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. 2026 Feb 2;4(6):1272–1284. doi: 10.1021/envhealth.5c00540

Gestational Arsenic Exposure Induces Metabolic Dysfunction-Associated Fatty Liver Disease–like Phenotypes in Middle-Aged Offspring

Qian-Qun Yang †,‡, Qing-Hua Qian †,‡, Wei-Wei Zhang †,‡, Ya-Ping Song †,‡, Qing-Yuan Huang †,‡, Xue Lu †,‡, Tao Wang †,‡, Yi-Chao Huang †,‡,*, De-Xiang Xu †,‡,*
PMCID: PMC13288237  PMID: 42344599

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) refers to hepatic steatosis accompanied by one of the following: type 2 diabetes, obesity, overweight, or metabolic dysfunction. Developmental arsenic (As) exposure is linked to metabolic disorders, but its effects on MAFLD-related phenotypes remain unclear. This study aims to assess the impact of prenatal As exposure on MAFLD-like phenotypes in adulthood offspring. Pregnant mice were given deionized water containing NaAsO2 at concentrations of 0, 1.5, and 15 mg/L throughout their pregnancy. Doses were selected based on environmentally relevant exposure (1.5 mg/L, L-As) and prior evidence of metabolic effects in rodents (15 mg/L, H–As). Gestational H–As exposure increased hepatic triglyceride content and enlarged hepatic lipid droplets in middle-aged offspring. Intraperitoneal glucose tolerance test and insulin tolerance test analyses revealed that prenatal L-As and H–As exposure impaired glucose and insulin tolerance in middle-aged offspring. White fat mass and adipocyte size were elevated in H–As exposed middle-aged offspring. Several metabolism-related hormones, including adiponectin, leptin, and insulin, were elevated, whereas glucagon-like peptide-1, a hormone secreted by the intestines, was reduced in H–As exposed female middle-aged offspring. These results demonstrate that prenatal As-alone promotes MAFLD-like phenotypes in middle-aged offspring, highlighting its role as an early life environmental trigger for metabolic disease.

Keywords: arsenic, MAFLD, hepatic steatosis, obesity, glucose tolerance, insulin resistance, metabolic hormone disorder

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1. Introduction

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a liver disease closely related to metabolic dysfunction. The diagnostic criteria are based on the histological examination results of liver biopsy, imaging examination results, or blood biomarkers. These results indicate the presence of hepatic steatosis while meeting one of three conditions: type 2 diabetes, obesity or overweight, and metabolic dysfunction. , Currently, the prevalence of MAFLD worldwide is approximately 32.4%. Given the rising trends of obesity and type 2 diabetes globally, the prevalence of MAFLD is expected to increase further in the next decade. The hazards of MAFLD are not limited to the liver itself but also affect multiple organs. It is known that MAFLD progression elevates risk of not only cardiovascular diseases but also chronic kidney diseases. − The etiology of MAFLD involves the interaction of multiple factors, including genetic predisposition, lifestyles, metabolic disturbances, and environmental exposure. Environmental toxicants have been linked to the initiation and advancement of MAFLD. Multiple studies demonstrated that chronic exposure to atmospheric contaminants increased the likelihood of MAFLD. − Therefore, investigating the effects of environmental exposure on MAFLD development is a critical public health research priority.

Arsenic (As), a metalloid with extensive environmental distribution, severely threatens human health, primarily through drinking water, skin contact, and other pathways. , Environmental As exposure causes multiorgan toxicity, including reproductive and developmental toxicity, neurotoxicity, and hepatotoxicity, and others. − Several epidemiological reports have confirmed that environmental As exposure is positively associated with risk of MAFLD. , The “Developmental Origins of Health and Disease (DOHaD)” theory assumes that metabolic diseases occur across entire lifecycle but originate in the early life. An early report showed that early life As exposure aggravated high-fat-diet-induced hepatic lipid deposition and metabolic disorders in later life. Although several studies indicated that gestational arsenic exposure induced hepatic lipid accumulation in offspring, , the impact of prenatal arsenic exposure on adulthood MAFLD-like phenotype needs to be explored.

The objective of this investigation was to develop a mouse model of maternal As exposure during gestation. This study focused on evaluating the impacts of As exposure during pregnancy on the MAFLD-like phenotypes in young adult and middle-aged offspring. We were to measure the influences of gestational As exposure on hepatic lipid droplet, glucose and insulin tolerance, metabolic hormones and obesity in young adult and middle-aged offspring. Our research findings provide new evidence suggesting that exposure to As during pregnancy causes MAFLD-like phenotypes in middle-aged offspring.

2. Materials and Methods

2.1. Chemicals and Reagents

Sodium arsenite (NaAsO2, S7400) and Oil Red O (O0625) were obtained from Sigma (USA). Hematoxylin-Eosin (H&E) stain kit (G1120), modified masson’s trichrome stain kit (G1346), triglyceride (TG) assay kit (BC0625), mouse adiponectin enzyme-linked immunosorbent assay (ELISA) kit (SEKM-0142), mouse leptin ELISA kit (SEKM-0105) and mouse insulin ELISA kit (SEKM-0387) were gained from Solarbio (China). Mouse cortisol ELISA kit (CSB-E05113m) was acquired from Cusabio (China). Mouse fibroblast growth factor 21 (FGF21) ELISA kit (E-EL-M0029) and mouse glucagon like peptide 1 (GLP-1) ELISA kit (E-EL-M3108) were from Elabscience (China). Reagent kits for measuring serum biochemical indicators were acquired from Zhejiang Yilikang Biotechnology Co., Ltd. The TRIzol reagent (15596026), Transcriptor First Strand cDNA Synthesis Kit (04897030001), and FastStart Essential DNA Green Master (06924204001) were supplied by Thermo Fisher Scientific and Roche Diagnostics, respectively.

2.2. Animal Treatment

The CD1 mice of 7 weeks were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). All mice were kept under standard specific-pathogen-free (SPF) conditions with a 12:12 h light-dark cycle and ad libitum access to food and water. Following a 7-day acclimation period, female and male mice were paired for mating at a 4:2 ratio during the nocturnal phase. Mice found with a vaginal plug 12 h later were considered pregnant and marked as gestational day (GD) 0. The pregnant mice were randomly divided into three groups: the control group, the low-As group (L-As), and the high-As group (H–As). Each group consisted of 10 pregnant mice. From GD0 to GD18, pregnant mice in the control group received deionized water, while those in the two As groups received water containing NaAsO2 at 1.5 or 15 mg/L. The 1.5 mg/L concentration of NaAsO2, equivalent to 0.87 mg/L inorganic arsenic, represented environmental concentration. , The 15 mg/L concentration of NaAsO2, equivalent to 8.7 mg/L inorganic arsenic, was based on evidence of metabolic effects in rodents from previous research. , On GD18, six pregnant mice per group were euthanized to collect fetal serum and fetal liver for arsenic determination. The remaining dams were permitted to deliver spontaneously, with the day of birth designated as postnatal day (PND) 0. At PND3, litter sizes were standardized to eight pups (4 males and 4 females) to provide sufficient animals for subsequent experiments. For pathological analysis, intraperitoneal glucose tolerance tests (IPGTT), insulin tolerance tests (ITT) and real-time reverse transcription polymerase chain reaction (real-time RT-PCR), 6 representative mice per group were randomly selected to ensure data representativeness. For serum index detection, the sample size was 4–6 per group due to limited serum volume for assaying multiple indicators, which still conforms to the statistical design of small-sample animal experiments. Offspring body weights were recorded every 2 weeks. Mice at postnatal week (PNW)­8 were defined as young adults, corresponding to the stage of sexual maturity with stabilized postnatal growth. Those at PNW28, approximately 7 months old, were categorized as middle-aged, a suitable time point for investigating early aging-related metabolic phenotypes. , IPGTT and ITT were conducted in young adult offspring at PNW8. After completion of the tests and a one-week recovery period, the mice were fasted overnight and subsequently euthanized. Serum samples were collected for biochemical assays and ELISA. Meanwhile, liver, inguinal white adipose tissue (iWAT), and interscapular brown adipose tissue (iBAT) were excised. Portions of liver tissue were allocated for real-time RT-PCR, hepatic TG measurement, and histopathological analysis. At PNW28, experimental procedures and sample collection were identical to those at PNW8. The detailed protocol is shown in Figure S1. All experimental procedures involving animals were reviewed and approved by the Animal Experimentation Committee of Anhui Medical University (LLSC 20190357).

2.3. Measurement of Inorganic Arsenic in Drinking Water Containing NaAsO2

Sodium arsenite powder was accurately weighed and dissolved in deionized water to prepare the stock solution. This stock solution was subsequently diluted to the target concentrations (1.5 and 15 mg/L) using a diluent composed of 1% HNO3 and 2.5% Triton X-100. The stock solution of 29e (ICP-MS-CAL2–1 AccuTrace Reference Standard) and the internal standard stock solution (ICP-MS-200.8-IS-1 AccuTrace Reference Standard) were purchased from AccuStandard, Inc. (USA). All reagents used in the experiment were of high purity. All used glass containers were immersed in 10% nitric acid for 24 h before experiment. The inorganic arsenic concentration in the sodium arsenite solution was then measured using Thermo Scientific iCAP TQ ICP-MS. The inorganic arsenic concentration in the sodium arsenite solution was expressed in mg/L.

2.4. Measurement of As in Fetal Serum and Fetal Liver

The samples were digested overnight with a mixture of nitric acid and hydrogen peroxide at a volume ratio of 3:1 (HNO3:H2O2). As concentrations in fetal serum and fetal liver were determined by hydride generation-atomic fluorescence spectrometry according to the previous study. The As levels were expressed as μg/L for serum and μg/kg for liver.

2.5. IPGTT and ITT

IPGTT and ITT were performed on adult offspring at 8 and 28 weeks of age. For IPGTT, mice were fasted for 16 h and then injected intraperitoneally with glucose (2.0 g/kg). For ITT, after a 4-hour fast, mice received an intraperitoneal injection of insulin (0.75 U/kg). In both tests, blood glucose levels were measured from the tail vein at 0, 15, 30, 60, 90, and 120 min using a glucometer (Roche Accu-Chek Performa).

2.6. Biochemical Analysis

Blood samples from 8-week and 28-week offspring were centrifuged, and the upper plasma was collected. The biochemical indicators were detected using a DIRUI CS-T300 Full-automatic biochemical analyzer (Changchun, China).

2.7. Hepatic TG Measurements

The livers from 8-week and 28-week offspring were weighed and then homogenized in a mixture of n-heptane and isopropanol (in a volume ratio of 1:1). The supernatant was collected after centrifugation for subsequent analysis. The hepatic TG levels were measured using a commercial test kit (Solarbio, TG: Cat# BC0625) according to the manufacturer’s protocols.

2.8. Hepatic Oil Red O Staining

Liver samples collected from 8-week and 28-week offspring were fixed in 4% paraformaldehyde for 24 h, cryoprotected in 30% sucrose for another 24 h, and subsequently embedded. From these embedded tissues, 9 μm-thick sections were prepared with a cryostat, stained with Oil Red O, and counterstained with Hematoxylin. After rinsing with distilled water, the sections were mounted and imaged using an upright fluorescence microscope. Quantitative analysis was carried out with Image-Pro Plus software.

2.9. H&E Staining

Liver tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections of 5 μm thickness were cut and sequentially deparaffinized to water. The sections were then sequentially stained with hematoxylin, differentiated, washed with water, stained with eosin, dehydrated, and mounted. Similarly, iWAT and iBAT samples were processed for H&E staining using the same protocol. The liver histopathological score based on HE staining was performed according to established criteria. , Adipocyte size in iWAT and iBAT was quantitatively analyzed using ImageJ software.

2.10. ELISA

The blood samples from 8-week and 28-week offspring were centrifuged at 4 °C, 3500 × g for 15 min to collect the supernatants. The levels of insulin, leptin, adiponectin, cortisol, FGF21, and GLP-1 were measured using commercial ELISA kits. And the concentrations were determined by plotting standard curves with ELISA Calc software.

2.11. Real-Time RT-PCR

Total RNA was extracted from frozen liver samples using TRIzol reagent. After homogenization and centrifugation, 1-bromo-3-chloropropane was added for phase separation. The upper aqueous phase containing RNA was collected into a nuclease-free tube. Isopropanol was added and mixed by gentle inversion. Following centrifugation, the RNA pellet was collected. The RNA pellet was washed twice with 75% ethanol, air-dried at room temperature, and finally dissolved in nuclease-free water. RNA concentration was quantified by ultraviolet spectrophotometry and adjusted to 500 ng/μL. Reverse transcription was performed using a PCR instrument. Subsequently, quantitative PCR analysis was conducted on the Roche Light Cycler 480 system, employing the SYBR Green I detection method. All primers were obtained from Sangon Biotech. Primers for all genes are listed in Table S1.

2.12. Modified Masson Staining

Liver tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections of 5 μm thickness were cut and deparaffinized to water. After treatment with a mordant solution, the sections were sequentially stained with celestine blue and Mayer’s hematoxylin according to established protocols. Subsequent steps were performed following the manufacturer’s instructions of the staining kit, and quantitative analysis was conducted by observing under a microscope using Image-Pro Plus software.

2.13. Statistical Analysis

Statistical analysis was conducted using SPSS 26.0, and graphs were generated with GraphPad Prism 8.0.2. Data are expressed as mean ± standard error of the mean (mean ± SEM). For normally distributed quantitative variables, intergroup comparisons were carried out by one-way analysis of variance (ANOVA) followed by LSD post hoc test. Non-normally distributed data were analyzed using the Kruskal–Wallis test. A threshold of P < 0.05 was applied to determine statistical significance.

3. Results

3.1. Measurement of External and Internal Exposure

The inorganic arsenic concentration in drinking water was presented in Table S2. The mean inorganic arsenic concentrations were measured as 0.01 mg/L in deionized water, 0.85 mg/L in the 1.5 mg/L NaAsO2 solution, and 8.18 mg/L in the 15 mg/L NaAsO2 solution. Inorganic arsenic levels in fetal serum (μg/L) and fetal liver (μg/kg) are presented in Table S3. Compared to the control group, serum As levels were significantly elevated in the exposed groups. Specifically, the L-As group showed nearly 3-fold higher levels in female fetuses (22.51 ± 2.52 vs 8.50 ± 0.60, P < 0.01) and over 3-fold higher levels in male fetuses (22.34 ± 1.28 vs 6.34 ± 0.97, P < 0.01), whereas the H–As group exhibited even greater increases of approximately 5-fold in females (39.57 ± 1.02 vs 8.50 ± 0.50, P < 0.01) and 7-fold in males (45.89 ± 3.40 vs 6.34 ± 0.97, P < 0.01). Similarly, hepatic As content was significantly increased. The L-As group displayed over 3-fold and 2-fold higher levels in females (38.65 ± 6.05 vs 12.81 ± 1.55, P < 0.01) and males (35.20 ± 6.64 vs 14.62 ± 2.30, P < 0.01), respectively. In the H–As group, the increase was more pronounced, reaching more than 7-fold in females (91.91 ± 6.42 vs 12.81 ± 1.55, P < 0.01) and over 5-fold in males (80.25 ± 5.45 vs 14.62 ± 2.30, P < 0.01).

3.2. Feed Consumption and Body Weight Changes

The influences of prenatal arsenic exposure on drinking water, feed consumption, and weight changes in offspring are presented in Figure S2. As shown in Figure S2A, female offspring in the high-dose group exhibited significant weight gain at PNW28. Similarly, male offspring showed more pronounced increases at both PNW26 and PNW28 (Figure S2B). No statistically significant differences in feed consumption were detected between groups (Figure S2C and D). In addition, no difference in drinking water was shown among different groups (Figure S2E and F). Furthermore, maternal body weight showed no significant differences at GD18, as illustrated in Figure S3A.

3.3. Serum Biochemical Parameters

The impact of arsenic exposure on maternal serum biochemical parameters was evaluated. As presented in Figure S3B–H, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (GLU), TG, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) showed no significant differences across the experimental groups. The effects of gestational arsenic exposure on serum biochemical parameters were assessed in PNW8 offspring. As presented in Table , no difference in serum HDL-C, ALT or AST was observed among different groups. Serum TG levels of both female and male offspring exposed to arsenic were reduced, but this difference was only observed between the L-As group and the control group. No alteration on serum TC and LDL-C was shown in female offspring, whereas a reduction on serum TC and LDL-C was observed in As-exposed male offspring. Next, the impacts of gestational As exposure on serum biochemical parameters were analyzed in PNW28 offspring. As shown in Table , no obvious change was detected in serum TG and ALT levels in arsenic exposed offspring. Serum HDL-C and TC were elevated not only in L-As female offspring but also in L-As and H–As male offspring. Serum LDL-C was elevated in L-As female offspring. Interestingly, LDL-C levels were increased in L-As males but decreased in H–As males. A significant reduction in serum AST was shown in L-As male and female offspring.

1. Serum Biochemical Parameters in Adult Offspring .

    Female
 Male
Stage Parameter 0 mg/L 1.5 mg/L 15 mg/L 0 mg/L 1.5 mg/L 15 mg/L
8W TG (mmol/L) 2.85 ± 0.16 2.24 ± 0.08 2.52 ± 0.15 2.42 ± 0.09 1.83 ± 0.07 2.35 ± 0.10
  TC (mmol/L) 3.54 ± 0.14 3.52 ± 0.16 3.852 ± 0.07 5.05 ± 0.03 4.48 ± 0.18 4.51 ± 0.2
  HDL-C (mmol/L) 1.81 ± 0.10 1.83 ± 0.04 1.88 ± 0.06 2.61 ± 0.05 2.60 ± 0.07 2.54 ± 0.07
  LDL-C (mmol/L) 0.57 ± 0.03 0.47 ± 0.03 0.48 ± 0.01 0.57 ± 0.02 0.41 ± 0.02 0.45 ± 0.03
  ALT (IU/L) 38.6 ± 4.16 34.7 ± 1.32 38.14 ± 1.77 43.03 ± 1.55 43.56 ± 2.82 46.5 ± 2.60
  AST (IU/L) 167.55 ± 10.50 143.1 ± 8.40 159.55 ± 4.04 166.85 ± 4.59 157.15 ± 9.11 170.4 ± 8.39
28W TG (mmol/L) 1.41 ± 0.09 1.23 ± 0.09 1.39 ± 0.05 0.98 ± 0.04 1.00 ± 0.12 1.27 ± 0.12
  TC (mmol/L) 2.74 ± 0.12 3.83 ± 0.11 2.79 ± 0.08 3.82 ± 0.11 4.89 ± 0.18 4.57 ± 0.07
  HDL-C (mmol/L) 1.66 ± 0.08 2.08 ± 0.04 1.64 ± 0.06 2.42 ± 0.04 3.01 ± 0.05 2.82 ± 0.06
  LDL-C (mmol/L) 0.40 ± 0.02 0.55 ± 0.01 0.41 ± 0.04 0.45 ± 0.02 0.53 ± 0.02 0.43 ± 0.02
  ALT (IU/L) 44.63 ± 2.27 36.7 ± 1.58 36.65 ± 0.75 47.38 ± 3.09 53.03 ± 2.78 50.18 ± 3.57
  AST (IU/L) 191.3 ± 8.91 162.4 ± 2.56 177.75 ± 3.18 195.80 ± 16.31 162.6 ± 5.54 187.9 ± 15.61
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a

Note: Abbreviations: TG, Triglyceride; TC, Total cholesterol; HDL-C, High-density lipoprotein cholesterol; LDL-C, Low-density lipoprotein cholesterol; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase. All data are presented as mean ± SEM, N = 8.

b

P < 0.05.

c

P < 0.01.

3.4. Hepatic Steatosis

The influence of gestational arsenic exposure on hepatic steatosis was examined in PNW8 offspring. Although liver weight was not altered (Figure A and G), the liver coefficient was unchanged in female offspring but decreased in L-As male offspring (Figure S4A and B). Hepatic TG was elevated in both L-As male and female offspring (Figure B and H). Hepatic MAFLD scores, determined by H&E staining (Figure C and I), were not altered in PNW8 offspring (Figure E and K). No hepatic lipid droplet deposition, quantified by Oil Red O staining (Figure D and J), was shown in arsenic exposed offspring (Figure F and L). Subsequently, the effects of prenatal arsenic exposure on liver steatosis were assessed in PNW28 offspring. Liver weight remained unchanged (Figure M and S); however, the liver coefficient decreased in L-As and H–As female offspring (Figure S4C), with a reduction seen only in H–As male offspring (Figure S4D). Hepatic TG levels were elevated in both female and male H–As offspring (Figure N and T). Quantitative assessment of H&E-stained sections demonstrated elevated MAFLD scores specifically in arsenic exposed female offspring (Figure O and Q), whereas obvious changes were only detected in H–As male offspring (Figure U and W). Oil Red O staining (Figure P and V) revealed hepatic lipid deposition in H–As offspring (Figure R and X).

1.

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Influence of gestational arsenic exposure on hepatic steatosis in adult offspring. Pregnant mice drank deionized water containing NaAsO2 (either 1.5 or 15 mg/L) from GD0 to GD18. Four dams per group delivered naturally and litters were standardized to 8 pups (four males and four females) for the subsequent study. (A–L) Eight offspring each group were euthanized and liver samples were collected on PNW8. (A and G) Liver weight. (B and H) Hepatic TG content. (C and I) Hepatic pathology was evaluated using H&E staining. Original magnification: × 400. (E and K) MAFLD score. (D and J) Oil Red O staining. Original magnification: × 400. (F and L) Quantitative analysis of hepatic lipid droplets. (M–X) The remaining offspring were euthanized and liver samples were collected on PNW28. (M and S) Liver weight. (N and T) Hepatic TG content. (O and U) Hepatic pathology was evaluated using H&E staining. Original magnification: × 400. (Q and W) MAFLD score. (P and V) Oil Red O staining. Original magnification: × 400. (R and X) Quantitative analysis of hepatic lipid droplets. Abbreviations: MAFLD, metabolic dysfunction-associated fatty liver disease. All data are presented as mean ± SEM (N = 6–8). *P < 0.05, **P < 0.01.

3.5. Glucose Tolerance and Insulin Resistance

The influence of gestational arsenic exposure on glucose tolerance was assessed in PNW8 offspring (Figure A and D). The glucose tolerance test-area under the curve (GTT-AUC) was increased in H–As-exposed female offspring but not altered in As-exposed male offspring (Figure B and E), whereas fasting blood glucose (FBG) was slightly elevated in arsenic exposed female offspring (Figure C) but not males (Figure F). The impact of prenatal arsenic exposure on insulin sensitivity was assessed in PNW8 offspring (Figure G and J). The insulin tolerance test-area under the curve (ITT-AUC) was increased in L-As female offspring (Figure H) but not in H–As females and all males (Figure H and K). For the offspring exposed to the arsenic, the homeostasis model assessment indicator of insulin resistance (HOMA-IR) was increased in L-As female offspring (Figure I), with no change in H–As females or all males (Figure I and L). Subsequently, the impact of arsenic exposure during pregnancy on glucose tolerance was assessed in PNW28 offspring (Figure M and P). The GTT-AUC was elevated in H–As-exposed female offspring and As-exposed male offspring (Figure N and Q). The FBG value was increased in As-exposed offspring (Figure O and R). The influence of gestational As exposure on insulin sensitivity was assessed in PNW28 offspring (Figure S and V). As shown in Figure T and W, the ITT-AUC was elevated in As-exposed female offspring and H–As-exposed males. HOMA-IR was elevated in both L-As and H–As exposed female offspring (Figure U); but was significantly increased only in H–As-exposed male offspring (Figure X).

2.

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Influence of gestational arsenic exposure on glucose tolerance and insulin resistance in adult offspring. Pregnant mice drank deionized water containing NaAsO2 (either 1.5 or 15 mg/L) from GD0 to GD18. Four dams per group delivered naturally and litters were standardized to 8 pups (four males and four females) for the subsequent study. (A–L) Six offspring each group were used for IPGTT and ITT on PNW8. Serum samples were collected to calculate HOMA-IR. (A and D) IPGTT. (B and E) GTT-AUC. (C and F) FBG. (G and J) ITT. (H and K) ITT-AUC. (I and L) HOMA-IR was calculated. (M–X) Six offspring each group were used for IPGTT and ITT on PNW28. Serum samples were collected to calculate HOMA-IR. (M and P) IPGTT. (N and Q) GTT-AUC. (O and R) FBG. (S and V) ITT. (T and W) ITT-AUC. (U and X) HOMA-IR was calculated. Abbreviations: IPGTT, intraperitoneal glucose tolerance test; GTT-AUC, glucose tolerance test - area under the curve; FBG, fasting blood glucose; ITT, insulin tolerance test; ITT-AUC, insulin tolerance test - area under the curve; HOMA-IR, homeostatic model assessment of insulin resistance. All data are presented as mean ± SEM (N = 6). *P < 0.05, **P < 0.01 indicate the difference between the control group and low-dose group; # P < 0.05, ## P < 0.01 indicate the difference between the control group and the high-dose group.

3.6. Obesity

The influence of gestational arsenic exposure on fat metabolism was assessed in PNW8 offspring. Although it was not altered in female offspring (Figure A), fasted body weight was gained in L-As male offspring (Figure D). iWAT weight was unchanged in arsenic exposed offspring (Figure B and E). Additionally, iBAT weight was not altered in both genders of As-exposed offspring (Figure C and F). Next, adipocyte size was evaluated using H&E staining (Figure G, I, K, M). As shown in Figure H, iWAT adipocyte size was enlarged in H–As females but not in males (Figure J). No alterations were detected in iBAT adipocyte size in either gender (Figure L and N). The impact of prenatal arsenic exposure on fat metabolism was assessed in PNW28 offspring. Fasted body weight was gained in arsenic exposed female offspring and H–As-exposed male offspring (Figure O and R). An obvious elevation of abdominal fats was showed in offspring following arsenic exposure (Figure S5A and B). Quantitative analysis showed that iWAT weight was increased in arsenic exposed female offspring and H–As-exposed male offspring (Figure P and S). Although it was not altered in female offspring (Figure Q), iBAT weight was slightly gained in arsenic exposed male offspring (Figure T). iWAT adipocyte size was evaluated using H&E staining (Figure U and W). As shown in Figure V and X, iWAT adipocyte size was enlarged in H–As female and male offspring. Finally, iBAT adipocyte size was evaluated in PNW28 offspring (Figure Y and AA). As shown in Figure Z and AB, iBAT adipocyte size was slightly enlarged in H–As female and male offspring.

3.

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Influence of gestational arsenic exposure on obesity in adult offspring. Pregnant mice drank deionized water containing NaAsO2 (either 1.5 or 15 mg/L) from GD0 to GD18. Four dams per group delivered naturally and litters were standardized to 8 pups (four males and four females) for the subsequent study. (A–N) Eight offspring each group were euthanized and fatty samples were collected on PNW8. (A and D) Body weight. (B and E) iWAT weight. (C and F) iBAT weight. (G and I) iWAT pathology was evaluated using H&E staining. Original magnification: × 400. (H and J) Quantitative analysis of iWAT adipocyte size. (K and M) iBAT pathology was evaluated using H&E staining. Original magnification: × 400. (L and N) Quantitative analysis of iBAT adipocyte size. (O–AB) The remaining offspring were euthanized and fatty samples were collected on PNW28. (O and R) Body weight. (P and S) iWAT weight. (Q and T) iBAT weight. (U and W) iWAT pathology was evaluated using H&E staining. Original magnification: × 400. (V and X) Quantitative analysis of iWAT adipocyte size. (Y and AA) iBAT pathology was evaluated using H&E staining. Original magnification: × 400. (Z and AB) Quantitative analysis of iBAT adipocyte size. Abbreviations: iWAT, inguinal white adipose tissue; iBAT, interscapular brown adipose tissue; Ad, adipocyte. All data are presented as mean ± SEM (N = 6–8). *P < 0.05, **P < 0.01.

3.7. Serum Metabolic Hormones

The influence of gestational arsenic exposure on serum metabolic hormones was detected in PNW8 offspring. No change in serum insulin was observed in both genders of arsenic exposed offspring (Figure A and D). Serum GLP-1 was mildly elevated in H–As-exposed female offspring but not males (Figure B and E). No significant change in serum leptin was observed in offspring following arsenic exposure (Figure C and F). Moreover, no change in serum adiponectin was observed in both genders of arsenic exposed offspring (Figure G and J). Serum cortisol was unchanged in arsenic exposed female offspring (Figure H) but reduced in H–As males (Figure K). Finally, serum FGF21 was not altered in female and male offspring following arsenic exposure (Figure I and L). Next, the impact of prenatal arsenic exposure on serum metabolic hormones was detected in PNW28 offspring. Insulin level in serum was slightly increased in female offspring from the H–As group (Figure M) but not in males (Figure P). Serum GLP-1 was reduced in As-exposed female offspring (Figure N) but not male offspring (Figure Q). Leptin levels in serum were significantly increased in H–As-exposed offspring (Figure O and R). Serum adiponectin was elevated exclusively in female offspring following arsenic exposure (Figure S and V). Similarly, serum cortisol was increased in H–As-exposed female offspring (Figure T) but not male offspring (Figure W). Finally, no difference in serum FGF21 was shown in both genders (Figure U and X).

4.

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Influence of gestational arsenic exposure on glucose metabolic hormones in adult offspring. Pregnant mice drank deionized water containing NaAsO2 (either 1.5 or 15 mg/L) from GD0 to GD18. Four dams per group delivered naturally and litters were standardized to 8 pups (four males and four females) for the subsequent study. (A–L) Eight offspring each group were euthanized and serum samples were collected on PNW8. (A and D) Serum insulin. (B and E) Serum GLP-1. (C and F) Serum leptin. (G and J) Serum adiponectin. (H and K) Serum cortisol. (I and L) Serum FGF21. (M–X) The remaining offspring were euthanized and serum samples were collected on PNW28. (M and P) Serum insulin. (N and Q) Serum GLP-1. (O and R) Serum leptin. (S and V) Serum adiponectin. (T and W) Serum cortisol. (U and X) Serum FGF21. Abbreviations: GLP-1, glucagon like peptide 1; FGF21, fibroblast growth factor 21; ADPN, adiponectin. All data are presented as mean ± SEM (N = 4–6). *P < 0.05, **P < 0.01.

3.8. Hepatic Inflammatory Cytokines and Fibrosis

The influence of gestational arsenic exposure on hepatic inflammatory cytokines was assessed in PNW8 offspring. Hepatic inflammatory cytokines, including Il6, Il1b, Tgfb1, and Cxcl15, were not altered in both genders of As-exposed offspring (Figure A and C). Hepatic fibrotic genes were analyzed using real-time RT-PCR. As illustrated in Figure B and D, hepatic Acta2, Cdh1, Cdh2, and Vim, four fibrotic genes, were not altered in As-exposed offspring. Hepatic fibrosis was evaluated using Masson staining (Figure E and G). No alteration in collagen area was detected in PNW8 offspring (Figure F and H). Subsequently, the influence of gestational arsenic exposure on hepatic inflammatory cytokines were measured in PNW28 offspring. No upregulation on hepatic Il6, Il1b, Tgfb1 and Cxcl15 mRNAs was shown in As-exposed females (Figure I). Similarly, no significant change on hepatic Il6, Il1b and Cxcl15 mRNAs was assessed in arsenic exposed male offspring (Figure K). Unexpectedly, hepatic Tgfb1 mRNA was downregulated in H–As-exposed male offspring (Figure K). The expression of fibrotic genes was analyzed in PNW28 offspring. Hepatic Acta2, Cdh1, Cdh2, and Vim mRNAs were not altered in arsenic exposed female offspring (Figure J). No significant change on hepatic Acta2 mRNA was shown in arsenic exposed male offspring (Figure L). A significant downregulation of hepatic Cdh1 and Vim was observed in the H–As male offspring (Figure L), whereas Cdh2 expression was decreased in both dose groups (Figure L). Finally, hepatic fibrosis was evaluated in PNW28 offspring (Figure M and O). No alteration in collagen area was detected in both genders of As-exposed offspring (Figure N and P).

5.

5

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Influence of gestational arsenic exposure on hepatic inflammatory cytokines and fibrotic markers in adult offspring. Pregnant mice drank deionized water containing NaAsO2 (either 1.5 or 15 mg/L) from GD0 to GD18. Four dams per group delivered naturally and litters were standardized to 8 pups (four males and four females) for the subsequent study. (A–H) Six offspring each group were euthanized and liver samples were collected on PNW8. (A and C) Hepatic Il6, Il1b, Tgfb1 and Cxcl15 mRNAs were measured using real-time RT-PCR. (B and D) Hepatic epithelial-mesenchymal transition (EMT) markers Acta2, Cdh1, Cdh2, and Vim mRNAs were measured using real-time RT-PCR. (E and G) Representative images of Masson staining. Original magnification: × 400. (F and H) Quantitative analysis of collagen area. (I–P) Six offspring each group were euthanized and liver samples were collected on PNW28. (I and K) Hepatic Il6, Il1b, Tgfb1 and Cxcl15 mRNAs were measured using real-time RT-PCR. (J and L) Hepatic EMT markers Acta2, Cdh1, Cdh2, and Vim mRNAs were measured using real-time RT-PCR. (M and O) Representative images of Masson staining. Original magnification: ×400. (N and P) Quantitative analysis of collagen area. All data are presented as mean ± SEM (N = 6). *P < 0.05, **P < 0.01.

4. Discussion

This study established a mouse model to evaluate the impact of prenatal arsenic exposure on adulthood MAFLD-like phenotypes. The novel results were as below: prenatal arsenic exposure induced hepatic lipid accumulation in middle-aged offspring, manifested as increased hepatic TG content and enlarged lipid droplet accumulation; gestational arsenic exposure caused obesity-like phenotype in middle-aged offspring, as evidenced by increased white fat mass and adipocyte size; gestational arsenic exposure induced glucose tolerance and insulin resistance in middle-aged offspring, as demonstrated by IPGTT, ITT and alterations of glucose metabolic hormones. It is widely accepted that hepatic steatosis accompanied by at least one of the following phenotypes: type 2 diabetes, obesity or overweight, and metabolic dysfunction, could be diagnosed as MAFLD. ,, These results suggest that arsenic exposure during pregnancy induces MAFLD-like phenotypes in middle-aged offspring.

Despite established international safety guidelines, arsenic concentrations in drinking water frequently exceed these limits in numerous regions globally, posing a significant public health concern. For instance, arsenic concentration in the groundwater of a typical smelter site in southwest China was extremely high, with an overall range of 0.00613–32.3 mg/L, and the highest contaminated site monitored was even as high as 111.64 mg/L during the dry season. Another report showed that arsenic concentration in the groundwater of a region in India ranged from 0.41 to 1.01 mg/L. In this experiment, the arsenic concentration in the L-As group was 1.5 mg/L, equivalent to 0.87 mg/L inorganic arsenic, which was considered comparable to the environmental concentration level reported in some regions. According to a Chinese birth cohort, the median serum arsenic content in pregnant women was 4.87 μg/L, with a range of 0.02 μg/L to 43.52 μg/L. A previous study showed that the mean serum arsenic level in pregnant mice was 55.47 μg/L in the L-As (1.5 mg/L NaAsO2) group, which was comparable to the higher serum arsenic concentrations reported for pregnant women in noncontaminated areas. Meanwhile, the arsenic level in fetal serum of our L-As exposure group was within the range of reported umbilical cord serum concentrations (19.83 ± 10.50 μg/L). We used the CD-1 mouse strain, a common model in developmental toxicology, and found that gestational arsenic exposure induced MAFLD-like phenotypes in middle-aged offspring. To confirm the generalizability of this effect, future studies should validate the finding in other strains such as C57BL/6.

Hepatic steatosis is an early phenotype of MAFLD. Numerous epidemiological data have confirmed that urinary arsenic levels exhibit a significant positive correlation with hepatic steatosis risk. , Animal studies indicate that long-term arsenic exposure causes lipid deposition in the liver. , Previous experiments have paid attention to the impacts of postnatal arsenic exposure on hepatic steatosis. − Two reports from our laboratory revealed that prenatal arsenic exposure led to hepatic lipid deposition in adulthood. , In this study, we investigated the influences of prenatal exposure to relatively lower concentrations of inorganic arsenic on hepatic steatosis in middle-aged offspring. Our results suggested that prenatal arsenic exposure caused hepatic lipid deposition in middle-aged but not young adult offspring. Even MAFLD scores were increased in middle-aged female offspring whose mothers were exposed to a relatively lower concentration of arsenic (1.5 mg/L). Moreover, hepatic lipid droplets were elevated in middle-aged male offspring. All the results show that fetal period is a sensitive time window for As-induced hepatic steatosis in later life.

Glucose tolerance and insulin resistance are the core incentives underlying development and progression of MAFLD. , Environmental arsenic exposure has been linked to type 2 diabetes. − Several animal experiments indicated that long-term arsenic exposure caused glucose tolerance and insulin resistance. , A recent experiment indicated that maternal exposure to arsenic from preconception to the entire pregnancy led to insulin resistance in their offspring. In this study, we found that maternal arsenic exposure throughout pregnancy caused glucose and insulin resistance in middle-aged offspring. GLP-1, a gut-derived incretin hormone, promotes glucose-dependent insulin secretion. Increasing data confirmed that serum GLP-1 level was decreased in patients with type 2 diabetes. This study showed that serum GLP-1 was elevated in As-exposed young adult offspring but reduced in middle-aged offspring. Two early reports indicated that leptin and insulin resistances suppressed the release of GLP-1 from enteroendocrine L cells. , A recent study found that statin drugs reduced serum GLP-1 by disrupting microbiota-mediated bile acid metabolism. The mechanism by which gestational arsenic exposure downregulates serum GLP-1 release in middle-aged offspring needs to be further explored.

Obesity or overweight is a phenotype associated with MAFLD. Epidemiological data have confirmed that urinary As concentration is associated with overweight and obesity. , An earlier experiment showed that exposure during adulthood to As-containing water for 9 weeks elevated fat mass and subcutaneous inguinal white adipose tissue in male mice. Another experiment found that postnatal exposure to As-containing water for 16 weeks aggravated high-fat-evoked obesity in male animals. Previous studies have focused more on obesity caused by arsenic exposure after birth. A recent experiment found that maternal drinking sodium arsenite water, beginning 15 days before pregnancy until the entire pregnancy, elevated fat mass in male offspring. Different from the above studies, our results revealed that gestational arsenic exposure interfered with adulthood fat metabolism, manifested as increased white fat mass and enlarged adipocyte size. It is known that white fat is an active endocrine organ that secretes glucose metabolic hormones, such as leptin and adiponectin. Leptin secretion was positively correlated with adipose tissue volume. Our study demonstrated that adipocyte size was significantly increased, accompanied by elevated serum leptin levels, suggesting that prenatal arsenic exposure might be a factor for adulthood overweight and obesity.

This study demonstrated that gestational arsenic exposure induced a MAFLD-like phenotype in middle-aged offspring, which was characterized by hepatic steatosis, glucose intolerance, insulin resistance and obesity. The potential underlying mechanisms may involve epigenetic reprogramming. As supported by previous research, arsenic can accelerate the progression of nonalcoholic fatty liver disease (NAFLD) either by activating the CD36-NLRP3 inflammasome axis via PDHA1-mediated H3K18 lactylation or by inhibiting S-adenosylmethionine dependent histone methylation. Notably, during early life, arsenic exposure suppressed DNA hydroxymethylation of key genes related to β-oxidation, which in turn led to hepatic lipid deposition in female offspring. Mechanisms beyond epigenetics include disruption of gut microbiota homeostasis. Arsenic may induce gut dysbiosis, thereby interfering with hepatic bile acid metabolism. It can also inhibit methionine synthase activity and perturbed one-carbon metabolism, this perturbation disrupted the structure and function of gut microbiota, ultimately impairing hepatic metabolic homeostasis. In conclusion, although our study confirmed that gestational arsenic exposure induced a MAFLD-like phenotype in middle-aged offspring, the precise molecular mechanisms which may involve the epigenetic and gut-liver axis pathways noted above await further systematic elucidation.

Collectively, our results provide direct evidence for the association between prenatal environmental exposure and the developmental origins of adulthood metabolic diseases. The key highlights of this study are summarized as follows: First, this study systematically characterized MAFLD-like phenotypes induced by gestational arsenic exposure, including hepatic steatosis, insulin resistance and obesity. Second, dynamic monitoring of adult offspring at 8 and 28 weeks further supported the DOHaD theory, highlighting the delayed-onset nature of developmental origin diseases. Finally, alterations in metabolic-related hormones were identified, providing critical clues for future mechanistic studies. Notably, two specific observations were made. First, a nonmonotonic dose–response relationship was observed in young offspring (8 weeks), with levels of indicators such as hepatic triglycerides increasing in the low-dose group and showing no significant change in the high-dose group. This phenomenon is potentially attributable to more severe intrauterine growth restriction (IUGR) in the high-dose group, which could mask subsequent lipid accumulation during the catch-up growth phase. , Second, this study found that female offspring exposed to arsenic during pregnancy exhibited more obvious MAFLD-like phenotypes, whereas epidemiological data demonstrated a higher MAFLD incidence in human males. Increasing evidence suggests that adverse maternal exposures during pregnancy disrupt the establishment of sexual dimorphism in offspring liver through epigenetic reprogramming, with more significant interference in metabolic reprogramming observed in female offspring. , Future studies should further investigate the specific mechanisms through which epigenetic reprogramming mediates sexual dimorphism in MAFLD-like phenotypes.

However, there are several shortcomings in this study. First, this study did not explore the underlying mechanism through which gestational arsenic exposure induced MAFLD-like phenotypes in middle-aged offspring. Recent research has revealed that prenatal exposure to perfluorinated compounds caused autism-like phenotype by disrupting colonization of intestinal microbiota. Indeed, the association between gut microbiota disorder and MAFLD has been widely confirmed. , Next study is to explore the role of gut microbiota colonization on early life As-induced adulthood MAFLD-like phenotypes. Second, this study did not track the long-term effects of prenatal arsenic exposure on hepatic lipid metabolism. Indeed, the present results found that prenatal arsenic exposure caused MAFLD-like phenotypes in middle-aged offspring but not in young adult offspring, suggesting a lag effect. It is known that hepatic steatosis induces steatohepatitis and ultimately progresses to liver fibrosis. In this study, we showed no significant increase in hepatic inflammatory cytokines and fibrotic indicators in As-exposed middle-aged offspring. Further work is required to extend the observation time to track the progression of As-induced MAFLD in middle-aged offspring.

5. Conclusions

Using a full-pregnancy exposure model, this study found that gestational arsenic exposure induced a MAFLD-like phenotype in middle-aged offspring. Different from the conventional view that emphasizes genetic predispositions and unhealthy lifestyle factors, this study revealed that early life environmental arsenic exposure acts as an independent risk factor for MAFLD. It provides an indispensable environmental perspective for understanding the etiology of MAFLD.

These findings not only deepen our understanding of MAFLD etiology research but also provide actionable guidance for public health interventions. From a public health perspective, our results highlight the importance of regarding pregnancy as a critical window for preventing long-term metabolic diseases in offspring. This provides a direct scientific basis for developing targeted public health strategies for pregnant women. In the future, systematic health education could enhance this population’s awareness of the long-term health risks posed to offspring by environmental pollutants such as arsenic. Guiding pregnant women to minimize arsenic exposure during pregnancy via practical strategies, including upgrading drinking water quality and refining dietary habits, may achieve early, source-oriented prevention of chronic metabolic disorders in their offspring.

Supplementary Material

eh5c00540_si_001.docx (692KB, docx)

Acknowledgments

This work was supported by National Natural Science Foundation of China (82330102, 82304186).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00540.

  • Sequences of the primers for real-time PCR; concentrations of inorganic arsenic in drinking water; concentrations of total arsenic in fetal serum and fetal liver; detailed experimental protocols; effects of gestational arsenic exposure on offspring weight, feed consumption, and water intake; impact of gestational arsenic exposure on maternal body weight and serum biochemical parameters at GD18; influence of gestational arsenic exposure on liver coefficient in adult offspring; effects of gestational arsenic exposure on abdominal fat deposition in offspring (DOCX)

§.

Q.-Q.Y., Q.-H.Q., and W.-W.Z. contributed equally. Qian-Qun Yang: Writing-original draft, Data curation, Formal analysis. Qing-Hua Qian: Project administration, Methodology. Wei-Wei Zhang: Visualization, Supervision. Ya-Ping Song: Funding acquisition. Qing-Yuan Huang: Methodology. Xue Lu: Visualization. Tao Wang: Validation. Yi-Chao Huang: Supervision, Writing-review and editing. De-Xiang Xu: Conceptualization, Funding acquisition, Resources, Writing-review and editing.

The authors declare no competing financial interest.

References

  1. Eslam M., Newsome P. N., Sarin S. K., Anstee Q. M., Targher G., Romero-Gomez M., Zelber-Sagi S., Wai-Sun Wong V., Dufour J. F., Schattenberg J. M.. et al. A new definition for metabolic dysfunction-associated fatty liver disease: An international expert consensus statement. J. Hepatol. 2020;73(1):202–209. doi: 10.1016/j.jhep.2020.03.039. [DOI] [PubMed] [Google Scholar]
  2. Nguyen V. H., Le M. H., Cheung R. C., Nguyen M. H.. Differential Clinical Characteristics and Mortality Outcomes in Persons With NAFLD and/or MAFLD. Clin Gastroenterol Hepatol. 2021;19(10):2172–2181.e6. doi: 10.1016/j.cgh.2021.05.029. [DOI] [PubMed] [Google Scholar]
  3. Shiha G., Korenjak M., Eskridge W., Casanovas T., Velez-Moller P., Hogstrom S., Richardson B., Munoz C., Sigurethardottir S., Coulibaly A.. et al. Redefining fatty liver disease: an international patient perspective. Lancet Gastroenterol Hepatol. 2021;6(1):73–79. doi: 10.1016/S2468-1253(20)30294-6. [DOI] [PubMed] [Google Scholar]
  4. Liu J., Ayada I., Zhang X., Wang L., Li Y., Wen T., Ma Z., Bruno M. J., de Knegt R. J., Cao W.. et al. Estimating Global Prevalence of Metabolic Dysfunction-Associated Fatty Liver Disease in Overweight or Obese Adults. Clin Gastroenterol Hepatol. 2022;20(3):e573–e582. doi: 10.1016/j.cgh.2021.02.030. [DOI] [PubMed] [Google Scholar]
  5. Lim G. E. H., Tang A., Ng C. H., Chin Y. H., Lim W. H., Tan D. J. H., Yong J. N., Xiao J., Lee C. W., Chan M.. et al. An Observational Data Meta-analysis on the Differences in Prevalence and Risk Factors Between MAFLD vs NAFLD. Clin Gastroenterol Hepatol. 2023;21(3):619–629.e7. doi: 10.1016/j.cgh.2021.11.038. [DOI] [PubMed] [Google Scholar]
  6. Pan Z., Shiha G., Esmat G., Mendez-Sanchez N., Eslam M.. MAFLD predicts cardiovascular disease risk better than MASLD. Liver Int. 2024;44(7):1567–1574. doi: 10.1111/liv.15931. [DOI] [PubMed] [Google Scholar]
  7. Wang T. Y., Wang R. F., Bu Z. Y., Targher G., Byrne C. D., Sun D. Q., Zheng M. H.. Association of metabolic dysfunction-associated fatty liver disease with kidney disease. Nat. Rev. Nephrol. 2022;18(4):259–268. doi: 10.1038/s41581-021-00519-y. [DOI] [PubMed] [Google Scholar]
  8. Zhou X. D., Targher G., Byrne C. D., Somers V., Kim S. U., Chahal C. A. A., Wong V. W., Cai J., Shapiro M. D., Eslam M.. et al. An international multidisciplinary consensus statement on MAFLD and the risk of CVD. Hepatol Int. 2023;17(4):773–791. doi: 10.1007/s12072-023-10543-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feng C., Yang B., Wang Z., Zhang J., Fu Y., Yu B., Dong S., Ma H., Liu H., Zeng H.. et al. Relationship of long-term exposure to air pollutant mixture with metabolic-associated fatty liver disease and subtypes: A retrospective cohort study of the employed population of Southwest China. Environ. Int. 2024;188:108734. doi: 10.1016/j.envint.2024.108734. [DOI] [PubMed] [Google Scholar]
  10. Guo B., Huang S., Li S., Han X., Lin H., Li Y., Qin Z., Jiang X., Wang Z., Pan Y.. et al. Long-term exposure to ambient PM2.5 and its constituents is associated with MAFLD. JHEP Rep. 2023;5(12):100912. doi: 10.1016/j.jhepr.2023.100912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Zhang J., Ran S., Wei S., Tian F., Chen L., Yang Z., Chen G., Lin H.. Associations of MAFLD subtypes and air pollutants with multi-system morbidity and all-cause mortality: A prospective cohort study. Ecotoxicol Environ. Saf. 2025;291:117893. doi: 10.1016/j.ecoenv.2025.117893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sultan M. W., Qureshi F., Ahmed S., Kamyab H., Rajendran S., Ibrahim H., Yusuf M.. A comprehensive review on arsenic contamination in groundwater: Sources, detection, mitigation strategies and cost analysis. Environ. Res. 2025;265:120457. doi: 10.1016/j.envres.2024.120457. [DOI] [PubMed] [Google Scholar]
  13. Tian C., Wu Q., Chen F., Wu Y., Wang J., Zhang A., Wang W.. Dynamic Telomere Length Response to Neurodevelopmental Arsenic Exposure: Insights into Transcriptional Regulation and Neuronal Morphogenesis. Environ. Health (Wash) 2025;3(9):1031–1042. doi: 10.1021/envhealth.5c00026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Niu F. W., Liu M. D., Yao K., Yang R., Gao L., Zhai J. X., Wang C., Zhang S. H., Xu D. X., Zhang Z. H.. Mitochondrial ROS-associated integrated stress response is involved in arsenic-induced blood-testis barrier disruption and protective effect of melatonin. Environ. Int. 2025;197:109346. doi: 10.1016/j.envint.2025.109346. [DOI] [PubMed] [Google Scholar]
  15. Lv Y., Chen X., Jia Z., Wang Y., Zhu Z., Li C., Xu S., Li Y.. Prenatal exposure to arsenic, umbilical cord blood DNA methylation, and child neurodevelopment: A prospective birth cohort study. Environ. Res. 2025;280:121914. doi: 10.1016/j.envres.2025.121914. [DOI] [PubMed] [Google Scholar]
  16. Zhao Y., Guo M., Pei T., Shang C., Chen Y., Zhao L., Lu Y., Liang C., Wang J., Zhang J.. alpha-Lipoic Acid Ameliorates Arsenic-Induced Lipid Disorders by Promoting Peroxisomal beta-Oxidation and Reducing Lipophagy in Chicken Hepatocyte. Adv. Sci. (Weinh) 2025;12(11):e2413255. doi: 10.1002/advs.202413255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Davydiuk T., Tao J., Lu X., Le X. C.. Effects of Dietary Intake of Arsenosugars and Other Organic Arsenic Species on Studies of Arsenic Methylation Efficiency in Humans. Environ. Health (Wash) 2023;1(4):236–248. doi: 10.1021/envhealth.3c00090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McKeon H. P., Chen W., Te Biesebeek J. D., Vrijenhoek N. G., Castenmiller J. J. M., Mengelers M. J. B.. Urinary concentrations of arsenic species in older Dutch adults and risk of chronic kidney disease. Environ. Int. 2025;196:109289. doi: 10.1016/j.envint.2025.109289. [DOI] [PubMed] [Google Scholar]
  19. Frediani J. K., Naioti E. A., Vos M. B., Figueroa J., Marsit C. J., Welsh J. A.. Arsenic exposure and risk of nonalcoholic fatty liver disease (NAFLD) among U.S. adolescents and adults: an association modified by race/ethnicity, NHANES 2005–2014. Environ. Health. 2018;17(1):6. doi: 10.1186/s12940-017-0350-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu Y., Li W., Zhang J., Yan Y., Zhou Q., Liu Q., Guan Y., Zhao Z., An J., Cheng X.. et al. Associations of arsenic exposure and arsenic metabolism with the risk of non-alcoholic fatty liver disease. Int. J. Hyg Environ. Health. 2024;257:114342. doi: 10.1016/j.ijheh.2024.114342. [DOI] [PubMed] [Google Scholar]
  21. Hoffman D. J., Powell T. L., Barrett E. S., Hardy D. B.. Developmental origins of metabolic diseases. Physiol Rev. 2021;101(3):739–795. doi: 10.1152/physrev.00002.2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ditzel E. J., Nguyen T., Parker P., Camenisch T. D.. Effects of Arsenite Exposure during Fetal Development on Energy Metabolism and Susceptibility to Diet-Induced Fatty Liver Disease in Male Mice. Environ. Health Perspect. 2016;124(2):201–209. doi: 10.1289/ehp.1409501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Qian Q. H., Song Y. P., Zhang Y., Xue H., Zhang W. W., Han Y., Wang Y., Xu D. X.. Gestational alpha-ketoglutarate supplementation ameliorates arsenic-induced hepatic lipid deposition via epigenetic reprogramming of beta-oxidation process in female offspring. Environ. Int. 2024;185:108488. doi: 10.1016/j.envint.2024.108488. [DOI] [PubMed] [Google Scholar]
  24. Song Y. P., Lv J. W., Zhao Y., Chen X., Zhang Z. C., Fan Y. J., Zhang C., Gao L., Huang Y., Wang H.. et al. DNA hydroxymethylation reprogramming of beta-oxidation genes mediates early-life arsenic-evoked hepatic lipid accumulation in adult mice. J. Hazard Mater. 2022;430:128511. doi: 10.1016/j.jhazmat.2022.128511. [DOI] [PubMed] [Google Scholar]
  25. Francisca F. M., Carro Perez M. E.. Assessment of natural arsenic in groundwater in Cordoba Province, Argentina. Environ. Geochem Health. 2009;31(6):673–682. doi: 10.1007/s10653-008-9245-y. [DOI] [PubMed] [Google Scholar]
  26. Smith A. H., Lingas E. O., Rahman M.. Contamination of drinking-water by arsenic in Bangladesh: a public health emergency. Bull. World Health Organ. 2000;78(9):1093–1103. [PMC free article] [PubMed] [Google Scholar]
  27. Li X., Li Y., Jin Y., Zhang Y., Wu J., Xu Z., Huang Y., Cai L., Gao S., Liu T.. et al. Transcriptional and epigenetic decoding of the microglial aging process. Nat. Aging. 2023;3(10):1288–1311. doi: 10.1038/s43587-023-00479-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Song Y. P., Lv J. W., Zhang Z. C., Qian Q. H., Fan Y. J., Chen D. Z., Zhang H., Xu F. X., Zhang C., Huang Y.. et al. Effects of Gestational Arsenic Exposures on Placental and Fetal Development in Mice: The Role of Cyr61 m6A. Environ. Health Perspect. 2023;131(9):97004. doi: 10.1289/EHP12207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Loomba R., Bedossa P., Grimmer K., Kemble G., Bruno Martins E., McCulloch W., O’Farrell M., Tsai W. W., Cobiella J., Lawitz E.. et al. Denifanstat for the treatment of metabolic dysfunction-associated steatohepatitis: a multicentre, double-blind, randomised, placebo-controlled, phase 2b trial. Lancet Gastroenterol Hepatol. 2024;9(12):1090–1100. doi: 10.1016/S2468-1253(24)00246-2. [DOI] [PubMed] [Google Scholar]
  30. Miao G. L., Zhang W. X., Xu Y. T., Liu Y. R., Lai P. P., Guo J. B., Chen G. L., Chen J. X., Zhou Z. H., Li Y. W.. et al. Motor protein KIF13B orchestrates hepatic metabolism to prevent metabolic dysfunction-associated fatty liver disease. Mil Med. Res. 2025;12(1):11. doi: 10.1186/s40779-025-00594-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Chen L., Tao X., Zeng M., Mi Y., Xu L.. Clinical and histological features under different nomenclatures of fatty liver disease: NAFLD, MAFLD, MASLD and MetALD. J. Hepatol. 2024;80(2):e64–e66. doi: 10.1016/j.jhep.2023.08.021. [DOI] [PubMed] [Google Scholar]
  32. Gofton C., Upendran Y., Zheng M. H., George J.. MAFLD: How is it different from NAFLD? Clin Mol. Hepatol. 2023;29(Suppl):S17–S31. doi: 10.3350/cmh.2022.0367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhang K., Mao K., Xue J., Chen Z., Du W., Zhang H.. Characteristics and risk assessment of heavy metals in groundwater at a typical smelter-contaminated site in Southwest China. Environ. Pollut. 2024;357:124401. doi: 10.1016/j.envpol.2024.124401. [DOI] [PubMed] [Google Scholar]
  34. Rahaman S., Sinha A. C., Pati R., Mukhopadhyay D.. Arsenic contamination: a potential hazard to the affected areas of West Bengal, India. Environ. Geochem Health. 2013;35(1):119–132. doi: 10.1007/s10653-012-9460-4. [DOI] [PubMed] [Google Scholar]
  35. Wang H., Li J., Zhang X., Zhu P., Hao J. H., Tao F. B., Xu D. X.. Maternal serum arsenic level during pregnancy is positively associated with adverse pregnant outcomes in a Chinese population. Toxicol. Appl. Pharmacol. 2018;356:114–119. doi: 10.1016/j.taap.2018.07.030. [DOI] [PubMed] [Google Scholar]
  36. Xu C., Tang M., Zhu S., Naranmandura H., Liu W.. Assessment of arsenic in colostrum and cord serum and risk exposure to neonates from an island population in China. Environmental science and pollution research international. 2016;23(22):22467–22476. doi: 10.1007/s11356-016-7265-1. [DOI] [PubMed] [Google Scholar]
  37. Fan B., Cheng C., Yang Y., Wang P., Xia H., Wu M., Li H., Manzoor Syed B., Liu Q.. Construction of an adverse outcome pathway framework based on integrated data to evaluate arsenic-induced non-alcoholic fatty liver disease. Environ. Int. 2024;183:108381. doi: 10.1016/j.envint.2023.108381. [DOI] [PubMed] [Google Scholar]
  38. Lu L., Hua W., Li F., Che Z., Tian M., Lu Y. Y., Chi Q., Zhang J., Huang Q.. Arsenic Exposure Triggers Nonalcoholic Fatty Liver Disease through Repressing S-Adenosylmethionine-Dependent Histone Methylation in Rats. Environ. Sci. Technol. 2025;59(1):268–278. doi: 10.1021/acs.est.4c10417. [DOI] [PubMed] [Google Scholar]
  39. Qiu T., Pei P., Yao X., Jiang L., Wei S., Wang Z., Bai J., Yang G., Gao N., Yang L.. et al. Taurine attenuates arsenic-induced pyroptosis and nonalcoholic steatohepatitis by inhibiting the autophagic-inflammasomal pathway. Cell Death Dis. 2018;9(10):946. doi: 10.1038/s41419-018-1004-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Chen X., Wu R., Wu H., Hu Y., Wang H., Fu J., Pi J., Xu Y.. Integrated miRNA-mRNA analysis reveals the dysregulation of lipid metabolism in mouse liver induced by developmental arsenic exposure. J. Hazard Mater. 2023;445:130459. doi: 10.1016/j.jhazmat.2022.130459. [DOI] [PubMed] [Google Scholar]
  41. Liu Q., Li P., Ma J., Zhang J., Li W., Liu Y., Liu L., Liang S., He M.. Arsenic exposure at environmentally relevant levels induced metabolic toxicity in development mice: Mechanistic insights from integrated transcriptome and metabolome. Environ. Int. 2024;190:108819. doi: 10.1016/j.envint.2024.108819. [DOI] [PubMed] [Google Scholar]
  42. Yang X., Weber A. A., Mennillo E., Secrest P., Chang M., Wong S., Le S., Liu J., Benner C. W., Karin M.. et al. Effects of Early Life Oral Arsenic Exposure on Intestinal Tract Development and Lipid Homeostasis in Neonatal Mice: Implications for NAFLD Development. Environ. Health Perspect. 2023;131(9):97001. doi: 10.1289/EHP12381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Li M., Chi X., Wang Y., Setrerrahmane S., Xie W., Xu H.. Trends in insulin resistance: insights into mechanisms and therapeutic strategy. Signal Transduct Target Ther. 2022;7(1):216. doi: 10.1038/s41392-022-01073-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sakurai Y., Kubota N., Yamauchi T., Kadowaki T.. Role of Insulin Resistance in MAFLD. Int. J. Mol. Sci. 2021;22(8):4156. doi: 10.3390/ijms22084156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Fan C., Zhan Z., Zhang X., Lou Q., Guo N., Su M., Gao Y., Qin M., Wu L., Huang W.. et al. Research for type 2 diabetes mellitus in endemic arsenism areas in central China: role of low level of arsenic exposure and KEAP1 rs11545829 polymorphism. Arch. Toxicol. 2022;96(6):1673–1683. doi: 10.1007/s00204-022-03279-1. [DOI] [PubMed] [Google Scholar]
  46. Grau-Perez M., Kuo C. C., Gribble M. O., Balakrishnan P., Jones Spratlen M., Vaidya D., Francesconi K. A., Goessler W., Guallar E., Silbergeld E. K.. et al. Association of Low-Moderate Arsenic Exposure and Arsenic Metabolism with Incident Diabetes and Insulin Resistance in the Strong Heart Family Study. Environ. Health Perspect. 2017;125(12):127004. doi: 10.1289/EHP2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ji S., Qu Y., Sun Q., Zhao F., Qiu Y., Li Z., Li Y., Song H., Zhang M., Zhang W.. et al. Mediating Role of Liver Dysfunction in the Association between Arsenic Exposure and Diabetes in Chinese Adults: A Nationwide Cross-Sectional Study of China National Human Biomonitoring (CNHBM) 2017–2018. Environ. Sci. Technol. 2024;58(6):2693–2703. doi: 10.1021/acs.est.3c08718. [DOI] [PubMed] [Google Scholar]
  48. Kuo C. C., Howard B. V., Umans J. G., Gribble M. O., Best L. G., Francesconi K. A., Goessler W., Lee E., Guallar E., Navas-Acien A.. Arsenic Exposure, Arsenic Metabolism, and Incident Diabetes in the Strong Heart Study. Diabetes Care. 2015;38(4):620–627. doi: 10.2337/dc14-1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Li W., Li Z., Yan Y., Zhang J., Zhou Q., Wang R., He M.. Association of urinary arsenic metabolism with type 2 diabetes and glucose homeostasis: Cross-sectional and longitudinal associations. Environ. Res. 2023;239(Pt 2):117410. doi: 10.1016/j.envres.2023.117410. [DOI] [PubMed] [Google Scholar]
  50. Huang C. F., Yang C. Y., Chan D. C., Wang C. C., Huang K. H., Wu C. C., Tsai K. S., Yang R. S., Liu S. H.. Arsenic Exposure and Glucose Intolerance/Insulin Resistance in Estrogen-Deficient Female Mice. Environ. Health Perspect. 2015;123(11):1138–1144. doi: 10.1289/ehp.1408663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Kirkley A. G., Carmean C. M., Ruiz D., Ye H., Regnier S. M., Poudel A., Hara M., Kamau W., Johnson D. N., Roberts A. A.. et al. Arsenic exposure induces glucose intolerance and alters global energy metabolism. Am. J. Physiol Regul Integr Comp Physiol. 2018;314(2):R294–R303. doi: 10.1152/ajpregu.00522.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Koshta K., Chauhan A., Singh S., Srivastava V.. Prenatal arsenic exposure alters EZH2-H3K27me3 occupancy at TNF-alpha promoter leading to insulin resistance and metabolic syndrome in a mouse model. Environ. Int. 2024;190:108929. doi: 10.1016/j.envint.2024.108929. [DOI] [PubMed] [Google Scholar]
  53. Drucker D. J., Holst J. J.. The expanding incretin universe: from basic biology to clinical translation. Diabetologia. 2023;66(10):1765–1779. doi: 10.1007/s00125-023-05906-7. [DOI] [PubMed] [Google Scholar]
  54. Vilsboll T., Krarup T., Deacon C. F., Madsbad S., Holst J. J.. Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients. Diabetes. 2001;50(3):609–613. doi: 10.2337/diabetes.50.3.609. [DOI] [PubMed] [Google Scholar]
  55. Anini Y., Brubaker P. L.. Role of leptin in the regulation of glucagon-like peptide-1 secretion. Diabetes. 2003;52(2):252–259. doi: 10.2337/diabetes.52.2.252. [DOI] [PubMed] [Google Scholar]
  56. Lim G. E., Huang G. J., Flora N., LeRoith D., Rhodes C. J., Brubaker P. L.. Insulin regulates glucagon-like peptide-1 secretion from the enteroendocrine L cell. Endocrinology. 2009;150(2):580–591. doi: 10.1210/en.2008-0726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. She J., Tuerhongjiang G., Guo M., Liu J., Hao X., Guo L., Liu N., Xi W., Zheng T., Du B.. et al. Statins aggravate insulin resistance through reduced blood glucagon-like peptide-1 levels in a microbiota-dependent manner. Cell Metab. 2024;36(2):408. doi: 10.1016/j.cmet.2023.12.027. [DOI] [PubMed] [Google Scholar]
  58. Abuawad A., Spratlen M. J., Parvez F., Slavkovich V., Ilievski V., Lomax-Luu A. M., Saxena R., Shahriar H., Nasir Uddin M., Islam T.. et al. Association between body mass index and arsenic methylation in three studies of Bangladeshi adults and adolescents. Environ. Int. 2021;149:106401. doi: 10.1016/j.envint.2021.106401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhong Q., Qin Q. R., Yang W. J., He J. L., Zhu J. L., Zhu Z. Y., Huang F.. Multiple metal exposure and obesity: A prospective cohort study of adults living along the Yangtze River, China. Environ. Pollut. 2021;285:117150. doi: 10.1016/j.envpol.2021.117150. [DOI] [PubMed] [Google Scholar]
  60. Castriota F., Zushin P. H., Sanchez S. S., Phillips R. V., Hubbard A., Stahl A., Smith M. T., Wang J. C., La Merrill M. A.. Chronic arsenic exposure impairs adaptive thermogenesis in male C57BL/6J mice. Am. J. Physiol Endocrinol Metab. 2020;318(5):E667–E677. doi: 10.1152/ajpendo.00282.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Calderon-DuPont D., Romero-Cordoba S. L., Tello J. K., Espinosa A., Guerrero B., Contreras A. V., Moran-Ramos S., Diaz-Villasenor A.. Impaired white adipose tissue fatty acid metabolism in mice fed a high-fat diet worsened by arsenic exposure, primarily affecting retroperitoneal adipose tissue. Toxicol. Appl. Pharmacol. 2023;468:116428. doi: 10.1016/j.taap.2023.116428. [DOI] [PubMed] [Google Scholar]
  62. Scheja L., Heeren J.. The endocrine function of adipose tissues in health and cardiometabolic disease. Nat. Rev. Endocrinol. 2019;15(9):507–524. doi: 10.1038/s41574-019-0230-6. [DOI] [PubMed] [Google Scholar]
  63. Perez-Perez A., Sanchez-Jimenez F., Vilarino-Garcia T., Sanchez-Margalet V.. Role of Leptin in Inflammation and Vice Versa. Int. J. Mol. Sci. 2020;21(16):5887. doi: 10.3390/ijms21165887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Li H., Li Z., Chen L., Peng X., Wang D., Sun Q., Chen Y., Zhang A., Liu Q., Tang H.. PDHA1-mediated H3K18 lactylation is involved in arsenic-induced nonalcoholic fatty liver disease by activating the CD36-NLRP3 inflammasome axis. J. Hazard Mater. 2025;498:139852. doi: 10.1016/j.jhazmat.2025.139852. [DOI] [PubMed] [Google Scholar]
  65. Yang Y., Chi L., Liu C. W., Hsiao Y. C., Lu K.. Chronic Arsenic Exposure Perturbs Gut Microbiota and Bile Acid Homeostasis in Mice. Chem. Res. Toxicol. 2023;36(7):1037–1043. doi: 10.1021/acs.chemrestox.2c00410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Li H., Ye F., Li Z., Peng X., Wu L., Liu Q.. The response of gut microbiota to arsenic metabolism is involved in arsenic-induced liver injury, which is influenced by the interaction between arsenic and methionine synthase. Environ. Int. 2024;190:108824. doi: 10.1016/j.envint.2024.108824. [DOI] [PubMed] [Google Scholar]
  67. Chang M., Shao Z., Wei W., Shen P., Shen G.. Sex-specific prevalence and risk factors of metabolic-associated fatty liver disease among 75,570 individuals in eastern China. Front Endocrinol (Lausanne) 2023;14:1241169. doi: 10.3389/fendo.2023.1241169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yadav A. K., Harris-Kawano A., Saxena R., Jiang G., Ji J., Gao H., Kua K. L., Morral N.. Establishment of sex-specific liver transcriptomes and H3K9me3 profiles during sexual maturity: the impact of maternal obesity. Biol. Sex Differ. 2025;16(1):81. doi: 10.1186/s13293-025-00767-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Xia Y., Hu L., Ren K., Han X., Sun Y., Li D.. Embryonic exposure to 6:2 fluorotelomer alcohol mediates autism spectrum disorder-like behavior by dysfunctional microbe-gut-brain axis in mice. J. Hazard Mater. 2025;484:136739. doi: 10.1016/j.jhazmat.2024.136739. [DOI] [PubMed] [Google Scholar]
  70. Nie Q., Luo X., Wang K., Ding Y., Jia S., Zhao Q., Li M., Zhang J., Zhuo Y., Lin J.. et al. Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway. Cell. 2024;187(11):2717–2734. doi: 10.1016/j.cell.2024.03.034. [DOI] [PubMed] [Google Scholar]
  71. Tan J., Hu R., Gong J., Fang C., Li Y., Liu M., He Z., Hou D. X., Zhang H., He J.. et al. Protection against Metabolic Associated Fatty Liver Disease by Protocatechuic Acid. Gut Microbes. 2023;15(1):2238959. doi: 10.1080/19490976.2023.2238959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Peiseler M., Schwabe R., Hampe J., Kubes P., Heikenwalder M., Tacke F.. Immune mechanisms linking metabolic injury to inflammation and fibrosis in fatty liver disease - novel insights into cellular communication circuits. J. Hepatol. 2022;77(4):1136–1160. doi: 10.1016/j.jhep.2022.06.012. [DOI] [PubMed] [Google Scholar]

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