Effects of polyvinyl chloride and low-density polyethylene microplastics on oxidative stress and mitochondria function of earthworm (Eisenia fetida).

Plastics are widely used worldwide due to their convenience. However, microplastics (MPs) accumulation poses a serious threat to ecosystem health. Therefore, understanding the effects of MPs on living organisms within their native ecosystem is crucial. Previous studies have primarily focused on the impacts of MPs in aquatic environments, whereas the effects of MPs on terrestrial ecosystems have remained largely understudied. Therefore, our study assessed the impacts of MPs on soil ecosystems by characterizing their toxic effects on earthworms (Eisenia fetida). Here, we exposed earthworms to two representative plastics within soil environments: polyvinyl chloride (PVC) and low-density polyethylene (LDPE). Given the known link between MPs and oxidative stress, we next quantified oxidative stress markers and mitochondrial function to assess the effects of MPs on the redox metabolism of earthworms. Mitochondria are crucial metabolic organelles that generate reactive oxygen species via uncontrolled ATP production. Our findings demonstrated that MPs exert different effects depending on their type. Neither the PVC-exposed groups nor the LDPE-exposed groups exhibited changes in oxidative stress, as worked by the action of superoxide dismutase (SOD) and glutathione (GSH). While treatment of the two types of MP did not significantly affect the amount of reactive oxygen species/reactive nitrogen species (ROS/RNS) generated, PVC exhibited a more pronounced effect on antioxidant system compared to LDPE. However, mitochondrial function was markedly decreased in the group exposed to high LDPE concentrations, suggesting that the examined LDPE concentrations were too low to activate compensatory mechanisms. Collectively, our findings demonstrated that exposure of MPs not only influences the antioxidant defense mechanisms of earthworms but also alters their mitochondrial function depending on their types.

Persistent Organic Pollutants released from decomposed adipose tissue affect mitochondrial enzyme function in the brain and eyes other than the liver.

Persistent organic pollutants (POPs) are toxic chemicals that can accumulate in the human body, and particularly in adipose tissue. POPs can induce metabolic diseases via mitochondrial dysfunction and can also cause cancer, obesity, and cardiovascular and neurodegenerative diseases. Although the effects of POPs were studied by evaluating mitochondrial function, which is fundamental in investigating the etiologies of various metabolic diseases, the physiological impact of POPs released by the decomposition of fat in adipose tissue is barely understood. Therefore, to investigate the mitochondrial dysfunction caused by POPs released from adipose tissue to other organs, zebrafish were exposed to POPs and placed into four groups: control (C), obesity control (OC), obesity control with POPs (OP), and POP exposure with obesity and caloric restriction (OPR). Next, the activities of the mitochondrial respiratory complexes and the levels of ATP production, reactive oxygen species/reactive nitrogen species (ROS/RNS), and antioxidants, such as glutathione and superoxide dismutase, were measured in the brain, eyes, and liver, as these are the major organs most susceptible to metabolic diseases. POPs released from adipose tissue showed a stronger effect than the direct effects of obesity and POPs on mitochondrial enzyme activity in the brain and eye. Released POPs increased mitochondrial complex I activity and decreased mitochondrial complex II activity compared with normal, obesity, and POP-treated conditions in the brain and eyes. However, the mitochondrial complexes' activities in the liver were affected more by obesity and POPs. In the liver, the mitochondrial enzyme activities of the OPR group seemed to recover to the control level, but it was slightly lowered in the OC and OP groups. Independently, the ROS/RNS and antioxidant levels were not affected by obesity, POPs, or the released POPs in the brain, eye, and liver. The results indicate that POPs stored in adipose tissue and released during fat decomposition did not affect oxidative stress but could affect mitochondrial respiratory enzymes in organ dependent manner. This study is meaningful in that it provides experimental evidence that stored POPs affect specific organs for prolonged periods and can be linked to various diseases in advance.

TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2.

BACKGROUND AND AIMS: Obesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. METHODS: The effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. RESULTS: TNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. CONCLUSIONS: This study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.

Differential mitochondrial dysregulation by exposure to individual organochlorine pesticides (OCPs) and their mixture in zebrafish embryos.

Organochlorine pesticides (OCPs) have been reported to cause mitochondrial dysfunction. However, most studies reported its mitochondrial toxicity with respect to a single form, which is far from the environmentally relevant conditions. In this study, we exposed zebrafish embryos to five OCPs: chlordane, heptachlor, p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), ß-hexachlorocyclohexane (ß-HCH), and hexachlorobenzene (HCB), as well as an equal ratio mixture of these OCPs. We evaluated mitochondrial function, including oxygen consumption, the activity of mitochondrial complexes, antioxidant reactions, and expression of genes involved in mitochondrial metabolism. Oxygen consumption rate was reduced by exposure to chlordane, and ß-HCH, linking to the increased activity of specific mitochondrial complex I and III, and decreased GSH level. We found that these mitochondrial dysfunctions were more significant in the exposure to the OCP mixture than the individual OCPs. On the mRNA transcription level, the individual OCPs mainly dysregulated the metabolic cycle (i.e., cs and acadm), whereas the OCP mixture disrupted the genes related to mitochondrial oxidative phosphorylation (i.e., sdha). Consequently, we demonstrate that the OCP mixture disrupts mitochondrial metabolism by a different molecular mechanism than the individual OCPs, which warrants further study to evaluate mitochondrial dysregulation by chronic exposure to the OCP mixture.

Tobacco etch virus (TEV) protease with multiple mutations to improve solubility and reduce self-cleavage exhibits enhanced enzymatic activity.

Tobacco etch virus (TEV) protease is a 27-kDa catalytic domain of the polyprotein nuclear inclusion a (NIa) in TEV, which recognizes the specific amino acid sequence ENLYFQG/S and cleaves between Q and G/S. Despite its substrate specificity, its use is limited by its autoinactivation through self-cleavage and poor solubility during purification. It was previously reported that T17S/N68D/I77V mutations improve the solubility and yield of TEV protease and S219 mutations provide protection against self-cleavage. In this study, we isolated TEV proteases with S219N and S219V mutations in the background of T17S, N68D, and I77V without the inclusion body, and measured their enzyme kinetics. The kcat of two isolated S219N and S219V mutants in the background of T17S, N68D, and I77V mutations was highly increased compared to that of the control, and S219N was twofold faster than S219V without Km change. This result indicates that combination of these mutations can further enhance TEV activity.

Bottom-line mechanism of organochlorine pesticides on mitochondria dysfunction linked with type 2 diabetes.

Environmental pollution by anthropogenic chemicals has become a considerable problem. Organochlorine pesticides (OCPs), a subclass of persistent organic pollutants, are used as insecticides and industrial chemicals. They are lipophilic and minimally degradable, and they easily accumulate in the environment and human body. Epidemiological studies have demonstrated that exposure to OCPs strongly correlates with the development of type 2 diabetes, which involves mitochondrial dysfunction. To clarify their effects, OCP mixtures (ß-hexachlorocyclohexane, heptachlor, hexachlorobenzene, 4,4'-DDT, and chlordane) were used to treat mitochondria from zebrafish livers. Results showed that as OCP concentrations increased, Ca2+ intake into the mitochondria rose, which increased the activity of mitochondrial complexes I, II, IV, and citrate synthase. Complex III yielded the opposite result because the OCP mixture mimicked decylubiquinol, a natural substrate of complex III. Our results reflect the actual state of toxins, non-monotonic, in the environment, which is important for determining the consequences of OCPs on mitochondrial dysfunction.

Steady-state kinetic mechanism of LodA, a novel cysteine tryptophylquinone-dependent oxidase.

LodA is a novel lysine-ε-oxidase which possesses a cysteine tryptophylquinone cofactor. It is the first tryptophylquinone enzyme known to function as an oxidase. A steady-state kinetic analysis shows that LodA obeys a ping-pong kinetic mechanism with values of kcat of 0.22±0.04 s(-1), Klysine of 3.2±0.5 µM and KO2 of 37.2±6.1 µM. The kcat exhibited a pH optimum at 7.5 while kcat/Klysine peaked at 7.0 and remained constant to pH 8.5. Alternative electron acceptors could not effectively substitute for O2 in the reaction. A mechanism for the reductive half reaction of LodA is proposed that is consistent with the ping-pong kinetics.