A 90-day toxicity study of tripeptide, arginine-alanine-lysine.

There is a growing global interest in using peptides in the health industry for pharmaceuticals, cosmetics, and natural food products. Peptides contain two or more linked amino acids, whereas more than 50 amino acids are classified as polypeptides. Although there is a growing level of interest in the use of peptides in the health and wellness industry, there is a lack of literature pertaining to a specific tripeptide derived from arginine, alanine, and lysine (RAK) that is of interest for human dietary use. Therefore, a 90-day repeated-dose toxicity study was performed in rats to evaluate the subchronic oral toxicity of RAK. Eighty Han:WIST rats were administered RAK by gavage at doses of 0, 250, 500, or 1000 mg/kg bw/day. There were no mortalities or other treatment related effects, and no target organs were identified. A no-observed-adverse-effect-level (NOAEL) of 1000 mg/kg bw/day, the highest dose tested, was determined. This study will contribute to the body of research in regard to the safety of the use of RAK.

Metabolomic Analysis of Microcystis aeruginosa After Exposure to the Algicide L-Lysine.

The widespread occurrence of cyanobacteria blooms damages the water ecosystem and threatens the safety of potable water and human health. Exogenous L-lysine significantly inhibits the growth of a dominant cyanobacteria Microcystis aeruginosa in freshwater. However, the molecular mechanism of how lysine inhibits the growth of M. aeruginosa is unclear. In this study, both non-target and target metabolomic analysis were performed to investigate the effects of algicide L-lysine. The results showed that 8 mg L- 1 lysine most likely disrupts the metabolism of amino acids, especially the arginine and proline metabolism. According to targeted amino acid metabolomics analysis, only 3 amino acids (L-arginine, ornithine, and citrulline), which belong to the ornithine-ammonia cycle (OAC) in arginine metabolic pathway, showed elevated levels. The intracellular concentrations of ornithine, citrulline, and arginine increased by 115%, 124%, and 19.4%, respectively. These results indicate that L-lysine may affect arginine metabolism and OAC to inhibit the growth of M. aeruginosa.

Diet-Derived Advanced Glycation End Products (dAGEs) Induce Proinflammatory Cytokine Expression in Cardiac and Renal Tissues of Experimental Mice: Protective Effect of Curcumin.

The beneficial effect of curcumin (CU) on dietary AGEs (dAGEs) involves blocking the overexpression of proinflammatory cytokine genes in the heart and kidney tissues of experimental mice. The animals were divided into six groups (n = 6/group) and were fed a heat-exposed diet (dAGEs) with or without CU for 6 months. Their blood pressure (BP) was monitored by a computerized tail-cuff BP-monitoring system. The mRNA and protein expression levels of proinflammatory genes were analyzed by RT-PCR and western blot, respectively. A marked increase in BP (108 ± 12 mmHg vs 149 ± 15 mmHg) accompanied by a marked increase in the heart and kidney weight ratio was noted in the dAGE-fed mice. Furthermore, the plasma levels of proinflammatory molecules (C5a, ICAM-1, IL-6, MCP-1, IL-1ß and TNF-α) were found to be elevated (3-fold) in dAGE-fed mice. mRNA expression analysis revealed a significant increase in the expression levels of inflammatory markers (Cox-2, iNOS, and NF-κB) (3-fold) in cardiac and renal tissues of dAGE-fed mice. Moreover, increased expression of RAGE and downregulation of AGER-1 (p < 0.001) were noticed in the heart and kidney tissues of dAGE-fed mice. Interestingly, the dAGE-induced proinflammatory genes and inflammatory responses were neutralized upon cotreatment with CU. The present study demonstrates that dietary supplementation with CU has the ability to neutralize dAGE-induced adverse effects and alleviate proinflammatory gene expression in the heart and kidney tissues of experimental mice.

The effect of ketoprofen lysine salt on mucosa of rat stomach after ethyl alcohol intoxication.

INTRODUCTION: Ketoprofen is a commonly used nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties. Side effects of ketoprofen occur mainly from the gastrointestinal tract due to the inhibition of cyclooxygenaze-1. Binge drinking at least once a week is reported by 80 million Europeans. On the day after many of them use NSAIDs. This increases the risk for damage of gastric mucosa. AIM: The aim of the study was to check if use of ketoprofen lysine salt (KLS) has any gastroprotective effect on mucosa of rat stomach after ethyl alcohol intoxication. MATERIALS AND METHODS: There were 6 groups of 6 male rats which received: RESULTS: In groups 1, 2 and 3 the histopathologic examination of the stomachs revealed normal picture, without signs of inflammation. In the group 4, 5 and 6 within the mucosa and submucosa there were visible numerous infiltrates of inflammatory cells, consisting mainly of lymphocytes, plasmocytes and eosinophilia. Total leukocyte count was elevated in group 3, 4, 6. There was a significant decrease of blood urea concentration in group 6 vs 2 and significant decrease of serum albumin in group 6 vs 1 and 2, and total protein vs group 1. CONCLUSION: Side effects of ketoprofen occur mainly from the gastrointestinal tract. KLS has no gastroprotective effect after ethanol-gastric injury and does not protect gastric mucosa from damage produced by binge drinking. Therefore it should not be used after drinking distilled spirits.

Determination of serum aflatoxin B1-lysine and biochemical parameters in broiler chicks fed an aflatoxin B1-contaminated diet.

In the present trial, the levels of serum aflatoxin B1 (AFB1)-lysine and their relationship with biochemical parameters in broiler chicks fed an AFB1-contaminated diet were determined. The experimental design was completely randomized with two treatments (control and 222.17 µg/kg AFB1) and 20 bird per treatment. Feeds were offered to broiler chicks for 14 days, from 28 to 42 days of age. Animals were vaccinated against Newcastle's and Marek's diseases on the 14th day of life, and were killed at 42 days of age. Broilers receiving AFB1 did not demonstrate any sign of toxicity. Compared with controls, aspartate aminotransferase and globulin levels were not affected in the AFB1-treated group. However, higher levels of gamma-glutamyl transferase and lower concentrations of total protein and albumin were observed in the group receiving AFB1 on days 35 and 42. AFB1-lysine were detected in the serum of all broilers fed the AFB1-contaminated diet, at mean levels of 56.52-77.83 ng/mg albumin on days 35 and 42 of age, respectively. These values indicated the internal dose of AFB1 in birds, which negatively correlated with total protein, albumin, and globulin levels. Data indicated that AFB1-lysine shows the potential to be a sensitive and specific biomarker for the evaluation of broiler exposure to dietary aflatoxin, as well as for diagnostic purposes. Further studies are necessary to determine physiologically-based toxicokinetics of serum AFB1-lysine in broilers.

The neurotoxicity of Nε-(carboxymethyl)lysine in food processing by a study based on animal and organotypic cell culture.

Nε-(carboxymethyl)lysine (CML) is a potentially noxious compound that is causing widespread concern due to its use in various food products. In this study, we investigated CML neurotoxicity via an in vivo experiment with mice, and an in vitro experiment using a 3D microvascular network model (with human brain vascular endothelial cell and human astrocyte) that simulated the blood-brain barrier. We found that CML could induce cell survival status variations, and histopathological changes to the brain. In addition, CML increased levels of oxidative stress, prompted the protein expression of the receptor for advanced glycation end-products (RAGE). CML up-regulated both the gene expression of RAGE, the activating protein-1 (AP-1), the inflammatory cytokines Interleukin-6 (IL-6), vascular cell adhesion molecule1 (VCAM-1), monocyte chemotactic protein1 (MCP-1). We, therefore, postulated that CML has the potential to deleteriously affect the nervous system through oxidative stress and that activation of the p38 MAPK-AP-1 signaling pathway might be implicated in this pathological process.

Identification of a 5-Hydroxymethylfurfural-Lysine Schiff Base and Its Cytotoxicity in Three Cell Lines.

5-Hydroxymethylfurfural (HMF) can undergo the Maillard reaction with amino acids. However, the safety of the products remains unknown. In this study, a HMF-lysine Schiff base named (E)-N6-((5'-(hydroxymethyl)furan-2'-yl)methylene)lysine (HML) was identified and detected for the first time in baked foods. HML formation significantly decreased the cytotoxicity (IC50) of HMF against GES-1 cells (81.81 versus 5.02 mM and 73.76 versus 2.94 mM for HML versus HMF at 24 and 48 h, respectively), EA.hy926 cells (86.05 versus 4.85 mM and 77.22 versus 0.71 mM, respectively), and Caco-2 cells (155.77 versus 36.84 mM and 112.70 versus 18.51 mM, respectively). Exposure of Caco-2 cells to HMF at 10.0 mM triggered cell apoptosis of 14.02% (versus 8.54% in the control), whereas exposure to HML at 10-15 mM hardly increased cell apoptosis. Moreover, the absorption capacities of HMF and HML by Caco-2 cells were equivalent (p > 0.05) at 7.23-12.57% after incubation at 2 mM for 30-150 min.

Furosine Posed Toxic Effects on Primary Sertoli Cells through Regulating Cep55/NF-κB/PI3K/Akt/FOX01/TNF-α Pathway.

As one of the Maillard reaction products, furosine has been widely reported in a variety of heat-processed foods, while the toxicity of furosine on the reproductive system and related mechanisms are unclear. Here, we constructed an intragastric gavage male mice model (42-day administration, 0.1/0.25/0.5 g furosine/Kg body weight per day) to investigate its effects on mice testicle index, hormones in serum, and mice sperm quality. Besides, the lipid metabonomics analysis was performed to screen out the special metabolites and relatively altered pathways in mice testicle tissue. Mice primary sertoli cells were separated from male mice testicle to validate the role of special metabolites in regulating pathways. We found that furosine affected testicle index, hormones expression level and sperm quality, as well as caused pathological damages in testicle tissue. Phosphatidylethanolamine (PE) (18:0/16:1) was upregulated by furosine both in mice testicle tissue and in primary sertoli cells, meanwhile, PE(18:0/16:1) was proved to activate Cep55/NF-κB/PI3K/Akt/FOX01/TNF-α pathway, and as a functional protein in dairy products, lactoferrin could inhibit expression of this pathway when combined with furosine. In conclusion, for the first time we validated that furosine posed toxic effects on mice sperms and testicle tissue through upregulating PE(18:0/16:1) and activating Cep55/NF-κB/PI3K/Akt/FOX01/TNF-α pathway.

Furosine, a Maillard Reaction Product, Triggers Necroptosis in Hepatocytes by Regulating the RIPK1/RIPK3/MLKL Pathway.

As one of the typical Maillard reaction products, furosine has been widely reported in a variety of heat-processed food. Though furosine was shown to be toxic on organs, its toxicity mechanism is still unclear. The present study aimed to investigate the toxicity mechanism of furosine in liver tissue. An intragastric gavage mice model (42-day administration, 0.1/0.25/0.5 g/kg of furosine per day) and a mice primary hepatocyte model were employed to investigate the toxicity mechanism of furosine on mice liver tissue. A metabonomics analysis of mice liver, serum, and red blood cells (RBC) was performed. The special metabolic mediator of furosine, lysophosphatidylcholine 18:0 (LPC (18:0)) was identified. Then, the effect of the upstream gene phospholipase A2 gamma (PLA2-3) on LPC (18:0), as well as the effect of furosine (100 mg/L) on the receptor-interacting serine/threonine-protein kinase (RIPK)1/RIPK3/mixed lineage kinase domain-like protein (MLKL) pathway and inflammatory factors, was determined in liver tissue and primary hepatocytes. PLA2-3 was found to regulate the level of LPC (18:0) and activate the expression of RIPK1, RIPK3, P-MLKL, and of the inflammatory factors including tumor necrosis factor α (TNF-α) and interleukin (IL-1ß), both in liver tissue and in primary hepatocytes. Upon treatment with furosine, the upstream sensor PLA2-3 activated the RIPK1/RIPK3/MLKL necroptosis pathway and caused inflammation by regulating the expression of LPC (18:0), which further caused liver damage.

Studies on mechanism of free Nε-(carboxymethyl)lysine-induced toxic injury in mice.

Nε-(carboxymethyl)lysine (CML), which is a compound produced when food is processed, has aroused concern in recent years because of its potentially dangerous effects. This study aimed to investigate the mechanism of free CML-induced toxic injury in mice. The inflammatory cytokine tumor necrosis factor-α, transforming growth factor-ß, vascular cell adhesion molecule-1 mRNA expression levels of CML-infected mice liver and kidney tissues significantly increased. While CML receptor-receptor for advanced glycation end products (RAGE) protein expression in male mice liver tissue had a more significant change than the control group, there was no significant difference in other dose groups compared with the control group. In conclusion, the foodborne free CML can be induced by oxidative stress and immune response to liver and kidney tissue injury in mice. Additionally, the free CML may also bind to RAGE, which activates the downstream inflammatory pathway.

Study of aflatoxicosis reduction: effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 exposed rats.

The toxicity of aflatoxins results in cancer and liver disease. Several natural substances such as plants exhibited their ability to inhibit the initiation of aflatoxin carcinogenesis. The aim of this study was to evaluate the effect of Alchornea cordifolia on biomarkers in an aflatoxin B1 (AFB1) exposed rats. The contents of polyphenols, flavonoids and the antioxidant activity of A. cordifolia ethanolic leaf extract (EELac) were assessed. Groups of rats were treated orally with a daily dose of a mixture of AFB1 at a dose of 150 µg/kg body weight and EELac (50, 100 and 300 mg/kg body weight) for 21 days. Biomarkers of AFB1, such as the AFB1-lysine adduct and aflatoxin M1 were assayed in blood and urine, respectively, using an HPLC system with a fluorescence detector. The contents of polyphenols and flavonoids were 6783.23 ± 272.76 µg EAG/g and 10.54 ± 3.15% of dry matter, respectively. EELac showed a good antioxidant activity (IC50 = 12.65 ± 0.13 µg/mL). The administration of the mixture (AFB1 + EELac) at different doses significantly reduced the level of AFB1-lysine adduct from 14.04 ± 2.1 to 4.13 ± 0.9 ng/mg albumin and that of Aflatoxin M1 (AFM1) from 456 ± 16 to 220 ± 24 ng/mL (p <0.05). The rate of reduction was 70.58% for AFB1-lysine adduct and 51.75% for AFM1. A. cordifolia could be used in the prevention of toxicity induced by AFB1 on account of its high content in phenolic compounds.

Effects of flavor enhancers on the survival and behavior of the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

To understand whether flavor enhancers pose potential risks to the environment, it is important to assess its effects on insects. Therefore, the objective of this study was to evaluate the toxicity of flavor enhancers on the survival and behaviors of the red imported fire ant, Solenopsis invicta. In this study, we found that the mortality of S. invicta workers that were fed glutamic acid monosodium salt hydrate, glycine, L-alanine, succinic acid, succinic acid disodium, inosinate 5'-monophosphate disodium salt hydrate, and GMP were significantly higher than the mortality rates of workers fed sucrose. Moreover, glycine and GMP exhibited the strongest toxicities and caused 100% mortality in workers after 84 h. LC50 values were 0.004 g/ml and 0.02 g/ml for GMP and glycine, respectively. Additionally, at sublethal doses, both GMP and glycine solutions decreased foraging and digging behaviors. Our results suggest that flavor enhancers are toxic to insects and also likely to have a negative impact at sublethal concentrations.

Genetic encoding of 2-aryl-5-carboxytetrazole-based protein photo-cross-linkers.

Three γ-heteroatom-substituted N-methylpyrroletetrazole-lysines (mPyTXKs) were synthesized and subsequently incorporated into proteins site-specifically via genetic code expansion. The γ-seleno-substituted derivative, mPyTSeK, showed excellent incorporation efficiency in Escherichia coli and allowed site-selective photo-cross-linking of the GST dimer. Furthermore, the mPyTSeK-cross-linked GST dimer can be cleaved under mild oxidative conditions. The incorporation of mPyTXKs into proteins in mammalian cells was also demonstrated. Lastly, the recombinantly expressed mPyTSeK-encoded Grb2 was shown to covalently capture its interaction partner, EGFR, in mammalian cell lysate, which was subsequently released after treatment with H2O2.

Novel α-Oxoamide Advanced-Glycation Endproducts within the N6-Carboxymethyl Lysine and N6-Carboxyethyl Lysine Reaction Cascades.

The highly reactive α-dicarbonyl compounds glyoxal and methylglyoxal are major precursors of posttranslational protein modifications in vivo. Model incubations of N2-t-Boc-lysine and either glyoxal or methylglyoxal were used to further elucidate the underlying mechanisms of the N6-carboxymethyl lysine and N6-carboxyethyl lysine reaction cascades. After independent synthesis of the authentic reference standards, we were able to detect N6-glyoxylyl lysine and N6-pyruvoyl lysine for the first time by HPLC-MS2 analyses. These two novel amide advanced-glycation endproducts were exclusively formed under aerated conditions, suggesting that they were potent markers for oxidative stress. Analogous to the well-known Strecker degradation pathway, leading from amino acids to Strecker acids, the oxidation of an enaminol intermediate is suggested to be the key mechanistic step. A highly sensitive workup for the determination of AGEs in tissues was developed. In support of our hypothesis, the levels of N6-glyoxylyl lysine and N6-pyruvoyl lysine in rat livers indeed correlated with liver cirrhosis and aging.

Toxicology studies of furosine in vitro/in vivo and exploration of the related mechanism.

AIM: Furosine is one of the Maillard reaction products (MRPs) and is found in a variety of heat-processed food. Yet its toxicity is still unclear. The present study was designed to assess furosine toxicity in cell models and in CD-1 mice, respectively. METHODS: In vitro, the effects of furosine on the cell viability, cell cycle and apoptosis (Hek293, HepG2, SK-N-SH and Caco2) were detected and evaluated, sensitive cell lines and proper dosage of furosine for further animal experiment were determined, and the mechanisms of toxicity were explored. In vivo, the acute toxicity studieswere performed, organ index, hematology parameters, functions of liver/kidney and pathological changes were detected and the target organs were uncovered. RESULTS: Hek293 cells and HepG2 cells were themost sensitive to furosine with respect to cytotoxicity and apoptosis. Furosine inhibited mice weight gain, and affected the functions of liver and kidney. CONCLUSIONS: Furosine posed toxic effects on mice liver and kidney, suggested thatthey were the target organs for furosine toxicity. This study for the first time provides evidence that high dosages of furosine pose adverse biological effects on the health of animals through induction of cell apoptosis and activation of inflammatory necrosis response.

Protein-Mediated Shape Control of Silver Nanoparticles.

Silver nanoparticles were grown in aqueous solution, without the presence of typical surfactant molecules, but under the presence of different proteins. The shape of the resulting silver nanoparticles could be tuned by the selection of the types of proteins. The amount of accessible lysine groups was found to be mainly responsible for the anisotropy in nanoparticle formation. Viability measurements of cells exposed to protein capped spherical or prism-shaped NPs did not reveal differences between both geometries. Thus, in the case of protein-only coated Ag NPs, no shape-induced toxicity was found under the investigated exposure conditions.

Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity.

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

Differential protein modulation by ketoprofen and ibuprofen underlines different cellular response by gastric epithelium.

Ketoprofen L-lysine salt (KLS), is widely used due to its analgesic efficacy and tolerability, and L-lysine was reported to increase the solubility and the gastric tolerance of ketoprofen. In a recent report, L-lysine salification has been shown to exert a gastroprotective effect due to its specific ability to counteract the NSAIDs-induced oxidative stress and up-regulate gastroprotective proteins. In order to derive further insights into the safety and efficacy profile of KLS, in this study we additionally compared the effect of lysine and arginine, another amino acid counterion commonly used for NSAIDs salification, in control and in ethanol challenged human gastric mucosa model. KLS is widely used for the control of post-surgical pain and for the management of pain and fever in inflammatory conditions in children and adults. It is generally well tolerated in pediatric patients, and data from three studies in >900 children indicate that oral administration is well tolerated when administered for up to 3 weeks after surgery. Since only few studies have so far investigated the effect of ketoprofen on gastric mucosa maintenance and adaptive mechanisms, in the second part of the study we applied the cMap approach to compare ketoprofen-induced and ibuprofen-induced gene expression profiles in order to explore compound-specific targeted biological pathways. Among the several genes exclusively modulated by ketoprofen, our attention was particularly focused on genes involved in the maintenance of gastric mucosa barrier integrity (cell junctions, morphology, and viability). The hypothesis was further validated by Real-time PCR.

In Vitro Evaluation of Cytotoxicity and Permeation Study on Lysine- and Arginine-Based Lipopeptides with Proven Antimicrobial Activity.

Owing to their excellent antimicrobial activities with a relatively low cost of production, lipopeptides are being intensively investigated as potential alternatives to popular antimicrobials. However, a critical obstacle for their application is a relatively high toxicity, hence a lot of attention has been paid to designing new molecules with optimal properties. In this study we synthesized the following lipopeptides: C16-KK-NH2, C16-KεK-NH2, C16-KKK-NH2, C16-KRK-NH2, C16-RR-NH2, C16-RRR-NH2, (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2. Their antimicrobial activity against representative strains of Gram-positive bacteria, Gram-negative bacteria and fungi has been confirmed. The compounds have been evaluated with regard to the safety of their application in dermatology. The cytotoxicity was determined in HaCaT keratinocytes using MTT assay, whereas Strat M membranes placed in Franz diffusion cells were used to assess their ability to skin permeation. The compounds containing one hexadecanoic acid chain turned out to be very toxic towards human keratinocytes, while lipopeptides containing two fatty acid chains (decanoic and dodecanoic) demonstrated much lower cytotoxicity. For the most promising lipopeptide, (C10)2-KKKK-NH2, the measured IC50 on HaCaT keratinocytes was few times higher as compared to MICs obtained for the tested bacteria. Both groups of lipopeptides did not permeate the model membranes and therefore lack of permeation through human skin could be expected. The results of this work encourage further research on the potential application of lipopeptides with two fatty acids as novel antimicrobials.

Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.