Zearalenone and Some Derivatives: Production and Biological Activities

Zearalenone and Some Derivatives: Production and Biological Activities P. H. HIDY, R. S. BALDWIN,R. L. GFWASHAM, C. L. KEITH, AND J. R. MCMULLEN I M C Chemical Group, Inc., Terre Haute, Indiana I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Chemistry.. ........................ A. Structure and Termi B. Assay and Recovery. 111. Fermentation . . . . . . . . . . . . . . . . A. Culture Development.. . . . B. Surface Fermentation ............................... C. Submerged Fermentation. ........................... D. Biosynthesis ..........................

..................................... A. General Drug Action. . . . . . . . . . . . . . . . ................ B. Endocrine Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... D. Safety Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ............................. . . . . . . . . . . . . . . .

59 61 61 62 63 63 64

68 73

74 74 75 77 78 81

I. Introduction During the early 195Os, Commercial Solvents Corporation1 (CSC) examined a number of microorganisms with the hope that they would produce, in controlled forage fermentations, substances having anabolic activity. This approach to a salable product was unsuccessful, but Dr. F. N. Andrews of Purdue University, who has served for many years as a consultant to CSC, called attention to reports (McNutt et al., 1928; Pullar and Lerew, 1937; Koen and Smith, 1945; McErlean, 1952) of estrogenic properties exhibited by moldy corn specimens. During 1957 and 1958, the Purdue University Agricultural Experiment Station had investigated complaints from several widely separated Indiana swine producers. These included vulvular enlargement and mammary gland stimulation in swine herds in Indiana in which the causative agent appeared to be moldy corn. In a joint effort, under an agreement between CSC and the Purdue Research Foundation, workers at Purdue and CSC began to investigate the microflora of these moldy corn specimens to determine whether estrogenicity was related to a particular genus and, if so, whether the active substance had utility in animal nutrition. 'International Minerals and Chemical Corporation acquired Commercial Solvents Corporation May 15, 1975.

60

P.

H. HIDY ET

AL

Among the cultures isolated by the Purdue group and grown on sterile moist ground corn, those of Gibberella produced a substance(s)that could be extracted with ethanol and concentrated by evaporation of the solvent. When this substance was fed to ovariectomized white mice, it induced uterine enlargement. The crude concentrate gave a similar response in mice when it was suspended in sesame oil and injected subcutaneously. Isolation and examination of additional cultures and production of larger quantities of moldy corn were undertaken. As a result of these early efforts, the symptoms observed in swine in the field were duplicated in gilts fed rations prepared from moldy corn. Partially purified extracts were shown to be effective in increasing the rate of gain and feed efficiency in sheep. The production of test material on corn was improved and, with the availability of what was then considered to be large quantities, one of us (R. S. Baldwin) began attempts to prepare the pure substance responsible for the physiologic effects of crude extracts. This work culminated in the isolation of a pure crystalline compound. Some of its properties were described in two publications (Stob et al., 1962; Andrews and Stob, 1965). The intervening years since this first work have seen the accumulation of publications describing the occurrence of the active principle, zearalenone, in stored grains (Eppley et al., 1974; Shotwell et al., 1970; Caldwell and Tuite, 1970); its synthesis by several species of Fusarium (Caldwell et al., 1970; Ishii et al., 1974);the development of techniques for its detection and quantitative estimation (Eppley, 1968; Vandenheuvel, 1968; Mirocha et al., 1968a); and the chemical and physical properties of zearalenone, its derivatives and related compounds. The literature related to the chemistry of zearalenone was reviewed by Shipchandler (1975) and again by Pathre and Mirocha (1976). Interest in the occurrence of zearalenone in feed grains was enhanced by an increasing awareness of the existence of substances produced by a wide variety of fungi that might directly or indirectly affect human health. Much of the work by others has been concerned with this aspect of zearalenone. Some of this information has been reviewed by Mirocha et al. (1971). In our laboratories, however, we were also interested in determining the potential value of this new class of compounds as anabolic substances in animals. This work has covered a period of nearly 15years, during which the production of zearalenone has evolved from the first moldy corn experiments to the present large-scale synthesis by submerged fermentation. The development and testing of a number of chemical derivatives of zearalenone for use in animal nutrition began as soon as adequate quantities of the parent compound became available. Interest in the derivatives extended beyond their potential value in animal nutrition to an examination of their general pharmacological properties. Two derivatives, the pair of dia-

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

61

stereoisomers obtained by catalytic reduction of the parent compound, exhibited properties suggesting possible value in treatment of the postmenopausal syndrome. One of these isomers is preferred for use as an anabolic agent in sheep and is currently marketed under the trademark RALGRO@. Pharmacological data have been obtained in several animal species in an effort to meet Food and Drug Administration requirements in connection with New Drug applications for these two isomers. The chemistry of zearalenone will be considered here only to the extent necessary to indicate the interrelationships of the parent compound, two derivatives currently being marketed, and the major products of metabolism of the parent compound and one derivative, which will be further considered in Section IV. The gaps in the literature related to zearalenone are in areas of the commercial production by fermentation and of the pharmacological and toxicological data accumulated by CSC. It is the intent of this summary to fill those gaps.

II. Chemistry A. STRUCTURE AND TERMINOLOGY When the structure of the active substance was established, the term zearalenone was selected as a convenient designation for the naturally occurTABLE I RELATED RESORCYLICACID LACTONES Melting point Compound

(“C)

(I)

Zearalenone

164-165

(IV)

Zearalanone

192-193

(V)

(Wb

Configurationu at C-6’

Trademarks

145-147

RALONE@

182-183

RALONEB, RALGROm

Zearalanol

~~

“From W. H. Urry, University of Chicago, Chicago, Illinois, unpublished results. *United States adopted name, zeranol.

62

P. H. HlDY ET AL.

Ni

c

no

\

(X.rn)

OH

FIG. 1. Structures and relationships of zearalenone and the derivatives considered in this paper. (See TabIe I for nomenclature.)

ring compound from which at least some of the early derivatives could be named, relating them to the parent compound. This terminology, especially for the parent material, has now found general acceptance. Prior to selection of this name, zearalenone had been referred to as FES or Fermentation Estrogenic Substance (Hodge et al., 1966) and as F-2 by Christensen et al. (1965).A more complete chemical designation applied by Urry et al. (1966)is 6-(10-hydroxy-6-oxo-trans-1-undeceny1)-P-resorcylicacid lactone. Johnson et al. (1970) indicated the size of the macrolide ring as a p-lactone. Chemical Abstracts indexes zearalenone as [S-(E)]-3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-lH -2-benzoxacyclotetradecin -1,7(8H)dione. Table I summarizes some information needed to i d e n e the compounds that will be considered in this paper. Figure 1, constructed mainly from Urry et al. (1966), indicates the relationships of the compounds which will be discussed. For the balance of this paper, specific compounds, with the exception of zearalenone, will be indicated by the Roman numeral designation assigned in Fig. 1.

B. ASSAYAND RECOVERY Zearalenone is practically insoluble in water, but is readily soluble in many organic solvents, as shown in Table 11, and in dilute alkali. These properties facilitate both assay and recovery of the compound from fermentations. Whether the zearalenone in fermentation broths is still contained within the mycelial cell or has been released by autolysis, its insol-

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

63

TABLE I1 SOLUBILITY OF ZEARALENONE Solvent

Gm/100 gma

Water Ethanol Methanol Acetonitrile Acetone Methylene chloride Benzene n-Hexane

0.002 24 18

8.6 58 17.5

1.13 0.05

"25°C

ubility in water ensures that simple filtration with a filter-aid such as Johns Manville Super-Cel will remove more than 99.8% of the total zearalenone in a 10-gm per liter broth. In our high-titer fermentations, the contribution of closely related metabolites to the assay has never been significant. In routine assays of fermentation broths, the zearalenone-containing solids were removed by filtration and the cake was thoroughly extracted with methanol. Ultraviolet absorption spectra were run on the methanol extracts, and titers were calculated by comparing absorbance at 236 or 274 nm with that of pure material. In fermentation samples, corrections may be applied for absorbance not due to zearalenone. Large-scale recovery could be accomplished similarly by extracting fermentation solids with an organic solvent, preferably methanol or acetone, and crystallizing the zearalenone by evaporating the solvent or by adding water to a concentrated solution of the compound. An alternative recovery process based on extraction with aqueous alkaline solutions has been described by Hidy and Young (1971). They slurried the fermentation solids in dilute (0.25%)sodium hydroxide solution (PH 11-12), filtered the slurry, and acidified the filtrate to precipitate the zearalenone. Solutions of crude zearalenone were decolorized with char when necessary and pure product was obtained by the usual methods of recrystallization, generally using isopropyl alcohol as the solvent. @

Ill. Fermentation A. CULTUREDEVELOPMENT As mentioned previously, initial culture isolation studies were conducted at Purdue University. Of the fungal cultures isolated from moldy corn from

64

P. H . HlDY ET A L .

Indiana farms which had reported estrogenic stimulation in swine, only those cultures identified as Gibberella zeae (the perithecial or perfect stage of Fusarium roseum “graminearum”),elicited an estrogenic response in sexually immature female pigs. Culture isolation studies were continued in CSC’s laboratories by examining various moldy corn and soil samples. Additionally, Fusarium cultures were procured from the American Type Culture Collection as well as the Northern Regional Research Laboratories, and their ability to produce zearalenone was tested under surface and submerged fermentation conditions. Three isolates of G. xeae-Dewar (ATCC 20028), Paul S (ATCC 20271), and H331 (ATCC 20272twere selected for further study. A mutation and selection program was initiated concurrently with media and solid support development. Various criteria for selection of improved strains were imposed on the selection process at different times during the program. The first priority was high-producing strains. Using the procedure of Eisenstark et al. (1965) to treat macroconidia of the Dewar strain with N-methyl-N‘-nitro-N-nitrosoguanidine (NTG), selected cultures were again tested under both surface and submerged fermentation conditions. From these studies, a strain (542, ATCC 20273) was selected that not only produced higher titers in surface fermentations, but also produced zearalenone in a suitable medium under aerobic submerged conditions (Keith, 1972). Improvement of this culture (strain 542) was continued using NTG as well as ethyl methane sulfonate as mutagens. This work has resulted in considerably higher zearalenone titers as described in Section II1,C. Other criteria for selection have been applied during the course of developing the submerged process. Because most fusaria produce pigments which can be difficult to remove in the recovery and purification process, some emphasis has been placed on finding colorless mutants with the ability to produce zearalenone. This goal has recently been attained in the laboratory. Strains having high rates of synthesis have been sought, the economic advantage of shortening a 2- to 3-week fermentation being obvious. Here we have been partially successful. Variation in ability of a Fusariurn strain to produce zearalenone has been discussed by Mirocha et al. (1971). Similar variability has not been encountered in our commercial production. Selected strains of G. zeae are preserved both by lyophilization and by submersion in liquid nitrogen.

B. SURFACEFERMENTATION There are few details reported of efforts in other laboratories to produce zearalenone in quantity. In every case, apparently only limited success has been attained utilizing the common grains for support and nutrient supply.

ZEARALENONE AND DERIVATIVES: PRODUCTION AND ACTIVITIES

65

Mirocha et al. (1971) have reviewed the work of the Minnesota group, which has used mainly corn or rice as a substrate either alone or with added glucose. From the data provided, it is difficult to calculate the efficiency of such fermentations, but it does not appear to be high. At the beginning of CSC investigations it was not known whether the active material sought was a metabolic product of the organism or was simply a constituent of grain that was altered by action of the fungus. For this reason our early efforts centered around the use of grains as media, but recovery of a product from the complex mixture including starch, partially digested protein and lipid of the grain base presented problems that were recognized early as barriers to commercial synthesis. Because of these barriers, efforts were directed toward cultivation of the fungus on defined media. Since there was no assurance that the product could be synthesized by Gibberella in submerged culture, a solid support was sought for defined media which could absorb considerable quantities of liquid without becoming waterlogged. Numerous possible supports were tested, but vermiculite, an expanded mica product, was found nearly ideal. Three or four milliliters of medium per gram of vermiculite could be loosely bound by the porous laminar structure so that no liquid separated from the moistened mass even on prolonged standing. This readily available mineral was essentially insoluble in water, dilute alkali, or organic solvents. These properties were valuable not only in the fermentation but also in the recovery process. For the first studies using a vermiculite support, a medium was selected containing a high level of glucose (30%). As later became evident, production of zearalenone was very difficult to initiate at carbohydrate levels less than 20%. It was determined that low initial temperatures were a requirement for a successful synthesis. This requirement has also been reported by others (Mirocha et al., 1971). While growth and production of zearaletione on grain is slow, requiring as much as 6 or 7 weeks to complete, utilization of the glucose on vermiculite was rapid with evolution of heat. Initially, fermentations were carried out in 2-liter Erlenmeyer flasks in which 240 ml of medium was held on 80 gm of vermiculite. Flasks were kept in an incubator room held at 21"-24"C. The temperature in the flasks rose rapidly to above 30"C, and it was obvious that heat transfer was a problem. For this reason, and to increase capacity to produce zearalenone, the flask fermentation was carried out by closely packing the flasks in a tray of running water that could be kept at about 16"-17°C. This provided better heat transfer from the flasks. The production of zearalenone in flasks was encouraging. Yields of 1.52.0 gm per flask (6.25-8.33 gm/liter) were readily obtained. Total production capacity in flasks was still limited and insufficient to provide the quantity of pure material required for structure work and pharmacological study.

66

P. H . HIDY ET AL.

The fermentation was next successfully transferred to 2 0 x 4 0 5-inch ~ aluminum trays with loosely fitted lids. The trays were immersed about 3 inches in a water bath maintained at about 17"-18°C. A single tray held a 3-inch layer of vermiculite weighing about 5 kg. After sterilization by autoclaving in the tray, the vermiculite layer was charged with 13.5 liters of inoculated medium and incubated for 4-5 weeks. Initially, glucose concentrations of 30% were used, but it was shown that 45% glucose could be tolerated and that tray loadings of as much as 17 liters did not greatly affect the efficiency of conversion of carbohydrate to zearalenone. A rack holding 16 trays in four levels was installed in a laboratory to allow increased attention to be given to variables of the process and a pilot plant operation was begun with two similar racks. Later, trays containing integral water baths and designed to be stacked without requirement for a rack were built and operated. Each of these trays had a capacity 2.5 times that of the original trays. Since inoculum preparation and medium composition for flask and tray fermentations were essentially identical, operation of a typical tray process will be described. For inoculum preparation, spores from a Bennett's agar slant or a lyophile culture were suspended in 5 ml of sterile water and added to 100 ml of Bennett's medium in a 500-ml Erlenmeyer flask. After incubation on a rotary shaker at 30°C for 24 hr, 10 ml of this first-stage inoculum was used to inoculate 300 ml of Bennett's medium in a 1-liter Erlenmeyer flask which was also incubated 24 hr at 30°C on a rotary shaker. One flask of second-stage inoculum sufficed to inoculate 13.5-17.0 liters of sterile production medium contained in a 5-gallon stainless steel milk can equipped with tubing to allow siphoning the contents of the can onto the vermiculite in one aluminum tray. Production medium was prepared as shown in Table 111. The concentration of every ingredient of the above medium was varied over a considerable TABLE I11 DEFINEDMEDIUMFOR PRODUCTION OF ZEARALENONE IN SURFACE CULTURE Component Glucose

BYF Yeast KCI

NaNO, N ~ N O S KzHPOi Distilled water to volume

Percent

37 0.1 0.05

0.05 0.2 1.0 0.1

67

ZEARALENONE AND DERIVATIVES: PRODUCTION AND ACTIVITIES

TABLE IV C O U R S E O F A TYPIC&

SURFACE

FERMENTATION^

Zearalenone (gmlliter)’ Tray

3 Weeks

4 Weeks

5 Weeks

1 2 3 4

9.25 9.62 10.36 4.07

13.32 12.95 12.58 10.73

15.91 14.06

-

17.02

“Strain Dewar. ‘13.5 Liters/tray

range without substantial improvement in yield of the product. No organic nitrogen source was as effective as the combination of ammonium and sodium nitrates shown. Titers of a typical fermentation, consisting of four trays, each containing 13.5 liters of medium, are shown in Table IV. The effect of the culture improvement program on the performance of the surface fermentation is demonstrated in Table V, where strain Dewar was compared to strain 542. A “microfermentation” was developed which proved to be a valuable device for the study of some variables and for evaluation of new cultures. Petri plates (20 x 1OO mm) containing 10 gm of dry vermiculite were autoclaved with lids in place. A total of 25 ml of preinoculated medium was applied evenly over the surface of the vermiculite by pipette. Inoculum volume was 10% of the medium volume. Plates were incubated with lids in place at 19.5”C and at 85% relative humidity. The fermentation was complete in 3 weeks and production of zearalenone on standard medium using strain Dewar was 12 gmfliter. Optimum performance required media containing 30% glucose. This fermentation procedure was easily adapted to the TABLE V COMPARISON OF PERFORMANCE O F STRAIN DEWAR A N D S T R A I N

542

TF114-tray

Strain

Gm/traya.’

Gm/liter

Gmigm CHO

1 2 3 4

Dewar 542 Dewar 542

274 438 247 486

13.5 27.4 15.4 30.37

0.036 0.074 0.042 0.082

“16.0 Literdtray. ’4 Weeks.

68

P . H. HIDY ET A L .

preparation of 14C-labeled zearalenone. Even smaller fermentations in 25-mm Petri plates and requiring only 5 ml of medium were successfully operated but never fully developed. Details of the surface fermentation have been previously described (Hidy, 1971).

C. SUBMERGEDFERMENTATION The discovery of mutant strains of G. zeae capable of producing zearalenone by submerged culture (Keith, 1972) was a major breakthrough in efforts to produce zearalenone on an industrial scale since earlier attempts had been unsuccessful. There have been no reports in the literature of appreciable zearalenone production by submerged fermentation; therefore, the information reported here represents work from our laboratories (Keith, 1972; Woodings, 1972; McMullen, 1972). Even with mutant strains, zearalenone titers from shake flasks were in the range of 1.0 to 1.5 gmhiter using a medium that gave up to 30 gmfliter in surface fermentation. It was obvious, therefore that the development of a successful submerged fermentation required a reinvestigation of the medium components as well as a study of other fermentation parameters. Early work on the submerged process was conducted in shake flasks and small fermentors. The fermentation was subsequently scaled up to 100gallon and 2000-gallon pilot plant fermentors and finally to 20,000-gallon production fermentors. Since the objective of the program was to obtain the highest zearalenone titers in the shortest time with the most economical nutrients, the medium was frequently changed as was the strain employed. Some of the various media and strains used during the course of the study are summarized in Table VI. Inoculum for the submerged fermentation was either a Bennett's broth culture grown for 24 hours at 30°C with aeration or an aliquot of a zearalenone producing fermentation (referred to hereinafter as a whole-beer inoculum). The volume of inoculum was generally 5%although volumes of about 2-20% could be used satisfactorily. Other inoculum media were investigated, but no advantage was seen for them. The optima1 temperature for the zearalenone fermentation was found to be between 21" and HOC-nearer 21°C being preferred for shake flasks and nearer 24°C for fermentors. Zearalenone was generally not produced in fermentations starting at temperatures above 28°C; however, once zearalenone production was established the temperature could be raised slowly to 32°C before production stopped. Temperatures below 20°C were not desirable because of the slower growth rate and lower rate of zearalenone production.

TABLE VI PROGRESSOF MEDIUM AND STRAINDEVELOPMENT PROGRAM Strain Titer (gm/liter) Glucose (grn/liter) NH4N03 NaN03 Urea N-Z-Amine A@ Yeast extract K,HPO, MgS04’ 7HzO KC1 ZnS04.7Hz0 (PP) Inoculum

542 4-5 21 days 300 10.0 2.0

542 7-8 21 days 300 5.0 1.0

542 20-22 21 days 300

542 21-23 21 days 300

4.0

4.0 3.0

RG-6C 26-28 14 days 200 -

-

4.0 3.0

4.0 3.0

4.0 3.0

0.5 0.25 0.25

0.5 0.25 0.25

0.9 Whole beer

0.9 Whole beer

-

-

-

-

1.0 1.0 0.5 0.5

1.0 0.5 0.25 0.25

1.0 0.5 0.25 0.25

Bennett’s broth

Bennett’s broth

Bennett’s broth

542 16-18 14 days 200

-

0.5 0.25 0.25

0.5 0.25 0.25

0.9 Bennett’s broth

0.9 Whole beer

-

RG-6C-88- 13 >32 14 days 200

-

70

P. H . HIDY ET AL

The fermentations were usually run for 14-21 days depending on the amount of sugar in the starting medium and the rate of sugar utilization. Zearalenone production declined shortly after the sugar was depleted but fermentations could be prolonged by feeding additional sugar before zearalenone synthesis completely ceased. Aeration requirement for the fermentation was not great, but maintaining a minimal amount of aeration was essential for production. Shake-flask studies were routinely conducted with 100 ml of medium per 500-ml flask incubated on a rotary shaker at 350 rpm. Reciprocal shakmg or larger volumes of medium per flask reduced titers markedly, as seen in Table VII. Laboratory fermentors were operated with 0.25 to 0.5 volumes of air per volume of medium per minute with a variety of impellor sizes and codigurations. In general, larger impellors at slower speeds (350-500 rpm) were preferred to smaller impellors at higher speeds (750-1000 rpm). Higher speeds also exhibited undesirable sheer on the mycelium. I t was possible to reduce aeration by 40-50% after 6-7 days, but continued zearalenone synthesis required a small amount of air. If fermentations were begun with reduced aeration, zearalenone production was prevented. Higher levels of aeration were not beneficial and caused excessive foaming. The pH of the fermentation was not critical and it was advantageous, from the standpoint of contamination, to allow it to drop naturally to about 3.54.0. Attempts to control pH never resulted in any significant increase in titer and in most cases reduced titers somewhat. The starting pH was about 6.8-7.2. Among the usual carbohydrates tested as carbon sources, glucose, fructose, and galactose were about equal in their ability to support zearalenone production. Xylose, sucrose, maltose, glycerol, and sorbitol performed best in combination with glucose. The more economical forms of glucose, such as CereloseB or Staleydex@,were selected for further development. The less TABLE VII AERATIONIN SHAKE FLASKFERMENTATIONS Mediuma volume

(4 50

100* 100 200

Shaker

Titer (gm/liter)

Rotary Rotary Reciprocal Rotary

19.6 19.8 0 0

"20%Glucose medium, 14-day fermentations *Standard conditions.

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

71

refined corn syrups and starch hydrolyzates were satisfactory energy sources, but they required alteration of the other medium components for their variability. Glucose concentration was a very important parameter in the fermentation. Concentrations above 20% were generally required for zearalenone synthesis in a standard medium inoculated with a Bennett’s broth inoculum. It was found, however, that by using a whole-beer inoculum from an established fermentation, zearalenone could be produced at glucose levels of 20% and below. Investigation of this peculiarity led to the observation that extracts of whole beer or a catalytic amount of pure zearalenone could initiate production at lower sugar levels even when a Bennett’s broth inoculum was used. As little as 10 mg of zearalenone per liter of medium could produce the desired fermentation. Table VIII shows the effect of added zearalenone in 20% and 30% glucose media using a Bennett’s broth inoculum. Use of a whole-beer inoculum, containing some zearalenone, eliminated the need to add the pure material and gave titers superior to those in media without added zearalenone. Various nitrogen sources were tested in the submerged fermentation, and in contrast to results from the surface fermentation, organic nitrogen sources gave higher titers than did inorganic sources. Urea was the best and cheapest source tested and, therefore, became a standard ingredient in subsequent fermentations. The optimum concentration was between 0.4and 0.6%. Proteins and protein hydrolyzates were not as satisfactory as urea for zearalenone production, but they did stimulate production when used in combination with urea. Highest titers were obtained with a urea concentration of 0.4% and an N-Z-Amine A* (Humko Sheffield casein hydrolyzate) concentration of 0.3%.Other protein hydrolyzates, casein, steepwater, corn gluten meal, and yeast fractions could also stimulate titers in combination with urea. TABLE VIII EFFECTOF ADDEDZEARALENONE Zearalenone

Percent glucose in medium

Amount added to medium (mgliter)

Amount produced after 14 days (gmiliter)

20 20 30 30

0 20 0 29

0 16.7 9.5 18.0

12

P. H . HIDY ET A L .

Ammonium salts of some organic acids and a number of amino acids, such as asparagine, glutamine, and glycine supported good zearalenone production but were not economically feasible for large-scale fermentations. Inorganic requirements for the fermentation were satisfied by adding dipotassium phosphate, magnesium sulfate, potassium chloride, and zinc sulfate. The optimum concentration of dipotassium phosphate was 0.05% and of magnesium sulfate, 0.025%. Increasing either of these beyond the optimum increased cell yield and sugar utilization but not zearalenone production. Potassium chloride had an optimum of around 0.025%, but varying the concentration beyond the optimum had little effect on the fermentation. Zinc sulfate was found, however, to have a marked effect on the fermentation (McMullen, 1972). Early fermentations contained a small amount of yeast extract, and all attempts to replace yeast extract with vitamins or amino acids were unsuccessful. I t was found that the ash of yeast extract satisfied the requirements. Subsequent experiments demonstrated that the component in yeast extract required by the fermentation was zinc. The amount required was found to be very low, and the optimum appeared to be around 1 ppm (as zinc sulfate heptahydrate). Higher levels of zinc stimulated growth, sugar utilization, and pigment production. The low tolerance for zinc may explain why some crude proteins and carbohydrates failed when tested. Another element for which a requirement was demonstrated was iron, but this held only when reagent grade chemicals were used in the fermentation. Semicontinuous fermentations were run using a variety of withdrawal and replacement levels. A process has been described (Woodings, 1972) wherein 75% of the spent medium from a zearalenone fermentation was withdrawn and an equal volume of fresh sterile medium was added. The replacement was carried out when the glucose was consumed and the zearalenone titer was 10-20 gmAiter. The time interval between replacements was 7-10 days depending on the rate of glucose utilization. The fresh medium added was a complete 20% glucose medium although it was reported that the glucose concentration could be as low as 10%. Other semicontinuous fermentations have been run ranging from a daily 10% replacement to a 75% replacement every 7-10 days. In essence, the use of a 5% whole-beer inoculum could be described as a 95% replacement. A successful semicontinuous fermentation depended on a withdrawal and replacement method that maintained an equilibrium between sugar utilization, growth, and zearalenone production. Once the fermentation had been stabilized, it could be continued for long periods before the production rate decreased significantly. A 2000-gallon pilot plant fermentor has been operated semicontinuously for over 6 months before being terminated for mechanical reasons. Parameters followed during the course of a typical fermentation are presented in Fig. 2. They represent a composite of data obtained from shake

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

73

Time (days)

FIG.2. Course of a typical fermentation

flasks containing 20% glucose medium inoculated with a 5% whole-beer inoculum and grown at 21°C. The initial level of zearalenone represents carryover from the inoculum. The same parameters followed in a 30% glucose medium would be very similar but would be extended to about 21 days to allow complete utilization of the extra sugar.

D. BIOSYNTHESIS

The zearalenone biosynthetic pathway has been investigated using radioactive potential precursors (Steele et al., 1974). Since the structure of zearalenone suggests a condensation of 9 acetate units, precursors such as acetate, diethyl malonate, senecioate, shikimate, and DL-mevalonic-2lactone have been tested using cultures of G. zeae grown on solid substrates. Only acetate and diethyl malonate were incorporated readily into zearalenone, indicating that zearalenone is synthesized via the polyketide pathway. From 14Cincorporation studies in the CSC laboratories, using cells grown in submerged culture, acetate was also found to be readily incorporated into M sodium acetate, a zearalenone. At an optimum concentration of 8.6 x linear incorporation rate of 8.6 ng/min/mg dry weight of cells for 30 minutes was observed. Acetate concentrations above this optimum proved to be inhibitory to 14C incorporation. This parallels unsuccessful attempts to feed acetate to the submerged fermentation as a technique for increasing zearalenone yields.

74

P. H . HIDY ET A 1

From thin-layer chromatographic examinations of methanol extracts of ['4C]-acetate-exposed cultures, a radioactive fraction other than zearalenone was detected. When this partially purified fraction was used in 14C incorporation studies in place of acetate, its radioactivity was found in zearalenone. At this time, the role, if any, this fraction has in the biosynthesis of zearalenone has not been determined.

IV. Pharmacology Biological studies, begun in 1958, were confined to mouse uterotropic assay of molded grain and fermentation products to establish and confirm that the estrogen syndrome observed in animals was due to a mold metabolite. The assay was selected to follow the course of zearalenone purification and production. Zearalenone and over 150 derivatives have been tested for various pharmacological activities since its initial isolation. A full range of studies to evaluate the potential usefulness of zearalenone and selected derivatives was undertaken following the isolation of the pure material. Data reported herein will be limited to the natural product and those derivatives having either scientific significance or useful application. The biological results reported here deal with the pure materials.

A. GENERALDRUGACTION Single oral doses of the compounds of interest were screened for drug activity in adult male and female mice using oral doses of 4644640 mg/kg. The usual tests for measuring and assessing a wide variety of drug actions were performed. In general, the outward appearance of the animals was unremarkable during the 7-day test period. Specifically, zearalenone and compounds (IV), (V), and (VI) displayed no drug-related effects except compound (VI), which showed a tendency to cause increased uterine weights. These compounds, when tested for central nervous system activity using oral doses of 328 mgkg in the mouse mental health screen test, displayed no significant CNS activity. In another series of tests the compounds were evaluated for their ability to produce behavioral changes in squirrel monkeys (Sidman Avoidance Test) and in food-deprived rats (Food Reinforcement Test). The compounds were without effect when a cumulative oral dose of 150 rng was given to monkeys and 48 mgkg was given to rats. Oral doses of 128 mgkg had no effect on urine output, electrolyte excretion, or propensity to produce gastric hemorrhage when administered to rats. These compounds had no significant effect on gastrointestinal motility when administered orally to mice at a maximum dose of 100 mgkg.

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TABLE IX BIOLOGICAL ACTIVITIES OF ZEARALENONE AND DERIVATIVES Compounda Activity

I

111

IV

v

VI

Anabolic (mice) Uterine weight (mice) (increase) Oviduct weight (chick) (increase) Vaginal cornification Antiestrogen Antiovulatory Implantation inhibition Gonadotropin inhibition Anti-FSH and HCG Anti-LH Progestational Androgenic Blood cholesterol (lowering) Antiinflammatory Antimicrobial Coccidiostatic Herbicide

+, Active; - , not active.

Zearalenone and compounds (V) and (VI) had no direct effect on the cardiovascular system when administered intravenously at doses of 5 m&g to the anesthetized dog. Other parameters monitored, such as blood pressure, electrocardiographic (ECG) and neurophysiologic activities, were not altered significantly by these compounds. Table IX summarizes certain of the biological activities of the parent material and those of the derivatives of current interest.

B. ENDOCRINE ACTIVITIES A total of 46 different classical as well as newly developed endocrine procedures were used to study the range of activities of these compounds. The initial objective of this program, i.e., evaluation for use as an anabolic agent for animals, was expanded to include an appraisal of use in clinical medicine. Zearalenone may be classified as a weak estrogen having anabolic properties when administered to certain animal species. The parent material and those derivatives that possess these properties produce a limited stimulation of the uterus. Thus they are classified as impeded estro-

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P. H. HlDY ET A L

gens. Perhaps the most remarkable property of these compounds is the difference in relative estrogenic activity displayed among different animal species. Dose-response slopes obtained with zearalenone and its estrogenic derivatives do not parallel those obtained with 17-P-estradiol and estrone. As a result of this divergence, any attempt to assign ratios of biological activities may be misleading. Zearalenone (I) and the derivatives (11), (111), (IV), (V), and (VI) produce estrogenic effects in a variety of assay systems using the mouse, rat, rabbit, chicken, ewe, domestic pig, and monkey. Their relative estrogenic activity listed in decreasing order is (VI), (IV), (I), (V), (111). In classical tests, i.e., uterine weight or vaginal smear, and using (VI) as an example, potency varies from approximately 0.1 to 0.7% of 17-0-estradiol and 1 to 20% of estrone depending upon the particular end point used. The 0ral:parenteral ratio for this compound varies from 0.2 to 3.0. In monkeys, (VI)will produce changes in the vaginal mucosa and the endometrium at a dose of 0.9 mgkg when administered orally. The clinically effective dose of (VI)to treat estrogen deficiency states, projected to be 50-75 mg daily based on animal studies, has been found to be correct. As a result of the advances made in the fermentation process, significant quantities of zearalenone became available in 1961. Evaluation of the anabolic properties of this material was then begun. In two preliminary studies, subcutaneous administration of zearalenone to growing sheep produced a moderate weight gain over untreated animals. Under the procedure of Hershberger et al. (1966),it was found that zearalenone and (VI)produced a positive myotropic effect in castrate male mice. This effect was subsequently confirmed in the Syntex Laboratories and has been reported (Mirocha et al., 196813).Subsequent animal growth trials, in which these two materials were compared as implants administered to steers and heifers, revealed that compound (VI) produced a significant improvement in growth and feed efficiency. The important aspects of the numerous growth trials conducted with (VI) have been reported (Brown, 1970). The same material when administered as an implant was found to produce a significant positive effect on nitrogen retention in sheep. When 6 mg of compound (VI) was compared with an equal weight dosage of diethylstilbestrol, nitrogen retention was improved 23.6%by the former versus 16.4%for the latter. These findings eventually led to the marketing of RALGROm (IMC brand of zeranol) implants for use as a growth promotor in cattle and sheep. Studies concerning metabolic changes produced in growing cattle and sheep have been reported by several investigators (Wilson et al., 1972; Borger et al., 1973a,b). Drug residue studies have shown that edible tissue from animals receiving 36 mg as an implant contain no detectable quantity of (VI) or metabolites 65 days after implantation. The procedure for drug residue

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analysis, consisting of tissue extraction and gas-liquid chromatography, has a detection limit of 20 ppb. Zearalenone inhibits synthesis and secretion of pituitary gonadotropins, an activity not unexpected for estrogenic substances. This activity was found to be in direct proportion to the uterotropic activity with the possible exception of compound (V). The finding that this compound was more active than anticipated has suggested certain clinical uses. Foreign clinical studies have indicated that effective control of the menopausal syndrome may be achieved with an oral dose of 25-50 mg/day or approximately one-half that of compound (VI). Zearalenone, (V), and (VI)significantly reduce blood lipids when administered to rats receiving a high fat diet. Blood cholesterol lowering appears to be greatest with zearalenone based on relative estrogenicity (5-1O:l). Triglycerides are unaffected by the administration of the above compounds. The oral administration of the parent material, (V), and (VI) to adrenalectomized, diabetic, and fasting rats at doses of 1-10 mg/kg produced no significant alteration of blood glucose levels. Glucose tolerance was likewise unaffected when doses of 1-10 mg/kg of these compounds were given orally to rats. None of the resorcylic acid 'lactones tested had progestational or androgenic activity. Zearalenone and compound (VI)showed antiinflammatory activity using the rat foot edema and the granuloma inhibition tests. However, in view of the thymolytic activity and body weight decreases observed, it was concluded that the activity observed had no practical significance. Compound (VI) showed a highly significant enhancement of cortical bone growth in the rat at an oral dose of 12.5 mg/kg. In summary, zearalenone is a nonsteroidal mold metabolite possessing weak and impeded estrogenic properties in all animals tested. Derivatives prepared from it may possess greater or less activity. The estrogenic nature of (VI) was hrther confirmed with the finding that it will displace tritiated estradiol from the isolated estrogen-binding uterine protein. All compounds tested were well tolerated, even by swine, which is perhaps the most sensitive species studied. It is more active orally than parenterally. The parent material and certain derivatives possess antigonadotropic properties, the effect being exerted at the pituitary level. Certain derivatives, particularly (IV) and (VI), possess significant anabolic properties. C. METABOLISM When biosynthetic 14C-labeled zearalenone was administered orally to rats, 7040% was excreted in the feces and 2030% in the urine. The only significant metabolite found in these studies was identified as (111) (R. S. Baldwin, unpublished results). This compound was found to be one-fourth as

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estrogenic as zearalenone. Similar metabolism was found to occur in sheep, where the ratio of metabolite to zearalenone in feces was 2:l. Extensive metabolism studies were carried out with (VI). The major metabolite found in all species studied (rat, sheep, cattle, monkey, dog, rabbit, and man) is (IV). Conjugates of PI)and (IV) have been isolated from the urine and feces of animals. The major route of excretion regardless of the mode of administration, with the exception of the rabbit, is via the feces. The rabbit excretes the major portion of the drug and metabolite by way of the urinary tract. Metabolism of (VI)in cattle, following administration of 72 mg in the ear, has been reported (Sharp and Dyer, 1972).

D. SAFETYEVALUATION Evaluation, in animals, of the effects produced by both single and repeated dose administration of zearalenone and certain derivatives was started by CSC in 1961. These materials have been remarkably well tolerated. The selection of the term “toxin” (Christensen-et d.,1965) used to describe the physiological action of this mold metabolite may have been unfortunate. The acute toxicity data obtained from single-dose experiments for zearalenone and the derivatives (IV), (V), and (VI) are presented in Table X. The oral administration of a single dose of 20,000 mgkg failed to produce deaths in mice or rats. Maximum doses produced slight irritability and hypoactivity during a 14-day observation period. Intraperitoneal doses proTABLE X SUMMARY OF ACUTETOXICITY DATAFOR ZEARALENONE AND DERIVATIVES

Oral Species Mouse

Rat

Guinea pig

Intraperitoneal

Compound

Male

Female

Male

Female

(1) (Iv)

-

>20,000

-

>SO0

3290 -

2530 4400

>20,OOo >lO,OOO >40,OOO

>500

(v) (VI)

>10,000 240,000

(1)

>10,000

>10,000 >10,OOo

5490

-

0

-

>10,OOO >40,OOO

4200 8900

3480

(VI)

>10,000 >40,000

10,900

(I)

-

>S,OOo

-

2500

(Iv)

-

-

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duce irritability, bradypnea, hypoactivity, and hypothermia. The responses were similar for all other compounds. Subacute studies of 2-14 weeks duration have been completed with zearalenone and derivatives (V) and (VI) using several species. Compounds (V) and (VI) have been given orally to rats, dogs, and monkeys for 2 years. Compound (VI) is currently the subject of long-term tests in dogs and monkeys projected to run 7 and 10 years, respectively. Periodic sacrifice and tissue histology are complete through 6 years. Table XI lists the subacute and chronic tests performed. These results have been briefly summarized for each compound. Zearalenone. An oral dose of 1 mgkglday given to rats for 13 weeks was without significant effect. Weight gain suppression but no histological changes were observed at a dosage of 5 mglkglday. A dose of 25 mglkglday produced suppressed weight gain and testicular size and reduced number of corpora lutea. Histological changes observed were similar to those following high dosages of estrogens and included endometrial stimulation and arrested maturation of spermatocytes. In a similar study in dogs, 1 rngkglday orally was without effect while 5 mglkglday produced reduced number of corpora TABLE XI

SUBACUTEAND CHRONICTOXICOLOGICAL STUDIESCONDUCTED WITH CERTAINRESORCVLIC ACID LACTONES

Species Rat

Chicken Swine Dog

Monkey

Sex

Drug

Dose range Route

(mgikg)

Duration

Subcutaneous Gavage Diet Gavage Diet Diet Diet Diet Intramuscular Diet Gavage Gavage Gavage Gavage Gavage Gavage Gavage

2.5-10.0 25-1600 1-25 1-30 0.25-6.25 1-25 0.1-20 110-220 ppmb 50-200 2.2-4.4 p p d 25-1600 1-30 0.25-12.5 0.025-25 15-37.5 1-30 15-75

2 Weeks 6 Weeks 13 Weeks 13 Weeks 13 Weeks

“Ongoing studies to continue for 7 years (dogs) and 10 years (monkeys). *In diet as parts per million (ppm).

2 Years 2 Years 4 Weeks 6 Days 61 Days 4 Weeks 13 Weeks 14 Weeks 2 Years 6 Year9 2 Years 6 Years’

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lutea and arrested spermatogenesisbut no uterine changes. Higher doses of up to 50 mg/kg/day produced similar effects. Growing poultry receiving up to 220 ppm of zearalenone in their diet for 4 weeks showed a slight, nonsignificant, weight improvement. Swine receiving diets containing 4.4 ppm showed signs of estrogenic stimulation in females but not in castrate males. A level of 12.5 ppm of zearalenone can be detected in diets when fed to immature female mice. Compound (V). Experiments of 90-day duration with rats and dogs at a maximum test level of 25 mg/kg/day orally failed to establish a true toxic effect with this compound. Two-year chronic rat and monkey studies with maximum oral doses of 30 mg/kg/day have been completed and data are being analyzed. Preliminary examination of the results have revealed no unexpected findings. Compound (VZ). Based upon the results obtained from the 2-year rat study the highest nontoxic oral dose lies between 6 and 20 mg/kg/day and has been set at the logarithmic mean of 11 mgkglday. Studies of similar duration in dogs have shown that the highest nontoxic oral dose lies between 2.5 and 25 mgkglday and has been set at 8 mgkglday. It is noteworthy that compound (VI)did not increase the incidence of neoplasms in the rat or induce malignant neoplasia in the mammary glands of dogs as has been reported for steroidal substances. Studies completed through 6 years have revealed no gross or histological drug-related evidence of malignant changes in dogs or monkeys. A series of studies was carried out with zearalenone and compounds (V) and (VI) to determine the effects of repeated oral dosage on the mammalian reproductive process. Breeding performance, embryo and teratogenic toxicity, perinatal and postnatal effects were evaluated. Ruddick et al. (1976) reported a no-effect zearalenone dosage of 0.3-1.0 mg/kg/day orally in rats in a teratological study. A CSC study indicated a slightly higher range, 1 . 0 3 . 0 mgkg/day. Higher doses of 5-10 mgkg occasionally produced skeletal anomalies, embryo toxicity, and resorptions. The no-effect dose of compound (VI) in these studies appeared to be between 0.31 and 1.25 mg/kg/ day. A rat three-generation study with compound (VI) showed no alteration of any parameter at a maximum dose of 200 ppb in the diet. This compound had no teratogenic effect in rabbits at a maximum dose of 5 mg/kg/day. Summarizing, safety studies have shown zearalenone and compounds (V) and (VI) are well tolerated upon single and short-term administration. In chronic studies using doses of 20-25 mg/kg/day these compounds may reduce food intake and weight gain in rats, dogs, and monkeys. Morphological changes in endocrine supported tissues, lowered hematocrit, and hemoglobin levels in rats, and elevated sedimentation rates in dogs may occur at oral doses of 20 mgkglday and are directly related to their

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relative estrogenicity. Rats treated with compound (VI) could not be differentiated from control rats on the basis of tumor incidence. Compound (VI) and, to a lesser extent compound (V), have been evaluated in estrogen deficiency states in women and have been found to be effective in a dose range of 0.5-1.0 mg/kg/day. Utian (1973)reported that compound (VI)was superior to natural conjugated estrogen in reducing plasma calcium levels and at least as effective as natural conjugated estrogens in treatment of the postmenopausal syndrome. This compound is currently marketed for this indication in certain foreign countries and is being clinically evaluated in the United States. The pharmacological data discussed were obtained in cooperation with The Endocrine Laboratories, Inc.; Merck, Inc.; and Sandoz, Inc. Safety data were obtained under CSC supervision and in cooperation with Woodard Research Corp.; International Research Corp.; Hill Top Research, Inc.; Institute of Experimental Pathology and Toxicology; and Bio/dynamics, Inc. ACKNOWLEDGMENT The authors are indebted to M. C. Bachman and Jerome Martin for their constructive criticism of our efforts to tell this story, and to Judy Jacobs for preparation of the manuscript. REFERENCES Andrews. F. N., and Stob, M. (1965). U.S. Patent 3,196,019. Borger, M. L., Wilson, L. L., Sink, J, D., Ziegler, J. H., and Davis, S. L. (1973a).J. Anim. Sci. 36, 706-711. Borger, M. L., Sink, J. D., Wilson, L. L., Ziegler, J. H., and Davis, S. L. (1973b).J. Anim. Sci. 36, 712-715. Brown, R. G. (1970).J . Am. Vet. Med. Assoc. 157, 1537-1539. Caldwell, R. W., and Tuite, J. (1970). Phytopathology 60, 1696-1697. Caldwell, R. W., Tuite, I., Stob, M., and Baldwin, R. S. (1970). Appl. Mierobiol. 20, 3 1 3 4 . Christensen, C. M., Nelson, G. H., and Mirocha, C. J. (1965). Appl. Microbiol. 13, 653-659. Eisenstark, A , , Eisenstark, R., and VanSicMe, R. (1965). Mutat. Res. 2, 1-10. Eppley, R. M. (1968).J. Assoc. OH. Anal. Chem. 51, 74-76. Eppley, R. M., Stoloff, L., Trucksess, M. W., and Chung, C. W. (1974)./. Assoc. Off. Anal. Chem. 57, 632-635. Hershberger, L. G., Thompson, C. R., and Clegg, M. T. (1966). Proc. SOC. Erp. Biol. Med. 121, 785-788. Hidy, P. H. (1971). U.S. Patent3,580,811. Hidy, P. H., and Young, V. V. (1971). U.S. Patent 3,580,929. Hodge, E. B., Hidy, P. H., and Wehrmeister, H. L. (1966). U.S. Patent 3,239,345. Ishii, K., Sawano, M., Ueno, Y., and Tsunoda, H. (1974). Appl. Microbiol. 27, 625-628. Johnson, D. B. R., Sawicki, C. A , , Windholz, T. B., and Patchett, A. A. (1970).J . Med. Chem. 13, 941-944. Keith, C. L. (1972). U.S. Patent 3,661,712. Koen, J. S., and Smith, H. C. (1945). Vet. Med. (Kansas City, Mo.) 40, 131-133. McErlean, B. A. (1952). Vet. Rec. 64, 539-540.

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