Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity / 生物工程学报

We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 µmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.

Toxicity induced by dieldrin and chlorpyrifos in the freshwater crayfish Cambarellus montezumae (Cambaridae) / Toxicidad inducida por dieldrín y clorpirifós en Cambarellus montezumae (Cambaridae)

The toxicity induced by insecticides in aquatic organisms is of utmost relevance because it may give a clue about the degree of health or damage of the involved ecosystem. In the present report, we determined the effect of dieldrin (DD) and chlorpyrifos (CP) on the freshwater crayfish, Cambarellus montezumae. The organisms (4-6cm in diameter) were collected in the Ignacio Ramírez Reservoir, situated at 50km Northeast of Mexico City, in the Rio Lerma Basin. Initially, we determined the LC50 value with the Probit method, then the DNA damage with single cell gel electrophoresis (comet assay applied at 24, 48, and 72h of exposure) to the brain and hepatopancreas of animals exposed (in reconstituted water) to 0.05 and 0.5µg/L of each insecticide. In the hepatopancreas of the same organisms, we determined the lipid peroxidation by applying the TBARS test. DNA damage and lipid peroxidation were also evaluated with the same methods to organisms exposed in water from the reservoir. In regard to the LC50 at 72h of exposure, we found a value of 5.1µg/L and a value of 5.62µg/L for DD and CP, respectively. The comet assay applied at different exposure times showed significant DNA damage to both organs, with respect to the control level. In the case of DD, statistical significance was observed for the two doses in the whole evaluated schedule. CP was genotoxic in the brain with the high dose at 72h, and in the hepatopancreas with the two tested doses at all evaluated exposure times. Also, a significant lipid peroxidation increase was detected with the two doses of insecticides. In the study with water from the reservoir, a more pronounced DNA damage was detected. Our results showed strong DNA damage induced by both insecticides in the crayfish, as well as a correlation with the lipid peroxidation effect, suggesting that oxidative stress is involved in the genotoxic alteration. Our results also showed the usefulness of the studied organism as well as the applied tests for the evaluation of toxicological effects, and suggested the pertinence of applying the comet assay to other freshwater organisms to evaluate the bioaccumulation of insecticides. Rev. Biol. Trop. 63 (1): 83-96. Epub 2015 March 01.

La toxicidad inducida por insecticidas en organismos acuáticos es de gran relevancia porque puede orientar sobre el grado de salud o daño del ecosistema involucrado. En el presente estudio determinamos el efecto del dieldrín (DD) y del clorpirifós (CP) en el acocil de agua dulce Cambarellus montezumae. Los organismos (4-6cm de diámetro) se recolectaron en la Represa Ignacio Ramírez, situada a 50km al Noreste de la Ciudad de México, en la cuenca del Río Lerma. Inicialmente determinamos la LC50 con el método de Probit y después el daño al ADN mediante la electroforesis unicelular en gel (ensayo cometa, aplicado a las 24, 48 y 72 h de exposición) en el cerebro y el hepatopáncreas de animales expuestos (en agua reconstituida) a 0.05 y 0.5µg/L de cada insecticida. En el hepatopáncreas de los mismos organismos determinamos la peroxidación lipídica aplicando la prueba de TBARS. El daño al ADN y la peroxidación lipídica también se evaluaron con los mismos métodos en organismos expuestos a los insecticidas en agua de la represa. En relación a la LC50, a las 72h de exposición encontramos un valor de 5.1µg/L y un valor de 5.62µg/L para DD y CP, respectivamente. El ensayo cometa aplicado a diferentes tiempos de exposición mostró un significativo daño al ADN en ambos órganos con respecto al valor del testigo. En el caso del DD se observó significancia estadística para las dos dosis en todo el horario evaluado. CP fue genotóxico en el cerebro con la dosis más alta a las 72 h y en hepatopáncreas con las dos dosis, en todos los tiempos de exposición evaluados. También se detectó un significativo aumento de la peroxidación lipídica con las dos dosis de los insecticidas. En el estudio con el agua de la represa se detectó un daño más pronunciado en el ADN. Nuestros resultados mostraron un fuerte daño al ADN en Cambarellus montezumae por ambos insecticidas, así como una correlación con el efecto de la peroxidación lipídica, lo que sugiere que el estrés oxidativo está involucrado en la alteración genotóxica. Nuestros resultados también mostraron la utilidad del organismo estudiado y de las pruebas aplicadas para evaluar efectos tóxicos, y sugieren la pertinencia de aplicar el ensayo cometa en otros organismos de agua dulce para evaluar la bioacumulación de insecticidas.

Neurotoxicidade de pesticidas organofosforados durante o desenvolvimento: alterações bioquímicas e comportamentais / Neurotoxicity of organophosphorus pesticides during development: behavioral and biochemical changes

Pesticidas organofosforados são amplamente usados e seu uso constitui um grave problema de saúde pública. A ação clássica destes compostos é a inibição irreversível da acetilcolinesterase, promovendo acúmulo de acetilcolina nas sinapses e hiperestimulação colinérgica. No entanto, as consequências da exposição a baixas doses podem se estender a outros mecanismos de ação e sistemas neurotransmissores. Considerando que crianças constituem um grupo particularmente vulnerável aos efeitos de pesticidas, neste trabalho investigamos os efeitos da exposição aos organofosforados metamidofós (MET) e clorpirifós (CPF) durante o desenvolvimento sobre os sistemas colinérgico e serotoninérgico e sobre o comportamento de camundongos. Para isso, camundongos suíços foram expostos a injeções subcutâneas de MET, clorpirifós ou veículo do terceiro (PN3) ao nono (PN9) dias de vida pós-natal. As doses de exposição foram previamente escolhidas através da construção de uma curva dose-resposta que identificou como mais adequadas para este estudo as doses de 1mg/kg de MET e 3mg/kg de CPF, as quais promoveram em torno de 20% de inibição da acetilcolinesterase. Em PN10, parte dos animais foi sacrificada e foram avaliados os sistemas colinérgico e serotoninérgico no tronco encefálico e córtex cerebral. De PN60 a PN63, os animais foram submetidos a uma bateria de testes comportamentais. Em seguida, estes animais também foram sacrificados tendo sido avaliados os sistemas colinérgico e serotoninérgico. Em PN10, MET e CPF causaram alterações que sugerem aumento da atividade colinérgica respectivamente no tronco e córtex em fêmeas. No sistema serotoninérgico, apenas CPF promoveu alterações, aumentando a ligação ao receptor 5HT1A e transportador 5HT em fêmeas e diminuindo na ligação ao 5HT2. Em PN63, a atividade da acetilcolinesterase foi reestabelecida em todos os grupos. Ainda assim, MET diminuiu a atividade da colina acetiltransferase no córtex e a ligação ao transportador colinérgico.

Organophosphate pesticides are widely used and its use consist on a severe public health problem. The classic effect of these compounds involve irreversible inhibition of the enzyme acetylcholinesterase, causing an accumulation of acetylcholine at cholinergic synapses and, consequently, cholinergic hyperstimulation. However, when the doses of exposure are low, other the mechanisms of action may play a role and other neurotransmitter systems may be affected. Considering that children are particularly vulnerable to effects of these compounds, in this study we investigated the effects of methamidophos and chlorpyrifos organophosphate exposure during development on cholinergic and serotonergic systems and behavior. For this purpose, Swiss mice received subcutaneous injections of methamidophos or chlorpyrifos, or vehicle from the third to the nineth postnatal day (PN3 - PN9). Initially, a dose-response study was performed and the doses of 1mg/kg methamidophos and 3mg/kg chlorphrifos, which promoted 20% inhibition of acetylcholinesterase activity in brain were chosen to be used in the next set of experiments. At PN10, one day after exposure, a group of animals was sacrificed and the brainstem and cortex collected and stored to further analysis of cholinergic and serotonergic systems. From PN60 to PN63 the animals were submitted to behavioral tests in order to evaluate: anxiety, locomotor activity, decision making, depressive-like behavior and learning/memory. After the last test, the animals were sacrificed and the brainstem and cortex collected and stored to further analysis of cholinergic and serotonergic systems. At PN10, methamidophos and chlorpyrifos promoted alterations that suggest an increase of cholinergic activity respectively on the brainstem and cortex of females. As for the serotonergic system: only chlorpyrifos elicited alterations: There were increases in 5HT1A receptor and 5HT transporter binding in females and a decrease in 5HT2 receptor binding.

Biochemical changes induced by chlorpyrifos, an organophosphate compound in sublethal concentrations to the freshwater fish Catla catla, Labeo rohita and Cirrhinus mrigala.

Freshwater fishes, Catla catla, Labeo rohita and Cirrhinus mrigala were exposed to sublethal concentration of the pesticide chlorpyrifos an organophosphate pesticide. Biochemical changes in total glycogen, total proteins, lactate dehydrogenase (LDH), alanine amino transferase (ALAT) and aspartate amino transferase (AAT) and nucleic acids, Deoxy and ribose, were estimated in the tissues of fishes exposed to the toxicant chlorpyrifos technical grade. The decrease in total glycogen and proteins over control and increase in selected enzymes, LDH, AAT, ALAT, DNA and RNA values were observed in the three fishes exposed to the toxicant.

Evaluation of the mutagenic potential of chlorpyrifos (CPF) using polytene chromosomes of Anopheles mosquito.

Diverse cytogenetic tests are employed for short term screening of suspect environmental mutagens by using insects and mammals as models. In the present paper the polytene chromosomes of a mosquito Anopheles maculatus were used to evaluate the mutagenic potential of a widely used organophosphate pesticide chlorpyrifos-[o, o-diethyl-o-(3, 5, 6-trichloro-2-pyridyl) phosphothioate]. The results are based on the frequency of various structural aberrations encountered in the polytene chromosomes of the larvae treated with LC20 of chlorpyrifos (CPF). These aberrations were dominated by inversions, stickiness of the chromosomes, heterochromatinization of the bands and lack of polyteny. The frequency of various aberrations was highest in the left arm of chromosome number 2L followed by 2R, 3L, 3R, and X-chromosomes i.e. 2.10 +/- 0.44, 1.84 +/- 0.44, 1.57 +/- 0.54, 1.31 +/- 0.50, and 0.22 +/- 0.27 respectively. The susceptibility of different chromosomal arms to this pesticide was 2L > 2R > 3L > 3R > X and the regions prone to these aberrations have been marked on the polytene chromosome map of Anopheles maculatus.

The effect of Chlorpyrifos, an organophosphate in acetyl cholinesterase activity in freshwater fishes.

The freshwater fish Catla catla, Labeo rohita and Cirrhinus mrigala are exposed to the toxicant chlorpyrifos, an organo phosphate commonly used in agriculture as well as in aquaculture. The effect was studied on the enzyme acetyl cholinesterase activity which was inhibited in the selected tissues of the fish. In different tissues and in brain, the maximum inhibition was observed.

Comparative effects of some insecticides as technical and formulated on male albino rats

The present study describes the effects of certain technical and formulated forms of chlorpyrifos, cypermethrin and deltamethrin on some haematological and biochemical parameters in rats. The tested insecticides were orally administered in single and repetitive doses. In single dose treatments, haemoglobin [Hb], RBCs and WBC's were highly affected [p

Genotoxicidad de tres plaguicidas utilizados en la actividad bananera de Costa Rica / Genotoxicity of three pesticides used in Costa Rican banana plantations

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.