Effect of UV irradiation on aflatoxin reduction: a cytotoxicity evaluation study using human hepatoma cell line

In this proof-of-concept study, the efficacy of a medium-pressure UV (MPUV) lamp source to reduce the concentrations of aflatoxin B₁, aflatoxin B₂, and aflatoxin G₁ (AFB_1, AFB₂, and AFG₁) in pure water is investigated. Irradiation experiments were conducted using a collimated beam system operating between 200 to 360 nm. The optical absorbance of the solution and the irradiance of the lamp are considered in calculating the average fluence rate. Based on these factors, the UV dose was quantified as a product of average fluence rate and treatment time. Known concentrations of aflatoxins were spiked in water and irradiated at UV doses ranging from 0, 1.22, 2.44, 3.66, and 4.88 J cm^?2. The concentration of aflatoxins was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB₁, AFB₂, and AFG₁. It was observed that UV irradiation significantly reduced aflatoxins in pure water (p < 0.05). Irradiation doses of 4.88 J cm^?2 reduced concentrations 67.22% for AFG₁, 29.77% for AFB₂, and 98.25% for AFB₁ (p < 0.05). Using this technique, an overall reduction of total aflatoxin content of ≈95% (p < 0.05) was achieved. We hypothesize that the formation of ˙OH radicals initiated by UV light may have caused photolysis of AFB₁, AFB₂, and AFG₁ molecules. In cell culture studies, our results demonstrated that the increase of UV dosage decreased the aflatoxin-induced cytotoxicity in HepG₂ cells. Therefore, our research finding suggests that UV irradiation can be used as an effective technique for the reduction of aflatoxins.     

Altered bacterial culture density following exposure to aflatoxins

Summary Eight species of bacteria were incubated in culture media containing 10 g/ml aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), or aflatoxin G₂ (AFG₂). Their culture density at 20°C was determined at four and eight days (d) after inoculation. In all species of bacteria studied (Bacillus cereus, Proteus mirabilis, Erysipylothrix rusiopathie (insidiosa), Streptococcus fecalis, Staphylococcus epidermis, Klebsiella pneumoniae, Micrococcus spp., andEscherichia coli), AFB₁, AFB₂ and AFG₂ substantially decreased culture sizes at 4 d, but not at 8 d. InB. cereus andP. mirabilis, culture sizes were increased by AFB₁, AFB₂, and AFG₂ at 8 d post inoculation. These results indicate that AFB₁, AFB₂, and AFG₂ suppressed initial growth of these species in vitro, while later growth in some species was either unaltered or enhanced.     

Natural occurrence of aflatoxins and ochratoxin a in corn and barley from mazandaran and golestan in north provinces of I. R. Iran

Fourteen barley and nine corn samples, destined for animal feed, collected from Golestan and Mazandaran provinces in the north of Islamic Republic of Iran (I. R. Iran) were analysed for aflatoxins (AF) and ochratoxin A (OA) by high performance liquid chromatography. In corn samples, aflatoxin B₁ (AFB₁) and aflatoxin B₂ (AFB₂) were detected in 8 (88.8%) and 6 (66.6%) samples at a mean level of 15.83 and 2.99 ppb (median 1.72 and 1 ppb), respectively. None of the corn samples contained detectable amounts of aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂). Only one of the AF-contaminated samples was co-contaminated with OA at a concentration of 0.35 ppb. This is the first report concerning natural occurrence of OA and co-occurrence with AF in corn samples of north of I. R. Iran.     

Transformation of sterigmatocystin and O-methylsterigmatocystin by aflatoxigenic and nonaflatoxigenic field isolates of the Aspergillus flavus group

Transformation of sterigmatocystin and O-methylsterigmatocystin (two metabolic aflatoxin precursors) to aflatoxins by aflatoxigenic and nonaflatoxigenic field isolates of Aspergillus flavus was studied. The 24 nonaflatoxigenic isolates investigated failed to transform both precursors. Among the 8 aflatoxin-producing isolates used, 7 transformed both precursors whereas the remaining failed to transform both. According to these results, the usefulness of the measurement of enzymatic activities related to aflatoxin production in understanding the true status of conflictive field isolates is discussed.Abbreviations ST sterigmatocystin - OMST O-methylsterigmatocystin - AFB₁ aflatoxin B₁ - AFB₂ aflatoxin B₂ - AFG₁ aflatoxin G₁ - AFG₂ aflatoxin G₂ - GM growth medium of Adye and Mateles - RM replacement medium of Adye and Mateles     

Effects of aflatoxin B, aflatoxin B, aflatoxin G and sterigmatocystin on viability, rates of development, and body length in two strains of Drosophila melanogaster (Diptera)

Two wild-type laboratory populations of Drosophila melanogaster, Florida-9 (sensitive to aflatoxin (AF) B₁-induced toxicity) and Lausanne-S (resistant to AFB₁-induced toxicity) were tested to determine relative degress of sensitivity to growth from the egg stage on media containing 0.2, 0.6, 2.0, and 4.0 ppm AFB₁, AFG₁, AFB₂, or sterigmatocystin (ST). Data indicate that strain Florida-9 is quite sensitive to AFG₁ toxicity at both the egg-pupa and egg-adult stages of development while Lausanne-S is quite resistant to such toxic effects. For Lausanne-S, AFB₁ > AFG₁ in relative toxicity, while for Florida-9, AFG₁ > AFB₁. The latter is noteworthy since vertebrate studies consistently show that AFB₁ is a significantly stronger carcinogen and mutagen than AFG₁. Possible explanations are discussed. Neither strain tested displayed toxic responses to the presence of AFB₂ or ST in the culture media; however, the 4.0-ppm Lausanne-S treatment displayed a significantly lower adult mortality rate than the control, indicating that Lausanne-S flies may benefit from the presence of ST in the culture medium.     

Aflatoxin B<Subscript>1</Subscript> levels in groundnut products from local markets in Zambia

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B₁ (AFB₁) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB₁ contamination levels in both types of groundnut products with maximum AFB₁ levels of 11,100 μg/kg (groundnut kernels) and 3000 μg/kg (milled groundnut powder). However, paired t test analysis showed that AFB₁ contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB₁ levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB₁ were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.     

Potential pathogenicity of Cladosporium tennuisimum,Phaeoisaria clematidis and Ramichloridium subulatum in a mouse model

In Sri Lanka, rice is the main staple which is mostly processed into parboiled rice. The levels of aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) in parboiled and raw milled rice collected from major rice producing areas and rice consuming townships were estimated. In almost all the samples of parboiled rice examined, the AFB₁ and AFG₁ contents were significantly higher than in raw milled rice. The highest AFB₁ content was 185 µg/kg and AFG₁ content 963 g/kg. These samples were collected from a major rice producing/milling district where the mean relative humidity is 78% and mean annual temperature 27 °C which is the highest amongst the rice growing areas in Sri Lanka. Raw rice was either free of aflatoxins or when toxins were detected, they occurred in less than 10% of the samples. The frequency of occurrence of surface fungal flora (Aspergillus/Penicillium) and aflatoxin content in market samples was closely related. Brownish or greenish moldy rice samples with fermented odour contained over 1000 g/kg of AFB₁.     

Bioconversion of Aflatoxin G1 to Aflatoxin B3 by selected fungi

Aflatoxin G₁ (AFG₁) was transformed into aflatoxin B₃ (AFB₃) by the fungiRhodotorula sp,Sporobolomyces sp,Rhizopus oryzae NRRL395,Pythium ultimum, Aspergillus terreus, A clavatus and Penicillium frequentans grown in a medium containing AFG₁, Difco potato dextrose broth, yeast extract, and peptone both in liquid shaken cultures and in solid static cultures at 25°C in the dark. A maximum rate of transformation of 10 % was obtained after 2 to 3 weeks of incubation. The transformation was correlated with an increase in the pH of the media from 5.7 –5.9 to 8.3 – 8.8.Saccharomyces cerevisiae also transformed AFG₁ into AFB₃, but at a slower rate; the pH of the media did not reach above 8.0 until 5 weeks after incubation. No transformation was observed whenA niger andP chrysogenum were tested; in both cases, no increase in pH was noticed. However, some transformation of AFG₁ to AFB₃ by both fungi was observed when the initial pH of the media was adjusted to 9.0. The rate of transformation increased to 15 – 20% in the static culture where the same medium was adsorbed onto vermiculite andRhizopus andAspergilli gave the highest increase in AFB₃ yield.     

Preliminary determination of mycotoxin binding to soil when leaching through soil with water

Mycotoxin-contaminated crops that are left in the field are potential contaminants of groundwater. Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) distribution in soil-water systems and the comparative response of aflatoxin-contaminated corn and pure aflatoxin when leached through soil were investigated using columns. Each experiment was repeated once. Eluates and soil extracts were analyzed for AFB₁ and its metabolites and FB₁ along with different amounts of pure FB₁ and water mixtures. AFB₁ was detected in water samples from columns containing 10% and 20% silty clay loam soil and aflatoxin-contaminated corn mixtures and in the upper (top) 2.5 cm of soil from the 10 cm soil column. Aflatoxin B₂ (AFB₂) was detected in eluates from the column containing 10% soil and aflatoxin-contaminated corn mixture and from the column containing aflatoxin-contaminated corn alone. No AFB₂ was detected in eluates from the column containing 20% soil and aflatoxin-contaminated corn mixture. No detectable amount of aflatoxin was observed in eluates from the containing 50% silty clay loam soil and aflatoxin contaminated corn. No detectable amount of FB₁ was observed in eluates or soil extracts, but FB₁ was detected in the mixtures of pure FB₁ and water.     

Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068

Aims: To prepare, purify and characterize an extracellular enzyme from Myxococcus fulvus ANSM068, designated as myxobacteria aflatoxin degradation enzyme (MADE), which possesses degradation activity against aflatoxin B₁ (AFB₁), G₁ (AFG₁) and M₁ (AFM₁) in solution. Methods and Results: The culture supernatant of strain M. fulvus demonstrated high degradation ability against AFB₁ (71·89%), AFG₁ (68·13%) and AFM₁ (63·82%) after 48 h of incubation. An enzyme was purified from the supernatant of M. fulvus using ethanol precipitation and chromatography on DEAE‐Sepharose and Superdex 75. An overall 166‐fold purification of the enzyme with a recovery of 57% and a final specific activity of 569·44 × 10³ U mg^?1 was obtained using the present purification protocol. The apparent molecular mass of MADE was estimated to be 32 kDa by SDS‐PAGE. AFG₁ and AFM₁ were significantly degraded, by 96·96 and 95·80%, respectively, when treated with pure MADE (100 U ml^?1) produced by strain ANSM068. MADE exhibited the largest amount of activity at 35°C and pH 6·0, with Mg²⁺ ions greatly promoting and Zn²⁺ strongly inhibiting MADE activity. Conclusions: An aflatoxin degradation enzyme from bacterial isolates can effectively remove aflatoxin B₁, G₁ and M₁ in solution. Significance and Impact of the Study: The high activity and wide temperature and pH range of MADE for the degradation of aflatoxin have promising applications in control of mycotoxins during food and feed processing.     

Aflatoxin B<Subscript>1</Subscript> Induced Systemic Toxicity in Poultry and Rescue Effects of Selenium and Zinc

Among many challenges, exposure to aflatoxins, particularly aflatoxin B₁ (AFB₁), is one of the major concerns in poultry industry. AFB₁ intoxication results in decreased meat/egg production, hepatotoxicity, nephrotoxicity, disturbance in gastrointestinal tract (GIT) and reproduction, immune suppression, and increased disease susceptibility. Selenium (Se) and zinc (Zn), in dietary supplementation, offer easy, cost-effective, and efficient ways to neutralize the toxic effect of AFB₁. In the current review, we discussed the impact of AFB₁ on poultry industry, its biotransformation, and organ-specific noxious effects, along with the action mechanism of AFB₁-induced toxicity. Moreover, we explained the biological and detoxifying roles of Se and Zn in avian species as well as the protection mechanism of these two trace elements. Ultimately, we discussed the use of Se and Zn supplementation against AFB₁-induced toxicity in poultry birds.     

Exposure to aflatoxin B<Subscript>1</Subscript> in Thailand by consumption of brown and color rice

This study assessed the aflatoxin B₁ (AFB₁) intake of the Thai population through consumption of contaminated brown and color rice. A total of 240 rice samples from two harvesting periods were collected in June/July 2012 (period I) and in December 2012/January 2013 (period II) and analyzed for AFB₁ by HPLC with fluorescence detection (limit of detection (LOD)?=?0.093 ng/g). Exposure assessment was based on AFB₁ levels in rice and food intake data for rice according to Thai National Consumption. Frequency and levels of AFB₁ were higher in period I (59 %, <LOD?=?26.61 μg kg^?1) than in period II (10 %, <LOD?=?3.51 μg kg^?1). Only one sample exceeded the Thai standard limit for total aflatoxin of 20 μg kg^?1, but 12 out of 240 rice samples exceeded the European Union maximum level for AFB₁ of 2 μg kg^?1. The data showed that the quality and safety of Thai rice largely comply with the requirement for both exports and domestic consumption. According to the Thai National Consumption data, the estimated AFB₁ intake via rice consumption in period I and period II was 0.80 and 0.12 μg kg^?1 bw day^?1, respectively. The potential risk for cancer, based on the recommendation of the JECFA, was estimated to be 0.011 person/year/100,000 people at a mean consumption. Although the risk via consumption of Thai rice seems to be low, the maximum levels of AFB₁ in this staple food suggest that careful monitoring and surveillance of AFB₁ contamination in rice is essential to ensure the safety of rice.     

Aflatoxins,discolouration and insect damage in dried cowpea and pigeon pea in Malawi and the effectiveness of flotation/washing operation in eliminating the aflatoxins

Aflatoxin contamination and biodeterioration were examined in 302 samples of dry cowpeas and pigeon peas that were randomly purchased from 9 districts of the Southern Region of Malawi during July and November 2015. Further, the impact of flotation/washing on aflatoxin levels on the pulses was elucidated. Aflatoxin analyses involved immunoaffinity column (IAC) clean-up and HPLC quantification with fluorescence detection (FLD) while legume biodeterioration assessments were done by visual inspection. Aflatoxins were frequently detected in cowpea (24%, max., 66 μg/kg) and pigeon pea (22%, max., 80 μg/kg) samples that were collected in the month of July. Lower aflatoxin incidence of 15% in cowpeas (max., 470 μg/kg) and 14% in pigeon peas (max., 377 μg/kg) was recorded in the November collection. Overall, aflatoxin levels were significantly higher in the pulses that were collected in November. However, there were no significant differences in the total aflatoxin (aflatoxin B₁ (AFB₁) + AFB₂ + AFG₁ + AFG₂) levels between the two types of pulses. Remarkably, in 76.2% of the aflatoxin positive cowpea and in 41.7% of the aflatoxin positive pigeon pea samples, aflatoxin G₁ concentration exceeded aflatoxin B_1. Insect damage percentage averaged at 18.1 ± 18.2% (mean ± SD) in the cowpeas and 16.1 ± 19.4% in pigeon peas. Mean discolouration percentage (number of pulses) of the cowpeas and pigeon peas was found to be at 6.7 ± 4.9 and 8.7 ± 6.2%, respectively. Washing and discarding the buoyant fraction was highly efficient in reducing aflatoxin levels; only 5.2 ± 11.1% of the initial aflatoxin level was found in the cleaned samples. In conclusion, cowpeas and pigeon peas sold on the local market in Malawi may constitute a hazard especially if floatation/washing step is skipped.     

Determination of serum aflatoxin B<Subscript>1</Subscript>-lysine to evaluate the efficacy of an aflatoxin-adsorbing feed additive in pigs fed an aflatoxin B<Subscript>1</Subscript>-contaminated diet

In this study, serum aflatoxin B₁ (AFB₁)-lysine was determined in order to evaluate the in vivo efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) in pigs fed AFB₁. Twenty-four 49-day-old crossbred barrows were maintained in individual cages and allowed ad libitum access to feed and water. A completely randomized design was used with six animals assigned to each of four dietary treatments for 21 days as follows: (A) basal diet (BD), (B) BD supplemented with 0.5 % HSCAS, (C) BD supplemented with 1.1 mg/kg AFB₁, and (D) BD supplemented with 0.5 % HSCAS and 1.1 mg/kg AFB₁. HSCAS was able to alleviate the toxic effects of AFB₁ on pigs and reduce (P < 0.05) the levels of serum AFB₁-lysine. Cumulative reductions of adduct yield values, calculated through the equation [(pg AFB₁-lysine/mg albumin) / (μg AFB₁/kg body weight)], were 53.0, 62.8, and 72.1 after 7, 14, and 21 days of oral exposure, respectively. AFB₁-lysine has potential as an AFB₁-specific biomarker for diagnostic purposes and for evaluating the efficacy of chemoprotective interventions in pigs.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

Mycobiota and mycotoxins in malted barley and brewer's spent grain from Argentinean breweries

Aims: To evaluate mycobiota and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁), G₂ (AFG₂) and fumonisin B₁ (FB₁) contamination in different malted barley types and brands and brewer’s grain collected from a major Argentinean brewery. Methods and Results: Total fungal counts were performed using the plate count method. Aflatoxin B₁, AFB₂, AFG₁, AFG₂ and Zearalenone (ZEA) analyses were performed by thin‐layer chromatography (TLC). Fumonisin B₁ was determined by HPLC. Eighty‐three percentage of the malted barley (100% M1, 50% M2 and 100% M3) and 61% of brewer’s grain samples had a count >1 × 10⁴ CFU g^?1. Yeasts were isolated from all malt and brewer’s grain samples. Genera containing some of the most important mycotoxin producer species –Fusarium ssp., Aspergillus ssp., Penicillium ssp. and Alternaria ssp. – were isolated from the analysed samples, along with other environmental saprophytic fungi such as Geotrichum ssp., Mucorales and Cladosporium ssp. All samples were contaminated with 104–145 μg kg^?1 FB₁. Eighteen per cent of brewer’s grain samples were contaminated with 19–44·52 μg kg^?1 AFB₁. Aflatoxin B₂, AFG₁, AFG₂ and ZEA were not detected in any of the analysed samples. Conclusions: Fungal and mycotoxin contamination in malt and brewer’s grain is an actual risk for animal and human health. Significance and Impact of the Study: This study may be useful for assessing the risk of mycotoxins in Argentinean beers and especially in animal feeds.     

Effects of mycotoxin pretreatment on aflatoxin B1 post-treatment toxicity in Drosophila melanogaster (Diptera)

Two wild-type laboratory strains of Drosophila melanogaster were used in this study: strain Flordia-9, which is sensitive to aflatoxin B₁ (AFB₁)-induced toxicity, and strain Lausanne-S, which is resistant. Eggs of these strains were deposited on medium containing either low or high doses of dietary AFG₁, AFB₂, or sterigmatocystin (ST) and allowed to develop into second instar larvae. After this pretreatment, the larvae were transferred onto medium containing either high or low doses of dietary AFB₁ (post-treatment) and allowed to complete development and eclose as adults. Viability and development data were analyzed to determine the effects of the various pretreatments on the level of AFB₁-induced toxicity in the post-treatments. In no case did any of the pretreatments reduce the toxic effects of AFB₁ post-treatment responses. However, for strain Florida-9, all high-dose pretreatments resulted in enhanced post-treatment toxicity, and all low-dose pretreatments also enhanced toxicity of high-dose post-treatments. For strain Lausanne-S, high-dose AFB₂ pretreatment significantly enhanced toxicity of both high- and low-dose post-treatments. These results indicate that, in strain Florida-9, pretreatment with relatively less toxic mycotoxins (ST and AFB₂) has an enhancing effect on AFB₁-induced toxicity, whereas in strain Lausanne-S, a similar but smaller enhancing effect is seen only with AFB₂ pretratment.     

Exposure assessment and risk characterization of aflatoxin B<Subscript>1</Subscript> in Malaysia

Aflatoxin B₁ (AFB₁) was detected in 57% of the nuts and nut products marketed in Penang, Malaysia using the liquid chromatography tandem mass spectrometry. The contamination levels ranged from 0.40 to 222 μg/kg and 17 out of 128 samples (13.3%) contained AFB₁ above the European Commission permitted level (2 μg/kg). Estimated dietary exposure of AFB₁ in nuts and nut products were 0.36 ng per kg body weight and day and 8.89 ng per kg body weight and day, representing the low and high-level of exposure, respectively. Dose-response modelling resulted in benchmark dose lower confidence limit (BMDL₁₀) values of 0.305 ng per kg body weight and day, with the best fitted from the log-logistic model. The derived margin of exposure (MoE) values ranged from 34 to 847 suggested that AFB₁ would be of public health concern and might reasonably be considered as a high priority for risk management actions.     

Toxicological effects of aflatoxin B<Subscript>1</Subscript> on the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> as determined in a contact paper test

In this study, aflatoxin B₁ (AFB₁) toxicity toward the earthworm Eisenia fetida (Savigny 1826) was evaluated in contact paper test systems containing distilled water and ethanol or 20 to 400 μg/ml of AFB₁ over 72 h of exposure. The results indicated that AFB₁ could induce significant damage to earthworms (coiling, curling, excessive mucus secretion, clitellum swelling) at greater than 75 μg/ml. Moreover, AFB₁ had harmful effects on E. fetida (degenerative changes such as bulging of the clitella regions) at levels higher than 150 μg/ml. The calculated LD₅₀ was 168.5 μg/ml. These findings confirm that E. fetida and standardized methods based on this organism (OECD 207 1984) are applicable and useful in mycotoxin related toxicity studies.     

Chemosensitization of Aflatoxigenic Fungi to Antimycin A and Strobilurin Using Salicylaldehyde,a Volatile Natural Compound Targeting Cellular Antioxidation System

Various species of fungi in the genus Aspergillus are the most common causative agents of invasive aspergillosis and/or producers of hepato-carcinogenic mycotoxins. Salicylaldehyde (SA), a volatile natural compound, exhibited potent antifungal and anti-mycotoxigenic activities to A. flavus and A. parasiticus. By exposure to the volatilized SA, the growth of A. parasiticus was inhibited up to 10–75% at 9.5 mM ≤ SA ≤ 16.0 mM, while complete growth inhibition was achieved at 19.0 mM ≤ SA. Similar trends were also observed with A. flavus. The aflatoxin production, i.e., aflatoxin B₁ and B₂ (AFB₁, AFB₂) for A. flavus and AFB₁, AFB₂, AFG₁, and AFG₂ for A. parasiticus, in the SA-treated (9.5 mM) fungi was reduced by ~13–45% compared with the untreated control. Using gene deletion mutants of the model yeast Saccharomyces cerevisiae, we identified the fungal antioxidation system as the molecular target of SA, where sod1Δ [cytosolic superoxide dismutase (SOD)], sod2Δ (mitochondrial SOD), and glr1Δ (glutathione reductase) mutants showed increased sensitivity to this compound. Also sensitive was the gene deletion mutant, vph2Δ, for the vacuolar ATPase assembly protein, suggesting vacuolar detoxification plays an important role for fungal tolerance to SA. In chemosensitization experiments, co-application of SA with either antimycin A or strobilurin (inhibitors of mitochondrial respiration) resulted in complete growth inhibition of Aspergillus at much lower dose treatment of either agent, alone. Therefore, SA can enhance antifungal activity of commercial antifungal agents required to achieve effective control. SA is a potent antifungal and anti-aflatoxigenic volatile that may have some practical application as a fumigant.