Regular ArticlesPiperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response
Introduction
Mycotoxins are secondary metabolites produced by some filamentous fungi. Among the 300 reported mycotoxins, AFB1 is the most dangerous one. In fact, AFB1 is a potent carcinogenic agent in humans inducing liver cancer (IARC, 1993, Wu et al., 2014). This mycotoxin is synthesized by at least 15 species belonging to the Flavi section of the Aspergillus genus, A. flavus considered as the most frequent source of aflatoxins in crops (Varga et al., 2015). To date, it is estimated that at least 500 million of people living in sub-Saharan Africa, Latin America and Asia, are exposed to increasing levels of mycotoxins, including aflatoxins, through contaminated commodities (Pitt et al., 2012). In 2010, analysis of several foodborne chemicals by the Chemical and Toxins Disease Task Force reported that aflatoxin was associated with the highest number of DALYs (deaths and disability adjusted life years) (636, 869) where one DALY represents the sum of years lived with disability and years of life lost (Gibb et al., 2015). Worryingly, surveys demonstrated that higher levels of aflatoxins in agricultural commodities were linked to upraising climatic conditions (Streit et al., 2013). Indeed, AFB1’s contamination was recently reported on crops from other temperate regions of Europe, demonstrating an increased distribution of this mycotoxin potentially related to global climate changes (Battilani et al., 2016, Perrone et al., 2014). Even if several approaches have been developed to limit AFB1’s contamination (Wild et al., 2015, Jalili, 2015), they remain insufficient to completely avoid mycotoxin contamination thus emphasizing the need to develop new strategies to guarantee food safety. The use of natural products such as spices, plant extracts or essential oils with anti-aflatoxinogenic properties represent a complementary or alternative strategy against mycotoxin contamination (Annis et al., 2000, El Khoury et al., 2017, Holmes et al., 2008, Sakuda et al., 2016). For instance, piperine, a major active component of black and long peppers (Piper nigrum L. and Piper longum L.) has been previously demonstrated as an effective inhibitor of aflatoxin production (Lee et al., 2002, Madhyastha and Bhat, 1984). However, the molecular mechanism of action by which this molecule inhibits AFB1 production has not been studied. In the last years, aflatoxin biosynthesis has been closely linked to fungal oxidative stress status (Montibus et al., 2013, Roze et al., 2013, Amare and Keller, 2014). Indeed, studies in Aspergilli demonstrated several molecules with antioxidant capacities are able to inhibit aflatoxin production (Jahanshiri et al., 2012, Liang et al., 2015). Furthermore, it has been demonstrated that a regulatory network of genes coding for stress response transcription factors (AtfB, SrrA, Ap-1 and MsnA) is directly involved in aflatoxin biosynthesis (Hong et al., 2013). Within this context, the present study aimed to elucidate the anti-aflatoxinogenic mechanism of action of piperine by studying its impact on the expression of 27 genes involved in AFB1 gene cluster and 20 genes coding for oxidative stress response in A. flavus, by using a large-scale q-PCR approach (Caceres et al., 2016).
We demonstrated that piperine resulted in a down-regulation of almost all genes of the AFB1 cluster leading to the subsequent inhibition of the mycotoxin biosynthesis. We also showed that exposure to piperine induced an inhibition by 50% of veA expression levels together with an over-expression of several bZIP transcription factors (atfA, atfB and ap-1) and genes coding for antioxidant defense belonging to catalase and superoxide dismutase families (sod1, catA and cat2). Simultaneously, a significant increase in catalase enzymatic activity was also observed.
Section snippets
Chemicals and reagents
Lyophilized piperine standard (purity ≥97.0%) was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Stock and work solutions were diluted in acetonitrile and stocked at 4 °C until use. All solvents were analytical grade and purchased from Thermo Fisher Scientific (Illkirch, France).
Fungal strain and culture conditions
A. flavus NRRL 62477 strain, uniquely producer of AFB1, was used in this study (El Mahgubi et al., 2013). All cultures were performed on Malt Extract Agar (MEA) medium (Biokar Diagnostics, Allone,
Statistics
All experiments were conducted in triplicate. Gene-expression and enzymatic tests were based on six biological replicates while piperine dose-dependent effect was based on three replicates. Data analyses were done using GraphPad Prism v4 software. A Mann-Whitney test was used to determine the differences between control and treated groups and differences were considered to be statistically significant when the p-value was lower than 0.05. All results are expressed as mean ± standard error of
Effect of piperine on AFB1 production and fungal growth in A. Flavus
Five different concentrations of piperine were tested for their effect on AFB1 production and fungal mycelia growth. Results demonstrated that piperine inhibits AFB1 production in a dose-dependent manner: 0.0006 mM of piperine inhibited toxin production by 30% while no detectable levels of toxin were observed using 0.17 mM (Fig. 1). Fungal mycelial growth was moderately impacted by piperine. Indeed, growth inhibition was of 12% using 0.04 mM of piperine and an inhibition of 35% was observed with
Discussion
Oxidative stress is an unbalanced status occurring when reactive oxygen species overcome the scavenging capacities of antioxidant mechanisms. This causes alteration of cellular functions by inducing damages to DNA, proteins or lipids (Montibus et al., 2013). Studies performed in Aspergilli, have demonstrated that several aflatoxin inhibitors can modulate the antioxidant system in fungi (Grintzalis et al., 2014, Reverberi et al., 2005, Sun et al., 2015). Thus, in the present study, we
Conclusion
Taken together, our results demonstrate that (i) piperine inhibits almost all genes of the aflatoxin biosynthetic pathway leading to an inhibition of the mycotoxin biosynthesis, (ii) piperine’s mechanism of action involves the inhibition of the global regulator veA together with enhanced levels of the stress response system including the over expression of bZIP transcription factors Ap-1, AtfA, AtfB and genes coding for antioxidant defense such as catalases and superoxide dismutases and (iii)
Acknowledgments
This work was financed by the projects Aflafree (ANR-11-ALID-0003), Aflared (joint project, 001-2012 STDF-AIRD) and ToxinFree (Campus France PHC Cèdre 32763). The authors would also like to thank to the Consejo Nacional de Ciencia y Tecnología (CONACYT) México, funding Isaura Caceres. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Author contributions
I.P.O., O.P. and J.D.B. conceived, supervised and designed the experiments. I.C. performed the experiments and wrote the paper. R.E.K. contributed to q-PCR molecular tool. S.B. contributed to the morphological and enzymatic tests.
Conflict of interest
The authors declare no conflict of interest.
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