BACKGROUND: As research progresses, there is a growing body of evidence indicating that urinary metallothionein (MT) levels may be elevated in individuals exposed to cadmium (Cd). This study aimed to investigate the potential association between urinary MT levels and causes of mortality among residents of the Kakehashi River Basin who have been exposed to Cd. METHOD: The study involved a total of 1,398 men and 1,731 women were conducted between 1981 and 1982, with follow-up until November 2016. The study employed the Cox proportional-hazards model to examine the association between higher urinary MT concentrations and the risk of all-cause or cause-specific mortality within the population. Furthermore, the Fine and Gray competing risks regression model was used to evaluate the links between specific causes of death. RESULTS: The findings revealed that elevated urinary MT concentrations were linked to increased all-cause mortality and higher mortality rates from renal and urinary tract diseases across all participants. Specifically, in men, higher urinary MT levels were associated with elevated all-cause mortality, while in women, increased concentrations were linked to higher mortality from endocrine, nutritional, and metabolic diseases, as well as cardiovascular diseases. Even after adjusting for competing risks, higher urinary MT concentrations were associated with tumor-related mortality in men and continued to be associated with cardiovascular disease mortality in women. CONCLUSIONS In conclusion, the results suggest that women may face a greater risk of adverse health effects due to prolonged exposure to Cd. Urinary MT levels could potentially serve as a biomarker for mortality from these diseases in populations chronically exposed to Cd.
Humans ; Male ; Female ; Cadmium/urine* ; Japan/epidemiology* ; Metallothionein/metabolism* ; Middle Aged ; Cause of Death ; Adult ; Follow-Up Studies ; Aged ; Environmental Exposure/analysis* ; Proportional Hazards Models

OBJECTIVE: Aloin, the main active component in Aloe vera (L.) Burm. f., has shown promising anti-tumor effects. This study investigated the impact of aloin in lung squamous cell carcinoma (LUSC) and explored its functional mechanism. METHODS: We analyzed the viability, migration, invasion, proliferation, and apoptosis of two LUSC cell lines after treatment with aloin. Target molecules of aloin and downstream target transcripts of nuclear receptor subfamily 3 group C member 2 (NR3C2) were predicted by bioinformatics. The biological functions of NR3C2 and metallothionein 1 M (MT1M) in the malignant properties of LUSC cells were determined. A co-culture system of LUSC cells with monocyte-derived macrophages was constructed. Mouse xenograft tumor models were generated to analyze the functions of aloin and NR3C2 in the tumorigenic activity of LUSC cells and macrophage polarization in vivo. RESULTS: Aloin suppressed malignant properties of LUSC cells in vitro. However, these effects were negated by the silencing of NR3C2. NR3C2 was found to activate MT1M transcription by binding to its promoter. Additional upregulation of MT1M suppressed the malignant behavior of LUSC cells augmented by NR3C2 silencing. Analysis of the M1 and M2 markers/cytokines in the macrophages or the culture supernatant revealed that aloin treatment or MT1M overexpression in LUSC cells enhanced M1 polarization while suppressing M2 polarization of macrophages, whereas NR3C2 silencing led to reverse trends. Consistent findings were reproduced in vivo. CONCLUSION This study demonstrated that aloin activates the NR3C2/MT1M axis to suppress the malignant behavior of LUSC cells and M2 macrophage polarization. Please cite this article as: Chen YN, Lu JY, Gao CF, Fang ZR, Zhou Y. Aloin blocks the malignant behavior of lung squamous cell carcinoma cells and M2 macrophage polarization by modulating the NR3C2/MT1M axis. J Integr Med. 2025; 23(2): 195-208.
Lung Neoplasms/metabolism* ; Humans ; Animals ; Cell Line, Tumor ; Carcinoma, Squamous Cell/metabolism* ; Mice ; Macrophages/drug effects* ; Emodin/analogs & derivatives* ; Metallothionein/genetics* ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Receptors, Glucocorticoid/genetics*

Cadmium/metabolism* ; Kidney ; Cells, Cultured ; Mitochondria ; Metallothionein/metabolism* ; Liver

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.
Metallothionein/metabolism* ; Elastin/chemistry* ; Recombinant Fusion Proteins/pharmacology* ; Mice ; Animals ; Peptides/metabolism* ; Escherichia coli/metabolism* ; NIH 3T3 Cells

OBJECTIVES: To investigate the time-dependent expression of metallothionein （MT） 1A mRNA and MT2A mRNA in contused skeletal muscle of rats. METHODS: A total of 54 Sprague-Dawley rats were used in this study. The rats were divided into two parts: control group （n=6） and contusion groups （0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion, n=6）. Total RNA was extracted from skeletal muscle. The expression levels of MT1A mRNA and MT2A mRNA were detected by SYBR Green I real-time PCR. RESULTS: The expression trends of the two potential marker genes were related to wound age. In addition to 0.5 h, there were significant contrasts between the control group and contused group （P<0.05）, about the expression levels of MT1A mRNA and MT2A mRNA in different phases. As the extension of wound age, the relative expression of MT1A mRNA and MT2A mRNA at 1 h, 6 h, 12 h and 18 h after contusion demonstrated upgrade tendency until its expression levels in 18 h peak with 239.41±15.20 and 717.42±50.76, respectively. When time extends to 24 h after injury, the expression of above two marks decreased, respectively. The MT1A mRNA and MT2A mRNA expression levels increased at 30 h and then decreased. CONCLUSIONS Determination of MT1A mRNA and MT2A mRNA levels by real-time PCR may be useful for the estimation of wound age.
Animals ; Contusions/pathology* ; Gene Expression Regulation ; Genetic Markers ; Metallothionein ; Muscle, Skeletal/metabolism* ; RNA, Messenger/metabolism* ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Time Factors ; Wound Healing

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.
Animals ; B-Lymphocytes ; Brain Neoplasms* ; Brain* ; Cell Line ; DNA, Complementary ; Drug Therapy ; Glioma* ; Humans ; Male ; Matrix Metalloproteinases ; Metallothionein ; Mice* ; Mice, Nude ; Nestin ; RNA

To evaluate the effect of diet on metabolic control and zinc metabolism in patients with type 2 diabetes mellitus (T2DM). One-week balanced diet was provided to 10 Brazilians patients with T2DM. Nutritional assessment, laboratorial parameters and expression of zinc transporter and inflammatory genes in peripheral blood mononuclear cells (PBMC) were performed. Healthy non-diabetic subjects of the same demographic were recruited to provide baseline data. Diabetic patients had higher body mass index and greater fasting plasma glucose, plasma tumor necrosis factor alpha (TNFalpha) and plasma interleukin 6 (IL6) levels compared with healthy subjects. In addition, the expression of transporters 4 (ZnT4) mRNA was lower and IL6 mRNA was higher in PBMC of these diabetic patients than in healthy subject. One week after a balanced diet was provided, fasting plasma glucose decreased significantly as did TNFalpha, IL6 and Metallothionein 1 (MT1) mRNAs. No change was observed in zinc transporter expression in PBMC after the dietary intervention. A healthy eating pattern maintained for one week was able to improve metabolic control of diabetic patients by lowering fasting plasma glucose. This metabolic control may be related to down-regulation of zinc-related transcripts from PBMCs, as TNFalpha, IL6 and MT1 mRNA.
Blood Glucose ; Body Mass Index ; Diabetes Mellitus, Type 2 ; Diet* ; Down-Regulation ; Eating ; Fasting ; Humans ; Inflammation* ; Interleukin-6 ; Metabolism* ; Metallothionein ; Nutrigenomics ; Nutrition Assessment ; Plasma ; RNA, Messenger ; Tumor Necrosis Factor-alpha ; Zinc*

BACKGROUND: Reactive oxygen species (ROS) and antioxidants are associated with maintenance of cellular function and metabolism. Nuclear factor-E2-related factor 1 (NFE2L1, Nrf1) is known to regulate the expression of a number of genes involved in oxidative stress and inflammation. The purpose of this study was to examine the effects of NFE2L1 on the response to oxidative stress in osteoblastic MC3T3-E1 cells. METHODS: The murine calvaria-derived MC3T3-E1 cell line was exposed to lipopolysaccharide (LPS) for oxidative stress induction. NFE2L1 effects were evaluated using small interfering RNA (siRNA) for NFE2L1 mRNA. ROS generation and the levels of known antioxidant enzyme genes were assayed. RESULTS: NFE2L1 expression was significantly increased 2.4-fold compared to the control group at 10 µg/mL LPS in MC3T3-E1 cells (P<0.05). LPS increased formation of intracellular ROS in MC3T3-E1 cells. NFE2L1 knockdown led to an additional increase of ROS (20%) in the group transfected with NFE2L1 siRNA compared with the control group under LPS stimulation (P<0.05). RNA interference of NFE2L1 suppressed the expression of antioxidant genes including metallothionein 2, glutamatecysteine ligase catalytic subunit, and glutathione peroxidase 1 in LPS-treated MC3T3-E1 cells. CONCLUSION: Our results suggest that NFE2L1 may have a distinct role in the regulation of antioxidant enzymes under inflammation-induced oxidative stress in MC3T3-E1 osteoblastic cells.
Antioxidants ; Catalytic Domain ; Cell Line ; Glutathione Peroxidase ; Inflammation ; Metabolism ; Metallothionein ; NF-E2-Related Factor 1 ; Osteoblasts* ; Oxidative Stress* ; Reactive Oxygen Species ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering

BACKGROUND: Increased oxidative stress has been suggested as one of the underlying mechanisms in iodide excess-induced thyroid disease. Metallothioneins (MTs) are regarded as scavengers of reactive oxygen species (ROS) in oxidative stress. Our aim is to investigate the effects of propylthiouracil (PTU), a thyroid peroxidase inhibitor, perchlorate (KClO4), a competitive inhibitor of iodide transport, and thyroid stimulating hormone (TSH) on mitochondrial superoxide production instigated by high concentrations of iodide in the thyroids of MT-I/II knockout (MT-I/II KO) mice. METHODS: Eight-week-old 129S7/SvEvBrd-Mt1(tm1Bri) Mt2(tm1Bri)/J (MT-I/II KO) mice and background-matched wild type (WT) mice were used. RESULTS: By using a mitochondrial superoxide indicator (MitoSOX Red), lactate dehydrogenase (LDH) release, and methyl thiazolyl tetrazolium (MTT) assay, we demonstrated that the decreased relative viability and increased LDH release and mitochondrial superoxide production induced by potassium iodide (100 µM) can be relieved by 300 µM PTU, 30 µM KClO4, or 10 U/L TSH in the thyroid cell suspensions of both MT-I/II KO and WT mice (P<0.05). Compared to the WT mice, a significant decrease in the relative viability along with a significant increase in LDH release and mitochondrial superoxide production were detected in MT-I/II KO mice(P<0.05). CONCLUSION: We concluded that PTU, KClO4, or TSH relieved the mitochondrial oxidative stress induced by high concentrations of iodide in the thyroids of both MT-I/II KO and WT mice. MT-I/II showed antioxidant effects against high concentrations of iodide-induced mitochondrial superoxide production in the thyroid.
Animals ; Antioxidants ; Iodide Peroxidase ; Iodides ; L-Lactate Dehydrogenase ; Metallothionein* ; Mice ; Mice, Knockout* ; Oxidative Stress ; Potassium Iodide ; Propylthiouracil* ; Reactive Oxygen Species ; Superoxides* ; Suspensions ; Thyroid Diseases ; Thyroid Gland* ; Thyrotropin*

OBJECTIVETo investigate the role of holoenzyme containing Protein Phosphatase 2A B56β in regulating CdCl2 induced cytotoxicity.

METHODCdCl2-induced cytotoxicity in normal human cell line L-02, AFB1-transformed hepatic cell line L-02 RT-AFB1 and tumor cell line Bel7402 was measured by modified MTT assay. Stable cell lines L-02 SHAKT, L-02 SHB56β, L-02 RT-AFB1-B56β and Bel7402-B56β were generated by infecting L-02 cells or Bel7402 cells with retroviral vectors encoding lentiviral AKT shRNA, lentiviral B56β shRNA and B56β. The relative cell viability was measured in normal human cell line AFB1-transformed hepatic cell line and tumor cell line when treated by CdCl2 (0, 20, 40, 80, 160 µmol/L). After treated by wortmannin (2.5, 5.0 µmol/L) combined with 40 µmol/L CdCl2, Western blot was applied to measure the expression of associated protein in L-02.Western blot was applied to measure the expression of B56β, MT (metallothionein), AKT, and p-AKT in these cell lines treated by CdCl2.

RESULTSThe levels of MT were 0.12 ± 0.02, 0.06 ± 0.06 in L-02 RT-AFB1 and Bel7402, which were lower than L02 (0.92 ± 0.14) (F = 1 148.16, P < 0.001) when treated by 40 µmol/L CdCl2. When treated by 40 µmol/L CdCl2, the expression of p-AKT in L-02 SHAKT-1 and L-02 SHAKT-2 were 0.08 ± 0.02, 0.08 ± 0.05, which levels were lower than L-02 SHGFP (0.18 ± 0.15) (F = 724.70, P < 0.001); and the expression of MT were both 0.62 ± 0.16 in L-02 SHAKT-1 and L-02 SHAKT-2, which levels were higher than L-02 SHGFP (0.22 ± 0.14) (F = 94.73, P < 0.001). After treated by wortmannin (2.5, 5.0 µmol/L) combined with 40 µmol/L CdCl2, the expression of p-AKT in L-02 were 0.28 ± 0.07, 0.15 ± 0.11, which levels were lower than wortmannin untreated cells (0.52 ± 0.11) (F = 578.57, P < 0.001); and the expreesion of MT were 1.62 ± 0.80, 1.08 ± 0.15, which levels were higher than wortmannin untreated cells (0.69 ± 0.18) (F = 12.34, P < 0.001). When treated by 40 µmol/L CdCl2, the levels of p-AKT in L-02 SHB56β-1 and L-02 SHB56β-2 were 0.57 ± 0.13, 0.59 ± 0.02, which were higher than L-02 SHGFP (0.32 ± 0.02) (F = 87.16, P < 0.001); and the levels of MT were 0.35 ± 0.07, 0.20 ± 0.03 in L-02 SHB56β-1 and L-02 SHB56β-2, which were lower than L-02 SHGFP (1.51 ± 0.13) (F = 2 457.10, P < 0.001). After treated by 40 µmol/L CdCl2, the expression of p-AKT in L-02 RT-AFB1-B56β and Bel7402-B56β were 0.10 ± 0.11, 0.09 ± 0.01, which were lower than L-02 RT-AFB1 (0.36 ± 0.01) and Bel7402 (0.43 ± 0.11) (F = 877.62, P < 0.001); and the levels of MT were 0.92 ± 0.13, 0.95 ± 0.08 in L-02 RT-AFB1-B56β and Bel7402-B56β,which were higher than L-02 RT-AFB1 (0.44 ± 0.12) and Bel7402 (0.77 ± 0.06) (F = 51.97, P < 0.001).

CONCLUSIONProtein phosphatase 2A complexes containing B56β participated in the regulation of MT expression through direct dephosphorylation of AKT, finally affected the cytotoxicity responding to CdCl2. Our study revealed a key signaling pathways of PP2A involved in heavy metals induced cytotoxicity.

Cadmium Chloride ; Cell Line ; Cell Line, Tumor ; Cell Survival ; Holoenzymes ; Humans ; Liver ; Metallothionein ; Metals, Heavy ; Protein Phosphatase 2 ; Signal Transduction