Burden of disease associated with dietary exposure to carcinogenic aflatoxins in Portugal using human biomonitoring approach.

Human biomonitoring is an important tool to assess human exposure to chemicals, contributing to describe trends of exposure over time and to identify population groups that could be under risk. Aflatoxins are genotoxic and carcinogenic food contaminants causing hepatocellular carcinoma, the third leading cause of cancer deaths worldwide. In Portugal, scarce data are available regarding exposure to aflatoxins and no previous study used human biomonitoring data to comprehensively characterize the associated burden of disease. 24 h urine and first-morning urine paired samples were collected by 94 participants and were analyzed by liquid chromatography-tandem mass spectrometry for the quantitative determination of aflatoxins (B1, B2, G1, G2 and M1). Deterministic and probabilistic models were developed to assess the Portuguese exposure to aflatoxins and to estimate the health impact of this exposure, estimating the attributed Disability-Adjusted Life Years (DALYs). Aflatoxins were detected in a maximum of 13% (AFB1), 16% (AFB2), 1% (AFG1), 2% (AFG2) and 19% (AFM1) of the urine samples. Data obtained through the probabilistic approach revealed an estimated mean probable daily intake of 13.43 ng/kg body weight per day resulting in 0.13 extra cases of hepatocellular carcinoma, corresponding to mean annual DALYs of 172.8 for the Portuguese population (10291027 inhabitants). The present study generated for the first time and within a human biomonitoring study, reliable and crucial data to characterize the burden associated to the exposure to aflatoxins of the Portuguese population. The obtained results constitute an imperative support to risk managers in the establishment of preventive policy measures that contribute to ensure public health protection.

Exposure assessment of Portuguese population to multiple mycotoxins: The human biomonitoring approach.

Mycotoxins constitute a relevant group of food contaminants with several associated health outcomes such as estrogenic, immunotoxic, nephrotoxic and teratogenic effects. Although scarce data are available in Portugal, human biomonitoring studies have been globally developed to assess the exposure to mycotoxins at individual level. In order to overcome this lack of data, the present study concerned the analysis of mycotoxins in 24h urine and first-morning urine paired samples from 94 participants enrolled within the scope of the National Food, Nutrition, and Physical Activity Survey of the Portuguese General Population (2015-2016). Following a salt-assisted matrix extraction, urine samples were analysed by liquid chromatography-mass spectrometry for the simultaneous determination of 37 urinary mycotoxins' biomarkers and data obtained used to estimate the probable daily intake as well as the risk characterization applying the Hazard Quotient approach. Results revealed the exposure of Portuguese population to zearalenone, deoxynivalenol, ochratoxin A, alternariol, citrinin and fumonisin B1 through the quantification in 24h urine and first-morning urine paired samples. Risk characterization data revealed a potential concern to some reported mycotoxins since the reference intake values were exceeded by some of the considered participants. Alternariol was identified for the first time in urine samples from a European country; however, risk characterization was not performed due to lack of reference intake value. These results confirmed mycotoxins as part of the human exposome of the Portuguese population reinforcing the need for further studies regarding the determinants of exposure.

Analysis of uni and bi-parental markers in mixture samples: Lessons from the 22nd GHEP-ISFG Intercomparison Exercise.

Since 1992, the Spanish and Portuguese-Speaking Working Group of the ISFG (GHEP-ISFG) has been organizing annual Intercomparison Exercises (IEs) coordinated by the Quality Service at the National Institute of Toxicology and Forensic Sciences (INTCF) from Madrid, aiming to provide proficiency tests for forensic DNA laboratories. Each annual exercise comprises a Basic (recently accredited under ISO/IEC 17043: 2010) and an Advanced Level, both including a kinship and a forensic module. Here, we show the results for both autosomal and sex-chromosomal STRs, and for mitochondrial DNA (mtDNA) in two samples included in the forensic modules, namely a mixture 2:1 (v/v) saliva/blood (M4) and a mixture 4:1 (v/v) saliva/semen (M8) out of the five items provided in the 2014 GHEP-ISFG IE. Discrepancies, other than typos or nomenclature errors (over the total allele calls), represented 6.5% (M4) and 4.7% (M8) for autosomal STRs, 15.4% (M4) and 7.8% (M8) for X-STRs, and 1.2% (M4) and 0.0% (M8) for Y-STRs. Drop-out and drop-in alleles were the main cause of errors, with laboratories using different criteria regarding inclusion of minor peaks and stutter bands. Commonly used commercial kits yielded different results for a micro-variant detected at locus D12S391. In addition, the analysis of electropherograms revealed that the proportions of the contributors detected in the mixtures varied among the participants. In regards to mtDNA analysis, besides important discrepancies in reporting heteroplasmies, there was no agreement for the results of sample M4. Thus, while some laboratories documented a single control region haplotype, a few reported unexpected profiles (suggesting contamination problems). For M8, most laboratories detected only the haplotype corresponding to the saliva. Although the GHEP-ISFG has already a large experience in IEs, the present multi-centric study revealed challenges that still exist related to DNA mixtures interpretation. Overall, the results emphasize the need for further research and training actions in order to improve the analysis of mixtures among the forensic practitioners.

The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

mtDNA single macrodeletions associated with myopathies: absence of haplogroup-related increased risk.

As for any non-recombining genome, any mutation at mtDNA, if not recurrent, appears on a particular haplotype background, allowing its detection by haplogroup association studies. It has been shown that the propensity for occurrence of single macrodeletions at a level beyond the pathological threshold is associated with super-haplogroup U/K. However, in this report, we present evidence for the absence of preferential haplogroup backgrounds for single macrodeletions. We have analysed how haplogroup diagnostic polymorphisms could disrupt direct repeats usually flanking the deleted segment, and we have concluded that for the Common Deletion, no such polymorphisms are observed in humans, but they do occur in other primates. Furthermore, we also report five new single macrodeletions.