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Abstract [Objectives] This study was conducted to investigate the toxicity of molds in contaminative fruits.
[Methods] Common fruits were used as materials to isolate contamination molds and screen the most virulent strain to study its pathogenic mechanism.
[Results] We isolated eight kinds of strains from red bayberries， pears， oranges and peaches， and the P2 strain from red bayberries was the most virulent strain， which was preliminarily identified as Aspergillus luchuensis. The toxin from the strain led to the bone marrow cell micronucleus phenomenon， and the greater the toxin dose， the greater the micronucleus rate. With the extension of time， the micronucleus rate increased. The SOD activity of the kidney was inhibited after the mice were exposed to the toxin， and the greater the toxin dose， the lower the SOD activity. With the extension of time， the SOD activity decreased， but increased instead after 48 h because the toxin was metabolized in the body. It was always lower than that of control group. The MDA content in kidney was also affected after the exposure， and the greater the toxin dose， the higher the MDA content. Specifically， it increased with the time prolonged and began to decrease until 48 h， but not less than the control group. It indicated mold contamination in fruits produced toxins， and the P2 strain was the most toxic in red bayberries， causing chromosome and kidney injury.
[Conclusions] This study provides a theoretical basis for the isolation and identification of molds in different fruits and toxicity tests， which is helpful to enhance people’s understanding of mycotoxins and their harm.
Key words Mycotoxin;Median lethal dose;Micronucleus; Cell apoptosis; Super oxide dixmutase
Received： August 5， 2020 Accepted： October 9， 2020
Supported by Public Welfare Technology Application Project of the Science and Technology Bureau of Lishui City （（2016GYX15）.
Qing’e LIU （1976-）， female， P. R. China， associate professor， devoted to research about development and utilization of biological resources.
*Corresponding author. E-mail： qinger91@126.com.
Molds have a strong ability to reproduce， often causing mildewing and rot of foods and growth of some visible fluffy， flocculent or cobweb-shaped colonies. Some molds produce toxic secondary metabolites， mycotoxins， during their growth and reproduction. In recent years， there have been frequent reports of poisoning incidents such as abdominal pain， diarrhea， abnormal liver function， dizziness， headache， drowsiness， coma， and even death caused by eating food contaminated by mycotoxins. Therefore， the problem of mycotoxin pollution has attracted people’s attention， especially the molds of some foods （such as fruits） that are commonly eaten in daily life and closely related to our lives. During long-term storage or under improper storage and transportation conditions， fruits are extremely susceptible to mold contamination which causes spoilage， and the contaminated fruits can cause harm to the human body after being eaten. For example， Liu et al.[1] in 1984 and Hou et al.[2] in 1989 carried out mold isolation and identification and toxicity tests on sugarcane that caused poisoning， and both confirmed that Arthrinium phaeospermum was the pathogen causing sugarcane poisoning. In recent years， scholars at home and abroad have conducted many studies on the isolation and identification of molds in contaminated fruits， but the studies on the toxic effects and pathogenic mechanisms of mycotoxins are relatively rare. Therefore， in this study， we isolated contamination molds from common contaminated fruits， and screened the most virulent strain to study its pathogenic mechanism， aiming to provide a theoretical basis for the isolation and identification of molds in different fruits and toxicity tests， which is beneficial to enhance people’s awareness of mycotoxins and their hazards and strengthen people’s attention to rotten fruits， which is of positive significance to human health. Materials and Methods
Experimental materials
Contaminated fruits： Contaminated fruits such as apples， oranges， red bayberries， pears， and peaches were randomly sampled.
Experimental animals： Healthy mice were from the mouse breeding center of Lishui City.
Instruments and reagents
Main instruments
Table type low-speed centrifuge （Hunan Xiangyi Laboratory Instrument Development Co.， Ltd.， L550）; table type high-speed centrifuge （SIGMA Laborzentrifugen GmbH， 3K 15）; spectrophotometer （Shanghai Xinmao Instrument Co.， Ltd.， 2C409035）.
Main reagents
Methanol， chloroform， ethanol and glacial acetic acid， all analytically pure; dimethyl sulfoxide （purchased from Beijing Dingguo Changsheng Biotechnology Co.， Ltd.）; calf serum （purchased from Zhejiang Tianhang Biotechnology Co.， Ltd.）; 1 mg/ml proteinase K （from Tritirachium Aibum）; MDA kit （Nanjing Jiancheng Bioengineering Institute）.
Medium
Czapek’s medium： Sodium nitrate 2 g， dipotassium phosphate 1 g， magnesium sulfate （MgSO4·7H2O） 0.5 g， potassium chloride 0.5 g， ferrous sulfate 0.01 g， sucrose 30 g， agar 20 g， distilled water 1 000 ml.
Isolation and purification of molds in fruits
Several pieces of fruit flesh with mildew stains were mixed with sterile water and shaken to make a microbial suspension， which was then diluted in a 10-fold grade[3]. The dilutions were 10-1， 10-2， 10-3， 10-4， 10-5， 10-6， and 10-7. From each dilution， 0.2 ml was taken and spread on Czapek’s medium， and two parallel groups were made. After culturing at a constant temperature of 28 ℃ for 3-5 d， single colonies that grew better were picked and streak-inoculated on Czapek’s medium for isolation. The molds were performed for 3-4 times， and preserved on slant.
Toxin extraction and strain screening
The purified strains were inoculated on liquid Czapek’s medium， shaken at 28 ℃ for 14-15 d， and sterilized at 121 ℃ for 20 min. Each culture was then filtered， extracted with an equal volume of chloroform twice and concentrated at 90 ℃ to dryness， and the weight of the crude toxin was calculated. The toxin was dissolved with dimethyl sulfoxide， and mice were injected intraperitoneally at 200 mg/kg. The control group was injected with dimethyl sulfoxide （DMSO）. Each group included 6 mice （3 female mice， 3 male mice）. The most virulent strains were selected based on the mortality of mice.
Identification of strains Referring to the Fungus Identification Manual[4]， the strains were preliminarily identified based on the culture characteristics of colonies， the hyphae morphology and the spore characteristics.
Determination of the median lethal dose of toxin in mice
The toxin was injected intraperitoneally into mice at doses of 320， 160， 80， 40， 20， 10， 5， 2.5， 1.25， 0.625 mg/kg， 8 mice per dose. It required that the death rate of mice with the highest dose was 100%， and the death rate of mice with the lowest dose was 0%. The median lethal dose was calculated according to the formula logLD50=Xk-i（∑p-0.5） （Xk is the highest logarithmic dose， i is the difference between two adjacent logarithmic doses， and p is the death rate of each dose group）.
Toxicity test of mycotoxins
The method of mycotoxin exposure to experimental animals
The toxin solution was formulated into four doses of LD50/2， LD50/4， LD50/8， and LD50/16. The mice were injected intraperitoneally at 0.01 ml/kg. The negative control group was injected with dimethyl sulfoxide， and each dose was injected with 12 mice. Three mice were dissected after 6， 24， 48 and 72 h of exposure， and the liver and kidney were taken out and washed with saline， dried with filter paper， and stored at -80 ℃.
The effect of the toxin on mouse cell DNA
A certain amount of liver （30 mg） was add with 600 μl TE buffer， and prepared into a homogenate， which was then added with 600 μl of extracting solution and 24 μl of RNase （final concentration 20 μg/ml）， and heated in a 37 ℃ water bath for 10 min. Then， 4.48 μl of proteinase K was added， followed by mixing and heating in a 37 ℃ water bath for 2.5 h. The obtained system was finally added with an equal volume of phenol chloroform-isoamyl alcohol and centrifuged at 10 000 r/min for 2 min after mixing well， which was repeated twice. The supernatant was added to pre-cooled anhydrous ethanol to precipitate the DNA， and the precipitate was washed twice with 75% ethanol and then dissolved in TE buffer， and electrophoresed in a 0.8% agarose gel.
Determination of SOD Activity in Mouse Kidney
The mouse kidney was made into a 10% kidney homogenate[5]， which was stored at -20 ℃ for later use. The SOD activity was determined by the pyrogallol autooxidation method[6]. The pyrogallol autooxidation rate was controlled at 0.71 min， and the reaction system （0.05 mol/L K2HPO4-KH2PO4 buffer， sample solution， and pyrogallol， 4.558 μl in total） was mixed and quickly measured for the OD value at 325 nm every 30 s. The sample volume was adjusted to control the oxidation rate of pyrogallol at 0.035（±0.002） OD/min， and the SOD activity was calculated according to the following formula： SOD activity （U/g）=（0.071-Pyrogallol autooxidation rate/0.071）×100%÷5%×（Total volume of system/Volume of reaction liquid）×Dilution ratio
Determination of MDA content in mouse kidney
The coomassie brilliant blue method was used to determine the protein content in mouse kidney homogenate. The standard curve was prepared with bovine serum albumin as the standard product， and the regression equation was y=0.007 6x-0.007 6 （R2=0.992 8）. MDA was determined by the thio-barbituric acid （TBA） method[7]， and the absorbance was determined according to the MDA kit. The MDA content was calculated according to the following formula：
MDA content （nmol/mg prot）=[6.45×（A532-A600）-0.56×A450]÷Concentration of standard protein to be detected（mg/ml）.
Results and Analysis
Isolation and purification of strains from fruits
Molds were isolated and purified from apples， red bayberries， pears， oranges， peaches and other fruits， and 8 different strains were preliminarily identified through observation of colony characteristics， and named P2， P3， L3， L4， J2， J3， J4， and T8， respectively. The specific culture characteristics are shown in Table 1.
Screening and identification of highly virulent strains
It can be seen from Table 2 that the death rate of mice exposed to the toxins from strains P2 and P3 isolated from red bayberries was 100% at 22 h， which was significantly higher than that of other strains. Because the death rate of mice exposed to the toxin from the P2 strain was 83.3% at 6 h， which was higher than the P3 strain， indicating that the P2 strain was the most virulent. The experimental results showed that the strains isolated from the contaminated fruits all produced toxins and all had certain toxicity， but the P2 strain isolated from red bayberries was significantly more toxic than other fruits， so this strain was used for the toxicity test.
The staining results of hyphae and spores of the P2 strain are shown in Fig. 1 and Fig. 2. The conidiophores of this strain are colorless and loose， unbranched， swollen at the top， and the conidia are clustered， and have thick membrane podocytes at the base. Combined with its culture characteristics， according to Fungus Identification Manual， the P2 strain was preliminarily identified as Aspergillus luchuensis of the Aspergillus niger group.
It can be seen from Table 3 that the death rates of mice injected at 0.625， 1.25， 2.5， 5， 10， 20， 40， 80， 160， and 320 mg/kg were 0， 12.5%， 25%， 37.5%， 25%， 37.5%， 62.5% and 100%， respectively. The death rates included 0 and 100%， which met the prerequisite for calculating the median lethal dose. Therefore， according to the calculation formula of the median lethal dose logLD50=Xk-i（∑p-0.5） （wherein Xk=2.505 1， i=0.301 0， ∑p=3.625 0）， the LD50 was calculated to be 36.683 9 mg/kg. Mouse bone marrow cell micronucleus test
Micronuclei appeared in mouse bone marrow cells at both 24 and 48 h after the exposure， and the morphology is shown in Fig. 3 and Fig. 4. The statistics of micronucleus rate （Table 4 and Table 5） showed that the micronucleus rates of the four toxin dose groups at 24 and 48 h were significantly lower than that of the cyclophosphamide positive control group （F= 4.937， P<0.01; F= 3.617， P<0.05）， but higher than the negative control group （dimethyl sulfoxide）， and the greater the toxin dose， the greater the micronucleus rate. The 48 h micronucleus rate was generally higher than 24 h， and the micronucleus rate of mice gradually increased with time. Micronucleus is the genetic material that remains outside the nucleus during the anaphase of mitosis due to chromosome breakage or influences on spindle fibers in the cell， and the micronucleus test can detect such two genetic end points as the chromosome integrity change and chromosome segregation change induced by chemical poisons or physical factors. Therefore， the experimental results showed that the P2 strain was genetically toxic and would cause certain damage to the chromosomes.
The effect of the toxin on mouse cell DNA
As can be seen from Fig. 5， numbers 1-5 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， and LD50/16 for 24 h， respectively， 6-10 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， LD50/16 doses for 48 h， respectively， and 11-15 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， and LD50/16 for 72 h， respectively. The results showed that there were no ladder-like or diffuse bands in the DNA of mice after 24 h， 48 h and 72 h exposure， indicating that the toxin of strain P2 had no effect on the DNA within 72 h， which might be that it takes a certain time for the drug to induce apoptosis or necrosis.
Effect of the toxin on SOD activity in mouse kidney
The effect of the toxin on SOD activity in mouse kidney is shown in Fig.9. At 6， 24 and 48 h after the exposure， each concentration had an extremely significant effect on mouse SOD activity （F=227.838， P<0.01; F=193.373， P<0.01; F=80.3， P<0.01）， and each concentration had a significant impact on SOD activity 72 h after the exposure （F=6.414， P<0.05）. When the mice were exposed for 6 h， the SOD activity of each concentration was extremely significantly lower than that of the negative control， and the greater the concentration， the lower the SOD activity， indicating that the toxin would cause the decline of the kidney’s antioxidant capacity. After 6 h of exposure， the SOD activity of mice in the LD50/8 and LD50/16 groups began to rise gradually over time， and approached the control group at 72 h. The SOD activity of mice in the LD50/2 and LD50/4 groups decreased with time from 6 to 48 h， and both decreased to about 3 000 U/g at 48 h， but gradually recovered after 48 h and began to approach the control group. It indicated that the toxin had a significant inhibitory effect on the SOD activity of mouse kidney， and the higher the toxin concentration， the stronger the inhibition on the SOD activity of mouse kidney， which might be due to that the body had certain ability to resist and remove the toxin from the A. luchuensis P2 strain at low concentrations， and its recovery speed was greater than that at high concentrations. In addition， the inhibition decreased with the increase of time， which might be that mycotoxins had been excreted and detoxified from organs such as liver and kidney after a long period of metabolism between 48 and 72 h. Effect of the toxin on MDA activity in mouse kidney
The effect of the toxin on the content of MDA in mouse kidney is shown in Fig. 10. Each concentration after exposure for 6， 24， 48 and 72 h had an extremely significant effect on the content of MDA in mice （F=73.761， P<0.01; F=294.176， P<0.01; F=4207.476， P<0.01; F=941.435， P<0.01）. When the mice were exposed for 6 h， the MDA content of each concentration was significantly higher than the negative control， and the greater the concentration， the higher the MDA content， which indicated that the degree of membrane lipid peroxidation increased and the biological membrane structure was damaged. After 6 h of exposure， the MDA contents of the mice in the LD50/8 and LD50/16 groups did not fluctuate much over time， and both remained approximately 3-4 nmol/mg prot. The MDA contents of mice in the LD50/2 and LD50/4 groups increased significantly from 6 to 48 h with time， reached the highest values of 9.1 and 7.9 nmol/mg prot at 48 h， respectively， and gradually decreased after 48 h. It indicated that the toxin significantly enhanced the MDA content of mouse kidney， and the higher the toxin concentration， the greater the MDA content， which was opposite to the change in SOD activity， which might be that the body had certain ability to resist the toxin of A. luchuensis. In addition， the MDA content gradually decreased with the increase of time. It might be that mycotoxins had been excreted and detoxified from the liver and kidney and other organs after a long period of metabolism between 48 and 72 h， which was consistent with the SOD activity determination result.
Conclusions and Discussion
Fruits are very susceptible to mold contamination during long-term storage or under improper storage and transportation conditions to cause spoilage. For example， Wang et al.[2] showed that apples were susceptible to contamination by Penicillium， Aspergillus and other molds to cause spoilage; Zhang et al.[8] reported that citrus is susceptible to contamination by various Penicillium species such as P. digitatum and P. italia. In this study， red bayberry， pear， peach， orange and other fruits that are susceptible to mold contamination were selected as the isolation materials， and a total of 8 different strains were screened. After the toxicity test， it was found that they were all lethal to mice. Among them， the P2 strain from red bayberries showed the highest death rate， which was as high as 100%. After identification， the P2 strain was A. luchuensis of the A. niger group. It indicates that there are many kinds of strong pathogenic microbes in contaminated fruits， and eating the fruit contaminated by molds by mistake will have an adverse effect on the human body. Therefore， moldy fruits should attract people’s attention. Mycotoxins are toxic and harmful substances produced by molds. Mycotoxins can cause changes in enzyme activity， cell and DNA activity， gene function and other indicators in the body， which will adversely affect animal organs， immune systems， and nervous systems. We found that the A. luchuensis strain P2 isolated from red bayberries can cause the increase of PCE micronucleus rate in mice， the decrease of SOD activity in mouse kidney and the increase of MDA content， but it did not produce a significant effect on apoptosis in mice. Micronucleus is the genetic material that remains outside the nucleus during the anaphase of mitosis due to chromosome breakage or influences on spindle fibers in the cell， and micronucleus tests can detect such two genetic end points as the chromosome integrity change and chromosome segregation change induced by chemical poisons or physical factors. For example， Song et al.[9] screened two strongly pathogenic mold strains from contaminated fruits and performed mouse PCE micronucleus tests on them. The results showed that their mycotoxins could increase the PCE micronucleus rate of mice， which was significantly higher than the negative control group. This study also found that the A. luchuensis strain P2 derived from red bayberries increased the micronucleus rate of mouse bone marrow. The micronucleus rate of each concentration was significantly higher than that of the negative control group （P<0.05）， and the greater the concentration， the higher the micronucleus rate. Moreover， with the prolongation of time， the micronucleus rate of each concentration increased. It indicated that mycotoxins caused certain damage to mouse chromosomes and were genetically toxic， and with the increase of concentration and time， the toxicity to the body got stronger. Ladder DNA electrophoresis is a classic biochemical characteristic of cell apoptosis[10]. The results of this study showed that there were no obvious apoptosis or necrosis bands in the DNA electrophoretogram after exposure for 24-72 h， which might be that although the toxin of strain P2 damaged the cells， the nucleus was still intact and the chromatin was only in a concentrated state rather than fragmented. Lei et al.[11] also found in the study of the toxic mechanism of microcystin damage to liver cells that even though the cell membrane vesiculation had been very severe， the nucleus was still intact， the chromatin was only in a concentrated state rather than fragmented， and cells undergoing apoptosis did not necessarily undergo DNA degradation. SOD is the only enzyme in the organism that can scavenge O-2 free radicals， and the decrease in SOD activity can lead to the decline of the body’s antioxidant capacity， which will cause O-2 accumulation[12]. The accumulated oxygen free radicals can attack the polyunsaturated fatty acids in the biological membrane， trigger lipid peroxidation， generate a large amount of MDA， and seriously damage the body’s tissues and organs[12]. Guo et al.[13] found that the toxin from P. cyclopium， penicillic acid， could reduce SOD activity and increase MDA content in mouse kidney tissues， showing a dose-related relationship， which is similar to the results of this study. After 6 h of exposure， the SOD activity of each concentration was significantly lower than that of the negative control， the greater the concentration， the lower the SOD activity， and the two were in a dose-effect relationship. The MDA content was opposite to the change of SOD activity. As the toxin concentration increased， the MDA content increased significantly， indicating that lipid peroxidation damage was aggravated. Moreover， we found that as time increased， the SOD activity gradually increased， and the MDA content gradually decreased. It was speculated that mycotoxins had been excreted from the liver and kidneys and other organs after a long period of metabolism between 48 and 72 h， and the body was in a state of recovery. Based on the above analysis， different fruits will be contaminated by molds and cause damage to the body. In this study， the P2 strain of A. luchuensis isolated from red bayberries caused the PCE micronucleus rate to increase， the kidney SOD activity to decrease and the MDA content to increase. Therefore， mold-contaminated fruits can damage the human kidneys and cause intracellular chromosome breakage when eaten by mistake， and thus have certain genetic toxicity. However， the effects of this strain on the cell cycle and genes and the physiological and biochemical properties of its toxin need further research.
References
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[5] ZHUANG JJ， LIU JD， WANG ZL， et al. Roles of apoptosis and iron in experimental immunological liver injury in rats[J]. Chinese Journal of Pathophysiology， 2006， 22（3）： 602-604. （in Chinese）
[6] LI CJ， LI Y， LI DG， et al. Determination of SOD activity in hepar of animal[J]. Chemical Engineer， 2006（7）： 14-15， 18. （in Chinese）
[7] WANG JH， ZHOU W， WANG SX， et al. Effects of lead on changes of SOD， MDA in the kidney[J]. Acta Academiae Medicinae CPAPF， 2002（3）： 146-148. （in Chinese）
[8] ZHANG CX， LI N， LI Q， et al. Identification and charateristic analysis about Penicillium spp.from Citrus[J]. Journal of Central China Normal University， 2014， 48（1）： 86-90. （in Chinese）
[9] TAO NG， WANG H， WANG CF， et al. Research on the isolating of two citrus postharvest pathogens and their biological characteristics[J]. Natural Science Journal of Xiangtan University， 2013， 35（3）： 75-78. （in Chinese）
[10] ZHANG HJ， WU YH， ZHAO J， et al. Toxic effect of nodularin on the apoptosis of grass carp （Ctenopharyngodon idellus） lymphocytes and related mechanisms[J]. Chinese Journal of Applied Ecology， 2013， 24（10）： 2977-2982. （in Chinese）
[11] LEI LM， SONG LR， HAN BP. Microcystin-LR induces apoptosis with the absence of DNA fragmentation[J]. Journal of Tropical Medicine， 2006， 6（7）： 749-751， 784. （in Chinese） [12] WANG JJ， LI CY， YU SY， et al. Effects of ephedrine on histological structure， activities of superoxide dismutase， catalase， and content of maleic dialdehyde in kidney of filial mice[J]. Chinese Journal of Zoology， 2011， 46（6）： 118-125. （in Chinese）
[13] GUO L， YUAN H. Renal injury and its mechanism on mice induced by penicillic acid from Penicillium cyclopium[J]. Journal of Hunan Agricultural University： Natural Sciences， 2010， 36（3）： 330-334. （in Chinese）
[14] YU GQ， YANG SL， HAN SY， et al. Micronucleus test with mold extracts from contaminated fruits[J]. Journal of Henan Medical University， 1994， 29（4）： 332-334. （in Chinese）
[15] HOU ZZ， LIU GH， SONG LJ， et al. Isolation and toxicity test of fungi from moldy sugarcane[J]. Chinese Journal of Public Health， 1989， 8（6）： 341-342. （in Chinese）
[16] ZHANG CX， LI N， LI Q， et al. Identification and characteristic analysis about Penicillium spp. from Citrus[J]. Journal of HuaZhong Normal University： Natural Sciences， 2014， 48（1）： 86-89. （in Chinese）
[17] ZHANG XB， LI XQ， LI J， et al. Microcystin toxicity and its median lethal dose on duck embryo[J]. Journal of Anhui Agricultural Sciences， 2011， 39（19）： 11582-11583. （in Chinese）
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[19] ZENG YS， XIANG MM， XIE YP. The pathogenic mechanism of toxin from Nimbya alternantherae to Alligator alternanthera[J]. Journal of Zhongkai University of Agriculture and Technology， 2005， 18（4）： 1-5. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] Common fruits were used as materials to isolate contamination molds and screen the most virulent strain to study its pathogenic mechanism.
[Results] We isolated eight kinds of strains from red bayberries， pears， oranges and peaches， and the P2 strain from red bayberries was the most virulent strain， which was preliminarily identified as Aspergillus luchuensis. The toxin from the strain led to the bone marrow cell micronucleus phenomenon， and the greater the toxin dose， the greater the micronucleus rate. With the extension of time， the micronucleus rate increased. The SOD activity of the kidney was inhibited after the mice were exposed to the toxin， and the greater the toxin dose， the lower the SOD activity. With the extension of time， the SOD activity decreased， but increased instead after 48 h because the toxin was metabolized in the body. It was always lower than that of control group. The MDA content in kidney was also affected after the exposure， and the greater the toxin dose， the higher the MDA content. Specifically， it increased with the time prolonged and began to decrease until 48 h， but not less than the control group. It indicated mold contamination in fruits produced toxins， and the P2 strain was the most toxic in red bayberries， causing chromosome and kidney injury.
[Conclusions] This study provides a theoretical basis for the isolation and identification of molds in different fruits and toxicity tests， which is helpful to enhance people’s understanding of mycotoxins and their harm.
Key words Mycotoxin;Median lethal dose;Micronucleus; Cell apoptosis; Super oxide dixmutase
Received： August 5， 2020 Accepted： October 9， 2020
Supported by Public Welfare Technology Application Project of the Science and Technology Bureau of Lishui City （（2016GYX15）.
Qing’e LIU （1976-）， female， P. R. China， associate professor， devoted to research about development and utilization of biological resources.
*Corresponding author. E-mail： qinger91@126.com.
Molds have a strong ability to reproduce， often causing mildewing and rot of foods and growth of some visible fluffy， flocculent or cobweb-shaped colonies. Some molds produce toxic secondary metabolites， mycotoxins， during their growth and reproduction. In recent years， there have been frequent reports of poisoning incidents such as abdominal pain， diarrhea， abnormal liver function， dizziness， headache， drowsiness， coma， and even death caused by eating food contaminated by mycotoxins. Therefore， the problem of mycotoxin pollution has attracted people’s attention， especially the molds of some foods （such as fruits） that are commonly eaten in daily life and closely related to our lives. During long-term storage or under improper storage and transportation conditions， fruits are extremely susceptible to mold contamination which causes spoilage， and the contaminated fruits can cause harm to the human body after being eaten. For example， Liu et al.[1] in 1984 and Hou et al.[2] in 1989 carried out mold isolation and identification and toxicity tests on sugarcane that caused poisoning， and both confirmed that Arthrinium phaeospermum was the pathogen causing sugarcane poisoning. In recent years， scholars at home and abroad have conducted many studies on the isolation and identification of molds in contaminated fruits， but the studies on the toxic effects and pathogenic mechanisms of mycotoxins are relatively rare. Therefore， in this study， we isolated contamination molds from common contaminated fruits， and screened the most virulent strain to study its pathogenic mechanism， aiming to provide a theoretical basis for the isolation and identification of molds in different fruits and toxicity tests， which is beneficial to enhance people’s awareness of mycotoxins and their hazards and strengthen people’s attention to rotten fruits， which is of positive significance to human health. Materials and Methods
Experimental materials
Contaminated fruits： Contaminated fruits such as apples， oranges， red bayberries， pears， and peaches were randomly sampled.
Experimental animals： Healthy mice were from the mouse breeding center of Lishui City.
Instruments and reagents
Main instruments
Table type low-speed centrifuge （Hunan Xiangyi Laboratory Instrument Development Co.， Ltd.， L550）; table type high-speed centrifuge （SIGMA Laborzentrifugen GmbH， 3K 15）; spectrophotometer （Shanghai Xinmao Instrument Co.， Ltd.， 2C409035）.
Main reagents
Methanol， chloroform， ethanol and glacial acetic acid， all analytically pure; dimethyl sulfoxide （purchased from Beijing Dingguo Changsheng Biotechnology Co.， Ltd.）; calf serum （purchased from Zhejiang Tianhang Biotechnology Co.， Ltd.）; 1 mg/ml proteinase K （from Tritirachium Aibum）; MDA kit （Nanjing Jiancheng Bioengineering Institute）.
Medium
Czapek’s medium： Sodium nitrate 2 g， dipotassium phosphate 1 g， magnesium sulfate （MgSO4·7H2O） 0.5 g， potassium chloride 0.5 g， ferrous sulfate 0.01 g， sucrose 30 g， agar 20 g， distilled water 1 000 ml.
Isolation and purification of molds in fruits
Several pieces of fruit flesh with mildew stains were mixed with sterile water and shaken to make a microbial suspension， which was then diluted in a 10-fold grade[3]. The dilutions were 10-1， 10-2， 10-3， 10-4， 10-5， 10-6， and 10-7. From each dilution， 0.2 ml was taken and spread on Czapek’s medium， and two parallel groups were made. After culturing at a constant temperature of 28 ℃ for 3-5 d， single colonies that grew better were picked and streak-inoculated on Czapek’s medium for isolation. The molds were performed for 3-4 times， and preserved on slant.
Toxin extraction and strain screening
The purified strains were inoculated on liquid Czapek’s medium， shaken at 28 ℃ for 14-15 d， and sterilized at 121 ℃ for 20 min. Each culture was then filtered， extracted with an equal volume of chloroform twice and concentrated at 90 ℃ to dryness， and the weight of the crude toxin was calculated. The toxin was dissolved with dimethyl sulfoxide， and mice were injected intraperitoneally at 200 mg/kg. The control group was injected with dimethyl sulfoxide （DMSO）. Each group included 6 mice （3 female mice， 3 male mice）. The most virulent strains were selected based on the mortality of mice.
Identification of strains Referring to the Fungus Identification Manual[4]， the strains were preliminarily identified based on the culture characteristics of colonies， the hyphae morphology and the spore characteristics.
Determination of the median lethal dose of toxin in mice
The toxin was injected intraperitoneally into mice at doses of 320， 160， 80， 40， 20， 10， 5， 2.5， 1.25， 0.625 mg/kg， 8 mice per dose. It required that the death rate of mice with the highest dose was 100%， and the death rate of mice with the lowest dose was 0%. The median lethal dose was calculated according to the formula logLD50=Xk-i（∑p-0.5） （Xk is the highest logarithmic dose， i is the difference between two adjacent logarithmic doses， and p is the death rate of each dose group）.
Toxicity test of mycotoxins
The method of mycotoxin exposure to experimental animals
The toxin solution was formulated into four doses of LD50/2， LD50/4， LD50/8， and LD50/16. The mice were injected intraperitoneally at 0.01 ml/kg. The negative control group was injected with dimethyl sulfoxide， and each dose was injected with 12 mice. Three mice were dissected after 6， 24， 48 and 72 h of exposure， and the liver and kidney were taken out and washed with saline， dried with filter paper， and stored at -80 ℃.
The effect of the toxin on mouse cell DNA
A certain amount of liver （30 mg） was add with 600 μl TE buffer， and prepared into a homogenate， which was then added with 600 μl of extracting solution and 24 μl of RNase （final concentration 20 μg/ml）， and heated in a 37 ℃ water bath for 10 min. Then， 4.48 μl of proteinase K was added， followed by mixing and heating in a 37 ℃ water bath for 2.5 h. The obtained system was finally added with an equal volume of phenol chloroform-isoamyl alcohol and centrifuged at 10 000 r/min for 2 min after mixing well， which was repeated twice. The supernatant was added to pre-cooled anhydrous ethanol to precipitate the DNA， and the precipitate was washed twice with 75% ethanol and then dissolved in TE buffer， and electrophoresed in a 0.8% agarose gel.
Determination of SOD Activity in Mouse Kidney
The mouse kidney was made into a 10% kidney homogenate[5]， which was stored at -20 ℃ for later use. The SOD activity was determined by the pyrogallol autooxidation method[6]. The pyrogallol autooxidation rate was controlled at 0.71 min， and the reaction system （0.05 mol/L K2HPO4-KH2PO4 buffer， sample solution， and pyrogallol， 4.558 μl in total） was mixed and quickly measured for the OD value at 325 nm every 30 s. The sample volume was adjusted to control the oxidation rate of pyrogallol at 0.035（±0.002） OD/min， and the SOD activity was calculated according to the following formula： SOD activity （U/g）=（0.071-Pyrogallol autooxidation rate/0.071）×100%÷5%×（Total volume of system/Volume of reaction liquid）×Dilution ratio
Determination of MDA content in mouse kidney
The coomassie brilliant blue method was used to determine the protein content in mouse kidney homogenate. The standard curve was prepared with bovine serum albumin as the standard product， and the regression equation was y=0.007 6x-0.007 6 （R2=0.992 8）. MDA was determined by the thio-barbituric acid （TBA） method[7]， and the absorbance was determined according to the MDA kit. The MDA content was calculated according to the following formula：
MDA content （nmol/mg prot）=[6.45×（A532-A600）-0.56×A450]÷Concentration of standard protein to be detected（mg/ml）.
Results and Analysis
Isolation and purification of strains from fruits
Molds were isolated and purified from apples， red bayberries， pears， oranges， peaches and other fruits， and 8 different strains were preliminarily identified through observation of colony characteristics， and named P2， P3， L3， L4， J2， J3， J4， and T8， respectively. The specific culture characteristics are shown in Table 1.
Screening and identification of highly virulent strains
It can be seen from Table 2 that the death rate of mice exposed to the toxins from strains P2 and P3 isolated from red bayberries was 100% at 22 h， which was significantly higher than that of other strains. Because the death rate of mice exposed to the toxin from the P2 strain was 83.3% at 6 h， which was higher than the P3 strain， indicating that the P2 strain was the most virulent. The experimental results showed that the strains isolated from the contaminated fruits all produced toxins and all had certain toxicity， but the P2 strain isolated from red bayberries was significantly more toxic than other fruits， so this strain was used for the toxicity test.
The staining results of hyphae and spores of the P2 strain are shown in Fig. 1 and Fig. 2. The conidiophores of this strain are colorless and loose， unbranched， swollen at the top， and the conidia are clustered， and have thick membrane podocytes at the base. Combined with its culture characteristics， according to Fungus Identification Manual， the P2 strain was preliminarily identified as Aspergillus luchuensis of the Aspergillus niger group.
It can be seen from Table 3 that the death rates of mice injected at 0.625， 1.25， 2.5， 5， 10， 20， 40， 80， 160， and 320 mg/kg were 0， 12.5%， 25%， 37.5%， 25%， 37.5%， 62.5% and 100%， respectively. The death rates included 0 and 100%， which met the prerequisite for calculating the median lethal dose. Therefore， according to the calculation formula of the median lethal dose logLD50=Xk-i（∑p-0.5） （wherein Xk=2.505 1， i=0.301 0， ∑p=3.625 0）， the LD50 was calculated to be 36.683 9 mg/kg. Mouse bone marrow cell micronucleus test
Micronuclei appeared in mouse bone marrow cells at both 24 and 48 h after the exposure， and the morphology is shown in Fig. 3 and Fig. 4. The statistics of micronucleus rate （Table 4 and Table 5） showed that the micronucleus rates of the four toxin dose groups at 24 and 48 h were significantly lower than that of the cyclophosphamide positive control group （F= 4.937， P<0.01; F= 3.617， P<0.05）， but higher than the negative control group （dimethyl sulfoxide）， and the greater the toxin dose， the greater the micronucleus rate. The 48 h micronucleus rate was generally higher than 24 h， and the micronucleus rate of mice gradually increased with time. Micronucleus is the genetic material that remains outside the nucleus during the anaphase of mitosis due to chromosome breakage or influences on spindle fibers in the cell， and the micronucleus test can detect such two genetic end points as the chromosome integrity change and chromosome segregation change induced by chemical poisons or physical factors. Therefore， the experimental results showed that the P2 strain was genetically toxic and would cause certain damage to the chromosomes.
The effect of the toxin on mouse cell DNA
As can be seen from Fig. 5， numbers 1-5 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， and LD50/16 for 24 h， respectively， 6-10 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， LD50/16 doses for 48 h， respectively， and 11-15 are the DNA electrophoresis results of mice exposed to the control group， LD50/2， LD50/4， LD50/8， and LD50/16 for 72 h， respectively. The results showed that there were no ladder-like or diffuse bands in the DNA of mice after 24 h， 48 h and 72 h exposure， indicating that the toxin of strain P2 had no effect on the DNA within 72 h， which might be that it takes a certain time for the drug to induce apoptosis or necrosis.
Effect of the toxin on SOD activity in mouse kidney
The effect of the toxin on SOD activity in mouse kidney is shown in Fig.9. At 6， 24 and 48 h after the exposure， each concentration had an extremely significant effect on mouse SOD activity （F=227.838， P<0.01; F=193.373， P<0.01; F=80.3， P<0.01）， and each concentration had a significant impact on SOD activity 72 h after the exposure （F=6.414， P<0.05）. When the mice were exposed for 6 h， the SOD activity of each concentration was extremely significantly lower than that of the negative control， and the greater the concentration， the lower the SOD activity， indicating that the toxin would cause the decline of the kidney’s antioxidant capacity. After 6 h of exposure， the SOD activity of mice in the LD50/8 and LD50/16 groups began to rise gradually over time， and approached the control group at 72 h. The SOD activity of mice in the LD50/2 and LD50/4 groups decreased with time from 6 to 48 h， and both decreased to about 3 000 U/g at 48 h， but gradually recovered after 48 h and began to approach the control group. It indicated that the toxin had a significant inhibitory effect on the SOD activity of mouse kidney， and the higher the toxin concentration， the stronger the inhibition on the SOD activity of mouse kidney， which might be due to that the body had certain ability to resist and remove the toxin from the A. luchuensis P2 strain at low concentrations， and its recovery speed was greater than that at high concentrations. In addition， the inhibition decreased with the increase of time， which might be that mycotoxins had been excreted and detoxified from organs such as liver and kidney after a long period of metabolism between 48 and 72 h. Effect of the toxin on MDA activity in mouse kidney
The effect of the toxin on the content of MDA in mouse kidney is shown in Fig. 10. Each concentration after exposure for 6， 24， 48 and 72 h had an extremely significant effect on the content of MDA in mice （F=73.761， P<0.01; F=294.176， P<0.01; F=4207.476， P<0.01; F=941.435， P<0.01）. When the mice were exposed for 6 h， the MDA content of each concentration was significantly higher than the negative control， and the greater the concentration， the higher the MDA content， which indicated that the degree of membrane lipid peroxidation increased and the biological membrane structure was damaged. After 6 h of exposure， the MDA contents of the mice in the LD50/8 and LD50/16 groups did not fluctuate much over time， and both remained approximately 3-4 nmol/mg prot. The MDA contents of mice in the LD50/2 and LD50/4 groups increased significantly from 6 to 48 h with time， reached the highest values of 9.1 and 7.9 nmol/mg prot at 48 h， respectively， and gradually decreased after 48 h. It indicated that the toxin significantly enhanced the MDA content of mouse kidney， and the higher the toxin concentration， the greater the MDA content， which was opposite to the change in SOD activity， which might be that the body had certain ability to resist the toxin of A. luchuensis. In addition， the MDA content gradually decreased with the increase of time. It might be that mycotoxins had been excreted and detoxified from the liver and kidney and other organs after a long period of metabolism between 48 and 72 h， which was consistent with the SOD activity determination result.
Conclusions and Discussion
Fruits are very susceptible to mold contamination during long-term storage or under improper storage and transportation conditions to cause spoilage. For example， Wang et al.[2] showed that apples were susceptible to contamination by Penicillium， Aspergillus and other molds to cause spoilage; Zhang et al.[8] reported that citrus is susceptible to contamination by various Penicillium species such as P. digitatum and P. italia. In this study， red bayberry， pear， peach， orange and other fruits that are susceptible to mold contamination were selected as the isolation materials， and a total of 8 different strains were screened. After the toxicity test， it was found that they were all lethal to mice. Among them， the P2 strain from red bayberries showed the highest death rate， which was as high as 100%. After identification， the P2 strain was A. luchuensis of the A. niger group. It indicates that there are many kinds of strong pathogenic microbes in contaminated fruits， and eating the fruit contaminated by molds by mistake will have an adverse effect on the human body. Therefore， moldy fruits should attract people’s attention. Mycotoxins are toxic and harmful substances produced by molds. Mycotoxins can cause changes in enzyme activity， cell and DNA activity， gene function and other indicators in the body， which will adversely affect animal organs， immune systems， and nervous systems. We found that the A. luchuensis strain P2 isolated from red bayberries can cause the increase of PCE micronucleus rate in mice， the decrease of SOD activity in mouse kidney and the increase of MDA content， but it did not produce a significant effect on apoptosis in mice. Micronucleus is the genetic material that remains outside the nucleus during the anaphase of mitosis due to chromosome breakage or influences on spindle fibers in the cell， and micronucleus tests can detect such two genetic end points as the chromosome integrity change and chromosome segregation change induced by chemical poisons or physical factors. For example， Song et al.[9] screened two strongly pathogenic mold strains from contaminated fruits and performed mouse PCE micronucleus tests on them. The results showed that their mycotoxins could increase the PCE micronucleus rate of mice， which was significantly higher than the negative control group. This study also found that the A. luchuensis strain P2 derived from red bayberries increased the micronucleus rate of mouse bone marrow. The micronucleus rate of each concentration was significantly higher than that of the negative control group （P<0.05）， and the greater the concentration， the higher the micronucleus rate. Moreover， with the prolongation of time， the micronucleus rate of each concentration increased. It indicated that mycotoxins caused certain damage to mouse chromosomes and were genetically toxic， and with the increase of concentration and time， the toxicity to the body got stronger. Ladder DNA electrophoresis is a classic biochemical characteristic of cell apoptosis[10]. The results of this study showed that there were no obvious apoptosis or necrosis bands in the DNA electrophoretogram after exposure for 24-72 h， which might be that although the toxin of strain P2 damaged the cells， the nucleus was still intact and the chromatin was only in a concentrated state rather than fragmented. Lei et al.[11] also found in the study of the toxic mechanism of microcystin damage to liver cells that even though the cell membrane vesiculation had been very severe， the nucleus was still intact， the chromatin was only in a concentrated state rather than fragmented， and cells undergoing apoptosis did not necessarily undergo DNA degradation. SOD is the only enzyme in the organism that can scavenge O-2 free radicals， and the decrease in SOD activity can lead to the decline of the body’s antioxidant capacity， which will cause O-2 accumulation[12]. The accumulated oxygen free radicals can attack the polyunsaturated fatty acids in the biological membrane， trigger lipid peroxidation， generate a large amount of MDA， and seriously damage the body’s tissues and organs[12]. Guo et al.[13] found that the toxin from P. cyclopium， penicillic acid， could reduce SOD activity and increase MDA content in mouse kidney tissues， showing a dose-related relationship， which is similar to the results of this study. After 6 h of exposure， the SOD activity of each concentration was significantly lower than that of the negative control， the greater the concentration， the lower the SOD activity， and the two were in a dose-effect relationship. The MDA content was opposite to the change of SOD activity. As the toxin concentration increased， the MDA content increased significantly， indicating that lipid peroxidation damage was aggravated. Moreover， we found that as time increased， the SOD activity gradually increased， and the MDA content gradually decreased. It was speculated that mycotoxins had been excreted from the liver and kidneys and other organs after a long period of metabolism between 48 and 72 h， and the body was in a state of recovery. Based on the above analysis， different fruits will be contaminated by molds and cause damage to the body. In this study， the P2 strain of A. luchuensis isolated from red bayberries caused the PCE micronucleus rate to increase， the kidney SOD activity to decrease and the MDA content to increase. Therefore， mold-contaminated fruits can damage the human kidneys and cause intracellular chromosome breakage when eaten by mistake， and thus have certain genetic toxicity. However， the effects of this strain on the cell cycle and genes and the physiological and biochemical properties of its toxin need further research.
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