dna-damaging activity of patulin in escherichia · dna strand breaks by patulin in e. coli 1047...

Vol. 52, No. 5APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1986, p. 1046-10540099-2240/86/111046-09$02.00/0Copyright © 1986, American Society for Microbiology

DNA-Damaging Activity of Patulin in Escherichia coliKIL-SOO LEEt AND ROBERT J. ROSCHENTHALER*

Institute of Microbiology, University of Muenster, D4400 Muenster, Federal Republic of Germany

Received 5 May 1986/Accepted 29 July 1986

At a concentration of 10 ,ug/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli.At 50 ,ug/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of10 ,ig of patulin per ml within 90 min when the cells were incubated at 37°C in M9-salts solution without acarbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophageof Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at10 ,ug/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strandbreaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesiswas less affected, and overall protein synthesis was not inhibited at 10 ,ug/ml. Only at higher concentrations(250 to 500 ,ug/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a

mycotoxin with selective DNA-damaging activity.

Patulin [4-hydroxy-4H-furol(3,2c)pyran-2(6H)-one], a sec-ondary metabolite produced by strains of Penicilliumpatulum and some other species of the genera Penicillium,Aspergillus, and Byssochlamys, is frequently found in highconcentrations in rotten apples and other fruits (5, 21, 23). Ithas been repeatedly demonstrated to be an important con-taminant of such fruit products as apple juice and cider (5,21, 23, 34).

Originally isolated as an antibiotic in the early 1940s (4),patulin did not fulfill the expectations placed upon it becauseof its toxicity, and it was subsequently classified as amycotoxin. One of the reasons for its occurrence in applejuice and cider is its stability at the lower pH of suchproducts. Under neutral or alkaline conditions it degradesrapidly (4).An unsaturated ot,4-lactone, patulin reacts readily with

sulfhydryl (SH) groups and more slowly with NH2 groups(23, 35; our observations). It might therefore interfere withmany cell functions, and the toxic effect of patulin in anorganism may depend on the concentration, the mode ofapplication, the presence or absence of any SH group-containing compounds, the pH, and other factors. A cleardefinition of the primary site of action may be quite difficultunder these circumstances.The number of different kinds of proteins or enzymes in an

organism such as Escherichia coli is estimated to be on theorder of 1,050 (11) and is still higher in eucaryotes. Many ofthese proteins may be inactivated by different concentra-tions of agents like patulin reacting with SH groups. Thus, anattempt to determine the most sensitive enzyme would likelybe fruitless, especially as the most sensitive enzyme neednot be an essential one and the enzyme may change witheach species of organism.

Since patulin is a food contaminant, another questionwhich concerns the carcinogenic or mutagenic activity is ofutmost importance. Unfortunately, this question also seemsdifficult to answer definitely. In the literature on patulin, asreviewed by Scott (21, 23) and Ciegler (5), several examplesof positive and negative results relating to the mutagenicityand carcinogenicity are presented. Thus, e.g., Umeda et al.

* Corresponding author.t Present address: Department of Biology, Hallym University, 1

Okchong-Dong, Chunchon, Kangwondo, 200, Republic of Korea.

(28, 29) were able to demonstrate DNA single- and double-strand breaks induced by patulin in HeLa and FM3A culturecells, whereas Becci et al. (2) have failed to showtumorigenic activity when patulin was administered orally torats in long-term experiments. Also in bacteria, the mutage-nicity test with Salmonella typhimurium TA98 and TA100 isnegative (14, 32), whereas the Rec test according to Uenoand Kubota (26) is positive (26; our observations).We attempted to investigate the hitherto unanswered

question of mutagenicity or DNA-attacking ability ofpatulin, using methods likely to provide data with which invivo and in vitro effects could be compared. By usingalkaline sucrose gradient analysis, in vivo DNA strandbreaks could be demonstrated directly. The lambda proph-age induction test gave indirect indications for DNA damagein living cells, whereas the effects on the syntheses ofpermeabilized cells allowed in vitro analysis of patulin actionon DNA replication, DNA repair, and the different steps ofprotein synthesis. We found a concentration correlation inE. coli of in vivo and in vitro DNA-attacking ability ofpatulin but no such correlation with inhibition of RNA andprotein syntheses at comparable concentrations. This sug-gests selective DNA-damaging activity of patulin.

(This report represents a portion of a thesis submitted byK.-S.L. to the Faculty of Natural Sciences, University ofMuenster, Muenster, Federal Republic of Germany, in par-tial fulfillment of the requirements for the Dr. rer. nat.degree.)

MATERIALS AND METHODS

Chemicals. The preparation of patulin was performedessentially as described by Norstadt and McCalla (19),except that instead of potato dextrose medium (Difco Lab-oratories) commercial apple juice was used and inoculatedwith 1.2 x 103 spores of P. patulum NRRL 5259 per ml.After incubation at room temperature patulin was extractedwith ethyl acetate from the apple juice culture, and forpurification alumina columns (2 by 15 cm; pH 4.5) wereused. Patulin was eluted with and crystallized from ethylether.Other samples of patulin were donated by H. K. Frank, V.

G. Engel, and K. Hofmann, and two patulin samples werepurchased from Sigma Chemical Co. and Serva.

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DNA STRAND BREAKS BY PATULIN IN E. COLI 1047

Purity was tested by thin-layer chromatography and de-tection by methyl-3-benzo-thiazolinone-2 reagent (24), bybioautography, by calculating the absorbance ratio(A276/A260), and by high-performance liquid chromatography,using a ,u-Bondapack CN column (Waters Associates) and0.1 M citrate-0.2 M disodium phosphate buffer (pH 6.0) forelution. All samples were tested for the prophage lambdainduction in E. coli X8011. Only those which showed apositive effect were used.

All other chemicals mentioned were from E. Merck AG,Sigma, or Serva and were of the purest available grade.Organisms and media. For the production of patulin the

toxigenic strain P. patulum NRRL 5259, obtained from A.Ciegler, was used. Instead of potato dextrose medium(Difco) as liquid medium for this strain, commercial applejuice was used. Static cultures were incubated at roomtemperature (ca. 22°C). The yields of patulin with apple juicewere 3 mg/ml, as opposed to 1 mg/ml with potato dextrosemedium.The bacterial strains used were as follows: E. coli D110,

polA endA Thy-, obtained from K. Muller; E. coli X8011,F- Alac-169 B1-(X cI857 h80 dlac) (X cI857 h80) originallyfrom J. Beckwith. This strain was also inducible by UV260and by mitomycin C. E. coli MRE600 was from our labora-tory collection. An E. coli X8011 strain as an indicator wasobtained at the nonpermissive temperature of 42°C and afterseveral recultivations of lac survivors at 42°C. Two isogenicstrains were designated as E. coli X8011(X) and E. coliX8011X-.The lysogenic Bacillus megaterium strain NRRL-B 6395

and its indicator strain NRRL-B 6394 were obtained fromL. K. Nakamura (Northern Regional Research Laboratory).These are the strains used previously by Whittaker andChipley (33) to demonstrate phage induction by aflatoxin B1.Both strains were grown in TGY medium (33).For experiments with alkaline and neutral sucrose gradi-

ent analysis, M9 medium (1) was supplemented with thymine(20 pug/ml) forE. coli D110. The induction of prophages in E.coli X8011 was carried out in 2YT medium (16). The plaquetests were done according to the double-layer technique ofAdams (1) with diagnostic sensitivity test agar (Oxoid Ltd.)as bottom agar and 0.6% soft agar. Bacteria for the prepa-ration of permeabilized cells were grown in M3 medium(Difco).

Analysis of DNA strand breaks by sucrose gradient centrif-ugation. The analysis of single-strand breaks in bacteriatreated with 10 ,ug of patulin per ml for 60 min was performedaccording to the method of McGrath and Williams (15) byradioactive labeling of the bacterial DNA, preparation ofprotoplasts, and lysis on 5 to 20% alkaline sucrose gradients(pH 12). Centrifugation was done at 100,000 x g for 2 h in anIEC-B60 ultracentrifuge. After fractionation of the gradientsthe radioactivity of the trichloroacetic acid-precipitable ma-terial was counted on fiber glass filters. The results arepresented as percentage of radioactivity in each fraction ofthe total acid-precipitable radioactivity.As a negative control, samples not treated with patulin

were run in parallel, and for a positive control, sampleswithout patulin were irradiated before analysis with UV254(1.2 J m2 s-1) for 30 or 60 s as indicated.

Neutral sucrose gradients were performed entirely by themethod of Krasin and Hutchinson (13). They were run for 1h at 100, 000 x g. For investigation of in vivo DNA repair inthe presence of 10 ,ug of patulin per ml, the bacteria werewashed and irradiated with UV254 in an M9-salts solutioncontaining 20,ug of thymine per ml and 37 mM MgSO4. The

irradiated bacteria were incubated in the presence of patulinat 37°C in the dark. Samples were withdrawn at differenttimes and analyzed by the alkaline sucrose gradient tech-nique.

Induction of lysogenic bacteria. A prophage X c1857tsinduction test similar to that of Ho and Ho (10) was usedwith the exception that the host was not an E. coli envA uvrBmutant but E. coli X8011(A) and the plaque-counting tech-nique with E. coli X8011X- as indicator for the estimation ofliberated phage was used. Also, in our experiments patulinwas not treated with an S-9 liver fraction for metabolicactivation.The lysis of patulin-, UV254-, and temperature-induced

cultures of E. coli X8011(X) was checked by optical densitymeasurements at 546 nm in an Eppendorf photometer.The ratio of phage counts from patulin-treated cells (T) to

phage counts from control cells (C) was calculated accordingto HIeinemann (9) except that the phage counts per milliliterwere related to the cell counts per milliliter as determinedwith a Helber chamber. This was done because the controlculture of E. coli X8011(X), having a constant spontaneousinduction rate yielding 1 plaque per 5 x i05 cells, wouldexceed the number of bacterial cells in the patulin-inhibitedculture by several orders of magnitude. In the case ofinduction the highest cell counts before the onset of lysiswere placed in relation. Thus, our T/C ratio gives rather apessimistic estimate of how many more phages per cell wereproduced in the treated culture compared with the control.

Permeabilized cells. Permeabilized E. coli cells are in vitrosystems which allow DNA and RNA syntheses to proceed ifthe respective substrates are provided externally (12, 17).We applied toluenized cells obtained according to the pro-cedures of Moses and Richardson (17) and Staudenbauer(24) and plasmolyzed cells by the method of Ben-Hamidaand Gros (3). Both systems allow for distinction of replica-tive DNA synthesis from DNA repair synthesis. The replica-tive synthesis occurred in the presence of the fourdeoxyribonucleotide triphosphates (0.02 mM), ATP (2 mM),and NAD (0.1 mM), whereas the repair synthesis requiredno ATP and NAD. The addition of DNase I or patulin to thecells and a PolA+ strain (E. coli MRE600) were necessary forthe demonstration of repair.RNA synthesis can also be measured in both systems if

the four ribonucleotide triphosphates (0.5 mM) are added.However, protein synthesis occurred in plasmolyzed cellsonly. It was dependent on the four ribonucleotide triphos-phates (0.5 mM) and on the 20 amino acids (0.25 mM),phosphoenolpyruvate, and phosphoenolpyruvate kinase. Inplasmolyzed cells mRNA synthesis can be distinguishedfrom synthesis of stable RNAs as the latter only occurs inthe presence of protein synthesis.For the measurements one of the nucleotides or amino

acids was always replaced by a radioactive one, and theincorporation of radioactivity into acid-precipitable materialwas measured in a liquid scintillation counter after precipi-tation with trichloroacetic acid on glass-fiber filters.

RESULTS

DNA strand breaks in vivo. In previous experiments (notshown) with three different bacterial species (E. coli, B.subtilis 168, B. stearothermophilus), we found that in eachspecies in vivo DNA synthesis was inhibited most stronglyby patulin, whereas RNA synthesis was somewhat lessinhibited. The severity of protein synthesis varied with therespective species. Protein synthesis was strongly inhibited

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FRA C TIONS fRAC TIONSFIG. 1. Sedimentation profiles of the DNA from E. coli D110 polA in alkaline (A and B) and neutral (C and D) sucrose gradients. (A)

Negative and positive control without patulin: 0, control cells; 0, cells irradiated with UV254 for 30 s. (B) Cells treated with patulin for 60min: O, 10 ,g/ml; A, 20 ,ug/ml. (C) Cells treated with 25 ,ug of patulin per ml for 60 min. (D) Cells treated with 50 ,ug of patulin per ml.

in E. coli, less so in B. stearothermophilus, and the least inB. subtilis.The sedimentation profiles of the chromosomal DNA of

repair-deficient (polA) E. coli D110 cells in alkaline sucrosegradients indicated single-strand breaks of DNA when thecells had been treated with 10 to 20 ,ug of patulin per ml. Theshift towards smaller DNA fragments was more pronouncedwith the higher concentration (Fig. 1B). At doses of 50 ,ug/mlalso, double-strand breaks could be demonstrated in neutralsucrose gradients (Fig. 1D). Thus, the DNA-damaging activ-ity of patulin was dose dependent.

Repair of DNA single-strand breaks in vivo. Single-strandbreaks were induced by UV254 irradiation of E. coli MRE600cells, and repair in the presence of 10 ,ug of patulin per mlwas analyzed by alkaline sucrose gradient centrifugation atdifferent times. Figure 2B shows the sedimentation profilesafter repair incubation of the cells in M9 solution without acarbon source at 37°C for 90 min. At this time the DNArepair was as complete as that of the control without patulin.Thus, no irreversible inhibition of DNA repair occurred.

Induction of prophage in lysogenic bacteria. E. coli cellslysogenic for the wild-type lambda phage could not beinduced by patulin.However, E. coli X8011(X), a temperature-sensitive, dou-

ble lysogenic strain was induced by patulin when incubatedat 35°C, a temperature below the inducing temperature. Thisstrain lysed with 10 p.g of patulin per ml (Fig. 3A), whereasthe isogenic strain E. coli X8011A- showed only growthinhibition at the same concentration (Fig. 3B). Virtually thesame curve was obtained when in addition to 10 ,ug of patulinper ml, 20 pLg of cysteine per ml was added to E. coliX8011(A), indicating inactivation of patulin by cysteine.

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FIG. 2. Sedimentation profiles of DNA from E. coli MRE600cells after irradiation with UV254 for 60 s. Alkaline sucrose gradientanalysis. (A) Negative and positive controls: 0, DNA from un-treated control cells; A, cells irradiated with UV254 for 60 s, norepair incubation. (B) Repair incubation in M-9 salts solution for 90min: *, without patulin; EJ, in the presence of 10 ,ug of patulin perml.

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INCUBA TON TIME(h)FIG. 3. Effect of patulin on growth of lysogenic E. coli X8011(X) and isogenic E. coli X8011 X-. (A) Effect of different concentrations of

patulin on E. coli X8011(X): 0, control; A, 5 Fg/ml; *, 10 ,ug/ml; A, 25 ,ug/ml; O, 50 Fxg/ml. (B) Effect of patulin on E. coli X8011 X-: 0,control; *, with 10 1kg of patulin per ml. OD, Optical density.

megaterium strain used previously by Whittaker andChipley (33) for testing prophage inducibility by aflatoxin B1.High concentrations of patulin are also able to cause lysis

in nonlysogenic strains, presumably by inhibition of pepti-doglycan synthesis (our observations). Therefore, it re-mained to be demonstrated that the lysis observed with thelysogenic strain could be attributed to the liberation ofphages. At 10 ,ug of patulin per ml an increase in the phagetiter and a simultaneous decrease in the Helber chamber cellcounts occurred, showing that the lysis could be ascribed tophage development and liberation. At concentrations above20 ,uglml, however, phage production was inhibited.To estimate the efficiency of patulin in the inductiQn of

prophage, we used the T/C ratio similar to the method ofHeinemann (9). These values show how many more phagesper cell are set free in the presence of patulin than in thecontrol. With 10 ,ug of patulin per ml at least 35 times more

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In vitro replicative DNA synthesis in permeabilized cells. Toavoid effects of patulin brought about by regulatory mecha-nisms in living cells, toluenized and plasmolyzed cells wereused. These are regarded as in vitro systems (3, 6, 12, 17).

Patulin at 10 ,ug/ml inhibited the replicative DNA synthe-sis. The inhibition was dose dependent in the concentrationrange of 10 to 50 ,ug/ml (Fig. 5). This inhibition was found intoluenized and plasmolyzed cells of E. coli D11O (PolA-) andMRE600 (PolA+). In E. coli D110 DNA synthesis ceased 30min after addition of patulin, whereas in E. coli MRE600 theinhibition was less pronounced and DNA synthesis contin-ued linearly for at least 100 min, albeit at a clearly reducedrate (not shown). As the inhibition kinetics resemble verymuch those of nalidixic acid, an indirect action on replicativeDNA polymerization is suggested.

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INCUBA TION TIIIE(h)FIG. 4. Release of phage after patulin treatment of E. coli X8011(A). (A) Dose dependence of phage release after 2 h in the presence of

different concentrations of patulin. Cells per milliliter were counted in a Helber chamber; phage titer per milliliter was determined by plaquecounting (1). (B) Ratio of phages per cell in a culture treated with 10 p.g of patulin per ml for various incubation times (T)/phages per cell inan untreated control culture (C).

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1050 LEE AND ROSCHENTHALER

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INCUBATION TIME (min)FIG. 5. In vitro DNA replication synthesis in toluenized (A and B) and plasmolyzed (C) cells of E. coli D110 (polA). (A) System control:

0, incorporation of 3H label from [3H]dTTP into acid-precipitable material; *, incorporation with 200 ,ug of nalidixic acid per ml present; A,without ATP; O, without ATP and NAD. (B) Incorporation in the presence of patulin: 0, control; 0, 10 ,ug/ml; A, 25 ,ug/ml; *, 50 ,ug/ml.(C) Incorporation in the presence of patulin: 0, control, plasmolyzed cells; O, in the presence of 10 ,ug of DNase I per ml; in the presenceof 0, 10 p.g, and A, 50 ,ug of patulin per ml.

In vitro DNA repair in permeabilized cells. The repairsynthesis mixtures containing E. coli MRE600 plasmolyzedcells were preincubated at 30°C for 60 min with patulin orDNase I before the synthesis reaction was started by addi-tion of radioactive thymidine triphosphate. The stimulationof repair synthesis by patulin was dose dependent, the higherdose being more stimulating. This may be interpreted as anindication of an increasing number of strand breaks at anincreasing concentration of patulin, leading to an increase inrepair synthesis. This would mean that patulin causes strandbreaks in vitro since in the absence of patulin or DNase Ionly little repair activity is observed, and these breaks arerepaired by an intact repair system (Fig. 6).With toluenized cells of E. coli MRE600 similar results

were obtained (not shown).In vitro RNA synthesis in permeabilized cells. Toluenized

and plasmolyzed cells of E. coli MRE600 were used. Inplasmolyzed cells the distinction between mRNA synthesisand synthesis of stable RNAs can be made (3), as the latteronly proceeds when protein is synthesized.At 10 jig of patulin per ml, the mRNA synthesis was

lowered by ca. 25% approximately 30 min after addition.However, the higher dose of 50 ,ug/ml caused less inhibition.Thus, this inhibition is not dose dependent. In toluenizedcells, in which the distinction between mRNA and stableRNA syntheses cannot be made, even a slight stimulation oftotal RNA synthesis by 50 ,ug of patulin per ml was repeat-edly observed (not shown). The meaning of these results arenot completely clear but as the rifampin system worked inthe expected manner, i.e., finishing already initiated chains

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FIG. 6. In vitro DNA repair synthesis in plasmolyzed cells of E.coli MRE600. The synthesis mixtures were incubated with patulinand DNase I at 30°C for 60 min. The synthesis reaction was startedby addition of [3H]dTTP at time zero. Symbols: 0, control; 0, 0.1,ug of DNase I per ml; *, 10 ,ug and *, 50 pug of patulin per ml.



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FIG. 7. Effect of patulin on in vitro synthesis of mRNA (A) and stable RNA (B) in plasmolyzed E. coli MRE600 cells. (A) Incorporationof 3H label from [3H]UTP into acid-precipitable material in absence of protein synthesis: 0, control; O, in the presence of 20 ,ug of rifampinper ml; with 0, 10 ,g and A, 50 p.g of patulin per ml. (B) Incorporation of 3H label into stable RNAs. The synthesis was started in absenceof protein synthesis at time zero. When the reaction came to a halt, additional [3H]UTP and 20 amino acids were added to allow proteinsynthesis. At that time also patulin was added. Symbols: 0, control; *, incorporation in the presence of 340 ,ug of chloramphenicol per ml;in the presence of 0, 10 ,g and A, 50 pg of patulin per ml.

and then blocking further synthesis, and as the synthesis ratewith patulin in the first 30 min does not differ significantlyfrom that of the control, it seems highly unlikely thatinhibition of mRNA synthesis is the only cause for growthinhibition in E. coli.The synthesis rate of stable RNAs was slightly more

inhibited by patulin concentrations in the range of 10 to 50,ug/ml. This synthesis was dependent on protein synthesis asis indicated by the effect of chloramphenicol.

In summary, patulin at 10 pug/ml inhibited the synthesis ofmRNA and stable RNAs (Fig. 7), although less effectivelythan DNA replication.

In vitro protein synthesis in plasmolyzed cells. For un-known reasons, only trace amounts of protein are synthe-sized in toluenized cells. However, plasmolyzed cells allowDNA-dependent in vitro protein synthesis (3).

Patulin at concentrations up to 50 ,ug/ml had no effect onoverall protein synthesis. Only at 250 to 500 ,ug/ml wasinhibition observed. This inhibition is much less severe thanthat caused by 200 ,ug of chloramphenicol per ml (Fig. 8).Although the inhibition of some specific protein cannot beexcluded, especially in view of some inhibition of mRNAsynthesis, the machinery of protein synthesis itself does notseem to be very sensitive to patulin.

DISCUSSION

Contradictory reports on DNA-damaging activity and onmutagenicity and carcinogenicity of patulin have appeared.A method by which DNA damage in living cells can bedirectly demonstrated is the analysis ofDNA by alkaline (15)and neutral (13) sucrose gradient centrifugation. Our resultswith E. coli clearly showed that patulin was able to causesingle-strand and double-strand breaks of the DNA in livingbacterial cells. The use of a repair-deficient (PolA-) mutant

in such an analysis is convenient because repair of thebreaks is much slower in strains lacking a functional DNApolymerase I.

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INCUBA TION TIME(min JFIG. 8. Effect of patulin on in vitro protein synthesis in

plasmolyzed cells of E. coli MRE600. Symbols: 0, control; A, in thepresence of 200 ,ug of chloramphenicol per ml; and 0, 5 ,ug; A, 50xLg; *, 250 ,ug; and O, 500 p.g of patulin per ml.

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Moreover, stimulation by patulin ofDNA repair synthesisat very low patulin concentrations in permeabilized cells hasto be interpreted as an indication for strand breaks in theDNA. Repair synthesis is only initiated if the DNA containsnicks (12). These can be brought about by DNase I as well asby patulin.

Single- and double-strand breaks of DNA caused bypatulin have also been reported in FM3A culture cells byUmeda et al. (28) and in HeLa cells by Umeda et al. (29).One report in which the inhibition zones caused by patulin

in an agar plate test were compared presents contrastingfindings. The PolA+ strain of Salmonella typhimurium waseven more sensitive to patulin than was the PolA- mutant(35). In E. coli D110 (PolA-) we found that the diameters ofthe inhibition zones at the same concentration of patulinwere twice as large as that of E. coli MRE600 (PolA+).There are controversial findings with other bacterial short-

term mutagenicity tests. In the Rec test of Ueno and Kubota(26) patulin was positive, whereas in the Ames test severalauthors reported negative results (14, 27, 32). We haverepeated the Rec test as has been described (26) and also inliquid medium at pH 6.0. In both cases the B. subtilis M45Rec- mutant was more sensitive than the isogenic Rec+strain when treated with >10 ,ug of patulin per ml, thusshowing a positive result.The reasons for negative results in such tests are usually

not known and most authors are concerned about thepossibility of false negatives (14, 32). Our results on theinduction of lambda prophage follow this line. For unknownreasons the wild-type phage could not be induced, whereasthe test according to Ho and Ho (10) with the temperature-sensitive prophage mutant was positive with patulin. Theinduction of prophage is usually interpreted as being indic-ative for DNA damage which induces the SOS repair sys-tem. One component of this system, the recA gene product,cleaves the lambda repressor and thus initiates the produc-tive cycle of the phage (30).

In the course of our work with this system we made anobservation which might partially explain the controversy onthe mutagenicity of patulin. Of seven different samples ofpatulin obtained from different sources or prepared our-selves, one sample was inactive in inducing the prophage,but otherwise it inhibited bacterial growth and DNA synthe-sis. Preliminary nuclear magnetic resonance and high-performance liquid chromatography analyses indicated purepatulin. We do not yet know whether this preparationconsisted of an isomer or an epimer, but isomers of somecarcinogenic substances such as benzo(e)pyrene have beenshown to be less mutagenic and cancerogenic (10). Thus, itwould be conceivable that using such a patulin preparationcould cause negative results in mutagenicity tests. All exper-iments reported here were done with patulin that was activein the induction of the lambda prophage mutant.The concentration necessary for in vivo inhibition ofDNA

synthesis varied somewhat with the patulin samples used.The most toxic one did not have the capacity to inducelambda prophage. Thus there seems to be no correlationbetween toxicity and the ability to induce lambda prophage,i.e., mutagenicity.Umeda et al. (29) found no repair of DNA damage caused

by patulin in HeLa cells. With E. coli MRE600 DNA repairin the presence of 10 ,ug of patulin per ml was completewithin 90 min. This discrepancy could simply reflect differ-ences in the repair systems. However, we found that theDNA fragments at 45 min of incubation were smaller than at30 min after addition of patulin and that only at 90 min of

incubation was coincidence of DNA sedimentation profileswith that of the control observed. This could mean that in thepresence of patulin DNA breakages and excision repair tookplace simultaneously until most of the patulin was inacti-vated by reaction with SH groups. Thereafter, the DNArepair may be completed. Thus, the discrepancy could be amatter of repair kinetics. Rihn et al. (20) describe a stimula-tion by low patulin doses of the incorporation of radioactivethymidine into DNA of hepatoma cells. This could probablybe explained in terms of DNA repair synthesis. Under invivo conditions, semiconservative DNA replication cannotbe distinguished from repair when measured by incorpora-tion of radioactive thymidine into DNA. Thymidine is incor-porated in both kinds of synthesis. The method of colorimet-ric measurements of increase in DNA content, on the otherhand, was too insensitive to allow a clear distinction.As the DNA repair was demonstrated in E. coli in the

presence of patulin, the repair system itself should not beirreversibly inhibited. Incubation of the intact cells duringDNA repair occurred in the absence of complete growthmedium and, therefore, the repair process should have beenlargely dependent on DNA polymerase I (36). As thisenzyme is also involved in DNA replication, our resultssuggest that the inhibition of patulin of replication is proba-bly not due to inhibition of the replicative enzyme system,but is rather a consequence of the DNA breakages inflictedby patulin, i.e., a secondary effect.A correlation exists between the patulin concentration

which causes in vivo and in vitro effects. In vivo, 5 to 10 jigof patulin per ml reduces the growth rate in E. coli (and B.subtilis), inhibits DNA synthesis, and produces single-strandbreaks of the DNA. In the in vitro systems the sameconcentration induced DNA repair synthesis and inhibitedsemiconservative DNA replication. The dose-dependent in-duction ofDNA repair can be interpreted as an indication ofan increasing number of nicks in the DNA (12) as the patulinconcentration is raised. This notion is in perfect agreementwith the dose-dependent increase in DNA strand breaks inintact cells as demonstrated by sedimentation analysis inalkaline and neutral sucrose gradients.

Furthermore, the same concentration of patulin inhibitsoverall incorporation of radioactive thymidine in vivo as wellas semiconservative DNA replication in permeabilized cells.This correlation would suggest that the in vivo inhibition bypatulin of growth and DNA synthesis may be attributed tothe induction of strand breaks in the DNA and to inhibitionof replication. This idea is supported by the fact that lowconcentrations of patulin (3 ,ug/ml) cause only a temporaryinhibition followed by noninhibited growth in E. coli (and B.subtilis). This could be explained by advancing inactivationof patulin and repair of DNA damage. Unfortunately nomutants exist which are completely deficient in DNA repair.Thus, in vivo the number of single-strand breaks cannot bedetermined in the complete absence of repair.However, it must be noted that these correlations are

probably not sufficient to answer the question of whether theDNA strand breaks cause cell death. It has long beensuspected that strand breaks may cause inhibition ofsemiconservative replication (6) or may be a "critical step onthe path to cell death" (31). But there are also reports whereeven UV-induced DNA strand breaks could not be attrib-uted to UV-lethality (25).

It has been shown that alkaline sucrose gradient condi-tions cause single-strand breaks in DNA containing apurinicsites (25). If apurinic sites in the DNA were the cause of thesingle-strand breaks produced by patulin, these sites would



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by mutagenic during replication and when repaired (30). Aswe have found that supercoiled ColEl plasmid DNA be-comes alkali and heat labile when treated with patulin invitro (unpublished observation), it would not be unlikely thatpatulin can depurinate DNA. However, further investigationin this field is needed.

It has been suggested that the primary target of patulinmay be the DNA-dependent RNA polymerase (18, 20; P.Thonart and J. Bechet, communication at the 4th IUPACSymposium on Mycotoxins and Phycotoxins, Lausanne,Switzerland, 1979). An in vitro effect on transcription hasbeen observed (18). We also found some inhibition of mRNAsynthesis in plasmolyzed cells and a much stronger inhibi-tion of total RNA synthesis in vivo. These results do notnecessarily conflict with our results on the DNA-attackingability of patulin, as one of the substrates of RNA polymer-ase is DNA. If the DNA is damaged, some effects on theactivity of RNA polymerase can also be expected. Cozarelli(6) pointed out that DNA synthesis inhibitors may alsoinhibit RNA synthesis.

Therefore, it cannot be completely ruled out that patulin atlow concentrations may inhibit the synthesis of some pro-teins in vivo, as there is some effect on mRNA synthesis. Inplasmolyzed cells, however, the rate of total protein synthe-sis was completely unaffected at 10 ,ug/ml, whereby theprotein-synthesizing system was not inhibited unless thepatulin concentration was raised to 250 to 500 jig/ml. In vivoand in vitro inhibition of protein synthesis has been de-scribed in rat liver and in a reticulocyte lysate protein-synthesizing system (7, 8).Thus, we found no in vivo-in vitro concentration correla-

tion of patulin with RNA and protein syntheses at lowconcentrations.The purpose of our experiments, however, was to find out

whether patulin is able to react with DNA or with thereplicative apparatus. Patulin induced DNA damages in E.coli. There was a concentration correlation between the invivo effects on DNA and in vitro DNA repair and inhibitionof replication. As these correlations were not observed withRNA and protein syntheses, a selective action of patulin onDNA is suggested.The notion that patulin may be inactivated on the surface

of living cells by reaction with SH groups is not supported byour results. Preliminary findings on the mechanism of theDNA strand breakages suggest an involvement of free radi-cals in this process.

ACKNOWLEDGMENTS

We thank H. K. Frank, V. G. Engel, and K. Hofmann forgenerous gifts of patulin and L. K. Nakamura for the B. megateriumstrains. We are also very grateful to J. Jeep for helping us to improveour English.We thank the Deutsche Forschungsgemeinschaft for supporting

this work.

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dna-damaging activity of patulin in escherichia · dna strand breaks by patulin in e. coli 1047...

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