WR2721 INTESTINAL RADIOPROTECTION: MORPHOMETRICAL ANALYSIS

RADIO-PROTECCION INTESTINAL POR EL WR2721: ANALISIS MORFOMETRICO

* Department of Morphology - UNIFESP-EPM, São Paulo, Brazil.
** Department of Pathology - UNIFESP-EPM, São Paulo, Brazil.
*** Department of Medicine - UNIFESP-EPM, São Paulo, Brazil.
**** Department of Preventive Medicine - UNIFESP-EPM, São Paulo, Brazil.

SUMMARY: Taking into account the great sensitivity of the intestinal mucosa to X irradiation we aimed at investigating the radioprotection afforded by the S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR2721) on the mice rectum exposed to a single whole body X ray dose of 7 Gy and sacrificed at 1, 3, 6 and 10 days after irradiation.

Comparing the data of protected group with those of the unprotected one the following features were observed in the first one: statistically significant increase of both crypt cells per crypt (CrC/Cr) at 1, 3, 6 and 10 days after irradiation and of mitotic figures per crypt (MF/Cr) at 3, 6 and 10 days post exposure.

In this way it became clear that these morphometric parameters allowed the estimation of WR2721 rectum protection through the crypt cellularity and epithelial cell kinetics.

KEY WORDS: 1. Mouse; 2. Rectum; 3. Radioprotector.

INTRODUCTION

The classic radioprotectant WR2721 has been shown to reduce the severity of intestinal injury when administered systemically shortly before irradiation has been delivered (DELANEY et al., 1994).

The rectum invariably sustains some acute injury during pelvic irradiation, the same happening to those portions of the small intestine that lie within the radiation field. The clinical consequence is an acute enteritislike syndrome with diarrhea, tenesmus and often bleeding. These symptons are always distressing to the patient and may be sufficiently severe so as to require alteration in the treatment plan.

Concerning the fact that most cases for radiation therapy are related to the destruction of tumoral tissue, effortts have been made to amplify differences in radiosensitiveness between normal and neoplastic tissue. One of the selective radioprotectants to normal tissue is WR2721 (S-2-(3 aminopropylamino) ethylphosphorothioic acid, a thiophosphate compound (PHILIPS, 1973; YUHAS, 1980) developed by Experimental Therapeutics Division at Walter Reed Army Medical Center, for the military staff in the event of nuclear warfare (CAPIZZI, 1993).

Systemic administration of WR2721 for intestinal radioprotection in patients seems to be very important because the drug might reduce not only the bowel mucosa damage but also at the same time protect several organs against toxicity of radiation. In previous studies we have had good results of radioprotection in bone marrow when 400 mg/kg of WR2721 were administered 30 min. before whole body exposure to X rays (SEGRETO et al., 1995).

The purpose of the present investigation is to test WR2127 for its ability to protect against the effects of X rays on the mouse rectum epithelium "in vivo". Mitotic figures and number of cells of Lieberkhün crypts were evaluated.

MATERIAL AND METHOD

Thirty Swiss mice weighting from 2326 g were assigned to the following groups:

· G1 (unprotected group): twelve mice received intraperitoneally (IP) 0,8 ml of physiological saline solution (PSS) 30 minutes prior to a whole body X ray dose of 7 Gy. They were assigned in four subgroups having three animals each and sacrificed 1, 3, 6 and 10 days after irradiation.

· G2 (protected group): twelve mice received IP, 400 mg/kg of WR2721, 30 minutes prior to irradiation and they were sacrificed at the same time as G1.

· G3 (control group): six mice were not irradiated; one did not receive PSS or WR2721; one received IP PSS and was sacrificed one day later, four received IP, 400 mg/kg of WR2721 and were sacrificed 1, 3, 6 and 10 days later.

The radiation was performed in a Westinghouse Duocondex unit with following radiation factors: 180 kV. 15 mA, 1 mm Cu + 1 mm Al filter at a mid target distance of 50 cm and HVL 1,8 mmCu at a dose rate of 0,295 Gy/mm in air.

For irradiation, mice were placed in special (20 x 20) cm wooden boxes without anesthesia.

Morphometrical analysis: during sacrifice, one animal of each group was anesthesized with IP injection of sodium thiopental (60 mg/kg) + cloral hydrate (10%). The segment of rectum were fixed by vascular perfusion with Karnovsky (KARNOVSKY, 1965) cut at 1 µm thickness after hidroxy ethylmethacrylate embbeding and stained with PAS + Gill hematoxylin. The total number of crypt cells per crypt (CrC/Cr) and the number of mitotic figures per crypt (MF/Cr) were counted in at least 6 sections of longitudinally cut crypts and were estimated quantitatively under light microscope.

Statistical analysis: performed by using the KruskalWallis Test (SIEGEL, 1975) to compare the result of number of CrC/Cr and MF/Cr at each period 1, 3, 6 and 10 days after irradiation. This test was applied in separate for the protected and unprotected groups. To assess the radioprotection effect of WR2721 we compared the results of number of Cr/C and MF/Cr between protected and unprotected groups by using Man Whitney Test (SIEGEL).

RESULTS

The average number of crypt cells/crypt (CrC/Cr) and mitotic figure/crypt (MF/Cr) of non irradiated mice rectum (control group) are presented in Table I.

Comparing the CrC/Cr and MF/Cr, protected versus unprotected, the values were significantly higher in the protected group at 1, 3, 6 and 10 days after irradiation for CrC/Cr and at 3, 6 and 10 days for MF/Cr (Table II and Fig. 1).

The photomicrographs in Fig. 2a and 2b show mucosal tissue from mice rectum given PSS group (2a) and WR 2721 group (2b), 10 days after being similar to the morphological feature of 3 and 6 days post irradiation for both groups.

At this radiation dose level a strike difference was seen in the rectum of the protected group, fact that which was revealed by significant increase of crypt height (Fig. 2b) in relation to PSS group (2a).

The photomicrograph in Fig. 3b (protected group) shows mitotic figures more frequent in the epithelium of intestinal crypts than in the unprotected ones (Fig. 3a), at 3, 6 and 10 days after irradiation.

Fig. 1 Average number of crypt cells/crypt (CrC/Cr) and mitotic figures/crypt (MF/Cr) of Swiss mice rectum after a single whole body Xray dose of 7 Gy of protected (WR2721 30 minutes prior irradiation) and unprotected animals (PSS) sacrificed at 1, 3, 6 and 10 days after irradiation.

DISCUSSION

The development of gastrointestinal syndrome and consequent death are related to the degenerative changes in the intestinal mucosa following irradiation (DeCOSSE et al., 1969). The present experiment showed that WR2721 protected the rectum mucosa from radiation damage, thus suppporting earlier studies by authors in other segments of intestinal mucosa of various species of mammals and humans (SIGDESTAD et al., 1975; HANSON, 1987; MURRAY et al., 1988; LIU et al., 1992; PRASANNA & DEVI, 1993; DELANEY et al.; CARROL et al., 1995).

The morphometrical analysis of intestinal mucosa of the exposed animals resulted in a significant (p < 0,01) decrease of approximately more than 30% of the number of crypt cells in the PSS (physiologycal saline solution) irradiated group at 3, 6 and 10 days than the number in the WR2721 group. The number of mitoses showed significant increase (p < 0,01 and p < 0,05) in the WR 2721 pretreated animals when compared with unprotected group, at 3, 6 and 10 days after irradiation.

The present finding indicating that WR2721 is effective against the radiation damage could mainly be related to the protection of stem cells as referred by PRASANNA & DEVI, not only to the intestine but also to hematopoietic stem cells (HANSON & AINSWORTH, 1985; ITO et al., 1994).

The mechanisms of radiation protection by thiol compounds, like WR2721, are thought to occur through hydrogen atom donation, competition of sulfhydryl groups with the free radicals (free radical scavengers) which would otherwise react with oxygen to form damaging oxygen radicals (ZHENG et al., 1988).

Taking these mechanisms into account it's widely accepted that WR2721 may predominantly protect the nucleus of the cell (HANSON & GRDINA, 1987; MEECHAN et al., 1991) specifically DNA (VAUGHAN et al., 1989; SMOLUK et al., 1981) even after receiving high doses of radiation (GUPTA & DEVI, 1986 and DEVI & PRASANNA, 1990).

Yet, considering the fact that most amino compounds with sulfhydryl groups such as WR2721 and its free thiol WR1065 are capable not only to delay the progression of cells through Sphase (GRDINA et al., 1988) but also to extend the celldoubling time (GRDINA & NAGY, 1986); it is reasonable to expect some alteration related to the renewal process of epithelial cells.

Fig. 3a. PSS + X ray. PAS + Gill Hematoxylin. ± 360 x. Crypts of the rectum mucosa showing a few mitotic figures (arrow).

Fig. 3b. WR2721 + X ray. PAS + Gill Hematoxylin. ± 360 x. Crypt of the rectum mucosa showing many mitotic figures (arrows).

Our results did show a significant increase of mucosal height of Lieberkühn crypts and a great number of mitotic figures in the rectum epithelium in the presence of WR2721, at the recovery phase. These data are consistent with the better preservation of viable stem cells in the protected group and their capacity of active proliferation to enhance the tissue repairing process.

RESUMEN: En base al elevado grado de radio-sensibilidad de la mucosa intestinal, ha sido estudiada la acción radioprotectora del acido S2(3aminopropilamino) etilfosforotioico (WR2721) sobre el epitelio de recto de ratones expuestos cuerpo entereo a los rayos X, dosis única de 7 Gy, y sacrificados después de 1, 3, 6 y 10 días. Al comparar las muestras de segmentos del recto de animales protegidos y no protegidos, fue observado, de manera general, que en los protegidos, ha ocurrido aumento estadísticamente significativo en el número de células de la crypta por crypta (CrC/Cr) después de 1, 3, 6 y 10 días luego de la irradiacón, y en el número de figuras de mitosis por crypta (MF/Cr) después de 3, 6 y 10 días. Se puede concluir que los parámetros morfométricos referidos, permitieron estimar cuantitativamente la radioprotección por el WR2721 en el recto de los ratones, evaluada por la celularidad de la cripta y por la cinética de las células epiteliales intestinales.

PALABRAS CLAVE: 1. Ratón; 2. Recto; 3. Radioprotector.

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Recibido : 02-10-1997
Aceptado : 16-10-1997