In Vivo Toxicity Evaluation of Sugar Adulterated Heterotrigona itama Honey Using Zebrafish Model.

Honey is prone to be adulterated through mixing with sugars, cheap and low-quality honey, and other adulterants. Consumption of adulterated honey may cause several health issues such as weight gain, diabetes, and liver and kidney dysfunction. Therefore, studying the impact of consumption of adulterated honey on consumers is critical since there is a lack of study in this field. Hence, the aims of this paper were: (1) to determine the lethal concentration (LC50) of adulterated honey using zebrafish embryo, (2) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish, (3) to determine the effects of adulterated honey on histological changes of zebrafish, and (4) to screen the metabolites profile of adulterated honey by using zebrafish blood serum. The LC50 of Heterotrigona itama honey (acacia honey) and its sugar adulterants (light corn sugar, cane sugar, inverted sugar, and palm sugar in the proportion of 1-3% (w/w) from the total volume) was determined by the toxicological assessment of honey samples on zebrafish embryos (different exposure concentrations in 24, 48, 72, and 96 h postfertilization (hpf)). Pure H. itama honey represents the LC50 of 34.40 ± 1.84 (mg/mL) at 96 hpf, while the inverted sugar represents the lowest LC50 (5.03 ± 0.92 mg/mL) among sugar adulterants. The highest concentration (3%) of sugar adulterants were used to study the toxicology of adulterated honey using adult zebrafish in terms of acute, prolong-acute, and sub-acute tests. The results of the LD50 from the sub-acute toxicity test of pure H. itama honey was 2.33 ± 0.24 (mg/mL). The histological studies of internal organs showed a lesion in the liver, kidney, and spleen of adulterated treated-honey groups compared to the control group. Furthermore, the LC-MS/MS results revealed three endogenous metabolites in both the pure and adulterated honey treated groups, as follows: (1) S-Cysteinosuccinic acid, (2) 2,3-Diphosphoglyceric acid, and (3) Cysteinyl-Tyrosine. The results of this study demonstrated that adulterated honey caused mortality, which contributes to higher toxicity, and also suggested that the zebrafish toxicity test could be a standard method for assessing the potential toxicity of other hazardous food additives. The information gained from this research will permit an evaluation of the potential risk associated with the consumption of adulterated compared to pure honey.

Miel alucinógena del Himalaya: ¿la próxima intoxicación? / Hallucinogenic Himalayan honey: the next poison?

No disponible

Carcinogenic and non-carcinogenic risk assessment of trace elements and POPs in honey from Shirak and Syunik regions of Armenia.

Honey is a highly nutritious natural product widely produced and consumed by people in Shirak and Syunik regions of Armenia. Unlike Shirak, Syunik is under the impact of mining industry. Since the environmental pollution can adversely impact the safety of honey and entail a probable risk to human health, it is important to evaluate the presence of potentially toxic trace elements in honey samples from both regions and draw comparisons. This study assesses the dietary exposure to trace elements and persistent organic pollutants through the intake of honey for the first time among people in Shirak and Syunik regions. 24-hour dietary recall method was used to investigate the consumption of honey. The presence of seven trace elements (Pb, Cd, As, Hg, Cu, Zn, and Ni) and persistent organic pollutants (hexachlorocyclohexane (HCH), dichlorodiphenyltrichloroethane (DDT) and its metabolites) were determined in honey samples using atomic absorption spectrometry and gas chromatography-mass spectrometry, respectively. In several samples, the concentrations of Cu were above the maximum allowable level. Non-carcinogenic risk values did not exceed the acceptable level, while carcinogenic risk values for Ni and As exceeded the risk level of 10-6 in both regions. Moreover, among the persistent organic pollutants, only the concentration of DDT in honey from Shirak was above the European Union maximum residue level.

Gelsedine-type alkaloids: Discovery of natural neurotoxins presented in toxic honey.

The fascinating collection and evaluation of natural products with enormous structural and chemical diversity can contribute to ensure human health and inspire potential drug discovery. We reported the identification of 14-(R)-hydroxy-gelsenicine (HGE), a new component from poisonous honey, which has recently caused multiple serious intoxications and deaths up on consumption. The prevalence, toxicity, toxicokinetics and metabolic profile of HGE were evaluated through in vitro and in vivo analyses. HGE is a very toxic substance and shows significant gender difference with LD50 of 0.125 mg kg-1 and 0.295 mg kg-1 for the female and male mice, respectively. Toxicokinetics test indicates that HGE has good bioavailability in rats, and is metabolized extensively, in which hydroxylation, reduction, N-demethyl ether and glucuronication are the major metabolic pathways. Additionally, HGE shows specific neurotoxicity by enhancing the binding of γ-aminobutyric acid (GABA) to its receptors. We found that flumazenil, a selective antagonist of GABA receptor, could effectively increase the survival of the tested animals, which provides a potential therapy for future clinical applications.

Determination of the dose-dependent toxic effects of mad honey on mouse liver using ATR-FTIR spectroscopy.

Mad honey (MH) is obtained from Rhododendron plants, which are extensively grown in some regions of the world such as Europe, North America, Tropical Asia and Turkey. Although it has been known that MH induces adverse effects in the body due to grayanotoxin (GTX) in it, it is widely used for some medical purposes by the public. In this study, the effects of MH (25, 50 and 75 mg/kg) and GTX-III (0.01 mg/kg), which is the pure form of the most toxic type of the GTXs in MH, were investigated on the mouse liver at molecular level via Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The results showed that 25 and 50 mg/kg of MH didn't cause any significant alterations in the liver tissue except a decrease in the glycogen amount. However, significant differences were observed between 75 mg/kg MH and GTX-III treated groups and control group. For example, the amounts of saturated lipids, nucleic acids and proteins increased in the 75 mg/kg MH and GTX-III treated groups. A decrease in the ratios of unsaturated/saturated lipid, CH2/lipid and carbonyl/lipid and an increase in the ratio of CH3/lipid were observed after the administration of 75 mg/kg MH and GTX-III, all of which may be a consequence of lipid peroxidation. Moreover, 75 mg/kg MH and GTX-III caused a decrease in the membrane order, an increase in the membrane fluidity and some important changes on the secondary structure of proteins indicating protein denaturation. In addition, Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA) confirmed these findings. These results revealed that MH induces significant dose-dependent toxic effects in the structure and function of the liver tissue. This study also showed that ATR-FTIR spectroscopy provides a rapid and sensitive monitoring of the changes induced by a toxic compound on biological tissues at molecular level.

Effect on oxidative stress, hepatic chemical metabolizing parameters, and genotoxic damage of mad honey intake in rats.

A total of 66 male Wistar rats were used and six groups (control: 10 animals and experimental: 12 animals) were formed. While a separate control group was established for each study period, mad honey application to the animals in the experimental group was carried out with a single dose (12.5 g kg-1 body weight (b.w.); acute stage), at a dose of 7.5 g kg-1 b.w. for 21 days (subacute stage), and at a dose of 5 g kg-1 b.w. for 60 days (chronic stage). Tissue and blood oxidative stress markers (malondialdehyde (MDA), nitric oxide (NO), 4-hydroxynonenal (HNE), superoxide dismutase, catalase, glutathione (GSH) peroxidase, and glucose-6-phosphate dehydrogenase), hepatic chemical metabolizing parameters in the liver (cytochrome P450 2E1, nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase, nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (CYTC), GSH S-transferase (GST), and GSH), and micronucleus and comet test in some samples were examined. Findings from the study showed that single and repeated doses given over the period increased MDA, NO, and HNE levels while decreasing/increasing tissue and blood antioxidant enzyme activities. From hepatic chemical metabolizing parameters, GST activity increased in the subacute and chronic stages and CYTC activity increased in the acute period, whereas GSH level decreased in the subacute stage. Changes in tail and head intensities were found in most of the comet results. Mad honey caused oxidative stresses for each exposure period and made some significant changes on the comet test in certain periods for some samples obtained. In other words, according to the available research results obtained, careless consumption of mad honey for different medical purposes is not appropriate.

A 28-day oral toxicity study of echimidine and lasiocarpine in Wistar rats.

Pyrrolizidine alkaloids (PAs) are a class of naturally-occurring plant toxins. Echimidine is one of the predominant PAs found in honeys produced in Australia and New Zealand. There is a lack of information on the oral toxicity of echimidine on which to base regulatory decisions concerning the risk to humans of these honeys. This GLP study was conducted to assess the subchronic dietary toxicity of echimidine to rats compared to that of lasiocarpine as a positive control. Wistar rats, 10/sex, were fed diets containing 0, 0.6, 1.2 or 2.5 mg/kg bw echimidine. Positive control groups, 10/sex, were fed diets containing 0.6, 1.2 or 2.5 mg/kg bw lasiocarpine. Neither PA had any effect on survival, food consumption, clinical signs, gross lesions, or histopathology. Consumption of lasiocarpine, but not echimidine, decreased bodyweight gain in males at ≥ 1.2 mg/kg bw, and in females at 2.5 mg/kg bw. Slight alterations in white cell counts and serum ALT concentrations at 2.5 mg/kg bw of both PAs were not clinically significant, had no histological correlates, and were considered to be of equivocal relevance. In conclusion, the subchronic No Observed Adverse Effect Level (NOAEL) for echimidine is 2.5 mg/kg bw/day, whereas, on the basis of a treatment-related decrease in bodyweight gain in males at 1.2 mg/kg bodyweight, the NOAEL for lasiocarpine is 0.6 mg/kg bw/day.

Effects of Mad Honey on Some Biochemical Parameters in Rats.

The aims of this study were to determine grayanotoxin (GTX-III) toxin level in mad honey using liquid chromatography-tandem mass spectrometry and examine the dynamic changes of certain biochemical parameters in blood serum of rats that consumed mad honey. For the experimental animal study, 20 Sprague-Dawley female rats were divided into 5 groups of 4 rats each, with one group being the control group (Group 1) and the others being the experimental groups (Groups 2-5). Groups 2, 3, 4, and 5 were, respectively, given mad honey extract at doses of 0.3, 0.6, 1.2, and 2.4 mg/g body weight/day via oral gavage for 8 days. According to results, the quantity of GTX-III found in the honey sample as 39.949 ± 0.020 µg GTX-III/g honey, and the biochemical analysis of the tested parameters (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, creatine kinase, and creatine kinase muscle and brain) showed a significant elevation with increasing concentration of honey. In conclusion, the use of increasing concentrations of Rhododendron honey was seen as a source of enzymatic symptoms.

A case of human poisoning by grayanotoxins following honey ingestion: elucidation of the toxin profile by mass spectrometry.

High-resolution mass spectrometry (HRMS) was applied for the detection of grayanotoxins (GrTx) in a contaminated honey sample. This sample was provided by a hospital due to a suspicion of intoxication after a patient had shown the typical symptoms of GrTx poisoning. Subsequent analysis proved the contamination with high amounts of GrTx and other toxins belonging to grayanane-type diterpenoids. This group of natural toxins is synthesised by the plant family Ericaceae and comprises more than 60 individual toxins, but only one compound is available as a reference standard. We applied a screening approach that easily confirms the presence or absence of GrTx without access to standards. By searching for predictable mass spectrometric fragment ions, including typical in-source fragments arising from collision-induced dissociation during electrospray ionisation, the complete toxin profile was screened and allowed the mass spectrometric identification of 15 individual GrTx. The potential of this approach is especially demonstrated by the fact that at least two of these toxins have not been previously described in the literature. A semi-quantitative estimation indicated a total toxin concentration of 358 mg kg(-1). An investigation of 49 honeys from the German retail market did not reveal the presence of GrTx.

Anti-HIV-1 activity of eight monofloral Iranian honey types.

Monofloral Iranian honeys from eight floral sources were analyzed to determine their anti-HIV-1 activities as well as their effects on lymphocyte proliferation. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were prepared from five healthy volunteers who were seronegative for HIV, HCV, HBV and TB. The anti-HIV-1 activity of eight different honeys was performed by quantitative polymerase chain reaction (PCR) assay and high pure viral nucleic acid kit. The results demonstrated that monofloral honeys from Petro selinum sativum, Nigella sativa, Citrus sinensis, Zataria multiflora, Citrus aurantium and Zizyphus mauritiana flowers had potent anti-HIV-1 activity with half maximal effective concentration (EC50) values of 37.5, 88, 70, 88, 105 and 5 µg/ml respectively. However, monofloral Iranian honeys from Astragalus gummifer and Chamaemelum nobile flowers had weak anti-HIV-1 activity. The frequency and intensity of CD4 expression on PBMCs increased in the presence of all honey types. CD19 marker were also increased after the treatment with monofloral honeys from Z. multiflora and N. sativa. The anti-HIV-1 agent in monofloral honeys from P. sativum, N. sativa, Z. multiflora and Z. mauritiana flowers was detected by spectroscopic analysis as methylglyoxal. Time of drug addition studies demonstrated that the inhibitory effect of methylglyoxal is higher on the late stage of HIV-1 infection. The result demonstrated that methylglyoxal isolated from monofloral honey types is a good candidate for preclinical evaluation of anti-HIV-1 therapies.

Mad honey intoxication mimicking acute coronary syndrome.

Mad honey intoxication or grayanotoxin poisoning is caused by consumption of grayanotoxin-containing toxic honey produced from leaves and flowers of the Rhododendron family. Despite the rarity of intoxication cases, the correct diagnosis and treatment are required because of the significance of haemodynamic disturbance and confounding of symptoms for disease identification. We report herein a case of a patient with mad honey intoxication mimicking acute non-ST segment elevation myocardial infarction and review the pathophysiology and diagnostic considerations.

A bitter-sweet tale from the land of milk and honey.

A sporadic seasonal neurotoxic food poisoning, unique to northern parts of New Zealand, especially The Bay of Plenty, has recurred-with implications for our primary produce industry, as well as human health.

Microscopic fungi recovered from honey and their toxinogenity.

The objective of this investigation was to contribute towards the knowledge of microbiology of honey, more than 50 samples of honey from Slovakia and other countries were mycologically investigated in terms of the overall fungal diversity and toxicological potential of isolated fungi from Penicillium genera. The study revealed that out of 13 genera recovered, Penicillium was the most frequent and diverse genus, followed by Aspergillus and Cladosporium being found in 65.91 % (29 samples), 34.1 % (15 samples) and 29.55 % (13 samples), respectively. The most frequently encountered taxa from Penicillium genera were Penicillium chrysogenum (found in 22.73 %), Penicillium brevicompactum (13.64 %), Penicillium crustosum (11.36 %) and Penicillium griseofulvum (11.36 %). In addition, the following genera were recorded (in descending order) Mycelia (18.18 %), Fusarium (11.36 %), Mucor (9.09 %), Acremonium (6.82 %), Alternaria (4.55 %), Epicoccum (4.55 %), and finally Botrytis, Eurotium Trichoderma and Phoma all were encountered in 2.27 % of the samples being represented. The mean value counts of total fungi ranged from 0.00 to 2 × 10(2) cfu.g(-1). Outcomes from mycotoxin screening within the appropriate potentially toxinogenic species from Penicillium genera showed a number of mycotoxin producers, namely those forming citrinin (n = 1), cyclopiazonic acid (n = 5), griseofulvin (n = 5), patulin (n = 5), penitrem A (n = 2) and roquefortin C (n = 13).

Otologic safety of manuka honey.

OBJECTIVE: To investigate the possible ototoxic effects of a 50% concentration of manuka honey in a chinchilla animal model. STUDY DESIGN: A prospective, controlled animal study. SETTING: The Research Institute of the Montreal Children's Hospital, McGill University Health Centre. SUBJECTS AND METHODS: Eight animals had myringotomy incisions in both ears. One ear was randomly assigned to receive the 50% manuka honey solution. The contralateral ear received saline and served as the control ear. OUTCOME MEASURES: Auditory brainstem evoked responses (ABRs) were measured bilaterally for a wide range of frequencies (between 8 and 25 kHz) before and 2 weeks after transtympanic manuka honey and saline application. The animals were sacrificed, and all cochleae were dissected out and processed for light and scanning electron microscopy (SEM). RESULTS: The measured ABR thresholds after the application of 50% concentration of manuka honey revealed severe ototoxicity in all honey-exposed ears. This was accompanied by gross physical changes and histologic evidence of hair cell toxicity on SEM and light microscopy. The control ears remained unchanged during the period of the experiment. CONCLUSION: Although 50% concentration of manuka honey is the proven concentration to have bactericidal properties against biofilms of Pseudomonas aeruginosa and Staphylococcus aureus, this concentration appeared to have caused severe or intense inflammatory changes that produced facial paralysis, vestibulotoxicity, and hearing loss.

Clinical events in mad honey poisoning: a single centre experience.

The aim was to evaluate the clinical findings of patients who admitted to the hospital with the diagnosis of grayanotoxin/mad honey poisoning. Thirty-three patients were included in this study. Three patients were female (9%) and the others male (91%). Median age of patients was 52 (42-68). The most frequently observed findings were sinus bradycardia (91%), nausea-vomiting (81.8%), and dizziness (78.8%). Average heart rate was 55.35 +/- 6.72 beats/min. Mean systolic and diastolic blood pressures were 77.86 +/- 16.64 mmHg and 46.42 +/- 12.30 mmHg, respectively. Mad honey poisoning is an important problem that is life-threatening in the Black Sea region of Turkey.

The intracellular effects of manuka honey on Staphylococcus aureus.

The purpose of this study was to investigate the effect of manuka honey on Staphylococcus aureus in order to identify the intracellular target site. The mode of inhibition of manuka honey against S. aureus NCTC 10017 was investigated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the effect of time on viability. Structural changes were observed by scanning (SEM) and transmission electron microscopy (TEM) of cells suspended for 4 h at 37 degrees C in 0.05 mM Tris buffer containing 10% (w/v) manuka honey and were compared to cells in buffer alone or buffer containing 10% (w/v) artificial honey (to assess osmotic damage). A bactericidal mode of inhibition for manuka honey on S. aureus was established. Marked structural changes in honey-treated cells were seen only with TEM, where a statistically significant increase in the number of whole cells with completed septa compared to untreated cells were observed (P < 0.05). Structural changes found with TEM suggest that honey-treated cells had failed to progress normally through the cell cycle and accumulated with fully formed septa at the point of cell division without separating. Sugars were not implicated in this effect. The staphylococcal target site of manuka honey involves the cell division machinery.

Effects of acacia honey on wound healing in various rat models.

Honey is a traditional remedy for the treatment of infected wounds, and is becoming more important as microbial resistance to conventional therapeutic agents increases. A study was conducted to assess the wound-healing activity of Acacia honey using incision, excision, burn and dead-space wound models in rats. Different formulations of honey were used and rats were treated topically as well as orally. Both the higher and lower doses of honey produced a significant effect on healing (p < 0.05). The area of epithelization was found to increase, followed by an increase in wound contraction, skin-breaking strength, tissue granulation. The hydroxyproline content also increased in the rats treated with higher doses of honey compared to control, indicating an increase in collagen formation.

Residues of antibacterial drugs in honey from the Italian market.

Antibacterial drugs are used worldwide for the control of American and, less often, European foulbrood. Their administration is mostly uncontrolled and applied without approved protocols and instructions for use as well as precautionary recommendations. Consequently, this practice is responsible for the contamination of beehive products and contributes to the problem of food safety. According to this situation, 4672 analyses were carried out on 5303 honeys collected from 2001 to 2007. These samples were investigated for antibacterial residues of tetracyclines, sulphonamides, streptomycin, chloramphenicol and tylosin. Honeys were classified according to their origin: imported honey and honey from the Italian market. In the last group (only for samples collected from 2001 to 2004), another type of honey was distinguished: that of local honey. A total of 6.3% of all samples were positive for the antibacterial drugs analysed; in particular, 6.8% of imported honeys and 6.1% of honeys on the Italian market. Only 1.7% of local honey had antibacterial residues. These results are indicative of a rather frequent presence of antibacterial drug residues in both Italian and imported honeys. Furthermore, the data showed that among the active substances analysed, sulphonamides are the most used antibacterial substance followed by tetracyclines, streptomycin, tylosin, and chloramphenicol. Finally, a continuous monitoring programme is needed, accompanied by an education programme to beekeepers on proper hive management.

Honeybee (Apis cerana) foraging responses to the toxic honey of Tripterygium hypoglaucum (Celastraceae): changing threshold of nectar acceptability.

To investigate honeybee foraging responses to toxic nectar, honey was collected from Apis cerana colonies in the Yaoan county of Yunnan Province, China, during June, when flowers of Tripterygium hypoglaucum were the main nectar source available. Pollen analysis confirmed the origin of the honey, and high-performance liquid chromatography showed the prominent component triptolide to be present at a concentration of 0.61 mug/g +/- 0.11 SD. In cage tests that used young adult worker bees, significantly more of those provided with a diet of T. hypoglaucum honey mixed with sugar powder (1:1) died within 6 d (68.3%) compared to control groups provided with normal honey mixed with sugar powder (15.8%). Honeybees were trained to visit feeders that contained honey of T. hypoglaucum (toxic honey) as the test group and honey of Vicia sativa or Elsholtzia ciliata as control groups (all honeys diluted 1:3 with water). Bees preferred the feeders with normal honey to those with toxic honey, as shown by significantly higher visiting frequencies and longer imbibition times. However, when the feeder of normal honey was removed, leaving only honey of T. hypoglaucum, the foraging bees returned to the toxic honey after a few seconds of hesitation, and both visiting frequency and imbibition time increased to values previously recorded for normal honey. Toxic honey thus became acceptable to the bees in the absence of other nectar sources.

Use of a spectrophotometric bioassay for determination of microbial sensitivity to manuka honey.

The antimicrobial activity of manuka honey has been well documented (Molan, 1992a,b,c, 1997) [Molan, P.C., 1992. The antibacterial activity of honey. 1: the nature of the antibacterial activity. Bee World 73 (1) 5-28; Molan, P.C., 1992. The antibacterial activity of honey. 2: variation in the potency of the antibacterial activity. Bee World 73 (2) 59-76; Molan, P.C., 1992. Medicinal uses for honey. Beekeepers Quarterly 26; Molan, P.C., 1997. Finding New Zealand honeys with outstanding antibacterial and antifungal activity. New Zealand Beekeeper 4 (10) 20-26]. The current bioassays for determining this antimicrobial effect employ a well diffusion (Ahn and Stiles, 1990) [Ahn, C., Stiles, M.E., 1990. Antibacterial activity of lactic acid bacteria isolated from vacuum-packed meats. Journal of Applied Bacteriology 69, 302-310], (Weston et al., 1999) [Weston, R.J., Mitchell, K.R., Allen, K.L., 1999. Antibacterial phenolic components of New Zealand manuka honey. J. Food Chem. 64, 295-301] or disc diffusion (Taormina et al., 2001) [Taormina, Peter J., Niemira, Brendan A., Beuchat, Larry R., 2001. Inhibitory activity of honey against food borne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. Int. J. Food Microbiol. 69, 217-225] assay using zones of inhibition as indicators of bacterial susceptibility. The development of a 24-h spectrophotometric assay employing 96-well microtiter plates, that is more sensitive and more amenable to statistical analysis than the assays currently employed, was undertaken. This simple and rapid assay permits extensive kinetic studies even in the presence of low honey concentrations, and is capable of detecting inhibitory levels below those recorded for well or disc diffusion assays. In this paper, we compare the assay to both well and disc diffusion assays. The results we obtained for the spectrophotometric method MIC values show that this method has greater sensitivity than the standard well and disc diffusion assays. In addition, inter- and intra-assay variance for this method was investigated, demonstrating the methods reproducibility and repeatability.