Genotoxic effects of caffeine in female mice exposed during pregnancy and lactation period and their offspring.

Caffeine is a widely consumed substance, and there is a discussion about its effects when ingested by women during pregnancy and lactation. We aimed to identify the genotoxic effects of caffeine in female mice that consumed it during pregnancy and lactation periods and its consequences in their offspring. Thirty-six couples of Swiss mice received water or caffeine (0.3 and 1.0 mg/mL) treatment during pregnancy and lactation. The male and female offspring were divided into 12 groups according to the treatment administered to the female mice. Genotoxicity was assessed using the comet assay and the micronucleus test. Both doses of caffeine showed genotoxic effects in pregnant and lactating mice groups compared to groups not administered caffeine. In relation to offspring, it can be observed that females and males of the offspring had low weight in early life. In both female and male offspring, genotoxicity was detected in the blood, liver, and kidney tissues. Thus, from the present study, we can suggest that the caffeine consumed by female mice during the periods of pregnancy and lactation led to genotoxic effects in their offspring.

Assessment of caffeine neurotoxicity using novel biomarkers of neural function in SH-SY5Y cells - Is there a need for environmental concern?

Worldwide usage of caffeine results in its constant release into the aquatic environment and growing concerns related to associated risks. We assessed (neuro)toxicity of environmentally relevant concentrations of caffeine, using novel biomarkers of neural function in SH-SY5Y cells and markers of general toxicity also in HepG2 cells. The RQ-PCR analyses showed that caffeine disturbs the expression of genes encoding several key elements of neurotransmitter pathways, with the most prominent responses observed for serotonin receptor 3A, dopamine receptor D2, monoamine oxidase B and GABA-transaminase. Expression of genes encoding synaptotagmin 10 involved in exocytosis of neurotransmitters and ATPase Na+/K+ transporting subunit alpha 3 was also disturbed. Caffeine stimulated the activity of monoamine oxidase, while cytotoxicity and effects on mitochondrial membrane potential were not observed. Our study points out the new possible molecular targets of caffeine and suggests that the raising concerns related to its growing environmental presence are justified.

The upregulation of glutamate decarboxylase 67 against hippocampal excitability damage in male fetal rats by prenatal caffeine exposure.

As a kind of xanthine alkaloid, caffeine is widely present in beverages, food, and analgesic drugs. Our previous studies have shown that prenatal caffeine exposure (PCE) can induce programmed hypersensitivity of the hypothalamic-pituitary-adrenal (HPA) axis in offspring rats, which is involved in developing many chronic adult diseases. The present study further examined the potential molecular mechanism and toxicity targets of hippocampal dysfunction, which might mediate the programmed hypersensitivity of the HPA axis in offspring. Pregnant rats were intragastrically administered with 0, 30, and 120 mg/kg/day caffeine from gestational days (GD) 9-20, and the fetal rats were extracted at GD20. Rat fetal hippocampal H19-7/IGF1R cell line was treated with caffeine, adenosine A2A receptor (A2AR) agonist (CGS-21680) or adenylate cyclase agonist (forskolin) plus caffeine. Compared with the control group, hippocampal neurons of male fetal rats by PCE displayed increased apoptosis and reduced synaptic plasticity, whereas glutamate decarboxylase 67 (GAD67) expression was increased. Moreover, the expression of A2AR was down-regulated, PCE inhibited the cAMP/PKA/CREB/BDNF/TrkB pathway. Furthermore, the results in vitro were consistent with the in vivo study. Both CGS21680 and forskolin could reverse the above alteration caused by caffeine. These results indicated that PCE inhibits the BDNF pathway and mediates the hippocampus's glutamate (Glu) excitotoxicity. The compensatory up-regulation of GAD67 unbalanced the Glu/gamma-aminobutyric acid (GABA)ergic output, leading to the impaired negative feedback to the hypothalamus and hypersensitivity of the HPA axis.

Prenatal caffeine exposure induced renal developmental toxicity and transgenerational effect in rat offspring.

Epidemiological studies revealed that prenatal caffeine exposure (PCE) is associated with adverse gestational outcomes and susceptibility to chronic diseases in offspring, yet the effects of PCE on glomerulosclerosis susceptibility in adult female offspring and its intergenerational transmission remain to be further investigated. Here, we found that PCE caused fetal kidney dysplasia and glomerulosclerosis of the female offspring. Besides, the kidney of F1 offspring in PCE group exhibited the "low expressional programming of AT2R" and "GC-IGF1 programming" alteration. Intergenerational genetic studies revealed that the renal defect and GC-IGF1 programming alteration was inherited to F2 adult female offspring derived from the female germ line, but Low expression of AT2R did not extend to the F2 female offspring. Taken together, PCE caused renal dysplasia and adult glomerulosclerosis in the F1 female offspring, which might be mediated by renal AT2R low expressional programming and GC-IGF1 axis alteration. Furthermore, PCE induced transgenerational toxicity on kidney, and GC-IGF1 programming alteration might be the potential molecular mechanism. This study provided experimental evidence for the mechanism study of the intergenerational inheritance of kidney developmental toxicity caused by PCE.

Sub-chronic and chronic toxicity evaluation of 7-methylxanthine: a new molecule for the treatment of myopia.

Myopia (nearsightedness) is a vision disorder with a blurring of far objects, affect millions worldwide. 7-methylxanthine (7-MX) is a molecule that is presently under clinical investigation for the treatment of myopia. In the present study, we have investigated sub-chronic and chronic toxicity of 7-MX in comparison to other clinically used methylxanthines i.e., caffeine and theobromine as per OECD guidelines 408 and 452. 7-MX was administered orally for 90 days at three different doses of 250, 500, and 1000 mg/kg for sub-chronic toxicity evaluation, and at a limit dose of 1000 mg/kg in 180 days chronic toxicity evaluation in rats. In sub-chronic treatment, 7-MX showed no mortality and signs for toxicity in any group, whereas 10% and 40% mortality with signs for toxicity were observed in caffeine and theobromine treated groups, respectively. A similar, safety profile was observed with 7-MX in 180 days of chronic toxicity study. Further, to confirm any morphological changes in organs; ultrasound and X-rays analysis were performed and no changes in the size of organs, cyst formation, fluid retention, or crystal formation was observed. Thus, the repeated dose study of 7-MX for 180 days may augment the possibility of using 7-MX clinically for the safe and effective treatment of myopia.

Caffeine-induced neurotoxicity mediated by Nrf2 pathway in PC12 cells and zebrafish larvae.

Caffeine is one of the most widely used psychostimulants in the world and possesses central excitative, anti-depressive, and neuroprotective properties. However, excessive ingestion or abuse of caffeine can lead to intoxication. Many toxic effects are attributed to oxidative damage, and nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical intracellular regulator of the oxidative stress response. Here, we investigated the neurotoxicity of caffeine in rat pheochromocytoma PC12 cells and zebrafish larvae. It was found that caffeine inhibited the viability of PC12 cells in a dose- and time-dependent manner. Furthermore, it induced PC12 cell apoptosis and elevated reactive oxygen species (ROS) production. Quantitative polymerase chain reaction (qPCR) and western blotting revealed that caffeine also inhibited the expression levels of Nrf2 mRNA and protein and its target genes (e.g., NADPH quinone oxidoreductase 1 [NQO1]). Furthermore, Nrf2 silencing attenuated the toxic effects of caffeine. In addition, zebrafish larvae were treated with different doses of caffeine. Behavioral experiments showed that a low dose of caffeine (0.05 to 0.3 mM) increased the average distance of movement and promoted excitation. Survivorship curves showed that caffeine (0.2 to 1.5 mM) caused lethality. Finally, qPCR revealed that a higher dose of caffeine inhibited mRNA levels in the Nrf2 pathway. Based on these results, this study identified for the first time that overuse of caffeine can induce neurotoxicity by inhibiting the Nrf2 pathway. These results will provide a new perspective for studies on caffeine toxicity.

Prenatal caffeine exposure caused H-type blood vessel-related long bone dysplasia via miR375/CTGF signaling.

Caffeine has developmental toxicity. Prenatal caffeine exposure (PCE) caused intrauterine growth retardation (IUGR) and multiple organ dysplasia. This study intended to explore the effect and mechanism of PCE on long bone development in female fetal rats. In vivo, the PCE group pregnant rats were given different concentrations of caffeine during the gestational Day 9-20. The mRNA expression of osteogenesis-related genes were significantly reduced in PCE group. In the PCE group (120 mg/kg·d), the length and primary center of fetal femur were shorter, and accompanied by H-type blood vessel abundance reducing. Meanwhile, connective tissue growth factor (CTGF) expression decreased in the growth plate of the PCE group (120 mg/kg·d). In contrast, the miR375 expression increased. In vitro, caffeine decreased CTGF and increased miR375 expression in fetal growth plate chondrocytes. After co-culture with caffeine-treated chondrocytes, the tube formation ability for the H-type endothelial cells was decreased. Furthermore, CTGF overexpression or miR375 inhibitor reversed caffeine-induced reduction of tube formation ability, and miR375 inhibitor reversed caffeine-induced CTGF expression inhibition. In summary, PCE decreased the expression of CTGF by miR375, ultimately resulting in H-type blood vessel-related long bone dysplasia.

Caffeine attenuates seizure and brain mitochondrial disruption induced by Tramadol: the role of adenosinergic pathway.

Tramadol (TR) is an analgesic drug used to treat moderate-to-severe pain but it induces seizure even at therapeutic doses. The exact mechanism of TR-inducing seizure is not clear but inhibition of the serotonin, GABA, and nitrous oxide (NOS) pathways are the commonly proposed mechanisms. Adenosinergic system has a crucial function in the modulation of seizure. Also, oxidative damage is an unavoidable effect of the seizure. This study was conducted to evaluate the role of the adenosinergic system on the seizure and oxidative stress biomarkers induced by TR using antagonist of the adenosinergic receptors in the Albino mice. For that purpose, generated clonic seizure, as seizure threshold, was evaluated by TR. Caffeine (CAF; 8 mg/kg, i.p.), a nonselective antagonist of adenosine receptors, was administered 1 hour before the seizure induction. The seizure threshold significantly increased by CAF-treated group when compared to TR group (p < 0.001). Oxidative stress biomarkers such as reactive oxygen species, protein carbonyl content, and lipid peroxidation significantly decreased and glutathione content significantly increased by CAF in brain mitochondria compared to the TR group, whereas oxidative biomarkers significantly increased in the TR group compared to the control group. The results of the present study suggested that the adenosinergic system is involved in seizure induced by TR and meanwhile, inhibition of adenosine receptors can decrease the TR seizure threshold and also decrease the induced oxidative damage in the brain mitochondria.

Diclofenac and caffeine inhibit hepatic antioxidant enzymes in the freshwater fish Astyanax altiparanae (Teleostei: Characiformes).

Although concentrations of pharmaceutical compounds in aquatic ecosystems are low, they can cause toxic effects on organisms. The aim of this study was to evaluate the effects of diclofenac (DCF), a non-steroidal anti-inflammatory drug, and caffeine (CAF), a central nervous system stimulant, both alone or combined, in Astyanax altiparanae males under acute exposure (96 h), measuring neurotoxicity biomarkers, antioxidant response and damage at biochemical and cellular levels. DCF concentration in water, separated and combined, was 3.08 mg L-1 and that of CAF was 9.59 mg L-1. To assess neurotoxicity, brain and muscle acetylcholinesterase (AChE) activities were measured. To evaluate oxidative stress, the enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione S-transferase (GST), as well as lipoperoxidation (LPO), were analyzed in liver and gills. Activity of hepatic cyclooxygenase (COX) was also evaluated. Genotoxicity was assessed in blood using comet assay and micronucleus test, as well as nuclear abnormalities. DCF and CAF, alone or combined, had neither effect on AChE activity, nor in the activity of SOD, CAT, GPx and GST in gills. In liver, DCF inhibited SOD and GPx activity, CAF inhibited CAT activity, the mixture inhibited SOD and GST activity; although only fish exposed to CAF showed increased hepatic LPO. Under these experimental conditions, no effect on COX activity was observed, nor cytotoxic and genotoxic damage. The most pronounced effects were caused by the drugs separately, since both compounds altered the enzymes, but only CAF triggered LPO, showing more harmful effects.

Comparing chromosome damage induced by mobile telephony radiation and a high caffeine dose: Effect of combination and exposure duration.

I recently reported induction of chromatid-type aberrations in human peripheral blood lymphocytes after a single 15 min exposure to universal mobile telecommunications system (UMTS) mobile telephony (MT) electromagnetic field (EMF) from a mobile phone. Lymphocytes from six healthy subjects were stimulated for mitosis, and exposed during the G2/M phase at 1 cm distance from the handset during an active phone call in "talk" mode. The same type of cells from the same subjects treated with a high caffeine dose (~ 290 times above the permissible single dose for an adult human) exhibited the same type of aberrations in a little smaller but comparable degree. The combination of this caffeine dose and the 15 min MT EMF exposure increased dramatically the number of aberrations in all subjects. The combined effect increased almost linearly with increasing duration of exposure to the MT EMF. Thus, MT EMF exposure ~ 136 times below the official limit (ICNIRP 2020) exerts a genotoxic action even greater than that of a caffeine dose ~ 290 times above the corresponding limit. Therefore, with a reasonable approximation, the limit for MT EMFs should be lowered by at least ~ 4 × 104 times (136 × 290) for short-term exposures, and ~ 4 × 106 times for long-term exposures.

The low-expression programming of 11ß-HSD2 mediates osteoporosis susceptibility induced by prenatal caffeine exposure in male offspring rats.

BACKGROUND AND PURPOSE: Prenatal caffeine exposure (PCE) can cause developmental toxicity of long bones in offspring, but the long-term effects and the underlying mechanism have not been fully clarified. Here, we investigated the effects of PCE peak bone mass accumulation and osteoporosis susceptibility in offspring and its intrauterine programming mechanism. EXPERIMENTAL APPROACH: Pregnant Wistar rats were administrated intragastrically with saline or caffeine (120 mg·kg-1 ·day-1 ) on gestational days 9-20. The serum and bone samples were collected from the fetal and postnatal offspring for bone mass, genes expression and corticosterone analysis. Then, rat bone marrow mesenchymal stem cells (BMSCs) were treated with corticosterone in vitro to confirm the molecular mechanism. KEY RESULTS: PCE caused fetal bone dysplasia in male and female offspring. In adulthood, PCE reduced peak bone mass and increased osteoporosis susceptibility in male offspring but not in females. Meanwhile, PCE only decreased the H3K9ac and expression levels of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) before and after birth in the male offspring but not in the females. Moreover, the high level of corticosterone induced by PCE down-regulated the H3K9ac and expression levels of 11ß-HSD2 through promoting glucocorticoid receptor (GR; NR3C1) into the nucleus of bone marrow mesenchymal stem cells (BMSCs) and recruiting histone deacetylase 11 (HDAC11) binding to 11ß-HSD2 promoter region, which further enhanced the effect of corticosterone on suppressing osteogenic function of BMSCs. CONCLUSION AND IMPLICATIONS: PCE caused osteoporosis susceptibility in male adult offspring, which attributed to the low-functional programming of 11ß-HSD2 induced by corticosterone via GR/HDAC11 signalling.

Articular damages in multi-generational female offspring due to prenatal caffeine exposure correlates with H3K9 deacetylation of TGFß signaling pathway.

Adverse environment during pregnancy could lead to maternal glucocorticoid overexposure in utero, and then induce the intrauterine growth retardation (IUGR) and the programmed change in cartilage development. The transforming growth factor ß (TGFß) signaling pathway plays a crucial role in the process of chondrogenesis, cartilage growth, development, maturation, and phenotype maintenance. Our previous results had shown that prenatal caffeine exposure (PCE) could result in the damaged articular cartilage in offspring rats. However, whether this change could transmit to multiple generations was still unknown. In this study, pregnant Wistar rats received either saline or caffeine (120 mg/kg, i.g.) once daily from gestational day 9-20 (GD9-20). The female offspring mated with normal male rats to generate the following generations. We obtained the articular cartilages in subsequent F1 to F3 female offspring. The H3K9 acetylation and expression of the TGFß signaling pathway were detected; the content of the cartilage matrix was detected. The results showed that PCE reduced the H3K9 acetylation and the expression of the TGFß signaling pathway, then reduced the extracellular matrix in F1, F2, and F3 generations. in vitro, corticosterone could induce the H3K9 deacetylation of the TGFß signaling pathway, thus inhibiting the expression of the TGFß signaling pathway and extracellular matrix. The overall results revealed that PCE induced a multi-generational damaged articular cartilage in female offspring rats, which was partially related to the maternal high glucocorticoid-induced H3K9 hypoacetylation of TGFß signaling pathway.

Case Report Stimulant-Induced Atrial Flutter in a Remote Setting.

Atrial flutter and atrial fibrillation are among the most commonly encountered cardiac arrhythmias; however, there is a dearth of clinical trials or case studies regarding its occurrence in the setting of stimulants such as caffeine and nicotine in otherwise healthy young patients. Described here is a case of a 29-year-old physically fit white man without significant past medical history who presented in stable condition complaining only of palpitations. He was found to have atrial flutter without rapid ventricular response on cardiac monitoring, most likely due to concomitant presence of high levels of nicotine and caffeine via chewing tobacco and energy drinks. He was treated conservatively with vagal maneuvers and intravenous fluids with complete resolution of symptoms and electrocardiographic abnormalities within 14 hours. This demonstrates an alternate conservative treatment strategy in appropriately risk stratified patients who present in an austere field setting with limited resources.

Neferine and lianzixin extracts have protective effects on undifferentiated caffeine-damaged PC12 cells.

BACKGROUND: The embryos of Nelumbo nucifera Gaertn seeds, lianzixin, are used in China as food and traditional herbal medicine. Principal therapeutic indications are insomnia, anxiety and pyrexia. Caffeine is a psychostimulant and excessive use predisposes to cell damage and neurotoxicity. We aimed to investigate the potential protect effect of Neferine and lianzixin extracts on undifferentiated caffeine-damaged phaeochromocytoma cells (PC12 cells). METHODS: A cell damage model based on undifferentiated PC12 was established with caffeine. Effect of Lianzixin extracts (total alkaloids, alcohol extract and water extract) and neferine on caffeine-damaged PC12 cells was evaluated. Cell viability was assessed using the methyl thiazolyl tetrazolium (MTT) assay, cellular morphology by inverted microscope, the nucleus by Hoechst 33342 staining and cleaved poly ADP-ribose polymerase (PARP) expression by western blot analysis. RESULTS: Lianzixin extracts (total alkaloids, alcohol extract and water extract) and neferine improved the viability of PC12 cells damaged by caffeine. The morphology of PC12 cells pretreated with neferine, or alcohol or water extract of lianzixin aggregated and attached better than caffeine-damaged cells, but cells pretreated with total alkaloids of lianzixin showed abnormal morphology. Compared with caffeine-damaged cells, cells pretreated with neferine, or alcohol or water extract of lianzixin showed a notable increase in nucleus staining and an obvious decrease in cleaved PARP expression. CONCLUSIONS: Lianzixin extracts and neferine have protective effects against caffeine-induced damage in PC12 cells, which laid a foundation for finding a new medicine value of Lianzixin.

Endocrine disruptive action of diclofenac and caffeine on Astyanax altiparanae males (Teleostei: Characiformes: Characidae).

Diclofenac (DCF) and caffeine (CAF) are persistent pharmaceuticals that occur in mixtures in the aquatic ecosystems causing effects in the reproductive physiology of aquatic organisms. This study evaluated the physiological reproductive responses of Astyanax altiparanae males exposed to nominal concentrations of DCF (3.08 mg L-1) and CAF (9.59 mg L-1) separately and combined, for 96 h. The steroids profile, estrogenic biomarker vitellogenin (vtgA), testes and liver morphology, and also mortality of males were assessed. DCF and CAF degradation was 5% of the initial concentration for 24 h. The LC50 of the DCF and CAF were 30.8 mg L-1 and 95.9 mg L-1, respectively. Males exposed to DCF and CAF exhibited a reduction of 17ß-Estradiol (E2) concentration compared to control (CTL). Similarly, testosterone (T) was also reduced in the DCF treatment, but this response was not observed in 11-Ketotestosterone (11-KT). Males exposed to DCF + CAF combined did not exhibit differences in T, E2 and 11-KT steroids. The vtgA gene expression and the sperm concentration did not change among the treatments. Moreover, acute exposure revealed a hypertrophy of hepatocytes cells in the DCF and DCF + CAF treatments. In conclusion, DCF and CAF, isolated, exhibit an endocrine disruptive activity in A. altiparanae male, an opposite response observed with the mixture of both compounds that abolishes the endocrine disruptive effects. DCF seems to be more toxic for this species, altering also hepatocytes morphology.

Caffeine Potentiates Ethanol-Induced Neurotoxicity Through mTOR/p70S6K/4E-BP1 Inhibition in SH-SY5Y Cells.

Caffeine is a popular psychostimulant, which is frequently consumed with ethanol. However, the effects of caffeine on neuronal cells constantly exposed to ethanol have not been investigated. Apoptosis and oxidative stress occurring in ethanol-induced neurotoxicity were previously associated with decreased phosphorylation of the mTOR/p70S6K/4E-BP1 signaling proteins. Evidence also suggested that caffeine inhibits the mTOR pathway. In this study, human SH-SY5Y neuroblastoma cells were exposed to caffeine after pretreatment for 24 hours with ethanol. Results indicated that both ethanol and caffeine caused neuronal cell death in a dose- and time-dependent manner. Exposure to 20-mM caffeine for 24 hours magnified reduced cell viability and enhanced apoptotic cell death induced by 200 mM of ethanol pretreatment. The phosphorylation of mTOR, p70S6K, and 4E-BP1 markedly decreased in cells exposed to caffeine after ethanol pretreatment, associated with a decrease of the mitochondrial membrane potential (ΔΨm). These findings suggested that caffeine treatment after neuronal cells were exposed to ethanol resulted in marked cell damages, mediated through enhanced inhibition of mTOR/p70S6K/4E-BP1 signaling leading to impaired ΔΨm and, eventually, apoptotic cell death.

Subchondral bone dysplasia mediates susceptibility to osteoarthritis in female adult offspring rats induced by prenatal caffeine exposure.

Our previous studies confirmed that prenatal caffeine exposure (PCE) could induce susceptibility to osteoarthritis in adult offspring rats due to poor chondrocyte differentiation, but its mechanism remains to be further investigated. This study aimed to explore whether subchondral bone dysplasia mediates susceptibility to osteoarthritis in adult offspring rats induced by PCE. Pregnant Wistar rats were treated with caffeine (120 mg/kg.d) or saline from gestational day (GD) 9 to 20. The female offspring were euthanized to collect femurs at GD20, postnatal week (PW) 6, and PW28 (non-ovariectomy and ovariectomy groups) to detect osteoarthritis-like phenotype, subchondral bone mass, ossification center development, and other evidence. The results showed that PCE increased the Mankin score of pathological articular cartilage, but decreased articular cartilage thickness and subchondral bone mass, which were more obvious after ovariectomy. Meanwhile, the correlation analysis results demonstrated that the Mankin score of articular cartilage was significantly negatively correlated with subchondral bone mass, and the thickness of articular cartilage was significantly positively correlated with subchondral bone mass. Further, the length and area of the primary and secondary ossification centers, the number of osteoblasts, and the related genes' expression of osteogenic differentiation (e.g., Runx2, BSP, ALP, and OCN) were all significantly decreased in the PCE group before and after birth. Taken together, PCE induced susceptibility to osteoarthritis in adult female offspring, which was likely related to the subchondral bone dysplasia and reduction of subchondral bone mass production due to developmental disorder of primary and secondary ossification centers caused by osteoblast differentiation disability before and after birth.

Decreased H3K9ac level of KLF4 mediates podocyte developmental toxicity induced by prenatal caffeine exposure in male offspring rats.

This study aimed to verify the toxic effects of prenatal caffeine exposure (PCE) on the podocyte development in male offspring, and to explore the underlying intrauterine programming mechanisms. The pregnant rats were administered with caffeine (30 to 120 mg/kg⋅d) during gestational day (GD) 9 to 20. The male fetus on GD20 and the offspring at postnatal week (PW) 6 and PW28 were sacrificed. The results indicated that PCE caused ultrastructural abnormalities on podocyte, and inhibited the expression of podocyte marker genes such as Nephrin, Wilms tumor 1 (WT1), the histone 3 lysine 9 acetylation (H3K9ac) level in the Kruppel-like factor 4 (KLF4) promoter and its expression in the male offspring from GD20 to PW28. Meanwhile, the expression of glucocorticoid receptor (GR) and histone deacetylase 7 (HDAC7) in the fetus were increased by PCE. In vitro, corticosterone increased GR and HDAC7 whereas reduced the H3K9ac level of KLF4 and KLF4/Nephrin expression. KLF4 over-expression reversed the reduction of Nephrin expression, knockdown of HDAC7 and GR antagonist RU486 partially reversed the inhibitory effects of corticosterone on H3K9ac level and KLF4 expression. In conclusion, PCE caused podocyte developmental toxicity in male offspring, which was associated with corticosterone-induced low-functional programming of KLF4 through GR/HDAC7/H3K9ac pathway.

The GC-IGF1 axis-mediated testicular dysplasia caused by prenatal caffeine exposure.

Prenatal caffeine exposure (PCE) can induce testicular developmental toxicity. Here, we aimed to explore the underlying mechanism of this process in reference to its intrauterine origin. Pregnant rats were intragastrically administrated caffeine (30 and 120 mg/kg/day) from gestational days 9 to 20. The results showed that the male fetuses exposed to high dose of caffeine (120 mg/kg/day) had a decreased bodyweight and inhibited testosterone synthetic function. Meanwhile, their serum corticosterone concentration was elevated and their testicular insulin-like growth factor 1 (Igf1) expression was decreased. Moreover, the histone 3 lysine 14 acetylation (H3K14ac) level in the Igf1 promoter region was reduced. Low-dose (30 mg/kg/day) caffeine exposure, however, increased steroidogenic enzymes expression in male fetuses. After birth, the serum corticosterone concentration gradually decreased in the PCE (120 mg/kg/day) offspring rats, whereas the expression and H3K14ac level of Igf1 gradually increased, with obvious catch-up growth and testicular development compensation. Intriguingly, when we subjected the offspring to 2 weeks of chronic stress to elevate the serum corticosterone concentration, the expression of Igf1 and testosterone synthesis were inhibited again in the PCE (120 mg/kg/day) group, accompanied by a decrease in the H3K14ac level in the Igf1 promoter region. In vitro, corticosterone (rather than caffeine) was proved to inhibit testosterone production in Leydig cells by altering the H3K14ac level and the expression of Igf1. These observations suggested that PCE-induced testicular developmental toxicity is related to the negative regulation of corticosterone on H3K14ac levels and the expression of Igf1.

Caffeine programs hepatic SIRT1-related cholesterol synthesis and hypercholesterolemia via A2AR/cAMP/PKA pathway in adult male offspring rats.

Clinical and animal studies have indicated that hypercholesterolemia has intrauterine developmental origin. Our previous studies showed that prenatal caffeine exposure (PCE) increased the serum total cholesterol (TCH) levels in adult offspring rats. This study investigates the intrauterine programming mechanism of PCE male offspring rats susceptible to adult hypercholesterolemia. Pregnant Wistar rats were intragastrically administered caffeine (30, 60, and 120 mg/kg∙d) from gestational days (GD) 9 to 20. Male offspring were sacrificed under anesthesia at GD20 and postnatal week (PW) 12, and the serum and liver were collected. The effects of caffeine (0-100 µM, 24 h) on the expression of cholesterol synthesis related genes and their epigenetic mechanisms were confirmed in L02 cells. The results showed that PCE induced higher levels of serum TCH, LDL-C and higher ratios of TCH/HDL-C and LDL-C/HDL-C. Furthermore, the high levels of histone acetylation (via H3K14ac and H3K27ac) and the expression of genes (Srebf2, Hmgcr, Hmgcs1) were responsible for cholesterol synthesis. The results of PCE offspring in utero and the data in vitro exhibited similar changes, and accompanied by the reduced expression of adenosine A2A receptor (A2AR), cyclic adenosine monophosphate (cAMP), sirtuin1 and protein kinase A (PKA). These changes could be reversed by A2AR agonist (CGS-21680), cAMP agonist (forskolin) and sirtuin1 agonist (resveratrol). Therefore, our results confirmed that caffeine could enhance histone acetylation and expression levels of genes responsible for cholesterol synthesis via inhibiting the A2AR/cAMP/PKA pathway and down-regulating sirtuin1, which continued throughout adulthood and elevated hepatic cholesterol synthesis and hypercholesterolemia in the male offspring rats.