Pathological Evaluation of WR Administered Orally in Irradiated and Non-Irradiated Male Mice

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 22, No. 3 Copyright > 1992, Institute for Clinical Science, Inc. Pathological Evaluation of WR Administered Orally in Irradiated and Non-Irradiated Male Mice LINDA STEEL-GOODW IN, Ph.D.,t JUDY M. KENDRICK,$ JUNE E. EGAN,t and JON M. ECKSTEIN, D.V.M. Departments o f fradiation Biophysics, tradiation Biochemistry, and Veterinary Sciences, Armed Forces Radiobiology Research Institute, Bethesda, MD ABSTRACT Studies were made on the radioprotective and toxic effects of orally adm inistered WR in male CD2F1 mice. The lowest dose of orally adm inistered drug perm itting probit analysis of data was 450 mg per kg. The calculated radioprotective dose reduction factors (DRF) at 450 mg per kg and 900 mg per kg of body weight (BW) WR were 1.2 and 1.3, respectfully. Pathological examination at 8, 30 or 90 days post administration of 100, 450, or 900 mg per kg of the drug dem onstrated that the major target organ for orally dosed mice was the testes. There was a decrease in the num ber of cells in the germinal cell layers of testes from animals administered 450 mg per kg WR or 10 Gy whole body irradiation after eight days. Moreover, there was a dramatic reduction in the germinal cells in mice seminiferous tubules treated with a combination of 450 mg per kg WR plus 10 Gy radiation after eight days. Introduction Chemical radioprotection is exhibited by certain su lfhydryl (-SH) co n tain ing compounds if they are adm inistered prior to ionizing radiation exposure.5 WR , S-3-(3-methylaminopropylamino)-propylphosphorothioic acid, is a w ater soluble phosphoroam inothiol com * Send reprint requests to: Dr. Linda Steel- Goodwin, Radiation Biophysics Department, Armed Forces Radiobiology Research Institute, Bethesda, MD pound which contains a -SH group upon dephosphorylation. 1 It has b e e n identified as an effective radioprotector when adm inistered orally8 and has b een dem onstrated to provide protection against neutron radiation effects in the gastrointestinal tract6 and bone marrow 7 w hen adm inistered intraperitoneally (ip). A recent report by van Beek et al9 indicated adverse pathological effects upon ip injection of WR to non-irradiated male mice. The purpose of this study was twofold: to determ ine an oral dose of WR providing radioprotection /92/ $01.20 Institute for Clinical Science, Inc.

2 WR-15I327 PATHOLOGY AND RADIOPROTECTION IN MICE 183 and to examine pathological changes in male mice following oral administration of the drug. Materials and Methods Male CD2F1 m ice were obtained from the National C ancer Institute, Frederick, MD. All mice were betw een eight and 12 weeks of age at the tim e of experimental use. Animals w ere provided w ith acidified water (ph 2.7) to prevent growth of Pseudom onas. T hey w ere fed m ouse chow,* housed eight to a cage, and m aintained on 12-hour light/dark cycle to prevent circadian rhythm s that could influence responses. D rugs w ere a d m in iste re d to m ice betw een 8:30 A.M. and 9:00 A.M. about one hour prior to irradiation. Mice were placed in plastic containers approxim ately 15 m inutes before total-body irradiation. T hey w ere irradiated in a bilateral 60BCo gamma-ray field at a dose rate of 1.0 Gy per min, receiving total doses of 7, 8, 9, 10, 11, 12, or 13 Gy. The control group was also irradiated at doses of 7.5, 7.75, 8.25, 8.5, or 8.75 Gy. The radioprotector W R was obtainedt and was adm inistered to mice using stainless steel oral animal feeders.t Mice were divided into groups of at least 1 0 anim als and given the following concentrations o f the drug dissolved in sterile saline ph 7.0: 100, 450, and 900 mg per kg body weight. The 900 mg per kg drug dosage represented approximately one half the L D 10 of orally adm inistered WR The drug concentration was adjusted so that 0.1 ml was adm inistered for each 0.01 kg body w eight. Approxim ately one hour prior to exposure, irradiated m ice w ere given the WR Control m ice received a sim ilar 0.1 ml * Wayne Rodent Lab BIox, Wayne Pet Food Division, Continental Grain Company, Chicago, IL. t US Bioscience, West Conshohocken, PA. i Popper and Sons, Inc., New Hyde Park, NY. volume of sterile saline ph 7.0 for each 0.01 kg of body w eight. Survival was m onitored for 30 days. Data w ere analy z e d by p ro b it u s in g th e F in n e y m ethod.2 Briefly, this m ethod is used to fit a dose-response curve to the observed proportion of animals surviving at each concentration of WR , assum ing an underlining normal distribution of tolerance to WR T he p ath o lo g ical effects of orally adm inistered W R w ere d e te r m in e d by h isto lo g ic a l co m p a riso n s betw een tissues from treated mice and controls. Surviving animals were euthanised on days 8, 30, or 90 by lethal inhalation of m ethoxyflurane. Gross necropsy was performed, and tissue samples of trachea, lung, heart, skeletal muscle, pancreas, salivary gland, duoden u m, ileum, jejunum, colon, kidney, liver, b rain, eye, sp le e n, and te ste s w ere rem oved im m ediately and p laced in saline formalin fixative. All tissues were em bedded in paraffin wax, cut into four micron sections, and stained w ith hem a toxylin and eosin. Light microscopy m easurem ents w ere m ade of the circum ference and the num ber of cells in the germinal cell layers of five seminiferous vesicles per testis for groups of eight treated m ice euthanised at day 8. All slides were analyzed using a Bioquant H ipad Digitiser attached to a microscope (Leitz Laborlux D). Slides were decoded into experim ental groups and raw data were statistically analyzed using two-way analysis of variance to com pare th e means of the num ber of cells and the circumferences of the testes in each group. Results A m odest enhancem ent in the length of tim e of survival of irradiated m ice com pared to controls was observed in groups Trademark: Metofane, Pitman-Moore, USA.

3 184 STEEL-GOODWIN, KENDRICK, EGAN, AND ECKSTEIN previously adm inistered WR at 100 mg per kg of body weight. However, the low est practical dose of drug resulting in survival of sufficient num bers of animals perm itting probit analysis of data was 450 mg per kg. In figure 1 are shown probit plots of percent mortality as a function of irradiation dose for controls and animals adm inistered either 450 mg per kg or 900 mg per kg. Each data point is an average of at least eight animals. The dose reduction factor for WR at 450 mg per kg and 900 mg per kg was (95 percent confidence limits 1.17 through 1.248) and (95 percent confid en ce lim its th ro u g h 1.355), respectively. This was calculated from p ro b it a n a ly sis u sin g th e e q u a tio n LD 50/30 = (LD 50/30 WR group)/ (LD50/30 control group). The slope of the probit of the control was significantly different from the slopes of the lines at each dose of WR For this reason the reported dose reduction factor is valid only for LD50/30. T he com bined pathological results observed in 72 mice dosed with either 100, 450, or 900 m g p e r kg of BW CO c o c a> o o CL L - v'o A WR WR and examined 8, 30, and 90 days following exposure indicated that 85 percent of the animals had focal atrophy of the testes. There were m inor changes in other organs w hich w ere not correlated w ith dosage. Approximately 35 percent of the livers presented subacute microgranuloma, 2 1 percent of the lungs show ed acute hem orrhage and congestion, eight p ercen t had epithelial sloughing of the gastrointestinal tract, 10 perc e n t p r e s e n t e d a c u te i n t e r s t i t i a l nephritis, 10 percent presented subacute pancreatitis, one percent of the spleens showed acute congestion, three percent of the thyroid glands had m ultifocal necrosis, th ree p ercen t of the lym ph nodes had hyperplasia, and four percent of the sebaceous glands had adenoma. T here w ere no pathological changes observed in brains, skeletal m uscles, salivary glands, or the eyes. Oral administration of the drug at 900 mg per kg caused diarrhea w ithin 24 hours in 77 percent (n = 8 8 ) of the mice treated. This observation was confirmed at higher concentrations of WR (results not shown). Tw enty-four control / «B A C /.A... Control mg/kg mg/kg F ig u r e 1. Percentage mortality after 30 days of groups of at least eight m ice fo llo w in g oral adm inistration of (A) saline (B) 450 mg per kg of WR151327, or (C) 900 mg per kg of WR orally one hour before irradiation in a 60Co gamma field at a rate of 1.0 Gy per min over the radiation dose range of 7 to 13 Gy. The data were analysed by probit, and the c a lc u la te d d ose reduction factor was for B and for C.

4 WR PATHOLOGY AND RADIOPROTECTION IN MICE 185 mice examined at days 8, 30, and 90 had one animal with a cyst in the kidney. No o th e r p a th o lo g ic a l c h a n g e s w e re observed. Following this broad survey of pathological results, the testes of the mice were studied in more detail. In table I is shown the percent of testic u la r in v o lv e m e n t at each dose of WR on days 8, 30, and 90. There is an increase in the percentage of animals presenting testicular damage when the dose was increased. In addition, there was an increase in the num bers of animals presenting testicular damage in groups adm inistered either 100 or 450 mg per kg of BW of the drug with the passage of time. The 900 mg per kg dosed animals rem ained at 100 percent involvem ent on days 8, 30, and 90. Pathological examination of groups of eight mice orally adm inistered 0, 100, 450, or 900 mg per kg of BW WR , subjected to 10 Gy total body gamma irradiation, and exam ined eight days later indicated focal atrophy of the testes in every animal. Control animals that were neith er adm inistered the drug nor subjected to irradiation presented normal testicular histology. A transverse section of several sem iniferous tubules of the testis from a mouse given saline orally and killed eight days later is shown in figure 2A. Each seminiferous tubule is surrounded by an outer compact connective tissue and an inner basem ent m em brane. E nclosed in the TABLE I Percentage of Groups of Eight Mice Presenting Testicular Pathological Changes Concentration WR Percent of Mice Presenting mg/kg Testicular Pathological Changes body weight Day 8 Day 30 Day basem ent m em brane is the specialized germinal epithelium and Sertoli s cells. Sertoli s cells are slender, elongated cells with very irregular outlines extending from the basem ent m em brane to the free luminal surface. The distinctive Sertoli s cell nucleus is ovoid or angular in shape, facilitating its discrim ination from the sperm atogenic cell that divides mitotically to p roduce several generations of cells. Oral administration of WR at a concentration of 450 mg per kg caused degeneration of the germinal cell layer. In figure 2B is shown a transverse section of the seminiferous tubules from a mouse treated with WR Both Sertoli s cells and the spermatogenic cells were affected in these mice. A transverse section of the sem iniferous tubules from a mouse adm inistered saline and irradiated one hour later with 10 Gy is shown in figure 2C. This photom icrograph indicated absence o f sperm a tozoa and a reduction of cells in the germ inal layer. T h e cells p re s e n t had necrosis with cytoplasmic swelling and v a c u o liz a tio n. M ice tr e a te d w ith WR had a loss of cells but no cytoplasmic swelling. The presence of cells in the testes of irradiated m ice eight days later indicates a degree of regeneration of cells after radiation injury. F ollowing a single 10-Gy exposure, radiosensitive proliferating cells could be replaced within eight days by previously resting stem cells. Combining treatm ent with WR at 450 mg per kg with exposure to 10-Gy total-body irradiation one hour later produced a pattern similar to treatm ent with WR alone. The photomicrograph of the seminiferous tubules of mice from this group (figure 2D) show ed loss of cells w ith no cytoplasm ic sw ellin g. There was a greater reduction in cell num ber in mice treated with WR and irradiated than in mice treated with WR alone.

5 F ig u r e 2. Photomicrographs of testes stained with hematoxylin and eosin eight days after oral admin istration of (A) saline, (B) mg per kg of W R , (C) saline and one hour later 10 Gy whole body irradiation from a 60Co gamma-ray source, and (D) mg per kg of W R and one hour later whole body irradiation from a 60Co gamma-ray source.

6 WR PATHOLOGY AND RADIOPROTECTION IN MICE 187 In table II are shown the m ean cell num bers in the germinal cell layer and the circum ference size of the sem iniferous tubules of the mice in each treatm ent group described for figure 2 A through D. There were eight animals used to collect data for each treatm ent group listed. Statistical analysis of the data revealed that the effect of irradiation alone and the adm inistration of WR alone produced similar effects: 47 percent decline in cell num ber (P < 0.001). There is a dramatic decrease in the num ber of cells in the germinal layers of mice given the com bined treatm ents of 450 mg per kg of WR plus 10 Gy irradiation (P < 0.001). T he circum ference size of the sem iniferous tubules show ed a 14 p ercent decrease in the group treated with WR only (P < ). Discussion W R was stu d ied as an oral radioprotector because it was identified as one o f th e six m ost effective com pounds to em erge from the National Cancer Institute-sponsored radioprotector screening program conducted from 1980 to WR also show ed protective capability against neutron radiation effects in g a stro in te stin al tra c t6 and b o n e m arro w.7 S w een ey 8 id e n tifie d WR as the most effective orally a d m in is te re d rad io p ro te c to r. In the p resen t experim ents, oral adm inistration TABLE II Hlstometric Data of Mice In Each Treatment Group' of WR provided radioprotection resulting in increased 30 day survival of mice after whole-body irradiation. The pathology results indicated that there were definite changes in the testes of mice orally adm inistered 100 mg per kg of WR at eight days post exposure. In addition, 450 mg per kg elicited diffuse changes in other organs, such as the liver, lungs, pancreas, and kidney. M oreover, diarrhea observed in m ice orally adm inistered WR at 900 mg per kg indicated a significant pathophysiological response in the gastrointestinal tract. The results of this study are supported by the data obtained by van Beek et al9 w hen th ey a d m in istered WR ip to mice at a dose of 540 mg per kg. The organs in w hich they found changes were the testes, salivary glands, and pancreas. The present experim ents d id not id en tify any changes in the salivary glands and this m ay reflect differences due to the route of drug administration. Oral administration o f450 mg per kg of WR to m ice provided m odest radioprotection as evidenced by a dose reduction factor (DRF) of H ow ever, oral adm inistration of this concentration of drug produced significant testicular damage. Frequency of testicular damage increased with tim e following oral administration of 450 mg per kg of WR ; by 90 days, 100 percent of the mice showed damage to these organs. WR Irradiation WR Control 450 mg/kg b w 10 Gy + Irradiation Germinal cell layer counts 177± ± ± 4 35 ± 9 Circumference of 0.82 ± ± ± ±0.02 seminiferous tubule (mm) * Mean ±S.D.

7 188 STEEL-GOODWIN, KENDRICK, EGAN, AND ECKSTEIN Both figure 2A (control) and figure 2B (drug treated) pictorially dem onstrate the effects of this drug dose on the testes. Oral administration of 450 mg per kg of WR to mice one hour prior to 10 Gy whole body gamma-irradiation exposure did not seem to afford protection to the germ inal cell layer of th e testes observed eight days after treatm ent. Ten Gy whole body irradiation alone reduced the germinal cell num bers in the testes, and the combination of 450 mg per kg of WR plus 10 Gy irradiation dramatically reduced the num bers of germinal cells. Figure 2A (controls), figure 2C (irradiated), and figure 2D (WR irradiation) provide pictorial evidence of this testicular damage. A nother sulfhydryl containing com p o u n d, W R-2721 (S -2 -[3 -am in o p ro - pylam ino] ethylphosphorothioic acid), has been shown to be cytotoxic to mice testes stem spermatogonia following ip injection.3 A subsequent paper by the same authors noted sperm atogonial survival enhancem ent by WR-2721 w hen animals were exposed to local high and low doses of irradiation of the testes.4 However, the authors carefully pointed out that drug toxicity may have enhanced spermatogonial stem cell toxicity at low radiation doses. Intraperitoneal injection of 540 mg per kg of WR resulted in m ouse testicular damage.9 Results presented in this paper did not demonstrate enhancem ent of sperm atogonial stem cell survival when 450 mg per kg of WR was introduced prior to 10 Gy whole body e x p o su re to ra d ia tio n. T h e lack of enhanced sperm atogonial stem cell survival using WR prior to irradiation (10 Gy) m ay reflect differences betw een this compound and WR In addition, the experim ental protocols differed b ecau se WR-2721 was ad m in iste re d ip and in th e p r e s e n t stu d y WR was given orally. Also, mice in the present study received whole body irradiation by a 60Co gamma-ray source not local irradiation of the testes by 137Cs as previously d escribed.4 Since WR has been shown to be a testicular toxin when introduced orally or by intraperitoneal injection in mice, further exam ination of the other sulfhydryl containing WR-compounds would appear to be efficacious. Acknowledgments The authors would like to thank the following for their valuable assistance with this manuscript: Dr. A. J. Carmichael, Dr. B. H. Gray, Mr. T. S. Kimmel, Mr. J. Andrews, Mr. W. E. Jackson, Dr. D. E. McClain, Dr. K. A. Cole, Mr. E. J. Golightly, Dr. G. I. Reeves, and the Information Services and Veterinary Sciences Departments, Armed Forces Radiobiology Research Institute. References 1. Brown, D. Q., Graham, W. J., Mackenzie, J. W., PlTTOCK, J. W., and Shaw, L.: Can WR-2721 be improved upon? Pharmacol. Ther. 39: , F i n n e y, D. J.: Probit Analysis, 3rd ed. Boston, Cambridge University Press, 1971, pp Meistrich, M. L., F inch, M. V., Hunter, N., and MlLAS, L.: Cytotoxic effects of WR-2721 on mouse testicular cells. Int. J. Rad. Oncol. Biol. Phys. 20: , M e i s t r i c h, M. L., F i n c h, M. V., H u n t e r, N., and M i l a s, L.: Protection of spermatogonial survival and testicular function by WR-2721 against high and low doses of radiation. Int. J. Rad. Oncol. Biol. Phy. 10: , N la S, A. H. W.: Radiosensitizers and radioprotectors. In: An Introduction to Radiobiology. New York, Wiley and Sons, 1990, pp S i g d e s t a d, C. P., G r d i n a, D. J., C o n n o r, A. M., and H a n s o n, W. R.: A comparison of radioprotection from three neutron sources and 60C o b y WR2721 and WR Rad. Res. 106: , St e e l, L. K., Jacobs, A. J., Giambarresi, L. I., and JACKSON, W. E.: Protection of mice against fission neutron irradiation by WR-2721 or WR Rad. Res. 109: , S w e e n e y, T. R.: A survey of compounds from the antiradiation drug development program of the U.S. Army Medical Research and Development Command, Walter Reed Army Institute of Research, Washington, DC, v a n B e e k, A. H. W., D o a k, R. L., S i g d e s t a d, C. P., and G r d i n a, A. J.: Pathological effects of the radioprotector WR in mice. Rad. Res. 124:79-84, 1990.