Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.     

Teratogenicity of patulin and patulin adducts formed with cysteine.

The mean lethal dose of patulin for the chicken embryo injected in the air cell before incubation was determined to be 68.7 mug and that for the 4-day-old embryo was 2.35 mug. Both patulin (1 to 2 mug/egg) and the reaction mixture between patulin and cysteine (15 to 150 mug of patulin equivalents) were teratogenic to the chicken embryo. At least two ninhydrin-negative and four ninhydrin-positive products were formed during the latter reaction. Our explanation of the reaction mechanism remains to be elaborated.     

Teratogenicity of patulin and patulin adducts formed with cysteine.

The mean lethal dose of patulin for the chicken embryo injected in the air cell before incubation was determined to be 68.7 mug and that for the 4-day-old embryo was 2.35 mug. Both patulin (1 to 2 mug/egg) and the reaction mixture between patulin and cysteine (15 to 150 mug of patulin equivalents) were teratogenic to the chicken embryo. At least two ninhydrin-negative and four ninhydrin-positive products were formed during the latter reaction. Our explanation of the reaction mechanism remains to be elaborated.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Use of activated charcoal for the removal of patulin from cider.

Penicillium urticae (NRRL 2159A) was grown in culture broth containing 1 muCi of [1-14C-A1acetate to produce [14C]patulin. [14C]patulin was purified from the broth and added to apple cider. After the patulin concentration of the cider was adjusted to 30 mug/ml with unlabeled patulin, the cider was subjected to various charcoal treatments. [14C]patulin was completely removed by shaking the cider with 20 mg of activated charcoal per ml and by eluting the cider through a 40- to 60-mesh charcoal column. Activated charcola at 5 mg/ml reduced patulin in naturally contaminated cider to nondetectable levels.     

The lack of mutagenic properties of patulin and patulin adducts formed with cysteine in Salmonella test systems.

The mutagenic properties of patulin and the patulin adducts formed with cysteine were tested with histidine auxotroph Salmonella typhimurium strains as indicator organisms. The tests were performed by microsomal activation and host-mediated assay. Neither patulin nor patulin--cysteine reaction mixture was mutagenic in these test systems.     

Identification of phyllostine as an intermediate of the patulin pathway in Penicillium urticae.

A patulin negative mutant (J1) of Penicillium urticae (NRRL 2159A) was found to accumulate large quantities (greater than 128 mg/L culture) of a reactive, photosensitive compound, which was isolated and identified as (-)-phyllostine (5,6-epoxygentisylquinone). This epoxyquinone possessed an antibiotic activity against Bacillus subtilis which was approximately 80% of that exhibited by patulin. In separate in vivo feeding experiments, [2-14C]acetate and [G-3H]gentisaldehyde were readily incorporated into phyllostine by mutant J1 and [14C]phyllostine was incorporated into patulin by the parent strain (NRRL 2159A). When fed to a washed-cell suspension of a second patulin negative mutant (J2) which produced gentisaldehyde but not phyllostine, unlabeled phyllostine was efficiently converted to patulin in yields of 33, 56, and 92% after 30 min, 1 and 5 h, respectively. The role of phyllostine as an intermediate of a new post-gentisaldehyde portion of the patulin biosynthetic pathway is discussed.     

Isolation of Haemophilus influenzae genes that suppress Escherichia coli polA mutations

Haemophilus influenzae was found to produce a DNA polymerase that was similar to polymerase I of Escherichia coli. E. coli polA mutants were used as backgrounds for the selection of H. influenzae polA suppressor genes. Six different H. influenzae fragments were isolated that could suppress E. coli polA mutations. None of the suppressors appeared to encode the H. influenzae equivalent of the E. coli polA gene. One type of clone, represented by pGW41, caused a polymerase I activity to appear in a suppressed polA1 mutant. Plasmids from the pGW41 class contained two genes (pol-2 and pol-3) that were both required for polA suppression. Mutated nonsuppressing derivatives of the pGW41 class were used to create H. influenzae mutants that were deficient in polymerase I.     

Disappearance of patulin during alcoholic fermentation of apple juice.

Eight yeast strains were used in three typical American processes to ferment apple juice containing 15 mg of added patulin per liter. Patulin was reduced to less than the minimum detectable level of 50 microgram/liter in all but two cases; in all cases, the level of patulin was reduced by over 99% during alcoholic fermentation. In unfermented samples of apple juice, the concentration of added patulin declined by only 10% when the juice was held for 2 weeks, a period equivalent to the time required for fermentation.     

Disappearance of patulin during alcoholic fermentation of apple juice.

Eight yeast strains were used in three typical American processes to ferment apple juice containing 15 mg of added patulin per liter. Patulin was reduced to less than the minimum detectable level of 50 microgram/liter in all but two cases; in all cases, the level of patulin was reduced by over 99% during alcoholic fermentation. In unfermented samples of apple juice, the concentration of added patulin declined by only 10% when the juice was held for 2 weeks, a period equivalent to the time required for fermentation.     

An evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity. A report of the U.S. EPA's Gene-TOX Program

The detection of DNA-damaging agents by repair-deficient bacterial assays is based on the differential inhibition of growth of repair-proficient and repair-deficient bacterial pairs. The various methodologies used are described and recommendations are made for their improved use. In a survey of the literature through April 1979, 91 of 276 papers evaluated contained usable data, resulting in an analysis of 611 compounds that had been assayed in 1 or more of 55 pairs of repair-proficient and repair-deficient strains. The results indicate that (1) a liquid suspension assay is more sensitive than a spot (diffusion) test. In a review of the Escherichia coli polA assay, 45 compounds that gave "No Test" in the spot test were clearly positive or negative in the liquid suspension assay. (2) Of the 21 compounds analyzed by the E. coli polA assay and by other E. coli repair-deficient strains (e.g., rec, uvr, hcr, and exr derivatives of WP2 and AB1157), 10 were in complete agreement in all strains except uvrA strains. This indicates that strains other than polA+/polA- are useful for detecting DNA-damaging agents. However, in selecting strains for use in these assays, care should be taken to consider repair pathway specificity for particular compounds. (3) There was a 78% correspondence between results obtained with E. coli polA and Bacillus subtilis (H17/M45, 17A/45T) rec assay and between E. coli polA and Proteus mirabilis. (4) In a comparison of test results with carcinogenicity data, 44 of 71 (62%) carcinogenic compounds assayed by the polA system were positive, 10 (14%) were negative, and 17 (24%) gave No Test or doubtful results. 7 carcinogens were assayed by other E. coli strains and all were positive. 56 carcinogens were assayed in B. subtilis: 24 (43%) were positive, 9 (16%) were negative, and 23 (41%) gave No Test or doubtful results. Of the 7 carcinogens assayed in P. mirabilis, 6 (86%) were positive and 1 (14%) was negative. (5) The results were analyzed with respect to chemical classes. E. coli polA detected the highest percentage of hydroxylamines and alkyl epoxides. The B. subtilis rec assay detected the highest percentage of nitrosamines and sulfur and nitrogen oxides. It is concluded that some of these test systems are effective tools for the detection of DNA-damaging and potentially carcinogenic compounds, especially if the assay is done in liquid suspension and if more than 1 pair of tester strains is used. Advantages and disadvantages of the assay are discussed and suggestions are made for improvements in the system.     

Growth and patulin formation byPenicillium urticae Bainier in pure and mixed cultures

Penicillium urticae Bainier synthesized patulin in potato-dextrose medium at temperatures ranging from 5 to 30°C. Maximum patulin yield was 2700 μg/ml of culture fluid in 14 days at 25°C. Two distinctive intervals affected patulin formation: 15 to 20°C and 30 to 35°C, the former favorable and the latter detrimental. An incubation period of 11 to 14 days made a nonsterile mixture of weathered wheat straw and soil a favorable medium for patulin formation. Autoclaved weathered wheat straw, inoculated withP. urticae alone, or in combination withTrichoderma sp., was a medium comparable to nonsterile, incubated weathered wheat straw in soil. Both carbon source and accessory growth factors were important for patulin formation. Of seven media tested, potato-dextrose was superior to potatodextrose supplemented with 70 ppm Zn-ions and 16 ppm Fe-ions, potatosucrose, Raulin-Thom, autoclaved weathered wheat straw in pure culture, weathered wheat straw in nonsterile soil, and autoclaved weathered wheat straw in mixed culture, in that order. Patulin production ranged from 337.5 to 0.2 mg/g of C in the medium. Contribution from the Northern Plains Branch, Soil and Water Conservation Research Division, Agricultural Research Service, U.S. Department of Agriculture, in cooperation with the Nebraska Agricultural Experiment Station, Lincoln. Published as Paper No.2621, Journal Series, Nebraska Agricultural Experiment Station.     

Effects of patulin on thymus capillary of rats

Patulin is a mycotoxin that is produced by species of Penicillum, Aspergillus, and Byssochylamys molds that may grow on a variety of foods including fruit, grains and cheese. Patulin, at a dose of 0.1 mg kg(-1) bw day(-1) was administered orally to growing male rats aged 5-6 weeks for a period of 60 or 90 days. The dose of patulin used in the present study was based on estimated human exposure levels. At the end of these periods, the thymus glands of patulin-treated and control Wistar rats were removed and ultrastructural changes in capillary cells of the thymus of patulin-treated Wistar rats were determined by electron microscopy. The walls of thymus capillaries of the 60-day patulin-treated rat groups (P-60) exhibited degeneration observable in electron microscopic sections. For example, loss of cytoplasm and mitochondrial cristae of cells, swollen endothelial cells, increased thickness of the basement membrane, closed lumen of capillaries, accumulation of fibrous material at the periphery of the capillaries and nuclear anomalies were seen in these sections. Such degeneration and changes were also observed in sections of capillaries of the 90-day patulin-treated rat groups (P-90). The levels of degeneration of endothelial cell nucleus of P-90 were greater than those of P-60. This study demonstrated the ultrastructural degeneration of thymus capillary cells of patulin-treated rats. The results obtained from this study may provide a guide to research dealing with the toxic effects of patulin on tissue and organ ultrastructure.     

X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T-->T:A transversion and G:C-->T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.     

Effect of preservatives on patulin production by Penicillium expansum.

Penicillium expansum has been grown on Capek-Dox medium using glucose and fructose as carbon source. Preservatives used in fruit processing and introduced in the medium were sorbic acid, formic acid, benzoic acid, SO2 and saccharose. Sulphur dioxide had a most inhibitory effect on mycelium growth and patulin production, formic acid concentration of 0.025% increased the amount of patulin by about 30% as compared to the culture with no preservatives. However its higher concentrations inhibited synthesis of this mycotoxin. Sorbic acid concentration of 0.1% stimulated the fungus strains examined in patulin synthesis but its highest amounts were detected using 0.0125% benzoic acid increased patulin secretion from 8 to 50% as compared to the control, depending on the strain examined. Saccharose concentration up to 50% clearly decreased patulin content in the medium until its total disappearance.     

Intergeneric conjugational crossing of Escherichia coli with Salmonella typhimurium. II. Transfer of a polA1 mutation from Escherichia coli to Salmonella typhimurium and its phenotypic expression in the salmonella genome]

The Escherichia coli structural gene for DNA polymerase I was inserted into Salmonella typhimurium chromosome by conjugal transfer. The genetic analysis of P1-mediated transduction of obtained hybrid showed that polA gene is located in it between metE and rha loci and is cotransduced with metE (about 50%) and rha (12%). The phenotypic properties of polA1 hybrid E. coliXS. typhimurium concerning UV-MMS-NG and gamma-ray sensitivity are similar to the polA1 mutants of E. coli.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.