LDL receptor-related protein 5 rs648438 polymorphism is associated with the risk of skeletal fluorosis.

To investigate the potential association between LRP5 rs648438 polymorphism and the risk of skeletal fluorosis (SF) was evaluated in a cross-sectional case-control study conducted in Shanxi, China, in 2019. A total of 973 individuals were enrolled in this study, in which cases and controls were 346 and 627, respectively. SF was diagnosed according to the standard WS/192-2008 (China). The LRP5 rs648438 was detected by the multiple PCR and sequencing. LRP5 rs648438 was found to follow a dominant genetic model using a web-based SNP-STATS software. Logistic regression analysis found that the TC/CC genotype of LRP5 rs648438 might be a protective factor for SF. When stratified by gender, this protective effect of TC/CC genotype in rs648438 was pronounced in males. There was an interaction between gender and rs648438 on risk of SF. Our study suggested that TC/CC genotype of rs648438 might be a protective factor for water-drinking-type skeletal fluorosis, especially in male participants.

ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes.

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 µmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 µmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 µmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

Association between arsenic (+3 oxidation state) methyltransferase gene polymorphisms and arsenic methylation capacity in rural residents of northern China: a cross-sectional study.

Arsenic is a toxic metal-like element. The toxic reaction of the body to arsenic is related to the ability of arsenic methylation metabolism. As the rate-limiting enzyme of arsenic methylation metabolism, the genetic single nucleotide polymorphisms (SNPs) of arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene are related to capacity of arsenic methylation. In this paper, we investigated the association of five SNPs (rs7085104, rs3740390, 3740393, rs10748835, and rs1046778) in AS3MT with arsenic methylation metabolizing using the data and samples from a cross-sectional case-control study of arsenic and Type 2 diabetes mellitus conducted in Shanxi, China. A total of 340 individuals were included in the study. Urinary total arsenic (tAs, µg/L) was detected by liquid chromatography-atomic fluorescence spectrometry (LC-AFS). According to "safety guidance value of urinary arsenic for population" as specified in WS/T665-2019 (China), participants were divided into the control group (tAs ≤ 32 µg/L, n = 172) and arsenic-exposed group (tAs > 32 µg/L, n = 168). iAs%, MMA%, and DMA% are as the indicator of arsenic methylation capacity. The genotypes of AS3MT SNPs were examined by Multiple PCR combined sequencing. Linear regression analysis showed that AG + GG genotype in rs7085104 was associated with decreased iAs% and increased DMA%. Moreover, AG + AA genotype in rs10748835 and TC + CC genotype in rs1046778 were associated with decreased iAs% and MMA% and increased DMA%. The interaction between rs7085104 and arsenic is associated with iAs% and DMA%. The interaction of rs3740390 and rs10748835 with arsenic is associated with iAs%. Haplotype CTAC (rs3740393-rs3740390-rs10748835-rs1046778) was associated with lower iAs% and higher DMA%, but this association disappeared after adjusting for age, gender, drink, smoking, BMI and tAs. Haplotype GCAC was associated with decreased MMA%. Our study provides additional support for revealing the factors influencing the metabolic capacity of arsenic methylation and might be helpful to identify the population susceptible to arsenic exposure through individualized screening in the future.

SIRT1/P53 pathway is involved in the Arsenic induced aerobic glycolysis in hepatocytes L-02 cells.

Arsenic is a known human carcinogen. Low doses of arsenic can induce cell proliferation, but the mechanism remains elusive. Aerobic glycolysis, also known as the Warburg effect, is one of the characteristics of tumour cells and rapidly proliferating cells. P53 is a tumour suppressor gene that has been shown to be a negative regulator of aerobic glycolysis. SIRT1 is a deacetylase that inhibits the function of P53. In this study, we found that P53 was involved in low dose of arsenic-induced aerobic glycolysis through regulating HK2 expression in L-02 cells. Moreover, SIRT1 not only inhibited P53 expression but also decreased the acetylation level of P53-K382 in arsenic-treated L-02 cells. Meanwhile, SIRT1 influenced the expression of HK2 and LDHA, which then promoted arsenic-induced glycolysis in L-02 cells. Therefore, our study demonstrated that the SIRT1/P53 pathway is involved in arsenic-induced glycolysis, thereby promoting cell proliferation, which provides theoretical basis for enriching the mechanism of arsenic carcinogenesis.

Malignant growth of arsenic-transformed cells depends on activated Akt induced by reactive oxygen species.

Arsenic is an identified carcinogen for humans.In this study, chronic exposure of human hepatocyte L-02 to low-doses of inorganic arsenic caused cell malignant proliferation. Meanwhile, compared with normal L-02 cells, arsenic-transformed malignant cells, L-02-As displayed more ROS and significantly higher Cyclin D1 expression as well as aerobic glycolysis. Moreover, Akt activation is followed by the upregulation of Cyclin D1 and HK2 expression in L-02-As cells, since inhibition of Akt activity by Ly294002 attenuated the colony formation in soft agar and decreased the levels of Cyclin D1 and HK2. In addition, scavenging of ROS by NAC resulted in a decreased expression of phospho-Akt, HK2 and Cyclin D1, and attenuates the ability of anchorage-independent growth ofL-02-As cells, suggested that ROS mediated the Akt activation in L-02-As cells. In summary, our results demonstrated that ROS contributes to the malignant phenotype of arsenic-transformed human hepatocyte L-02-As via the activation of Akt pathway.

The ROS/NF-κB/HK2 axis is involved in the arsenic-induced Warburg effect in human L-02 hepatocytes.

Arsenic has been identified as a carcinogen, although the molecular mechanism underlying itscarcinogenesis has not been fully elucidated. To date, only a few studies have attempted to confirm a direct link between oxidative stress and the Warburg effect . This study demonstrated that 0.2 µmol/L As3+ induced the Warburg effect to contribute to abnormal proliferation of L-02 cells, that was mediated by upregulation of hexokinase 2 (HK2), a key enzyme in glycolysis. Further study indicated that arsenic-induced accumulation of reactive oxygen species (ROS) activated the nuclear factor kappa B (NF-κB) signaling pathway by phosphorylation of p65 at the Ser536 and Ser276 sites, leading to upregulated expression of HK2. We therefore concluded that the ROS/NF-κB/HK2 axis contributes to the Warburg effect and cell proliferation induced by low doses of arsenic.AbbreviationsROS, Reactive oxygen species; NAC, N-acetyl-L-cysteine; 2-DG, 2-deoxy-D-glucose; 2-NBDG, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose.

Research for type 2 diabetes mellitus in endemic arsenism areas in central China: role of low level of arsenic exposure and KEAP1 rs11545829 polymorphism.

Type 2 diabetes mellitus (T2DM) is one of the major public health problems worldwide; both genetic and environmental factors are its risk factors. Arsenic, an environmental pollutant, might be a risk factor for T2DM, but the association of low-to-moderate level arsenic exposure with the risk of T2DM is still inconsistent. Single nucleotide polymorphisms (SNPs) can affect the development of T2DM, but the study on KEAP1 rs11545829 (G>A) SNP is few. In this paper, we explored the effect of KEAP1 rs11545829 (G>A) SNP and low-to-moderate level arsenic exposure on risk of T2DM in a cross-sectional case-control study conducted in Shanxi, China. Total of 938 participants, including 318 T2DM cases and 618 controls, were enrolled. Blood glycosylated haemoglobin (HbA1c) was detected by Automatic Biochemical Analyzer, and participants with HbA1c≧6.5% were diagnosed as T2DM. Urinary total arsenic (tAs, mg/L), as the indicator of arsenic exposure, was detected by liquid chromatography-atomic fluorescence spectrometry (LC-AFS). Genomic DNA was extracted and the genotypes of KEAP1 rs11545829 SNP were examined by multiplex polymerase chain reaction (PCR). The urinary tAs concentration in recruited participants was 0.075 (0.03-0.15) mg/L, and was associated with an increased risk of T2DM (OR = 8.45, 95% CI 2.63-27.17); rs11545829 mutation homozygote AA genotype had a protective effect on risk of T2DM (OR = 0.42, 95 % CI 0.25-0.73). Although this protective effect of AA genotype was found in participants with higher urinary tAs level (>0.032 mg/L) (OR = 0.48, 95% CI 0.26-0.86), there was no interaction effect for arsenic exposure and rs11545829 SNP on risk of T2DM. In addition, BMI modified the association between rs11545829 SNP and the risk of T2DM (RERI = -1.11, 95% CI -2.18-0.04). The present study suggest that low-to-moderate level arsenic exposure may be a risk factor, while KEAP1 rs11545829 SNP mutation homozygote AA genotype may be a protective factor for risk of T2DM, especially for T2DM patients with urinary tAs level>0.032 mg/L.

miR-21-5p and canonical Wnt signaling pathway promote osteoblast function through a feed-forward loop induced by fluoride.

Long-term excessive exposure to fluoride from environmental sources can cause serious public health problems such as dental fluorosis and skeletal fluorosis. The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis and canonical Wnt signaling pathway participate in the progress. However, the specific mechanism that how canonical Wnt signaling pathway was mediated is not yet clear. In this study, we found that miR-21-5p induced the activation of canonical Wnt signaling pathway via targeting PTEN and DKK2 during fluoride induced osteoblasts activation and firstly demonstrated the forward loop between canonical Wnt signaling and miR-21-5p in the process. These findings suggested an important regulatory role of miR-21-5p on canonical Wnt signaling pathway during skeletal fluorosis and miR-21-5p might be a potential therapeutic target for skeletal fluorosis.

Association between fluoride exposure and kidney function in adults: A cross-sectional study based on endemic fluorosis area in China.

BACKGROUND: The kidney toxicity of fluoride exposure has been demonstrated in animal studies, and a few studies have reported kidney function injury in children with fluoride exposure. However, epidemiological information for the effects of long-term fluoride exposure on adult kidney function remains limited. METHODS: We conducted a cross-sectional investigation in Wenshui County, Shanxi Province to examine the association between fluoride exposure and kidney function in adults, and a total of 1070 adults were included in our study. Urinary fluoride concentrations were measured using the national standardized ion selective electrode method. And markers of kidney function injury (urinary NAG, serum RBP, serum Urea, serum C3, serum UA and serum αl-MG) were measured using automatic biochemical analyzer. Multivariate linear regression analysis and binary logistic regression model were used to assess the relationship between urinary fluoride and markers of kidney function injury. RESULTS: Urinary fluoride was positively correlated with urinary NAG and serum Urea, negatively correlated with serum C3. In multivariate linear regression models, every 1 mg/L increment of urinary fluoride was associated with 1.583 U/L increase in urinary NAG, 0.199 mmol/L increase in serum Urea, 0.037 g/L decrease in serum C3 after adjusting for potential confounding factors. In the binary logistic regression model, higher levels of urinary fluoride were associated with an increased risk of kidney function injury. Determination of kidney function based on urinary NAG, every 1 mg/L increment in the urinary fluoride concentrations was associated with significant increases of 22.8% in the risk of kidney function injury after adjusting for potential confounding factors. Sensitivity analysis for the association between urinary fluoride concentrations and markers of kidney function (urinary NAG, serum Urea, and serum C3) by adjusting for the covariates, it is consistent with the primary analysis. CONCLUSIONS: Our study suggests that long-term fluoride exposure is associated with kidney function in adults, and urinary NAG is a sensitive and robust marker of kidney dysfunction caused by fluoride exposure, which could be considered for the identification of early kidney injury in endemic fluorosis areas.

Association between ALOX15 gene polymorphism and brick-tea type skeletal fluorosis in Tibetans, Kazaks and Han, China.

To evaluate the association between ALOX15 gene polymorphism and skeletal fluorosis (SF), a case-control study was conducted. A total of 1023 individuals, including 308 Tibetans, 290 Kazaks and 425 Han, were enrolled in this study, in which cases and controls were 278 and 745, respectively. SF was diagnosed by X-ray absorptiometry. SNPs were genotyped using the Sequenom Mass ARRAY system. The genotypes of ALOX15 rs7220870, rs2664593 and rs1107852 were not associated with the risk of SF. After reconstructing the haplotype of rs7220870 and rs11078528, the risk effect of haplotype CA was found in Han participants aged ≤45 years or with moderate fluoride intake. Diplotype of CC/CC had a protective effect on SF risk in Han participants; whereas, CA/CC diplotype showed a risk effect on SF risk in participants aged ≥65; Our results provide the first evidence of an association between ALOX15 gene polymorphism and SF risk in Han participants.Abbreviation: SF: Skeletal fluorosis; SNP: Single Nucleotide polymorphism.

sKlotho is associated with the severity of brick tea-type skeletal fluorosis in China.

The change of serum soluble Klotho (sKlotho) content is related to a variety of osteoarthropathy. However, its association with the severity of skeletal fluorosis (SF) is not clear. Here, the association of tea fluoride exposure with serum sKlotho levels and the severity of SF were investigated and further verified in a rat model of fluorosis. A cross sectional case control study was conducted in residents over 50 years old from brick-tea drinking areas in Qinghai and Xinjiang Provinces, China. Concentrations of fluoride in brick tea water and urine were determined by ion selective electrode method, and the levels of serum sKlotho were determined by ELISA method. Linear regression and ordered logistic regression models were constructed to examine the relationship among fluoride exposure, serum sKlotho levels and the severity of SF. The kidney and small intestine of Wistar rats were isolated for detection of Klotho by immunohistochemistry (IHC), and femoral artery blood was sampled to measure the serum levels of sKlotho. An increase of 1 mg/day in tea fluoride intake (TFI) was associated with a 12.070 pg/mL (95% CI: 0.452-23.689) increase in serum sKlotho levels and a 1.163-fold (95% CI: 1.007-1.342) increase in the severity of SF after adjusting for age, gender, and ethnicity. Serum sKlotho levels were also positively associated with the severity of SF (P < 0.05). The mediation analysis showed that serum sKlotho levels mediated 17.76% of the increase in the severity of SF caused by an increase of 1 mg/day of TFI. Moreover, a significant increase of serum sKlotho levels in fluoride-exposed groups was also seen in the rat model. The present study suggests that serum sKlotho may be a potential mediator of SF in brick tea-type fluorosis endemic areas.