Prenatal caffeine exposure induces autism-like behaviors in offspring under a high-fat diet via the gut microbiota-IL-17A-brain axis.

Prenatal caffeine exposure (PCE) is a significant contributor to intrauterine growth retardation (IUGR) in offspring, which has been linked to an increased susceptibility to autism spectrum disorder (ASD) later in life. Additionally, a high-fat diet (HFD) has been shown to exacerbate ASD-like behaviors, but the underlying mechanisms remain unclear. In this study, we first noted in the rat model of IUGR induced by PCE that male PCE offspring exhibited typical ASD-like behaviors post-birth, in contrast to their female counterparts. The female PCE offspring demonstrated only reduced abilities in free exploration and spatial memory. Importantly, both male and female PCE offspring displayed ASD-like behaviors when exposed to HFD. We further observed that PCE + HFD offspring exhibited damaged intestinal mucus barriers and disturbed gut microbiota, resulting in an increased abundance of Escherichia coli (E. coli). The induced differentiation of colonic Th17 cells by E. coli led to an increased secretion of IL-17A, which entered the hippocampus through peripheral circulation and caused synaptic damage in hippocampal neurons, ultimately resulting in ASD development. Our strain transplantation experiment suggested that E. coli-mediated increase of IL-17A may be the core mechanism of ASD with a fetal origin. In conclusion, PCE and HFD are potential risk factors for ASD, and E. coli-mediated IL-17A may play a crucial role in fetal-originated ASD through the gut-brain axis.

A novel multi-omics data analysis of dose-dependent and temporal changes in regulatory pathways due to chemical perturbation: a case study on caffeine.

Comprehensive analysis of multi-omics data can reveal alterations in regulatory pathways induced by cellular exposure to chemicals by characterizing biological processes at the molecular level. Data-driven omics analysis, conducted in a dose-dependent or dynamic manner, can facilitate comprehending toxicity mechanisms. This study introduces a novel multi-omics data analysis designed to concurrently examine dose-dependent and temporal patterns of cellular responses to chemical perturbations. This analysis, encompassing preliminary exploration, pattern deconstruction, and network reconstruction of multi-omics data, provides a comprehensive perspective on the dynamic behaviors of cells exposed to varying levels of chemical stimuli. Importantly, this analysis is adaptable to any number of omics layers, including site-specific phosphoproteomics. We implemented this analysis on multi-omics data obtained from HepG2 cells exposed to a range of caffeine doses over varying durations and identified six response patterns, along with their associated biomolecules and pathways. Our study demonstrates the effectiveness of the proposed multi-omics data analysis in capturing multidimensional patterns of cellular response to chemical perturbation, enhancing understanding of pathway regulation for chemical risk assessment.

Evaluation of subchronic toxicity of the compound of diphenhydramine hydrochloride and caffeine after 28 days of repeated oral administration in Sprague-Dawley rats and beagle dogs.

This study was designed to evaluate the subchronic toxicity of the compound of diphenhydramine hydrochloride (DH) and caffeine in Sprague-Dawley (SD) rats and beagle dogs. A total of 180 SD rats (15/sex/group) were randomly divided into the compound low-, medium- and high-dose groups (51, 102, 204 mg/kg), DH group (60 mg/kg), caffeine group (144 mg/kg) and the vehicle control group. Sixty beagle dogs (5/sex/group) were randomly divided into the compound low-, medium- and high-dose groups (male: 14.20, 28.30, 56.60 mg/kg, female: 5.66, 14.20, 28.30 mg/kg), DH group (male: 16.60 mg/kg, female: 8.30 mg/kg), caffeine group (male: 40.00 mg/kg, female: 20.00 mg/kg) and the vehicle control group. Rats and dogs were given continuous oral administration for 28 days following a 28-day recovery period. The adverse effects of the compound on rats and beagle dogs mainly included anorexia and liver function impairment. Most adverse effects induced by administration were reversible. Under the experimental conditions, the no-observed-adverse-effect level (NOAEL) of the compound of DH and caffeine was 51 mg/kg/day for SD rats and 28.30 mg/kg/day (male) and 5.66 mg/kg/day (female) for beagle dogs.

Time-resolved quantitative phosphoproteomics reveals cellular responses induced by caffeine and coumarin.

Protein phosphorylation is a critical way that cells respond to external signals and environmental stresses. However, the patterns of cellular response to chemicals at different times were largely unknown. Here, we used quantitative phosphoproteomics to analyze the cellular response of kinases and signaling pathways, as well as pattern change of phosphorylated substrates in HepG2 cells that were exposed to caffeine and coumarin for 10 min and 24 h. Comparing the 10 min and 24 h groups, 33 kinases were co-responded and 32 signaling pathways were co-enriched in caffeine treated samples, while 48 kinases and 34 signaling pathways were co-identified in coumarin treated samples. Instead, the percentage of co-identified phosphorylated substrates only accounted for 4.31% and 9.57% between 10 min and 24 h in caffeine and coumarin treated samples, respectively. The results showed that specific chemical exposure led to a bunch of the same kinases and signaling pathways changed in HepG2 cells, while the phosphorylated substrates were different. In addition, it was found that insulin signaling pathway was significantly enriched by both the caffeine and coumarin treatment. The pattern changes in phosphorylation of protein substrates, kinases and signaling pathways with varied chemicals and different time course shed light on the potential mechanism of cellular responses to endless chemical stimulation.

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-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.

Intrauterine programming of cartilaginous 11ß-HSD2 induced by corticosterone and caffeine mediated susceptibility to adult osteoarthritis.

Our previous study reported that prenatal caffeine exposure (PCE) could induce chondrodysplasia and increase the susceptibility to osteoarthritis in offspring rats. However, the potential mechanisms and initiating factors remain unknown. This study aims to investigate whether 11ß-HSD2, a glucocorticoid-metabolizing enzyme, is involved in the susceptibility of osteoarthritis induced by PCE and to further explore its potential mechanisms and initiating factors. Firstly, we found that PCE reduced cartilage matrix synthesis (aggrecan/Col2a1 expression) in male adult offspring rats and exhibited an osteoarthritis phenotype following chronic stress, which was associated with persistently reduced H3K9ac and H3K27ac levels at the promoter of 11ß-HSD2 as well as its expression in the cartilage from fetus to adulthood. The expression of 11ß-HSD2, aggrecan and Col2a1 were all decreased by corticosterone in the fetal chondrocytes, while overexpression of 11ß-HSD2 could partially alleviate the decrease of matrix synthesis induced by corticosterone in vitro. Furthermore, the glucocorticoid receptor (GR) activated by glucocorticoids directly bonded to the promoter region of 11ß-HSD2 to inhibit its expression. Meanwhile, the activated GR reduced the H3K9ac and H3K27ac levels of 11ß-HSD2 by recruiting HDAC4 and promoting GR-HDAC4 protein interaction to inhibit the 11ß-HSD2 expression. Moreover, caffeine could reduce the expression of 11ß-HSD2 by inhibiting the cAMP/PKA signaling pathway but without reducing the H3K9ac and H3K27ac levels of 11ß-HSD2, thereby synergistically enhancing the corticosterone effect. In conclusion, the persistently reduced H3K9ac and H3K27ac levels of 11ß-HSD2 from fetus to adulthood mediated the inhibition of cartilage matrix synthesis and the increased susceptibility to osteoarthritis. This epigenetic programming change in utero was induced by glucocorticoids with synergistic effect of caffeine.

Sublethal effects of environmental concentrations of caffeine on a neotropical freshwater fish.

Caffeine is a contaminant frequently detected in water bodies. Growth trends in both human population and caffeine consumption per capita are expected to exacerbate the occurrence of caffeine in freshwaters. Yet the effects of caffeine on native fish fauna are poorly understood. We exposed larvae of an endemic Neotropical catfish (Rhamdia quelen) to a range of caffeine concentrations for 30 days. We found that larvae exposed to the highest concentration (16 mg L-1) showed skeletal deformations and reduced growth. We further compiled measured environmental concentrations of caffeine in surface freshwater globally and performed a risk assessment. Our analysis points to a low risk to R. quelen and equally sensitive fish species in ~90% of the freshwater ecosystems considered in our analysis. The risk quotient is higher in freshwater ecosystems of South and Central America, where R. quelen is endemic. Although the ecotoxicological risk is currently low in most places, increased caffeine consumption, exacerbated by the lack of sanitation, is expected to increase caffeine concentrations in many parts of the world, posing a threat of sublethal morphological effects to local fish species.

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.

Effect of simultaneous coffee/caffeine and ethanol administration on sperm quality and reproductive hormones: an experimental study in Sprague Dawley rats.

Chronic alcohol or coffee/caffeine consumption has been associated with negative effects on male reproductive system. The present study evaluated the possible interaction (combination treatment) of coffee/caffeine and alcohol and its resultant consequence on sperm parameters in rats. Ten groups of adult Sprague Dawley rats (n = 5 per group) were orally gavaged with different quantities of coffee (≈5, 10, and 20 mg/kg caffeine); 30% v/v ethanol (0.5, 1, and 2 g/kg); coffee + ethanol or vehicle (distilled water) daily for 60 days. Sperm parameters, serum sex hormone levels, together with antioxidants and lipid peroxidation status of testis homogenate were evaluated. Single and combined administration of coffee/caffeine and ethanol caused a reduction in sperm count dose-dependently (p = 0.0002), but this effect was highest in the combined treatment. Sperm viability, motility, and morphology were unaffected by coffee or ethanol administration, but the combination produced detrimental effects on them. While changes in follicle stimulating hormone were not significant, testosterone and luteinizing hormone levels were decreased significantly and dose-dependently in all experimental groups (p < 0.05-0.0001). This effect was most pronounced in the groups that received the combination. Testicular superoxide dismutase and catalase activities, and reduced glutathione and malondiadehye (MDA) levels were not affected by coffee/caffeine, but ethanol decreased (p < 0.05) the antioxidants and elevated MDA levels mostly at 2 g/kg. The combination reduced (p < 0.0001) the antioxidants and increased MDA levels in testis homogenates at all doses. Combination of coffee/caffeine and ethanol potentiates their detrimental effects on sperm quality and reproductive hormones via reduction in testicular antioxidants activities and elevation of lipid peroxidation in rats.

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.

How temperature can alter the combined effects of carbon nanotubes and caffeine in the clam Ruditapes decussatus?

Nowadays, multi-walled carbon nanotubes are considered to be emerging contaminants and their impact in ecosystem has drawn special research attention, while other contaminants, such as caffeine, have more coverage in literature. Despite this, the effects of a combination of the two has yet to be evaluated, especially considering predicted temperature rise. In the present study a typical bioindicator species for marine environment, the clam Ruditapes decussatus, and classical tools, such as biomarkers and histopathological indices, were used to shed light on the species' response to these contaminants, under actual and predicted warming scenarios. The results obtained showed that both contaminants have a harmful effect at tissue level, as shown by higher histopathological index, especially in digestive tubules. Temperatures seemed to induce greater biochemical impacts than caffeine (CAF) and -COOH functionalized multi-walled carbon nanotubes (f-MWCNTs) when acting alone, namely in terms of antioxidant defences and energy reserves content, which were exacerbated when both contaminants were acting in combination (MIX treatment). Overall, the present findings highlight the complex response of clams to both pollutants, evidencing the role of temperature on clams' sensitivity, especially to mixture of pollutants.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Caffeine as a contaminant of periphyton: ecological changes and impacts on primary producers.

Every day, tons of caffeine is consumed by humans in beverages, medications or supplements, and a significant amount of this stimulant is released in domestic sewage. Once in aquatic environments caffeine interacts directly with the periphytic community, which is responsible for a significant part of primary production in aquatic ecosystems. However, the effects of exposure to caffeine are mostly unknown for both the periphyton and their predators. Aiming to comprehend the interaction between caffeine and the periphytic community, ecotoxicological experiments were performed by exposing a periphytic biofilm cultivated in the laboratory to different concentrations of caffeine, following concentrations found in domestic sewers. The impact of exposure to this contaminant was observed on the structure of the community through taxonomic evaluation, as well a set of physiological variables linked to primary production. After exposure to the highest caffeine concentration (300 µg L-1), the density of the genus Scenedesmus was severely affected, leading to an increase in cyanobacteria and diatoms. Both richness and diversity decreased after exposure, and there was lower photosynthetic activity, with light saturation point changing from 186 µmol m-2 s-1 in the control treatment to 108 µmol m-2 s-1 after exposure. Caffeine accumulation within the biofilm was also observed during the first 24 h, in the concentration of 0.14 µg /cm².

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.

Neuromotor activity inhibition in zebrafish early-life stages after exposure to environmental relevant concentrations of caffeine.

Caffeine (CAF), a neuroactive compound, has been found in surface waters at concentrations ranging from few nanograms up to micrograms and may induce adverse effects in aquatic vertebrates. Thus, the aim of this study was to evaluate the potential of CAF in affecting fish early-life stages in a wide concentration range, including occurring levels in surface waters. Specimens of zebrafish in early-life stages were exposed to CAF for 168 h and survival, developmental alterations, locomotor activity and acetylcholinesterase activity were evaluated. CAF induced mortality in embryos unable to hatch or in larvae after hatching (LC50 - 168 h = 283.2 mg/L). Tail deformities were observed in organisms exposed to concentrations ≥ 40 mg/L, while edemas were found at concentrations of 100 mg/L. CAF also decreased the total swimming time and distance moved of exposed organisms (LOEC = 0.0006 mg/L). Locomotor inhibition may be associated with an acetylcholinesterase inhibition observed at concentration ≥ 0.0088 mg/L. Therefore, the hazard of CAF for fish populations deserves further attention since unexpected effects on neuro-behavioral parameters occurs at concentrations often detected in natural aquatic ecosystems.

Trait specific modulatory effects of caffeine exposure on compulsive-like behaviors in a spontaneous mouse model of obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by recurring intrusive thoughts and repetitive compulsive behaviors, ultimately interfering with their quality of life. The complex heterogeneity of symptom dimensions across OCD patient subgroups impedes diagnosis and treatment. The core and comorbid symptomologies of OCD are thought to be modulated by common environmental exposures such as consumption of the psychostimulant caffeine. The effect of caffeine on the expression of obsessions and compulsions are unexplored. The current study utilized mouse strains (HA) with a spontaneous, predictable, and stable compulsive-like phenotype that have face, predictive, and construct validity for OCD. We demonstrate that an acute high dose (25 mg/kg) of caffeine decreased compulsive-like nest-building behavior in the HA strains in the first hour after injection. However, nest-building scores increased in hours 3, 4, and 5 after administration finally decreasing over a 24 h period. In contrast, a high dose of chronic caffeine (25 mg/kg/d) increased nest-building behavior. Interestingly for compulsive-like digging behavior, acute exposure to a high dose of caffeine decreased the number of marbles buried, while chronic exposure had little effect. An acute high dose of caffeine decreased anxiety-like and motor activity in open field behaviors whereas chronic caffeine administration did not have any overall effect on open field activity. The results, therefore, suggest a complex role of caffeine on compulsive-like, anxiety-like, and locomotor behaviors that is dependent on the duration of exposure.

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.

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.

Toxic doses of caffeine are needed to increase skeletal muscle contractility.

Discrepant results have been reported regarding an intramuscular mechanism underlying the ergogenic effect of caffeine on neuromuscular function in humans. Here, we reevaluated the effect of caffeine on muscular force production in humans and combined this with measurements of the caffeine dose-response relationship on force and cytosolic free [Ca2+] ([Ca2+]i) in isolated mouse muscle fibers. Twenty-one healthy and physically active men (29 ± 9 yr, 178 ± 6 cm, 73 ± 10 kg, mean ± SD) took part in the present study. Nine participants were involved in two experimental sessions during which supramaximal single and paired electrical stimulations (at 10 and 100 Hz) were applied to the femoral nerve to record evoked forces. Evoked forces were recorded before and 1 h after ingestion of 1) 6 mg caffeine/kg body mass or 2) placebo. Caffeine plasma concentration was measured in 12 participants. In addition, submaximal tetanic force and [Ca2+]i were measured in single mouse flexor digitorum brevis (FDB) muscle fibers exposed to 100 nM up to 5 mM caffeine. Six milligrams of caffeine per kilogram body mass (plasma concentration ~40 µM) did not increase electrically evoked forces in humans. In superfused FDB single fibers, millimolar caffeine concentrations (i.e., 15- to 35-fold above usual concentrations observed in humans) were required to increase tetanic force and [Ca2+]i. Our results suggest that toxic doses of caffeine are required to increase muscle contractility, questioning the purported intramuscular ergogenic effect of caffeine in humans.