an animal model to study toxicity of central nervous ... · (1 xrt, 1 rpm, 1 xrt-c, and 3 rpm-c)...

[CANCER RESEARCH 50, 6455-6460, October 15. 1990|

An Animal Model to Study Toxicity of Central Nervous System Therapy forChildhood Acute Lymphoblastic Leukemia: Effects on Growth andCraniofacial Proportion1

Ann Schunior, Anna E. Zengel, Phyllis J. Mullenix,2 Nancy J. Tarbell, Anthony Howes, and Melissa S. Tassinari

Department of Toxicology, Forsyth Research Institute, Boston, Massachusetts 02115 [A. S., A. E. Z., P. J. M.]; Department of Radiation Therapy, Harvard MedicalSchool, Boston, Massachusetts 02115 [N. J. T., A. H.J; and Department of Cell Biology, University of Massachusetts Medical Center, H'orcester, Massachusetts 01655

[M. S. T.I

ABSTRACT

Many long term survivors of childhood acute lymphoblastic leukemiahave short stature, as well as craniofacial and dental abnormalities, asside effects of central nervous system prophylactic therapy. An animalmodel is presented to assess these adverse effects on growth. Cranialirradiation (1000 cGy) with and without prednisolone (18 mg/kg i.p.) andmethotrexate (2 mg/kg i.p.) was administered to 17- and 18-day-oldSprague-Dawley male and female rats. Animals were weighed 3 times/week. Final body weight and body length were measured at 150 days ofage. Femur length and craniofacial dimensions were measured directlyfrom the bones, using calipers. For all exposed groups there was apermanent suppression of weight gain with no catch-up growth or normaladolescent growth spurt. Body length was reduced for all treated groups,as were the ratios of body weight to body length and cranial length tobody length. Animals subjected to cranial irradiation exhibited microcephaly, whereas those who received a combination of radiation andchemotherapy demonstrated altered craniofacial proportions in additionto microcephaly. Changes in growth patterns and skeletal proportionsexhibited sexually dimorphic characteristics. The results indicate thatcranial irradiation is a major factor in the growth failure in exposed rats,but chemotherapeutic agents contribute significantly to the outcome ofgrowth and craniofacial dimensions.

INTRODUCTION

Successful treatment of ALL3 in young children has resulted

in a population of patients with greatly increased life expectancy(1, 2). There are, however, side effects of the treatment whichdo not fully manifest themselves until many years after initialtherapy. These include persistent short stature (3-7), increasedweight for height (5, 7), altered cognitive development (7, 8),and craniofacial abnormalities including microcephaly (7), mid-facial hypoplasia (4), and mandibular retrognathia with arresteddental development (9).

Treatment protocols for ALL include an initial period ofsystemic chemotherapy, with or without radiotherapy and/orintrathecal chemotherapy as CNS prophylaxis against recurrence of the disease. This is followed by a course of maintenancechemotherapy lasting up to 3 years. Clinical studies have generally assumed that the CNS radiotherapy (given as cranial,cranial/spinal, or cranial/spinal/abdominal) was the most likelycause of statura! growth reduction (3, 5, 10,11). Chemotherapyhas been reported as having an effect on long term growth (5,6), possibly through synergism with cranial radiation (4).

Received 11/28/89; accepted 7/19/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1The authors are grateful to the Amoco Foundation and the Mobil Foundation

for partial support of this project.2To whom requests for reprints should be addressed, at Forsyth Research

Institute. 140 Fenway, Boston, MA 021 IS.3The abbreviations used are: ALL, acute lymphoblastic leukemia; CNS, central

nervous system; RPM, radiation, prednisolone. methotrexate; RPM-C, radiation,prednisolone, methotrexate control; XRT, cranial radiation; XRT-C. cranialradiation control.

To determine which variable of the combined treatmentmodality is responsible for abnormal growth and other negativeeffects, we developed a rat model that provides a detaileddescription of changes in growth and behavior after CNS prophylaxis. Because of their inclusion in most therapeutic protocols (1, 4, 6, 7), we chose to study the effects of cranialirradiation, prednisolone, and methotrexate. Each has beenimplicated as causing changes in behavior and/or growth (11-14). This paper reports abnormal growth patterns and alterations in the craniofacial proportion resulting from CNS prophylactic treatment. The behavioral effects in this model arereported in the accompanying paper (15).

MATERIALS AND METHODS

One hundred-sixty pathogen-free Sprague-Dawley male and femalerats with dams were obtained from the Charles River Laboratories(Kingston, RI) at 9 days of age (day of birth = 0). These animals werespecified nonlittermates and housed 12 pups/dam. Pups were assignedrandomly to one of two experimental groups or their matching controls.Each group contained 15-20 rats/sex. They were given standard PurinaRat Chow and tap water ad libitum, except during the short behavioralobservation periods. Light cycles were maintained as 12-h light (6:00a.m. to 6:00 p.m.)/dark periods. The four treatment groups were: RPM,pups received a combined therapy of radiation, prednisolone, andmethotrexate; RPM-C, the matched control group for RPM; XRT,pups received a single therapy of radiation only; and XRT-C, thematched control group for XRT.

On day 17 the rats in the RPM group were given i.p. injections of18 mg/kg body weight prednisolone sodium succinate (Sigma) in sterilesaline. RPM-C pups were given sham i.p. injections of an equal volume/body weight of sterile saline.

On day 18, 2-3 h prior to radiation exposure, the RPM pups wereagain given injections of prednisolone, as on day 17. The rats were thengiven i.p. injections of 2 mg/kg body weight methotrexate (+ amethop-terin; Sigma) in sterile saline. RPM-C animals received two sham salineinjections.

On day 18, radiation procedures were identical for the RPM andXRT groups. Rats were immobilized using pentobarbital anesthesia (25mg/kg, i.p.) and exposed six or seven at a time to radiation. Eachanimal was shielded with 2 mm lead placed over the body and face toexpose only the skull from behind the eyes to the mid-posterior neck.Care was taken to protect the eyes, oropharyngeal mucosa, and salivarytissues, so that the animal's ability to eat was not affected (16). Treat

ment was delivered by a lateral beam of X-rays produced by a PhillipsX-ray machine operating at 250 kVp with 0.35-mm copper filtration.The calculated mid-brain dose rate was 125 cGy/min. Each irradiatedbrain was given a total dose of 1000 cGy Â±5% in a single fraction over8 min. Control (RPM-C and XRT-C) animals were sham-irradiatedafter pentobarbital anesthesia by similar placement in the radiationbeam but with complete shielding by 2 mm lead. All pups receivedanesthesia and radiation procedures at approximately the same time(9:00 a.m. to noon) of day and then were returned to their dams.

The doses for each agent in both the XRT and RPM groups wereselected to be therapeutically relevant. The doses of prednisolone and

6455

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GROWTH EFFECTS OF CNS THERAPY FOR ALL

methotrexate avoided lethality and obvious neurotoxicity (14, 17). The18 mg/kg/day prednisolone dose, after applying a species conversionfactor of 4.1 (18), was approximately equal to 74 mg/nr/day Â¡nhumans,which is within the range used to treat ALL (19). The single wholebrain dose of 1000 cGy approximated a biological equivalent to theclinical dose of 2400 cGy given in 12-14 fractions. The equivalentsingle dose was calculated using the linear-quadratic model applied totolerance of central nervous tissue (20). The total effect (TE) of a courseof radiation is given by the formula TE = D(d + a/fi), where D = totalradiation dose; d = radiation dose for each fraction; and a/ÃŸ= aconstant specific for the tissue of interest. For CNS tissue, the a/ÃŸratiohas a value of approximately 3 (21).

On day 21 all pups were weaned and housed 2/cage/sex, and bodyweights were recorded 3 times weekly for each rat from 14 to 120 daysof age. At 150 days of age the rats were weighed and asphyxiated withCOj, and body lengths (tip of snout to base of tail) were measured. Thehead and left hind limb were removed, skinned, and placed in a beetlecolony (Dermestes vulpinas) to remove soft tissue. The defleshed boneswere then washed in 50% ethanol and allowed to air dry. Craniofacialmeasurements were performed on at least 10 rats/sex/treatment group(Fig. 1 and Table 1). The accuracy of individual measurements for eachskeletal measure was analyzed and percentage of error was at the 1%level. Data were statistically analyzed using unpaired i tests for theparameters of weight, body length, and cranial measures. Statisticalsignificance of cranial proportions was determined using the Mann-Whitney test at the 0.05 and 0.01 levels of confidence.

RESULTS

Mortality and Morbidity. The deaths observed in both groupsappeared to be unrelated to experimental treatment. Six deaths(1 XRT, 1 RPM, 1 XRT-C, and 3 RPM-C) occurred as a resultof anaesthetic overdose on day 18 during the radiation treatment. Additionally, 2 RPM rats and 1 RPM-C rat died 5 daysafter treatment, and 1 RPM-C rat died 13 days after treatment(31 days old). Two XRT rats were killed on day 50 because ofextreme weight loss, dehydration, and lethargy. No pathologywas observed in any of the animals. RPM treatment also causedtransient alopecia on the neck and shoulders of the rats, but atthe end of the experiment the fur had grown back on theanimals. One XRT rat had agenesis of both maxillary incisorsand was removed from the study.

Body Weight. Prior to treatment there was no significant

Table 1 Cranial measurements

Measurement Definition

con| max

ocw biz m'n

Fig. 1. Skull measurements. See Table 1 for abbreviations and definitions ofdimensions. OCW, occipital width.

Femur length

Cranial length (era)

Nasal length (nsl)

Frontal length (fro)

Facial length (fai)

Nasal height (nsh)

Calvarial length (cal)

Facial height (Tan)

Occipital height (och)

Bizygomatic width (biz)

Transnasal width (tnw)

Transfrontal maximumwidth (max)

Transfrontal minimumwidth (min)

Mandibular length (man)

Condylar width (con)

Distance from superior pole of greatertrochanter to inferior pole of lateral con-dyleDistance from anterior tip of nasal boneat midline to interparietal/supraoccipitalsuture at midlineDistance from anterior tip of nasal boneat midline to nasal/frontal sutureMidline distance from nasal/frontal suture to frontal/parietal sutureMidline distance from anterior tip of nasal bone to frontal/parietal sutureTransnasal distance (dorsal/ventral) ofsnout from incisive foramen in midline ofpalatine process of premaxilla to midlineof nasal bones when calipers are at aright angle to the long axis of skullMidline distance from nasal/frontal suture to interparietal/supraoccipital sutureTranscranial distance from midpoint ofmullinc of frontal bones and midpoint ofmidline of palate at the level of the second molar when the calipers are at aright angle to the long axis of the skullMidline distance from interparietal/supraoccipital suture to most ventral pointof foramen magnumMaximum span between lateral surfacesof right and left zygomatic processes oftemporal boneWidth of snout at most anterior junctionof nasal process of the premaxilla andzygomatic process of the maxilla on lateral surface of snoutDistance between lateral borders of frontal bones at right angle to the midline;tips of calipers approximate the lateralsurfaces at the superior aspect of thefrontal/temporal sutureNarrowest distance between lateral borders of frontal bones at right angle tomidline: tips of calipers approximate thelateral frontal surfaces just posterior tofacial surface of frontal bonesDistance from inferior junction of labialalveolar bone and incisor to posteriorprojection of angular process of mandibleTransverse distance between midpoints ofcondylar fossae

weight difference between the exposed groups and their controls. The growth trajectories for all treated animals had asimilar pattern. By 24 days of age there was a significantdifference between the RPM and XRT animals and their respective controls (Fig. 2 and Table 2). Neither group displayedevidence of catch-up growth and the initial suppression ofweight persisted until the termination of the experiment. At theonset of puberty (observed around day 42 in controls), neitherthe XRT- nor RPM-exposed animals exhibited the characteristic pattern of weight gain coincident with the adolescentgrowth spurt. This lack of a growth spurt was particularlydramatic in the males (Fig. 2, A and C). There was no differencein growth patterns between the XRT and RPM treatments (Fig.3A). On day 150 the XRT and RPM males had achieved 69.8%and 72.2%, respectively, of their matched control weights. TheRPM females had a greater suppression of growth immediatelyfollowing dosing than the XRT females (Fig. 3Ã„).The XRTand RPM females' postpubertal weight gain trajectories were

parallel to those of the control females (Fig. 2, B and D), butthe RPM females never completely recovered from the greaterinitial insult to their weight. On day 150 the XRT and RPMfemales had attained 89.5% and 79.6% of their respectivecontrols' weight.

Body Proportions. RPM and XRT exposures resulted in6456



E

500-

400

â€ž 300-

200-

100-

600

500

400-

â€ž 300-

200-

100


4001

a

20 40 60 80

Age (days)

Â« 200-.Cg>

100-

100 120

I

300

200

100-

20 40 60 80

Aga (day.)

B

100 120

20 40 60 80

Aga (days)

100 120 20 100 12040 60 80

Aga (daya)

Fig. 2. Patterns of weight gain after exposure to 1000 cGy radiation (XRT) or 1000 cGy radiation. 2 mg/kg methotrexate. and 18 mg/kg prednisolone (RPM) at17 and 18 days of age. A, XRT males; B, XRT females; C, RPM males; D. RPM females. O, controls; â€¢,exposed. Note the flattening of the growth trajectoriesduring puberty (days 40-60) for all exposed groups.

Table 2 Body si:e and proportionsRats were exposed to 1000 cGy radiation (XRT) or 1000 cGy radiation. 2 mg/kg methotrexate. and 18 mg/kg prednisolone (RPM) at 17 and 18 days of age.

Weights of animals in all treatment groups were significantly reduced by day 24 and remained below control weights through day 150. Adult XRT and RPM animalshad significantly reduced body length and femur length, but these were not proportional, indicating a change in body shape as well as body size.

RPMMaleWeight

(g)24 daysn150 days

nBody

length (cm)nFemur

length (cm)nBody

length/body weightnCranial

length/body lengthnControl52.8

Â±6.612

629.8 Â±77.01326

1.5 Â±6.4134.22

Â±0.09120.42Â±0.05

130.186Â±0.006

13Exposed44.3

Â±7.3Â°

19454.8 Â±73.2*

13235.1

Â±8.2'

113.83Â±0.10C

130.5

1Â±0.06*

110.

19 1Â±0.006''

11FemaleControl50.1

Â±7.120

313.9Â± 25.218226.3

Â±24.7183.51

Â±0.11180.71

Â±0.04180.204

Â±0.00517Exposed37.8

Â±6.6*

20249.9 Â±25.4*

19205.8

Â±8.1C

193.37

Â±0.08'

190.83Â±0.08r

190.206

Â±0.00719XRTMaleControl85.5

Â±7.026

612.8 Â±50.320266.9

Â±5.9194.18

Â±0.10190.44Â±0.03

190.

186 Â±0.00313Exposed7I.4Â±

8.3*

33427.6 Â±62.9*

17237.9

Â±8.8f

173.90Â±O.I4r

140.56Â±0.06f

170.

194 Â±0.004r

IIFemaleControl80.2

Â±8.225

331.5 + 38.819229.8

Â±5.0193.65

Â±0.07190.70Â±0.09

190.201

Â±0.00414Exposed68.6

Â±7.5*

29297.4 Â±18.1*

202

16.5 Â±4.7'

193.59

Â±0.07Â°

200.73Â±0.04

190.205Â±0.004"'

14Â°P < 0.01, compared to controls.* P < 0.001, compared to controls.' P< 0.0001. compared to controls.d P < 0.05. compared to controls.

smaller skeletal dimensions, compared to controls. Body lengthwas significantly reduced for all experimental animals (Table2). The proportion of body length to body weight was significantly different for the RPM males. RPM females, and XRTmales (Table 2). The proportional difference was greatest forthe XRT and RPM males, with approximately a 30% reduction

in weight and only a 10% reduction in length. The female RPManimals had a 20% reduction in weight and 9% reduction inlength. In contrast, the XRT females had only a 10% reductionin weight and a 6% reduction in length, a ratio which was notsignificantly different from their controls. While femur lengthsalso were reduced for all experimental groups (Table 2), the

6457




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Age (days)Fig. 3. Weight of animals expressed as a proportion of their control's weight.

A, males: B, females. Rats were exposed to 1000 cGy radiation (XRT) or 1000cGy radiation. 2 mg/kg methotrexate, and 18 mg/kg prednisolone (RPM) at 17and 18 days of age. O. XRT exposure;*. RPM exposure. Note the weight of themale rats is affected similarly by both RPM and XRT exposures. The RPMfemales had a greater suppression of weight after dosing.

experimental groups had proportionately longer femurs, compared to body length, except for the RPM males.

Craniofacial Proportions. The heads of the XRT animals weresignificantly smaller than XRT-C for all bony dimensions measured, except the transfrontal maximum and facial height in theXRT females.4 Proportionately, however, the XRT animals,

male and female, had longer cranial lengths relative to theirbody lengths, when compared to the control animals (Table 2),due to a greater reduction in total body length than the reduction in cranial length.

All cranial dimensions in the RPM animals were significantlyless than in the RPM-C animals, except for those of thetransfrontal area.4 Unlike the XRT animals, the female RPM

animals did not have longer cranial lengths for their bodylengths, when compared to controls (Table 2).

To assess changes in craniofacial proportion, all the individual bony dimensions were analyzed relative to cranial length togive a basis for comparison between control and exposed animals (Table 3). The ratios of facial length, calvarÃa!length,transnasal width, and condylar width to cranial length wereunchanged by either treatment. The RPM and XRT groupsshared the following changes in craniofacial proportions relative to cranial length: (a) reduction in occipital height, (b)reduction in bizygomatic width, and (c) for the females only,increased nasal length. Additional craniofacial pattern changesin the area of the midface were seen in the RPM group. Relativeto cranial length, the RPM animals had (a) increased width ofthe transfrontal area, (b) decreased facial height and frontallength in the males, and (c) increased nasal height in thefemales. All XRT animals had shortened mandibles and the

4 Data are not shown but will be provided upon request.

females had a decreased transfrontal minimum relative to cranial length. The RPM animals had normally proportionedmandibles and increased transfrontal area relative to craniallength.

DISCUSSION

In studies of immature animals, somatic reaction to earlystress may be seen either as an immediate interruption in growth(decrease in weight or body length), later as a temporary changein body proportion (head sparing or change in body length/weight ratio), or much later in life as a permanent deformity inbody size or shape or as an inability of the animal to functionnormally (behavioral abnormalities). Both the XRT and RPMtreatments produced permanent alterations in many of thesegrowth parameters. Both treatments reduced final body size asmeasured by weight and length. The XRT and RPM growthpatterns were similar, indicating that cranial irradiation was amajor factor in the inhibition of normal body growth in this ratmodel.

The addition of methotrexate and prednisolone to the irradiation caused a further inhibition of growth, which was mostapparent in the RPM females. These animals had the greatestsuppression of growth immediately after treatment and, unlikethe other groups, lacked head sparing. Head sparing, increasedcranial length in relation to body length, frequently is accompanied by an increase in the leg length to body length ratio anddemonstrates the biological priority given to growth of thecranium and long bones when young animals are stressed (22,23). The RPM males had marginal increases in cranial length/body length proportions. The XRT animals and only the RPMfemales maintained this long leg body proportion. The additionof chemotherapeutic components, therefore, further disruptednormal growth in these animals. Interestingly, head size wasnot an indicator of the behavioral alterations observed in theseanimals (15).

The XRT and RPM treatments clearly lowered growth velocities in the rats immediately after exposure and later duringpuberty. No exposed group exhibited catch-up growth. Generally, after growth suppression, an animal will experience aperiod of accelerated growth velocity in order to return to itsnormal size for age. This absence of catch-up growth is anindicator of severe toxicity leading to a loss of growth potential,resulting in permanent stunting. Additionally, the adolescentgrowth spurt was not observed in any of the treated animals. Inboth treatments male rats experienced a greater percentage ofweight loss than the females, which is consistent with theobservation that the young male animal is more vulnerable topermanent weight loss after early stress (24).

Other studies using animals treated with chemotherapeuticagents have shown prednisolone to delay growth in rats (14),and methotrexate has been shown to inhibit bone growth (25).The stunting effects of irradiation on growing animals havebeen well documented (26-29) and occur without significantchanges in pituitary hormone secretion (30, 31).

Children treated for ALL tend to have short stature (3-7) asa result of decreased growth, similar to what was observed inour rat model, with growth suppression both during treatment(32) and at puberty (32-34). Likewise, these children appearnot to experience catch-up growth (32). While growth velocitiesin these children return to normal, they lack the compensatoryresponse of accelerated growth.

Factors such as age at onset of treatment, course of treatment,6458




Table 3 Changes in craniofacial proportionsRats were exposed to 1000 cGy radiation (XRT) or 1000 cGy radiation. 2 mg/kg methotrexate. and 18 mg/kg prednisolone (RPM) at 17 and 18 days of age.

Abbreviations of cranial dimensions are listed in Table 1. Regardless of treatment, exposed animals had disproportionate reductions in occipital height and bizygomaticwidth, resulting in reduced cranial size.

RPM XRT

Male Female Male Female

Control Exposed Control Exposed Control Exposed Control Exposed

nNsl/craFro/eraMan/eraB12/craMax/eraMin/eraNsh/craFah/craOch/craMan/fal130.405Â±0.0160.323Â±0.0110.568

Â±0.0220.538Â±0.0180.229

Â±0.0130.132+0.0070.197+0.0070.270+0.0060.232Â±0.0090.781Â±0.023130.410

+0.0110.311Â±0.012"0.554

Â±0.0150.494+0.013Â°0.250

Â±0.011Â°0.137+0.0080.194+0.0040.262+0.012*0.220Â±0.008Â°0.768

Â±0.024180.3900.3190.5530.4990.2390.1370.1800.2590.2340.0080.0120.0150.0070.0120.0110.0080.0050.0080.7780.024190.402

Â±0.009Â°0.319

Â±0.0140.549+0.0130.484+0.015Â°0.256+0.009Â°0.147

Â±0.012*0.187Â±0.006Â°0.261

Â±0.0090.223+0.008Â°0.764+ 0.022140.404

Â±0.0090.318Â±0.0110.543

+0.0090.515Â±0.0110.240

+0.0130.127Â±0.0110.190Â±0.0040.264

Â±0.0090.249Â±0.0040.761+0.014110.411

Â±0.0060.319Â±0.0070..523

+0.013"0.497+0.015*0.244

Â±0.0090.125Â±0.0080.192

+0.0050.261Â±0.0080.236Â±0.003Â°0.733Â±0.024Â°140.3890.3250.5430.5060.2450.1360.1850.2600.2480.0140.0080.0090.0110.0120.0090.0050.0160.0040.7670.0120.40014+

0.009*0.320

Â±0.0100.5330.4950.2460.1280.1870.2600.2400.752Â±0.018*Â±0.012*+

0.007Â±0.012*Â±

0.005Â±0.005+0.006Â°+

0.031' P < 0.01, compared to controls.* P < 0.05, compared to controls.

and sex of the child contribute to the wide variations in responseto ALL treatment. Body proportion is affected and a stockybuild (a greater weight/height ratio) is frequently reported (5,7). Girls tend to be affected more than boys (7). Some ALLpatients, particularly girls, experience precocious puberty andgrowth hormone deficiency (33, 34). Other investigators havereported normal pubertÃ ! timing (32) with an overall decreasein height. Growth hormone deficiency can be a factor in theshort stature of ALL patients (4,34,35) but cannot fully explainthe short stature seen after treatment, since in many casesgrowth hormone secretion is normal (10). An early age at timeof treatment is a consistent factor in the severity of growthfailures (4, 10).

One outcome of pediatrie CNS therapy which includes cranialirradiation is microcephaly. In one study, head circumferencebelow the second percentile was found in 52% of the girls and42% of the boys (7). In this animal model, both the XRT andRPM animals had disproportionate reductions in occipitalheight and bizygomatic width, resulting in reduced cranial size.Microcephaly, therefore, occurs as a reproducible marker oftoxicity of CNS therapy. Other craniofacial and dental abnormalities have been reported but, because the children comprisea heterogenous population who were given a variety of treatments at differing ages, further generalization about their craniofacial alterations is difficult (4, 9, 36). In irradiated animals,significant changes in craniofacial proportions, including areasoutside the irradiated field, were found in the present study andhave been reported previously (37). Alterations in craniofacialproportions were specific to the treatment. Significant reductions in mandibular length were observed after XRT treatment.In contrast, the RPM treatment group produced altered midfacemorphology (increased transfrontal area, reduced facial height),an effect more evident in the female RPM rats.

Many of the changes in growth patterns after XRT and RPMtreatments were sex related. The most distinct sex-relatedchange was in the behavior of the RPM animals (15), butdifferences were also noted in body proportion and cranialdimensions. One reason for these differences may be that thediffering developmental timetables between male and femaleanimals influence the long term toxic effect of therapeuticagents. Growth retardation with midfacial alterations similarto those seen in the RPM group has been observed in ratsprenatally exposed to phenytoin (38). Phenytoin also eliminatedthe normal sexually dimorphic geometry of the face. Only slightsexually dimorphic effects were observed in the XRT- and

RPM-exposed rats, but the phenytoin model would suggest thattreatment affecting cranial growth may produce sexually dimorphic facial alterations, which should be explored more fullyboth in an animal model and in ALL children.

In conclusion, we have developed a model to study the effectsof CNS prophylactic treatments (cranial irradiation with orwithout chemotherapeutic agents) on growth and developmentusing the young growing rat. Results in the XRT animalsshowed that radiation was the major factor in the reduction inoverall growth. The addition of chemotherapeutic agents further decreased the growth rate and caused significant changesin craniofacial growth not seen with radiation alone. In bothtreatments, the sex of the rat appeared to modulate the response. Further studies with this model using methotrexatealone, prednisolone alone, and combinations of these individualagents with radiation, as well as other variables such as age attreatment, are necessary to understand the effects of CNSprophylaxis on long term growth.

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1990;50:6455-6460. Cancer Res Ann Schunior, Anna E. Zengel, Phyllis J. Mullenix, et al. on Growth and Craniofacial ProportionTherapy for Childhood Acute Lymphoblastic Leukemia: Effects An Animal Model to Study Toxicity of Central Nervous System

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