Current approaches and challenges of sample preparation procedures for the safety assessment of paper and cardboard food contact materials: A comprehensive review.

In the European Union (EU), Regulation (EC) 1935/2004 provides a harmonized legal EU framework and sets out the general principles for safety and inertness for all Food Contact Materials (FCMs) and Food Contact Articles. From a food safety point of view, however, specific EU legislation for paper and cardboard FCMs is lacking, while at Member State level, national legislation differs among countries. More than 11,000 chemicals have been identified in all types of FCMs, most of them without any information on toxicity or migration potential from FCM to food. The present review shows a wide variability of protocols, approaches, and conditions used in scientific studies, which are difficult to compare. In this regard, procedures and conditions laid down in EU legislation for plastics and European Standards (EN protocols) may serve as a good basis for the future sample preparation procedures in the framework of paper and cardboard FCMs safety assessment. Challenges on sample preparation procedures are presented involving the interlinked steps of sample preparation, conditions used and their impact in chemical analysis and in vitro bioassay testing. Currently, there is no general consensus on the criteria for structuring, evaluating, and tuning sample preparation procedures for paper and cardboard FCMs. For this purpose, a set of modified criteria and a decision tree are proposed based on the literature. Along this, mass transfer processes occurring in paper and cardboard FCMs and parameters affecting chemical migration need to be accounted for prior to reaching general consensus on criteria for sample preparation procedures.

Red yeast rice (Monascus purpureus) supplements: Case series assessment of spontaneously reported cases to The Netherlands Pharmacovigilance Centre Lareb.

Serious concerns are expressed on the safe use of red yeast rice (RYR) supplements. The aim of the present study was to analyse cases received by Lareb on RYR-related adverse health events. These cases were analysed for the number of reports, number of adverse drug reactions (ADRs), causality, seriousness of the reaction, latency-period, age and sex of the patients, concomitant medication and type of reporter. A total of 94 individual reports were collected by Lareb, corresponding with 187 ADRs. The analysis showed most reported ADRs were labelled musculoskeletal and connective tissue disorders (n = 64), gastrointestinal disorders (n = 33) and general disorders and administration site conditions (n = 23). The use of RYR supplements should be considered as a significant safety concern. Exposure to monacolin K could lead to serious adverse effects. To fully assess the safety profile of RYR supplements, more research is necessary to compose a complete ADR profile of RYR supplements.

Role of antioxidants in the treatment of gastroesophageal reflux disease-associated idiopathic pulmonary fibrosis.

PURPOSE OF REVIEW: Idiopathic pulmonary fibrosis (IPF) is a terminal lung disease of largely unknown cause. Gastroesophageal reflux disease (GERD) was recently discovered to be a trigger for the development of IPF. The current pharmaceutical approach to IPF falls short and there is a pressing need for improved therapeutic options. The present review describes the currently available knowledge regarding the role of oxidative stress and inflammation in the pathophysiology of IPF and GERD and determines the potential use of antioxidants as a treatment option for GERD-associated IPF. RECENT FINDINGS: IPF and GERD share a similar pathophysiology, as oxidative stress and inflammation play a pivotal role in both conditions. This raises the question whether antioxidant treatment could be a well-tolerated and effective means to alleviate at least some of the symptoms of both conditions. In IPF, antioxidant supplementation complements the inadequately working antioxidant defense system of the lung, reducing oxidative stress and inflammation. In GERD, antioxidants increase levels of endogenous antioxidants, decrease pepsin and gastric acid production, lipid peroxidation, and ulceration, and alleviate subsequent damage to the gastric mucosa. SUMMARY: The increased comorbidity of GERD in IPF patients makes it clear that there is a connection between GERD and IPF. As current treatment options are still inadequate to improve the condition and increase the survival rate of IPF patients, alternative treatment options are crucial. Based on the reviewed scientific evidence, antioxidant supplementation could complement standard IPF treatment, certainly in GERD-associated IPF.

The minor structural difference between the antioxidants quercetin and 4'O-methylquercetin has a major impact on their selective thiol toxicity.

Antioxidants act as intermediates by picking up the high unselective reactivity of radicals and transferring it to other molecules. In this process the reactivity is reduced and becomes selective. This channeling of the reactivity can cause selective toxicity. The antioxidant quercetin is known to channel the reactivity towards thiol groups. The present study compares the thiol reactivity of quercetin with that of 4'O-methylquercetin (tamarixetin) towards creatine kinase (CK), a vital protein that contains a critical thiol moiety. Our results showed that oxidized quercetin and oxidized tamarixetin both adduct CK, which then loses its enzymatic function. Ascorbate, an important representative of the antioxidant network, is able to prevent adduction to and thus the inhibition of the enzyme by tamarixetin but not by quercetin. Apparently, tamarixetin is less thiol toxic than quercetin, because--rather than adduction to CK--tamarixetin quinone prefers to pass reactivity to the antioxidant network, i.e., to ascorbate. The findings exemplify that radical scavenging flavonoids pick up the reactivity of radicals and act as a pivot in directing the way the reactivity is channeled. A mere minor structural difference of only one methyl moiety between quercetin and tamarixetin appears to have a high impact on the selective, thiol toxicity.

In Vitro Activation of Human Adrenergic Receptors and Trace Amine-Associated Receptor 1 by Phenethylamine Analogues Present in Food Supplements.

Pre-workout supplements are popular among sport athletes and overweight individuals. Phenethylamines (PEAs) and alkylamines (AA) are widely present in these supplements. Although the health effects of these analogues are not well understood yet, they are hypothesised to be agonists of adrenergic (ADR) and trace amine-associated receptors (TAARs). Therefore, we aimed to pharmacologically characterise these compounds by investigating their activating properties of ADRs and TAAR1 in vitro. The potency and efficacy of the selected PEAs and AAs was studied by using cell lines overexpressing human ADRα1A/α1B/α1D/α2a/α2B/ß1/ß2 or TAAR1. Concentration-response relationships are expressed as percentages of the maximal signal obtained by the full ADR agonist adrenaline or the full TAAR1 agonist phenethylamine. Multiple PEAs activated ADRs (EC50 = 34 nM-690 µM; Emax = 8-105%). Almost all PEAs activated TAAR1 (EC50 = 1.8-92 µM; Emax = 40-104%). Our results reveal the pharmacological profile of PEAs and AAs that are often used in food supplements. Several PEAs have strong agonistic properties on multiple receptors and resemble potencies of the endogenous ligands, indicating that they might further stimulate the already activated sympathetic nervous system in exercising athletes via multiple mechanisms. The use of supplements containing one, or a combination of, PEA(s) may pose a health risk for their consumers.

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Androgen Receptor and Cardiovascular Disease: A Potential Risk for the Abuse of Supplements Containing Selective Androgen Receptor Modulators.

The androgen receptor (AR) is a member of the family of ligand-activated transcription factors. Selective androgen receptor modulators (SARMs) exert their biological function through complex interactions with the AR. It has been speculated that overexertion of AR signaling cascades as a result of SARM abuse can be a risk factor for the development of various cardiovascular diseases. The present literature review explores the implications of the interaction between SARMs and the AR on cardiovascular health by focusing on the AR structure, function, and mechanisms of action, as well as the current clinical literature on various SARMs. It is shown that SARMs may increase the risk of cardiovascular diseases through implications on the renin-angiotensin system, smooth muscle cells, sympathetic nervous system, lipid profile, inflammation, platelet activity, and various other factors. More research on this topic is necessary as SARM abuse is becoming increasingly common. There is a noticeable lack of clinical trials and literature on the relationship between SARMs, cardiovascular diseases, and the AR. Future in vivo and in vitro studies within this field are vital to understand the mechanisms that underpin these complex interactions and risk factors.

Mixture effects of tetrodotoxin (TTX) and drugs targeting voltage-gated sodium channels on spontaneous neuronal activity in vitro.

Tetrodotoxin (TTX) potently inhibits TTX-sensitive voltage-gated sodium (NaV) channels in nerve and muscle cells, potentially resulting in depressed neurotransmission, paralysis and death from respiratory failure. Since a wide range of pharmaceutical drugs is known to also act on NaV channels, the use of medicines could predispose individuals to a higher susceptibility towards TTX toxicity. We therefore first assessed the inhibitory effect of selected medicines that act on TTX-sensitive (Riluzole, Chloroquine, Fluoxetine, Valproic acid, Lamotrigine, Lidocaine) and TTX-resistant (Carbamazepine, Mexiletine, Flecainide) NaV channels on spontaneous neuronal activity of rat primary cortical cultures grown on microelectrode arrays (MEA). After establishing concentration-effect curves, binary mixtures of the medicines with TTX at calculated NOEC, IC20 and IC50 values were used to determine if pharmacodynamic interactions occur between TTX and these drugs on spontaneous neuronal activity. At IC20 and IC50 values, all medicines significantly increased the inhibitory effect of TTX on spontaneous neuronal activity of rat cortical cells in vitro. Subsequent experiments using human iPSC-derived neuronal co-cultures grown on MEAs confirmed the ability of selected medicines (Carbamazepine, Flecainide, Riluzole, Lidocaine) to inhibit spontaneous neuronal activity. Despite the need for additional experiments using human iPSC-derived neuronal co-cultures, our combined data already highlight the importance of identifying and including vulnerable risk groups in the risk assessment of TTX.

Dietary Advanced Glycation Endproducts and the Gastrointestinal Tract.

The prevalence of inflammatory bowel diseases (IBD) is increasing in the world. The introduction of the Western diet has been suggested as a potential explanation of increased prevalence. The Western diet includes highly processed food products, and often include thermal treatment. During thermal treatment, the Maillard reaction can occur, leading to the formation of dietary advanced glycation endproducts (dAGEs). In this review, different biological effects of dAGEs are discussed, including their digestion, absorption, formation, and degradation in the gastrointestinal tract, with an emphasis on their pro-inflammatory effects. In addition, potential mechanisms in the inflammatory effects of dAGEs are discussed. This review also specifically elaborates on the involvement of the effects of dAGEs in IBD and focuses on evidence regarding the involvement of dAGEs in the symptoms of IBD. Finally, knowledge gaps that still need to be filled are identified.

Haemodynamic effects of the flavonoid quercetin in rats revisited.

BACKGROUND AND PURPOSE: The flavonoid quercetin increased the in vitro potency of the α1 -antagonist tamsulosin to reduce phenylephrine-dependent arterial contractions by 10-fold. To examine if this supplement-drug interaction luxates hypotensive and orthostatic events in vivo, several set of studies were conducted in spontaneously hypertensive (SHR) and normotensive (Wistar Kyoto [WKY]) rats. EXPERIMENTAL APPROACH: First, in rats pretreated with quercetin or its vehicle, responses to phenylephrine and tamsulosin were examined. Second, tamsulosin-induced changes in renal, mesenteric, hindquarter and carotid conductance were compared in quercetin- and vehicle-treated rats instrumented with Doppler flow probes. Animals were also placed on a tilt table to record regional haemodynamic changes to orthostatic challenges. Third, adult SHR were instrumented with telemeters to measure 24-hr patterns of BP. Recordings were made before and during a 5-week oral treatment of quercetin. Finally, pre-hypertensive SHR were treated with quercetin from 4 to 8 weeks of age and arterial pressure was measured at 8 and 12 weeks. KEY RESULTS: Pretreatment with quercetin did not influence the responses to phenylephrine and tamsulosin, in neither WKY nor SHR. While tamsulosin treatment and tilting lowered BP and increased conductance in all vascular beds, effect size was not influenced by pretreatment with quercetin. Prolonged treatment with quercetin, in either prehypertensive SHR or adult SHR with established hypertension did not lower BP. CONCLUSIONS AND IMPLICATIONS: Cumulatively, these data demonstrate that quercetin does not amplify haemodynamic effects of tamsulosin or tilting in vivo in rats and has no effect on BP development in SHR.

Dietary Advanced Glycation Endproducts Decrease Glucocorticoid Sensitivity In Vitro.

Glucocorticoids are very effective anti-inflammatory drugs and widely used for inflammatory bowel disease (IBD) patients. However, approximately 20% of IBD patients do not respond to glucocorticoids and the reason for this is largely unknown. Dietary advanced glycation endproducts (AGEs) are formed via the Maillard reaction during the thermal processing of food products and can induce a pro-inflammatory reaction in human cells. To investigate whether this pro-inflammatory response could be mitigated by glucocorticoids, human macrophage-like cells were exposed to both LPS and AGEs to induce interleukin-8 (IL8) secretion. This pro-inflammatory response was then modulated by adding pharmacological compounds interfering in different steps of the anti-inflammatory mechanism of glucocorticoids: rapamycin, quercetin, and theophylline. Additionally, intracellular reactive oxygen species (ROS) were measured and the glucocorticoid receptor phosphorylation state was assessed. The results show that AGEs induced glucocorticoid resistance, which could be mitigated by quercetin and rapamycin. No change in the phosphorylation state of the glucocorticoid receptor was observed. Additionally, intracellular ROS formation was induced by AGEs, which was mitigated by quercetin. This suggests that AGE-induced ROS is an underlying mechanism to AGE-induced glucocorticoid resistance. This study shows for the first time the phenomenon of dietary AGE-induced glucocorticoid resistance due to the formation of ROS. Our findings indicate that food products with a high inflammatory potential can induce glucocorticoid resistance; these results may be of great importance to IBD patients suffering from glucocorticoid resistance.

Gastrointestinal digestion of dietary advanced glycation endproducts using an in vitro model of the gastrointestinal tract (TIM-1).

Protein- and sugar-rich food products processed at high temperatures contain large amounts of dietary advanced glycation endproducts (dAGEs). Our earlier studies have shown that specifically protein-bound dAGEs induce a pro-inflammatory reaction in human macrophage-like cells. To what extent these protein-bound dAGEs survive the human gastrointestinal (GI) tract is still unclear. In this study we analysed gastric and small intestinal digestion of dAGEs using the validated, standardised TNO in vitro gastroIntestinal digestion model (TIM-1), a dynamic in vitro model which mimics the upper human GI tract. This model takes multiple parameters into account, such as: dynamic pH curves, peristaltic mixing, addition of bile and pancreatic digestive enzymes, and passive absorption. Samples of different digested food products were collected at different time points after (i) only gastric digestion and (ii) after both gastric plus small intestinal digestion. Samples were analysed for dAGEs using UPLC-MS/MS for the lysine derived Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL), and the arginine derived methylglyoxal-derived hydroimidazolone-1 (MG-H1), and glyoxal-derived hydroimidazolone-1 (G-H1). All AGEs were quantified in their protein-bound and free form. The results of this in vitro study show that protein-bound dAGEs survive gastrointestinal digestion and are additionally formed during small intestinal digestion. In ginger biscuits, the presence MG-H1 in the GI tract increased with more than 400%. This also indicates that dAGEs enter the human GI tract with potential pro-inflammatory characteristics.

Dietary Advanced Glycation Endproducts Induce an Inflammatory Response in Human Macrophages in Vitro.

Advanced glycation endproducts (AGEs) can be found in protein- and sugar-rich food products processed at high temperatures, which make up a vast amount of the Western diet. The effect of AGE-rich food products on human health is not yet clear and controversy still exists due to possible contamination of samples with endotoxin and the use of endogenous formed AGEs. AGEs occur in food products, both as protein-bound and individual molecules. Which form exactly induces a pro-inflammatory effect is also unknown. In this study, we exposed human macrophage-like cells to dietary AGEs, both in a protein matrix and individual AGEs. It was ensured that all samples did not contain endotoxin concentrations > 0.06 EU/mL. The dietary AGEs induced TNF-alpha secretion of human macrophage-like cells. This effect was decreased by the addition of N(ε)-carboxymethyllysine (CML)-antibodies or a receptor for advanced glycation endproducts (RAGE) antagonist. None of the individual AGEs induce any TNF-alpha, indicating that AGEs should be bound to proteins to exert an inflammatory reaction. These findings show that dietary AGEs directly stimulate the inflammatory response of human innate immune cells and help us define the risk of regular consumption of AGE-rich food products on human health.

The vitamin B6 paradox: Supplementation with high concentrations of pyridoxine leads to decreased vitamin B6 function.

Vitamin B6 is a water-soluble vitamin that functions as a coenzyme in many reactions involved in amino acid, carbohydrates and lipid metabolism. Since 2014, >50 cases of sensory neuronal pain due to vitamin B6 supplementation were reported. Up to now, the mechanism of this toxicity is enigmatic and the contribution of the various B6 vitamers to this toxicity is largely unknown. In the present study, the neurotoxicity of the different forms of vitamin B6 is tested on SHSY5Y and CaCo-2 cells. Cells were exposed to pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5-phosphate or pyridoxamine-5-phosphate for 24h, after which cell viability was measured using the MTT assay. The expression of Bax and caspase-8 was tested after the 24h exposure. The effect of the vitamers on two pyridoxal-5-phosphate dependent enzymes was also tested. Pyridoxine induced cell death in a concentration-dependent way in SHSY5Y cells. The other vitamers did not affect cell viability. Pyridoxine significantly increased the expression of Bax and caspase-8. Moreover, both pyridoxal-5-phosphate dependent enzymes were inhibited by pyridoxine. In conclusion, the present study indicates that the neuropathy observed after taking a relatively high dose of vitamin B6 supplements is due to pyridoxine. The inactive form pyridoxine competitively inhibits the active pyridoxal-5'-phosphate. Consequently, symptoms of vitamin B6 supplementation are similar to those of vitamin B6 deficiency.

Iron Supplements and Magnesium Peroxide: An Example of a Hazardous Combination in Self-Medication.

The use of self-medication, which includes dietary supplements and over-the-counter drugs, is still on the rise, while safety issues are not well addressed yet. This especially holds for combinations. For example, iron supplements and magnesium peroxide both produce adverse effects via the formation of reactive oxygen species (ROS). This prompted us to investigate the effect of the combination of three different iron supplements with magnesium peroxide on ROS formation. Hydroxyl radical formation by the three iron supplements either combined with magnesium peroxide or alone was determined by performing a deoxyribose assay. Free iron content of iron supplements was determined using ferrozine assay. To determine hydrogen peroxide formation by magnesium peroxide, a ferrous thiocyanate assay was performed. Finally, electron spin resonance spectroscopy (ESR) was performed to confirm the formation of hydroxyl radicals. Our results show that magnesium peroxide induces the formation of hydrogen peroxide. All three iron supplements induced the formation of the extremely reactive hydroxyl radical, although the amount of radicals formed by the different supplements differed. It was shown that combining iron supplements with magnesium peroxide increases radical formation. The formation of hydroxyl radicals after the combination was confirmed with ESR. All three iron supplements contained labile iron and induced the formation of hydroxyl radicals. Additionally, magnesium peroxide in water yields hydrogen peroxide, which is converted into hydroxyl radicals by iron. Hence, iron supplements and magnesium peroxide is a hazardous combination and exemplifies that more attention should be given to combinations of products used in self-medication.

The supplement-drug interaction of quercetin with tamsulosin on vasorelaxation.

The food supplement quercetin is used as self-medication for prostate disorders and is known to induce vasorelaxation. The drug tamsulosin is used in the treatment of benign prostatic hyperplasia. A major side effect of tamsulosin is orthostatic hypotension, mediated by vasorelaxation resulting from α1-adrenoceptor blockade. The overlapping profile prompted us to investigate the pharmacodynamic interaction of quercetin with tamsulosin. Since quercetin is extensively metabolized in the intestines and the liver, the metabolites quercetin-3-glucuronide and 4'O-methyl-quercetin were also examined. Vasorelaxation induced by the compounds was tested in rat mesenteric arteries (average diameter: 360±µm) constricted by the α1-adrenoceptor agonist phenylephrine. Tamsulosin (0.1nM) decreased phenylephrine sensitivity 17-fold (n=10). Quercetin (5, 10 and 20µM) also caused a decrease (2-, 4- and 6-fold respectively) of phenylephrine sensitivity, while 10µM of quercetin-3-glucuronide and 4'O-methyl-quercetin decreased this sensitivity (1.5- and 2-fold) only slightly (n=6). The combination of tamsulosin with quercetin or quercetin metabolites proved to be far more potent than the compounds in isolation. The combination of quercetin, quercetin-3-glucuronide or 4'O-methyl-quercetin with tamsulosin decreased the phenylephrine sensitivity approximately 200-, 35- and 150-fold (n=6). The strong pharmacodynamic interaction between the food supplement quercetin and tamsulosin underlines the potential of the impact of supplement-drug interactions that warrant more research.

Anticholinergic Accumulation: A Slumbering Interaction between Drugs and Food Supplements.

Many compounds display anticholinergic effects which might give rise to cognitive impairment and even delirium. These side effects are caused by their ability to bind to muscarinic receptors in our brain. Especially with combination of compounds, these serious effects are seen. This phenomenon, known as anticholinergic accumulation, is especially seen in the elderly. A classification of drugs for anticholinergic side effects has been made based on clinical observations, the ACB score. Here, we aimed to substantiate this classification by comparing the affinity of numerous drugs for the muscarinic receptors to the ACB score. Additionally, a number of supplements were screened. The affinity of the compounds was determined by their ability to displace the radioligand [(3)H]pirenzepine of the muscarinic receptor induced by these compounds. Our results show that the affinity of a compound for the muscarinic receptors correlated with its ACB score. Also food supplements appeared to bind to these muscarinic receptors. Moreover, several drug-drug, supplement-supplement and supplement-drug combinations had an affinity that is higher than the affinity of single compounds. This explains the phenomenon of anticholinergic accumulation. In conclusion, care should be taken to drug-drug and supplement-drug combinations with respect to anticholinergic accumulation.

The shifting perception on antioxidants: the case of vitamin E and ß-carotene.

Antioxidants are vital for aerobic life, and for decades the expectations of antioxidants as health promoting agents were very high. However, relatively recent meta-analyses of clinical studies show that supplementation of antioxidants does not result in the presumed health benefit, but is associated with increased mortality. The dilemma that still needs to be solved is: what are antioxidants in the end, healthy or toxic? We have evaluated this dilemma by examining the presumed health effects of two individual antioxidants with opposite images i.e. the "poisonous" ß-carotene and the "wholesome" vitamin E and focused on one aspect, namely their role in inducing BPDE-DNA adducts. It appears that both antioxidants promote DNA adduct formation indirectly by inhibition of the protective enzyme glutathione-S-transferase π (GST π). Despite their opposite image, both antioxidants display a similar type of toxicity. It is concluded that, in the appreciation of antioxidants, first their benefits should be identified and substantiated by elucidating their molecular mechanism. Subsequently, the risks should be identified including the molecular mechanism. The optimal benefit-risk ratio has to be determined for each antioxidant and each individual separately, also considering the dose.