Min-Kyoung Paik

Chlorpyrifos induces NLRP3 inflammasome and pyroptosis/apoptosis via mitochondrial oxidative stress in human keratinocyte HaCaT cells.

Chlorpyrifos (CPF) has been widely used around the world as a pesticide for both agricultural and residential application. Although various studies have reported toxicity and health-related effects from CPF exposure, the molecular mechanism of CPF toxicity to skin has not been well-characterized. The present study determined the potential mechanism involved in skin toxicity of CPF using the HaCaT human skin keratinocyte cell line. After treating to HaCaT cells, CPF triggered reactive oxygen species (ROS) generation and mitochondrial oxidative stress. We focused on NLRP3 inflammasome, known to induce innate immune response. We used mitochondrial ROS (mROS) scavenger mitoTEMPO to demonstrate a role for mROS in NLRP3 inflammasome and programmed cell death induced by CPF. Our results showed that CPF provoked NLRP3 inflammasome and pyroptosis/apoptosis via an increase of mROS in HaCaT cells. This study proposes that CPF induces innate immune response and skin inflammation through activating the NLRP3 inflammasome in skin epithelial cells. CPF may lead to cutaneous disease conditions and antioxidants could be proposed for therapy against skin exposure to CPF.

Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells.

Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH‐SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN‐induced neuronal cell death. Treatment of SH‐SY5Y cells with FPN induced apoptosis via activation of caspase‐9 and ‐3, leading to nuclear condensation. In addition, FPN induced oxidative stress and increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Pretreatment of cells with meloxicam enhanced the viability of FPN‐exposed cells through attenuation of oxidative stress and inflammatory response. FPN activated mitogen activated protein kinase (MAPK) and inhibitors of MAPK abolished FPN‐induced COX‐2 expression. Meloxicam also attenuated FPN‐induced cell death by reducing MAPK‐mediated pro‐inflammatory factors. Furthermore, we observed both nuclear accumulation of p53 and enhanced levels of cytosolic p53 in a concentration‐dependent manner after FPN treatment. Pretreatment of cells with meloxicam blocked the translocation of p53 from the cytosol to the nucleus. Together, these data suggest that meloxicam may exert anti‐apoptotic effects against FPN‐induced cytotoxicity by both attenuating oxidative stress and inhibiting the inflammatory cascade via inactivation of MAPK and p53 signaling. Copyright

Comparison of International Guidelines of Dermal Absorption Tests Used in Pesticides Exposure Assessment for Operators

The number of farmers who have suffered from non-fatal acute pesticide poisoning has been reported to vary from 5.7% to 86.7% in South Korea since 1975. Absorption through the skin is the main route of exposure to pesticides for farmers who operate with them. Several in vitro tests using the skins of humans or animal and in vivo tests using laboratory animals are introduced for the assessment of human dermal absorption level of pesticides. The objective of this study is to evaluate and compare international guidelines and strategies of dermal absorption assessments and to propose unique approaches for applications into pesticide registration process in our situation. Until present in our situation, pesticide exposure level to operator is determined just using default value of 10 as for skin absorption ratio because of data shortage. Dermal absorption tests are requested to get exposure level of pesticides and to ultimately know the safety of pesticides for operators through the comparison with the value of AOEL. When the exposure level is higher than AOEL, the pesticide cannot be approved. We reviewed the skin absorption test guidelines recommended by OECD, EFSA and EPA. The EPA recommends assessment of skin absorption of pesticides for humans through the TPA which includes all the results of in vitro human and animal and animal in vivo skin absorption studies. OECD and EFSA, employ a tiered approach, which the requirement of further study depends on the results of the former stage study. OECD guidelines accept the analysis of pesticide level absorbed through skin without radioisotope when the recovery using the non-labeled method is within 80~120%. Various factors are reviewed in this study, including the origin of skin (gender, animal species and sites of skin), thickness, temperature and, etc., which can influence the integrity of results.

Study of Kidney Toxicity of Azadirachta Indica Extract for Oral Administration in Rats

BACKGROUND: Azadirachta indica has been widely used as environment-friendly organic materials because of its insecticidal properties. This study was carried out to investigate the acute toxicity and the subacute toxicity of Azadirachta indica extract(AIE) in rats. METHODS AND RESULTS: For the oral acute toxicity test, Sprague-Dawley rats were gavaged with 2.0 g/Kg bw of AIE. The value was greater than 2.0 g/Kg bw for both male and female rats. For the subacute toxicity study, rats were treated with AIE at doses of 0.5, 1.0, 2.0 mg/Kg bw once a day for 4 weeks(n=10 animals per each group). There were no significant changes in body weight, food intake and water consumption observed during the experimental duration. In addition, no difference of relative kidney weight was observed among all treated groups. Serum creatinine level in the AIE 2.0 g/Kg group increased significantly compared with that of control group in male rats, but serum blood urea nitrogen was significantly decreased in a dose-dependent manner (p

Evaluation of Dermal Absorption Rate of Pesticide Chlorpyrifos Using In Vitro Rat Dermal Tissue Model and Its Health Risk Assessment

All pesticides must be assessed strictly whether safe or not when agricultural operators are exposed to the pesticides in farmland. A pesticide is commonly regarded as safe when estimated dermal absorption amount is lower than the acceptable operators exposure level (AOEL). In this study, dermal absorption rate of chlorpyrifos, a widely used organophosphate insecticide, was investigated using rat dermal tissue model. Chlorpyrifos wettable powder solved in water (250, 500 and 2,500 ppm) was applied to freshly excised rat dermal slices (341~413 μm thickness) on static Franz diffusion cells at 32℃ for 6 hours. After exposure period of 6 hours, and then washing-at residual amount of chlorpyrifos was analyzed in dermal tissues, tape strips, washing solution, washing swabs of receptor bottles and receptor fluids at 1, 2, 4, 8 and 24 hours. Chlorpyrifos was only detected in dermal tissue but not found in receptor fluid at each concentration and time point, and the absorption rate of 250, 500 and 2,500 ppm was 2.36%, 1.96% and 1.69%, respectively. The estimated exposure level of chlorpyrifos was calculated as 0.012 mg/kg bw/day. The health risk for farmers in this condition is a level of concern because the estimated exposure level is 12 times higher than AOEL 0.001 mg/kg bw/day. However, actual health risk will be alleviated than estimated because absorbed chlorpyrifos is not permeated into internal body system and only retained in skin layer.

Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells.

Abstract As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.

Effects of Aqueous Azadirachta indica Extract on Hepatotoxicity in Rats

BACKGROUND: Azadirachta indica Extract(AIE) containing azadirachtin as active ingredient have been used worldwide as environment-friendly organic material having pest control properties. However, the extracts prepared with different solvent and from different plant site is very diverse and have different toxicity. METHODS AND RESULTS: In this study, the four week repeated oral dose toxicity test of aqueous AIE in Sprague-Dawley rats was carried out to investigate the toxic effect of liver, main toxicity target organ of AIE. The male and female rats were divided into 4 groups, respectively; control(0 g/Kg bw), low-dose group(0.5 g/Kg bw), middle-dose(1.0 g/Kg bw) and high-dose group(2.0 g/Kg bw). As a results, relative liver weight increased with dose-dependent of AIE(p<0.05). Serum LDH in all AIE-treated groups were significantly lower than the control in male rats(p<0.05). However, serum GOT and GPT were significantly increased in all male AIE-treated groups in male rats(p<0.05) and, in particular, increase of serum GPT in dose-dependent manner raise the possibility of liver damage. Even through serum GLU was increased significantly in high-dose group in male rats compared to control, there were no significant differences of urinary GLU among all groups(p<0.05). In addition, histopathological examination of the liver did not reveal any lesions in all AIE-treated groups. CONCLUSION: In conclusion, 4 weeks of the repeated oral administration of AIE 2.0 g/Kg to rats has resulted no toxic response in liver. Therefore, AIE was no indicated to have any toxic effect in the SD rats, when it was orally administrated below the dosage 2.0 g/Kg/day for 4weeks.

Genotoxicity of Environment-friendly Organic Materials of Plant Origin in the Micronucleus Test Using Chinese Hamster Lung Cells

Abstract BACKGROUND: Azadirachta Indica extract(AIE) and Sophorae radix extract(SRE) are widely used as environment-friendly organic materials of plant origin in South Korea. METHODS AND RESULTS: In this study, the in vitro micronucleus(vitMN) tests of two samples of AIE and SRE were conducted to evaluate their genotoxicity using the Chinese hamster lung(CHL) cell. This study was composed of two parts; cytochalasin B(cyto B) test and non-cyto B test. Mitomycin C and colchicine were used as positive controls. As a result, the incidence of micronucleus(MN) in all AIE and SRE treated groups increased in dose- dependent manner, but were less than 2.2% in 1,000 binucleated cells. In addition, there were no significant increases of MN incidence in all AIE and SRE treated groups, compared with the negative control group. CONCLUSION: Therefore, we suggest that AIE samples and SRE samples used in this study may have no genotoxicity in the in vitro micronucleus test using the CHL cells. In our previous study, we reported that AIE and SRE did not cause genotoxicity in Ames test. According to the genotoxicity battery system, we concluded that AIE and SRE used in this study have no genotoxic effects to humans.