Biological monitoring in workers in a nitrobenzene reduction plant : Haemoglobin versus serum albumin adducts

The high priority of monitoring workers exposed to nitrobenzene is a consequence of clear findings of experimental carcinogenicity of nitrobenzene and the associated evaluations by the International Agency for Research on Cancer. Eighty male employees of a nitrobenzene reduction plant, with potential skin contact with nitrobenzene and aniline, participated in a current medical surveillance programme. Blood samples were routinely taken and analysed for aniline, 4-aminodiphenyl (4-ADP) and benzidine adducts of haemoglobin (Hb) and human serum albumin (HSA). Also, levels of methaemoglobin (Met-Hb) and of carbon monoxide haemoglobin (CO-Hb) were monitored. Effects of smoking were straightforward. Using the rank sum test of Wilcoxon, we found that very clear-cut and statistically significant smoking effects (about 3-fold increases) were apparent on CO-Hb (P = 0.00085) and on the Hb adduct of 4-ADP (P = 0.0006). The mean aniline-Hb adduct level in smokers was 1.5 times higher than in non-smokers; the significance (P = 0.05375) was close to the 5% level. The strongest correlation was evident between the Hb and HSA adducts of aniline (rs = 0.846). Less pronounced correlations (but with P values < 0.02) appeared between aniline-Hb and 4-ADP-Hb adducts (rs = 0.388), between 4-ADP and 4-ADP-HSA adducts (rs = 0.373), and between 4-ADP-Hb and aniline-HSA adducts (rs = 0.275). In view of the proposal for additional use of the aniline-HSA adduct for biological monitoring, particularly in cases of acute overexposures or poisonings, the strong correlation of the Hb and HSA conjugates is noteworthy; the ratio aniline-HSA:aniline-Hb was 1:42 for the entire cohort.

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B-1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

TP53 R249S Mutations, Exposure to Aflatoxin, and Occurrence of Hepatocellular Carcinoma in a Cohort of Chronic Hepatitis B Virus Carriers from Qidong, China

Hepatocellular carcinoma (HCC) has a high mortality in East Asia and Sub-Saharan Africa, two regions where the main etiologic factors are chronic infections with hepatitis B vir-us and dietary exposure to aflatoxin. A single base substitution at the third nucleotide of codon 249 of TP53 (R249S) is common in HCC in these regions and has been associated with aflatoxin-DNA adducts. To determine whether R249S may be detected in plasma DNA before HCC diagnosis, we conducted a case-control study nested in a cohort of adult chronic hepatitis B virus carriers from Qidong County, People's Republic of China. Of the 234 plasma specimens that yielded adequate DNA, only 2 (0.9%) were positive for R249S by restriction fragment length polymorphisms, and both of them were controls. Of the 249 subjects tested for aflatoxin-albumin adducts, 168 (67%) were positive, with equal distribution between cases and controls. Aflatoxin-albumin adduct levels were low in the study, suggesting an overall low ongoing exposure to aflatoxin in this cohort. The R249S mutation was detected in 11 of 18 (61%) available tumor tissues. To assess whether low levels of mutant DNA were detectable in pre-diagnosis plasma, 14 plasma specimens from these patients were analyzed by short oligonucleotide mass analysis. Nine of them (64%) were found to be positive. Overall, these results suggest that HCC containing R249S can occur in the absence of significant recent exposure to aflatoxins. The use of short oligonucleotide mass analysis in the context of low ongoing aflatoxin exposure may allow the detection of R249S in plasma several months ahead of clinical diagnosis. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1638-43)

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.

Determining mycotoxins and mycotoxigenic fungi in food and feed:Developing biomarkers of human exposure to mycotoxins

Exposure assessment is a critical part of epidemiological studies into the effect of mycotoxins on human health. Whilst exposure assessment can be made by estimating the quantity of ingested toxins from food analysis and questionnaire data, the use of biological markers (biomarkers) of exposure can provide a more accurate measure of individual level of exposure in reflecting the internal dose. Biomarkers of exposure can include the excreted toxin or its metabolites, as well as the products of interaction between the toxin and macromolecules such as protein and DNA. Samples in which biomarkers may be analysed include urine, blood, other body fluids and tissues, with urine and blood being the most accessible for human studies. Here we describe the development of biomarkers of exposure for the assessment of three important mycotoxins; aflatoxin, fumonisin and deoxynivalenol. A number of different biomarkers and methods have been developed that can be applied to human population studies, and these approaches are reviewed in the context of their application to molecular epidemiology research.

Dietary exposure to aflatoxin and fumonisin among Tanzanian children as determined using biomarkers of exposure

SCOPE: The study aims to evaluate the status of dietary exposure to aflatoxin and fumonisin in young Tanzanian children, using previously validated biomarkers of exposure. METHODS AND RESULTS: A total of 148 children aged 12-22 months, were recruited from three geographically distant villages in Tanzania; Nyabula, Kigwa, and Kikelelwa. Plasma aflatoxin-albumin adducts (AF-alb) and urinary fumonisin B1 (UFB1) were measured by ELISA and LC-MS, respectively. AF-alb was detectable in 84% of children, was highest in fully weaned children (p <0.01) with higher levels being associated with higher maize intake (p <0.05). AF-alb geometric mean (95% CI) was 43.2 (28.7-65.0), 19.9 (13.5-29.2), and 3.6 (2.8-4.7) pg/mg albumin in children from Kigwa, Nyabula, and Kikelelwa, respectively. UFB1 was detectable in 96% of children and the level was highest in children who had been fully weaned (p <0.01). The geometric UFB1 mean (95% CI) was 327.2 (217.1-493.0), 211.7 (161.1-278.1), and 82.8 (58.3-117.7) pg/mL in Kigwa, Nyabula, and Kikelelwa, respectively. About 82% of all the children were exposed to both mycotoxins. CONCLUSION: Young children in Tanzania are chronically exposed to both aflatoxin and fumonisin through contaminated diet, although the level of exposure varies markedly between the three villages studied.

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.

A Prospective Study of Growth and Biomarkers of Exposure to Aflatoxin and Fumonisin during Early Childhood in Tanzania

BACKGROUND: Aflatoxin and fumonisin are toxic food contaminants. Knowledge about effects of their exposure and co-exposure on child growth is inadequate.

OBJECTIVE: To investigate the association between child growth and aflatoxin and fumonisin exposure in Tanzania.

METHODS: A total of 166 children were recruited at 6 to 14 months of age and studied at recruitment, and at the sixth and twelfth month following recruitment. Blood and urine samples were collected and analysed for plasma aflatoxin albumin adducts (AF-alb) using ELISA, and urinary fumonisin B1 (UFB1) using LC-MS, respectively. Anthropometric measurements were taken and growth index Z-scores were computed.

RESULTS: AF-alb geometric mean concentrations (95% confidence intervals) were 4.7 (3.9, 5.6), 12.9 (9.9, 16.7) and 23.5 (19.9, 27.7) pg/mg albumin at recruitment, six months, and 12 months from recruitment, respectively. At these respective sampling times, geometric mean UFB1 concentrations (95% CI) were 313.9 (257.4, 382.9), 167.3 (135.4, 206.7) and 569.5 (464.5, 698.2) pg/mL urine, and the prevalence of stunted children were 44%, 55% and 56%, respectively. UFB1 concentrations at recruitment were negatively associated with length for age Z-scores (LAZ) at six months (p = 0.016) and at 12 months from recruitment (p = 0.014). The mean UFB1 of the three sampling times (at recruitment, at six and 12 months from recruitment) in each child was negatively associated with LAZ (p < 0.001) and length velocity (p = 0.004) at 12 months from recruitment. The negative association between AF-alb and child growth did not reach statistical significance.

CONCLUSIONS: Exposure to fumonisin alone, or co-exposure with aflatoxins may contribute to child growth impairment.

Seasonal and geographical differences in aflatoxin exposures in Senegal

The aim of the study was to determine the geographical and seasonal variations in aflatoxin dietary exposure levels in adults from Senegal. A total of 168 adults (50% male) were recruited from three districts: Nioro du Rip (n=90), located in the Sudan Savannah agro-ecological zone where rainfall is sufficient for groundnut growth; Saint-Louis (n=40) and Mboro (n=38), located in the Sahel zone where groundnuts are produced under irrigated conditions. Diet information and samples were collected at groundnut harvest and post-harvest seasons. Plasma aflatoxin-albumin adducts (AF-alb) and total aflatoxin in household groundnut samples were measured by ELISA and a quantitative thin layer chromatography method, respectively. The blood AF-alb geometric mean was 45.7 pg/mg albumin (range 5.5-588.2 pg/mg). Nioro du Rip had a higher AF-alb level at harvest than Saint-Louis and Mboro (80.0 vs 15.6 and 33.3 pg/mg, P<0.001). Similar trends were observed at post-harvest (P<0.05). Seasonal trends were not consistent across the districts as Nioro du Rip had a higher AF-alb level at harvest than post-harvest (80.0 vs 58.6 pg/mg, P=0.026), whereas Saint-Louis had a higher level at post-harvest than harvest (25.6 vs 15.6 pg/mg, P=0.032). It is clear that aflatoxin exposure is prevalent in adults from Senegal and that season and geographical location are strong determinants of aflatoxin exposure.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.

Study of aflatoxin exposure in a Brazilian population using an aflatoxin - albumin biomarker

The evaluation of exposure to aflatoxins (AF) by measurement of the level of contamination in food is hampered due to the heterogeneous distribution of AF in food. Therefore, an alternative is to estimate the exposure using specific biological markers (biomarkers) based on an understanding of the metabolism of the compound. For AF, these include aflatoxin-N-7-guanine in the urine, or AFB(1)-albumin (AF-alb) in the blood. This study assessed the level of exposure to AF in Brazilian individuals using a biomarker approach, i.e. the AF-alb adducts. Blood samples were collected from urban residents (n=50; aged 18-52) in June 1999, at the Blood Center of Antonio Carlos de Camargo Hospital, Sao Paulo, Brazil. AF-alb adduct levels were determined, by ELISA following serum albumin extraction and digestion. AF-alb adducts were detected in 31/50 (62%) samples [range 0-57.3 pg AFB(1)-lys adducts/mg of blood albumin (pg/mg)]. The mean level of positives was 14.9 pg/mg and males had the two highest levels measured (57.1 and 57.3 pg/mg). There was no correlation with age or profession. This is the first study of Brazilian, or indeed South American, individuals that has determined exposure to AF at the individual level using a biomarker approach. These levels are similar to those observed in the Philippines. These data warrant further investigation of both the sources and consequences of exposure to this potent toxin in Brazil.

FoodCAP: acid-labile protein adducts of heterocyclic amines in human blood are not viable biomarkers of HCA dietary exposure

Intake of heterocyclic amines (HCAs, carcinogens produced during cooking of meat/fish, the most abundant being PhIP, DiMeIQx and MeIQx) is influenced by many factors including type/thickness of meat and cooking method/temperature/duration. Thus, assessment of HCA dietary exposure is difficult. Protein adducts of HCAs have been proposed as potential medium-term biomarkers of exposure, e.g. PhIP adducted to serum albumin or haemoglobin. However, evidence is still lacking that HCA adducts are viable biomarkers in humans consuming normal diets. The FoodCAP project, supported by World Cancer Research Fund, developed a highly sensitive mass spectrometric method for hydrolysis, extraction and detection of acid-labile HCAs in blood and assessed their validity as biomarkers of exposure. Multiple acid/alkaline hydrolysis conditions were assessed, followed by liquid-liquid extraction, clean-up by cation-exchange SPE and quantification by UPLC-ESI-MS/ MS. Blood was analysed from volunteers who completed food diaries to estimate HCA intake based on the US National Cancer Institute’s CHARRED database. Standard HCAs were recovered quantitatively from fortified blood. In addition, PhIP/MeIQx adducts bound to albumin and haemoglobin prepared in vitro using a human liver microsome system were also detectable in blood fortified at low ppt concentrations. However, except for one sample (5pg/ml PhIP), acid-labile PhIP, 7,8-DiMeIQx, 4,8-DiMeIQx and MeIQx were not observed above the 2pg/ml limit of detection in plasma (n=35), or in serum, whole blood or purified albumin, even in volunteers with high meat consumption (nominal HCA intake >2µg/day). It is concluded that HCA blood protein adducts are not viable biomarkers of exposure. Untargeted metabolomic analyses may facilitate discovery of suitable markers.

Albumin causes a synergistic increase in the antioxidant activity of green tea catechins in oil-in-water emulsions

Model oil-in-water emulsions containing epicatechin (EC) and epigallocatechin gallate (EGCG) showed a synergistic increase in stability in emulsions containing added albumin. EGCG showed a stronger synergy (35%) with ovalbumin than did EC. Oxidation of the oil was monitored by determining peroxide values and hexanal contents. The effect of bovine serum albumin (BSA) on model oil-in-water emulsions containing each of the green tea catechins [epicatechin gallate (ECG), EGCG, EC and epigallocatechin (EGC)] was studied during storage at 30 degrees C. The green tea catechins showed moderate antioxidant activity in the emulsions with the order of activity being ECG approximate to EGCG > EC > EGC. Although BSA had very little antioxidant activity in the absence of phenolic antioxidants, the combination of BSA with each of the catechins showed strong antioxidant activity. BSA, in combination with EC, EGCG or EGC, showing the strongest antioxidant activity with good stability after 45 days storage. Model experiments with the catechins stored with BSA in aqueous solutions confirmed that protein-catechin adducts with antioxidant activity were formed between the catechins and protein. The antioxidant activity of the separated protein-catechin adducts increased strongly with storage time and was stronger for EGCG and ECG than for EC or EGC. (c) 2006 Elsevier Ltd. All rights reserved.

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014