Hydrochemical characteristics of surface waters and their relationships to the Kashin-Beck Disease in Longzi County, Tibet.

Although previous studies have been reported between the Kashin-Beck Disease (KBD) epidemic and the hydrochemical characteristics of surface waters, the etiology of the disease remains unclear. In the present study, we comprehensively investigated the relationship between the KBD and the hydrochemical characteristics of surface waters in Longzi County. Results show that, the pH (mean = 7.27 ± 0.30), total hardness (TH, mean = 57.08 ± 45.74 mg L-1), total dissolved solids (TDS, mean = 67.56 ± 44.00 mg L-1) and oxidation-reduction potential (ORP, mean = 84.11 ± 23.55 mV) of surface waters in KBD endemic areas are lower than those in the non-KBD endemic areas (means of pH = 7.49 ± 0.30; TH = 262.06 ± 123.29 mg L-1; TDS = 253.25 ± 100.39 mg L-1; ORP = 215.90 ± 55.99 mV). These results suggest that long-term consumption of low TDS, essential trace elements (e.g., nickel, cobalt, iron, selenium, zinc, molybdenum, and iodine) deficient, and potential toxic elements (e.g., arsenic) enriched waters by humans likely causes the KBD. Environmental factors such as the geology and geomorphology may produce biogeochemical imbalance, geomorphic, vegetation types and local climatic conditions may have significant impact on food fungi toxin poisoning and water organic compound poisoning, and these also impact the KBD occurrence.

Reduction of Smad2 caused by oxidative stress leads to necrotic death of hypertrophic chondrocytes associated with an endemic osteoarthritis.

OBJECTIVE: The occurrence and development of an endemic OA, Kashin-Beck disease (KBD), is closely related to oxidative stress induced by free radicals. The aim of the study was to find the key signalling molecules or pathogenic factors as a potential treatment strategy for KBD. METHODS: Real-time PCR and western blotting were performed to detect the mRNA and protein expression levels in cells and tissues. Immunohistochemical staining was assayed in rat models and human samples obtained from children. The type of cell death was identified by annexin V and propidium iodide staining with flow cytometry. RESULTS: Oxidative stress decreased levels of Smad2 and Smad3 in hypertrophic chondrocytes both in vitro and in vivo. In the cartilage of KBD patients, the expression of Smad2 and Smad3 proteins in the middle and deep zone was significantly decreased with an observed full deletion in the deep zone of some samples. Reduction of Smad2 protein induced necrotic death of hypertrophic chondrocytes, while reduction of Smad3 protein induced apoptosis. The reduction of Smad2 protein was not accompanied by Smad3 protein reduction in hypertrophic chondrocyte necrosis. Furthermore, the reduction of Smad2 also impaired the construction of tissue-engineered cartilage in vitro. CONCLUSION: These studies reveal that oxidative stress causes necrosis of hypertrophic chondrocytes by downregulating Smad2 protein, which increases the pathogenesis of KBD cartilage. The importance of Smad2 in the development of KBD provides a new potential target for the treatment of KBD.

Assessing the Health Loss from Kashin-Beck Disease and Its Relationship with Environmental Selenium in Qamdo District of Tibet, China.

Kashin-Beck Disease (KBD) is one of major endemic diseases in China. In this study, we estimated the health loss from KBD in Qamdo district of Tibet using the years lived with disability (YLD) metric and investigated the influence of environmental selenium (Se) on it by multiple regression model. The results showed that YLD rates produced a different ranking of health loss of KBD from that produced by prevalence rates between Basu and Luolong County, with higher health loss from KBD (43.61 YLD/1000) but lower prevalence (17.86%) in Basu County. YLD rates in two counites were both highest for the 45-64 years age group. Compared with the prevalence rate, the YLD rate had a closer relation to environmental Se and was significantly negatively correlated with Se in both soil and highland barley. The multiple linear regression further revealed that Se contents in cultivated soil and highland barley were main influencing factors for the health loss of KBD, which could explain 90.5% of the variation in YLD rates. The information obtained highlights the significance of the YLD metric in exploring the environmental etiology of KBD and provides important information on which to base decisions on future prevention and control of endemic diseases.

Prevention and control strategies for children Kashin-Beck disease in China: A systematic review and meta-analysis.

BACKGROUND: To evaluate prevention and control strategies for children with Kashin-Beck disease (KBD) in China through a systematic review and meta-analysis. METHODS: We conducted literature searches of articles indexed in Web of Knowledge, PubMed, Springerlink, Elsevier, the Chinese National Knowledge Infrastructure, and Wanfang data until February 2019. Search terms included "Kashin-Beck disease" or "KBD," and "improvement of water" or "change of grain" or "salt-rich selenium" or "comprehensive measures." Eligible studies were prospective trials of interventions in endemic area. Data extraction was performed by 2 independent authors using predefined data fields that also included quality evaluation. RESULTS: We screened 1183 potentially relevant articles, and included 22 studies that reported 24 trials, with data from 3700 healthy children and 2961 children KBD. The pooled odds ratios (ORs) and confidence intervals (95% CIs) for primary prevention new incidence in healthy children following interventions to comprehensive measures, change of grain, salt-rich selenium, and improvements of water were 0.15 (0.02, 0.95), 0.15 (0.03, 0.70), 0.19 (0.09, 0.38), and 0.20 (0.09, 0.42), respectively. The OR (95% CI) for clinical improvement in children KBD following interventions to improvement of water, salt-rich selenium, comprehensive measures, and change of grain were 5.03 (3.21, 7.89), 4.39 (3.15, 6.11), 2.98 (1.61, 5.52), and 2.35 (1.59, 3.47), respectively. All interventions showed significant differences and were effective (P < .05). CONCLUSION: Comprehensive measures and change of grain were the most effective measures in preventing new case, whereas improvement of water and salt-rich selenium resulted in clinical improvements in children KBD.

Selenium Content and/or T-2 Toxin Contamination of Cereals, Soil, and Children's Hair in Some Areas of Heilongjiang and Gansu Provinces, China.

It has been strongly suggested that selenium deficiency and T-2 contamination in cereals are responsible for the development of Kashin-Beck disease (KBD). In order to assess these risk factors of KBD in the internal and external environments, our team undertook a two-stage survey in some areas of Heilongjiang and Gansu Provinces, China. The selenium content in children's hair (293), cereal (192), and soil (46) samples were determined using the 2, 3-diamino-naphthalene fluorometric assay technique. The T-2 toxin contamination level in the cereal samples (704) was assayed using an ELISA kit. There were no clinical KBD cases identified in this survey. The selenium statuses of the children in all the investigated regions during the first phase were at the medium selenium nutrition level. During the second phase, the selenium status of the children in Weiyuan County, Ning County, and Shangzhi City was at the medium selenium nutrition level, at the edge of selenium deficiency, and selenium deficient, respectively. Furthermore, the selenium contents in the cereal and soil samples were low. During the first phase, the average T-2 toxin contamination level in the family staple food samples for all the investigated regions was about 10 ng/g. However, the T-2 toxin contamination levels in eight homegrown corn samples were higher than 100 ng/g. During the second phase, all the average T-2 toxin contamination levels in the flour and corn samples from the three investigated regions were less than 10 ng/g. Risk factors that affect the prevalence of KBD still remain in the internal and external environments of some areas in Heilongjiang and Gansu Provinces.

Increased Chondrocyte Apoptosis in Kashin-Beck Disease and Rats Induced by T-2 Toxin and Selenium Deficiency.

OBJECTIVE: To investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model. METHODS: Cartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction. RESULTS: Increased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet. CONCLUSION: T-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.

Zinc: the Other Suspected Environmental Factor in Kashin-Beck Disease in Addition to Selenium.

Kashin-Beck disease (KBD) is an endemic chronic osteochondral disease characterized by high prevalence, disability, and morbidity and is distributed from the northeast to the southwest in China, in some regions of Eastern Siberia in Russia, and in North Korea. Although the selenium deficiency etiological hypothesis for KBD has been proposed by scientists for decades, the idea that selenium deficiency is one of the most important environmental factors but not the primary and sole pathogenic factor for KBD has been widely accepted. Zn2+, which is closely involved in the synthesis of enzymes, nucleic acids, and proteins, is an essential microelement in vivo. A conundrum still exists in research on the relationship between Zn2+ and KBD due to inconsistent results, but it has been confirmed that Zn2+ can help repair metaphyseal lesions in patients with KBD, indicating that Zn2+ might play a key role in the pathogenesis of KBD, although the mechanism is unknown. The zinc-ZIP8-MTF1 axis in chondrocytes forms a catabolic cascade that promotes upregulation of the crucial effector matrix-degrading enzymes MMP3, MMP13, and ADAMTS5, thereby leading to osteoarthritis (OA) cartilage destruction. Zinc finger protein-related genes, the ZNT family, and the ZIP family of Zn2+ transporter genes have been found to be differentially expressed in KBD by high-throughput screening. Therefore, Zn2+ could play a key role in the pathogenesis of KBD.

The roles of selenium, insulin-like growth factor binding protein 2 and suppressor of cytokine signaling 3 in the pathogenesis of Kashin-Beck disease.

We aimed to verify the levels of IGFBP2 and SOCS3 in cartilage and chondrocytes of Kashin-Beck disease (KBD) patients and the effects of different selenium concentrations on the protein expression levels. Chondrocytes were cultured with sodium selenite in vitro. Immunohistochemistry and western blotting were used to verify the protein expressions. IGFBP2 and SOCS3 were up-regulated in KBD chondrocytes and decreased with increasing selenium concentrations. IGFBP2 expressed highest in the middle zone of KBD cartilage, SOCS3 expressed higher in the middle and deep zone. IGFBP2 and SOCS3 may be the biomarkers for KBD diagnosis and evaluating the effect of selenium supplement.

Integrative Multivariate Logistic Regression Analysis of Risk Factors for Kashin-Beck disease.

To determine the current evidence on risk factors for Kashin-Beck disease (KBD) using an integrative meta-analysis. We searched five English and three Chinese databases from inception to September 2015, to identify case-control studies that examined risk factors for KBD using multivariate logistic analysis. DerSimonian and Laird effective models are applied in processing data using pooled odds ratios (ORs) and 95 % confidence intervals (CI). Seven studies were identified with 3087 cases and 6402 controls. The main risk factors found to be significantly associated with the onset of KBD were age (OR 1.19, 95 % CI 1.10-1.28), parents prevalence (OR 5.16, 2.51-7.80), family hygiene (OR 1.68, 1.42-1.93), food source (OR 3.29, 2.38-4.19), wheat (OR 1.12, 1.08-1.16), wheat germ necrosis rate (OR 6.03, 1.87-12.92), total volatile basic nitrogen (OR 6.85, 1.01-28.67), low selenium in hair (OR 2.29, 1.08-3.50) were found to be significant risks factors. The pooled ORs (95 % CI) of protein intake and rice were 0.79 (0.66-0.93) and 0.90 (0.86-0.95), respectively, indicating that the two factors may be protective for KBD. We found that the combination of low protein intake, polluted grain, and selenium deficiency may contribute to be onset of KBD together.

Is It the Appropriate Time to Stop Applying Selenium Enriched Salt in Kashin-Beck Disease Areas in China?

We aimed to identify significant factors of selenium (Se) nutrition of children in Kashin-Beck disease (KBD) endemic areas and non-KBD area in Shaanxi Province for providing evidence of whether it is the time to stop applying Se-enriched salt in KBD areas. A cross-sectional study contained 368 stratified randomly selected children aged 4-14 years was conducted with 24-h retrospective questionnaire based on a pre-investigation. Food and hair samples were collected and had Se contents determined with hydride generation atomic fluorescence spectrometry. Average hair Se content of 349.0 ± 60.2 ng/g in KBD-endemic counties was significantly lower than 374.1 ± 47.0 ng/g in non-KBD counties. It was significantly higher in the male children (365.2 ± 52.3 ng/g) than in the female (345.0 ± 62.2 ng/g, p = 0.002) and significantly higher in the 4.0-6.9 years group (375.2 ± 58.9 ng/g) than the 7.0-14.0 years group (347.0 ± 56.1 ng/g, p < 0.01). Gender, living area, Se intake without supplements, Se-enriched salt, oil source and protein intake were identified as significant factors of hair Se contents. Cereals, meat and milk were commonly included as significant food categories that mainly contributed to Se intake without supplement of the whole population. Balanced dietary structure without Se supplement could effectively enhance and maintain children's Se nutrition. It may be the time to stop applying Se-enriched salt in KBD areas in Shaanxi Province.

An animal model of Kashin-Beck disease induced by a low-nutrition diet and exposure to T-2 toxin.

OBJECTIVE: We investigated the combined roles of a low-nutrition diet (low levels of protein, iodine, and selenium) and T-2 toxin in bone development and to establish an experimental animal model of Kashin-Beck disease (KBD) that reliably mimics the disease's pathological changes for further study of the pathogenesis and prevention of the disease. METHODS: Sprague-Dawley rats were randomly divided among four groups: group A, normal diet; group B, normal diet plus T-2 toxin; group C, low-nutrition diet; and group D, low-nutrition diet plus T-2 toxin exposure. The radiographic and histopathological changes in the tibial growth zone, plate cartilage and metaphysis were examined. RESULTS: In group D, all epiphyseal plates were blurred, thin, and irregular. Tibias were significantly shorter in group D than in groups A and B. After 4 weeks, epiphyseal plates showed chondrocyte necrosis, with the more obvious necrosis appearing in groups C and D. The positive rate of lamellar necrosis was significantly higher in group D than in groups B and A (P < 0.01). In group D, metaphyseal trabecular bone was sparse, disordered, and disrupted, and massive transverse trabecular bone appeared in the metaphysis at 12 weeks. CONCLUSIONS: A rat model of KBD induced by a low-nutrition diet and T-2 toxin exposure demonstrated radiographic and histopathological abnormalities of the proximal epiphyseal plate and the tibial metaphysis that are very similar to the bone changes found in patients with KBD. This animal model will be helpful for further study of the pathogenesis and prevention of KBD.

T-2 toxin contamination in grains and selenium concentration in drinking water and grains in Kaschin-Beck disease endemic areas of Qinghai Province.

It has been strongly suggested that two factors are involved in the development of Kaschin-Beck Disease (KBD), namely grains contamination with T-2 toxin and selenium deficiency. So our team undertook a survey about grains and drinking water in three rural KBD endemic villages and one non-KBD village in Qinghai Province. The level of T-2 toxin contamination in 364 grain samples was assayed using an ELISA kit. The selenium concentration in these grains and 15 drinking water samples from three KBD endemic villages were determined using the 2,3-diaminonaphthalene fluorometric assay. The results revealed that the level of T-2 toxin contamination in the samples from three KBD endemic villages was relatively high with an average level of 78.91 ng/g in wheat and 47.47 ng/g in flour. The T-2 toxin level in samples from the non-KBD village (12.23 ng/g) was significantly lower than that of local grains from the three KBD endemic villages. The average selenium content in wheat and flour from KBD areas was 0.0045 and 0.0067 µg/g, respectively. The selenium concentration in local grain samples was significantly lower than that in samples from the non-KBD village (0.0604 µg/g). In addition, the selenium concentration in drinking water from three KBD endemic villages was also low (0.156 µg/L). These results support a potential role of T-2 toxin contamination and selenium deficiency in KBD. Compared with non-KBD endemic areas, health hazards in grains and in the environment of KBD endemic areas were observed.

The effects of mycotoxins and selenium deficiency on tissue-engineered cartilage.

OBJECTIVE: To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments. MATERIALS AND METHODS: Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M). RESULTS: Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins. CONCLUSION: Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.

Growth, nutritional status, and signs of rickets in 0-5-year-old children in a Kashin-Beck disease endemic area of Central Tibet.

UNLABELLED: In order to describe the growth of 0-5-year-old Tibetan children living in a Kashin-Beck disease (KBD) endemic rural area and to examine the relationship between anthropometric indicators and clinical signs of rickets, we analyzed the baseline data of a cohort of 668 children enrolled in a prospective program of calcium and vitamin D supplementation. Tibetan children suffer from growth retardation. Z score of weight-for-age, height-for-age, weight-for-height was below -2 in 32.5%, 27.7%, and 12.1% of the children, respectively. Clinical signs of severe rickets are highly prevalent. Underweight, stunting, and clinical rickets increases with age. Prevalence of malnutrition was higher in the presence of signs of rickets. The proportion of children with a head circumference Z score < -2 was lowest when signs of rickets were observed. CONCLUSION: Stunting and underweight are frequent and probably associated with rickets.

SNP and mRNA expression for glutathione peroxidase 4 in Kashin-Beck disease.

Kashin-Beck disease (KBD) is a chronic endemic osteoarthropathy, which mainly occurs in West and Northeast China. Epidemiological studies suggest that Se deficiency is an important environmental factor for the incidence of KBD. Glutathione peroxidase 4 (GPx4) belongs to the glutathione peroxidase family, which is crucial for optimal antioxidant defences. Our purpose is to investigate the putative association between GPx4 polymorphisms and the risk of KBD. Restriction fragment length polymorphism-PCR was used to detect two SNP (rs713041, rs4807542) in 219 cases and 194 controls in Han Chinese subjects, and quantitative analysis for the GPx4 mRNA level was performed by the real-time PCR method. The results revealed that linkage disequilibrium existed in the two SNP. A significant difference was observed in the haplotype A-T (P = 0·0066) of GPx4, which was obviously lower in the KBD cases (0·006 v. 0·032 %). Correlation analysis based on a single locus showed no association between each SNP and KBD risk. Furthermore, the GPx4 mRNA level was dramatically lower in the blood of KBD patients. Overall, our finding indicated GPx4 polymorphisms and decreased mRNA level may be related to the development of KBD in the Chinese population, suggesting GPx4 as a possible candidate susceptibility gene for KBD.

[Interaction of plasma selenium levels and D12S304 gene site in the pathogenesis of Kashin-Beck disease analyzed by multifactor dimensionality reduction software].

OBJECTIVE: To explore the interaction of plasma selenium levels and D12S304 gene site in the pathogenesis of Kashin-Beck disease (KBD). METHODS: Case-control design was taken to compare the difference of plasma selenium levels and genotype of D12S304 between KBD patients and non-patients, and the interactions were analyzed by MDR software. RESULTS: Plasma selenium levels was lower in the case group than in the control group, while the D12S304 gene site was not different between the two groups, and no interaction between plasma selenium and genotype was observed. CONCLUSION: There was no interaction between plasma selenium and genotype at D12S304. Enlarging sample size or selecting another gene site might be needed in exploring the gene-environment interactions in the pathogenesis of KBD.

[Transmission disequilibrium test for 15 short tandem repeat loci in Kashin-Beck disease and their interaction with low selenium].

OBJECTIVE: To identify the genetic susceptibility to Kashin-Beck disease (KBD) and explore the interaction between low selenium (Se) and the susceptibility gene loci in KBD. METHODS: The DNA samples collected from 23 KBD nuclear families were analyzed using PCR and GeneScan Analysis 3.7 and Genotyper3.7 software. The haplotype relative risk (HRR) and transmission disequilibrium test (TDT) were used to test the data of the genotypes. The serum selenium (Se) concentration was measured by atomic fluorescence spectrometry, and the interaction between low Se and the susceptibility loci was calculated using a binary logistic regression. RESULTS: In the 23 nuclear families, the alleles of D2S151 (248 bp), D2S305 (320 bp), and D11S4094 (194 bp) showed significant correlation to KBD (P<0.05). Serum Se concentrations in the studied individuals was 0.037 µg/ml. No significant statistical interaction was observed between low Se exposure and the susceptibility loci (P>0.05). CONCLUSION: The polymorphisms in the STR loci D2S305, D2S151, and D11S4094 or the polymorphism loci near them might been related to KBD susceptibility. Low Se exposure shows no significant interaction with the susceptibility loci.

Environmental selenium in the Kaschin-Beck disease area, Tibetan Plateau, China.

Now, there is a decreasing trend for the prevalence rate of Kaschin-Beck disease (KBD) in most parts of China, but the disease is still active and severe in the Tibetan Plateau for some reason. To further explore the role of selenium in the occurrence of KBD, We collected samples including drinking water, cultivated topsoil, Highland Barley grains, and tsamba in Rangtang County and Aba County, Sichuan Province and determined concentrations of selenium by Hydride Generation Atomic Fluorescence Spectrometry. Levels of selenium in the environment were analyzed in detail. Selenium in the soil-plant-food system and their relationship with prevalence rate of KBD were also discussed. The results indicate: (a) the levels of environmental selenium are very low and the study area belongs to a selenium-deficient ecological landscape; (b) the KBD becomes much more severe with decreasing environmental selenium under the selenium-deficient condition. Namely, the lower the environmental selenium is, the more severe the disease is; (c) soil selenium deficiency plays a critical role for the prevalence of local KBD, and more factors inducing selenium deficiency should be more concerned in the future.