Reduction in exposure to carcinogenic aflatoxins by postharvest intervention measures in west Africa: a community-based intervention study

Background Aflatoxins are fungal metabolites that frequently contaminate staple foods in much of sub-Saharan Africa, and are associated with increased risk of liver cancer and impaired growth in young children. We aimed to assess whether postharvest measures to restrict aflatoxin contamination of groundnut crops could reduce exposure in west African villages.

Methods We undertook an intervention study at subsistence farms in the lower Kindia region of Guinea. Farms from 20 villages were included, ten of which implemented a package of postharvest measures to restrict aflatoxin contamination of the groundnut crop; ten controls followed usual postharvest practices. We measured the concentrations of blood aflatoxin-albumin adducts from 600 people immediately after harvest and at 3 months and 5 months postharvest to monitor the effectiveness of the intervention.

Findings In control villages mean aflatoxin-albumin concentration increased postharvest (from 5.5 pg/mg [95% CI 4.7-6.1] immediately after harvest to 18.7 pg/mg [17.0-20.6] 5 months later). By contrast, mean aflatoxin-albumin concentration in intervention villages after 5 months of groundnut storage was much the same as that immediately postharvest (7.2 pg/mg [6.2-8.4] vs 8.0 pg/mg [7.0-9.2]). At 5 months, mean adduct concentration in intervention villages was less than 50% of that in control villages (8.0 pg/mg [7.2-9.2] vs 18.7 pg/mg [17.0-20.6], p<0.0001). About a third of the number of people had non-detectable aflatoxin-albumin concentrations at harvest. At 5 months, five (2%) people in the control villages had non-detectable adduct concentrations compared with 47 (20%) of those in the intervention group (p<0.0001). Mean concentrations of aflatoxin B1 in groundnuts in household stores in intervention and control villages were consistent with measurements of aflatoxin-albumin adducts.

Interpretation Use of low-technology approaches at the subsistence-farm level in sub-Saharan Africa could substantially reduce the disease burden caused by aflatoxin exposure.

Determinants of aflatoxin exposure in young children from Benin and Togo, West Africa: the critical role of weaning

Background Dietary exposure to high levels of the fungal toxin, aflatoxin, occurs in West Africa, where long-term crop storage facilitates fungal growth.

Methods We conducted a cross-sectional study in Benin and Togo to investigate aflatoxin exposure in children around the time of weaning and correlated these data with food consumption, socioeconomic status, agro-ecological zone of residence, and anthropometric measures. Blood samples from 479 children (age 9 months to 5 years) from 16 villages in four agro-ecological zones were assayed for aflatoxin-albumin adducts (AF-alb) as a measure of recent past (2-3 months) exposure.

Results Aflatoxin-albumin adducts were detected in 475/479 (99%) children (geometric mean 32.8 pg/mg, 95% CI: 25.3-42.5). Adduct levels varied markedly across agro-ecological zones with mean levels being approximately four times higher in the central than in the northern region. The AF-alb level increased with age up to 3 years, and within the 1-3 year age group was significantly (P=0.0001) related to weaning status; weaned children had approximately twofold higher mean AF-alb adduct levels (38 pg AF-lysine equivalents per mg of albumin [pg/mg]) than those receiving a mixture of breast milk and solid foods after adjustment for age, sex, agro-ecological zone, and socioeconomic status. A higher frequency of maize consumption, but not groundnut consumption, by the child in the preceding week was correlated with higher AF-alb adduct level. We previously reported that the prevalence of stunted growth (height for age Z-score HAZ) and being underweight (weight for age Z-score WAZ) were 33% and 29% respectively by World Health Organziation criteria. Children in these two categories had 30-40% higher mean AF-alb levels than the remainder of the children and strong dose- response relationships were observed between AF-alb levels and the extent of stunting and being underweight.

Conclusions Exposure to this common toxic contaminant of West African food increases markedly following weaning and exposure early in life is associated with reduced growth. These observations reinforce the need for aflatoxin exposure intervention strategies within high-risk countries, possibly targeted specifically at foods used in the post-weaning period.

Heterogeneous oxidation of pyrimidine and alkyl thioethers in ionic liquids over mesoporous Ti or Ti/Ge catalysts

Heterogeneous catalytic oxidation of a series of thioethers (2-thiomethylpyrimidine, 2-thiomethyl-4,6-dimethyl-pyrimidine, 2-thiobenzylpyrimidine, 2-thiobenzyl-4,6-dimethylpyrimidine, thioanisole, and n-heptyl methyl sulfide) was performed in ionic liquids by using MCM-41 and UVM-type mesoporous catalysts containing Ti, or Ti and Ge. A range of triflate, tetrafluoroborate, trifluoroacetate, lactate and bis(trifluoromethanesulfonyl)imide-based ionic liquids were used. The oxidations were carried out by using anhydrous hydrogen peroxide or the urea-hydrogen peroxide adduct and showed that ionic liquids are very effective solvents, achieving greater reactivity and selectivity than reactions performed in dioxane. The effects of halide and acid impurities on the reactions were also investigated. Recycling experiments on catalysts were carried out in order to evaluate Ti leaching and its effect on activity and selectivity.

Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia reperfusion injury and rheumatoid arthritis. Chemical modification of amino acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative stress in vivo. The focus of the studies described here was initially to identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (HNE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays for the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diimine cross-link and the Michael addition product of HNE to lysine. Using these assays, we showed that the concentrations of all three compounds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine (CML) also increased during LDL oxidation, while that of its putative carbohydrate precursor the Amadori compound N epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating that CML is a marker of both glycoxidation and lipoxidation reactions. These results suggest that MDA and HNE adducts to lysine residues should serve as biomarkers of lipid modification resulting from lipid peroxidation reactions, while CML may serve as a biomarker of general oxidative stress resulting from both carbohydrate and lipid oxidation reactions.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus

Glycation, oxidation, and browning of proteins have all been implicated in the development of diabetic complications. We measured the initial Amadori adduct, fructoselysine (FL); two Maillard products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine; and fluorescence (excitation = 328 nm, emission = 378 nm) in skin collagen from 39 type 1 diabetic patients (aged 41.5 +/- 15.3 [17-73] yr; duration of diabetes 17.9 +/- 11.5 [0-46] yr, [mean +/- SD, range]). The measurements were related to the presence of background (n = 9) or proliferative (n = 16) retinopathy; early nephropathy (24-h albumin excretion rate [AER24] > or = 20 micrograms/min; n = 9); and limited joint mobility (LJM; n = 20). FL, CML, pentosidine, and fluorescence increased progressively across diabetic retinopathy (P <0.05, P <0.001, P <0.05, P <0.01, respectively). FL, CML, pentosidine, and fluorescence were also elevated in patients with early nephropathy (P <0.05, P <0.001, P <0.01, P <0.01, respectively). There was no association with LJM. Controlling for age, sex, and duration of diabetes using logistic regression, FL and CML were independently associated with retinopathy (FL odds ratio (OR) = 1.06, 95% confidence interval (CI) = 1.01-1.12, P <0.05; CML OR = 6.77, 95% CI = 1.33-34.56, P <0.05) and with early nephropathy (FL OR = 1.05, 95% CI = 1.01-1.10, P <0.05; CML OR = 13.44, 95% CI = 2.00-93.30, P <0.01). The associations between fluorescence and retinopathy and between pentosidine and nephropathy approached significance (P = 0.05). These data show that FL and Maillard products in skin correlate with functional abnormalities in other tissues and suggest that protein glycation and oxidation (glycoxidation) may be implicated in the development of diabetic retinopathy and early nephropathy.

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of diabetes and aging on carboxymethyllysine levels in human urine

Carboxymethyllysine (CML) has been identified as a modified amino acid that accumulates with age in human lens proteins and collagen. CML may be formed by oxidation of fructoselysine (FL), the Amadori adduct formed on nonenzymatic glycosylation of lysine residues in protein, or by reaction of ascorbate with protein under autoxidizing conditions. We proposed that measurements of tissue and urinary CML may be useful as indices of oxidative stress or damage to proteins in vivo. To determine the extent to which oxidation of nonenzymatically glycosylated proteins contributes to urinary CML, we measured the urinary concentrations of FL and CML in diabetic (n = 26) and control (n = 28) patients. The urinary concentration of FL correlated strongly with HbA1 measurements and was significantly higher in diabetic compared with control samples (9.2 +/- 6.5 and 4.0 +/- 2.8 micrograms/mg creatinine, respectively; P less than 0.0001). There was also a strong correlation between the concentrations of CML and FL in both diabetic and control urine (r = 0.67, P less than 0.0001) but only a weakly significant increase in the CML concentration in diabetic compared with control urine (1.2 +/- 0.5 and 1.0 +/- 0.3 micrograms/mg creatinine, respectively; P = 0.05). The molar ratio of CML to FL was significantly lower in diabetic compared with control patients (0.25 +/- 0.12 and 0.43 +/- 0.16, respectively; P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

The Maillard reaction in vivo

The Maillard or browning reaction between reducing sugars and protein contributes to the chemical deterioration and loss of nutritional value of proteins during food processing and storage. This article presents and discusses evidence that the Maillard reaction is also involved in the chemical aging of long-lived proteins in human tissues. While the concentration of the Amadori adduct of glucose to lens protein and skin collagen is relatively constant with age, products of sequential glycation and oxidation of protein, termed glycoxidation products, accumulate in these long-lived proteins with advancing age and at an accelerated rate in diabetes. Among these products are the chemically modified amino acids, N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxymethyl)hydroxylysine (CMhL), and the fluorescent crosslink, pentosidine. While these glycoxidation products are present at only trace levels in tissue proteins, there is strong evidence for the presence of other browning products which remain to be characterized. Mechanisms for detoxifying reactive intermediates in the Maillard reaction and catabolism of extensively browned proteins are also discussed, along with recent approaches for therapeutic modulation of advanced stages of the Maillard reaction.

A pilot study to evaluate aflatoxin exposure in a rural Ugandan population

Objectives: The fungal metabolite aflatoxin is a common contaminant of foodstuffs, especially when stored in damp conditions. In humans, high levels can result in acute hepatic necrosis and death, while chronic exposure is carcinogenic. We conducted a pilot study nested within an existing population cohort (the General Population Cohort), to assess exposure to aflatoxin, among people living in rural south-western Uganda. Methods: Sera from 100 adults and 96 children under 3 years of age (85 male, 111 female) were tested for aflatoxin-albumin adduct (AF-alb), using an ELISA assay. Socio-demographic and dietary data were obtained for all participants; HIV serostatus was available for 90 adults and liver function tests (LFTs) for 99. Results: Every adult and all but four children had detectable AF-alb adduct, including five babies reported to be exclusively breastfed. Levels ranged from 0 to 237.7 pg/mg albumin and did not differ significantly between men and women, by age or by HIV serostatus; 25% had levels above 15.1 pg/mg albumin. There was evidence of heterogeneity between villages (P = 0.003); those closest to trading centres had higher levels. Adults who consumed more Matooke (bananas) had lower levels of AF-alb adduct (P = 0.02) than adults who did not, possibly because their diet contained fewer aflatoxin-contaminated foods such as posho (made from maize). Children who consumed soya, which is not grown locally, had levels of AF-alb adduct that were almost twice as high as those who did not eat soya (P = 0.04). Conclusions: Exposure to aflatoxin is ubiquitous among the rural Ugandans studied, with a significant number of people having relatively high levels. Sources of exposure need to be better understood to instigate practical and sustainable interventions. © 2014 John Wiley & Sons Ltd.

Quantitative correlation of aflatoxin biomarker with dietary intake of aflatoxin in Tanzanian children

The association between aflatoxin intake from maize-based weaning food and aflatoxin albumin adducts (AF-alb) was investigated in 148 Tanzanian children aged between 12 and 22 months, at 2 visits 6 months apart. At the first visit (storage season) there was a significant correlation at the individual level between AF-alb (geometric mean 43.2 pg/mg albumin) and aflatoxin intake (geometric mean 81.7 ng/kg b.w./d) through maize-based weaning food (r = 0.51, p < 0.01). Overall, this correlation was r = 0.43 (p < 0.01). The AF-alb level in weaning-age children in Tanzania closely reflects aflatoxin intake from maize in weaning food. Exposure levels suggest children may be at risk from aflatoxin associated health effects.

Aflatoxin exposure is inversely associated with IGF1 and IGFBP3 levels in vitro and in Kenyan schoolchildren

SCOPE: This study explores the relationship between aflatoxin and the insulin-like growth factor (IGF) axis and its potential effect on child growth.

METHODS AND RESULTS: One hundred and ninety-nine Kenyan schoolchildren were studied for aflatoxin-albumin adduct (AF-alb), IGF1 and IGF-binding protein-3 (IGFBP3) levels using ELISA. AF-alb was inversely associated with IGF1 and IGFBP3 (p < 0.05). Both IGF1 and IGFBP3 were significantly associated with child height and weight (p < 0.01). Children in the highest tertile of AF-alb exposure (>198.5 pg/mg) were shorter than children in the lowest tertile (<74.5 pg/mg), after adjusting for confounders (p = 0.043). Path analysis suggested that IGF1 levels explained ∼16% of the impact of aflatoxin exposure on child height (p = 0.052). To further investigate this putative mechanistic pathway, HHL-16 liver cells (where HHL-16 is human hepatocyte line 16 cells) were treated with aflatoxin B1 (0.5, 5 and 20 μg/mL for 24-48 h). IGF1 and IGFBP3 gene expression measured by quantitative PCR and protein in culture media showed a significant down-regulation of IGF genes and reduced IGF protein levels.

CONCLUSION: Aflatoxin treatment resulted in a significant decrease in IGF gene and protein expression in vitro. IGF protein levels were also lower in children with the highest levels of AFB-alb adducts. The data suggest that aflatoxin-induced changes in IGF protein levels could contribute to growth impairment where aflatoxin exposure is high.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.

Dietary exposure to aflatoxin and micronutrient status among young children from Guinea

SCOPE: Aflatoxin exposure coincides with micronutrient deficiencies in developing countries. Animal feeding studies have postulated that aflatoxin exposure may be exacerbating micronutrient deficiencies. Evidence available in human subjects is limited and inconsistent. The aim of the study was to investigate the relationship between aflatoxin exposure and micronutrient status among young Guinean children.

METHOD AND RESULTS: A total of 305 children (28.8 ± 8.4 months) were recruited at groundnut harvest (rainy season), of which 288 were followed up 6 months later post-harvest (dry season). Blood samples were collected at each visit. Aflatoxin-albumin adduct levels were measured by ELISA. Vitamin A, vitamin E and β-carotene concentrations were measured using HPLC methods. Zinc was measured by atomic absorption spectroscopy. Aflatoxin exposure and micronutrient deficiencies were prevalent in this population and were influenced by season, with levels increasing between harvest and post-harvest. At harvest, children in the highest aflatoxin exposure group, compared to the lowest, were 1.98 (95%CI: 1.00, 3.92) and 3.56 (95%CI: 1.13, 11.15) times more likely to be zinc and vitamin A deficient.

CONCLUSION: Although children with high aflatoxin exposure levels were more likely to be zinc and vitamin A deficient, further research is necessary to determine a cause and effect relationship. 

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.