEOkg(-1) BW (FEO group), 2.5mgEBkg(-1) BW (EB group), and 0.5mlFEOkg(-1) BW+2.5mgEBkg(-1) BW (FEO+EB group) for 28 days. The obtained results showed that EB treatment resulted in a significant decrease in body weight, body weight gain, RBC count, Hb concentration, % PCV, MCV and MCHC. Moreover, EB significantly decreased total leukocyte, lymphocyte, monocyte and platelet count but significantly increased granulocyte count. EB markedly decreased total protein, albumin, globulin, IgG and IgM concentrations with a significant increase in TNF-α secretion. EB had a negative impact on the liver as it significantly increased ALT, ALP, and MDA, while decreasing SOD activity. Regarding to the histopathological examination, EB treatment induced coagulative necrosis and blood vessels congestion of the liver in treated rats. Furthermore, it resulted in depletion and necrosis of the white pulp of the spleen in treated rats. The co-administration of FEO with EB, however, improved the majority of parameters studied, suggesting that FEO is an important substance in decreasing toxic effects of EB.

Determination and analysis of the genome sequence of Spodoptera littoralis multiple nucleopolyhedrovirus.

The Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV), a pathogen of the Egyptian cotton leaf worm S. littoralis, was subjected to sequencing of its entire DNA genome and bioassay analysis comparing its virulence to that of other baculoviruses. The annotated SpliMNPV genome of 137,998 bp was found to harbor 132 open reading frames and 15 homologous repeat regions. Four unique genes not present in SpltMNPV were identified, as were 14 genes that were absent or translocated by comparison. Bioassay analysis of experimentally infected Spodoptera frugiperda revealed an extended killing time for SpliMNPV as compared to S. frugiperda MNPV (SfMNPV), but a level of mortality similar to that caused by infection with SfMNPV and superior to that of Autographa californica MNPV (AcMNPV). Although extensive similarity was observed between the genome structure and predicted translation products of SpliMNPV and Spodoptera litura MNPV (SpltMNPV), genetic distances between isolates of SpliMNPV and SpltMNPV suggest that they are in fact different species of genus Alphabaculovirus.

Biosafety of recombinant and wild type nucleopolyhedroviruses as bioinsecticides.

The entomopathogenic Autographa californica (Speyer) nucleopolyhedrovirus (AcMNPV) has been genetically modified to increase its speed of kill. The potential adverse effects of a recombinant AcMNPV (AcAaIT) as well as wild type AcMNPV and wild type Spodoptera littoralis NPV (SlNPV) were studied. Cotton plants were treated with these viruses at concentrations that were adjusted to resemble the recommended field application rate (4 x 10(12) PIBs/feddan, feddan = 4,200 m2) and 3rd instar larvae of S. littoralis were allowed to feed on the contaminated plants. SDS-PAGE, ELISA, and DNA analyses were used to confirm that larvae that fed on these plants were virus-infected. Polyhedra that were purified from the infected larvae were subjected to structural protein analysis. A 32 KDa protein was found in polyhedra that were isolated from all of the viruses. Subtle differences were found in the size and abundance of ODV proteins. Antisera against polyhedral proteins isolated from AcAaIT polyhedra were raised in rabbits. The terminal bleeds from rabbits were screened against four coating antigens (i.e., polyhedral proteins from AcAaIT, AcAaIT from field-infected larvae (AcAaIT-field), AcMNPV, and SlNPV) using a two-dimensional titration method with the coated antigen format. Competitive inhibition experiments were conducted in parallel to optimize antibody and coating antigen concentrations for ELISA. The IC50 values for each combination ranged from 1.42 to 163 microg/ml. AcAaIT-derived polyhedrin gave the lowest IC50 value, followed by those of SlNPV, AcAaIT-field, and AcMNPV. The optimized ELISA system showed low cross reactivity for AcMNPV (0.87%), AcAaIT-field (1.2%), and SlNPV (4.0%). Genomic DNAs isolated from AcAaIT that were passaged in larvae of S. littoralis that were reared in the laboratory or field did not show any detectable differences. Albino rats (male and female) that were treated with AcAaIT, AcMNPV or SlNPV (either orally or by intraperitoneal injection at doses of 1 x 10(8) or 1 x 10(7) PIBs/rat, respectively) appeared to be healthy and showed increased body weight at 21 days posttreatment. The effect of virus administration on hematological, serum biochemical, and histopathological parameters were determined. Slight to moderate differences were observed in most of the hematological parameters. Specifically, serum proteins were decreased markedly in female rats treated orally with SlNPV, and in male rats injected with AcAaIT. SDS-PAGE analysis also showed some changes in serum protein profiles. No marked changes in acetylcholine esterase (AChE) activity were found. Changes in serum glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, creatinin, and urea were also observed. Immunohistochemical observation of tissues from stomach, intestine, liver, kidney, brain, spleen, and lung also showed slight changes. Fish (Tilapia nilotica) were also exposed to AcAaIT, AcMNPV or SlNPV by incorporating each of the viruses into diet (1 x 10(9) PIBs/group). No mortality was found in treated or untreated fish during the experimental period (28 days). Macrophage phagocytic activity of fish head kidney cells increased with time, reaching maximum values at 180 min for both treated and control fish.