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[CANCER RESEARCH 28, 2296-2311, November 1968]

Mycotoxins in Fermented Food1

R. Kinosita, T. Ishiko, S. Sugiyama, T. Seto, S. Igarasi, and I. E. Goetz

City of Hope Medical Center, Duarte, California 91010

SUMMARY

Selected for study fields were several distinct provincialregions of Japan which are noted to be stable in populationcomposition, generally conservative in the mode of life, anddistinguished by especially high figures in biometrie statisticsrepresenting the Japanese characteristics of extraordinarilyhigh incidence in death by cardiovascular diseases, stomachcancer, and hepatoma. From these regions, samples of popularfermented foodstuffs, consumed daily in quantity, were obtained as materials of study. They included "miso" or fermented soy bean paste, "katsuobushi" or fermented drybonitos, and "starters" used for their fermentation processes.

They were either commercial products from local plants orhome-made ones. From 24 samples, 37 strains of fungi wereisolated, of which 21 were found to produce toxic culturefiltrates and extracts. Many of them produced kojic acid andÃŸ-nitropropionic acid. None, however, produced aflatoxin,although some strains produced blue and green fluorescentmetabolites which have not yet been identified. Biologic testscarried out with the culture preparations revealed significantbiologic activities in bacteriocidal effects, induction of bacteri-ophage, and influence on propagation of hamster cells in vitro.Moreover, short-term animal experiments showed developmentof miscellaneous remarkable pathologic changes, such as extensive hemorrhagic erosion in the glandular stomach, necrosis inthe liver, and injuries in the kidneys, pancreas islets, andmyocardium. The changes in the stomach and liver drewspecial attention in association with high incidence of chronicgastric and hepatic diseases in the biostatistics. Of the otherchanges, glomerulotubular nephritis, renal tubular oxalosis,pancreas-islet hemorrhage and degeneration, and myocardialhemorrhage and scar formation were impressive in relation tothe high incidence of cardiovascular diseases. To preventoccurrence of toxic agents in fermented food, it was recommended to avoid the use of toxic strains of fungi and toeliminate contamination of undesirable strains of fungi.

INTRODUCTION

The present investigation was undertaken to find possiblenoxious agents in foodstuffs prepared by fermentation, such as"miso" and similar foodstuffs, consumed daily and in quantity

by almost all people in Japan, and to contribute to etiologicanalysis of such diseases especially high in incidence amongthese people as shown by biometrie statistics.

'This study was supported by NIH Contract No. PH43-65-1036.

The biometrie statistics in Japan are quite reliable. Thewhole population is over 100 million, large enough to offerreliable statistic figures. Moreover, practically all the peopleare of the same race, similar in habit and custom, literate, andlive close to each other on the small well-developed land.Practically no one is left out of the official registration andcensus. According to a recent report issued from the Department of Statistical Investigation, Japanese Ministry of Health(Japanese Medical News, No. 2282, p. 108, 1968), the threehighest causes of death in 1967 in Japan were apoplexy,neoplasms, and heart diseases in that order, or more precisely,25.6, 16.7, and 11.0 percent of the total death cases (about670,000) respectively. The percentage of total death cases bycardiovascular diseases, including those by apoplexy and byhypertension, was as high as 39.3 percent. Compared with the1966 figures, the total death cases by cardiovascular diseaseswere higher, especially so in heart diseases, but those forapoplexy were slightly decreased. The death cases by neoplasms were generally increased, except for those by uterinecancer. Of neoplasms, stomach cancer and hepatoma stillremained leading the list. It was remarkable, however, thathepatoma showed a slight decline in the rural areas, wherevarious environmental conditions including daily food wereconsiderably changing. Thus, an approach was suggested fromthe epidemiologie angle to study prevailing environmentalnoxae, especially the popular but possibly injurious foodstuffs,such as fermented foodstuffs. These were represented by"miso" or fermented soy bean paste and "katsuobushi" or

fermented dry bonito, consumed daily in quantity by mostJapanese.

MATERIALS AND METHODS

Fermented foodstuffs usually are prepared by the use of astarter, namely "tane-koji," commercially obtained or indi

vidually prepared in a traditional way and preserved. In fact, itis a batch of dried fermented rice containing a lot of spores offungi, usually strains of Aspergillus.

For a preliminary study, tane-koji was locally obtainedwhich had been produced in a plant in Los Angeles, probablyoriginally started many years ago with imported tane-koji fromJapan. Pathologic effects were examined by administering tomice and rats tane-koji itself, rice gruel fermented with it, andpreparations prepared from cultures in Czapek medium of A.oryzae isolated from the tane-koji (11). Since kojic acid wasfound to be a metabolite of the fungus, effects of a commercially obtained preparation of kojic acid also were similarlyinvestigated (10). Remarkable findings were (a) massive necrosis in the guinea pig liver by s.c. injections of fermented rice

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Mycotoxins in Fermented Food

gruel extract, (b) tubular dilatation, epithelial degeneration,and focal atypical epithelial hyperplasia with abnormal mitosisin the convoluted tubules in the mouse kidneys by i.p. injections of a preparation from methanol extract of A. oryzaecultures, (c) focal necrosis and circumscript hyperplasia withmany mitotic figures in the mouse liver by i.p. injections ofkojic acid solution, (d) marked inhibition of the body growthand cirrhotic changes in the liver of rats by s.c. injections ofkojic acid solution, and (e) various abnormalities such asacrania (Fig. 1), ataxia, demyelinization, and neuron degeneration in the midbrain, etc., in newly hatched chicks by injection of kojic acid in embrionated eggs on the 5th day ofincubation. Furthermore, interesting findings obtained fromthe additional experiments were (d) bile duct proliferation andfibrosis in the liver of ducklings given a concentrated culturefiltrate of A. oryzae, and (b) asymmetric mitosis, chromosomestickiness and breaks, and reversion of mitosis in the rat livercells in culture treated with kojic acid.

On the basis of these findings, the study was designed toexpand to an extensive scale. Katsuobushi, miso, etc., weresampled in several distinct provincial regions in Japan whichare noted to be stable in population composition, (havingscarcely any settlers from other regions), generally conservative in the mode of life, and extraordinarily high in death casesof cardiovascular diseases, stomach cancer, and hepatoma.

Miso is consumed, on the average, 32.7 gm in the countryand 26.1 gm in towns per person every day in soups or mixedwith vegetables and fish (19). It is prepared from soy beansand barley by fermentation and then mixed with a largeproportion of salt, in about 12 or more percent, to stop theprogress of fermentation for preservation. In urban areas it isproduced mainly at modern plants on a large scale in a more orless scientific way by the use of selected tane-koji, preparedfrom known culture strains of A. oryzae, whereas in rural areasit is usually produced at local small shops or at homes in atraditional way by the use of commercial or home-madetane-koji, prepared from wild strains inherited generation aftergeneration; or it is often produced without using any startersand depending on natural contamination by useful strains. Therural ways of uncontrolled fermentation therefore can bedangerous. In this connection "nukamiso" should be men

tioned, which is a soft paste of fermented rice bran used forpreparing pickles of radishes and vegetables. No starters areused at all. Since salt is mixed from the beginning, microbesresponsible in this case usually are some wild strains of fungiother than Aspergini.

The next important foodstuff is katsuobushi. Its scrapingsand extracts are daily used to add flavors to soups and sidedishes. The consumption is fairly large, although in towns it isbecoming little by little substituted by glutamate and inosi-nate. Katsuobushi is produced in a traditional way at smallplants near some fishermen's ports on the warm southern

Pacific coast with enough supply of fresh bonitos. From eachfish the skin, organs, and intestines are removed to obtain largeleft and right halves of muscle pieces. These pieces are boiledfor disinfection and placed in warm humid cellars, to fermentwith preexisting fungi, usually wild strains of Aspergillus. Theyare taken out in the sun about once every two months to dry thealready fermented superficial portions. When they are thor

oughly fermented, they are put in the sun to dry completely.The entire procedure usually takes about 10 months. Furthermore, at katsuobushi plants the removed intestines are cleanlywashed with running water and fermented similarly by naturalcontamination of fungi and then mixed with salt. It is awell-known delicious appetizer called "shuto" or "shiokara."

Another fermented product consumed in a large quantity is"shoyu" or soy sauce. Its production has been greatly indus

trialized and known cultured strains of fungi are usually used.In small scale production at rural areas, however, wild strainsare still used for starters. "Sake" or rice wine is in a similar

situation. In any case, the starter used by each brewery is animportant part of a secret formula to assure the special qualityand flavor for its products. A starter, therefore, is a specialstrain or a certain combination of strains. Thus, numerousstrains of fungi are concerned with the essential process offermentation for preparing fermented food. Often, however,some starters carry passenger strains, actually useless for thepurpose. Furthermore fungal contamination sometimes occurs.For instance, A. tamarii has been found in soy sauce in certainareas of Middle Japan. It was shown in another experiment(unpublished) that a culture filtrate of the fungi given orally toC57BL/6J mice caused atypical epithelial proliferation in theglandular stomach (Fig. 2) resembling the features oftenobserved in gastric biopsy specimens and described as earlychanges of stomach cancer.

To screen harmful fermented foodstuffs in this complicatedcircumstance, it was very important to plan carefully wherewhat sorts of materials were to be sampled for the study. First,three distinct provincial regions, namely Nara, Wakayama, andYamagata, were selected, which are noted in the nation forvery high figures in the death statistics of the diseases previously referred to. Within the regions, certain counties especiallyhigh in incidence of the diseases were of special interest. Theywere Tsuruoka City and its suburb in Yamagata, Shimoichi inNara, and Wakayama City and Mitogowa in Wakayama. Atthese places, samples of starters and fermented foodstuffs werebought from small local makers, and samples of home-madefermented foodstuffs, especially those prepared withoutadding starters, were obtained. The additional materials weresampled for other reasonsâ€”for instance, commercial startersfrom Nagoya, which were suspected of being contaminated byA. tamarii, cycad miso from Oomi Ooshima, which wasprepared without using any starter in an area prevalent with A.awamorii, starters from Honolulu and Los Angeles, which wereprepared from the strains imported many years ago fromJapan, and fermented soy bean paste from South Korea(received from Dr. Falk), which, according to the Koreanmethod of not adding a starter, was fermented for manymonths until naturally arrested by air drying. There werealtogether 24 different samples of foodstuffs. From them, 37strains of fungi were isolated by the use of malt extract andpotato-dextrose agar as media. For convenience they werenumbered and marked by K for katsuobushi and by M formiso and shoyu (Table 1).

To obtain test preparations, these strains of fungi werecultured for 9 to 14 days at 25-30Â°C without shaking in the

following 3 kinds of media: potato-carrot (A), glucose ammonium nitrate (B), and Czapek plus ZnSO4 (C) (Table 2). To

NOVEMBER 1968 2297




Table l

Sampled foodstuffs (places)

1. Katsuo-shuto (Kochi)

2. Katsuobushi No. 1 (Kochi)

3. Shin-bushi (Kochi)

4. Katsuobushi No. 2 (Kochi)

5. Kinzanzi-miso (Wakayama)

6. Shiro-miso (Hawaii)

7. Miso (Mitogawa. Wakayama)

8. Nukamiso (Mitogawa. Wakayama)

9. Cycad miso (Oomi. Ooshima)

10. Miso (Korea)

11. Miso (Los Angeles)

12. Koji starter (Los Angeles)

13. Miso starter (Wakayama)

14. Miso starter 1 (Nagoya)

15. Shoyu starter 1 (Nagoya)


17. Miso starter 2 (Nagoya)



20. Miso koji 1 (Shimoichi. Nara)

21. Miso koji (Tsuruoka. Yamagata)

22. Wasabi-zuke (Tsuruoka. Yamagata)

23. Miso koji 2 (Shimoichi. Nara)

24. Nukamiso (Tsuruoka. Yamagata)

Isolated fungi

K-l, Aspergillus oryzae

K.-2.PÃ©nicilliumterrestre seriesK-3, A. flavus groupK-4, Aspergillus sp.K-5. A. flavus groupK-6. P. chrysogenum

K-7. P. cyclopiumK-8. A. cremeus group

K-9. P. cyclopium

M-l. A. oryzae

M-2, Aspergillus sp.

M-3. Alternada sp.M-4. PÃ©nicilliumsp.M-5. Blastomyces sp.M-6. A. glaucus group

M-8. Aspergillus sp.M-9. Mucor sp.

M-10, A. versicolor group

M-ll. P. chrysogenumM-l2, Scopulariopsis sp.M-13, Mucor sp.M-14, Pestalotia sp.

M-16. Aspergillus sp.

M-l7. A. oryzae

M-l8, A. oryzae

M-l9, A. oryzae

M-20. A. soyae

M-21. A. soyae

M-22, A. oryzae

M-23, A. soyae

M-24. A. soyae

M-25. A. oryzaeM-21, A. flams group

M-28, A. candidus group


M-30, A. oryzae

M-33, A. flavus group

Strains of fungi isolated from samples of fermented foodstuffs.

each flask of the potato-carrot medium was added 0.1 ml of10 percent streptomycin. One hundred milliliters of B and Cmedia were dispensed into 500-ml flasks for culturing eachstrain. Of the A medium cultures, after 14 days of incubation,600 ml of distilled water was added and homogenized by theuse of a Waring blender. The resulting homogenates were usedfor animal tests. Besides the culture homogenate, 5 ml wasmixed with 15 ml of methanol and filtered. The filtrate wasused for determination of kojic acid and aflatoxin and for theâ€¢detectionof aflatoxin-like fluorescent compounds. Of the B

Table2Diced

potatoDicedcarrotD-glucoseSucroseNH4NO3NaNO3KHiPO4K2HPO4MgSO4

â€¢7H2OFeSO4â€¢7H2OZnSO4â€¢7H2OCuSO4â€¢5H2OCo

(NO3)7-6H2OKC1CaCl2A

B1001005.00.241.00.20.0020.00020.00010.006Câ€¢3.00.20.10.050.0010.020.05

Standard nutrient media for fungus cultures. A, potato-carrot medium;B. glucose ammonium nitrate medium: C. supplemented Czapek Doxmedium.

medium cultures, the culture broth was filtered after 14 daysof incubation. The filtrate was used for determination of kojicacid and j3-nitropropionic acid (j3-NPA) and for animal experiments. Of the C medium cultures, the culture broth wasfiltered after 9 days of incubation. The filtrate was used foranimal tests and for chemical determinations. Furthermore,each filtrate (15 ml) was extracted with 40 ml of chloroformusing a continuous extractor. The crude extract obtained afterevaporation was used for determination of aflatoxin and otherfluorescent compounds.

Before going into the general screening tests for toxic fungusstrains, it was examined whether known toxic fungal metabolites, such as aflatoxins and kojic acid were produced. Qualitative determination of kojic acid and aflatoxin was conductedby paper chromatography and thin-layer chromatographyusing the methods shown in Table 3. An absorption spectrumof each sample was taken from 420 to 220 mju with aBeckman DB spectrophotometer before and after the additionof 0.1 ml of 0.5 N NaOH in order to determine the quantity ofkojic acid present. For determination of aflatoxins, methanolextracts (for A medium) or chloroform extracts (for C medium) of culture broth were used as samples.

Furthermore, /3-NPA was also considered important forsimilar study. Unusual toxic effects of the filtrate fromcultures of M-28 A. candidus group from Tsuruoka misostarter were suggested partially due to 0-NPA, since it wasfound in a high concentration in culture broth when M-28 wascultured in B media. For quantitative determination of/3-NPA,the culture filtrate (20 ml) was mixed with 40 ml of 0.1 N HC1solution and boiled gently for 1 hour. The solution was thencooled, adjusted to pH 6 with 0.5 N NaOH, and made 60 ml involume with distilled water. The resulting solution was thenused in Matsumoto's procedure (13). It has been reported that

ÃŸ-NPAis produced in culture broth by certain strains of A.flavus (6, 7), A. oryzae (14), PÃ©nicillium atrovenetum G.Smith (9, 15, 18) and Streptomyces sp. (1). Nothing has beenknown about the formation of ÃŸ-NPAby any strains of A.candidus. In spite of several investigations with rabbits (8), rats




Filter paper or thin-layer

chromatography plate

Developing solvents

Developing conditions

Detection of compounds

Table 3

Kojic acid (paper chromatography)

Whatman No. 1

Â»-BuOH: glacial acetic acidt

(18:2:5) upper layer

Ascending (room temperature)

UV absorption, FeCl Â¡reaction

Procedures for determination of kojic acid and aflatoxin.


Aflatoxin (thin-layer chromatography}

Eastman silica gel chromatogramsheet, K301 R-2

1) Benzcne:cthyl acctate:ethanol(30:19:1)

2) CHCl3:acctone(9:l)

Ascending (room temperature)

UV fluorescence

(12), and chicks (4, 5), pathologic effects of the compoundhave not been well clarified. Recently, however, K. Sawai andothers (16, 17) suggested that it has inhibitory effects oncatalase activity and oxidative phosphorylation of mammaliancells.

To study biologic effects of the previously mentionedcompounds and test preparations from fungus cultures, experiments were started at the cellular level. First, bacteriocidaleffects were investigated against the Gram-negative and Gram-positive bacteria. Each strain of fungus was grown on maltextract agar in Petri dishes for 3 days at 25Â°C.The base layer

was then overlayed with 10 ml of soft agar (0.8 percentnutrient broth, 0.5 percent NaCl, 0.5 percent agar) inoculatedwith 2 drops of E. coli K-12 or Bacillus megatherium ATCC15561 which had been cultured for 16 hours at 37Â°Cwith

shaking. The culture plates, after overnight incubation at37Â°C,were examined for zones of inhibited bacterial growth.

Secondly, cytotoxic effects, specially proliferation-inhibitory effects, on animal cells in culture were studied.Cells from 12-day-old hamster embryos were grown in enriched Earle's medium with 10 percent calf serum. The cells

first were cultured for 2 days in the nutrient medium withkojic acid or ÃŸ-NPAin varying concentrations and then weretransferred to the standard medium without any compounds.Every 6 days thereafter the cell count and the transfer to freshmedia were repeated to investigate changes in the rate of cellpropagation. Also, microscopic preparations were made atintervals to study cytologie changes.

Thirdly, activity of X-bacteriophage induction was tested. Ithas been noted that many induction-positive compounds arecarcinogenic. For the test, the lysogenic strain E. coli K-12 (X)was kept refrigerated on nutrient agar slants. Bacto-peptonebroth (1 percent Bacto-peptone, 0.2 percent yeast extract, and0.2 percent NaCl) was made with tap water and adjusted to apH of 7.0-7.2 with l N NaOH. The basal layer medium wasBacto-peptone agar (1.5 percent agar) and the top layer softagar (1 percent Bacto-peptone, 0.75 percent agar, 0.2 percentyeast extract, 0.2 percent NaCl, and tap water) were dispended3 ml per tube. One mg of test material was placed for diffusionin the center of the base layer agar in the plate. ApproximatelyIO7 cells of E. coli K-12 (X) Fl 192 were mixed with 3 ml of

melted soft agar and poured over the base layer. The platesthus prepared were incubated at 37Â°Covernight and then

checked for lytic zones. A clear zone around the test material

was caused by lysis due to liberation of phages, if the testsample is active.

Finally, animal experiments were carried out. C3H/HeJ malemice weighing 18 to 20 gm were fed with 8 percent lowprotein diet (Nutritional Biochemicals Corp.), starting about 4weeks previously, to make experimental animals more sensitivein response to pathogenic agents. Usually 5 or occasionally 4mice were used for each experimental group. The test materials were the homogenates and filtrates of fungus culturesprepared as described previously. A dose, 0.5 ml per 20 gmbody weight, was given intraperitoneally or orally. In the i.p.series, designed to study toxicity in general, mice were givenone dose of a homogenate of an A medium culture and oneday later sacrificed for autopsy or were given a filtrate of a Bor C medium culture once a day for 5 days and sacrificed onthe 7th day for autopsy. Gross and microscopic examinationswere carried out routinely of the liver, kidneys, heart, lungs,spleen, stomach, small and large intestines, pancreas, adrenalglands, testes, thymus, submandibular glands, and mesenterallymph nodes. In the series of oral administration, planned tostudy gastric changes in particular, mice after 18 hours fastingwere given homogenate of a medium A culture by stomachtube, and 6 to 24 hours later are sacrificed for autopsy.Animals in many categories show inactiveness, crouching,bristling, fall of body temperature, and decrease in bodyweight, and they often did not survive long.

RESULTS AND DISCUSSION

From 24 collected samples of foodstuffs, 37 strains of fungiwere isolated (Table 1). of which 21 strains were foundpathologically significant. From the katsuobushi group, 4strains of Aspergillus sp. and 4 strains of PÃ©nicilliumsp. wereisolated. Although certain strains of A. glaucus or A. gymuno-sardae had been referred to with regard to some samples ofkatsuobushi, no such strains were found this time. Earlier itwas reported that strains of A. ochraceus were isolated fromkatsuobushi by Yukawa (21). Recently, some strains of thesame species isolated from maize were found to produce"ochratoxin," which was highly toxic and even hepatocarcino-

genic (20). In the present investigation, however, this specieswas not found. The fungus most frequently found in misopreparations and starters was A. oryzae, primarily known forbrewing miso. Occasionally, however, some other strains and

NOVEMBER 1968 2299




Table 4

FungiisolatedK-l,

AspergillusoryzaeK-3,A. flavusgroupK-5,A, flavusgroupM-2,Aspergillussp.M-6,A. glaucusgroupM-8,Aspergillussp.M-9,Mucorsp.M-16,

Aspergillussp.M-17, A.oryzaeM-18, A.oryzaeM-19, A.oryzaeM-20,

A.soyaeM-21,A.soyaeM-23,A.soyaeM-25,A.oryzaeM-27,A. flavusgroupM-28,A. candidusgroupM-30,A.oryzaeM-33,A. flavus groupKMedium

A(mg/gin)1.311.018.68.415.66.89.51.824.01.40.01.42.43.86.24.510.50.0ojie

acid producedby:Medium

11(nig/ml)4.49.56.44.99.73.00.00.00.00.00.20.514.60.20.10.60.10.30.2Medium

C(mg/ml)1.92.01.01.40.00.00.00.00.00.00.00.01.70.00.00.00.00.00.0

Production of kojic acid.

species were found, for example, M-10 A. versicolor groupfrom Oomi-Ooshima cycad miso, M-27 A. flavus group fromShimoichi miso koji I, M-33 A. flavus group from Tsuruokanukamiso, and M-28 A. candidus group from Tsuruoka misokoji. Moreover, secondary contaminations were notedâ€”forinstance, 4 species of fungi including M-6 A. glaucus groupfrom Mitogawa miso, prepared without adding any starter, and4 miscellaneous species of fungi from Korean miso. Little hasbeen known about toxicity of metabolites from strains offungi involved in these kinds of fermented food. As previouslystated, however, kojic acid was shown as a toxic compoundproduced by a sample of tane-koji.

The present laboratory investigations of the 37 strains offungi revealed that 18 of them produced kojic acid, allbelonging to the Aspergillus sp. except M-9 Mucor sp. Itsproduction, however, distinctly varied according to the mediaused.

M-16 A. flavus group from Los Angeles miso, M-17 A.oryzae from Los Angeles koji starter, and M-18 A. oryzae from

Wakayama miso starter produced kojic acid in A medium butnot in B and C media. M-21 A. soyae from Nagoya shoyustarter 2 produced kojic acid in B and C media, but not in Amedium. K-l A. oryzae from Kochi katsuo-shuto,and K-3 A.flavus group and K-5 A. flavus group both from Kochikatsuobushi 1 produced a large amount of kojic acid in A andB media but not much in C medium. This suggested that thequantity of kojic acid produced in actual fermented foodstuffsalso could considerably vary according to compositions ofmaterials used besides other fermentation conditions.

Strains of fungi, known to produce aflatoxin or a group ofstrongly toxic and even carcinogenic compounds, were notedto be taxonomically closely related to many of the fungipresently considered. By the thin-layer chromotographymethod for aflatoxin determination, K-3 A. flavus group fromKochi katusobushi 1 and M-10 A. versicolor group fromOomi-Ooshima cycad miso were found to produce bluish orviolet fluorescent compounds, like aflatoxin. The RF values ofblue fluorescent spots of K-3 and M-10 in the chloroform-acetone solvent system resembled that of the standard aflatoxin B group (Table 5). Results of thin-layer chromatogramand RF values from K-3 and M-10 in several other solvent

systems also were found comparable with those from aflatoxin. Absorption curves of chloroform extracts of bothsamples, however, definitely differed from that of aflatoxin(Chart 1), suggesting that the fluorescent compounds produced by the fungi were not aflatoxin. They were not yetidentified. Anyway, the culture broth of K-3 and M-10 werevery toxic as described later.

It was previously stated that M-28 A. candidus groupcultured in B medium produced ÃŸ-NPA.The production of|3-NPA, as measured by the Matsumoto's method, was very low

for the first 4 days of incubation, then rose gradually andstarted to rise steeply from 7 days, reaching the peak at 17days (Chart 2). The toxic effects of (3-NPA have not yet beenwell clarified. Nevertheless, mice 5 days after injection of8-day culture broth of M-28 started to show a gradual decreasein body weight, whereas animals injected with 15- to 17-dayculture broth containing the largest quantity of ÃŸ-NPAwerekilled within 5 days. Thus, it was suggested that these resultswere caused by 0-NPA produced, but probably not exclusivelyif complicated by other potential agents. At any rate, so far asthe period of fermentation does not exceed 4 days, as usuallypracticed at duly controlled miso breweries, the danger of

Table 5

Thin-layerchromatography

solventsAflatoxin

B(standardsolution)K-3,

AspergillusflavusgroupM-10,

A.versicolorgroupCHCl3:acctone

(9:1)0.820.800.75Benzene:ethylacctate:cthanol

(30:19:1)0.410.600.42Butanohaceticacid:H->O

(18:2:5)0.710.800.70

values on thin-layer chromatography of bluish fluorescent compounds produced by fungi.





0.4

0.3

S 0.2

0.1

240 260 280 300

WAVE LENGTH

320

m\Â¡340 360 380

Chart 1. UV absorption spectra. Comparison of fluorescent preparations from cultures of K-3 (A) and M-10 (B) and a solution of chemically defined aflatoxin B group (C).

1.31.2U1.0

0.90.80.70.6

0.30.2ai

1 2 3456 78 9 1011 12 13 1415 1617 18 19 20CULTURE (DAYS)

Chart 2. Production of beta-nitropropionic acid in B medium byM-28; Aspergilluscandidusgroup.

ÃŸ-NPAis minimal. For screening ÃŸ-NPA-produring strains offungi, however, it is reasonable to have a longer period ofincubation. In the present research, therefore, the culturefiltrate from 9 to 19 days, incubation was pooled, yielding atotal of 500 ml. This fluid was extracted with 1000 ml ofchloroform by the use of a continuous extractor. The chloroform layer was evaporated, leaving 300 mg of straw-coloredmaterial (F-l). This crude chloroform extract, 250 mg, wasdissolved in 20 ml of ethyl acetate and reextracted 3 times

with 25 ml of 5 percent NaHCO3 (pH 8.5). The water layerwas adjusted to pH 2.5 with HC1 and extracted with 20 ml ofethyl acetate. The ethyl acetate solution was washed with asmall amount of water and evaporated completely, yielding200 mg of slightly colored powder (F-2). Highly concentratedÃŸ-NPAwas detected in F-2. Besides the aforementioned M-28A. candidus group, M-24 A. soyae from Nagoya shoyu starter4, M-8 Aspergillus sp. from Mitogawa nukamiso, M-10 A.versicolor group from Oomi-Ooshima cycad miso, M-20 A.Soyae from Nagoya shoyu starter 1, and M-23 A. soyae fromNagoya shoyu starter 3 produced 0-NPA in a considerableamount, from 1.1 to 0.1 mg/ml in decreasing order, whencultured in B medium (Table 6). Many others, namely K-7 P.cyclopium from Kochi shinbushi, K-9 P. cyclopium fromKochi katsuobushi, M-2 Aspergillus sp. from Hawaii shiromiso, M-6 A. glaucas group from Mitogawa miso, M-l 1 P.chrysogenum from Korean miso, M-17 A. oryzae from LosAngeles koji starter, M-21 A. soyae from Nagoya shoyustarter 2, and M-22 A. oryzae from Nagoya miso starter 2produced a trace in B medium. Production of 0-NPA in Cmedium, however, was very infrequent and low. Only M-23 A.soyae from Nagoya shoyu starter 3, M-28 A. candidus groupfrom Tsuruoka miso starter, and M-33 A. flavus group fromTsuruoka nukamiso produced a trace.

Of the in vitro biologic tests, the bacteriocidal or antibioticeffects against Gram-negative and Gram-positive bacteria werecarried out. The positive results against E. coli K-12 wereobtained from culture preparations of K-2 P. terrestre series,K-4 Aspergillus sp., and K-6 P. chrysogenum from Kochikatsuobushi 1, K-8 A. cremeus group from Kochi shinbushi,M-3 AlternarÃa sp. and M-4 PÃ©nicilliumsp. from Mitogawamiso, M-l2 Scopulariopsis sp. from Korean miso, and M-3CM.oryzae from Shimoichi miso starter 2. The positive resultsagainst B. megatherium ATCC 15561 were obtained fromculture preparations of K-6 P. chrysogenum and K-7 P.cyclopium from Kochi shinbushi, K-9 P. cyclopium fromKochi katsuobushi 2, M-8 Aspergillus sp. from Mitogawanukamiso, M-l 1 P. chrysogenums from Korean miso, and M-25

Table 6

MediumB

MediumC

Fungi isolated 0-NPA produced (mg/ml)

K-7, PÃ©nicilliumcyclopiumK-9,P-cyclopiumM-2,Aspergillussp.M-6,A, glaucusgroupM-8,Aspergillussp.M-10,

A. versicolorgroupM-ll,P.chrysogenumM-

17, A.oryzaeM-20,A.soyaeM-21,A.soyaeM-22,A.oryzaeM-23,A.soyaeM-24,A.soyaeM-28,A. candidusgroupM-33,A. flavus groupTraceTraceTraceTrace0.30.2TraceTrace0.2TraceTrace0.10.41.1000000000000Trace0TraceTrace

fi-nitroproprionic acid (0-NPA) production by isolated fungi.

NOVEMBER 1968 2301




A. oryzae from Shimoichi miso koji l. It was remarkable thatK-6 P. chrysogenum was effective either against the gram-negative or -positive microorganisms.

Cytotoxic effects on mammalian cells in culture werestudied by examining the population growth of hamster cellsin the control nutrient media following 2-day pretreatmentwith nutrient media containing kojic acid and /3-NPA. Eithercompound was shown toxic. At a high concentration, however, /3-NPA was more toxic than kojic acid. For instance, at10~2 M concentration, the former caused a steep linear fall in

the cell population curve (Chart 3), whereas the latter showeda slight temporary recovery after an initial fall. At a lowconcentration, the response was considerably different. Thecultures pretreated with 10~6M /3-NPA showed a rapid

recovery from inhibition of cell increase, followed by amarked growth in cell population rising even higher than thecontrols, while those pretreated with 10~7M kojic acid showed

a slower and less vigorous recovery, remaining considerablylower than the controls. /3-NPA at this concentration apparently initiated some stimulatory effect on the cell multiplication.

In the X-bacteriophage induction tests, kojic acid and /3-NPAproduced the positive results like aflatoxin (Fig. 3). Not all,but many, chemical carcinogens were similarly positive (Table7). Almost all nucleotoxic antiobiotics tested also were positive. The preparations prepared by methanol or chloroformfraction from cultures of the toxic fungus strains were similarly tested. Those of K-5 A. flavus group from Kochikatsuobushi 1 and M-28 A. candidas group from Tsuruokamiso koji caused the positive results (Table 8).

Finally, to study pathologic effects on animals of A, B, andC culture preparations of all the strains of fungi, short-termexperiments ranging from several hours to several days werecarried out employing male C3H/HeJ mice about 20 gm. Theresults shown by 5 or 4 animals in a group used for each testwere similar. Animals which survived and were repeatedlytreated at intervals showed a marked loss of body weight. Thepathologic changes, however, were multifarious, complicatedlyvarying according to strains of fungi used for preparing testpreparations and even often different according to A, B, and C

IO"M

O 6

Chart 3. The cell population of hamster embryo cells in culture pretreated for 2 days with ÃŸ-nitropropionicacid (/3-NPA)added to nutrientmedia in varying concentrations: 10~2 M ( - x - x -) ; 10~4 M( - 0 - 0 - ); and 1CT6 M ( - â€¢- â€¢- ) /3-NPA,compared with theunpretreated controls ( â€”â€¢â€”â€¢â€”).Means of 5 experiments.

preparations from the same strain of fungus, as shown summarized in Table 9. These differences probably were due todifferences in a noxious agent or agents involved and also tovarieties in combinations of potential metabolites, and allthese suspected compounds were unknown, except for a few.

Table 7

Compound Effect Compound Effect

p-Benzoquinonel ,4-Naphthoquinone4-Nitroquinoline-N-oxideDime thy Ini trosoamineN-NitrosodibutylamineN-Me thy lure thaneN-EthylurethancN-Nitrosomethyl urea2,7-DiacetaminofluoreneN-2-Fluorcnylacctamide3,4-Benzpyrcne3-Mcthylcholanthrenc

20-Methy!cholanthrene3'-Methyl-4-dimethylaminoazobenzeneN,N-Dimethyl-p-phenylazoaniline HC1l-Methyl-3-nitroguanidine

Mitomycin CNovobiocinToyomycinGriseofulvinActinomycin D

Induction of X-bacteriophage in E. coli K-12(X) by carcinogenic compounds and related compounds and by nucleotoxic antibiotics.





Table 8

Culture filtrates Effect

K-5, Aspergillus flavus group, 2-week surface culture; +methanol-soluble fraction

M-4, PÃ©nicilliumsp., 2-week surface culture;methanol-insoluble fraction

M-22, A. oryzae, 2-week surface culture;methanol-insoluble fraction

M-27, A. flavus group, 2-week surface culture ; chloroform ?

extract fraction

M-28, A. candidas group, 2-week surface culture ; chloroform +

extract fraction

Induction of X-bacteriophage in E. coli K-12 (d) by preparations fromsome culture filtrates of fungi isolated in the present investigation.

Naturally it was difficult to describe all the cases categoricallyclassified. Accordingly, the way chosen was first to look intostrains of fungi which produced culture preparations capableof causing very remarkable changes but showed the positiveresults only in the least categories of the in vitro biologic tests.

K-6 P. chrysogenum from Kochi katsuobushi 1 produced

neither kojic acid nor ÃŸ-NPAin any of the three media, andalso did not show blue or green fluorescence, but was anti-/T.coli as well as B. megatherium. Its A preparation given p.o. ori.p. caused with_jn 3 to 24 hours marked extensive hemor-rhagic erosion in the glandular stomach (Fig. 4). The Cpreparation injected i.p. caused renal tubular oxalosis mainlyat the corticomedullary junction in 7 days.

M-10 A. versicolor group from Oomi-Ooshima cycad misoalso did not produce kojic acid in any of the 3 media and alsodid not produce anti-Â£".coli and anti-A. megatherium sub

stance, but produced a fair amount of 0-NPA (0.2 mg/ml) inMedium B and, furthermore, produced blue fluorescent substance in Media A and C. By i.p. injection of the A preparation, the kidneys became enormously swollen within 24 hours(Fig. 5). Microscopically, hydrops, necrobiosis, karyorrhexis,and hyaline cast were evident in the proximal tubules (Fig. 6).Five i.p. injections of the C preparation caused tubularoxalosis at the corticomedullary junction and spleen atrophy.

M-22 A. oryzae from Nagoya miso koji 2 did not produceanti-ZT,coli and anti-A megatherium substance, either blue orgreen fluorescent substance, and kojic acid, but produced atrace of 0-NPA in B medium. Its A preparation, injected i.p.,caused in 24 hours severe exfoliative enteritis (Fig. 7),sinusoidal congestion and karyorrhexis in the germinal centersin the spleen, and lung congestion. Its B preparation injectedi.p. caused hyaline degeneration, karyorrhexis, proliferation in

Table 9

Strains(sources)K-l

, Aspergillusoryzae(Kochishuto)Culturemedia

RoutesA

p.o.I.p.B

i.p.Doses113BodyweightPeriods

(percent)Toxicity3

hours6hours3

days -11 ++AutopsyKidney: degeneration and hyaline casts

7 days -12

in proximal tubules.Pancreas: islet hemorrhage.Glandular stomach: hemorrhagic erosion.Kidney: tubular oxalosis at corticomed

ullary junction; dilation of proximaltubules.

K-2, PÃ©nicilliumterrestreseries (Kochikatsuobushi 1) B

C

p.o.Â¡.p.

3 hours24 hours

7 days7 days -10 Kidney: tubular oxalosis at

corticomedullary junction, dilationof proximal tubules.

K-3, A. flavus group(Kochi katsuobushi 1)

p.o.

up.

3 hours18 hours

24 hours

7d ays

-11

-23

Kidney: necrobiosis and hyaline castsin proximal tubules.

Kidney: necrosis and hyaline castsin proximal tubules.

Pancreas: islet hemorrhage.Glandular stomach: hemorrhagic erosion.Spleen: karyorrhexis in perifollicular

area.Kidney: tubular oxalosis at

corticomedullary junction, dilationof proximal tubules.

NOVEMBER 1968 2303



R. Kinosita, T. Ishiko, S. Sugiyama, T. Seto, S. Igarasi,and I. E. Goetz

Table 9 (continued)

Strains(sources)K-5,

A. flavusgroup(Kochikatsuobushi1)K-6,

P.chrysogenum(Kochikatsuobushi1)K-7,

P. cyclopium(Kochishinbushi)K-9,

P.cyclopium(Kochikatsuobushi2)M-2,

Aspergillussp.(Hawaiishiromiso)M-4,

PÃ©nicilliumsp.(Mitogawamiso)M-6,

A. glaucasgroup(Mitogawamiso)Culture

mediaRoutesA

p.o.i.p.B

i.p.C

Lp.A

p.o.i.p.B

i.p.Ci.p.A

p.o.i.p.Bi.p.Ci.p.A

p.o.i.p.B

i.p.Ci.p.A

p.o.i.p.B

i.p.C

i.p.A

i.p.B

i.p.C

Â¡.p.A

i.p.Bi.p.C

i.p.Doses11151165114511451125115135Bodyweight

Periods (percent)Toxicity3

hours24hours -10+++24

hours -11-t-H-7

days3

hours+24hours+8

days7days -22+3

hours24hours7

days7days -26+2.5

hours6hours8days7

days -26+23

hours18hours+++2

days -9+++7

days -18+24

hours++24

hours++7

days -10+24

hours3days -18+++5

days -20 +AutopsyKidney:

necrobiosis ofjuxtaglomerulartubules,increase in glomerularnuclei.Kidney:

degeneration and hyalinecastsinproximaltubules.Glandular

stomach: markedhemorrhagicerosion.Pancreas:

islet hemorrhageanddegeneration.Glandular

stomach: hemorrhagicerosion.Glandularstomach: hemorrhagicerosion.Kidney:

tubular oxalosisatcorticomedullaryjunction.Kidney:

tubular oxalosisatcorticomedullaryjunction.Liver:

fibroticatrophy.Kidney:

tubular oxalosisatcorticomedullaryjunction.Liver:

fibroticatrophy.Kidney:

necrobiosis ofproximaltubules.Kidney:

hyaline degeneration andmanymitosesin proximaltubules.Pancreas:islethemorrhage.Heart:

myocardialhemorrhage.Glandularstomach: mucosalhemorrhagicerosion.Kidney:


hyaline casts in tubulesatcorticomedullaryjunction.Kidney:

tubular degenerationÂ¡increaseofglomerularnucleiKidney:


degeneration and hyalinecastsinproximaltubules.Pancreas:

islethemorrhage.Glandularstomach: hemorrhagicerosion.Heart:

myocardialhemorrhage.Kidney:anemic swelling,tubularoxalosis

at corticomedullaryjunction.Spleen:atrophy.





Table 9 (continued)

Strains(sources)M-10,

A. versicolorgroup(Oomi-Ooshimacycad

miso)M-14,

Pestalotiasp.(Koreanmiso)M-17,

A.oryzae(LosAngeleskoji)M-21,

A. soyae(Nagoyashoyu

starter2)M-22,A.

oryzae(Nagoyamisostarter2)M-24,

A. soyae(Nagoyashoyu

starter4)M-25,

A.oryzae(Shimoichimisokoji

1)Culture

mediaRoutesA

p.o.i.p.B

i.p.CLp.A

p.o.i.p.B

Lp.C

i.p.A

i.p.Bi.p.Ci.p.B

i.p.Ci.p.A

i.p.B

p.o.i.p.C

i.p.A

i.p.Bi.p.C

Lp.B

i.p.C

Lp.Doses11751155155521114514535Body

weightPeriods (percent)Toxicity3

hours24hours+++8

days5days -33-H-3

hours24hours5

days -14++7

days24

hours+7daysâ€”7

days7

days2days -26+++24

hours+++17

days4days -10+++7

days24

hours5days -16++7

days4

days -15-H-7

daysAutopsyKidney:

marked swelling,necrobiosis,hyalinecasts in proximaltubules.Kidney:

marked swelling;tubularoxalosisat corticomedullaryjunction.Spleen:

markedatrophy.Kidney:

degeneration ofproximaltubules.Heart:

myocardial hemorrhage, granulation with mononuclearcellproliferation.Acute

severeperitonitis.Kidney:

epithelial destructionandtubularoxalosisatcorticomedullary

junction.Spleen:atrophy.Small

intestines: severeexfoliativeenteritis.Spleen:

sinusoidalcongestion,karyorrhexisin germinalcenters.Lung:

congestion.Kidney:

epithelialnecrosis,karyorrhexis,regenerationofproximal

tubules.Liver:dissociation and pleomorphismofliver

cells.Kidney:

hyaline degeneration ofproximaltubules,many mitoses; increaseinnuclei

inglomerulus.Kidney:

necrosis of proximaltubules,hyalinecasts.Pancreas:

islet hemorrhageanddegeneration.Glandular

stomach: mucosal hemorrhage.

NOVEMBER 1968 2305



R. Kinosita, T. Ishiko, S. Sugiyama, T. Seto, S. Â¡garosi,and I. E. Goetz

Table 9 (continued)

Strains(sources)M-27,

A. flavusgroup(Shimoichimisokojil)BodyCulture

weightmediaRoutes Doses Periods (percent)ToxicityB

p.o. 11 17daysi.p.3 3 days -16 +++AutopsyKidney: necrosis of proximaltubules,hyaline

casts.Glandularstomach: mucosalhemorrhage.Salivary

glands: hemorrhage.

M-28, A. candidas group(Tsuruoka miso koji)

C

B


(Tsuruoka,Wasabi-zuke)

up.

p.o.If.

up.

113

7 days

17 days4 days

7 days

3 days

-17

-10

Kidney: hyaline degeneration andepithelial hyperplasia with markedmitoses in proximal tubules,glomerular necrosis.

Pancreas: islet hemorrhage.Glandular stomach: hemorrhagic erosion.Heart: subepicardial and myocardial

hemorrhage.

Kidney: degeneration of proximaltubules.

Glandular stomach: hemorrhagic erosion.

M-30, A. oryzae(Shimoichi misokoji 2)

Lp. 7 days

7 days

-21 Kidney: degeneration of proximaltubules, many mitoses.

Glandular stomach: hemorrhagic erosion.

Summarized results of short-term animal experiments.

the proximal tubules in the kidneys (Fig. 8), and dissociationand pleomorphism in the liver cells.

K-7 P. cyclopium from Kochi shinbushi, like M-22, did notproduce anti-Â£".coli substance, either blue or green fluorescent

substance, and kojic acid, but produced a trace of 0-NPA in Bmedium. However, unlike M-22, it produced anti-B. megatherium substance. Its A preparation, unlike M-22, did not causeenteritis, and the C preparation injected i.p. 5 times in 7 dayscaused renal tubular oxalosis at the corticomedullary junction(Fig. 9) instead of tubular necrobiosis and epithelial proliferation in the kidneys, and fibrotic atrophy in the liver instead ofdissociation and pleomorphism in the liver cells.

Thus, a certain change was found commonly in theexperiments treated with culture preparations from differentstrains of fungi. Renal tubular oxalosis was noted in K-6,M-10, and K-7 experiments, but not for M-22. Furthermore,strains of fungi which yielded similar results, as shown in Table9, were K-l A. oryzae from Kochi shuto, K-2 P. terrestre seriesfrom Kochi Katsuobushi 1, K-3 A. flavus group from Kochikatsuobushi 1, K-9 P. cyclopium from Kochi katsuobushi 2,M-2 Aspergillus sp. from Hawaii shiromiso, M-4 PÃ©nicilliumsp.from Mitogawa miso, M-6 A. glaucus group from Mitogawamiso, and M-21 Nagoya shoyu starter 2. Altogether this was11 of 21 strains. It was remarkable that the renal change wasalways caused by C culture preparation from every one of

these fungi. Concerning pathologic significance of renal oxalosis, it has been suggested that the renal failure is a problem,rather than simple mechanical obstruction of tubular luminaby oxalate crystals (3). Further, a report about possibleassociation of hypertension with oxalate nephritis (2) isimpressive.

K-5 A. flavus group from Kochi katsuobushi 1 did notproduce anti-jE".coli or anti-A megatherium substance, blue or

green fluorescent substance, and 0-NPA, but produced aquantity of kojic acid in A and B culture preparations. Its Apreparation injected i.p. caused changes in juxtaglomerulartissues and increase in glomerular nuclei. Its B preparationgiven i.p. caused degeneration and hyaline casts in proximaltubules in the kidneys, hemorrhagic erosion in the glandularstomach, and islet hemorrhage and degeneration in thepancreas (Fig. 10).

M-2 Aspergillus sp. from Hawaii shiromiso did not produceanti-2f. coli or anti-5. megatherium substance and blue or greenfluorescent substance. Unlike K-5, it produced a trace ofÃŸ-NPA.The changes caused by A and B preparations in thekidneys, stomach, and pancreas were very much like those ofK-5. The only differences noted were myocardial hemorrhageby B and renal tubular oxalosis by C preparation. The latterchange was previously discussed in relation to 0-NPA.

M-6 A. glaucus group from Mitogawa miso showed similar





characteristics, such as negative in bacteriocidal effects onGram-positive and -negative bacteria, negative in production ofblue or green fluorescent substance, a trace in ÃŸ-NPAproduction, and production of kojic acid in quantity. Pathologiceffects on the kidneys, stomach, pancreas, and heart by Bpreparation and renal tubular oxalosis by C preparation werecomparable to those in the case of M-2. Thus, it was suggestedthat kojic acid probably was responsible for the changes foundin the kidneys, stomach, and pancreas in the experiments withB preparations from K-5, M-2, and M-6. This possibility wasparticularly supported by the previously cited preliminarystudy with kojic acid. It was remarkable that myocardialhemorrhage was observed in the experiments with B preparations from M-2 and M-6, and that renal tubular oxalosis wasfound in the experiments with C preparations also from M-2and M-6.

Finally, M-28 A. candidas group from Tsuruoka miso kojipresented a complicated case. It produced a large amount ofÃŸ-NPA(1.1 mg/ml) in B medium and a fair amount of kojicacid in A medium, and did not produce blue fluorescentsubstance in A and C media. The B preparation given i.p.caused glomerular necrosis (Fig. 11), tubular degeneration, andround cell infiltration in the cortex (Fig. 12), hyaline bodyformation (Fig. 13), and hyperplasia of tubular epithelial cells,many in mitosis, in the kidneys (Fig. 14), hemorrhage anddegeneration in the pancreas islets, hemorrhagic erosion in theglandular stomach, and subepicardial and myocardial hemorrhage (Fig. 15).

ACKNOWLEDGMENTS

The authors wish to thank Dr. H. Klein, Mrs. C. Bosi, Miss J.Pemberton, Mrs. P. Epps and Mr. J. Hargrove for proficient technicalassistance and also express their appreciation to Mrs. D. Wein for hervaluable assistance in preparation of the manuscript.

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2. Arnott, W. M., and Dellar, R. J. Hypertension Associated withExperimental Oxalate Nephritis. Brit. i. Exptl. Pathol., 16:265-270, 1935.

3. Bove, K. E. Ethylene Glycol Toxicity. Am. J. Clin. Pathol., 45:46-50, 1966.

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7. Bush, M. T., Touster, O., and Early, J. The Production ofÃŸ-NitropropionicAcid by a Strain of Aspergillus Flavus. J. Biol.Chem., Â¡88:685-693, 1951.

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13. Matsumoto, H., Unrau, A. M., Hylin, J. W., and Tempel, B.Spectrometric Determination of 3-Nitropropionic Acid in Biological Extracts. Anal. Chem., 33: 1442-1444, 1961.

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NOVEMBER 1968 2307




Fig. l. A newly hatched chick after injection of 0.5 mg of kojic acid in 0.1 ml physiologic saline into the yolk sac on the 5th day of incubation.Note a large defect in cranial bone formation, exposing a part of brain.

Fig. 2. A section of the stomach wall from a C57BL/6J mouse, given a concentrated filtrate of Aspergillus tornarti culture in Czapek Doxmedium once every 3 days 8 times by stomach tube. Note downgrowth of atypical epithelial structures, epithelial hyperplasia, and submucosal scarformation. H & E, x 200.

Fig. 3. Induction tests of X-bacteriophages in E. coli K-12(X) F1192. The positive results: 3a by kojic acid, 3ft by /3-nitroproprionic acid (ÃŸ-NPA)and 3c by aflatoxin. The control experiments with nonlytic/f. coli K-12 W3110 show negative results: 3a'by kojic acid, 36'by (3-NPA,and 3c'by

aflatoxin.Fig. 4. Stomach. Hemorrhagic erosion of the glandular stomach 3 hours after oral administration of A preparation of K-6 PÃ©nicilliumchryzo-

genum.Fig. 5. Kidneys (right). Enormous swelling, anemia, and hydropsis 24 hours after i.p. injection of A preparation of M-\0 Aspergillus versicolor

group. Kidneys (left), the controls.Fig. 6. Tissue from the experimented kidney in Fig. 5. Marked swelling, necrobiosis, karyorrhexis, and hyaline casts in the proximal tubules.

H & E, x 400.Fig. 7. Small intestine. Acute exudative and exfoliative enteritis, 24 hours after i.p. inoculation of A preparation of M-22 Aspergillus oryzae.

H&E, x 100.Fig. 8. Kidney. Hyaline degeneration and hyperplasia of the proximal tubular epithelium, 2 times i.p. injection in four days of B preparation of

M-22 Aspergillus oryzae. H & E, x 400.Fig. 9. Kidney. Tubular oxalosis at the corticomedullary junction, 5 times i.p. injection in 7 days of C preparation of K-7 PÃ©nicilliumcyclopium.

H & E, x 400.Fig. 10. Pancreas. Islet hemorrhage and degeneration, 24 hours after i.p. inoculation of B preparation of K-5 Aspergillus flavus group. H & E, x

200.Fig. 11. Kidney. Glomerular necrosis, three times i.p. injection, 3 days of B preparation of M-28 Aspergillus candidus group. H & E, x 400.Fig. 12. Kidney. Tubular degeneration and round cell infiltration in the cortex from the same specimen used for Fig. 11. H & E, x 100.Fig. 13. Kidney. Appearance of spherical hyaline bodies in the degenerating tubular structure from the same specimen used for Fig. 11. H & E,

x400.Fig. 14. Kidney. Hyplasia of tubular epithelial cells, many in mitosis, from the same specimen used for Fig. 11. H & E, x 400.Fig. 15. Heart. Subepicardial and myocardial hemorrhage from the same specimen used for Fig. 11. H & E, x 100.





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NOVEMBER 1968 2309



R. Kinosita, T. Ishiko, S. Sugiyama, T. Seto, S. Â¡garosi,and I. E. Goetz

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1968;28:2296-2311. Cancer Res R. Kinosita, T. Ishiko, S. Sugiyama, et al. Mycotoxins in Fermented Food

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