Carcinogenic Food Contaminant Research Articles

Electrochemical/photoelectrochemical dual-mode aptasensor for sensitive aflatoxin B1 assay based on distance-modulation strategy using Au NPs/PCZIF-8-ZnO as sensing substrate

Aflatoxin B1 (AFB1), a hepatotoxic and carcinogenic food contaminant, is commonly found in agricultural food. Herein, Au NPs anchored ZIF-8-derived porous carbon-ZnO (Au NPs/PCZIF-8-ZnO) was firstly synthesized to act as the sensing substrate. Then, a ratiometric electrochemical (EC) and “off–on” photoelectrochemical (PEC) dual-mode paper-based aptasensor was presented for AFB1 detection based on a distance-modulation sensing strategy. The independent signal transduction mechanisms and output mode not only broaden the dynamic detection range but also provide a self-verification to assay results, improving the sensitivity and reliability. The wide detection ranges of 0.1 pg/mL-100 ng/mL (EC mode) and 0.02 pg/mL-100 ng/mL (PEC mode) were obtained using dual-mode aptasensor, with detection limits of 36.7 and 9.3 fg/mL, respectively. The fabricated aptasensor exhibited excellent selectivity, reproducibility and stability. Furthermore, it exhibited good practicability for AFB1 assays in real samples, demonstrating great potential applications for food safety evaluation.

Rationally designed peptide conjugated to gold nanoparticles for detection of aflatoxin B1 in point-of-care dot-blot assay

Aflatoxin B1 (AFB1) is a hepatotoxic and carcinogenic food contaminant. Although on-site paper-based detection is sensitive it depends on expensive antibodies which are difficult to raise against mycotoxins. Here, we rationally designed a high binding octapeptide, N-KSGKSKPR-C peptide for AFB1 detection, by molecular docking, as confirmed by indirect ELISA (Kd 323 nM). Further, conjugation of octapeptide with gold nanoparticles (26 nm) permitted its use as a visual detection agent in rapid, sensitive dot-blot assay (LOD 0.39 μg/kg). The assay displayed negligible cross-reactivity with co-contaminating mycotoxins. AFB1 recovery from spiked wheat sample was comparable by dot-blot (78–91 %) and HPLC (65–87 %). Evaluation of dot-blot using certified reference material and 146 food and feed samples showed high correlation R2 = 0.87 with HPLC. The assay displayed high accuracy (91 %), sensitivity (71 %) and specificity (96.5 %). Therefore, the developed dot-blot assay holds promise for monitoring AFB1 contamination in food and feed.

Experimental–theoretical study of laccase as a detoxifier of aflatoxins

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π–π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Burden of disease of dietary exposure to aflatoxins in European countries

Abstract Aflatoxins (AFTs) are genotoxic and carcinogenic food contaminants causing hepatocellular carcinoma, the third leading cause of cancer deaths worldwide. In 2015, WHO estimated the burden of disease associated with exposure to AFTs and concluded that this exposure corresponds to 636,869 Disability-adjusted Life Years (DALYs) at global level. Particularly for Portugal and recently, there were estimated 0.08-0.30 DALYs/100,000 considering consumption and occurrence in food data, and 1.7 DALYs/100,000 based on human biomonitoring data. The present study considered the exposure data presented in the EFSA scientific opinion “Risk assessment of aflatoxins in food” where data from occurrence of AFTs in food and from food consumption were gathered to estimate the exposure to AFTs in 19 European countries. A deterministic and a bottom-up approach was developed to estimate the health impact of the exposure to AFTs for adult population through the calculation of DALYs associated to the number of estimated extra-cases of hepatocellular carcinoma (HCC). Results showed similar values for all the 19 European countries considered. For adult population and considering the data provided by EFSA using the upper-bound approach (worst-case scenario), the estimated number of DALYs ranged from 0.32 (Spain) to 1.10 (United Kingdom) DALYs/100,000. A total of 11.76 DALYs/100,000 was globally estimated for the considered European countries. The highest burden was estimated for the United Kingdom, that together with The Netherlands, Ireland, Czech Republic, and Austria were the highest contributors to the global estimated European burden associated to the exposure to AFTs. The present study generated reliable and crucial data to characterize the burden associated to exposure to AFTs in the European population. The obtained results constitute an important contribution to define priorities and support the need for further policy actions to protect European citizen's health.

Chemoprotective Effects of Xanthohumol against the Carcinogenic Mycotoxin Aflatoxin B1.

The present study addresses the chemoprotective effects of xanthohumol (XN), a prenylated flavonoid found in the female inflorescences (hops) of the plant Humulus lupulus L., against the carcinogenic food contaminant aflatoxin B1 (AFB1). The chemical reactions of XN and its derivatives (isoxanthohumol (IXN), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN)) with the AFB1 metabolite, aflatoxin B1 exo-8,9-epoxide (AFBO), were investigated in silico, by calculating activation free energies (ΔG‡) at the Hartree–Fock level of theory in combination with the 6-311++G(d,p) basis set and two implicit solvation models. The chemoprotective effects of XN were investigated in vitro in the metabolically competent HepG2 cell line, analyzing its influence on AFB1-induced cytotoxicity using the MTS assay, genotoxicity using the comet and γH2AX assays, and cell cycle modulation using flow cytometry. Our results show that the ΔG‡ required for the reactions of XN and its derivatives with AFBO are comparable to the ΔG‡ required for the reaction of AFBO with guanine, indicating that XN, IXN, 8-PN, and 6-PN could act as scavengers of AFBO, preventing DNA adduct formation and DNA damage induction. This was also reflected in the results from the in vitro experiments, where a reduction in AFB1-induced cytotoxicity and DNA single-strand and double-strand breaks was observed in cells exposed to combinations of AFB1 and XN, highlighting the chemoprotective effects of this phytochemical.

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.

Gut Microbiota Profiling of Aflatoxin B1-Induced Rats Treated with Lactobacillus casei Shirota.

Aflatoxin B1 (AFB1) is a ubiquitous carcinogenic food contaminant. Gut microbiota is of vital importance for the host’s health, regrettably, limited studies have reported the effects of xenobiotic toxins towards gut microbiota. Thus, the present study aims to investigate the interactions between AFB1 and the gut microbiota. Besides, an AFB1-binding microorganism, Lactobacillus casei Shirota (Lcs) was tested on its ability to ameliorate the changes on gut microbiota induced by AFB1. The fecal contents of three groups of rats included an untreated control group, an AFB1 group, as well as an Lcs + AFB1 group, were analyzed. Using the MiSeq platform, the PCR products of 16S rDNA gene extracted from the feces were subjected to next-generation sequencing. The alpha diversity index (Shannon) showed that the richness of communities increased significantly in the Lcs + AFB1 group compared to the control and AFB1 groups. Meanwhile, beta diversity indices demonstrated that AFB1 group significantly deviated from the control and Lcs + AFB1 groups. AFB1-exposed rats were especially high in Alloprevotella spp. abundance. Such alteration in the bacterial composition might give an insight on the interactions of AFB1 towards gut microbiota and how Lcs plays its role in detoxification of AFB1.

Biological Activity of the Carrier as a Factor in Immunogen Design for Haptens.

Immunoanalytical methods are frequently employed in the detection of hazardous small molecular weight compounds. However, antibody development for these molecules is a challenge, because they are haptens and cannot induce a humoral immune response in experimental animals. Immunogenic forms of haptens are usually prepared by conjugating them to a protein carrier which serves as an immune stimulator. However, the carrier is usually considered merely as a bulk mass, and its biological activity is ignored. Here, we induced an endocytic receptor, transferrin receptor, by selecting its ligand as a carrier protein to enhance antibody production. We conjugated aflatoxin, a potent carcinogenic food contaminant, to transferrin and evaluated its potential to stimulate antibody production with respect to ovalbumin conjugates. Transferrin conjugates induced aflatoxin-specific immune responses in the second immunization, while ovalbumin conjugates reached similar antibody titers after 5 injections. Monoclonal antibodies were successfully developed with mice immunized with either of the conjugates.

Identification and evaluation of potentially mutagenic and carcinogenic food contaminants.

Heat processing of food gives rise to a plethora of chemical compounds whose toxicological effects are largely unknown. Due to a general lack of experimental toxicological data, assessing the risks associated with the consumption of these substances remains a challenge. Computer models that allow for an in silico prediction of physicochemical and toxicological characteristics, may be able to fill current data gaps and facilitate the risk assessment of toxicologically uncharacterised chemicals, their transformation products and their biological metabolites. The overall aims of the present project were for the fellow: (i) to get acquainted with the application of computational toxicological analyses tools in risk assessment based on results and experiences from previous research performed at the German Federal Institute for Risk Assessment (BfR); and (ii) to apply the newly gained skills on historic and novel data using updated and additional in silico tools. The project contributed to the continuous further education of the fellow in the use of computational toxicology tools, corroborated findings related to the safety of heat‐induced contaminants and laid the foundations for future collaborations between the fellow's home institution, the Institute of Marine Research (IMR) in Norway, and the BfR in Germany.

Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans.

Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine.

There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1-M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4-M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4-M7) that could make humans more susceptible to lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.

3-MONOKLORO-1,2-PROPANDIOL PADA KEMASAN KERTAS DUPLEKS SERTA MIGRASINYA KE DALAM SIMULAN PANGAN

3-Monochloro-1,2-propandiol (3-MCPD) is a carcinogenic food contaminant. 3-MCPD is formed during food processing, and can also be derived from food contact packaging materials, including paper. Wet-strength resin is often added into paper food packaging to provide moisture resistance and thus enhancing food shelf-life and consumer usage. The wet-strength resins which are manufactured from epichlorohydrin-based starting materials, are known to initiate the formation of 3-MCPD. Thus, the objectives of this study were: 1) to validate an analytical method for the analysis 3-MCPD in duplex paper packaging, 2) to analyze3-MCPD content in duplex paper, and 3) to analyze 3-MCPD migration from duplex paper packaging into food simulants. 3-MCPD content in duplex paper was analyzed by a validated GC-MS method with linearity value (R2) of 0.993, limit of detection (LOD) of 6.65 ppb, limit of quantification (LOQ) of 22.15 ppb, and recovery range of 83.00–114.13%. The 3-MCPD content of five sample duplex papers obtained from different packaging paper manufacturers were ranging from 753.43 to 825.36 ppb, and there was no significant differences between the samples. Direct contact between food simulants and duplex paper for 24 hours at 40°C generated migration of 3-MCPD at levels of 40.55 to 57.61%.

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin-mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure-function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.

Risk Assessment Approaches for Carcinogenic Food Contaminants

Health Canada has identified the need for a standardized department-wide approach for the risk assessment of carcinogens in foods (e.g., pesticides, food chemical contaminants, veterinary therapeutics). A standardized approach would better facilitate and inform risk management strategies for the control of human exposure to food sources of carcinogens. Within the post- market regulatory context, directly DNA-reactive carcinogens are of most concern because any exposure is theoretically assumed to be associated with a risk of producing a carcinogenic effect in proportion to the dose. Such non-threshold carcinogens, as well as carcinogens in which a non-linear dose response has not been demonstrated, require different approaches for risk characterization. In order to contribute to Health Canada's department-wide discussions regarding the development of risk management strategies for carcinogens, a general overview was conducted on international approaches for post-market risk assessments of carcinogenic contaminants in food. In this review, areas in the risk assessment paradigm which are identified for development of further standardized guidance include the weight-of-evidence determination for whether a compound should be considered a non-threshold carcinogen, the technical criteria for choosing the appropriate dose-response assessment approach, and a consistent approach for interpreting and prioritizing risk.

Carcinogens in Food: Opportunities and Challenges for Regulatory Toxicology

The risk of developing cancer from carcinogens occurring in food is of widespread interest to scientific researchers, food policymakers and food surveillance institutions, as well as to the general public. When evaluating the risk of carcinogenic food contaminants or carcinogenic foodstuff per se (e.g., alcoholic beverages), the level of scientific evidence should be reflected more clearly. In the past, interest often focused on ‘fashionable’ agents with only moderate levels of evidence of their carcinogenicity (e.g., acrylamide or furan); whereas agents with the highest level of evidence (e.g., substances classified by the International Agency for Research on Cancer (IARC) as group 1, being ‘carcinogenic to humans’) were sometimes disregarded. For example, important carcinogens such as arsenic (a contaminant in drinking water), cadmium and other heavy metals, but also benzene, were not even mentioned in a recent review article about carcinogenic food contaminants. Research, control and prevention strategies for carcinogenic agents in food should comprise a risk-oriented approach and should not lose sight of agents that pose an immediate and scientifically quantifiable threat. Suitable strategies include the use of quantitative risk assessments, for example the use of the Margin of Exposure (MOE)

Diverse inhibitors of aflatoxin biosynthesis

Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

A Critical Analysis of the Testing of Potentially Carcinogenic Food Contaminants

A Critical Analysis of the Testing of Potentially Carcinogenic Food Contaminants

Volatile N-nitrosamines in selected Italian cheeses.

Volatile N-nitrosamines are a small part of the larger class of nitroso compounds, which are widespread and often carcinogenic food contaminants. Since Freund’s discovery (1937) of human liver damage due to exposure to N-nitrosodimethylamine and Magee and Barnes’ demonstration of the toxic and carcinogenic action of this nitrosamine in animals (1956), many studies have set up to measure environmental contamination with these molecules. Beer and meats were found to be an important exogenous source, but other foods such as fish, vegetables and dairy products also made significant contribution to the total dietary intake of N-nitrosamines (MAFF, 1987 and 1992; Gangolli, 1994). Only few research projects have dealt with the measurement of nitrosamines in cheese (Goodhead et al., 1976; Gough et al., 1977; Sen et al., 1978 ; Elgersma et al., 1978; Stephany et al., 1978; Pedersen et al., 1980, Mavelle et al., 1991). The Italian food market has rarely been studied and no recent data are available about N-nitrosamines in cheeses in Italy (Cantafora et al, 1975; Cerutti et al., 1975).

High-performance liquid chromatographic analysis of the mycotoxin penicillic acid and its application to biological fluids

Penicillic acid (PA) is a carcinogenic food contaminant produced by several food-borne fungi. PA was resolved as a sharp peak by reversed-phase-high-performance liquid chromatography on a small-particle (10 μm) column in 3–4 min by an elution system composed of acetonitrile (AN), water and glacial acetic acid (AC) with detection by ultraviolet (UV) absorbance at 254 nm. Peak height and peak area were related linearly to the amount of PA injected over a range of 5–500 ng. Reproducibility of retention time, peak height and peak area was demonstrated. The lower detection limit was 5 ng in two elution systems [AN-H 2-O-AC (45:55:2), flow-rate ( F) 1 ml/min; AN-H 2O-AC (40:60:2), F = 1.2 ml/min] and 10 ng in a third system [AN-H 2O-AC (25:75:2), F = 1.6 ml/min]. Based on the sensitivity and separation of PA from interfering peaks, the following systems were optimal: plasma and bile, AN-H 2O-AC (40:60:2), F = 1.2 ml/min; urine, AN-H 2O-AC (25:75:2), F = 1.6 ml/min. Good recovery (89–98 %) over a range of 1–50 μg/ml of PA was obtained from PA-spiked plasma samples treated first with 25 % HPO 3 followed by extraction with chloroform. A single peak detected either by UV absorbance or by radioactivity was obtained when plasma samples spiked with [ 14C]PA were extracted. Good recovery (92–105 %) of PA also was obtained from spiked urine and bile samples.

Dose-response studies and 'no-effect-levels' of N-nitroso compounds: some general aspects.

One major problem in the evaluation of potential carcinogenic food additives and contaminants is that of thresholds or, better, of 'no-adverse-effect-levels'. Arguments in favor of the postulated 'irreversibility' of carcinogenic effects are based on dose-response studies, single dose and multigeneration experiments as well as on the concept of somatic mutation as the first step in carcinogenesis with subsequent transmittance of induced defects during cell replication. The problem of extrapolation of results of animal experiments using high doses to low exposure and low incidences in man is not yet solved satisfactorily. Possible practical consequences include zero tolerance, acceptable thresholds at low risk and safety factors. Acceptable intakes should never be considered constants but should be changeable as soon as new facts in regard to the safety evaluation are available.