The impact of hydroxyapatite crystal structures and protein interactions on bone's mechanical properties.

Hydroxyapatite (HAP) constitutes the primary mineral component of bones, and its crystal structure, along with the surface interaction with proteins, significantly influences the outstanding mechanical properties of bone. This study focuses on natural hydroxyapatite, constructing a surface model with calcium vacancy defects. Employing a representative model of aspartic acid residues, we delve into the adsorption mechanism on the crystal surface and scrutinize the adsorption forms of amino acid residues on HAP and calcium-deficient hydroxyapatite (CDHA) surfaces. The research also explores the impact of different environments on adsorption energy. Furthermore, a simplified sandwich structure of crystal-polypeptide-crystal is presented, analyzing the distribution of amino acid residue adsorption sites on the crystal surface of the polypeptide fragment. This investigation aims to elucidate how the stick-slip mechanism of polypeptide molecules on the crystal surface influences the mechanical properties of the system. By uncovering the interface mechanical behavior between HAP and osteopontin peptides, this article offers valuable theoretical insights for the construction and biomimetic design of biocomposites.

ß -Cypermethrin promotes the adipogenesis of 3T3-L1 cells via inducing autophagy and shaping an adipogenesis-friendly microenvironment.

The toxicity of synthetic pyrethroids has garnered attention, and studies have revealed that pyrethroids promote fat accumulation and lead to obesity in mice. Nevertheless, the effect of ß-cypermethrin (ß-CYP) on adipogenesis and its underlying mechanism remains largely unknown. In this study, mouse embryo fibroblasts 3T3-L1 cells were exposed to ß-CYP, and the cell viability, intracellular reactive oxygen species (ROS) level, autophagy, and adipogenesis were assessed to investigate the roles of oxidative stress and autophagy in the toxic effects of ß-CYP on adipogenesis. The results demonstrated that treatment with 100 µΜ ß-CYP elevated the ROS level, decreased mitochondrion membrane potential, stimulated autophagy, and enhanced the adipogenesis induced by the mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. However, co-treatment with N-acetyl-L-cysteine partially blocked the abovementioned effects of ß-CYP in 3T3-L1 cells. In addition, co-treatment with rapamycin, an autophagy agonist, enhanced the inductive effect of ß-CYP on adipogenesis, whereas co-treatment with 3-methyladenine blocked the enhancement of adipogenesis caused by ß-CYP. Moreover, ß-CYP also altered the microenvironment of 3T3-L1 cells to an adipogenesis-friendly one by reducing the extracellular expression of miR-34a, suggesting that the culture media of ß-CYP-treated 3T3-L1 cells could shift macrophages to M2 type. Taken together, the data obtained in the present study demonstrated that ß-CYP promoted adipogenesis via oxidative stress-mediated autophagy disturbance, and it caused macrophage M2 polarization via the alteration of miR-34a level in the microenvironment. The study demonstrated the adipogenesis-promoting effect of ß-CYP and unveiled the potential mechanism.

Pesticides-induced energy metabolic disorders.

Metabolic disorders have become a heavy burden on society. Recently, through excessive use, pesticides have been found to be present in environmental matrixes and sometimes even accumulate in humans or other mammals through the food chain, which then causes health concerns. Evidence has indicated that pesticides have the potential to induce energy metabolic disorders by disturbing the physical process of energy absorption in the intestine and energy storage in the liver, adipose tissue and skeletal muscle in humans or other mammals. In addition, the homeostasis of energy regulation by the pancreas and immune cells is also affected by pesticides. These pesticide-induced disruptions ultimately cause abnormal levels of blood glucose and lipids, which in turn induce the development of related metabolic diseases, including overweight, underweight, insulin resistance and even diabetes. In this review, the results of previous studies focused on the induction of metabolic disorders by pesticides are summarized. We hope that this work will facilitate the discovery of a potential strategy for the treatment of diseases caused by pesticides.

ß-Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3-L1 Cell Maturation into Adipocytes.

Studies have elucidated that pyrethroids induce adipogenesis. It is also known that macrophages can affect the homeostasis of adipose tissue. However, whether and how the ß-cypermethrin (ß-CYP)-mediated inhibition of the macrophages affects adipogenesis remain unknown. To explore the effects of ß-CYP on adipogenesis through modulating the function of macrophages, 3T3-L1 cells, a preadipocyte cell line, were exposed to culture medium from either RAW 264.7 cells, a macrophage cell line (RM), or ß-CYP-treated RAW 264.7 cells (CRM). CRM decreased the inhibitory effects of RM treatment on cell proliferation and adipogenesis, as lipid accumulation, the CEBPA content, and Fasn and Acaca expression in 3T3-L1 cells were higher following CRM treatment than following RM treatment through the higher levels of the demethylated CEBPA promoter in 3T3-L1 cells. However, the medium from ß-CYP- and N-acetyl-L-cysteine-cotreated RAW 264.7 cells (CNRM) partially restored the inhibitory effects of RAW 264.7 cells on 3T3-L1 cells that had been reduced by CRM, indicating that ß-CYP might reduce the cytotoxicity and inhibitory effects of RAW 264.7 cells on the adipogenesis of 3T3-L1 cells through elevating ROS levels in RAW 264.7 cells. Moreover, exposure to ß-CYP downregulated the TNF-α secretion in RAW 264.7 cells. In conclusion, these data demonstrated that ß-CYP affected the function of RAW 264.7 cells, alleviating their inhibitory effects on adipogenesis and CEBPA demethylation in 3T3-L1 cells. ß-CYP might achieve these effects through downregulating the secretion of TNF-α via elevating ROS levels in RAW 264.7 cells. Our experiments provide a new perspective on the obesogenic effect of pyrethroids.

Tetrabromoethylcyclohexane (TBECH) exhibits immunotoxicity in murine macrophages.

Tetrabromoethylcyclohexane (TBECH) has been linked to endocrine disruption, hepatotoxicity, and reproductive toxicity. However, its immunotoxicity remains largely unknown. In the present study, RAW 264.7 cells, mouse macrophage cell line, were exposed to TBECH. MTT assays showed that TBECH significantly enhanced lactate dehydrogenase (LDH) release in RAW 264.7 cells. The mRNA expression of some proapoptotic genes was upregulated by TBECH. Accordingly, TBECH elevated caspase-3 activity. In addition, TBECH upregualted the mRNA levels of some pro-inflammatory cytokines, whereas it downregulated LPS-stimulated mRNA expression of these cytokines. Moreover, TBECH downregulated the mRNA expression of selected antigen presenting-related genes. Furthermore, TBECH increased reactive oxygen species level, reduced glutathione content and the activities of superoxide dismutase and catalase, and upregulated the mRNA expression of selected oxidative stress-related genes. The obtained data demonstrated that TBECH exhibits immunotoxicity in macrophages, and will help to evaluate its health risks.

Environmentally relevant doses of tetrabromobisphenol A (TBBPA) cause immunotoxicity in murine macrophages.

TBBPA is one of the main brominated flame retardants and is ubiquitous in the environment. TBBPA can directly encounter immune cells via the bloodstream, posing potential immunotoxicity. To understand the immunomodulating effect of TBBPA on macrophages, the murine macrophages, RAW 264.7, were exposed to TBBPA at environmentally relevant concentrations (1-100 nM). The results showed that TBBPA at the selected concentrations did not alter cell viability of RAW 264.7 cells with or without LPS stimulation. TBBPA upregulated the expression of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, whereas it attenuated the LPS-stimulated expression of these pro-inflammatory cytokines, and the expression of anti-inflammatory cytokines, including IL-4, IL-10, and IL-13. In addition, TBBPA reduced the mRNA levels of antigen-presenting-related genes, including H2-K2, H2-Aa, Cd80, and Cd86. Moreover, TBBPA impaired the phagocytic activity of macrophages. Furthermore, exposure to TBBPA significantly elevated the protein levels of phosphorylated NF-κB p65 (p-p65), while it reduced LPS-stimulated p-p65 protein levels. DCFH-DA staining assays showed that TBBPA caused a slight but significant elevation in reactive oxygen species levels. The data obtained in the present study demonstrated that exposure to environmentally relevant concentrations of TBBPA posed immunotoxicity in macrophages and unveiled a potential health risk of TBBPA.

C9-13 chlorinated paraffins cause immunomodulatory effects in adult C57BL/6 mice.

Short-chain chlorinated paraffins (SCCPs, C10-13) were listed as persistent organic pollutants (POPs) by the Stockholm Convention in 2017 and pose extensive exposure risks to humans. To our knowledge, there have been no studies reporting the immmunomodulatory effects of SCCPs until now. C9-CPs have also been shown to be present in the environment. In this study, adult male C57BL/6 mice were exposed to 1, 10, or 100 mg/kg/d C9-13-CPs by gavage for 28 d. The results showed that compared to those of the controls, exposure to C9-13-CPs led to increased spleen weight, delimited germinal centers, enhanced energy metabolism, and elevated glutathione content, but no variation in the malonaldehyde level in the spleen was observed. Exposure to C9-13-CPs also increased the populations of splenic lymphocytes, T lymphocytes, NK cells, and the ratio of the CD3+/CD19+ subsets and CD4+/CD8+ subsets compared to those of the controls. RNA-seq revealed 424 differentially expressed genes (DEGs) (fold change ≥ 1.5, FDR < 0.05) in the spleen between the control group and the 100 mg/kg/d C9-13-CPs-treated group. KEGG analysis demonstrated that folate biosynthesis, pathways in cancer and thyroid hormone signaling were the three most significantly enriched pathways, and despite not reaching statistical significance, some immune-related pathways were also enriched in the KEGG functional enrichment analysis, including the chemokine signaling pathway (FDR < 0.0584), the NF-κB signaling pathway (FDR < 0.0663), Th17 cell differentiation (FDR < 0.0839), and the Jak-STAT signaling pathway. Moreover, compared to those of the controls, exposure to C9-13-CPs enhanced the Concanavalin A (Con A)-stimulated cultured splenocyte proliferation, while the exposure showed no effect on the splenocyte proliferation that was stimulated by lipopolysaccharides (LPS). Taken together, these results demonstrated that subacute exposure to C9-13-CPs could have immunomodulatory effects in mice. The present study helps to provide an understanding of the comprehensive health risks posed by C9-13-CPs.

Autophagy protects murine macrophages from ß-cypermethrin-induced mitochondrial dysfunction and cytotoxicity via the reduction of oxidation stress.

The immunotoxicity of synthetic pyrethroid (SPs) has garnered much attention, and our previous research demonstrated that ß-CYP causes immunotoxicity and oxidative stress in macrophages. Nevertheless, the underlying mechanism remains largely unknown. In this study, the murine macrophage RAW 264.7 cells and murine peritoneal macrophages (PMs) were exposed to ß-CYP. The results showed that ß-CYP elevated intracellular ROS levels in both RAW 264.7 cells and PMs. Exposure to ß-CYP also caused mitochondrial dysfunction with reduced mitochondrial membrane potential (MMP), intracellular ATP level and mitochondrial DNA (mtDNA) content in the two cell types. In addition, exposure of RAW 264.7 cells to ß-CYP for 12 h and 24 h enhanced autophagy, with elevated Beclin1, Rab7, Lamp1 and LC3-II expression levels, while 48 h of exposure attenuated autophagy. In contrast, exposure of PMs to ß-CYP for 12 h promoted autophagy, whereas exposure for 24 h and 48 h impaired autophagy. Cotreatment with an antioxidant, N-acetyl-L-cysteine (NAC), partially blocked the reduced MMP, intracellular ATP level and autophagy disturbance. Moreover, cotreatment with an autophagy agonist, rapamycin (RAPA), partially blocked mitochondrial dysfunction and oxidative stress in the two cell types, whereas cotreatment with an autophagy inhibitor, 3-methyladenine (3-MA), augmented the abovementioned toxic effects. Furthermore, mitochondrial ROS levels in both RAW 264.7 cells and PMs were elevated by exposure to ß-CYP, and molecular docking showed that ß-CYP docked with mouse respiratory chain complex I by binding to the ND2, ND4, and ND5 subunits of the protein complex. Taken together, the data obtained in the present study demonstrate that oxidative stress partially mediates mitochondrial dysfunction and autophagy disturbance upon exposure to ß-CYP in macrophages, and autophagy plays a protective role against the toxic effects.

Evaluation of development, locomotor behavior, oxidative stress, immune responses and apoptosis in developing zebrafish (Danio rerio) exposed to TBECH (tetrabromoethylcyclohexane).

Tetrabromoethylcyclohexane (TBECH), as one emerging brominated flame retardants, is ubiquitous in the environment, including water and aquatic organisms. TBECH was found to exhibit endocrine-disrupting effects in different models, whereas a survey of comprehensive toxic effects of TBECH on zebrafish is limited. In the present study, zebrafish (Danio rerio) were waterborne exposed continuously to TBECH from embryonic stage (3 h post-fertilization (hpf)) to the time when the respective parameters were evaluated. Exposure to TBECH reduced hatchability of zebrafish embryos at 72 and 96 hpf, diminished heart rate of zebrafish larvae at 48 hpf, and increased malformation in zebrafish larvae at 96 hpf. In addition, exposure to TBECH diminished free swimming distance both in the light and under a photoperiod of 10 min light/10 min dark cycles in zebrafish larvae at 6 days post-fertilization (dpf). Moreover, exposure to TBECH elevated activities of superoxide dismutase (SOD) and catalase (CAT), malondialdehyde (MDA) content, whereas it reduced glutathione (GSH) content, in zebrafish larvae at 6 dpf. Accordingly, RT-qPCR analysis demonstrated that TBECH exposure increased the mRNA levels of sod1, sod2, cat, and gpx1 in zebrafish larvae at 6 dpf. With respect to the immune aspect, the mRNA levels of pro-inflammatory genes, including il-1b, il-6, il-8, and tnfa, in larval zebrafish at 6 dpf were increased by exposure to TBECH, while pretreatment with TBECH inhibited 24 h of exposure to LPS-stimulated elevation in the mRNA levels of the abovementioned four pro-inflammatory genes in zebrafish larvae at 6 dpf. Furthermore, TBECH treatment increased caspase-3 enzyme activities and regulated apoptosis-related genes in larval zebrafish at 6 dpf. Taken together, the data obtained in this study demonstrated that TBECH caused developmental and locomotor behavioral toxicity, immunotoxicity, oxidative stress and proapoptotic effects in early life zebrafish. The present study will help to understand the comprehensive toxicity of TBECH in zebrafish.

8:2 fluorotelomer alcohol inhibited proliferation and disturbed the expression of pro-inflammatory cytokines and antigen-presenting genes in murine macrophages.

Fluorotelomer alcohols (FTOHs, F(CF2)nCH2CH2OH) are members of per- and polyfluoroalkyl substances (PFASs) and are increasingly used in surfactant and polymer industries. FTOHs pose hepatotoxicity, nephrotoxicity and endocrine-disrupting risks. Nevertheless, there is limited research on the immunotoxic effects of FTOHs. In this study, we examined the immunotoxicity of 8:2 FTOH (n = 8) on murine macrophage cell line RAW 264.7. The results showed that 8:2 FTOH exposure reduced cell viability in dose- and time-dependent manners, inhibited cell proliferation and caused cell cycle arrest. Exposure to 8:2 FTOH downregulated the mRNA expression of some cell cycle-related genes, including Cdk4, Ccnd1, Ccne1, and p53, but also upregulated the mRNA expression of other cell cycle related genes, including Ccna2, p21, and p27. Additionally, exposure to 8:2 FTOH under unstimulated and LPS-stimulated conditions downregulated the mRNA expression of pro-inflammatory genes, including Il1b, Il6, Cxcl1, and Tnfa, and secreted levels of IL-6 and TNF-α. Treatment with 8:2 FTOH upregulated the mRNA expression of antigen-presenting-related genes, including H2-K1, H2-Ka, Cd80, and Cd86. The abovementioned immunotoxic effects caused by 8:2 FTOH in RAW 264.7 cells were partially or completely blocked by co-treatment with hydralazine hydrochloride (Hyd), a reactive carbonyl species (RCS) scavenger. However, exposure to 8:2 FTOH did not exhibit any effects on intracellular reactive oxygen species (ROS) level with or without LPS stimulation. Taken together, these results suggest that 8:2 FTOH may have immunotoxic effects on macrophages and RCS may underlie the responsible mechanism. The present study aids in understanding the health risks caused by FTOHs.

ß-Cypermethrin and its metabolite 3-phenoxybenzoic acid induce cytotoxicity and block granulocytic cell differentiation in HL-60 cells.

The most widely used type II pyrethroid is ß-cypermethrin (ß-CYP), and 3-phenoxybenzoic acid (3-PBA) is one of its primary metabolites. Although CYP has been shown to pose toxic effects in some immune cells, as of now the immunotoxicity of CYP on immune progenitor cells has not been well studied. In this study, we evaluated the immunotoxicity of ß-CYP and 3-PBA on the human promyelocytic leukemia cell line, HL-60. Both ß-CYP and 3-PBA reduced cell viability. In addition, both ß-CYP and 3-PBA stimulated the intrinsic apoptotic pathway in a dose- and time-dependent manner, while only ß-CYP induced cell cycle arrest in G1 stage. Moreover, exposure to ß-CYP and 3-PBA at 100 µM inhibited all-trans retinoic acid (ATRA)-induced mRNA expressions of the granulocytic differentiation-related genes, CD11b and CSF-3R. Furthermore, exposure to ß-CYP and 3-PBA resulted in a downregulation of the granulocytic differentiation promoting transcriptional factors, PU.1 and C/EBPε. Furthermore, we found that ß-CYP and 3-PBA exposure led to elevated levels of cellular reactive oxygen species (ROS), and that pretreatment with N-acetylcysteine (NAC) blocked the toxic effects caused by ß-CYP and 3-PBA. The results obtained in the present study provide evidence showing the immunotoxic effects of ß-CYP and 3-PBA on promyelocytic cells as well as its possible underlying mechanism.

ß-Cypermethrin and its metabolite 3-phenoxybenzoic acid exhibit immunotoxicity in murine macrophages.

ß-Cypermethrin (ß-CYP), one of most important pyrethroids, is widely used to control insects, and has been detected in organisms, including human. Pyrethroids have been shown to pose neurotoxicity, hepatotoxicity, endocrine disruption and reproductive risks in mammals. However, research in immunotoxicity of pyrethroids, especially their metabolites, is limited. A common metabolite of pyrethroids is 3-phenoxybenzoic acid (3-PBA) in mammals. Thus, in this study, we evaluated the immunotoxicity of ß-CYP and 3-PBA in mouse macrophages, RAW 264.7 cells. MTT assays showed that both ß-CYP and 3-PBA reduced cell viability in a concentration- and time-dependent manner. Flow cytometry with Annexin-V/PI staining demonstrated that both ß-CYP and 3-PBA induced RAW 264.7 cell apoptosis. Furthermore, our results also showed that N-acetylcysteine partially blocked ß-CYP- and 3-PBA-induced cytotoxicity and apoptosis. Intrinsic apoptotic pathway was stimulated by both ß-CYP and 3-PBA exposure. In addition, we found that ß-CYP and 3-PBA inhibited mRNA levels of pro-inflammatory cytokines with or without LPS stimulation. Phagocytosis assay showed that both ß-CYP and 3-PBA inhibited phagocytic ability of macrophages. Moreover, it was also found that both ß-CYP and 3-PBA increased reactive oxygen species (ROS) levels in RAW 264.7 cells. Accordingly, both ß-CYP and 3-PBA were found to regulate the mRNA levels of oxidative stress-related genes in RAW 264.7 cells. Taken together, the results obtained in this study demonstrated that ß-CYP and 3-PBA may have immunotoxic effect on macrophages and that elevated ROS may underlie the mechanism. The present study will help to understand the health risks caused by ß-CYP and other pyrethroids.

Cis-bifenthrin induces immunotoxicity in adolescent male C57BL/6 mice.

Bifenthrin (BF) is an important synthetic pyrethroid. Previous studies have demonstrated that cis-BF exhibits toxic effects on development, the neurological, reproductive and endocrine system. In this study, we evaluated the immunotoxicity caused by cis-BF in adolescent male C57BL/6 mice. Mice were exposed orally to 0, 5, 10, and 20 mg/kg/d for 3 weeks. The results showed that body weight, spleen weight, and splenic cellularity decreased in mice exposed to 20 mg/kg/d cis-BF. Additionally, we found that the mRNA levels of the pro-inflammatory factors IL-1ß, IL-6, CXCL-1, and TNF-α, in peritoneal macrophages, the spleen, and the thymus were inhibited in the cis-BF-treated groups. Moreover, MTT assays demonstrated that cis-BF inhibited splenocyte proliferation stimulated by LPS or Con A, as well as the secretion of IFN-γ on Con A stimulation. Collectively, the results of this study suggest that exposure to cis-BF has the potential to induce immunotoxicity in adolescent male C57BL/6 mice.

Cis-bifenthrin causes immunotoxicity in murine macrophages.

Synthetic pyrethroids (SPs) are commonly used insecticides that have been detected in mammals, including humans, indicating a potential threat to human health. Bifenthrin (BF), as well as other pyrethroids, has been shown to possess neurotoxic, reproductive, hepatotoxic and nephrotoxic potential in mammals. However, studies regarding the immunotoxicity of BF and its mechanism are limited. In this study, we aim to exam the immunotoxicity of cis-BF on the murine macrophage cell line, RAW 264.7. MTT assay results demonstrated that cis-BF exposure induced apoptosis in RAW 264.7 cells in a concentration-dependent manner. We found that the expression of p53 and caspase-3 was up-regulated, while the expression of Bcl-2 was down-regulated during cis-BF-induced apoptosis. In addition, we also found that cis-BF exposure caused oxidative stress in RAW 264.7 cells in a dose-dependent manner. Interestingly, cis-BF exposure was found to inhibit the increase in transcription levels of IL-1ß, IL-6 and TNF-α responding to LPS stimulation. We also found that the induced increase in IFN-ß mRNA levels upon Sendai virus infection was blocked with cis-BF exposure. Finally, we found that cis-BF exposure increased ROS levels and dysregulated mRNA levels of oxidative stress-related genes in RAW 264.7 cells. The present study elucidates the immunotoxicity effect of cis-BF on macrophages and its possible underlying mechanism. The results from this study support the necessity to evaluate immune dysfunction in the risk assessment of cis-BF exposure.