Article series: Safety of esterified propoxylated glycerol (EPG), a nonabsorbable fat replacer

This article introduces a series of articles addressing the safety of esterified propoxylated glycerols (EPGs), a family of fat- and oil-like substances that resemble triglycerides in structure and appearance, but have been modified to prevent or limit their digestion when consumed in food. A general summary of the history, composition, metabolism, and safety of EPGs is provided.     

90-Day dietary toxicity study with esterified propoxylated glycerol (EPG) in Micropigs

The subchronic (90-day) toxicity of esterified propoxylated glycerol (EPG) was assessed in micropigs. Animals (5/sex/group) received feed containing 5%, 10%, and 17% EPG, mixed accordingly throughout the study to deliver 1.5, 3, and 5 g/kg bw/day of EPG, respectively. Corn oil served as the vehicle control (0 g/kg bw/day). Subsets of animals were evaluated at Week 6; the remainder between Weeks 12 and 14. With the exception of liver and serum vitamin levels, statistically significant difference between control and EPG groups were seen sporadically, and with no apparent connection to treatment and/or no consistency across time intervals. EPG intakes of 3 and 5 g/kg bw/day, but not at 1.5 g/kg bw/day were associated with significantly lower serum 25-OH vitamin D levels. Serum total vitamin D levels were significantly lower across all EPG groups. There were also trends toward lower levels of liver vitamins A and E among EPG-treated animals, but the effects were less consistent. The effects on vitamin levels observed in EPG-treated animals were not accompanied by any signs of vitamin deficiency (e.g., effects on growth, clinical signs, or clinical pathology), and might have been related to the larger mass of EPG acting as a lipid “sink” during transit in the gastrointestinal tract.     

90-Day dietary toxicity study with esterified propoxylated glycerol (EPG) in rats

The subchronic (90-day) toxicity of a “core” version of EPG was assessed in rats. Crl:CD-1®(ICR)BR rats (70/sex) received diets containing a constant level of 5% EPG (w/w) or adjusted to deliver 0 (control), 0.5, 1, or 2 g/kg of body weight/day (g/kg bw/day). Subsets of animals from each group (20/sex) were evaluated after 30 days (interim sacrifice); the remainder after 90 days. EPG intake at all dose levels was associated with lower mean liver vitamin E levels; liver vitamin A and serum vitamin D were also lower, but less consistently. Animals given 5% EPG had higher fecal output (males) and cholesterol (males and females) without corresponding changes in serum cholesterol. Urinary pH was also mildly lower in males given 5% EPG. However, detailed evaluation of general health and assessment of blood, organs and tissues showed no evidence that EPG administration compromised the nutritional requirements of the animals, caused a state of fat-soluble vitamin deficiency, or caused‘ toxicity to any organ system. Based on the results of this study, it was not possible to establish a no-observable-effect level (NOEL). The possible effect of EPG on vitamin levels in the absence of any clinical signs of deficiency was not considered “adverse” per se. As such, the 2 g/kg and 5% EPG level were considered to represent a no-observable-adverse-effect levels (NOAELs).     

Developmental toxicity evaluation of esterified propoxylated glycerol (EPG) administered in the diet to New Zealand white rabbits

The safety of a “core” version of esterified propoxylated glycerols (EPGs) was assessed in a developmental toxicity study in New Zealand white rabbits, Hra:(NZW)SPF. Four groups each of 18 inseminated female rabbits received diets ad libitum containing concentrations of 0%, 2.5%, 5%, and 10% EPG (w/w) with 6% corn oil (w/w). No treatment-related effects were observed in any maternal toxicity parameter, including maternal body weight and weight gain, feed consumption, or clinical signs of toxicity. There were no statistically significant treatment-related effects in gestational parameters, including pre- and post-implantation loss, litter size, sex ratio, fetal body weight, and crown–rump length. The incidences of fetal external, visceral, and skeletal malformations or variations were also comparable across groups. A no-observable-adverse-effect level (NOAEL) of 10% EPG (approximately 4.76 g/kg bw/day) for both maternal and developmental toxicity is proposed based on the results of this study.     

Assessment of the effect of esterified propoxylated glycerol (EPG) on the status of fat-soluble vitamins and select water-soluble nutrients following dietary administration to humans for 8 weeks

This double-blind, randomized, controlled study assessed the effect of esterified propoxylated glycerol (EPG) on fat-soluble vitamins and select nutrients in human subjects. For 8 weeks, 139 healthy volunteers consumed a core diet providing adequate caloric and nutrient intakes. The diet included items (spread, muffins, cookies, and biscuits) providing EPG (10, 25, and 40 g/day) vs. margarine alone (control). EPG did not significantly affect circulating retinol, α-tocopherol, or 25-OH D₂, but circulating β-carotene and phylloquinone were lower in the EPG groups, and PIVKA-II levels were higher; 25-OH D₃ increased but to a lesser extent than the control. The effect might be related to EPG acting as a lipid “sink” during gastrointestinal transit. No effects were seen in secondary endpoint measures (physical exam, clinical pathology, serum folate, RBC folate, vitamin B₁₂, zinc, iron, calcium, phosphorus, osteocalcin, RBP, intact PTH, PT, PTT, cholesterol, HDL-C, LDL-C, triglycerides). Gastrointestinal adverse events (gas with discharge; diarrhea; oily spotting; oily evacuation; oily stool; liquid stool; soft stool) were reported more frequently by subjects receiving 25 or 40 g/day of EPG. In general, the incidence and duration of these symptoms correlated directly with EPG dietary concentration. The results suggest 10 g/day of EPG was reasonably well tolerated.     

One-generation reproduction study of esterified propoxylated glycerol (EPG) administered in the feed to CD® (Sprague-Dawley) rats

This one-generation study assessed the potential of esterified propoxylated glycerol (EPG) to affect reproduction and offspring development in rats. Male and female Crl:CD(SD)BR rats (30/sex/group) were exposed to EPG at 0, 0.5, 1, and 2 g/kg bw/day or at 5% (w/w) in the diet prior to (13 weeks), during, and after two consecutive matings. For dams, exposure continued through gestation and lactation; F_1a and F_1b pups were weaned to the respective diet (for up to 91 days). No consistent treatment-related effects were observed in: body weights/gains; feed consumption; clinical observations; mating indices; survival, growth and development of litters, litter sizes, body weights, sex ratios (lower % males/litter at 1 and 2 g/kg bw/day), acquisition of developmental landmarks, behavioral indices, or histology of selected organs. Lower serum vitamin D, liver vitamin A, and liver vitamin E levels were seen in some EPG-treated groups. None of the reductions were judged to be biologically significant. A/G ratio was greater among males receiving 2 g/kg bw/day and 5%. In the absence of any other related effects, the biological significance of this finding is doubtful.     

Genotoxicity studies on licorice flavonoid oil (LFO)

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.     

A Bacterial DNA Repair Test Evaluating the Genotoxicity of Light Sources

A DNA repair test was used in order to assess its applicability for detecting the genotoxicity of sunlight and of the light emitted by halogen lamps and fluorescent lamps. This experimental system compares the lethality of test agents in the Escherichia coli wild-type WP2 and its isogenic counterparts lacking, either individually or in combination, various DNA repair mechanisms. DNA repair-deficient strains included WP2uvrA (uvrA^-), WP67 (uvrA^- polA^-), CM561 (lexA^-), CM571 (recA^-), WP100 (uvrA^- recA^-), and CM871 (uvrA^- recA^- lexA^-). All light sources produced a substantial killing of repair-deficient strains, with a maximum activity in the triple mutant CM871, at doses that did not affect survival of the wild type. The genotoxicity of uncovered quartz halogen bulbs was particularly potent, compared to fluorescent lamps and sunlight. Moreover, the mechanisms involved in repairing the DNA damage induced by halogen lamps were similar to those of a 254 nm UV source. The spectrum of genetic damage produced by sunlight and fluorescent lamps was conversely more comparable to that of a 365 nm UV source. These data demonstrated a harmful emission of appreciable amounts of genotoxic far-UV wavelengths by halogen lamps, thereby confirming our previous results in the his^-Salmonella typhimurium mutagenicity test. Genotoxicity of halogen lamps could be easily prevented in both experimental systems by suitable glass or plastic covers. Compared to the mutagenicity end point, the differential lethality end point provided even more clear-cut results in detecting the DNA-damaging ability of all light sources. Moreover, parallel assays provided evidence that the bacterial DNA repair test was far more sensitive than the mutagenicity test in evaluating the genotoxicity of the light produced by halogen lamps. On the whole, the DNA repair test in E. coli is even simpler and faster (24 vs. 48 h) than the Salmonella mutagenicity test, and compares favorably in terms of sensitivity to genotoxic light sources.     

Use of the excluded protecting group (EPG) method for peptide synthesis

Abstract: The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin‐1, [Ala¹⁹, Asn²²]magainin‐1. In the approach reported, the C‐terminal amino acid is esterified to the 2‐position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl‐steroid as the Fmoc‐pentafluorophenyl‐ester. The Fmoc group is removed with Et₂NH/DMF (～15% v/v) and, after evaporation to ～10 mL, the solution chromatographed on Sephadex LH‐20 in DMF. The dipeptidyl‐steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc‐amino acid‐pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn°/AcOH to yield the peptide with intact α‐amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C‐terminal amino acids in other EPG syntheses.     

Genotoxicity assessment of chromium(III) propionate complex in the rat model using the comet assay

The aim of the study was to assess genotoxicity of a chromium(III) propionate complex in rat’s peripheral blood lymphocytes by the comet assay. The study was carried out on 18 12-weeks old female Wistar rats that were divided into three equal groups (six animals each): control (0), control-Cr(VI) and Cr(III)-tested rat fed ad libitum a basal diet and the diet supplemented either with 10 mg Cr(VI)/kg diet (given as K₂Cr₂O₇, equivalent of 1 mg/kg body mass/day) or 1000 mg Cr(III)/kg diet (given as [Cr₃O(O₂CCH₂CH₃)6(H₂O)₃]NO₃), equivalent of 100 mg Cr/kg body mass/day) for 4 weeks. High doses of supplementary Cr(III) were found to not affect body mass gain, feeding efficiency ratio and internal organ masses. Treatment of rats with the Cr(III) propionate complex, in contrast to Cr(VI), did not affect significantly the comet assay results in lymphocytes, which suggests that the compound does not exert genotoxic effects in rats.     

Immunohistochemical study of proliferating cell nuclear antigen (PCNA) in duckling liver fed with aflatoxin B1 and esterified glucomannan

The effect of esterified glucomannan on aflatoxin B₁ toxicity in ducklings was studied by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in hepatic cells on formalin-fixed paraffin-embedded liver samples. Cherry Valley ducklings were divided into five groups, 20 birds in each. One of the groups was fed with conventional feed, and the other groups were fed with diet containing 100 ppb aflatoxin B₁, that containing 0.05% esterified glucomannan, or that containing 100 ppb aflatoxin B₁ supplemented with 0.05 or 0.1% esterified glucomannan, from five days of age for one month, and subsequently all the groups were fed with conventional feed for 20 days. Four birds of each group were sacrificed on the 30th, 35th, 40th, 45th and 50th day of feeding, and PCNA on the liver tissue sections was quantitatively analyzed by immunohistochemical staining. The percentage of PCNA-positive hepatocytes was significantly higher in the group given diet containing aflatoxin B₁ than in the other groups, which were not significantly different from each other. The results demonstrate that supplementation of feed with esterified glucomannan is effective in reduction of aflatoxin B₁-induced hepatic injury in ducklings.     

Polyelectrolyte complex nanoparticles of amino poly(glycerol methacrylate)s and insulin

Amino poly(glycerol methacrylate)s (PGOHMAs) were synthesized from linear or star-shaped poly(glycidyl methacrylate)s (PGMA)s via ring opening reactions with 1,2-ethanediamine, 1,4-butanediamine and diethylenetriamine, respectively. The resulting cationic polymers were employed to form polyelectrolyte complexes (PECs) with insulin. Parameters influencing complex formation were investigated by dynamic light scattering (DLS). PECs in the size range of 100-200 nm were obtained under optimal conditions, i.e., the pH value of PECs was 5.58-6.27, the concentration of NaCl was 0.02 mol/L, and insulin-polymer weight ratio was 0.8. The insulin association efficiency (AE) of current system increased with zeta potentials of PECs. Circular dichroism (CD) analysis corroborated that the structure of insulin in the PEC nanoparticles was preserved after lyophilization. Fourier transform infrared (FT-IR) and X-ray diffraction (XRD) experiments demonstrated that weak physical interactions between insulin and amino PGOHMAs play an important role in the formation of PECs. The release of insulin depends on both structure and architecture of amino PGOHMAs. These PECs would be potentially useful for mucosal administration.     

The Assesment of Genotoxicity of Carbamazepine Using Cytokinesis-Block (CB) Micronucleus Assay in Cultured Human Blood Lymphocytes

The genotoxic effect of CBZ has been investigated in few studies. There is little evidence linking carbamazepine (CBZ) with any genotoxic effects, particularly in vitro micronucleus test using cytogenesis-block technique. In this study, the genotoxicity of the antiepileptic drug, carbamazepine, was tested using cytokinesis-block (CB) micronucleus assay. In vitro analysis was performed in human blood lymphocytes from four healthy persons at five different concentrations of carbamazepine (6, 8, 10, 12, 14 μg/mL). Genotoxic potential and cytotoxic effects of carbamazepine were evaluated by using micronucleus assay and cytokinesis-block proliferation index (CBPI), called the parameter of cytotoxicity in human peripheral blood lymphocyte cultures, respectively. The results of this study indicate that CBZ caused the genotoxic effect under in vitro conditions, except at the dose of 6 μg/mL, and cytotoxic effects of carbamazepine were revealed by a decrease in the cytokinesis-block proliferation index at all the concentrations.     

Poly(glycerol methacrylate)-based degradable nanoparticles for delivery of small interfering RNA

Abstract

Nucleic acids therapeutic efficiency is generally limited by their low stability and intracellular bioavailability, and by the toxicity of the carriers used to deliver them to the target sites. Aminated poly(glycerol methacrylate) polymers are biodegradable and pH-sensitive polymers that have been used previously to deliver antisense oligonucleotide and show high transfection efficiency. The purpose of this study is to compare the efficiency and toxicity of aminated linear poly(glycerol methacrylate) (ALT) biodegradable polymer to the most commonly used cationic degradable (i.e. chitosan) and non-degradable (i.e. polyethylenimine (PEI)) polymers for delivery of short interfering RNA (siRNA). ALT, PEI and chitosan polymers were able to form nanosized particles with siRNA. Size, size-distribution and zeta-potential were measured over a wide range of nitrogen-to-phosphate (N/P) ratios, and the stability of the formed nanoparticles in saline and upon freeze-drying was also assessed. No significant cytotoxicity at the range of the tested concentrations of ALT and chitosan nanoparticles was observed, whereas the non-degradable PEI showed significant toxicity in huh-7 hepatocyte-derived carcinoma cell line. The safety profiles of the degradable polymers (ALT and chitosan) over non-degradable PEI were demonstrated in vitro and in vivo. In addition, ALT nanoparticles were able to deliver siRNA in vivo with significantly higher efficiency than chitosan nanoparticles. The results in the present study give evidence of the great implications of ALT nanoparticles in biomedical applications due to their biocompatibility, low cytotoxicity, high stability and simple preparation method.     

Development of poly(glycerol adipate) nanoparticles loaded with non-steroidal anti-inflammatory drugs

The aim of this study was to assess acylated and non-acylated poly(glycerol adipate) polymers (PGA) as suitable nanoparticulate systems for encapsulation and release of ibuprofen, ibuprofen sodium salt (IBU-Na) and ketoprofen as model drugs. Drug encapsulated nanoparticles were prepared using the interfacial deposition method in the absence of surfactants. Physicochemical characterisation studies of the produced loaded nanoparticles showed that drug–polymer interactions depend on the characteristics of the actual active substance. IBU-Na showed strong interactions with the polymers and it was found to be molecularly dispersed within the polymer matrix while ibuprofen and ketoprofen retained their crystalline state. The drug release profiles showed stepwise patterns which involve an initial burst release effect, diffusion of the drug from the polymer matrix and eventually drug release possibly via a combined mechanism. PGA polymers can be effectively used as drug delivery carriers for various active substances.     

Genotoxicity and antigenotoxicity of cashew (Anacardium occidentale L.) in V79 cells

The use of plants for the treatment of diseases continues to rise although there are few studies providing proof of these effects. One of these plants is the Anacardium occidentale, popularly known as the cashew. The present study evaluated the possible genotoxic and protective activities of cashew stem bark methanolic extract, in vitro, using methyl methanesulfonate (MMS) as a positive control, to compare possible mechanisms of DNA damage induction in the Comet assay. The antigenotoxicity protocols used were pre, simultaneous and post-treatment in relation to MMS. In genotoxicity and antigenotoxicity assessments, besides MMS, PBS was used as the negative control and three concentrations of the A. occidentale extract (500 μg/mL, 1000 μg/mL and 2000 μg/mL) were used on Chinese hamster lung fibroblasts (V79 cells). The Comet assay revealed that the two lowest concentrations tested presented no genotoxic activity, whereas the highest presented genotoxicity. All of the concentrations showed protective activity in simultaneous and post-treatment in relation to MMS. Further studies are required to identify the substances that comprise the extract and more clearly comprehend the antigenotoxic mechanism detected in this study.     

Genotoxicity of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™)

The genotoxic potential of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) was evaluated in a battery of genotoxicity tests. The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in 2 separate in vitro chromosomal aberration test in Chinese hamster lung (CHL) fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity of PQQ was noted. In the in vivo micronucleus assay in mice, PQQ at doses up to 2000 mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. The weak responses in the in vitro test CHL cells were considered of little relevance under conditions of likely human exposure. PQQ disodium was concluded to have no genotoxic activity in vivo.     

P-postlabeling analysis of DNA adducts formed in vitro and in rat skin by methylenediphenyl-4,4'-diisocyanate (MDI)

The ³²P-postlabeling method was adapted for the detection of DNA adducts formed by methylenediphenyl-4,4'-diisocyanate (MDI). Incubation of the 3'-phosphates of the deoxyribosides of cytosine (C), adenine (A), guanine (G) and thymine (T) with MDI in Tris buffer resulted in the formation of 5, 7, 8, and 2 reaction products, respectively. Incubation of DNA with MDI resulted in detectable levels of 5, 2, and 1 adducts attributable to C, A, and G. Analysis of DNA isolated from the epidermis of rats treated dermally with 9 mg MDI showed an adduct pattern similar to the one seen in the in vitro DNA incubation. A total adduct level of 7 per 10⁸ nucleotides was measured, the limit of detection was 2 adducts per 10¹⁰ nucleotides. The data indicate that a minute fraction of MDI can reach DNA in vivo in a chemically reactive form. In comparison with the genotoxic skin carcinogen 7,12-dimethylbenz[a]anthracene on the other hand, the DNA-binding potency of MDI was more than 1000-fold lower.     

Antimutagenic and anticlastogenic effects of Turkish Black Tea on TA98 and TA100 strains of Salmonella typhimurium (in vitro) and mice (in vivo)

Context: Black tea has been reported to have significant antimutagenic and anticarcinogenic properties associated with its polyphenols theaflavins (TF) and thearubigins (TR). Similarly, Turkish black tea (TBT) also contains a considerable amount of TF and TR.

Objective: This study investigated the mutagenic, antimutagenic and anticlastogenic properties of TBT.

Materials and methods: The mutagenic and antimutagenic effects of TBT (10 to 40000?μg/plate) were investigated in vitro on Salmonella strains TA98 and TA100 with and without S9 fraction. Anticlastogenic effect was studied at concentrations of 300–1200?mg/kg TBT extract by chromosomal aberrations (CA) assay from bone marrow of mice.

Results: The results of this study did not reveal any mutagenic properties of TBT. On the contrary, TBT extract exhibited antimutagenic activity at >1000?μg/plate concentrations in TA98 strain with and without S9 activation (40% inhibition with S9 and 27% without S9). In TA100 strain, the antimutagenic activity was observed at?>20,000?μg/plate TBT extracts without S9 activation (28% inhibition) and at >1000?μg/plate with S9 activation (59% inhibition). A significant decrease in the percentage of aberrant cells (12.33%?±?1.27) was observed in dimethylbenz(a)anthracene (DMBA) plus highest concentration (1200?mg/kg) of TBT extract-treated group when compared to only DMBA-treated group (17.00%?±?2.28).

Discussion and conclusion: Results indicated that TBT can be considered as genotoxically safe, because it did not exert any mutagenic and clastogenic effects. As a result, TBT exhibited antimutagenic effects more apparently after metabolic activation in bacterial test system and had an anticlastogenic effect in mice.     

Shiitake (Lentinula edodes (Berkeley) Pegler) extracts as a modulator of micronuclei induced in HEp-2 cells

Shiitake (Lentinula edodes (Berkeley) Pegler) is one of the most consumed mushrooms, for both therapeutic purposes and as food, therefore, the study of its biological properties is of great interest for producers and consumers. Aqueous extracts of the shiitake mushroom (L. edodes (Berkeley) Pegler) were evaluated by the micronucleus test (MN) in HEp-2 cells in vitro, to analyze their possible mutagenic and antimutagenic activities. None of the three extract concentrations tested (0.5, 1.0 and 1.5 mg/mL) presented mutagenicity at any of the preparation temperatures (4 °C, 22 ± 2 °C and 60 °C). In the antimutagenicity evaluation, all extract concentrations at all preparation temperatures presented a strong protective activity for the HEp-2 cells in response to the alkylating agent methyl methanesulfonate (MMS) in the different treatment protocols: pre-treatment, simultaneous treatment and post-treatment. The extracts prepared at 22 ± 2 °C presented the lowest frequencies of MN in the evaluations of mutagenicity and antimutagenicity, indicating these as the best option for potential therapeutic use.