Induction of insulin resistance in female mice due to prolonged phenanthrene exposure: unveiling the low-dose effect and potential mechanisms.

Phenanthrene (Phe) is a commonly occurring polycyclic aromatic hydrocarbon (PAH) found in various food sources and drinking water. Previous studies have shown that long-term exposure to Phe in male mice leads to insulin resistance in a dose-dependent manner. However, the effect of Phe on glucose homeostasis in female mice remains unknown. To address this knowledge gap, female Kunming mice were exposed to Phe through their drinking water at concentrations of 0.05, 0.5, and 5 ng/mL. After 270 d of exposure, we surprisingly discovered a low-dose effect of Phe on insulin resistance in female mice, which differed from the effect observed in male mice and showed sexual dimorphism. Specifically, insulin resistance was only observed in the 0.05 ng/mL treatment, and this low-dose effect was also reflected in the concentration of Phe in white adipose tissue (WAT). Differences in metabolic enzyme activities in the liver may potentially explain this effect. The observed sexual dimorphism in Phe exposure could be attributed to variations in estrogen (E2) level and estrogen receptor beta (ERß) expression in WAT. These findings highlight the association between environmental factors and the development of insulin resistance, emphasizing the pathogenic effect of even low doses of Phe. Moreover, sex dependent-effect should be given more attention when studying the toxic effects of environmental pollutants.

Prenatal exposure to a mixture of PAHs causes the dysfunction of islet cells in adult male mice: Association with type 1 diabetes mellitus.

Polycyclic aromatic hydrocarbons (PAHs) have been detected throughout the human body. Whether exposure to PAHs is associated with the incidence of type 1 diabetes mellitus should be investigated. To this end, pregnant mice were exposed to mixed PAHs (5, 50, or 500 µg/kg) once every other day during gestation. The adult male offspring displayed impaired glucose tolerance and reduced serum levels of glucagon and insulin. Immunohistochemical staining revealed increased numbers of apoptotic ß-cells and a reduced ß-cell mass in these males. The downregulated expression of pancreatic estrogen receptor α, androgen receptor, and transcription factor PDX1 was responsible for impacting ß-cell development. The relatively reduced α-cell area was associated with downregulated ARX expression. The transcription of Isn2 and Gcg in pancreatic tissue was downregulated, which indicated that the function of ß-cells and α-cells was impaired. Methylation levels in the Isn2 promotor were significantly elevated in mice prenatally exposed to 500 µg/kg PAHs, which was consistent with the change in its mRNA levels. The number of macrophages infiltrating islets was significantly increased, indicating that prenatal PAH exposure might reduce islet cell numbers in an autoimmune manner. This study shows that prenatal exposure to PAHs may promote the pathogenesis of type 1 diabetes mellitus.

In utero exposure to mixed PAHs causes heart mass reduction in adult male mice.

Polycyclic aromatic hydrocarbons (PAHs) are a risk factor for the occurrence of cardiac diseases. The present study was conducted to investigate the influence of prenatal exposure to a mixed PAHs on heart and the underlying mechanism. Pregnant mice were orally administered with a mixture of 8 kinds of PAHs (0, 5, 50, 500 µg/kg body weight) once every 2 days for a total of 8 dosages. The mixed PAHs contained naphthalene, acenaphthylene, phenanthrene, fluoranthene, pyrene, benzo[a]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene at a weight ratio of 10: 10: 10: 10: 10: 1: 1: 1. The adult males, not females, showed significantly decreased heart/body weight ratio, which was attributed to the loss of cardiac fiber and the increase of cell apoptosis. The protein expression of transforming growth factor ß1 and its downstream transcription factors, Smad3 and Smad4, was significantly downregulated, which caused the loss of cardiac fiber. The downregulated phosphatidylinositol 3-kinase and AKT led to increased expression of caspase3, caspase9, BAX and reduced expression of Bcl-2, which was responsible for the increased cell apoptosis. Different levels of aromatic hydrocarbon receptor and sex hormone receptors between males and females were associated with the distinct effect on heart.

Chronic Exposure to Environmental Level Phenanthrene Induces Non-Obesity-Dependent Insulin Resistance in Male Mice.

Epidemiological evidence shows that the body burden of polycyclic aromatic hydrocarbons (PAHs) is related to the disruption of glucose homeostasis. However, the contribution of PAHs to the development of diabetes remains poorly documented. In the current work, male Kunming mice received phenanthrene (Phe) (5, 50, and 500 ng/kg) by gavage administration once every 2 days for 28 weeks. The significant elevation of homeostasis model assessment-insulin resistance (HOMA-IR) and HOMA-ß cell, accompanied by hyperinsulinemia, indicated the occurrence of insulin resistance. The suppression of the insulin receptor signaling pathway in skeletal muscle might be responsible for glucose intolerance. Under the nonobese state, the serum levels of resistin, tumor necrosis factor-α, and interleukin-6 were elevated, whereas the levels of adiponectin were reduced. These changes in adipocytokine levels were consistent with their transcription in white adipose tissue. The promoter methylation levels of Retn (encoding resistin) and Adipoq (encoding adiponectin) were inversely correlated with their mRNA levels, indicating that Phe exposure could cause the disruption of adipocytokine secretion via epigenetic modification. The results would be helpful for understanding the pathogenesis in the development of T2DM caused by nonobesogenic pollutants.

Exposure to Aroclor 1254 differentially affects the survival of pancreatic ß-cells and α-cells in the male mice and the potential reason.

Previous works showed that chronic exposure to Aroclor 1254 disrupted glucose homeostasis and induced insulin resistance in male mice. To further observe the different effects of Aroclor 1254 exposure on the pancreatic α-cells and ß-cells, male mice were exposed to Aroclor 1254 (0, 0.5, 5, 50, 500 µg/kg) for 60 days, the pancreas was performed a histological examination. The results showed that the percentage of apoptosis cell (indicated by TUNEL assay) was increased in both α-cells and ß-cells, as the Aroclor 1254 dose was increased; the proliferation (indicated by PCNA expression) rate of ß-cells was elevated while that of α-cells was not affected, resulting in an increased ß-cell mass and a decreased α-cell mass in a dose-depend manner. The number of Pdx-1 positive ß-cells was significantly increased whereas that of Arx positive α-cells was markedly decreased, indicating an enhanced ß-cell neogenesis and a weakened α-cell neogenesis. The drastically reduction of serum testosterone levels in all the treatments suggested an anti-androgenic potency of Aroclor 1254. The up-regulation of estrogen receptors (ERα and ERß) and androgen receptor in ß-cells might be responsible for the increased ß-cell mass and neogenesis.

The effects of EGCG supplementation on pancreatic islet α and ß cells distribution in adult male mice.

Tea and tea products are widely used as the most popular beverage in the world. EGCG is the most abundant bioactive tea polyphenol in green tea, which has positive effects on the prevention and treatment of diabetes. However, the impact of EGCG exposure on glucose homeostasis and islets in adult mice have not been reported. In this study, we studied glucose homeostasis and the morphological and molecular changes of pancreatic islet α and ß cells in adult male mice after 60 d of exposure to 1 and 10 mg/kg/day EGCG by drinking water. Glucose homeostasis was not affected in both EGCG groups. The expression of pancreatic duodenal homebox1 (Pdx1) in ß cells was upregulated, which might be related to increased insulin level, ß cell mass and ß cell proliferation in 10 mg/kg/day EGCG group. The expression of aristaless-related homeobox (Arx) in α cells did not change significantly, which corresponded with the unchanged α-cell mass. The significant reduction of musculoaponeurotic fibrosarcoma oncogene homolog B (MafB) positive α-cells might be associated with decreased glucagon level in both EGCG groups. These results suggest that EGCG supplementation dose-dependent increases ß cell mass of adult mice and affects the levels of serum insulin and glucagon. Our results show that regular tea drinking in healthy people may have the possibility of preventing diabetes.

Chronic exposure to low levels of phenanthrene induces histological damage and carcinogenic risk in the uterus of female mice.

Phenanthrene (Phe), a polycyclic aromatic hydrocarbon with low molecular weight, is detected in the environment at high frequency. To study the toxic effects of Phe on the uterine structure and function, female Kunming mice were exposed to Phe (0.05, 0.5, 5 ng/mL) for 270 days by drinking water. Pathological alterations and their action pathways were analyzed using immunohistochemical and biomolecular technology. Phe significantly increased the percentage of blood vessel area, the number of endometrial neutrophils (indicating the occurrence of inflammation), collagen deposition (indicating fibrosis), and the percentage of Ki-67-positive cells (indicating carcinogenesis) in the uterus. Transcriptome sequencing identified differentially expressed genes that were mainly enriched in some signaling pathways, including inflammation and carcinogenesis, suggesting a carcinogenic risk in the Phe-exposed uterus. Elevated serum estrogen levels and decreased progesterone levels exhibited a disturbance of steroid hormone balance, which might be related to uterine damage. Upregulated protein levels of uterine androgen receptor and estrogen receptor α were linked to the pathological effects. Most of the effects exhibited a nonmonotonic dose response, which might be attributed to the corresponding change in the serum levels of Phe. The results suggest that exposure to low levels of Phe could exert adverse effects on the uterus.

Prenatal EGCG consumption impacts hepatic glycogen synthesis and lipid metabolism in adult mice.

In this study, the impact of prenatal exposure to Epigallocatechin gallate (EGCG) on the liver of adult offspring mice was investigated. While EGCG is known for its health benefits, its effects of prenatal exposure on the liver remain unclear. Pregnant C57BL/6 J mice were exposed to 1 mg/kg of EGCG for 16 days to assess hepatotoxicity effects of adult offspring. Transcriptomics and metabolomics were employed to elucidate the hepatotoxicity mechanisms. The findings revealed that prenatal EGCG exposure led to a decrease in liver somatic index, enhanced inflammatory responses and disrupted liver function through increased glycogen accumulation in adult mice. The integrated omics analysis revealed significant alterations in key pathways involved in liver glucose lipid metabolism, such as gluconeogenesis, dysregulation of insulin signaling, and induction of liver inflammation. Furthermore, the study found a negative correlation between the promoter methylation levels of Ppara and their mRNA levels, suggesting that EGCG could reduce hepatic lipid content through epigenetic modifications. The findings suggest that prenatal EGCG exposure can have detrimental impacts on the liver among adult individuals and emphasize the need for a comprehensive evaluation of the potential risks associated with EGCG consumption during pregnancy.

Prenatal EGCG consumption causes obesity and perturbs glucose homeostasis in adult mice.

Epigallocatechin gallate (EGCG) has a wide consumption for its health advantages. The current study investigates the effects of prenatal EGCG administration on glucose metabolism and obesity in adulthood. Pregnant C57BL/6J mice were supplemented with EGCG in drinking water (3 µg/mL) for 16 d. Abdominal obesity was observed in both male and female adult mice, which was associated with the upregulation of adipose-specific genes, including C/ebpα and Srebf1 (Srebf1 only in males), and the downregulation of genes related to lipolysis, such as Acox1, Atgl and Pdk4 (only in males) in visceral adipose tissue. Elevated fasting glucose levels and hyperinsulinemia were observed in adult males, while females exhibit lower glucose level in glucose tolerance test, which might be due to reduced glucagon levels. Though hepatic expression of the insulin receptor signaling pathway was upregulated in males and was not altered in females, prenatal treatment with EGCG downregulated the expression of this signaling pathway in the skeletal muscle of adult mice, which was further demonstrated in primary human skeletal muscle cells treated with EGCG. The methylation levels in promotor of genes related to the insulin receptor signaling were matched with their transcription in mice, while the expression of acetylated histones was downregulated in human skeletal muscle cells. These results suggest that EGCG consumption during pregnancy should be a risk factor for the disruption of glucose homeostasis in adulthood.

EGCG exposure during pregnancy affects uterine histomorphology in F1 female mice and the underlying mechanisms.

Although epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to have many benefits, the effect of EGCG exposure in utero on adult uterine development is unclear. In this study, pregnant C57BL/6 mice were exposed to 1 mg/kg body weight (bw) EGCG dissolved in drinking water from gestational days 0.5-16.5. A significant decrease in uterine weight was observed in the adult female mice, accompanied by uterine atrophy, inflammation, and fibrosis in the endometrium. Uterine atrophy was attributed to the thinning of the endometrial stromal layer and a significant reduction in endometrial cell proliferation. The expression levels of related proteins in the NF-κB and RAF/MEK/ERK signaling pathways were significantly increased, which might be responsible for the occurrence of inflammation. Activation of the transforming growth factor beta (TGF-ß1)/Smad signaling pathway might be involved in the development of endometrial fibrosis. The changes in the expression of estrogen receptor α, ß (ERα, ERß), progesterone receptor (PGR), and androgen receptor (AR) might lead to changes in the aforementioned signaling pathways. The promoter region methylation level of Esr2 was increased, and the expression of DNMT3A was evaluated. Our study indicates a risk of EGCG intake during pregnancy affecting uterine development in offspring.

Long-term exposure to environmental levels of phenanthrene induces emaciation-thirst disease-like syndromes in female mice.

Phenanthrene (Phe) is a polycyclic aromatic hydrocarbon widely present in foods and drinking water. To explore the detrimental effects of Phe on body metabolism, female Kunming mice were treated with Phe in drinking water at concentrations of 0.05, 0.5 and 5 ng/mL. After exposure for 270 d, the animals exhibited dose-dependent reduced body weight and increased water consumption. The dose-dependent accumulation of Phe in the brain decreased hypothalamic neuron numbers, upregulated hypothalamic expression of anaplastic lymphoma kinase, elevated norepinephrine levels in white adipose tissue (WAT) and further activated lipolysis in WAT, leading to a reduction in fat mass. Brown adipose tissue formation was reduced, accompanied by the inhibition of the bone morphogenetic protein signaling pathway. A simultaneous reduced serum levels of antidiuretic hormone (arginine vasopressin) might be one of the reasons for increased water consumption. The present results indicate an environmental etiology and prevention way for the development of emaciation-thirst disease.

Long-term exposure to environmental levels of phenanthrene disrupts spermatogenesis in male mice.

Phenanthrene (Phe) is a tricyclic polycyclic aromatic hydrocarbon with high bioavailability under natural exposure. However, there are few studies on the reproductive toxicity of Phe in mammals. In this study, male Kunming mice were gavaged once every two days with Phe (5, 50, and 500 ng/kg) for 28 weeks. The accumulation levels of Phe in the testis were dose-dependently increased. Histopathological staining showed that Phe exposure reduced the number of spermatogonia, sperm and Sertoli cells. The percentage of testicular apoptotic cells was significantly increased, which was further verified by the upregulated BAX protein. The expression of the GDNF/PI3K/AKT signaling pathway was downregulated, which might suppress the self-renewal and differentiation of spermatogonial stem cells. Meanwhile, Phe exposure inhibited the expression of Sertoli cell markers (Fshr, WT1, Sox9) and the Leydig cell marker Cyp11a1, indicating damage to the function of Sertoli cells and Leydig cells. Serum estrogen and testicular estrogen receptor alpha were significantly upregulated, while androgen receptor expression was downregulated. These alterations might be responsible for impaired spermatogenesis. This study provides new insights for evaluating the reproductive toxicity and potential mechanisms of Phe in mammals.

Prenatal EGCG exposure-induced heart mass reduction in adult male mice and underlying mechanisms.

Epigallocatechin-3-gallate (EGCG), which is a major polyphenol in tea, has an unclear effect on cardiac development. In the present study, mice (C57BL/6) were exposed in utero to EGCG dissolved in drinking water (3 µg/ml) for 16 days. A significant decrease in the heart/body weight ratio was observed in adult males but not in adult females. The protein expression levels of TGF-ß1 and its downstream transcription factors SMAD3 and SMAD4 were significantly decreased in male hearts. The PI3K/AKT signaling pathway was inhibited, the expression of proapoptotic proteins, such as BAX, Cleaved Caspase3 and Cleaved Caspase9, was elevated, and the level of antiapoptotic proteins, such as BCL-2, was decreased. A reduced heart/body weight ratio may be associated with the loss of cardiac fibers and an increase in myocardial apoptosis. The cardiac levels of aromatic hydrocarbon receptor and androgen receptor were elevated only in males, which may explain the sexual dimorphism in the effects. The promoter methylation levels of pik3r1, tgf-ß, smad4 were elevated, and those of ahr were reduced, explaining the mechanism underlying the cardiac histological alteration caused by prenatal exposure to EGCG. The results suggest that ingestion of EGCG during pregnancy may be a risk factor for cardiac development in offspring.

Tributyltin exposure disturbs hepatic glucose metabolism in male mice.

Some previous studies showed that organotin compounds induced diabetes in animal models. The underlying mechanisms should be further revealed. In this study, male KM mice were exposed to tributyltin (TBT) at 0.5, 5 and 50 µg/kg once every three days for 45 days. The TBT-treated mice exhibited an elevation of fasting blood glucose level and glucose intolerance. The fasting serum insulin levels were increased and reached a significant difference in the 50 µg/kg group; the glucagon levels were significantly decreased in all the treatments. Pancreatic ß-cell mass was significantly decreased in all the treatments; α-cell mass showed a significant decrease in the 5 and 50 ug/kg groups. The transcription of pancreatic insulin gene (Ins2) showed an up-regulation and reached a significant difference in the 5 and 50 µg/kg groups, which would be responsible for the increased serum insulin levels. The transcription of glucagon gene (Gcg) in the pancreas was significantly down-regulated in the 5 and 50 ug/kg groups. The protein expression of hepatic glucagon receptor was down-regulated, while the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was up-regulated accompanied by increased hepatic glycogen content. These results indicated that hepatic gluconeogenesis was enhanced during insulin resistance stage caused by TBT exposure, which would exert a potential risk inducing the development of diabetes mellitus.

Embryonic exposure to benzo(a)pyrene inhibits reproductive capability in adult female zebrafish and correlation with DNA methylation.

This study was conducted to investigate the effects of embryonic short-term exposure to benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, on ovarian development and reproductive capability in adult female zebrafish. In 1-year-old fish after embryonic exposure to BaP for 96 h, the gonadosomatic indices and the percentage of mature oocytes were significantly decreased in the 0.5, 5 and 50 nmol/L treatments. The spawned egg number, the fertilization rate and the hatching success were significantly reduced, while the malformation rate of the F1 unexposed larvae were increased. The mRNA levels of follicle-stimulating hormone, luteinizing hormone, ovarian cytochrome P450 aromatase cyp19a1a and cyp19b, estrogen receptor esr1 and esr2, and hepatic vitellogenin vtg1 and vtg2 genes, were down-regulated in adult female zebrafish that were exposed to BaP during embryonic stage. Both 17ß-estradiol and testosterone levels were reduced in the ovary of adult females. The methylation levels of the gonadotropin releasing hormone (GnRH) gene gnrh3 were significantly increased in the adult zebrafish brain, and those of the GnRH receptor gene gnrhr3 were elevated both in the larvae exposed to BaP and in the adult brain, which might cause the down-regulation of the mRNA levels of gnrh3 and gnrhr3. This epigenetic change caused by embryonic exposure to BaP might be a reason for physiological changes along the brain-pituitary-gonad axis. These results suggest that short-term exposure in early life should be included and evaluated in any risk assessment of pollutant exposure to the reproductive health of fish.

Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors.

A green fluorescent protein (GFP)-based bacterial biosensor Escherichia coli DH5alpha (pVLCD1) was developed based on the expression of gfp under the control of the cad promoter and the cadC gene of Staphylococcus aureus plasmid pI258. DH5alpha (pVLCD1) mainly responded to Cd(II), Pb(II), and Sb(III), the lowest detectable concentrations being 0.1 nmol L(-1), 10 nmol L(-1), and 0.1 nmol L(-1), respectively, with 2h exposure. The biosensor was field-tested to measure the relative bioavailability of the heavy metals in contaminated sediments and soil samples. The results showed that the majority of heavy metals remained adsorbed to soil particles: Cd(II)/Pb(II) was only partially available to the biosensor in soil-water extracts. Our results demonstrate that the GFP-based bacterial biosensor is useful and applicable in determining the bioavailability of heavy metals with high sensitivity in contaminated sediment and soil samples and suggests a potential for its inexpensive application in environmentally relevant sample tests.

Development and testing of a green fluorescent protein-based bacterial biosensor for measuring bioavailable arsenic in contaminated groundwater samples.

A green fluorescent protein (GFP)-based bacterial biosensor for the detection of bioavailable As(III), As(V), and Sb(III) was developed and characterized. The biosensor strain Escherichia coli DH5alpha (pVLAS1) was developed based on the expression of gfp under the control of the ars promoter and the arsR gene of Staphylococcus aureus plasmid pI258. Strain DH5alpha (pVLAS1) responded mainly to As(III), As(V), and Sb(III), with the lowest detectable concentrations being 0.4, 1, and 0.75 microM, respectively, during a 2-h exposure and 0.1 microM for all three metal ions with an 8-h induction period. To assess its applicability for analyzing environmentally relevant samples, the biosensor was field-tested on shallow-well groundwater for which contaminant levels were known. Our results demonstrate that the nonpathogenic bacterial biosensor developed in the present study is useful and applicable in determining the bioavailability of arsenic with high sensitivity in contaminated groundwater samples, and they suggest a potential for its inexpensive application in field-ready tests.