Dataset Information

Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis.

ABSTRACT: The diffusion of the legalization of cannabis for recreational, medicinal and nutraceutical uses requires the development of adequate analytical methods to assure the safety and security of such products. In particular, aflatoxins are considered to pose a major risk for the health of cannabis consumers. Among analytical methods that allows for adequate monitoring of food safety, immunoassays play a major role thanks to their cost-effectiveness, high-throughput capacity, simplicity and limited requirement for equipment and skilled operators. Therefore, a rapid and sensitive enzyme immunoassay has been adapted to measure the most hazardous aflatoxin B1 in cannabis products. The assay was acceptably accurate (recovery rate: 78-136%), reproducible (intra- and inter-assay means coefficients of variation 11.8% and 13.8%, respectively), and sensitive (limit of detection and range of quantification: 0.35 ng mL^-1 and 0.4-2 ng mL^-1, respectively corresponding to 7 ng g^-1 and 8-40 ng g^-1 ng g^-1 in the plant) and provided results which agreed with a HPLC-MS/MS method for the direct analysis of aflatoxin B1 in cannabis inflorescence and leaves. In addition, the carcinogenic aflatoxin B1 was detected in 50% of the cannabis products analyzed (14 samples collected from small retails) at levels exceeding those admitted by the European Union in commodities intended for direct human consumption, thus envisaging the need for effective surveillance of aflatoxin contamination in legal cannabis.

SUBMITTER: Nardo FD

PROVIDER: S-EPMC7232199 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis.

Nardo Fabio Di FD Cavalera Simone S Baggiani Claudio C Chiarello Matteo M Pazzi Marco M Anfossi Laura L

Toxins 20200420 4

The diffusion of the legalization of cannabis for recreational, medicinal and nutraceutical uses requires the development of adequate analytical methods to assure the safety and security of such products. In particular, aflatoxins are considered to pose a major risk for the health of cannabis consumers. Among analytical methods that allows for adequate monitoring of food safety, immunoassays play a major role thanks to their cost-effectiveness, high-throughput capacity, simplicity and limited re ...[more]

PMID: 32326118

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Project description:Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by Aspergillus flavus and A. parasiticus, and prevalent in food and feed. Microbial degradation is a promising strategy which can be performed in mild and environmental friendly condition. This work is a step towards identifying the enzyme responsible for biodegradation of AFB1 by P. putida. Experiments were performed with P. putida lysate and compared with commercial lipase to see the degradation efficiency and the temperature stability. The cell free lysate of P. putida efficiently degraded AFB1 in a range of temperature from 20 to 90 °C. The lysate is thermostable and could retain its activity on pre-incubation up to 90 °C. Highest rate of degradation was observed at 70 °C. These observations show that the P. putida lysate is not only stable at higher temperatures but its enzymatic activity increases after incubation. Similarly, the commercial lipase degraded AFB1 efficiently. However, both, the P. putida lysate and lipase ceased degradation in presence of a lipase inhibitor, HgCl2. The Hill function accurately predicted enzyme activity at various times and temperatures. Like lipase, the lysate also hydrolyses the p-nitrophenyl palmitate to p-nitrophenol. Kinetic parameters such as Vmax, Km and n values are good measures to characterize the lysate response with respect to changing paranitro phenyl palmitate levels. The substrate specificity test of lipase showed linear correlation between the absorbance at 410 nm vs amount of product paranitro phenol. The value of Km, Vmax and n are 0.62 mM, 355.7 μmol min-1 and 1.29, respectively. The lipase gene presence in P. putida was confirmed using PCR technique. These observations indicate that the main enzyme responsible for AFB1 degradation by P. putida is lipase. Thus, lipase as a multifunctional biocatalyst provides a promising future for a variety of industries and may also help to ensure the food safety by degrading the mycotoxins.

Project description:Cytochrome P450 enzymes are often responsible for the toxic and carcinogenic effects of toxicants, such as aflatoxin B1 (AFB1). The human hepatic CYP2A6 enzyme mediates the oxidative metabolism of several procarcinogens. In this study, we characterized a partial sequence of CYP2A6 gene from Arbor Acres (AA) broiler and studied its role in AFB1 bioactivation. Moreover, the effect of curcumin on CYP2A6 is illustrated. Six groups of AA broiler were treated for 28 days including the control group (fed only basal diet), curcumin alone-treated group (450 mg/kg feed), the group fed AFB1-contaminated feed (5 mg/kg feed) plus the low (150 mg), medium (300 mg) or high (450 mg) of curcumin, and the group fed AFB1-contaminated diet alone (5 mg/kg feed). After the end of treatment period, liver samples were collected for different analyses. The results revealed that the histopathological examination showed clear signs of liver toxicity in AA broliers in AFB1-fed group, but curcumin-supplementation in feed prevented partially AFB1-induced liver toxicity. Liver and body weights were recorded to study the AFB1 harmful effects. We noted an obvious increase in liver weight and decrease in body weight in AFB1-fed group. But, the administration of curcumin partially ameliorated the increase in liver weight and decrease in body weight in a dose-dependent manner. The results (RT-PCR and Elisa) revealed that mRNA and protein expression level enhanced in AFB1-fed group. Consistently, CYP2A6 enzyme activity also increased in AFB1-fed group, suggesting that AA broiler CYP2A6 actively involved in bioactivation of AFB1. However, curcumin treatment inhibited CYP2A6 at mRNA and protein levels in AFB1 treated AA broiler in a dose-dependent manner. Maximum inhibition of liver CYP2A6 enzyme activity in AA broiler has been achieved at a dose of 450 mg/kg curcumin. This is the first study identifying and confirming the role of CYP2A6 enzyme in AFB1 bioactivation in AA broiler liver (in vivo), and the hepatoprotective role of curcumin via inhibiting CYP2A6 expression and enzyme activity. The study contributed to identify an important CYP enzyme involved in AFB1 bioactivation in broilers and thus could pave the way for the prevention of the harmful effects of AFB1 in broilers.

Dataset Information

Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis.

Publications

Enzyme Immunoassay for Measuring Aflatoxin B1 in Legal Cannabis.

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