Assessing the potential value and mechanism of Ginkgo biloba L . On coal-fired arsenic-induced skin damage: In vitro and human evidence

Abstract

Exposure through arsenic-contaminated air and food caused by the burning of coal is a major environmental public health concern in Guizhou Province of China. Previous studies have shown that immunological dysfunction is involved in the pathogenesis and carcinogenesis of arsenic; however, knowledge regarding effective prevention measures have not been fully examined. The effect of Ginkgo biloba extract (EGb761) on arsenic-induced skin damage of human immortalized keratinocyte cells (HaCaT) was first evaluated in this study. The results showed that 200 μg/mL EGb761 can reduce the expression of miR-155-5p, and the indicators reflecting arsenic-induced skin damage (Krt1, Krt6c and Krt10) in arsenic-exposed cells (P < 0.05), the expression levels of NF-AT1; the indicators reflecting arsenic-induced immunological dysfunction (IL-2, IFN-γ) in cells; and the levels of secreted IL-2 and IFN-γ in cell supernatants were significantly increased (P < 0.05). Further randomized controlled double-blind experiments showed that compared to the placebo control group, the expression level of miR-155-5p in the plasma of the Ginkgo biloba intervention group, the indicators in the serum reflecting arsenic-induced skin damage (Krt1, Krt6c, and Krt10) and the epithelial-mesenchymal transformation (EMT) vimentin were significantly reduced (P < 0.05), but the levels of NF-AT1 and the indicators reflecting arsenic-induced immunological dysfunction (IL-2, IFN-γ) and EMT (E-cadherin) in serum were significantly increased (P < 0.05). Our study provides some limited evidence that Ginkgo biloba L. can increase the expression of NF-AT1 by downregulating the level of miR-155-5p, alleviating immunological dysfunction, and decreasing the expression of EMT biomarkers, thus indirectly improving arsenic-induced skin damage.

Keywords

Arsenic skin damage immunological dysfunction

Introduction

Chronic exposure to arsenic is a major environmental public health concern worldwide¹ affecting hundreds of millions of people.² In addition to common drinking water exposure routes in the world,^3–7 arsenic poisoning caused by exposure to large amounts of arsenic-contaminated air and food is one of the key prevention and treatment diseases in Guizhou Province, China.⁸ Although the previous research shows that the endemic arsenic poisoning in Guizhou Province of China has reached the highest level in the world,⁸ very little is known about effective preventive measures for patients, which has limited the development of methods to control endemic arsenic poisoning in recent times.

Skin is the main target organ for arsenic exposure. Previous studies^9–13 have shown that immunological dysfunction was involved in arsenic-induced skin lesions. Our recent study¹⁴ provides evidence that miR-155-5p regulates the NF-AT1-mediated immunological dysfunction involved in the pathogenesis and carcinogenesis of arsenic. Ginkgo is an ancient edible and medicinal plant with a long history of use; its main active ingredients are flavonoid glycosides and terpene lactones. Many studies^15–21 have shown that Ginkgo biloba L. have many functions such as anti-oxidation, anti-inflammatory, platelet aggregation inhibition, and immune regulation and have been widely used for treating cardiovascular and nervous system diseases.^22–24 Ginkgo biloba L. attenuates the disruption of pro-and anti-inflammatory T-cell balance in peripheral blood in the coal-burning type of endemic arsenic poisoning patients.²⁵ Moreover, Ginkgo biloba L. can reduce kidney damage induced by coal-burning arsenic by improving the immune function in rats.²¹ Immunological dysfunction, which is a common mechanism of disease occurrence, is involved in arsenic-induced skin damage,¹⁴ and Ginkgo biloba L. can improve immune function^18,19,21,25 along with the positive curative effects of Ginkgo biloba L. in kidney damage of coal-burning arsenic poisoning rats²¹; therefore, it is very necessary to study the role and mechanism of Ginkgo biloba L. in arsenic-induced skin damage in rats.

In this study, by observing the expression changes in mRNA and protein of miR-155-5p and NF-AT1 and the indicators reflecting the arsenic-induced immunological dysfunction (IL-2, IFN-γ), skin damage (Krt1, Krt6c and Krt10) in cells, the goal was to study the potential mechanism of Ginkgo biloba L. in arsenic-induced skin damage. A 3-month randomized, controlled, double-blind experiment was then conducted to observe the changes in the above indicators in the serum/plasma of the Ginkgo biloba L. treatment group and the placebo control group, combined with changes in EMT indicators (E-cadherin, Vimentin) to explore the potential application value of Ginkgo biloba L. in arsenic-induced skin damage. This study will contribute to a deeper understanding of the mechanisms of Ginkgo biloba L. in arsenic-induced skin damage and our results will identify a possible natural medicinal plant that can be used to design more effective prevention and control strategies.

Methods

Cell culture and treatments

Human immortalized keratinocyte cells (HaCaT) were used for the in vitro study uses, and the cell line source and culture method were the same as those described in the previous study.¹⁴ Ginkgo biloba L. extract (EGb761) was purchased from Dr. Willmar Schwabe GmbH & Co. KG (Germany). According to the design of this study, HaCaT cells were treated with various concentrations (0∼1600 μg/mL) of EGb761 for 24 hours, and the cell viability was determined by Cell Counting Kit-8 (CCK8). HaCaT cells in the logarithmic growth phase were collected and treated with 10 M NaAsO₂ for 24 h. EGb761 concentration (0∼400 μg/mL) without cytotoxicity was used to treat arsenic-exposed HaCaT cells. The CCK8 method was used to detect the effect of EGb761 on the viability of arsenic-exposed HaCaT cells. The concentration of EGb761 (200 μg/mL) that can significantly restore the viability of arsenic-exposed HaCaT cells was selected as the treatment dose. There were four groups in the intervention experiment which included the control group, EGb761 treatment group, NaAsO₂ (10 μM) treatment group and EGb761 combined with NaAsO₂ (10 μM) treatment group. Each group had three complex holes, and the experiment was repeated six times.

Population-based study

In this study, 75 patients diagnosed with arsenic poisoning have overt skin damage were selected as the subjects from a proved endemic area of coal-burning type of arsenic poisoning, based on the Chinese Standard of Diagnosis for Endemic Arsenism (WS/T 211-2015).²⁶ According to the block random design, the participants were randomly divided into the Ginkgo biloba L. treatment group (36 cases) and a placebo control group (39 cases). First, the participants divided into block groups according to gender and age (one interval for every 5 years); then, evenly distribute the participants in the same block group into the above groups. The inclusion criteria were that all participants had to be residents of Jiaole village, aged 30–65 years, and the age and gender of the two groups had to match exactly. Exclusion criteria included occupational arsenic poisoning, suffering from immunodeficiency or autoimmune-related diseases, a recent history of infection, seafood intake, or taking drugs that can affect arsenic metabolism. A randomized, double-blind, placebo-controlled trial was designed to assess the potential value of Ginkgo biloba L. on coal-fired arsenic-induced skin damage. The study was approved by the Ethics Committee of Guizhou Medical University, and all participants provided written informed consent. The characteristics of the study participants are provided in Supplementary Table S1.

Ginkgo biloba L. was purchased from Guizhou Xinbang Pharmaceutical Co., Ltd. (the national medicine permission number is Z20028023, and the production lot number is 20170101), and each tablet contained 19.2 mg of total flavonol glycosides and terpene lactone 4.8 mg; placebo was produced by Guizhou Xinbang Pharmaceutical Co., Ltd., the color, weight and size are the same as those of Ginkgo biloba L., but the main component is starch. Oral administration was used, with a dose of 40 mg each time, 3 times a day for 90 days. To ensure the patient’s compliance and the reliability of the study, the entire study process was treated by full-time doctors from the 44th hospital of the Chinese People’s Liberation Army, Guiyang, China, and telephonic follow-up and on-site supervision were arranged every day.

The polyethylene plastic bottle was soaked with 10% nitric acid overnight, washed with deionized water and dried for use. Following informed consent, fasting morning urine was collected from subjects on the spot, and 1 mL concentrated hydrochloric acid was added to 100 mL urine samples to make the pH value less than 2 for the determination of urinary arsenic (UAs). The fasting ethylene diamine tetraacetic acid (EDTA) anticoagulated and non-anticoagulated blood of the surveyed subjects were collected aseptically before and after the treatment, centrifuged at 3,000 g for 10 min at 4°C, plasma or serum was separated, and stored at −80°C for testing.

Cell proliferation assay

The detection method of CCK8 was the same as that used in a previous study.¹⁴ In short, according to the manufacturer’s protocol (Dojindo, Kumamoto, Japan), the absorbance of HaCaT cells was measured at OD = 450 nm, after 24 h of treatment with various concentrations of EGb761 (0, 25, 50, 100, 200, 400, 800, or 1600 μg/mL). The same method was also used to detect the recovery effect of EGb761 on the decreased cell viability caused by arsenic exposure. Each group had six complex holes, and the experiment was repeated 6 times.

Quantitative real-time PCR

As described in the previous literature,¹⁴ after RNA extraction, the relative expression levels of miR-155-5p and other genes (Krt1, Krt6c, Krt10, IL-2, IFN-γ and NF-AT1) were examined using a Bio-Rad CFX96 machine (BIO-RAD, USA) and calculated with the analysis software (Bio-Rad CFX Manager 3.1, USA), based on the cycle threshold (Ct) values. All primers are presented in Supplementary Table S2 and were synthesized by RiBoBio (Guangzhou, China). U6 snRNA and -actin were used to assess the relative expressions of miR-155-5p and other genes.

Western blot analysis

The total protein extraction, quantification and Western blot analysis were performed as described previously.¹⁴ Briefly, the enhanced chemiluminescence (Cell Signaling Technology) was used to measure the immune complexes in the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by using a ChemiDoc™ Imaging System (ChemiDoc, BIO-RAD, USA). By comparing the difference between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the target protein in the same sample, the relative expression of the target protein was evaluated. All antibodies were purchased from Abcam (Cambridge, USA).

ELISA assays

The ELISA analysis was performed as described previously.¹⁴ Human-specific E-cadherin and vimentin were purchased from Abcam (Cambridge, USA), IL-2 and IFN-γ from CUSABIO Technology LLC (Wuhan, China), Krt6c from MyBioSource, Inc. (San Diego, USA), and others (NF-AT1 and Krt10) were purchased from Elabscience, Inc. (Wuhan, China). All samples were detected in parallel, repeated three times, and the average absorbance was calculated.

Statistical analysis

Excel 2019 and SPSS 22.0 software were used for data entry and data analysis, respectively. Kolmogorov-Smirnov test was used to examine normality of the distribution, and one-factor analysis was used for the measurement data, including miR-155-5p, keratins (Krt1, Krt10, and Krt6c), IL-2, IFN-γ, NF-AT1 and cell viability in HaCaT cells between the groups; multiple comparisons were performed using the least significant difference test. The data of UAs and miR-155-5p in human plasma were skewed. The Mann-Whitney U test was used to analyze the differences in miR-155-5p and UAs between the groups. The data were separated by median and quartile. The Ginkgo biloba L. intervention group and placebo control group were compared using the independent sample T test before or after the intervention of the above indicators (unless UAs and miR-155-5p). And the paired T-test is mainly used to compare the above indicators of the Ginkgo biloba L. intervention group or placebo control group before and after the intervention. The level of test standards was set at α = 0.05.

Results

Ginkgo biloba L. can increase the expression of NF-AT1 by down-regulating the level of miR-155-5p in cells, improve the inhibitory effect of arsenic-induced immunological dysfunction indicators, thereby reducing the expression of biological markers of skin damage caused by arsenic

This study first evaluated the intervention effect of EGb761 on HaCaT cells. The results showed that a low concentration of EGb761 (25, 50 μg/mL) can stimulate cell growth (P < 0.05), when the concentration is greater than 100 μg/mL, the cell viability gradually decreases (P < 0.05), and the cell viability of the 800 and 1600 μg/mL EGb761 group decreased to below 80% (Figure 1(A)). Subsequently, 25∼400 μg/mL EGb761 without cytotoxicity was selected to treat arsenic-exposed HaCaT cells and the restoration of cell viability by EGb761. The results showed that the restoration of cell viability was most obvious in the 200 μg/mL EGb761 intervention group (P < 0.05), but the cell viability gradually decreased in the 400 μg/mL EGb761 intervention group (P < 0.05) (Figure 1(B)). Further, 200 μg/mL EGb761 can reduce the expression of miR-155-5p (Figure 1(C)) and the indicators reflecting arsenic-induced skin damage (Krt1, Krt6c and Krt10) in arsenic-exposed cells (P < 0.05) (Figure 1(F) to (J)), but the expression levels of NF-AT1 (Figure 1(E), (G) and (I)), the indicators reflecting the arsenic-induced immunological dysfunction (IL-2, IFN-γ) in cells (Figure 1(E), (G) and (I)), and the levels of secreted IL-2 and IFN-γ in cell supernatants (Figure 1(D)) were significantly increased (P < 0.05).

Figure 1.

Ginkgo biloba can increase the expression of NF-AT1 by down-regulating the level of miR-155-5p in cells, improve the inhibitory effect of arsenic-induced immunological dysfunction indicators, thereby reducing the expression of biological markers of skin damage caused by arsenic. The in vitro results were based on 6 independent experiments. All data are presented as mean ± standard deviation. *p < 0.05. (A) Cell viability for EGb761. The Cell Counting Kit-8 assay was used to assess cell viability. (B) Effect of EGb761 on the cell vitality of arsenic-exposed cells. (C) The levels of miR-155-5p in HaCaT cells of each group. (D) The concentration of secreted IL-2 and IFN-γ for each group. (E) The mRNA levels of NF-AT1, IL-2 and IFN-γ in the different groups. (F) The mRNA levels of Krt1, Krt6c and Krt10 in the different groups. (G) Western blots of NF-AT1, IL-2 and IFN-γ. (H) Western blots of Krt1, Krt6c and Krt10. (I) Relative protein levels of NF-AT, IL-2 and IFN-γ. (J) Relative protein levels of Krt1, Krt6c and Krt10.

Ginkgo biloba L. intervention can downregulate the expression level of miR-155-5p in the plasma of arsenic-exposed subjects, increase the secretion of serum NF-AT1, alleviate immunological dysfunction caused by arsenic, and improve the expression of biological markers reflecting skin damage and EMT caused by arsenic

Although Ginkgo biloba L. did not directly improve skin damage, a randomized controlled double-blind experiment showed that compared to the placebo control group, the contents of UAs (Figure 2(A)), the expression level of miR-155-5p (Figure 2(B)) in the plasma, the indicators in the serum reflecting arsenic-induced skin damage (Krt1, Krt6c and Krt10) (Figure 2(E)) and the EMT (vimentin) (Figure 2(D)) of the Ginkgo biloba L. intervention group were significantly reduced (P < 0.05), but the levels of NF-AT1 (Figure 2(C)), and the indicators reflecting arsenic-induced immunological dysfunction (IL-2, IFN-γ) (Figure 2(C)) and EMT (E-cadherin) (Figure 2(D)) in serum were significantly increased (P < 0.05). The changes of the above indicators before and after treatment in the Ginkgo biloba L. intervention group were statistically significant (P < 0.05), but the above indicators did not change significantly before and after treatment in the placebo control group (P > 0.05).

Figure 2.

Ginkgo biloba intervention can down-regulate the expression level of miR-155-5p in the plasma of arsenic-exposed people, and increase the secretion of serum NF-AT1, alleviate immunological dysfunction caused by arsenic, and improve the expression of biological markers reflecting skin damage and EMT caused by arsenic. The number of participants the Ginkgo biloba treatment group and the placebo control group are 36, 39. The results of Urinary arsenic and miR-155-5p were separated by median and quartile, and other data are presented as mean ± standard deviation. *p<0.05. (A) The concentration of urinary arsenic for each group. (B) The levels of miR-155-5p in human plasma of each group. (C) The concentration of NF-AT1, IL-2 and IFN-γ in human serum. (D) The concentration of E-cadherin and Vimentin in human serum. (E) The concentration of Krt1, Krt6c and Krt10 in human serum.

Discussion

China has conducted comprehensive research on the prevention and treatment of endemic arsenic poisoning since 1980. The intervention methods mainly include environmental intervention, behavioral intervention, and drug intervention. Thus far, it has achieved fruitful results in the prevention of arsenic poisoning. In terms of medical interventions for endemic arsenic poisoning, vitamins (Vitamins A, B2, B6, C, E, and folic acid),^27–29 trace elements (Zinc, selenium),^28,29 and natural medicine plants (Ginkgo biloba L. and Rosa roxburghii fruit extract)^21,25,30 can reduce the risk of endemic arsenic poisoning. However, to date there is a lack of research on the mechanism of arsenic-causing medical interventions; all research is only at the observation stage, and the lack of effect verification in the arsenic poisoning population makes it difficult to promote the transformation and application of medical intervention programs.

In recent years, “conventional drug in new use” has received increasing attention as a drug development strategy, and many new indication drugs have been derived. As a traditional natural medicine plant, Ginkgo biloba L. has been widely used in the treatment of diseases such as those in the cardiovascular and nervous systems due to its antioxidant, anti-inflammatory, platelet aggregation and immune regulation functions.^22–24 Guizhou Province, China, has very rich resources of Gingko. The oldest Ginkgo tree and the largest ancient Ginkgo tree in the world are located in Guizhou province. To evaluate the intervention effect and possible mechanism of Ginkgo biloba L. on skin damage caused by arsenic, this study used EGb761 to establish a cell model of Ginkgo biloba L. extract treatment for arsenic exposure. The results showed that Ginkgo biloba L. extract can reduce the expression of miR-155-5p induced by arsenic. Previous studies showed that the upregulated expression of miR-155-5p can increase the risk of skin damage caused by coal-burning arsenic³¹; combined with the epigenetic reversibility characteristics, it is suggested that Ginkgo biloba L. is expected to reduce miR-155-5p expression and improve arsenic-induced skin lesions and even cancer. Our recent study ¹⁴ indicated that miR-155-5p regulates NF-AT1-mediated immunological dysfunction involved in the pathogenesis and carcinogenesis induced by arsenic. This study found that EGb761 can restore the level of mRNA and protein expression of NF-AT1 and the genes related to immunological dysfunction (IL-2 and IFN-γ) caused by arsenic to a certain extent, and the secretion of IL-2 and IFN-γ in the cell supernatant. Additionally, EGb761 can reduce the expression of arsenic-induced biomarkers that reflect arsenic-induced skin damage (Krt1, Krt6c, and Krt10). The above results indicate that Ginkgo biloba L. has potential application value because it can induce increased expression of NF-AT1 by downregulating the level of miR-155-5p in cells, improve the inhibitory effect of arsenic-induced immunological dysfunction indexes, and thus reduce the expression of biological markers of arsenic-induced skin damage.

Although Ginkgo biloba L. do not directly improve the skin damage caused by arsenic, endemic arsenic poisoning is a systemic disease, and our previous study²¹ showed good effects of Ginkgo biloba L., which can reduce kidney damage caused by arsenic exposure by improving the systemic immune function; meanwhile, considering the reversibility of epigenetics, Ginkgo biloba L. can downregulate the expression of miR-155-5p, which is related to arsenic-induced skin damage in in vitro studies. In view of this, it is very necessary to explore the intervention value of Ginkgo biloba L. on arsenic-induced skin damage in the population.

The randomized controlled trial is currently the design scheme with the least possibility of bias in the prevention and treatment of traditional Chinese medicine. The double-blind trial means that neither the experimenter nor the test subject are aware of the group to which the test subject belongs (control group or experimental group). The New England Journal of Medicine has published an article³² that explains the important role of double-blind testing in reducing the influence of human factors in the experimenters and subjects. This study used randomized, double-blind experiments to show that Ginkgo biloba L. can downregulate the expression level of miR-155-5p in exposed plasma, increase serum NF-AT1 secretion, and reduce immunological dysfunction caused by arsenic. Furthermore, Ginkgo biloba L. improved the positive effect of arsenic exposure to reflect the biological signs of arsenic-induced skin damage. Based on the difficulty in obtaining skin samples, in order to better understand the intervention effect of Ginkgo biloba L. on coal-fired arsenic-induced skin damage, the changes in serum E-cadherin and vimentin of the subjects were analyzed in this study, which reflect EMT molecular markers. E-cadherin is the most classic epithelial cell marker, expressed in epithelial cells, and its expression decreases during various types of EMT, and the loss of E-cadherin can further promote the occurrence of EMT.^33,34 Vimentin is a scaffold protein that can increase expression in response to a variety of lesions and has a significant positive correlation with tumor invasion and metastasis ability.³⁴ In vitro studies^35–38 have shown that long-term arsenic exposure can promote the occurrence of EMT by reducing the expression of E-cadherin and increasing the expression of Vimentin in cells. The results showed that the intervention of Ginkgo biloba L. reduced the content of EMT molecular marker vimentin in the serum of arsenic poisoning patients, but E-cadherin was significantly increased in this study. Combined with EMT can be used as an early molecular marker for the development of arsenic skin damage and even cancer, the results further provide evidence to support that Ginkgo leaves can improve arsenic-induced skin damage to a certain extent. Additionally, EMT is involved in various biological processes such as the occurrence, invasion and metastasis of skin cancers,^39,40 and Ginkgo biloba L. can improve EMT, suggesting that Ginkgo biloba L. also has certain auxiliary effects for skin cancer caused by arsenic exposure.

The strengths of our study include the use of randomized controlled, double-blind experiments, and the full use of a coal-burning type of endemic arsenic poisoning area population resources to explore the potential application value of Ginkgo biloba L. in arsenic-induced skin damage from the perspective of the population. At the same time, on the basis of the advantages of in vitro study, preliminary exploration of the possible intervention mechanism of Ginkgo biloba L. on skin damage caused by arsenic was performed. Our study also has limitations. In view of the difficulty of obtaining skin samples from the population, the findings in this study are mainly based on the expression changes of biological markers reflecting arsenic-induced skin damage and EMT, and there is no direct population evidence that Ginkgo biloba L. can improve arsenic-induced skin damage. Therefore, it is very necessary to conduct more in-depth studies on the biological significance of Ginkgo biloba L. treatment of arsenic-induced skin damage.

Conclusions

Overall, our study provides some limited evidence that Ginkgo biloba L. can increase the expression of NF-AT1 by downregulating the level of miR-155-5p, alleviate the immunological dysfunction, and decrease the expression of EMT biomarkers, and thus indirectly improve arsenic-induced skin damage (Figure 3). This study is the first to comprehensively assess the value and mechanism of Ginkgo biloba L. in the treatment of skin damage caused by coal-fired arsenic. This study provides scientific basis for further understanding a possible natural medicinal plant, Ginkgo biloba L., as a more effective measure to prevent and control arsenic poisoning induced by burning of coal.

Figure 3.

Ginkgo biloba can increase the expression of NF-AT1 by down-regulating the level of miR-155-5p, alleviate the immunological dysfunction and decrease the expression of EMT biomarkers, and indirectly improve arsenic-induced skin damage.

Supplemental material

Supplemental Material, sj-pdf-1-het-10.1177_09603271211021887 - Assessing the potential value and mechanism of Ginkgo biloba L. On coal-fired arsenic-induced skin damage: In vitro and human evidence

Supplemental Material, sj-pdf-1-het-10.1177_09603271211021887 for Assessing the potential value and mechanism of Ginkgo biloba L. On coal-fired arsenic-induced skin damage: In vitro and human evidence by Qibing Zeng, Shaofeng Wei, Baofei Sun and Aihua Zhang in Human & Experimental Toxicology

Footnotes

Acknowledgments

We are very grateful to Xiaoxin Huang, Ducai Cen, Zhongyi Liu, Xuexin Dong, Guicheng Liu (the 44th hospital of the Chinese People’s Liberation Army, Guiyang, China) for their invaluable cooperation in the epidemiological investigation, physical examination, diagnosis and drug intervention.

Author contributions

Q.Z.: Methodology, Validation, Investigation, Data Curation, Writing—Original Draft, Writing—Review & Editing, Visualization. S.W.: Methodology, Investigation. B.S.: Methodology, Investigation, Data Curation. Z.Z.: Methodology, Investigation. A.Z.: Conceptualization, Methodology, Investigation, Resources, Writing—Review & Editing, Supervision, Project administration, Funding acquisition.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundations of China (81430077, U1812403).

ORCID iD

Aihua Zhang

Supplemental material

Supplemental material for this article is available online.

References

Carlin

Naujokas

Bradham

, et al. Arsenic and environmental health: state of the science and future research opportunities. Environ Health Perspect 2016; 124: 890–899.

Naujokas

Anderson

Ahsan

, et al. The broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem. Environ Health Perspect 2013; 121: 295–302.

Nardone

Ferreccio

Acevedo

, et al. The impact of BMI on non-malignant respiratory symptoms and lung function in arsenic exposed adults of Northern Chile. Environ Res 2017; 158: 710–719.

Kippler

Skroder

Rahman

, et al. Elevated childhood exposure to arsenic despite reduced drinking water concentrations—a longitudinal cohort study in rural Bangladesh. Environ Int 2016; 86: 119–125.

Bretzler

Lalanne

Nikiema

, et al. Groundwater arsenic contamination in Burkina Faso, West Africa: Predicting and verifying regions at risk. Sci Total Environ 2017; 584-585: 958–970.

Hsu

Hsieh

Wang

, et al. Arsenic exposure from drinking water and the incidence of CKD in low to moderate exposed areas of Taiwan: a 14-year prospective study. Am J Kidney Dis 2017; 70: 787–797.

Cheng

Chang

Cheng

, et al. Associations between arsenic in drinking water and occurrence of end-stage renal disease with modifications by comorbidities: a nationwide population-based study in Taiwan. Sci Total Environ 2018; 626: 581–591.

Wang

Luo

Zou

, et al. Alterations of arsenic levels in arsenicosis residents and awareness of its risk factors: a population-based 20-year follow-up study in a unique coal-borne arsenicosis County in Guizhou, China. Environ Int 2019; 129: 18–27.

Zeng

Luo

, et al. PKC theta-mediated Ca(2+)/NF-AT signalling pathway may be involved in T-cell immunosuppression in coal-burning arsenic-poisoned population. Environ Toxicol Pharmacol 2017; 55: 44–50.

10.

Raqib

Ahmed

Sultana

, et al. Effects of in utero arsenic exposure on child immunity and morbidity in rural Bangladesh. Toxicol Lett 2009; 185: 197–202.

11.

Soto-Pena

Luna

Acosta-Saavedra

, et al.Assessment of lymphocyte subpopulations and cytokine secretion in children exposed to arsenic. FASEB J 2006; 20: 779–781.

12.

Rahman

Vahter

Ekstrom

, et al. Arsenic exposure in pregnancy increases the risk of lower respiratory tract infection and diarrhea during infancy in Bangladesh. Environ Health Perspect 2011; 119: 719–724.

13.

Liao

Lan

, et al. Differential effects of arsenic on cutaneous and systemic immunity: focusing on CD4+ cell apoptosis in patients with arsenic-induced Bowen’s disease. Carcinogen 2009; 30: 1064–1072.

14.

Zeng

Zhang

. Assessing potential mechanisms of arsenic-induced skin lesions and cancers: human and in vitro evidence. Environ Pollut 2020; 260: 113919.

15.

Bridi

Crossetti

Steffen

, et al. The antioxidant activity of standardized extract of Ginkgo biloba (EGb 761) in rats. Phytother Res 2001; 15: 449–451.

16.

Kotakadi

Jin

Hofseth

, et al. Ginkgo biloba extract EGb 761 has anti-inflammatory properties and ameliorates colitis in mice by driving effector T cell apoptosis. Carcinogen 2008; 29: 1799–1806.

17.

Akiba

Kawauchi

Oka

, et al. Inhibitory effect of the leaf extract of Ginkgo biloba L. on oxidative stress-induced platelet aggregation. Biochem Mol Biol Int 1998; 46: 1243–1248.

18.

Sochocka

Zaczynska

Tabol

, et al. The influence of donepezil and EGb 761 on the innate immunity of human leukocytes: effect on the NF-kappaB system. Int Immunopharmacol 2010; 10: 1505–1513.

19.

Puebla-Perez

Lozoya

Villasenor-Garcia

. Effect of Ginkgo biloba extract, EGb 761, on the cellular immune response in a hypothalamic-pituitary-adrenal axis activation model in the rat. Int Immunopharmacol 2003; 3: 75–80.

20.

Tian

Zhang

, et al. Effects of Ginkgo biloba extract (EGb 761) on hydroxyl radical-induced thymocyte apoptosis and on age-related thymic atrophy and peripheral immune dysfunctions in mice. Mech Ageing Dev 2003; 124: 977–983.

21.

Zeng

Yao

, et al. A possible new mechanism and drug intervention for kidney damage due to arsenic poisoning in rats. Toxicol Res (Camb) 2016; 5: 511–518.

22.

Ude

Schubert-Zsilavecz

Wurglics

. Ginkgo biloba extracts: a review of the pharmacokinetics of the active ingredients. Clin Pharmacokinet 2013; 52: 727–749.

23.

Le Bars

Katz

Berman

, et al. A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. North American EGb Study Group. JAMA 1997; 278: 1327–1332.

24.

Ran

Yang

Chang

, et al. Ginkgo biloba extract postconditioning reduces myocardial ischemia reperfusion injury. Genet Mol Res 2014; 13: 2703–2708.

25.

Xia

Sun

Zou

, et al. Ginkgo biloba extract attenuates the disruption of pro-and anti-inflammatory T-cell balance in peripheral blood of arsenicosis patients. Int J Biol Sci 2020; 16: 483–494.

26.

NHSPRC (National Health Commission of the People’s Republic of China). WS/T 211-2015 Diagnosis of endemic arsenicosis. Beijing: National Health Commission of the People’s Republic of China, 2015, pp. 1–3.

27.

Zablotska

Chen

Graziano

, et al. Protective effects of B vitamins and antioxidants on the risk of arsenic-related skin lesions in Bangladesh. Environ Health Perspect 2008; 116: 1056–1062.

28.

Mahata

Argos

Verret

, et al. Effect of selenium and vitamin e supplementation on plasma protein carbonyl levels in patients with arsenic-related skin lesions. Nutr Cancer 2008; 60: 55–60.

29.

Chen

Hall

Graziano

, et al. A prospective study of blood selenium levels and the risk of arsenic-related premalignant skin lesions. Cancer Epidemiol Biomarkers Prev 2007; 16: 207–213.

30.

Zeng

, et al. Assessing the potential value of Rosa Roxburghii Tratt in arsenic-induced liver damage based on elemental imbalance and oxidative damage. Environ Geochem Health 2020; 43(3): 1165–1175.

31.

Zeng

Zou

Wang

, et al. Association and risk of five miRNAs with arsenic-induced multiorgan damage. Sci Total Environ 2019; 680: 1–9.

32.

Wechsler

Kelley

Boyd

, et al. Active albuterol or placebo, sham acupuncture, or no intervention in asthma. N Engl J Med 2011; 365: 119–126.

33.

Buda

Pignatelli

. E-cadherin and the cytoskeletal network in colorectal cancer development and metastasis. Cell Commun Adhes 2011; 18: 133–143.

34.

Chaw

Abdul Majeed

Dalley

, et al. Epithelial to mesenchymal transition (EMT) biomarkers—E-cadherin, beta-catenin, APC and Vimentin—in oral squamous cell carcinogenesis and transformation. Oral Oncol 2012; 48: 997–1006.

35.

Liu

Ling

Chen

, et al. Impaired autophagic flux and p62-mediated EMT are involved in arsenite-induced transformation of L-02 cells. Toxicol Appl Pharmacol 2017; 334: 75–87.

36.

Fan

, et al. Arsenic trioxide inhibits EMT in hepatocellular carcinoma by promoting lncRNA MEG3 via PKM2. Biochem Biophys Res Commun. 2019; 513: 834–840.

37.

Wang

Luo

, et al. Effects of long term low- and high-dose sodium arsenite exposure in human transitional cells. Am J Transl Res 2017; 9: 416–428.

38.

Person

Ngalame

Makia

, et al. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells. Toxicol Appl Pharmacol 2015; 286: 36–43.

39.

Walsh

, et al. Cyclosporine a mediates pathogenesis of aggressive cutaneous squamous cell carcinoma by augmenting epithelial-mesenchymal transition: role of TGFβ signaling pathway. Mol Carcinog 2011; 50: 516–527.

40.

Papanikolaou

Bravou

Gyftopoulos

, et al. ILK expression in human basal cell carcinoma correlates with epithelial-mesenchymal transition markers and tumour invasion. Histopathology 2010; 56: 799–809.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.16 MB