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Camp. Biochem. Physiol.Vol. 98A, No. 2, pp. 259-263, 1991
‘0 1991 Pergamon
Printed in Great Bntain
Press plc
THE EFFECT OF IONIZING RADIATION ON THE VIABILITY OF TRZCHOMONAS VAGINALIS JAMESJ. DALY, MAX L. BAKER,* TERRY L. HOSTETLER and PATRICK L. GUTHRIE *Departments
of Microbiology
and Immunology
and Radiology, Little Rock, AR 72205, USA Fax: (501) 686-5359
University of Arkansas for Medical Sciences, 4301 W. Markham, Telephone:
(501) 686-5144.
(Received 12 June 1990) Abstract-l. The effects of continuous gamma radiation on the viability of Trichomonas vaginalis (ATCC 30001) were assessed by a colony count technique. 2. A triphasic survival curve showed an initial shoulder (Do) of 3 Gy followed by three linear curves with D, values of 34, 300, and 90Gy. 3. Sterilization of 106cells/ml occurred from 1600 to IgOOGy of radiation. 4. Population growth, subsequent to radiation exposure of 17-100 Gy, showed an increased lag time followed by a faster rate of growth, compared with unirradiated cells. 5. Trichomonas vnginalis is more sensitive to ionizing radiation than free-living protozoa and appears as radiosensitive as those parasitic protozoa examined in radioattenuation experiments.
INTRODUCTION Studies regarding the effect of ionizing radiation on protozoa have been hampered by the lack of easy and efficient techniques for estimating their survival. Investigations of irradiated populations of free-living protozoa have successfully used a variety of metabolic and morphological changes that are associated with loss of viability. Radiation effects on the more difficult to manipulate parasitic protozoa have produced much less exact data. This has been due to a reliance on the survival of inoculated hosts as an indirect criterion for the viability of the irradiated parasites. Survival of a host animal, injected with a usually lethal number of irradiated parasites, indicates a large reduction in the number of parasites capable of reproducing after radiation. Such data, although useful for host-parasites studies, are difficult to use for comparisons of radiosensitivity with other organisms. It would be desirable to obtain more precise and standardized radiobiologic values that could be used in such comparisons. A high efficiency, agar-plating method is available to accurately measure the viability of Trichomonas wzginalis, the human urogenital parasite. This plating technique has been used to measure the effects of ultraviolet radiation, cryopreservation, isotonicity, semen, seminal fluid and certain salts on the survival of several different species of trichomonads (Daly et al., 1981, 1989, 1990; Ivey, 1975; Matthews and Daly, 1974; Sherman et al., 1988; Krieger and Rein, 1982a, b). The main objective of this study was to utilize the plating technique to follow the response of T. cuginalis to gamma radiation. Data obtained was used to construct a dose-response curve from which the standard parameters of Do, D, (D3,), and D,, (LDSO) could be estimated. These values were then used for comparisons with similar standard values obtained with other organisms. Also, populations of T. uuginnlis surviving sublethal dosages of radiation
were examined as to the effect of the radiation on subsequent growth by subculturing techniques and the aforementioned agar-plating methods. MATERIALSAND
METHODS
Strain and culture maintenance The strain of Trichomonas vaginalis used was ATCC 30001. The organisms were grown and maintained in Diamond’s TYM medium (Diamond, 1957), pH 6.5, without agar, at 37°C and supplemented with 5% heat-inactivated human serum. Irradiation of Trichomonas
vaginalis
Irradiation of trichomonads was carried out as follows: Cells in the late or logarithmic or early stationary phases of growth (approximately 106cells/ml) were taken from maintenance medium and washed twice with 0.6% saline using low speed (5OOg) centrifugation. These cells were then diluted into 100 ml of TYM medium contained in 125 ml screwcap Erlenmeyer glass flasks or 1Oml of medium in screwcap glass test tubes (15 x 125 mm). Cell populations taken from the irradiated medium were adjusted with sterile 0.6% saline to 200 or 1000 cells/ml for radiation dosages up to 500 Gy, IO4 cells/ml for dosages from 500 to 1000 Gy, and IO6 cells/ml for dosages over 1000 Gy. These dilutions were used after initial experiments had determined what approximate numbers of survivors would be left after each given radiation dosage to produce from 25 to 200 colonies in the plating medium. If Erlenmeyer flasks were used, the flasks were removed at predetermined radiation exposures, the contents of the flasks swirled, 5 ml samples were removed for assaying viability, and the flasks then placed back into the irradiator for further exposure. If test tubes were used, groups of tubes were placed together in the gamma irradiator and individually removed after each desired radiation exposure. The tube was then agitated with a vortex mixer and an appropriate sample removed for assaying. Test tubes were not returned for re-exposure to gamma radiation. Test tubes and flasks containing unirradiated cells as controls were kept in the dark at ambient temperatures. Samples were removed for viability testing at time intervals corresponding to the times
JAMESJ.DALY etai.
260
required for the desired radiation dosages. Irradiation was performed using a 35OCi Cs-137 irradiator delivering 100 Gy/hr at ambient temperature. Deveiupmentof colonies in semi-solid medium Colony count methodology was performed as described by Iv;ey (1961) and Matthews and Daly (1974). Tubes contammg TYM with Noble agar were placed in a boiling water bath to melt the medium and then removed to a water bath at 40°C. After temperature equilibration, inactivated human serum (5% v/v) and an inoculum containing trichomonads were added to the tubes. Saline (0.6%) was then added to adjust the medium to contain the Noble agar at 0.45%. The contents of the tubes were then mixed and poured into plastic Petri dishes where the medium was allowed to solidify. The Petri dishes were then placed in GasPak” jars and anaerobic conditions prepared with CO, and Hz generator envelopes. The jars and their contents were incubated at 37C for 5 days after which the dishes were removed and the colonies in the medium were counted. Total cell counts, when required, were made with a Neubauer hemocytometer chamber. .?@ect of radiation on the subsequentgrowthof Trichomonas vaginalis Experiments determining the lag and generation times of cells surviving radiation were performed as follows: Tubes of melted piating medium, containing serum. were placed in a 38C water bath and inoculated with irradiated cells. The number of surviving cells to give an initial 100-200 colonies plate was determined from the previously constructed dose-response curves according to the radiation dosage received. Unirradiated control cells were diluted to give the same number of colonies. The tubes of inoculated medium were incubated in the water bath for predetermined time periods (up to 20 hr), removed, and the contents of the tubes poured into Petri dishes to be further incubated in anaerobe jars for colony development. Calculations and statistics
Dose-response curves were constructed according to Travis (1989). Regression analyses on the linear survival curves were performed with a program for continuous simple linear regression. To evaluate the results in the growth experiments the Student’s t-test was carried out.
differences between lag times, generation times, or total populations obtained between cells in irradiated or unirradiated medium. Based on these results TYM, without serum, was used as the suspending medium for all experiments, E&ct oj gamma radiation on the sl~~f~iz)~i of Trichomonas vaginalis The effect of gamma
radiation
on T. vaginalis is
seen in Fig. 1, a composite curve obtained from nine experiments that were done with radiation doses up to 900 Gy. Three different linear curves were seen that were preceeded by an inital shoulder, or Do. The Do was estimated to be only about 3 Gy and is too small to be accurately depicted in Fig. 1. The first linear death curve (A) seen in Fig. 1 was obtained from measurements on 12.5. 25, 50, 75, and 100 Gy. These data were subjected to linear regression for each experiment. The D, (LI+,)and D, (DX7)values were derived by inspection after graphic fit for each experiment and averaged for the nine experiments. The second linear curve (B) was more extensive than the first and continued to about 500 Gy. Assuming 100% survival at 100 Gy, and that curve B stopped at 500 Gy, regressions were performed and average D, and D,, values determined. Linear regressions were also done on the last three points that constituted curve C and the D, and D,, values calculated as before. A comparison of these values for curves A, B, and C can be found in Table I. Figure 1 shows an initial radiosensitivity by T. vaginalis to 1OOGy of gamma radiation that reduces the viable cell number to less than 20%. The surviving organisms are relatively radioresistant up to 500 Gy with only 13% more of the initial cell population being unable to reproduce. After 500Gy the population again becomes more radiosensitive with a
marked increase in the mortality curve. The change
RESULTS Either 0.6% saline or a Kreb’s Ringer Phosphate buffer solution was used as the suspending media in the first experiments performed to determine the effects of gamma radiation on T. vaginitis. However, after 1.5 hr in these solutions the unirradiated control cells began to show a marked decrease in survival. After 2.5 hr only 50% of the cells were viable. Such a decrease in viability in the control cells could not provide a reliable baseline for determining per cent survival values, especially since viable trichomonads had also been detected after 2.5 hr in the gamma irradiator. Since the output of the irradiator could not be increased (100 Gy/hr), TYM medium, without the serum additive, was used as the supporting milieu. To test if irradiated medium would contain trichomonad growth-inhibiting or tidal activity, and therefore influence survival, flasks of TYM medium, without serum, were exposed to gamma radiation doses of ZOO, 1000, and 2400 Gy. Serum and T. vuginalis from maintenance cultures were then added to the flasks. These flasks were incubated and growth parameters were determined by cell count as described by Daly, 1970. There were no significant
01234567
8 GAMMA-RAY
DOSE
(Gy
9
10
11
12
x 100)
Fig. I. Dose-reponse
curve to gamma radiation for Trithe first-, B the second-, and C the third linear curve. Points represent averages for nine experiments.
chomonas vaginalis ATCC 30001. A represents
261
Effect of ionizing radiation on T. vaginalis Table I. D,, (LD~~) and D, (D,,) values for the linear portions of the survival curve (Fig. I) for Trichomonas caginalis exposed to gamma radiation Linear section of curve
D,
D>n
A B C
23 200 60
34 300 90
Values represent
mean (in Grays)
of nine experiments.
in radiosensitivity can also be seen by comparing the Do and D,, values in Table 1. Values for the radioresistant population (B) are ten times greater than those of A and approximately four times that of the final radiosensitive population (C). Radiosensitivity high dosages
of Trichomonas
vaginalis surviving
Extrapolation of the third linear curve gives a hypothetical 100% kill at 1600 Gy for IO6cells/ml. Using radiation dosages ranging from 1000 to 2400 Gy, 100% kill for this number was found experimentally to average 1800Gy. To determine if cells surviving in the tail of the third curve were more radioresistant than the initial unirradiated population, colonies were picked from plates subcultured from 10 ml liquid medium with lO”cells/ml that had been exposed to 1800 Gy of gamma radiation. Five colonies were subcultured into liquid medium, and the medium incubated until a population of 106cells/ml was reached. These cells were irradiated with 300Gy and the per cent survivors determined. Per cent survival was not greater than seen for unirradiated control cells. Survival ranged from 6.7 to 38.3% with a mean of 22.5%. This experiment was repeated using 10 colonies and the results were similar. Based on these re-isolation techniques, cloned cells from the tail of the third survival curve were not inherently more radioresistant than the unselected population. Effect of radiation on subsequent growth
In order to study the after-effects of gamma radiation on surviving trichomonads, cells that were exposed to a range of 15 to 25 and from 50 to 100 Gy were incubated in melted plating medium. After predetermined intervals the inoculated medium was poured into Petri dishes and colonies allowed to develop after further incubation in anaerobe jars. Reproduction of trichomonads in the medium (before plating) would result in increasing numbers of colonies from which growth parameters could be determined. The data from three experiments are summarized in Table 2. Although the difference between the unirradiated control lag time and the highest dosage lag time were not significantly differTable 2. EKecectof low dose Ionizing radiation on subsequent growth of Trichomonas wginolis
Radiation
dose
Unirradiated control I7-25 Grays 50-100 Grays
ent, a tendency can be seen toward an increased lag time as the radiation dose increases. However, generation times of unirradiated cells and cells exposed to 17-25 Gy are significantly different from cells exposed to 50-100 Gy with P = ~0.05. A tendency toward a faster growth rate of survivors exposed to increasing doses of radiation is also seen.
Length of lag (hr)
Generation time (hr/generation)
2.0 i: I.1 3.0 k I .6 4.2 i: 2.2
5.5 i 0.7 4.1 f 1.3 2.9 k 0.4’
Values represent mean + SEM; ‘significanlly different from unirradiated control and 17-25 Gy (P = ~0.05); Number of experiments = 3.
DISCUSSION
A major effect of high-dosage ionizing radiation on microorganisms is a decrease in the number of cells capable of reproducing, We have been able to follow this decrease in viable cells of T. vaginalis by using a colony-forming technique. This agar-plating procedure allows construction of dose-response curves not easily or accurately done with other methods of assessing trichomonad survival, such as motility or subculturing into liquid medium. Specific and precise data obtained in this study on gamma radiation can be compared with information in a short report by Jedrzejczak (1969) which was not based on a strict criterion for viability of trichomonad populations. To assess radiation effect in that study, cells were radiated in liquid medium, followed by incubation for the growth of survivors. Jedrzejczak found that dosages of 80, 100, 240 and 320 Gy did not have much effect on survival of T. vaginalis. Radiation with 2000-3200 Gy produced sterility in those cultures. Dosages between 1200 and 1600 Gy delayed division for 10 days. The present work found a 90% reduction in viability after exposure to 300 Gy that was undetected by Jedrzejczak. The extensive delayed division found by Jedrzejczak is puzzling and may have been due, in part, to erroneous total cell counts which did not eliminate those cells unable to reproduce. We have found that trichomonads exposed to high dosages of radiation are still intact and motile but few are capable of reproducing upon subculture. We did find an increased lag time due to increased radiation but there was also an increase in growth rate by the irradiated survivors which would offset the delayed growth. However, trichomonads tested for subsequent growth effects in the present study had been exposed to radiation dosages in the range of the second death curve. The prolonged delay in growth seen by Jedrzejczak may be a characteristic of cells surviving the higher dosages in the third curve. We are presently evaluating this possibility using the agar-plating technique. We also observed lower sterilizing dosages (1600-I 800 Gy) but this smaller difference may be due to strain variability or culture history. The plating methodology used in the present study allows direct comparisons of the radiosensitivity of T. vaginalis with other organisms where an endpoint of death can also be measured. Certain morphologic and physiologic characteristics that are associated with cell death with free-living protozoa have been used to determine dose-response parameters. Representative data for free-living protozoa can be seen in Table 3. When T. vaginalis is compared to free-living protozoa, it appears to be much more radiosensitive. The presence of three identifiable death curves to gamma radiation complicates analysis but even so, the most sensitive of the free-living organisms are
262
JAMESJ. Table 3. Sensitivity
Organism Amoeba Chaos
of free-living
protozoa
Wichterman and Honegger Wichterman and Honegger Wichterman and Honegger Wichertman (1948) Elliot (I 954) Gebicki et al. (1980)
I200 320
grncilis
Paramecium
bursaria
3400
Tetrahymena
pyriformis
4000
Terrahvmena
ovriformis
1200-6000
*Values representative
still more radioresistant than T. uaginalis. If T. is representative of parasitic protozoa or, if trichomonads are more radioresistant than other more obligate parasitic protozoa, such as the sporozoa, this may indicate a loss of radiation repair mechanisms as an adaptive sacrifice to a parasitic existence. A general comparison of radiosensitivity can be made with other parasitic protozoa (Table 4) although methods strictly defining viability have not been used for those types of protozoa. Values in Table 4 are the lowest dose of radiation that attenuated the parasites with the average attenuating dose being about 250 Gy. It can be assumed that at 250Gy some of the irradiated parasites will survive. This assumption is based on a study where immunocompetent mice survived inoculation with Plasmodium berghei that had been radiated with 400 Gy, but one of five similarly vaccinated athymic nude mice developed parasitemia (Waki et al., 1982). Based on this evidence and that few trichomonads survive after 500 Gy, it would seem that the radiosensitivity of T. vaginalis and other parasitic protozoa are probably similar. The finding of three separate death curves was a surprise, although the second curve might be interpreted as a second shoulder. The appearance of a resistant population with 5-20% of the cells still surviving is also unusual for microorganisms since such a break generally occurs with only 1% or less viability. These survivors are usually explained on the basis of statistical distribution of resistance in the general population. However, isolation of surviving clones and radiation of their progeny did not indicate that these organisms possessed inherently greater radioresistance than cells that did not survive. The most likely explanation for the different curves is that there are two (or three) distinct physiological cell types in the irradiated population which would have different radiosensitivities. These cell types may be a characteristic of the phase of growth (late log-early stationary) from which the cells were taken. Evidence for this comes from two studies. The first, on the Table 4. Attennuation
effect of near-UV on T. vaginalis (Daly et al., 1981) showed that older cells were more resistant than younger cells to near-UV. The second, on the effect of gamma radiation on different culture stages of the sporozoan Plasmodium falciparum, showed different radiation responses by different cell types in its life cycle (Waki et al., 1983). In conclusion, the effect of gamma radiation on the viability of the parasitic flagellate T. vaginalis resulted in three measurable death curves with a very small initial Do. Compared to free-living protozoa, T. vaginalis appears to be radiosensitive. Radiation sensitivity seems to be similar to other parasitic protozoa that have used indirect techniques to measure viability. Gamma radiation in the dose range which allows approximately 20% survivors produces a tendency for an increased lag time and an increased generation time in growth subsequent to radiation exposure.
of parasitic
Acknowledgements-We
thank Professor Jerome K. Sherman, Department of Anatomy, University of Arkansas for Medical Sciences for reviewing this manuscript and his positive comments for its improvement. REFERENCES
Chhabra M. B., Mahajan R. C. and Gangluly N. K. (1979) Effects of Co@’irradiation on virulent Toxoplasma gondii and its use in experimental immunization. Int. J. Radial. Biol. 35, 433440.
Daly J. J. (1970) The maltose metabolism of Trichomonas gallinae (Rivolta): I. Growth studies. J. Purasitol. 56, 8833888.
Daly J. J., Baker M. L. and Burton S. B. (1981) The sensitivity of Trichomonas vaginalis and Trichomonas gallinae to ultraviolet radiation. Photochem. Photobiol. 33, 191-195.
Daly J. J., Sherman J. K., Green L. and Hostetler T. L. (1989) Survival of Trichomonas vaginalis in human semen. Genitourin. Med. 65, 1066108.
Daly J. J., Sherman J. K., Haley T. and Hostetler T. L. (1990) Differences in the effect of dog seminal fluid and human seminal fluid and human semen on in uitro survival of Trichomonas vaginalis. Sex. Transmitt. Dis. 17, 106-109. protozoa
Minimum attenuating dose (Gy)
Organism Plasmodium
berghei
170
Plasmodium
yoelii
200
Plasmodium
gallinaceum
260
Plasmodium
falciparum
Toxoplasma
gondii
Eimt-ria
necalrix rhodhaini
250
400
rhodesiense
representative
200 150-200 2ols400
Nosema algerae ‘Values
(1958) (1958) (1958)
of the literature.
vaginalis
Babesia
radiation’
Reference
2400
proteus
Trypnnosoma
to ionizing
L% (GY)
Chaos
Euglena
DALY et al.
of the literature.
200
with ionizing
radiation* Reference
Wellde and Sadun (1967) Wells el al. (1977) Hughes and Dixon (1980) Waki er al. (1983) Chhbara et al. (1979) Singh and Gill (1975) Irvin and Young (1979) Undeen er al. (1984) Duxbury and Sadun (1969)
Effect of ionizing radiation on T. vaginalis Diamond L. S. (1957) The establishment of various trichomonads of animals and man in axenic cultures. J. Parasitol. 41, 488490.
Duxbury R. E. and Sadun E. H. (1969) Resistance produced in mice and rats by inoculation with irradiated Trypanosoma rhodesiense. J. Parasitol. 55, 859-865.
Elliot A. M., Hayes R. E. and Byrd J. R. (1954) X-irradiation of sexual strains of Tetrahymena. Biol. Bull. 107, 309-310. Gebicki J. M., Darlington J. L. and Baumgartner S. (1980) The effect of extracellular environment on the sensitivity of two strains of Tetrahymena to gamma rays. ht. J. Radial. Biol. 38, 473479.
Hughes H. P. and Dixon B. (1980) Vaccination of chicks against Plasmodium gallinaceum by erythrocytic and exoerythrocytic parasites attenuated by gamma irradiation. Ann. Trop. Med. Hyg. 74, 115-126. Irvin A. D. and Young E. R. (1979) The effects of irradiation on Babesia maintained in vitro. Res. Vet. Sci. 27, 200-204. Ivey M. H. (1961) Growth characteristics of clones of Trichomonas in solid medium. J. Parasitol. 47, 539-544. Ivey M. H. (1975) Use of solid medium to evaluate factors affecting the ability of Trichomonas vaginalis to survive freezing. J. Parasitol. 61, 1101-l 103. Jedrzejczak W. (1969) Dzialanie promieni jonizujacych no hodowie rzesistka pochowego. Wad. Parazytol. 15, 277-288. Krieger J. N. and Rein M. F. (1982a) Canine prostatic secretions kill Trichomonas vaginalis. Infect. Immun. 37, 77-8
1.
Krieger J. N. and Rein M. F. (1982b) Zinc sensitivity of_Trichomonas vaginalis: in vitro studies and clinical implications. J. Infect. Dis. 146, 341-345. Matthews R. S. and Daly J. J. (1974) Trichomonas gallinae: Use of solid medium to test survival under
various environmental
263 conditions.
Exptl Parasitol. 36,
288-298.
Sherman J. K., Daly J. J., Hostetler T. L. and McHenry K. (1988) Survival of Trichomonas vaginalis during cryopreservaton of human semen. Cryobiology 25, 567. Singh J. and Gill B. S. (1975) Effect of gamma-irradiation on oocysts of Eimeria necatrix. Parasitology 71, 117-124. Travis E. L. (1989) Primer of Medical Radiobiology, pp. 53-59. Yearbook Medical Publisher, Chicago. Undeen A., Vander Meer R. K., Smittle B. J. and Avery S. W. (1984) The effect of gamma radiation on Nosema algerae (Microspora: Nosematidae) spore viability, germination, and carbohydrates. J. Protorool. 31, 479-482. Waki S., Tamura J., Imanaka M., Ishikawa S. and Suzuki M. (1982) Plasmodium berghei: Isolation and maintenance of an irradiation attenuated strain in the nude mouse. Exptl Parasitol. 53, 335-340.
Waki S., Yonome I. and Suzuki M. (1983) Plasmodium falciparum: Attenuation by irradiation. Exptl Parasitol. 56, 339-345.
Wellde B. T. and Sadun E. H. (1967) Resistance produced in rats and mice by exposure to irradiated Plasmodium berghei. Expfl Parasitol. 2.3, 31&324.
Wells R. A., Martin L. K., Stutz D. R. and Diggs C. L. (1977) Plasmodium yoeli: An inbred model for protective immunization against malaria in BALB/c mice. Exptl Parasitol. 41, 472479.
Wichterman R. (1948) Action of X-rays on mating types and conjugation of Paramecium bursaria. Biol. Bull. 94, 113-127.
Wichterman R. (1955) Survival and other effects following X-irradiation of the flagellate, Euglena gracilis. Biol. Bull. 109,371. Wichtennan R. and Honegger C. M. (1958) Action of X-rays on the two common amebas, Chaos digpuens and Chaos chaos. Proc. Penn. Acad. Sci. 32, 240-253.
Camp. Biochem. Physiol.Vol. 98A, No. 2, pp. 259-263, 1991
‘0 1991 Pergamon
Printed in Great Bntain
Press plc
THE EFFECT OF IONIZING RADIATION ON THE VIABILITY OF TRZCHOMONAS VAGINALIS JAMESJ. DALY, MAX L. BAKER,* TERRY L. HOSTETLER and PATRICK L. GUTHRIE *Departments
of Microbiology
and Immunology
and Radiology, Little Rock, AR 72205, USA Fax: (501) 686-5359
University of Arkansas for Medical Sciences, 4301 W. Markham, Telephone:
(501) 686-5144.
(Received 12 June 1990) Abstract-l. The effects of continuous gamma radiation on the viability of Trichomonas vaginalis (ATCC 30001) were assessed by a colony count technique. 2. A triphasic survival curve showed an initial shoulder (Do) of 3 Gy followed by three linear curves with D, values of 34, 300, and 90Gy. 3. Sterilization of 106cells/ml occurred from 1600 to IgOOGy of radiation. 4. Population growth, subsequent to radiation exposure of 17-100 Gy, showed an increased lag time followed by a faster rate of growth, compared with unirradiated cells. 5. Trichomonas vnginalis is more sensitive to ionizing radiation than free-living protozoa and appears as radiosensitive as those parasitic protozoa examined in radioattenuation experiments.
INTRODUCTION Studies regarding the effect of ionizing radiation on protozoa have been hampered by the lack of easy and efficient techniques for estimating their survival. Investigations of irradiated populations of free-living protozoa have successfully used a variety of metabolic and morphological changes that are associated with loss of viability. Radiation effects on the more difficult to manipulate parasitic protozoa have produced much less exact data. This has been due to a reliance on the survival of inoculated hosts as an indirect criterion for the viability of the irradiated parasites. Survival of a host animal, injected with a usually lethal number of irradiated parasites, indicates a large reduction in the number of parasites capable of reproducing after radiation. Such data, although useful for host-parasites studies, are difficult to use for comparisons of radiosensitivity with other organisms. It would be desirable to obtain more precise and standardized radiobiologic values that could be used in such comparisons. A high efficiency, agar-plating method is available to accurately measure the viability of Trichomonas wzginalis, the human urogenital parasite. This plating technique has been used to measure the effects of ultraviolet radiation, cryopreservation, isotonicity, semen, seminal fluid and certain salts on the survival of several different species of trichomonads (Daly et al., 1981, 1989, 1990; Ivey, 1975; Matthews and Daly, 1974; Sherman et al., 1988; Krieger and Rein, 1982a, b). The main objective of this study was to utilize the plating technique to follow the response of T. cuginalis to gamma radiation. Data obtained was used to construct a dose-response curve from which the standard parameters of Do, D, (D3,), and D,, (LDSO) could be estimated. These values were then used for comparisons with similar standard values obtained with other organisms. Also, populations of T. uuginnlis surviving sublethal dosages of radiation
were examined as to the effect of the radiation on subsequent growth by subculturing techniques and the aforementioned agar-plating methods. MATERIALSAND
METHODS
Strain and culture maintenance The strain of Trichomonas vaginalis used was ATCC 30001. The organisms were grown and maintained in Diamond’s TYM medium (Diamond, 1957), pH 6.5, without agar, at 37°C and supplemented with 5% heat-inactivated human serum. Irradiation of Trichomonas
vaginalis
Irradiation of trichomonads was carried out as follows: Cells in the late or logarithmic or early stationary phases of growth (approximately 106cells/ml) were taken from maintenance medium and washed twice with 0.6% saline using low speed (5OOg) centrifugation. These cells were then diluted into 100 ml of TYM medium contained in 125 ml screwcap Erlenmeyer glass flasks or 1Oml of medium in screwcap glass test tubes (15 x 125 mm). Cell populations taken from the irradiated medium were adjusted with sterile 0.6% saline to 200 or 1000 cells/ml for radiation dosages up to 500 Gy, IO4 cells/ml for dosages from 500 to 1000 Gy, and IO6 cells/ml for dosages over 1000 Gy. These dilutions were used after initial experiments had determined what approximate numbers of survivors would be left after each given radiation dosage to produce from 25 to 200 colonies in the plating medium. If Erlenmeyer flasks were used, the flasks were removed at predetermined radiation exposures, the contents of the flasks swirled, 5 ml samples were removed for assaying viability, and the flasks then placed back into the irradiator for further exposure. If test tubes were used, groups of tubes were placed together in the gamma irradiator and individually removed after each desired radiation exposure. The tube was then agitated with a vortex mixer and an appropriate sample removed for assaying. Test tubes were not returned for re-exposure to gamma radiation. Test tubes and flasks containing unirradiated cells as controls were kept in the dark at ambient temperatures. Samples were removed for viability testing at time intervals corresponding to the times
JAMESJ.DALY etai.
260
required for the desired radiation dosages. Irradiation was performed using a 35OCi Cs-137 irradiator delivering 100 Gy/hr at ambient temperature. Deveiupmentof colonies in semi-solid medium Colony count methodology was performed as described by Iv;ey (1961) and Matthews and Daly (1974). Tubes contammg TYM with Noble agar were placed in a boiling water bath to melt the medium and then removed to a water bath at 40°C. After temperature equilibration, inactivated human serum (5% v/v) and an inoculum containing trichomonads were added to the tubes. Saline (0.6%) was then added to adjust the medium to contain the Noble agar at 0.45%. The contents of the tubes were then mixed and poured into plastic Petri dishes where the medium was allowed to solidify. The Petri dishes were then placed in GasPak” jars and anaerobic conditions prepared with CO, and Hz generator envelopes. The jars and their contents were incubated at 37C for 5 days after which the dishes were removed and the colonies in the medium were counted. Total cell counts, when required, were made with a Neubauer hemocytometer chamber. .?@ect of radiation on the subsequentgrowthof Trichomonas vaginalis Experiments determining the lag and generation times of cells surviving radiation were performed as follows: Tubes of melted piating medium, containing serum. were placed in a 38C water bath and inoculated with irradiated cells. The number of surviving cells to give an initial 100-200 colonies plate was determined from the previously constructed dose-response curves according to the radiation dosage received. Unirradiated control cells were diluted to give the same number of colonies. The tubes of inoculated medium were incubated in the water bath for predetermined time periods (up to 20 hr), removed, and the contents of the tubes poured into Petri dishes to be further incubated in anaerobe jars for colony development. Calculations and statistics
Dose-response curves were constructed according to Travis (1989). Regression analyses on the linear survival curves were performed with a program for continuous simple linear regression. To evaluate the results in the growth experiments the Student’s t-test was carried out.
differences between lag times, generation times, or total populations obtained between cells in irradiated or unirradiated medium. Based on these results TYM, without serum, was used as the suspending medium for all experiments, E&ct oj gamma radiation on the sl~~f~iz)~i of Trichomonas vaginalis The effect of gamma
radiation
on T. vaginalis is
seen in Fig. 1, a composite curve obtained from nine experiments that were done with radiation doses up to 900 Gy. Three different linear curves were seen that were preceeded by an inital shoulder, or Do. The Do was estimated to be only about 3 Gy and is too small to be accurately depicted in Fig. 1. The first linear death curve (A) seen in Fig. 1 was obtained from measurements on 12.5. 25, 50, 75, and 100 Gy. These data were subjected to linear regression for each experiment. The D, (LI+,)and D, (DX7)values were derived by inspection after graphic fit for each experiment and averaged for the nine experiments. The second linear curve (B) was more extensive than the first and continued to about 500 Gy. Assuming 100% survival at 100 Gy, and that curve B stopped at 500 Gy, regressions were performed and average D, and D,, values determined. Linear regressions were also done on the last three points that constituted curve C and the D, and D,, values calculated as before. A comparison of these values for curves A, B, and C can be found in Table I. Figure 1 shows an initial radiosensitivity by T. vaginalis to 1OOGy of gamma radiation that reduces the viable cell number to less than 20%. The surviving organisms are relatively radioresistant up to 500 Gy with only 13% more of the initial cell population being unable to reproduce. After 500Gy the population again becomes more radiosensitive with a
marked increase in the mortality curve. The change
RESULTS Either 0.6% saline or a Kreb’s Ringer Phosphate buffer solution was used as the suspending media in the first experiments performed to determine the effects of gamma radiation on T. vaginitis. However, after 1.5 hr in these solutions the unirradiated control cells began to show a marked decrease in survival. After 2.5 hr only 50% of the cells were viable. Such a decrease in viability in the control cells could not provide a reliable baseline for determining per cent survival values, especially since viable trichomonads had also been detected after 2.5 hr in the gamma irradiator. Since the output of the irradiator could not be increased (100 Gy/hr), TYM medium, without the serum additive, was used as the supporting milieu. To test if irradiated medium would contain trichomonad growth-inhibiting or tidal activity, and therefore influence survival, flasks of TYM medium, without serum, were exposed to gamma radiation doses of ZOO, 1000, and 2400 Gy. Serum and T. vuginalis from maintenance cultures were then added to the flasks. These flasks were incubated and growth parameters were determined by cell count as described by Daly, 1970. There were no significant
01234567
8 GAMMA-RAY
DOSE
(Gy
9
10
11
12
x 100)
Fig. I. Dose-reponse
curve to gamma radiation for Trithe first-, B the second-, and C the third linear curve. Points represent averages for nine experiments.
chomonas vaginalis ATCC 30001. A represents
261
Effect of ionizing radiation on T. vaginalis Table I. D,, (LD~~) and D, (D,,) values for the linear portions of the survival curve (Fig. I) for Trichomonas caginalis exposed to gamma radiation Linear section of curve
D,
D>n
A B C
23 200 60
34 300 90
Values represent
mean (in Grays)
of nine experiments.
in radiosensitivity can also be seen by comparing the Do and D,, values in Table 1. Values for the radioresistant population (B) are ten times greater than those of A and approximately four times that of the final radiosensitive population (C). Radiosensitivity high dosages
of Trichomonas
vaginalis surviving
Extrapolation of the third linear curve gives a hypothetical 100% kill at 1600 Gy for IO6cells/ml. Using radiation dosages ranging from 1000 to 2400 Gy, 100% kill for this number was found experimentally to average 1800Gy. To determine if cells surviving in the tail of the third curve were more radioresistant than the initial unirradiated population, colonies were picked from plates subcultured from 10 ml liquid medium with lO”cells/ml that had been exposed to 1800 Gy of gamma radiation. Five colonies were subcultured into liquid medium, and the medium incubated until a population of 106cells/ml was reached. These cells were irradiated with 300Gy and the per cent survivors determined. Per cent survival was not greater than seen for unirradiated control cells. Survival ranged from 6.7 to 38.3% with a mean of 22.5%. This experiment was repeated using 10 colonies and the results were similar. Based on these re-isolation techniques, cloned cells from the tail of the third survival curve were not inherently more radioresistant than the unselected population. Effect of radiation on subsequent growth
In order to study the after-effects of gamma radiation on surviving trichomonads, cells that were exposed to a range of 15 to 25 and from 50 to 100 Gy were incubated in melted plating medium. After predetermined intervals the inoculated medium was poured into Petri dishes and colonies allowed to develop after further incubation in anaerobe jars. Reproduction of trichomonads in the medium (before plating) would result in increasing numbers of colonies from which growth parameters could be determined. The data from three experiments are summarized in Table 2. Although the difference between the unirradiated control lag time and the highest dosage lag time were not significantly differTable 2. EKecectof low dose Ionizing radiation on subsequent growth of Trichomonas wginolis
Radiation
dose
Unirradiated control I7-25 Grays 50-100 Grays
ent, a tendency can be seen toward an increased lag time as the radiation dose increases. However, generation times of unirradiated cells and cells exposed to 17-25 Gy are significantly different from cells exposed to 50-100 Gy with P = ~0.05. A tendency toward a faster growth rate of survivors exposed to increasing doses of radiation is also seen.
Length of lag (hr)
Generation time (hr/generation)
2.0 i: I.1 3.0 k I .6 4.2 i: 2.2
5.5 i 0.7 4.1 f 1.3 2.9 k 0.4’
Values represent mean + SEM; ‘significanlly different from unirradiated control and 17-25 Gy (P = ~0.05); Number of experiments = 3.
DISCUSSION
A major effect of high-dosage ionizing radiation on microorganisms is a decrease in the number of cells capable of reproducing, We have been able to follow this decrease in viable cells of T. vaginalis by using a colony-forming technique. This agar-plating procedure allows construction of dose-response curves not easily or accurately done with other methods of assessing trichomonad survival, such as motility or subculturing into liquid medium. Specific and precise data obtained in this study on gamma radiation can be compared with information in a short report by Jedrzejczak (1969) which was not based on a strict criterion for viability of trichomonad populations. To assess radiation effect in that study, cells were radiated in liquid medium, followed by incubation for the growth of survivors. Jedrzejczak found that dosages of 80, 100, 240 and 320 Gy did not have much effect on survival of T. vaginalis. Radiation with 2000-3200 Gy produced sterility in those cultures. Dosages between 1200 and 1600 Gy delayed division for 10 days. The present work found a 90% reduction in viability after exposure to 300 Gy that was undetected by Jedrzejczak. The extensive delayed division found by Jedrzejczak is puzzling and may have been due, in part, to erroneous total cell counts which did not eliminate those cells unable to reproduce. We have found that trichomonads exposed to high dosages of radiation are still intact and motile but few are capable of reproducing upon subculture. We did find an increased lag time due to increased radiation but there was also an increase in growth rate by the irradiated survivors which would offset the delayed growth. However, trichomonads tested for subsequent growth effects in the present study had been exposed to radiation dosages in the range of the second death curve. The prolonged delay in growth seen by Jedrzejczak may be a characteristic of cells surviving the higher dosages in the third curve. We are presently evaluating this possibility using the agar-plating technique. We also observed lower sterilizing dosages (1600-I 800 Gy) but this smaller difference may be due to strain variability or culture history. The plating methodology used in the present study allows direct comparisons of the radiosensitivity of T. vaginalis with other organisms where an endpoint of death can also be measured. Certain morphologic and physiologic characteristics that are associated with cell death with free-living protozoa have been used to determine dose-response parameters. Representative data for free-living protozoa can be seen in Table 3. When T. vaginalis is compared to free-living protozoa, it appears to be much more radiosensitive. The presence of three identifiable death curves to gamma radiation complicates analysis but even so, the most sensitive of the free-living organisms are
262
JAMESJ. Table 3. Sensitivity
Organism Amoeba Chaos
of free-living
protozoa
Wichterman and Honegger Wichterman and Honegger Wichterman and Honegger Wichertman (1948) Elliot (I 954) Gebicki et al. (1980)
I200 320
grncilis
Paramecium
bursaria
3400
Tetrahymena
pyriformis
4000
Terrahvmena
ovriformis
1200-6000
*Values representative
still more radioresistant than T. uaginalis. If T. is representative of parasitic protozoa or, if trichomonads are more radioresistant than other more obligate parasitic protozoa, such as the sporozoa, this may indicate a loss of radiation repair mechanisms as an adaptive sacrifice to a parasitic existence. A general comparison of radiosensitivity can be made with other parasitic protozoa (Table 4) although methods strictly defining viability have not been used for those types of protozoa. Values in Table 4 are the lowest dose of radiation that attenuated the parasites with the average attenuating dose being about 250 Gy. It can be assumed that at 250Gy some of the irradiated parasites will survive. This assumption is based on a study where immunocompetent mice survived inoculation with Plasmodium berghei that had been radiated with 400 Gy, but one of five similarly vaccinated athymic nude mice developed parasitemia (Waki et al., 1982). Based on this evidence and that few trichomonads survive after 500 Gy, it would seem that the radiosensitivity of T. vaginalis and other parasitic protozoa are probably similar. The finding of three separate death curves was a surprise, although the second curve might be interpreted as a second shoulder. The appearance of a resistant population with 5-20% of the cells still surviving is also unusual for microorganisms since such a break generally occurs with only 1% or less viability. These survivors are usually explained on the basis of statistical distribution of resistance in the general population. However, isolation of surviving clones and radiation of their progeny did not indicate that these organisms possessed inherently greater radioresistance than cells that did not survive. The most likely explanation for the different curves is that there are two (or three) distinct physiological cell types in the irradiated population which would have different radiosensitivities. These cell types may be a characteristic of the phase of growth (late log-early stationary) from which the cells were taken. Evidence for this comes from two studies. The first, on the Table 4. Attennuation
effect of near-UV on T. vaginalis (Daly et al., 1981) showed that older cells were more resistant than younger cells to near-UV. The second, on the effect of gamma radiation on different culture stages of the sporozoan Plasmodium falciparum, showed different radiation responses by different cell types in its life cycle (Waki et al., 1983). In conclusion, the effect of gamma radiation on the viability of the parasitic flagellate T. vaginalis resulted in three measurable death curves with a very small initial Do. Compared to free-living protozoa, T. vaginalis appears to be radiosensitive. Radiation sensitivity seems to be similar to other parasitic protozoa that have used indirect techniques to measure viability. Gamma radiation in the dose range which allows approximately 20% survivors produces a tendency for an increased lag time and an increased generation time in growth subsequent to radiation exposure.
of parasitic
Acknowledgements-We
thank Professor Jerome K. Sherman, Department of Anatomy, University of Arkansas for Medical Sciences for reviewing this manuscript and his positive comments for its improvement. REFERENCES
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