APPUED MICROBOLOGY, Mar., 1967, p. 431-440Copyright © 1967 American Society for Microbiology

Release of Inorganic Phosphate from IrradiatedYeast: Radiation Biodosimetry and Evaluation

of Radioprotective CompoundsHILLEL S. LEVINSON AND ESTHER B. GARBER

Pioneering Research Division, U.S. Army Natick Laboratories, Natick, Massachusetts

Received for publication 5 December 1966

When cells of bakers' yeast, Saccharomyces cerevisiae, were irradiated withionizing radiation, inorganic phosphate, ninhydrin-reactive material, and sub-stances absorbing at 260 m,u were released into the suspending medium. The amountof inorganic phosphate released depended on the radiation dose and on the temper-ature and pH during irradiation. The concentration of yeast cells did not affect thephosphate yield per milligram of yeast. It is suggested that the release of phosphatemay serve as an index of the total radiation environment (i.e., as a biodosimeter)where radiation inactivation of microrganisms is of primary importance, e.g., inradiation preservation of foods. The somewhat limited range of the yeast bio-dosimeter (ca. 0.5 to 1.75 Mrad) may be extended by use of other more resistantmicroorganisms, such as bacterial spores. Compounds which have been reported asprotecting microorganisms and mammals against the lethal effect of ionizing radia-tion also inhibited the radiation-induced release of inorganic phosphate from yeast.This phosphate release system is proposed as the basis for an economical, rapid sup-plement to screening procedures in the evaluation of radioprotective compounds.

A radiation dosimeter and test substanceshould not only be irradiated under similar con-ditions, but the physicochemical makeup of thedosimeter and of the substance being irradiatedshould resemble each other as closely as possible(5, 31). Where irradiation of microorganisms isinvolved, as in radiation preservation of foods,it would thus be desirable to use microorganismsas a dosimeter substance. Quantitation of dosewith most of the large, and ever-expanding, num-ber of biological effects of radiation (4, 5, 15, 16,22, 25, 26) is impractical for routine use. However,we felt that the release of an easily detectable sub-stance, such as inorganic phosphate, from ir-radiated yeast cells (19, 23, 28, 30) would providea basis for a practical system of biodosimetry withmicroorganisms. Such a system might be suffi-ciently rapid, reproducible, simple, and inexpen-sive for routine use in the radiation preservationof food. A biodosimeter of this type would meas-ure not simply the dose of radiation absorbed bymicroorganisms, but would also reflect the im-portant influence of the total radiation environ-ment (8, 9, 13, 31).

In this paper, we describe some of the condi-tions governing the release of inorganic phosphatefrom irradiated yeast into the suspending fluid.Further, on the basis of the inhibition of phos-

phate release by radioprotective compounds, wesuggest use of the system as a supplement to othermethods for the rapid evaluation of such com-pounds.

MATERIALS AND METHODS

Organism. Bakers' yeast, Saccharomyces cere-visiae, was obtained as 1-lb cakes from a local baker.On the day prior to irradiation, a portion of the yeastcake was washed three times by centrifugation fromwater suspension (5,000 rev/min, 3 C). The washedcells were resuspended in water, and the cell concen-tration was adjusted by dilution with water so that 1.5ml, diluted to 100 ml, gave a reading of 145 in a Klett-Summerson photoelectric colorimeter (560 m,). Thisstock suspension (2Y), ca. 410 ml of which was pre-pared from 115 g of yeast cake, contained 40.0 0.5mg of yeast (dry weight) and ca. 2 X 109 cells permilliliter. Suspensions for irradiation were preparedby suitable dilution of 2Y with water, buffer, or addi-tive. Unless otherwise stated, the concentration ofyeast cells in irradiated suspensions was one-half thatof the stock (i.e., 20 mg/ml). Yeast suspensions pre-pared for irradiation were stored overnight at 4 C.

Irradiation. Suspensions were irradiated instoppered "Hi-temp" polyethylene test tubes (17 by100 mm; Falcon Plastics, Los Angeles, Calif.), eachcontaining 7 ml of yeast suspension. Duplicate aque-ous suspensions were used as controls in experimentsinvolving irradiation of yeast in the presence of addi-

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FIG. 1. (A) Radiation-produced release of inorganicphosphate from yeast cells suspended in water and inTris (0.05 M, pH 7.0). (B) Effect ofpH on the releaseof inorganic phosphate from irradiated (1.0 Mrad)yeast cells suspended in Tris (0.05 M). Temperaturewas not controlled and rose from 22 to ca. 42 C duringirradiation.

tive. The tubes were supported on a conveyor in Luciteracks in aluminum troughs, usually containing an icebath the level of which was ca. 1.0 cm above that ofthe liquid in the tubes. The conveyor carried samplesinto the radiation area, and, during irradiation,plaques containing rods of 60Co were raised out ofthe pool-one on each side of the conveyor.The 60Co facility (1.0 Mcurie, 4.2 Mrad per hr) at

the U.S. Army Natick Laboratories, Natick, Mass.,was the -y-radiation source. The total radiation dose,monitored by Fricke-type (ferrous sulfate-coppersulfate) dosimeters (14), was divided into equalincrements usually of 250,000 rad. Appropriate tubeswere removed after each increment, and the remainingtubes were well shaken to ensure uniform suspensionof the yeast cells before continuing with the radiation.For a dose increment of 250,000 rad, the samples wereexposed in the source for ca. 4.5 mir. An additional2 min was required to lower the source into the pool,to bring samples out of the source area, to removesamples, and to shake the remaining samples.

Survival. Appropriate dilutions of yeast suspensionswere plated on Mycophil Agar (BBL), pH 4.7. Yeastcolonies were counted after incubation for 6 days at28 C. Percentage survival was calculated by compar-ing colony counts of irradiated and of unirradiatedyeast suspensions.

Analyses. Suspensions were centrifuged promptlyafter irradiation, and supernatant fluids were analyzedfor inorganic phosphate (12) and for amino nitrogen(21). The absorbance of the supernatant fluid at 260miA was determined in a Beckman model DU spec-trophotometer.The capacities of various additives to protect

yeast against the radiation-induced release of phos-phate were compared by means of the dose-reductionfactor (DRF), the ratio of the dose required to effectthe release of a certain amount of phosphate in thepresence of protector, compared with the dose re-

quired to effect the release of the same amount ofphosphate in the absence of protector. We consider aDRF > 1.5 to represent significant protection.

RESULTS

In preliminary experiments, aqueous suspen-

sions of yeast were irradiated in polyethylene testtubes supported in wooden racks. Under theseconditions, without temperature control, thetemperature of samples rose from ca. 22 to ca.

42 C during the course of the experiment (totaldose, 1.8 Mrad, in 0.1-Mrad increments). Ap-proximately the same amount (50 ,ug) of inorganicphosphate was released into the suspending me-dium as was released by complete breakage of thecells in a Mickle tissue disintegrator (20). Whenthe yeast was suspended in 0.05 M tris(hydroxy-methyl)aminomethane (Tris), the pattern of re-lease was altered and phosphate release increasedwith increasing pH levels (Fig. 1). Thus, for thissystem to be useful in quantitating the dose ofirradiation, the conditions of irradiation must berigidly controlled so that variations in phosphateyield would be minimized. Such factors as pH,temperature, cell concentration, and additiveswere considered.

Temperature. More phosphate was releasedfrom yeast suspensions immersed in a water bathat ambient temperature (ca. 22 C) than from simi-lar yeast suspensions maintained at 0.4 C duringirradiation (Fig. 2). Although more inorganicphosphate was released as a result of irradiationat ambient temperatures, the inability to repro-duce this release under conditions of uncontrolledtemperature was undesirable for radiodosimetry.When irradiation was conducted at 0.4 C, releasewas more reproducible (Fig. 2), and, in the rangefrom approximately 0.5 to 1.75 Mrad, the phos-phate released approximated a linear function ofthe logarithm of the dose of radiation (Fig. 2B).

Yeast concentration. The release of phosphateper milligram of yeast irradiated was constantover the range of 2 to 40 mg of yeast per ml.

Buffer and pH. The amount of inorganic phos-phate released from irradiated yeast dependedon the pH of the suspending medium and on theparticular buffer used (Fig. 1 and 3). The order ofresistance of yeast suspensions to radiation-in-duced release of phosphate at pH 6.0 was phthal-ate > citrate > acetate. At pH 8.0, maleatemarkedly suppressed phosphate release from Tris-suspended yeast cells. In a sense, then, the bufferprotected the yeast from the radiation-inducedloss of inorganic phosphate.

Other additives. In view of the demonstration ofthe protectiveness of certain buffers, the sensi-tivity of the phosphate release system to thepresence of other substances was examined. Theeffect, on the system, of substances known toprotect animals or microbes against the lethaleffect of radiation was of particular interest. Of

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INORGANIC PHOSPHATE FROM IRRADIATED YEAST

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FIG. 2. Effect of temperature during irradiation on

the release of inorganic phosphate from aqueous

suspensions of yeast. Yeast suspensions were irradiatedat the temperature of a water bath (which rangedfrom20 to 26 C during irradiation) and of an ice bath(samples maintained a temperature of 0.4 C). Verticallines represent range of valuesfrom several experiments.B represents same data as A, but with dose plotted on a

logarithmic scale.

such substances, mercaptoethylamine (MEA)and aminoethylisothiuronium bromide hydro-bromide (AET) are virtual standards. Generally,compounds (Table 1) which are protective formice are also effective in the yeast system. Forexample, for protection of mammals, AET mustbe adjusted to pH 7 to form the protective mer-captoethylguanidine (MEG); unadjusted AET(pH 4 to 4.5) does not protect mice against thelethal effect of ionizing radiation (27). Similarly,in the yeast phosphate release system, the DRFof 1.0 mm AET (pH 7.0) is ca. 2.0 (Fig. 4), com-paring favorably with reported DRF values insystems involving mouse kill (4, 10), but unad-justed AET is not protective (Fig. 4) in our sys-tem or in the mouse. In most cases (Table 1),compounds which are protective for yeast arealso protective for mice, but some compoundswhich protect yeast against the radiation-inducedrelease of phosphate have questionable radiopro-tective value for mammals. Of the 30 compoundsfor which complete data are available, onlytyramine, which does not protect yeast againstphosphate loss, and tryptamine and dimethyl-sulfoxide, which sensitize yeast to phosphate loss,have been reported to protect animals againstirradiation. We suggest that the economy, ease,and rapidity with which the phosphate releasesystem can be handled make it a potentially use-ful supplement in large-scale preliminary evalu-ation of compounds for their radioprotectivecapacity. A compound which affects the radio-sensitivity of the yeast release system (either pro-

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tective or sensitizing) must be further tested foranimal toxicity and protection.

Survival. The ability of yeast cells to formcolonies was lost after a dose of 0.5 Mrad (Fig. 5),before there was appreciable loss of inorganicphosphate (Fig. 2). Although ascorbic acid, so-dium thioglycolate, and MEA protected yeastagainst phosphate loss (Table 1), these com-pounds had no protective effect on yeast survival(Fig. 5).Other parameters. Supernatant fluids of ir-

radiated yeast cells were also assayed for aminonitrogen and for 260 m,u-absorbing substances.Approximately equal percentages of the totalreleasable phosphate, ninhydrin-reactive material,and 260 m,u-absorbing substance were releasedat each dose level (Fig. 6).

DIscussIoN

We propose a biodosimeter based on the radia-tion-induced release of inorganic phosphate fromuniformly suspended yeast. Under standardizedconditions of temperature, yeast concentration,and pH, the amount of phosphate released wasreproducible and was proportional to dose overthe range from 0.5 to ca. 1.75 Mrad. The proposeddosimeter is inexpensive, and simple to prepareand assay. Moreover, the similarity of the dosim-eter substance (yeast) to other microorganismsmakes the proposed system superior to chemicaldosimeters for application to radiation steriliza-tion problems. With this biodosimeter, we meas-

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against the radiation-produced release of inorganicphosphate. AET used either atpH ofunadjusted aqueoussolution (pH 4.5) or adjusted to neutrality. DRF ofAET(pH 7.0) calculated graphically (dashed lines). Irradia-tion was at temperature of melting ice.

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suspended in Tris (0.05 M), were irradiated at ambienttemperature in the absence of further additive (0);and in the presence of 0.001 M MEA (0), 0.05 msodium thioglycolate (O), or 0.05 M sodium ascorbate(U); all at pH 7.0. Identical results were obtainedwhen yeast cells were suspended in water and in phos-phate buffer (0.05 &f, pH 7).

ure not simply the applied dose of radiation, butrather an integrated effect of the radiation en-vironment. We suggest that variations in theenvironment during irradiation may be more

easily detected with a biological than a chemicaldosimeter. Extension of the range of radiationbiodosimetry to include the higher doses appli-cable in radiation sterilization of foods may bepossible through selection of microorganisms(such as bacterial spores) which are more re-sistant to the release of their internal substances.For example, recalculation of our earlier data(17) indicates that, when dry spores were ir-radiated with a Van de Graaff electron accelera-tor, the amount of dipicolinic acid released fromBacillus megaterium was proportional to dose inthe range from 0.8 to 1.6 Mrad and that spores ofClostridium sporogenes (putrefactive anaerobe3679) released no dipicolinic acid at doses lowerthan 2 Mrad. Thereafter, release from C. sporo-genes was a linear function of dose until ca. 10

Mrad (Fig. 7). The basis for the release of in-ternal substances is unclear, but may be due todestruction of the semipermeable character of themembranes, to depolymerization and subsequentleakage of high molecular weight intracellularlybound or surface-bound substances, or to a com-bination of these effects. The similarity in thepattern of release of inorganic phosphate, ninhy-drin-reactive material, and 260 m,u-absorbing ma-terial suggests a common mechanism-perhapsdestruction of the semipermeable character of thecell membrane-for the loss of all of these ma-

terials from the irradiated yeast cell.The resistance of yeast cells to the release of

inorganic phosphate depended upon the partic-

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irradiated spores of Bacillus megaterium and Clostrid-ium sporogenes. Dry, lyophilized spores were irradiatedwith electrons (2 million electron volt Van de Graaffaccelerator). Points were obtainedfrom data of Levin-son and Hyatt (17), with permission.

ular substance added to the irradiation medium.Compounds which protected yeast against phos-phate release did not necessarily protect yeastagainst radiation kill. The two effects, lethalityand release of phosphate, appear to be orderedevents with different sensitivities to irradiationand with different susceptibilities to protectionby added compounds. Various sulfur-bearingcompounds had a wide range of effect in alteringthe sensitivity of yeast cells to the radiation-produced loss of phosphate (Table 1). Differencesin the effect of specific compounds in alteringsensitivity to loss of intracellular substances andin protecting yeast (and multicellular organisms)against radiation kill might provide a useful toolin the exploration of the mechanism of radiationdamage and protection.

Furthermore, the loss of the ability to divideand to form colonies, which is often equated withloss of viability in a single-celled organism, is notnecessarily equivalent to death in higher forms.In general, agents which inhibit the release ofphosphate from yeast are associated with theradiation protection of animals. The correlationof release with mammalian protection warrantsfurther investigation, not only to elucidate differ-ences in the mechanisms of radiation kill andprotection in microorganisms and in multicellularanimals, but also with the idea of using the phos-phate release system in the screening of water-soluble radioprotective compounds which are nothighly colored. Mangina (18), too, has suggestedthat yeast cells are excellent tools in mass testingof the radioprotective properties of chemicals,

since the radiobiology of yeasts and their reactionto irradiation have been well studied. One mustnot, however, expect complete agreement be-tween the protection of yeast against loss of phos-phate and the protection of mammals. To beprotective, the activity of a compound must notbe destroyed during metabolism, must be presentat the sensitive site in sufficient concentration,and must be nontoxic at these levels. As Bacq (4)has stated, "When ... yeast cells are irradiated,it is not difficult to know exactly what the phys-ical and chemical conditions are. When a mammalis irradiated after injection of MEA or AET, onedeals with a most heterogeneous system: varioustype of cells, responding differently to ionizingradiation, concentrate the protector and reactto it in various ways.... No wonder if the de-tail of this complicated mosaic and the relativeimportance of its different patterns are not fullyunderstood even after 15 years of relentless re-search." The yeast system, however, may afford auseful supplement to more time-consuming andcostly methods for evaluation of radioprotectivechemicals.

ACKNOWLEDGMENTS

We thank A. Brynjolffson, R. Jarrett, J. Halliday,and B. MacDonald for operation of the radiationfacility, and Mrs. M. T. Hyatt, P. Angelini, G. R.Mandels, and E. T. Reese for their reviews of the man-uscript.

LITERATURE CITED

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2. ALEXANDER, P. 1963. Chemical protection inchemical systems, p. 255-271. In M. Ebert andA. Howard [ed.], Radiation effects in physics,chemistry and biology. North-Holland Pub-lishing Co., Amsterdam.

3. ALEXANDER, P., Z. M. BACQ, S. F. COUSENS, M.Fox, A. HERVE, AND J. LAZAR. 1955. Mode ofaction of some substances which protect againstthe lethal effects of x-rays. Radiation Res. 2:392-415.

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6. BALABUKHA, V. S. 1963. Present state of chemicalprotection against penetrating radiation, p.3-10. In V. S. Balabukha [ed.], Chemical pro-tection of the body against ionizing radiation.Pergamon Press Inc., New York.

7. BREGADZE, I. F. 1965. Reducing the radiation

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damage to isolated cells by a number of aminoacids. Radiobiologiya 5:97-102.

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