INT. J. RADIAT. BIOL ., 1978, VOL. 34, NO 1, 81-90

Radioprotective effect of 5-hydroxytryptamine and5-hydroxytryptophan on mammalian cells irradiated in vitro

TOKIKO NAKAYAMA and WATARU NAKAMURADepartment of Experimental Radiology,Aichi Cancer Center Research Institute,Chikusa-ku, Nagoya 464, Japan

(Received 21 February 1978)

The radioprotective effect in vitro of 5-hydroxytryptamine (5-HT) and 5-hydroxy-tryptophan (5-HTP) was studied with cultured mammalian cells of three celllines : 5-HT-synthesizing FMA and 5-HT-non-synthesizing FM3A and B16-C2W. In these cells, the addition of 5-HT to the suspending medium inducedonly a weak protection or no protection at all . The increase in the 5-HTcontent of these cells at the time of irradiation was negligible . Pre-incubationof cells for 40 min in a 5-HTP-containing medium resulted in an elevation ofthe 5-HT content concomitantly with an increase in the radioresistance ofFMA cells, where the DRF at 1 per cent was 1 .8 . In FM3A and B16-C2W cells such an effect was not observed . The same relationship between5-HT content and radioresistance was also observed in FMA cells which were cul-tured in different densities or with reserpine . These results strongly suggest thatthe substance playing the main role in the induction of radioresistance in cells invitro is the 5-HT that exists in the cells .

1. IntroductionSince the pioneering work of Bacq and Herve (1952) and Gray, Tew and

Jensen (1952), many reports of the potent radioprotective ability of 5-hydroxy-tryptamine (5-HT) in mammals have appeared (Bacq 1954, Langendorff andKoch 1957, Langendorff, Melching and Ladner 1959, Van der Meer andVan Bekkum 1959) . The pronounced radioprotective effect of 5-HT's meta-bolic precursor, 5-hydroxytryptophan (5-HTP), was also confirmed in miceby Kobayashi, Nakamura and Eto (1966 a, b), but the data published on the effectof 5-HT in mammalian cells in vitro are still conflicting . Booz and Betz (1960)reported that the administration of 5-HT did not protect thymocytes from thenuclear pycnosis observed after an irradiation of 300 R, while a considerableradioprotection was observed in cells of a human kidney cell line (Vos, Budkeand Vergroesen 1962) and Ehrlich's ascites tumour (Koch, Onderka and Seiter1962) in vitro. Therefore it seemed very important to us to take the differentialcell susceptibility into consideration in developing a general model of the radio-protective effect of 5-HT on various mammalian cells in vitro . In the presentstudy, three different mammalian cell lines established in culture were used .The FMA cells originated from Furth's mastocytoma cell and have the capacityto incorporate 5-HTP and synthesize from it 5-HT . The cells of the other twocell lines, FM3A and B16-C2W, are not able to do this .

2. Materials and methods2 1 `Cell culture

FM : cells were originally established in our laboratory from a solid tumourof Furth's mastocytoma cells of LAF I mice (Furth, Hagen and Hirsch 1957) by

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adapting the cells to growth in the ascites prior to transfer to an in vitro culture .They had been maintained for about 3 years as a suspension culture (Nakamura,Fujii, Nakayama and Nishimoto 1976) . The culture medium was Moor'sRPMI No. 1640 supplemented with 10 per cent foetal calf serum and antibiotics .The capacity of the cells to synthesize 5-HT in vitro as well as in the abdominalcavity of host animals (F l hybrids between C57L and A) is well maintained, asin the original mastocytoma cells .

FM3A cells were originally established from a spontaneous mammarycarcinoma in a C3H mouse and maintained as a suspension culture (Nakano1966) . The culture medium was Eagle's MEM plus 10 per cent calf serumand 1 mM L-glutamine .

B16-C2W cells (Claunch, Oikawa, Tchen and Hu 1969) originated frommurine melanoma cells and were maintained in vitro in Ham's F12 mediumplus 10 per cent foetal calf serum and antibiotics .

Cells of these cell lines near the final stage of exponential growth were usedfor the experiments .

2.2 . Chemicals5-HT and 5-HTP were obtained from the Sigma Chemical Co . Ltd., U.S.A .

2.3 . Analytical methods o f 5-HT and 5-HTPCells were separated from the culture medium by centrifugation (at 1000g

for 5 min) at 4 °C . The technical methods of assay for 5-HT and 5-HTP areessentially the same as those described by Bogdanski, Pletscher, Brodie andUdenfriend (1956), and Udenfriend, Titus and Bogdanski (1957), respectively .

2.4. Preparation o f cells and irradiationIn FMA and FM3A cells, a few pipettings were sufficient to obtain a suspen-

sion of isolated cells. The cell suspension was introduced into plastic dishes6 cm in diameter and subjected to irradiation in air at a room temperature ofaround 20 ° C . In the case of B16-C2W, cells growing attached to the bottom ofthe plastic dishes were washed twice with a mixed solution of 0 . 1 per cent trypsinand 0 .01 per cent EDTA which were dissolved in phosphate buffered salinewithout Mgt+ or Cat+. They were incubated for a few minutes in the samemixed solution and separated to single cells by several pipettings . After onewashing with culture medium, the cells were replated into plastic dishes andincubated overnight . The next day they were subjected to irradiation innew culture medium both with and without 5-HT or 5-HTP .

A conventional deep-therapy X-ray generator (Toshiba Co . Ltd., Tokyo,Japan) was operated at 200 kVp and 15 mA with 0 .5 mm aluminium and0.5 mm copper filtration (h.v .l . of 1 . 13 mm copper) . The dose-rate employedwas 113 rad/min, with a target-to-surface distance of 40 cm .

2.5 . Determination o f the surviving cell fractionThe surviving fraction of treated cells was determined by the method of

colony counting . FMA and FM3A cells were transferred into soft agar plateswhich contained 0 .5 per cent Seaplaque agar, manufactured by Marine Colloid

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Inc ., U.S.A ., in culture medium . The colonies consisting of more than 50 cellswere counted on day 12 and 8 of culture for FMA and FM3A respectively .The plating efficiency was between 70 and 80 per cent in FMA and between90 and 100 per cent in FM3A . For the studies with B16-C2W, Falcon plasticdishes 6 cm in diameter were used . On day 13 of the culture, colonies whichhad grown attached to the bottom of the dish and which consisted of more than50 cells were counted . The plating efficiency was between 60 and 70 per cent .

Each point in the dose-survival curves was obtained from triplicate experi-ments . The surviving fraction was calculated as a percentage of each control,which was treated in the same way except for irradiation . The straight-lineparts of the dose-survival curves were obtained as regression lines which werethe best fits to 4-6 points on the curves .

2.6 . Calculation o f the dose reduction factor (DRF)In order to evaluate the protective effects of various treatments, the DRF

(1 per cent) was calculated . It is the ratio of the radiation doses in the experi-mental group to those in the control group which corresponded to the 1 per centlevel of cell survival .

3 . Results3 .1 . Relationship between the amount o f endogenous 5-HT and the radiosensitivity

o f FMA cellsThe amount of endogenous 5-HT was controlled in two ways : by changing

the inoculum size of the cell culture, and by adding thea-HT releaser, reserpine,to the culture medium .

3.1 .1 . Radiosensitivity o f FMA cells which were cultured starting with differentinoculum size

An example of cell growth-rate and the time-dependent 5-HT content ofcells is presented in figure 1 . In this experiment, the inoculum size on day zerowas 105 cells per ml of culture medium . Exponential growth continued up to

Protection of cultured cells by 5-HTP

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1

2

3

4

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7TIME AFTER IMPLANTATION (DAYS)

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, 106

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Figure 1 . The 5-HT content of FMA cells incubated both with and without renewedculture medium . The arrow in the figure indicates the time of renewal . 41 : celldensity, culture medium not renewed . 0 : 5-HT content of 10 8 cells, culturemedium not renewed . A : 5-HT content of 10 8 cells after renewal of theculture medium .

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day 6, but the 5-HT content of cells began to decline early on day 2 of the culture,as the culture medium was not renewed at any time during the experiment . Asindicated in figure 1, the depressed level of 5-HT was raised by renewing theculture medium . In another experiment, cells in the final stage of exponentialgrowth increased their 5-HT content by a factor of 1 .71 in 3 hours after theaddition of 1 mg of tryptophan per ml of medium (data not shown) . Thesefacts indicate that the 5-HT biosynthesis is sensitively affected by a slightdepletion of tryptophan which does not influence the cell growth-rate . Takingthese findings into consideration, an attempt was made to prepare cells containingdifferent amounts of 5-HT by starting the culture with different sizes ofinoculum . As shown in figure 2, the 5-HT content of cells, measured 24 hoursafter inoculation, depended on the inoculum size . The denser the cell popula-tion, the lower the 5-HT-content of the cells . Note that the amount of 5-HT

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3 4 5

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INOCULUM SIZE AT START OF CULTURE W0 51ml)

Figure 2 . Relation between the 5-HT content of FMA cells and the inoculum size at thestart of the culture . The 5-HT was measured 24 hours after the culture was started .•

: 5-HT content of 10 6 cells . A : 5-HT concentration in 1 ml of culture medium .

1500

DOSE (rad)

Figure 3 . Radiosensitivity of FMA cells cultured under different cell densities . The cellswere irradiated 24 hours after the culture was started. The inoculum size at startof culture is indicated on each dose-survival curve .

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which accumulated in the medium was higher in the culture which was startedwith the larger inoculum . Cells prepared in this way were subjected to irradia-tion 24 hours after the start of the culture . The dose-survival curves of thesecells are presented in figure 3 . The cells obtained from a dense culture showeda radiosensitivity higher than that observed in cells of more sparsely populatedcultures .

3 .1 .2 . Effect o f reserpine on the 5-HT content and the radiosensitivity o f FMA cellsFMA cells of two cell densities, 10 5 and 5 x 10 5 cells per ml, were incubated

in a CO 2 incubator for 6 hours, both with and without reserpine, the finalconcentration of which was 0 . 02 mM . The cells were chemically analysed for5-HT and their radiosensitivity estimated . In order to minimize physicalshock to the cells the medium was not changed before X-irradiation . Thedose-survival curves obtained are shown in figure 4 . By incubating withreserpine, the 5-HT contents of cells were depressed to 9 .5 and 39 per cent ofeach control level at cell densities of 10 5 and 5 x 10 ,1 respectively. In this casealso, a tendency towards a reverse relationship between the 5-HT content andradiosensitivity of cells was noticed .

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1500

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Figure 4 . Radiosensitivity of FMA cells pre-incubated in reserpine-containing culturemedium. The cells were irradiated 6 hours after the start of pre-incubation . Thefinal concentration of reserpine was 0 .02 mM . The cell density and 5-HT contentof 1011 cells at the time of irradiation are indicated in parentheses on each dose-survival curve .

3.2. Effect o f exogenously added 5-HT on the 5-HT content and the radiosensi-tivity of FMA, FM3A and B16-C2W cells

3.2.1 . 5-HT content o f cellsAn aqueous solution of 5-HT creatinine sulphate was added to a cell sus-

pension to make the final concentration of free 5-HT 2 .5 mM . Cells incubated

for 30 min both with and without 5-HT were washed three times with Hank'ssolution by centrifugation and chemically analysed for 5-HT . The results are

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86 T. Nakayama and W. Nakamura

presented in table 1 . After incubating with 5-HT for 30 min, only a traceamount of 5-HT was detected in the FM3A and B16-C2W cells . This amountof 5-HT was similar to that found as a difference between the FMA cells incu-bated both with and without 5-HT .

Table 1 . 5-HT content of cells incubated both with and without 5-HT . The finalconcentration of 5-HT was 2 .5 mM, and the incubation time 30 min . Beforechemical analysis, the cells were washed three times with Hank's solution by centri-fugation .

Table 2 . Effect of 5-HT on the plating efficiency of three cell lines in culture . Theplating efficiency of control cells DRF (1 per cent) was regarded as 100 per cent .

t Significant with a 5 per cent probability of error .Table 3 . DRF (1 per cent) obtained from treatment of 5-HT in three cell lines . The cells

incubated for 30 min both with and without 5-HT were subjected to irradiation .Experiments of the same number (1-9) were carried out at the same time . Thefigures in parentheses indicate the mean value of the DRF (1 per cent) obtained fromthree separate experiments .

5-HT (mM)DRF (1 per cent)

FMA FM3A B16-C2W

0. 3 Exp. 1 0 . 997 Exp. 4 1 . 094 Exp. 7 0 .9892 1 . 032 5 1 . 062 8 0 . 9943 1 . 025 6 1 . 148 9 1 . 175

(1 . 018) (1 . 101) (1 . 053)

1 . 0 Exp. 1 1 . 178 Exp. 4 1 . 057 Exp. 7 0 .7892 1 . 146 5 1 . 120 8 1 .0383 1 .384 6 1 . 126 9 1 . 150

(1 . 236)t (1 . 101) (0 .992)

2 . 5 Exp . 1 1 . 082 Exp. 4 1 . 127 Exp. 7 1 . 0662 1 . 225 5 1 . 161 8 1 . 0853 1 . 338 6 1 . 236 9 1 . 085

(1 . 215)t (1 . 175) (1 . 079)

Plating efficiency (per cent)Concentration of5-HT (mM) FMA FM3A B16-C2W

0 . 3 92+_ 4. 6 103+_4.8 101+_8 . 81 . 0 88+6. 3 98+_ 2 . 5 97+6 .72 . 5 73±9 .4 97±5 .8 94±3 . 5

5-HT content (µg/106 cells)Cell lines

Without 5-HT With 5-HT Increase

FMA 1 .405 1 . 500 0 . 095FM3A 0 .0 0 . 102 0 . 102B16-C2W 0 . 0 0 . 098 0 . 098

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3.2.2 . Radiosensitivity o f cellsCells were incubated for 30 min as described in § 3 .2 .1 . They were then

subjected to X-irradiation at room temperature and in room air without changingthe medium . At the time of plating, the cell suspension was diluted with 5-HT-free medium to obtain an adequate number of cell colonies . The effect of added5-HT on plating efficiency is shown in table 2. The surviving fraction in eachsample was obtained at a dose range between 0 and 1200 rad . The DRFs(1 per cent), which were calculated from the dose-survival curves, are sum-marized in table 3 . A slight protective effect was observed at higher concentra-tions of 5-HT in FMA and FM3A cells, but it was statistically insignificant .

3 .3 . Effect o f exogenously added 5-HTP on the 5-HT content and the radio-sensitivity o f FMA, FM3A and B16-C2W cells

3.3 .1 . 5-HT content o f cellsAn aqueous solution of 5-HTP was added to a cell suspension to make the

final concentration of 5-HTP 1 .25 or 2.5 mM . The cells, incubated for variousperiods of time, were washed three times with Hank's solution by meansof centrifugation and chemically analysed for 5-HTP and 5-HT . The5-HT content of FMA cells is shown in figure 5 . The 5-HTcontent of cells increased gradually, reaching, within 40 min, about five and sixtimes higher than the control levels for 1 .25 and 2 .5 mM concentrations of5-HTP respectively . In FM3A and B16-C2W cells, no 5-HT was detected .5-HTP itself was not detected at any time during incubation, even in FMA cells .

7'

6'

5'

4-

3 '

0 60

120

TIME OF INCUBATION (min)

180

Figure 5. The 5-HT content of FMA cells pre-incubated in the culture medium supple-mented with 5-HTP . The final concentration of 5-HTP in the culture mediumis indicated on each curve .

3 .3.2 . Radiosensitivity o f cellsThe radiosensitivity of cells was examined 40 min after incubating with

5-HTP. The final concentrations of 5-HTP were 1 .25 and 2 .5 mM . Dose-survival curves obtained with the three cell lines are shown in figure 6. Thecalculated DRF values (1 per cent) in FMA cells were 1 .48 and 1 .81 for 1 .25and 2 .5 mM 5-HTP respectively. In FM3A and B16-C2W cells, they were0 .90 and 0 .93 respectively .

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4. DiscussionFrom the data presented in figure 5, it is clear that, under the conditions used,

the 5-HT content of FMA cells is increased to a level several times higher thanthe controls 40 min after the addition of 5-HTP to the culture medium . Thosecells showed a higher resistance to X-irradiation, as indicated in figure 6, whereasin FM3A and B16-C2W cells, such changes in 5-HT content or radiosensitivityare not induced . As for the exogenously added 5-HT, none of these cellsshowed a capacity to incorporate significantly . At the same time, the radio-sensitivity of these cells was not modified appreciably by the addition of 5-HTto the medium. These results indicate that the 5-HT existing at the cellmembrane or in the surrounding medium has little or no effect on the reductionof radiosensitivity of cells . From the fact that there exists a relationship between5-HT content and radioresistance in FMA cells incubated with 5-HTP, it ispossible to postulate two alternative causes of the resistance : the increase of5-HT molecules in cells, and some altered physiological state of cells such asmetabolic activity or an arrangement of the cell constituents which is inducedby 5-HTP penetrating into the cells . Results obtained in the experiment usinga 5-HT releaser, reserpine, seem unhelpful in choosing between the two possi-bilities because this compound might cause a sort of chemical shock to cells,as well as stimulating their release of 5-HT . However, the results presented infigure 3 support the first possibility, for, in the experiment, no treatment orchemical which has a capacity to alter the growth rate of cells was applied .No modification worthy to be called ' shock ' was induced . In connectionwith this argument, the evidence reported by Lohmann, Moss, Sanders,Porter and Woodall (1966) concerning the radioprotection of catalase by 5-HTis important . They investigated the formation of a complex between Fe3+ inthe active site of the catalase and the 5-HT molecule by optical absorption ande.s .r . techniques. The existence of such a complex might explain the mechanismsof the radioprotection afforded by 5-HT in catalase as well as in cells in vitro .

Figure 6 . Radiosensitivity of FMA, FM3A and B16-C2W cells pre-incubated in theculture medium supplemented with 5-HTP . (A) FMA cells, (B) FM3A cells,(C) B16-C2W cells . The cells were irradiated 40 min after the start of pre-incuba-tion .

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As for the radioprotective effect of 5-HT and 5-HTP at the somatic level inmammalian species, many complex problems still exist . It has long beenbelieved that the target of whole-body irradiation in the dose range of haemato-poietic death is the haematopoietic stem cell . Extending this aspect, Nakamura,Kojima, Minamizawa, Kankura, Kobayashi and Eto (1969) and Nakamura(1973) proposed the idea that a radiation-induced intense thrombocytopeniamight be a key event which is responsible for the initiation of a serial reactionfatal to the animal . On the other hand, very important evidence was presentedby Streffer and Flugel (1972) . They have shown that only a small fraction ofi.p.-injected 5-HT penetrates into special regions of the central nervous system .They also found that the same brain regions were intensively labelled after theintracerebral injection of radioactive 5-HT. In the case of intracerebral injec-tion, about one-tenth the amount of 5-HT used in the i .p. injection was sufficientto induce protection . Thus they thought it very probable that the brain regionsmentioned were the sites through which the protective effect of the amine waseffected . As for the change induced by 5-HT through the brain regions in theperipheral organs, Streffer (1977) studied a shock syndrome of cells in theperipheral organs as evidenced by the change of the redox equilibria in liver cells(Wollenberger, Ristan and Schoffa 1960) .

Taking this evidence into consideration, including the findings obtained in thepresent study, it might be valuable to compare the mechanisms of protectionexhibited by 5-HT and 5-HTP . The general condition of mice is quite differentafter the administration of 5-HT and 5-HTP . The possibility might existthat different cells are acting in those cases. It seems highly probable that thereexist several types of cell in brain and bone marrow which respond differentlyto 5-HT and 5-HTP ; some cells incorporate 5-HT itself, some incorporate5-HTP without converting it into 5-HT, some incorporate 5-HTP and convert itinto 5-HT, and some incorporate neither 5-HTP nor 5-HT but have receptorsfor them on the cell membrane. In this connection, we are considering a studyof the behaviour of cells in thrombocytopoiesis which have a special affinity for5-HT derivatives .

ACKNOWLEDGMENTS

This work was supported in part by a Grant-in-Aid for Cancer Researchfrom the Ministry of Science and Culture, Japan .

On a etudie l'effet radioprotecteur de la 5-hydroxytryptamine (5-HT) et de la 5-hydroxy-tryptophane (5-HTP) sur les cellules mammiferes cultivees de trois souches ; la FMAqui a la capacite de synthetiser 5-HT et la FM3A et la B16-C2W qui n'en ont pas . Auxcellules etudiees, rien qu'une faible protection ou pas du tout a ete induite par l'additionde 5-HT dans le milieu . L'augmentation de la teneur des cellules en 5-HT au momentde l'irradiation a ete negligeable . L'incubation prealable de cellules pendant 40 mndans un milieu contenant de la 5-HTP a cause une augmentation importante de la teneuren 5-HT en meme temps que celle de la radioresistance des cellules de la FMA, oil leFRD (10-2 ) a ete 1,8 . Un tel effet n'a pas ete observe pour les cellules de la FM3A etde la B16-CM Pour les cellules de la FMA cultivees a la densite de cellule variable ouavec de la reserpine, ce parallelisme entre la teneur en 5-HT et la radioresistance de cellulea ete observe . Ces faits nous suggerent fortement que la substance qui joue le role principaldans l'induction de la radioresistance de cellule in vitro est la 5-HT qui existe dans lescellules .

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Protection o f cultured cells by 5-HTP

Es wurde die Schutzwirkung in vitro des 5-Hydroxytryptamins (5-HT) and des 5-Hydroxytryptophans (5-HTP) gegen Rontgenstrahlen bei Kulturzellen von drei Stammen(5-HT-synthesierender FMA and 5-HT-nichtsynthesierender FM3A and B16-C2W)untersucht . Bei diesen Zellen gibt es nur einen schwachen oder keinen Schutzeffekt durchden Zusatz von 5-HT ins Kulturmedium. Eine Zunahme des 5-HT-Gehaltes der Zellenwahrend der Bestrahlungszeit war vernachlassigbar . Eine Vorinkubation der Zellen fur40 min in 5-HTP-haltigen Medien induzierte in den FMA-Zellen eine auffallende Zunahmedes 5-HT-Gehalts, die parallel mit der Zunahme der induzierten Radioresistenz verlief,dabei lag der Dosisreduktionsfaktor (10 -2 ) bei 1,8 . Ein solcher Effekt war bei FM3A- andB16-C2W-Zellen nicht zu beobachten . Es ergab sich eine Parallelitat zwischen dem 5-HT-Gehalt and der Radioresistenz bei FMA-Zellen, die bei verschieder Zelldichte oder mitZusatz von Reserpin im Kulturmedium inkubiert wurden . Diese Ergebnisse weisendarauf hin, dal3 5-HT eine Hauptrolle bei der Induzierung der Radioresistenz von Zellenin vitro spielt .

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exp. Ther ., 117, 82.Booz, G., and BETS, E . H., 1960, C. r. Seanc. Soc. Biol ., 155, 197 .CLAUNCH, C ., OIKAWA, A ., TCHEN, T. T., and Hu, F., 1969, Advances in Biology of Skin,

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