Chemical protection of the alimentary tract of whole-body X-irradiated mice

Experimental

Cell Research 21, 317-352

CHEMICAL PROTECTION OF WHOLE-BODY II.

CHROMOSOME

Division,

OF THE ALIMENTARY X-IRRADIATED MICE

BREAKS

J. MAISINI Biology

347

(2960)

AND

MITOTIC

TRACT

ACTIVITY

and J. MOUTSCHEN*

Oak Ridge National

Laboratory,3

Oak Ridge, Tennessee, U.S.A.

Received November 6, 1959

SOME chemical compounds can protect the mucosa of the small intestine against radiation damage. Conard [2] found that, after exposure in the 550 to 950 r range, weight loss in the rat intestine was less if glutathione was given to rats before exposure. The mitotic count of the small intestine was shown to be higher in MEAtreated, irradiated rats [l, 41 and mice [3] than in irradiated controls. Glutathione and p-aminopropiophenone had a protective effect in irradiated rats Br . HBr) gave similar pro[lOI, and AET (S,2-aminoethylisothiuronium. tection to irradiated mice [5]. Protection of DNA synthesis by MEA (/3mercaptoethylamine) has been observed in irradiated rats and mice [S, 91 and by AET in irradiated mice [5]. Mikaelson [6, 71 showed that glutathione and cysteine reduced the number of radiation-induced chromosome aberrations in Tradescantia root tips. A similar observation was made for AET in irradiated Vicia faba (S. Wolff, personal communication) and in the intestine of irradiated mice [5]. To further correlate the biological mechanisms that are protected by AET, we studied the mitotic activity and chromosome damage in the intestinal crypt cells of protected and nonprotected irradiated mice. MATERIALS Animals.-Male

(21-29

g) A-i

mice

AND

METHODS

were

used.

They

were

housed

5 to 10 in a

cage and had free access to food and water. Irradiation.-A Philips constant-potential X-ray machine was used, and the radiation factors were 250 kv at 15 ma; 1.0 mm of Al added filtration; hvl 0.5 mm of Cu; target-object distance 60 cm; dose rate in air -160 r/minute. The mice were 1 WHO Fellow from Belgium. The opinions expressed by the author in this paper do not necessarily represent the view of the WHO. ’ International Cooperation Administration Fellow. 3 Operated by Union Carbide Corporation for the United States Atomic Energy Commission. Experimental

Cell Research 21

Jean Maisin

and Jean Moutschen

exposed in groups of 10 or 12 in a partitioned, revolving lucite cage. The LD,,/30 days was 650 r. of compounds.-In separate experiments, one of the following comPreparafion pounds was injected, intraperitoneally 15 minutes before irradiation: 8.8 mg of AET, 6.0 mg of APMT (S,3-aminopropyl-W-methyl isothiourea) or 1.2 mg of 2-ABT (D-S,2-aminobutylisothiourea). AET was neutralized with NaOH, and APMT and 2-ABT were neutralized with Na(HCO,),. Microscopic examination.-Treated and nontreated irradiated mice were killed at intervals by dislocating the neck. Two fragments of the second part of the duodenum were fixed in Zenker-Formol, sectioned, and stained with hematoxylin-eosin. Microscopic fields of the crypt region and neck of the small intestinal villi were studied, and mitotic counts were made at intervals up to 9 days after irradiation. The intervals ranged from 4 to 24 hours. In each section, 60-120 fields were examined. Cells from late prophase to early telophase were counted. The numbers of chromosome fragments and bridges per 300 cells in anaphasc were determined at daily intervals. RESULTS Chromosome nherrrrtioJls.--Visible chromosome aberrations consistetl esof anaphase fragments and bridges, excluding some types of very sentially rare aberrations that did not alter the total frequency of anaphases. Fig. 1

shows an anaphase fragment (A) and an anaphase bridge longitudinal sections of cells in anaphase were scored.

Fig. IA.-Anaphase X irradiation.

fragments

Fig. lB.-Anaphase irradiation.

bridge

Experimental

Cell Research

in intestinal

in intestinal

21

crypt

crypt

cell of an A-l

cell of an A-l

(13). Only good

mouse after 900 r whole-body

mouse after

900 r whole-body

X

Chemical protection of alimentary tract. II

Fig. 2.-The protective effect of AET on the number of anaphase fragments as a function of X-ray dose in intestinal crypt cells of A-l mice 4 days after exposure. Fig. 3.-The protective effect of AET on the number of anaphase fragments in the intestinal crypt cells of A-l mice.

6

Fig. 4.-The effect of protective compounds on the number of anaphase fragments crypt cells of A-l mice 4 days after 900 r of whole-body X irradiation. Fig. 5.-The A-1 mice.

protective

effect

of 4ET

Fig. B.-The protective effect of AET, lethally irradiated (900 r) A-l mice.

on mitosis 2.4BT,

in the intestinal

and APMT

epithelium

on mitosis of intestinal

E.rperimenfnl

in intestinal

of X-irradiated epithelium

of

CeZI Research 21

Jean Maisin

and Jean Moutschen

The number of fragments in the intestines of AET-treated and nontreated mice on day 4 after 225-1500 r is shown in Fig. 2. It can be seen that the higher the dose, the greater the difference between the number of fragments in the treated and nontreated animals. It was not possible to count aberrations in the nontreated 1500-r group because regeneration did not occur. In a second set of experiments, the number of fragments was determined in AET-treated and nontreated 900-r mice at daily intervals from days 2 to 9 after irradiation. Results in Fig. 3 indicate that the number of fragments was higher in the intestines of nontreated mice than in the treated ones for 2 to 5 days after irradiation. After 5 days the curves were similar. These results were essentially unchanged when both anaphase fragments and bridges were considered because most aberrations were fragments. AET, however, did not appreciably reduce the bridge frequency per cell after 900 r. Fig. 3 also shows the number of fragments in the intestine of AETtreated mice on days 3 to 9 after 1500 r. The curve is S-shaped and has a plateau between days 4 and 6. On day 6, the number of fragments in the 1500-r group was higher than those of the two 900-r groups, but all three were essentially the same on day 9. Data from nontreated mice exposed to 1500 r could not be obtained because the intestinal epithelium did not regenerate. The fourth day after irradiation was chosen as the time to compare the efficiency of the three compounds in reducing the number of chromosome fragments. The results (Fig. 4) indicate that (1) the three compounds signiticantly reduced the number of fragments and (2) the ability to prevent fragment formation varies from one compound to another. The efficiency of AET was significantly higher than that of 2-ABT or APMT. Mitotic acitiuity.-Fig. 5 shows the results for AET-treated and nontreated animals exposed to 225 and 1500 r. A marked protective action of AET on the number of mitoses per microscopic field is apparent at the 1500-r exposure level. At 225 r, however, there is essentially no difference between treated and nontreated mice. Fig. 6 shows the results for AET-, 2-ABT-, and APMTtreated mice and for nontreated animals at 900 r. It is seen that return of the number of mitoses per field to normal values occurred earlier in the treated mice than in the 900 r controls. In this respect, AET appeared to be better than 2-ABT or APMT. In all treated mice, at all X-ray doses studied, there was a temporary overshoot in the number of mitoses per held. DISCUSSION

Since it is very difficult to determine accurately the metaphase damage in the mouse intestine, we used the chromosome aberrations observable in Experimental

Cell Research 21

Chemical protection of alimentary tract. I1

351

anaphase as the criterion for X-ray damage. Clearly, by this method, an absolute measurement of chromosome damage was not obtainable. In our experiments, only anaphase fragments and bridges were considered, not those aberrations described in the literature as clumping or sticking of chromosomes. Whether chromosome lesions consisted of one- or two-hit aberrations could not be ascertained from our preparations because metaphase fragments could not be accurately counted and because a relation between X-ray dose and the number of aberrations was not determined. It is possible that some treatments specifically influence one class of aberrations and not others. Our results (Fig. 2) show that, on the fourth day in both treated and nontreated mice, the number of fragments increases with the X-ray dose, and that the higher the dose the greater the difference between treated and nontreated mice. Many investigators have shown, however, that it is not possible to compare mitotic activity after different X-ray doses without a long period of observation [ll]. For this reason, anaphase damage was compared in treated and nontreated 900-r mice for a long period (i.e., 9 days). Our results also indicate that the number of fragments decreases with time after exposure. This decrease could have resulted from a gradual loss of damaged cells and a subsequent recovery of the intestinal epithelium by proliferation of viable cells. Radiation may have caused gene deficiencies in some of these viable cells; in which case, damage would be present but would not be observed as fragmentation. There is an inverse relation between mitotic activity and the number of fragments during recovery of the intestinal mucosa in mice receiving 900 r of X rays. This relation is particularly evident on day 4 in irradiated mice (900 r) protected with 2-,4BT or APMT. The plateau between days 4 and 6 observed for chromosome fragments in the AET-1500 r group could result from longer inhibition of mitoses and a consequent accumulation of lesions. It should be mentioned that the number of fragments and bridges is higher in the control mice in,jected with AET or 2-ABT and APRIT than in the normal controls. Recovery of mitotic activity after 225 r was about the same in treated and nontreated mice. Whether this observation reflects insensitivity of the method or there is virtually no protective action of AET on mitosis at low X-ray doses cannot be determined from these data. On the other hand, a comparison of the number of chromosome fragments on day 4 in treated and nontreated mice after 450, 900, or 1500 r suggests that, as the X-ray dose increases, the degree of protection also increases. Experimental

Cell Research 21

Jean Maisin

and Jean Moutschen SUMMARY

The protective effect of AET on mitotic activity and chromosome damage in intestinal crypt cells of X-irradiated mice has been studied. It was found that the number of chromosome fragments in the crypt cells of both treated and nontreated mice is dose dependent. In lethally X-irradiated mice given AET the number of chromosome fragments was less than that of nontreated mice; at supralethal X-ray doses, AET markedly protected mitotic activity. The data indicate that, in treated and nontreated mice, recovery of mitotic activity was inversely related to the number of chromosome fragments. We wish to thank in this study.

Dr. D. G. Doherty

for preparing

the protective

compounds

used

REFERENCES R. P., KEREIAKES, J. G. and KREBS, A. T., .7. Sofl. Cancer Inst. 22, 1045 (1959). R. A., Radiation Research 1, 492 (1954). DESAIVE, P., and VARETTO-DEN~EL, Experientia 11, 242 (1955). MANN, H. and FIEVEZ, C., in Radiobiology Symposium, 1954, p. 304. Ed. by BACQ, %. M. and ALEXASDER, P. Butterworths, London, 1955. RAISIN, .J., MOUTSCAE~, J., NOVELLI, G. D. and DOIIERTY, D. G., (Abstr.), Rudiation Research in press. RIIKAELSOS, Ii., Science 116, 172 (1952). -Proc. Natl. Acad. Ski. U.S. 40, 171 (1954). MOLE, R. RI. and TEMPLE, D. M., Nature 180, 1278 (1957).

1. BELILES,

2. CONARD, 3. 4. 5.

6. 7. 8.

9. __ Intern. J. Radiation Riot. 1, 28 (1959). 10. WILLIAMS, R. B. and LONG, R. P., Federation

11.

WILLIAMS,

R. B., JR., To.&L,

21, 17 (1958).

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J. N., \\'HITE,

Proc. 12, 1338 (1953). J. and

CARPENTER,

H. ll/I., J. s~l[.

Cuncer Inst.