The alkaline phosphatase level in mice with experimentally induced osteosarcomas by Janice Marie Bailey A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Microbiology Montana State University © Copyright by Janice Marie Bailey (1971) Abstract: Experimentally induced osteosarcomas are detected roentgenographically in mice. In order to develop a more practical and earlier means of osteosarcoma detection the alkaline phosphatase level of CFl/Anl mice was studied. Plasma samples obtained from the retro-orbital sinus were assayed using the Bessey-Lowry-Brock procedure. Osteosarcomas were induced in 55 mice with FBJ virus and in 26 mice with strontium-90. In the FBJ virus study group weekly enzyme assays were begun on mice 4 weeks of age and followed over an 8 week period. No rise in alkaline phosphatase values occurred in mice with FBJ virus induced osteosarcomas. Enzyme assays of the strontium-90 test group were begun on mice near 3 months of age and were continued for 8 months. Twenty-one of the strontium-90 injected mice which developed osteosarcomas had significantly elevated alkaline phosphatase levels compared with normal controls. Elevation of values occurred as much as 2 to 8 weeks prior to roentgenographic tumor detection. Five other mice with strontium-90 induced osteosarcomas failed to show elevated alkaline phosphatase levels until 2 to 6 weeks after radiographic detection of tumors.  Statement of Permission to Copy- I n p r e s e n t in g t h i s t h e s i s i n p a r t i a l f u l f i l lm e n t o f th e r e q u i r e ­ m en ts f o r an advanced d e g re e a t M ontana S t a te U n iv e r s i ty , I a g re e t h a t th e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r i n s p e c t i o n . I f u r t h e r a g re e t h a t p e rm is s io n f o r e x te n s iv e co py in g o f t h i s t h e s i s f o r s c h o l a r l y p u rp o se s may b e g r a n te d b y my m a jo r p r o f e s s o r , , o r , i n h i s a b s e n c e , b y th e D i r e c to r o f L i b r a r i e s . I t i s u n d e rs to o d t h a t any co py in g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l , n o t be a llow e d w i th o u t my w r i t t e n p e rm is s io n . J1 /? ? /Date f> b \ oiai S \ THE ALKALINE PHOSPHATASE LEVEL IN MICE WITH EXPERIMENTALLY INDUCED OSTEOSARCOMAS by JANICE MARIE BAILEY A t h e s i s s u bm it te d t o th e G radu a te F a c u l ty i n p a r t i a l f u l f i l lm e n t o f th e r e q u i r em e n ts f o r th e d e g re e o f MASTER OF SCIENCE in M ic ro b io lo g y A pp roved : 'H ead, M a jo r D epa rtm en t C ha irm an , Exam in ing Comm ittee MONTANA STATE UNIVERSITY Bozeman, Montana December, 1971 ACKNOWLEDGMENT The a u th o r w ish e s t o e x p re s s h e r g r a t i t u d e and a p p r e c i a t i o n t o D r. A lv in G. F is c u s and D r. W illiam D. H i l l f o r t h e i r tim e and g u id ­ a n c e . A ls o , t h i s p r o j e c t c o u ld n o t h ave been c om p le te d w i th o u t th e c o o p e r a t io n and a s s i s t a n c e o f A rgonne N a t io n a l L a b o r a to ry . The a s s i s t a n c e o f D r. J . E . G a t l i n w i th x - r a y p ro c e d u re s and D r. S . R. Chapm an 's a d v ic e on s t a t i s t i c a l d a ta a n a ly s i s a r e a p p r e c i a t e d . The s u g g e s t io n s o f D r. F ran k S . Newman r e g a r d in g th e p r e p a r a t i o n o f t h i s m a n u s c r ip t a r e g r a t e f u l l y acknow ledged . T h is r e s e a r c h was s u p p o r te d by P u b l ic H e a lth S e r v ic e G ran t AHT-68 -03 l| and G ra n t 319, D ep a rtm en t o f B o tany and M ic ro b io lo g y , M ontana S t a te U n iv e r s i t y , Bozeman, M on tana . • TABLE OF CONTENTS Page VITA . ......................................................... i i ACKNOWLEDGMENT ............................................................................. i l l TABLE OF CONTENTS....................................... ................................................................... i v LIST OF TABLES .......................................................................................................... . v i i LIST OF FIGURES . . . . . . ....................................... . . . . . . . . . I x ABSTRACT . . . . . ....................................... . . . . . . . . . . . . . x INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . I REVIEW OF LITERATURE . ................................................. .... ...................................... 2 R a d io n u c lid e I n d u c t io n o f O s teo sa rcom as . . 2 FBJ V iru s I n d u c t io n o f O s teo sa rcom as ............................................ .... 5> The E f f e c t o f O s teo sa rcom as on A lk a l in e P h o sp h a ta se A c t i v i t y . . . . . ............................. . ...................................................... ? A d d i t io n a l F a c to r s W hich A f f e c t A lk a l in e P h o sp h a ta se A c t i v i t y ................................................................................................................... . 8 C h a r a c t e r i s t i c s o f A lk a l in e P h o sp h a ta s e . . 11 MATERIALS AND METHODS . ............................. .............................................................. ' 13 Sou rce and M a in te n an c e o f A n i m a l s ........................................................ 13 FBJ V iru s S tu d y G roups . . . . . . ................................................. . 13 U n in je c te d a n im a ls .............................................................................. 13 U n in je c te d a n im a ls w i th x - r a y ex po su re ................................. 13 i v VFBJ v i r u s i n j e c t e d a n im a ls ........................................................... , 13 S tro n t iu m -9 0 S tu d y G r o u p s ...................................... I i i B n in je c te d a n im a ls .w ith m in im a l x - r a y e x p o su re . . . . I l i U n in je c te d a n im a ls w i th m axim al x - r a y e x p o su re . . . . Ili S tro n t iu m -9 0 i n j e c t e d a n i m a l s ........................................................... i l l S ou rce and P r e p a r a t i o n o f FBJ V iru s ..................................................... I l i X-Ray P r o c e d u r e ..................................................................................................... Ij? A lk a l in e P h o sp h a ta s e A n a ly s is . ................................................ 16 P r e p a r a t i o n o f r e a g e n ts and s t a n d a r d i z a t i o n o f t e s t . . 16 P r i n c i p l e and p e rfo rm an c e o f t e s t ..................................................I ? Isoenzym e d i f f e r e n t s t i o n .................................................................... Io R E SU L T S ..................................................... 19 A lk a l in e P h o sp h a ta s e L e v e ls i n U n in je c te d G roups . . . . . 19 FBJ V iru s Base L in e G r o u p ................................................................................... 19 FBJ V iru s I n j e c t e d Group .............................................................................. 2U Mean AU caline P h o sp h a ta s e L e v e ls R e l a t i v e t o Time f o r FBJ V iru s I n j e c t e d and U n in je c te d M i c e ...........................................................31 S tro n t iu m -9 0 Base L in e G r o u p ......................................................................... 31 S tro n t iu m -9 0 I n j e c t e d Group . .................................................................... 31 H ea t L a b i l i t y o f P lasm a A lk a l in e P h o sp h a ta s e .............................. 37 Mean A lk a l in e P h o sp h a ta s e L e v e ls R e la t i v e t o Time f o r S tro n t iu m -9 0 I n j e c t e d and U n in je c te d M ice ....................................... 37 Page DISCUSSION . . .............. . . . . . . . . . . . . . . . . . h2 V a r ia n c e i n A lk a l in e P h o sp h a ta s e V a lu e s Due t o Age . . . . h2 E f f e c t o f X -Hej E xposu re on A lk a l in e P h o sp h a ta se L e v e ls « . 1$2 FBJ V iru s I n j e c t e d A nim als .......................................................................... 1)3 S tro n t ium -9 0 I n j e c t e d A nim als . . . . . . . . . . . . . . . L3 C on c lu s io n - ........................ 1)5 SUMMARY ....................................................................................... 1)6 REFERENCES CITED.............................. lj.8 v i Page v i i LIST OF TABLES Page T ab le I W eekly a l k a l i n e p h o s p h a ta s e l e v e l s in IU o f u n in je c te d m ice b e g in n in g a t I* weeks o f age . . . . . 2 0 T ab le I I W eekly a l k a l i n e p h o s p h a ta s e l e v e l s i n IU o f un ­ i n j e c t e d m ice w ith x - r a y e x p o s u re . b e g in n in g a t U weeks o f age . .................................................................... .... . . 22 T ab le I I I P a t t e r n o f mean a l k a l i n e p h o sp h a ta s e v a r i a n c e i n 8 u n in j e c t e d m ice b e g in n in g a t U w eeks o f age w i th no x - r a y e x p o su re ..................................................... T ab le IV A com pa riso n o f v a r i a n c e o f mean a l k a l i n e p h o s p h a ta s e l e v e l s be tw een x - ra y e d and non x - r a y e d a n im a ls o f u n i n j e c t e d g ro up s b e g in - • n in g a t U weeks o f a g e ........................ ................................. .... . ?? T ab le V C a lc u la te d nt n v a lu e s f o r mean a l k a l i n e p h o sp h a ­ t a s e l e v e l s o f x - ra y e d and non x - r a y e d a n im a ls o f u n in j e c t e d g roup b e g in n in g a t h w eeks o f a g e . . ............................. ................................................................... .... . 26 T ab le VI W eekly a l k a l i n e p h o s p h a ta s e l e v e l s i n IU o f FBJ v i r u s i n j e c t e d m ice b e g in n in g a t it w eeks o f a g e . . . 27 T ab le V II p Mean, Sj?, and " t " v a lu e s c a l c u l a t e d f o r a l k a l i n e p h o s p h a ta s e l e v e l s o f non x - r a y e d a n im a ls o f u n i n j e c t e d g roup and FBJ v i r u s i n j e c t e d g roup b e g in n in g a t I* weeks o f a g e . ................................................. T ab le V I I I A lk a l in e p h o sp h a ta s e l e v e l s i n IU o f m ice b e g in ­ n in g a t mean age o f 97 days w i th m in im al x - r a y e x p o s u r e ......................................................................... .... . . . . CMC"\ T ab le IX A lk a l in e p h o s p h a ta s e l e v e l s i n IU o f m ice b e g in ­ n in g a t mean age o f 97 d ay s w ith m axim al x - r a y e x p o su re ................................................. .... ................................. viii T ab le X C a lc u la te d " t " v a lu e s f o r mean a l k a l i n e p h o s ­ p h a ta s e l e v e l s o f m ice w i th m in im a l and m aximal x - r a y e x p o su re b e g in n in g a t 97 days o f a g e ............................. ............................................................................ 314- T ab le XI A lk a l in e p h o sp h a ta s e l e v e l s i n IU o f m ice w h ich r e c e iv e d s t r o n t iu m -90 b e g in n in g a t mean age o f 97 d a y s ..................................................... ............................ .... 35 T ab le X I I Sample m ean, s |, and ’’t " v a lu e s c a l c u l a t e d from mean a l k a l i n e p h o s p h a ta s e l e v e l s o f s t r o n t iu m -90 i n j e c t e d g ro up s and combined c o n t r o l g ro u p s b e g in n in g a t 97 day s o f age .................................. .... 38 T ab le X I I I C a lc u la te d H n v a lu e s - f o r mean a l k a l i n e p h o s ­ p h a ta s e l e v e l s o f s t r o n t iu m -90 i n j e c t e d g roup and n o rm a l g roup from 2 t o 8 weeks b e fo r e ro e n tg e n o g ra p h ic tum or d e t e c t i o n . . . . . . . . . . 39 T ab le XIV A com p a r iso n o f p la sm a a l k a l i n e p h o sp h a ta s e l e v e l s i n IU b e fo r e and a f t e r a 16 m in u te e x p o su re o f 55° C ....................................... .... . .........................1*0 Page Ix LIST OF FIGURES Page F ig u re I Mean a l k a l i n e p h o sp h a ta s e l e v e l s v e r s u s tim e o f FBJ V iru s g ro u p s . . . . . . ............................................ 30 F ig u r e 2 Mean a l k a l i n e p h o s p h a ta s e l e v e l s v e r s u s t im e o f s t r o n t iu m -90 g r o u p s ............................. ................................. I i i ABSTRACT E x p e r im e n ta l ly in d u ced o s te o s a rc om a s a r e d e te c te d ro e n tg e n o g ra p h - i c a l l y i n m ic e . I n o r d e r t o d e v e lo p a more p r a c t i c a l and e a r l i e r means o f o s te o sa rc om a d e t e c t i o n th e a l k a l i n e p h o sp h a ta s e l e v e l o f C F l/A n l m ice was s t u d i e d . P lasm a sam p les o b ta in e d from th e r e t r o - o r b i t a l s in u s w ere a s s a y e d u s in g th e B essey -L ow ry -B rock p ro c e d u r e . O steo sa rcom as w ere in d u ced i n 55 m ice w i th FBJ v i r u s and i n 2 6 -m ice w i th s t r o n t iu m -9 0 . I n th e FBJ v i r u s s tu d y g roup w eek ly enzyme a s s a y s w ere begun on m ice I4 w eeks o f a g e and f o l lo w e d o v e r an 8 week p e r i o d . No r i s e i n a l k a l i n e p h o s p h a ta s e v a lu e s o c c u r r e d i n m ice w i th FBJ v i r u s in d u c ed o s t e o ­ s a rc om as . Enzyme a s s a y s o f th e s t r o n t iu m -9 0 t e s t g roup w ere begun on m ice n e a r 3 months o f ag e and w ere c o n t in u e d f o r 8 m on th s . Tw enty-one o f th e s t r o n t iu m -90 i n j e c t e d m ice w h ich d ev e lo p ed o s te o s a rc om a s had s i g n i f i c a n t l y e l e v a te d a l k a l i n e p h o s p h a ta s e l e v e l s compared w i th no rm a l c o n t r o l s . E l e v a t io n o f v a lu e s o c c u r r e d a s much a s 2 t o 8 w eeks p r i o r t o ro e n tg e n o g ra p h ic tum or d e t e c t i o n . F iv e o th e r m ice w i th s tro n t iu m -9 0 ■ in d u c ed o s te o s a rc om a s f a i l e d to show e l e v a te d a l k a l i n e p h o sp h a ta s e l e v e l s u n t i l 2 t o 6 w eeks a f t e r r a d io g r a p h ic d e t e c t i o n o f tu m o rs . ' INTRODUCTION The o n ly c h em ic a l a n a l y s i s o f b lo o d p e r t i n e n t t o o s te o sa rc om a s i s th e serum a l k a l i n e p h o s p h a ta s e l e v e l ( J a f f e , 1 9 5 9 ) . V a lu e s from 2 t o HO t im e s th e u p p e r no rm a l l i m i t have b e en r e p o r te d i n humans w ith o s te o g e n ic sa rcom as (S c hw a r tz , e t a l . , 1969) . In m ice a s t r o n g h i s t o - c h em ic a l a c t i v i t y o f a l k a l i n e p h o sp h a ta s e h a s b een n o te d w i th o s t e o ­ s a rc om a s , a l th o u g h b lo o d l e v e l s o f a l k a l i n e p h o sp h a ta s e have n o t b e en c o r r e l a t e d w i th th e p r e s e n c e o f o s te o s a rc om a s (T immer, e t a l . , 1968) . A com pa riso n o f p lasm a a l k a l i n e p h o s p h a ta s e l e v e l s and o s t e o s a r ­ coma d ev e lo pm en t h a s b e e n made i n t h i s s tu d y t o p ro v id e a more p r a c t i c a l and e a r l i e r m eans o f tum or d e t e c t i o n i n e x p e r im e n ta l m ice t h a n . i s p o s s i b l e by th e u s e o f r o e n tg e n o g ra p h s . I n te rm s o f expense a l k a l i n e p h o s p h a ta s e t e s t s a r e a d v a n ta g e o u s , f o r t h e y a r e o n e - t h i r d th e c o s t o f x - r a y s . F o r ex am p le , m a t e r i a l s f o r 5(X) a l k a l i n e p ho spha ­ t a s e a s s a y s c o s t a p p ro x im a te ly $23 compared w i th $69 f o r 500 x - r a y s . O s teo sa rcom as i n C F l/A n l m ice w ere in d u ced w i th FBJ v i r u s o r s t r o n t iu m -9 0 . A lk a l in e p h o s p h a ta s e l e v e l s were d e te rm in e d u s in g th e B essey -L ow ry -B rock m ethod . Tumors w e re d e te c te d r a d i o g r a p h i c a l l y and t h e i r a p p e a ra n c e was c o r r e l a t e d w i th a l k a l i n e p h o s p h a ta s e l e v e l s . U n in j e c t e d a n im a ls w ere t e s t e d c o n c u r r e n t l y to e s t a b l i s h a b a s e l i n e o f no rm a l v a lu e s . 2REVIEW OF LITERATURE N e o p la s t i c p r o c e s s e s a r e fo und i n a l l v e r t e b r a t e s ( S ch lum berger and Luckfe, 1 9 b 8 ) . The m ost common p r im a ry m a l ig n a n t n eop la sm o f bone i s th e o s te o s a rc om a , w h ich i s f a t a l i n m ost i n s t a n c e s , i r r e s p e c t i v e o f t r e a tm e n t (B e n n e t t , 1 9 6 1 ) . O s te o g en ic sa rcom as a r e h i g h ly m a l ig n a n t tum o rs o f o s t e o b l a s t c e l l s and u s u a l l y a r i s e i n th e p e r io s te u m (M ou lto n , 1961 ; R e i f e n s t e i n , 1 9 6 2 ) . Most tum ors o r i g i n a t e from o s s e o u s t i s s u e d i r e c t l y , a l th o u g h some may b eg in i n s o f t t i s s u e by m e ta p la s t i c d i f f e r e n t a t i o n o f m esenchym al t i s s u e t o p ro d u ce an o s t e o i d (R obb in s , 19610 . An o s te o sa rc om a u s u a l l y fo rm s a l a r g e w h ite f i b r o u s mass w ith v a r i a b l e a r e a s r e s em b lin g c a r t i l a g e o r bone (G o tc h in , 1 9 5 7 ) . The n e o ­ p lasm s ' a r e ro u n d , o v o id , o r s p in d le - s h a p e d and have i n t im a te a s s o c i ­ a t i o n w i th th e b o n e s from w h ich th e y a r i s e . From o n e - f o u r th to o n e - h a l f o f th e a f f e c t e d bone may be r e p l a c e d by n e o p l a s t i c t i s s u e (M ou lton , 1 9 6 1 ) . RADIONUCLIDE INDUCTION OF OSTEOSARCOMAS R a d io a c t iv e s u b s ta n c e s w i th m e ta b o lic a c t i v i t y s im i l a r t o c a lc ium a r e e x trem e ly e f f e c t i v e i n p ro d u c in g bone tum ors (B ru e s , 1 956 j B i s k is and F in k e I , 1 9 7 0 ) . One o f th e f i r s t e lem en ts t o be im p l ic a te d i n th e p r o d u c t io n o f bone tum ors was rad ium (B e n n e t t , 1 9 6 1 ) . S t r o n t iu m -90 , 3a n o th e r b o n e -s e e k in g r a d i o n u c l i d e , h a s come i n t o p rom in en ce due t o i t s p r e s e n c e i n r a d i o a c t i v e f a l l o u t (McLean, I 96J4) . C a r c in o g e n ic i ty o f r a d io n u c l i d e s i s r e l a t e d t o t h e i r p h y s i c a l and c h em ic a l p r o p e r t i e s ( F in k e l , e t a l . , 1 9 6 b ) . The em is s io n o f h ig h - e n e rg y b e ta p a r t i c l e s and a lo n g h a l f - l i f e o f 2?.-7 y e a r s a r e two f e a t u r e s w h ich make s t r o n t iu m -90 a n e x c e l l e n t c a rc in o g e n (McLean, 19610 » The s o l u b i l i t y o f s t r o n t iu m en h an ce s i t s a b s o r p t io n from th e g a s t r o ­ i n t e s t i n a l t r a c t and e n t r y i n t o t h e b lo od s tre am ( P e t r o v , e t a l . , 1 9 6 6 ) . S tro n t iu m l o c a t e s in a r e a s w here bone i s b e in g fo rm ed o r r e ­ c o n s t r u c te d (McLean, I 96U ). The in c id e n c e o f bone tum ors c a u se d by r a d io s t r o n t iu m can a p p ro a c h 100% ( I t o , e t a l . , 1969) . C h ro n ic o r a l e x p o su re t o s t r o n t iu m -9 0 g iv e s a more u n ifo rm d i s t r i ­ b u t io n o f r a d i a t i o n i n th e bone th a n a s i n g l e dose (Howard, e t a l . , 1 969 ; McLean, I 96I1) . D e p o s i t io n o f s t r o n t iu m o c c u rs m a in ly i n th e m in e r a l p h a se o f bone th ro u g h d i f f u s i o n e x ch an g e , r e c r y s t a l l i z a t i o n , and new c r y s t a l f o rm a tio n (B ru e s , 1 9 5 6 ) . R e s u l ts from e x p e r im en ts u s in g in t r a v e n o u s i n j e c t i o n s o f s t r o n t iu m -90 show t h a t a s in g l e dose i s more c a r c in o g e n ic th a n f r a c t i o n a t e d am oun ts o f th e same dose g iv e n o v e r a number o f day s ( F in k e l , e t a l . , 196b ) . / s in g l e i n t r a v e n o u s i n j e c ­ t i o n y i e l d s an uneven s k e l e t a l d e p o s i t io n and may l e a d t o tum ors i n one o r more b o n e s . Hot s p o ts i n th e bone r e s u l t i n f o c i o f s e v e re n e c r o s i s w h i le o t h e r r e g io n s o f th e same bone a r e r e l a t i v e l y u n a f f e c t e d . hS u b l e th a l am ounts o f i r r a d i a t i o n a r e n e c e s s a r y f o r t h e inducem en t o f n e o p l a s t i c c h a n g e s , s in c e a n im a ls d ie b e fo r e tum or a p p e a ra n c e w i th h ig h d o sag e s (B ru e s , 1 9 5 6 ) . A l a t e n c y tim e h a s b een n o te d b e tw een th e a d m in i s t r a t i o n o f a r a d i o a c t i v e m a t e r i a l and th e a p p e a ra n c e o f tum o rs , E x p e r im en ta l e v id e n c e i n d i c a t e s t h a t tum or l a t e n c y t im e i s t h e same f o r a l l a g e s o f m ice r e c e iv in g s t r o n t iu m -9 0 i n j e c t i o n s . The e a r l i e s t r a d io g r a p h ic i d e n t i f i c a t i o n o f tum ors was 98 days a f t e r th e optimum c a rc in o g e n ic amount o f s t r o n t iu m -9 0 h ad b een i n j e c t e d ( F in k e l , e t a l . , 1 9 6 6 a ) . W ith in th e s u b l e t h a l r a n g e th e i n c id e n c e o f sarcom a f o rm a t io n i n c r e a s e s w i th th e am ount o f r a d i o a c t i v e m a t e r i a l a d m in is t e r e d . There i s u s u a l l y an a b ru p t r i s e i n th e in c id e n c e o f bone sarcom as among a n im a ls s u r v iv ­ in g l o n g e s t w i th r e l a t i v e l y h ig h d o se s ( J a f f e , 1959.) • The in c id e n c e o f bone tum o rs i n m ice i s r e d u c e d a s s m a l l e r d o ses o f s t r o n t iu m a r e adm in­ i s t e r e d (B ru e s , 1 9 5 6 ) . T h e re a p p e a r s t o be a t h r e s h o ld below w h ich sa rcom as a r e n o t in d u ced s in c e no m a l ig n a n t bone tum o rs have been p ro d u ced b y a do se o f 8 .9 m ic ro C u r ie s (p C i) /k g body w e ig h t ( F in k e l , e t a l . , 1 9 5 9 )• An in t r a v e n o u s i n j e c t i o n o f 1 .0 p C i /g body w e ig h t a p p e a r s to be an optimum dose o f s t r o n t iu m -90 f o r tum o r i n d u c t io n ( F in k e l , e t A l . , 1966a ) . T y p ic a l r a d i a t i o n - i n d u c e d o s te o s a rc om a s u s u a l l y a p p e a r f i r s t a s a r e a s o f i n c r e a s e d o r d e c re a s e d d e n s i t y w i t h in bone i n th e m e ta p h y se a l 5o r e p ip h y s e a l r e g io n . The tum or b r e a k s th ro u g h th e bone c o r t e x and s p re a d s b y d i r e c t e x te n s io n and in v a s io n o f a d ja c e n t t i s s u e (Finkel, e t a l . , 1 9 6 6 a ; F in k e l and B i s k i s , I 969) . R a d io g r a p h ic a l ly th e l e s i o n s a r e q u i t e o s t e o l y t i c , r e s u l t i n g i n e x te n s iv e a r e a s o f bone d e s t r u c t i o n (Howard, e t a l . , 1969) . S t r e a k s , p a tc h e s , o r ev en l a r g e b lo tc h y a r e a s o f r a d i o p a c i t y r e f l e c t t h e p r e s e n c e o f bone n e c r o s i s and i t s c o n se ­ q u e n c e s . S c a t t e r e d a r e a s o f r a d io lu c e n c y a r e a l s o p r e s e n t ( J a f f e , 1 959 ; D un lap , 1966) . It h a s b een s u g g e s te d t h a t r a d io s t r o n t iu m u s u a l l y in d u c e s o s te o ­ g e n ic sa rcom as by i n a c t i v a t i n g a v i r a l i n h i b i t o r ( F in k e l and B i s k i s , 1969) . T he re i s e v id e n c e t h a t a v i r u s may r e s i d e i n t h e mouse a t th e t im e o f i r r a d i a t i o n (H uebner and T od a ro , 1969) . G e rm -fre e m ice a r e r e n d e r e d le u k em ic by i r r a d i a t i o n i n th e ab sen ce o f c o n tam in a t io n b y m ic ro o rg an ism s from th e e n v iro nm en t. T is s u e s o f g e rm - f r e e m ice w i th x - r a y in d u ced leu k em ia a s w e l l a s n o rm a l g e rm -fre e m ice c o n ta in leu k em ia v i r u s - l i k e p a r t i c l e s (K ajim a and P o l l a r d , 1 9 6 5 ) . All s t r o n ­ t ium -90 in d u ced tum o rs exam ined in t h i n s e c t i o n have c o n ta in e d v i r u s ­ l i k e p a r t i c l e s ( F in k e l and B i s k i s , 1969) . FBJ VIRUS INDUCTION OF OSTEOSARCOMAS In 1966 a unique viral agent was isolated from a spontaneous osteosarcoma of a CFl/Anl mouse by Finkel, Biskis, and Jinkins. Tumor extracts injected into newborn mice caused the development of 6o s te o s a rc om a s . T h is a g e n t h a s b een named th e FBJ v i r u s . I t d i f f e r s from o th e r o n co g en ic v i r u s e s i n t h a t i t p ro d u ce s o n ly o s te o g e n ic sa rcom as ( F in k e l , e t a l . , 1966b ) . I t i s a Type C ENA v i r u s w i th a • c om p lem en t- f ix in g a n t ig e n c h a r a c t e r i s t i c o f th e m u rin e le u k em ia - sa rcom a v i r u s e s ( F in k e l , e t a l . , 1 9 7 0 ) . E l e c t r o n m ic ro sc o p y demon­ s t r a t e s Type C v i r a l p a r t i c l e s b o th i n c e l l - f r e e e x t r a c t s and i n u l t r a t h i n s e c t i o n s o f o s te o s a rc om a s in d u ced by th e FBJ v i r u s ( F in k e l , e t a l . , 1 9 6 6 a ) . Newborn m ice a r e more s u s c e p t i b l e t o FBJ v i r u s - in d u c e d tum o rs th a n o ld e r a n im a ls (G ro s s , 1 9 7 0 ) . The a p p e a ran ce o f an o s teo sa rcom a i n one mouse i s u s u a l l y f o l lo w e d s h o r t l y by th e a p p e a ra n c e o f tum o rs among o th e r members o f th e i n j e c t e d l i t t e r . However, a n im a ls in th e same l i t t e r may n o t be e q u a l ly s e n s i t i v e t o th e v i r u s ( F in k e l , e t a l . , 1 9 6 6 b ) . I n 1969 K e l lo f f and h i s cow o rk e rs found t h a t CFW and NIH Sw iss m ice w ere h ig h ly s u s c e p t i b l e (kO~hS%), and C^yBl was w eak ly s u s c e p t i b l e (10%) t o th e FBJ v i r u s . No tum ors w ere in d u ced i n n ew ­ b o rn NIH h am s te rs o r newborn F i s h e r r a t s (K e l l o f f , e t a l . , 1 9 6 9 ) . The r i b s w ere t h e m ost f r e q u e n t s i t e o f tum o r f o rm a t io n w ith FBJ v i r u s - in d u c e d o s te o s a rc om a s ( F in k e l , e t a l . , 1 9 66 a ). Tumors a p p e a red s u b c u ta n e o u s ly a t o r n e a r th e s i t e o f i n o c u l a t i o n ( K e l l o f f , e t a l , 1 9 6 9 ) . The t i b i a had tum o r g row th p r im a r i l y i n th e p e r io s te u m and th e marrow c a v i t y was i n f r e q u e n t l y in v o lv e d , and th e n o n ly s l i g h t l y 7( F i n k e l j . e t a l , , 1 9 6 6 a ) . O s teo sa rcom as may o c c u r anyw here a lo n g th e b o n e , f i r s t a s c o r t i c a l th ic k e n in g and a s sm a ll a r e a s o f in c r e a s e d d e n s i t y i n s o f t t i s s u e s a d j a c e n t to th e bone ( F in k e l , e t a l . , 1966b), The p r i n c i p l e g row th i s p e r i o s t e a l , and a s th e tum o r e n l a r g e s , i t a p p e a r s t o evoke p e r i o s t e a l g row th i n n e a rb y bone ( F in k e l , e t a l . , 1 9 6 6 a ) . Growth p ro c e e d s p e r i p h e r a l l y w i th d e la y ed in v o lv em en t o f d eep c o r t i c a l bone ( F in k e l , e t a l . , 1 9 6 6 b ) . I n d u c t io n tim e f o r tum or a p p e a ra n c e a f t e r v i r u s i n j e c t i o n d e c re a s e d from 280 day s i n th e f i r s t p a s s a g e o f th e v i r u s t o 21 days a f t e r s u b s e q u e n t p a s s a g e s ( F in k e l , e t a l . , 1 9 6 6 a ) . The r o e n tg e n o g ra p h ic a p p e a ra n c e o f v i r u s - in d u c e d tum o rs i s some­ w hat d i f f e r e n t from o s te o sa rc om a s in d u ced w i th s t r o n t iu m -9 0 . In con ­ t r a s t to r a d io n u c l id e - in d u c e d l e s i o n s , FBJ v i r u s tum o rs f i r s t a p p e a r a s a r e a s o f p e r i o s t e a l g row th o r a s r e g io n s o f bone fo rm a tio n i n s o f t t i s s u e a d j a c e n t t o th e b o n e . They may o c cu r anyw here a lo n g th e bone s u r f a c e s . R ad io g rap h s may show a s u n b u r s t p a t t e r n w i th o ld e r l e s i o n s due t o an i n v a s io n o f s o f t t i s s u e a d j a c e n t t o th e bone and s u b s e q u e n t c a l c i f i c a t i o n o f th e s u r ro u n d in g t i s s u e . S u c c e s s iv e p a s s a g e s o f th e v i r u s have n o t changed t h e r a d io g r a p h ic a p p e a ra n c e o f tum ors ( F in k e l , e t a l . , 1 9 6 6 b ) . THE EFFECT OF OSTEOSARCOMAS ON ALKALINE PHOSPHATASE ACTIVITY H is to c h em ic a l a n a l y s i s o f o s te o s a rc om a s r e g u l a r l y shows a h ig h 8s c o n c e n t r a t io n o f a l k a l i n e p h o s p h a ta s e , w i th th e enzyme a c t i v i t y b e in g g r e a t e r in th e more r a p id ly g row ing p o r t i o n s o f th e tum o r . The c o n c e n t r a t i o n o f a l k a l i n e p h o sp h a ta s e i s g r e a t e s t i n t h e v i c i n i t y o f th e b lo o d v e s s e l s . The b lo o d l e v e l o f a l k a l i n e p h o s p h a ta s e p a r a l l e l s t h e c o u rs e o f th e tum o r, b e in g red u c ed by tumor; e x c i s i o n and i n ­ c r e a s e d b y e x te n s io n o r m e t a s t a s i s t o o t h e r t i s s u e s ( R e i f e n s t e i n , 1962) . ■ ADDITIONAL FACTORS WHICH AFFECT ALKALINE PHOSPHATASE ACTIVITY Bone a l k a l i n e p h o s p h a ta s e , f i r s t d is c o v e re d i n 1923 by R ob iso n , i s h i g h ly c o n c e n t r a te d in th e o s t e o b l a s t s w i th m icrosom e f r a c t i o n s y i e l d in g th e g r e a t e s t am ount o f th e enzyme (Vaes and J a c q u e s , 1965). The e l e v a t i o n o f a l k a l i n e p h o sp h a ta s e i s a s s o c i a t e d w i th a number o f bone d i s e a s e s o t h e r th a n o s te o s a rc om a . Such c o n d i t io n s may i n c l u d e ; r i c k e t s , P a g e t 's d i s e a s e , p r im a ry h y p e rp a r a th y ro id i sm , o s te o m a la s ia , Von R e c k l in g h a u s e n 's d i s e a s e w ith bone in v o lv em en t , o s t e i t i s d e fo rm ans j u v e n i l i a due t o v i tam in D d e f i c i e n c y , m a la b s o rp t io n o f c a lc iu m , o r r e n a l t u b u l a r d y s t r o p h ie s (Damm, 1 965 , D av id sohn and V ie l l s , 1963 j Kay, 1 9 3 0 ) . Under no rm a l p h y s io lo g ic c o n d i t io n s th e b lood l e v e l o f bone a l k a l i n e p h o sp h a ta s e may f l u c t u a t e ( F e n n e l ly , e t a l . , 1 9 6 9 ) . Kuan and h i s a s s o c i a t e s (1966) n o te d a change i n th e a l k a l i n e p h o sp h a ta s e l e v e l o f d e v e lo p in g c h ic k s . O th e rs have a l s o n o te d a v a r i a t i o n o f 9a lk a l in e .p h o s p h a ta s e l e v e l s i n humans o f d i f f e r e n t a g e s . I n n e o n a te s and c h i l d r e n , n o rm a l v a lu e s r a n g e from u p p e r a d u l t l e v e l s t o a p p r o x i ­ m a te ly two t im e s th e u p p e r n o rm a l l e v e l s o f a d u l t s . I n th e 3 t o 10 y e a r o ld g ro u p , a. s te a d y d e c l in e o c c u r s . W ith t h e o n s e t o f p u b e r t y , v a lu e s b e g in t o r i s e a g a in , w i th a r e t u r n to no rm a l a d u l t l e v e l s a f t e r p u b e r ty (D av id sohn and W e l l s , 1963) . E x p e r im en ta l e v id e n c e i n d i c a t e s t h a t t h e s e p h y s io l o g i c a l v a r i a t i o n s a r e due t o th e o s t e o b l a s t i c a c t i v i t y o f b o n e s (E is e n b e rg , 1 9 7 0 ) . G e n e t i c a l l y endowed v a r i a t i o n s may a l s o o c c u r . R a ts have a h ig h a l k a l i n e p h o s p h a ta s e a c t i v i t y com­ p a re d t o t h a t o f o t h e r s p e c i e s ( S a in i and P o sen , 1969) . I n in b re d s t r a i n s o f m ic e , a l k a l i n e p h o sp h a ta s e l e v e l s v a ry a s much a s 12 K in g - A rm strong u n i t s among d i f f e r e n t mouse s t r a i n s (Y uhas, e t a l . , 1 9 6 7 ) . O th e r t i s s u e s b e s id e s bone w h ich c o n ta in a h ig h l e v e l o f a l k a l i n e p h o s p h a ta s e in c lu d e t h e i n t e s t i n a l m ucosa , p l a c e n t a , k id n e y , and l i v e r (P o se n , e t a l . , 1 967 ; Damm 196£ ; F ishm an , e t a l . , 1 9 6 2 ) . . Even w i th in . t h e same t i s s u e m u l t i p l e fo rm s o f a l k a l i n e p h o sp h a ta s e may be p ro ­ d u ced . T h ree d i f f e r e n t iso enzym es h ave b een d e te c te d i n t h e mouse duodenum a t d i f f e r e n t s t a g e s o f m a tu r i t y ( E t z l e r and Moog, 1968 ; Moog, e t a l . , 1969) . A lk a l in e p h o s p h a ta s e c o n t r ib u te d b y th e i n t e s t i n e may c a u se a v a r i a t i o n o f t h e serum p h o sp h a ta s e l e v e l (Madsen and T uba, 1 9 5 2 ) . D i f f e r e n c e s i n t h e c i r c u l a t i n g l e v e l o f i n t e s t i n a l a l k a l i n e 10 p h o sp h a ta s e may be m ed ia te d by changes i n d i e t ( Tadayyon and L u tw ak , 1969 j K e e l in g , 1969) . F a s t in g r a t s showed low e r l e v e l s th a n no rm al (Bodansky and J a f f e , . 1932) and r a t s on f a t d i e t s showed h ig h e r l e v e l s th a n n o rm a l ( Sai n i and P o sen , 19695 J a c k s o n , 1 9 5 2 ) , I n humans th e a l k a l i n e p h o s p h a ta s e l e v e l itiay be r a i s e d by a p l a c e n t a l iso en zym e . D uring th e t h i r d t r im e s t e r o f p re g n a n c y , a s p i l l o v e r i n t o th e b lo o d s tr e am o c c u rs (K i tc h e n e r , e t a l . , 1 9 6 5 ) . No com­ p a r a b l e s p i l l o v e r o c c u rs i n r a t s (P o s e n , e t a l . , 1 9 6 9 ; M anning, e t a l . , 1 9 6 9 ) . A bnorm al c o n d i t io n s o f th e l i v e r w hich may c o n t r i b u t e to an e l e ­ v a te d b lo o d a l k a l i n e p h o s p h a ta s e l e v e l in c lu d e b i l i a r y t r a c t o b s t r u c ­ t i o n and h e p a t i c i n f i l t r a t i o n s w i th n e o p l a s t i c o r g ran u lom a to u s p r o c e s s e s (T i c k t i n and T r u j i l l o , I 97O5 T a sw e ll and J e f f e r s , 1 9 6 3 ) . E x tra h e p a t i c b i l e o b s t r u c t i o n a s s o c i a t e d w i th e l e v a te d a l k a l i n e p h o s p h a ta s e may be due t o im p a ire d e x c r e t i o n o f p r o d u c t s . I n b i l i a r y t r a c t d i s e a s e s an i n c r e a s e d a l k a l i n e p h o sp h a ta s e may be due t o o v e r ­ p ro d u c t io n o r im p a irm en t o f s e c r e t o r y f u n c t io n o f th e l i v e r (Damm, 1 9 6 5 ) . Through e x p e r im e n ts o f b i l e d u c t l i g a t i o n , i t was co n c lu d ed t h a t i n c r e a s e d a l k a l i n e p h o sp h a ta s e v a lu e s in o b s t r u c t i v e l i v e r d i s e a s e w ere th e r e s u l t o f de novo s y n th e s i s o f a l k a l i n e p h o sp h a ta se i n th e l i v e r and s u b s e q u e n t l e a k a g e o f t h i s in d u ced enzyme i n to th e serum (K ap lan and R i g h e t t i , 1 9 6 9 ) . 11 The p r o l i f e r a t i o n o f m a l ig n a n t tum ors may a l s o c o n t r i b u t e t o an in c r e a s e d serum p h o sp h a ta s e a c t i v i t y (A n n o ta t io n , 1 9 6 9 ) . The Regan iso en zym e , fo u n d i n t h e serum o f p a t i e n t s w i th v a r io u s m a l ig n a n t tum o rs , i s b io c h em ic a l ly and im im m o lo g ic a lly i n d i s t i n g u i s h a b l e from p l a c e n t a l a l k a l i n e p h o s p h a ta s e (F ishm an , 1 969 ; ‘Ghosh, 1969) . I t a l s o o c c u rs i n tum or t i s s u e and m a l ig n a n t e f f u s i o n f l u i d s (K e l le n , 1 9 7 0 ) . Q u a n t i t a t i v e a n a ly s i s o f serum l e v e l s i s u s e f u l i n m o n ito r in g th e p r o g r e s s io n o r r e g r e s s i o n o f tum o rs ( S to lb a c h , e t a l . , 1 969 ; N a th an so n , and F ishm an , 1971 )• Types o f a l k a l i n e p h o sp h a ta s e o th e r th a n Regan isoenzym e may be p ro d u ced by tum ors ( S to lb a ch , e t a l . , 1 9 6 9 ) . CHARACTERISTICS OF ALKALINE PHOSPHATASE A lk a l in e p h o s p h a ta s e i s a z in c met a llo en zym e (P o se n , e t a l . , 1969) w i th a m o le c u la r w e ig h t r a n g in g from 8 6 ,0 0 0 t o 2 0 0 ,0 0 0 ( S c u t t . and M oss, 1 9685 S c h l e s i n g e r , e t a l . , 1969) . E x p e r im en ts w i th b a c ­ t e r i a and t i s s u e c u l t u r e s have shown a lk a l i n e p h o s p h a ta s e t o be a c o n s t i t u t i v e enzyme i n some i n s t a n c e s and an in d u c ib le enzyme in o th e r s ( S c h le s in g e r , e t a l . , 1 9 69 ; G r i f f i n and B o ttom ley , 1969 ; M a r t in , e t a l . , 1 9 6 9 ) . The n a t u r a l s u b s t r a t e o f a l k a l i n e p h o sp h a ta se and i t s p h y s io l o g i c a l r o l e a r e unknown (Kachmar, 1 9 7 0 ) . O p tim al a c t i v i t y o c c u rs a ro u n d a pH o f 9 (Damm, 1 9 6 ? ) . A number o f r e l i a b l e p ro c e d u re s have b een d ev e lo p ed f o r th e a s s a y o f a l k a l i n e p h o sp h a ta s e 12 o f b lo o d (N a th an son and F ishm an , 1 9 71 )• M ost t i s s u e fo rm s o f a l k a l i n e p h o sp h a ta s e may be d i f f e r e n t i a t e d by t h e i r i n d iv i d u a l p h y s i c a l and c h em ic a l p r o p e r t i e s (K e l l e n , 1 9 7 0 ) . E l e c t r o p h o r e t i c m ig r a t i o n p a t t e r n s show l i v e r i n th e f i r s t p o s i t i o n fo l lo w e d by b on e , p l a c e n t a l , and i n t e s t i n a l p h o s p h a ta s e , r e s p e c t i v e l y (S u z u k i, e t a l . , 1 9 6 9 ) . H ea t l a b i l i t y i s d e te c te d by h e a t in g serum t o $5)0 C f o r 16 m in u te s . L iv e r p h o s p h a ta s e i s i n a c t i v a t e d $0 -70 p e r ­ c e n t , i n t e s t i n a l p h o s p h a ta s e $ 0 -60 p e r c e n t , and bone 90-100 p e r c e n t , w h ile t h e p l a c e n t a l isoenzym e i s n o t a f f e c t e d . F iv e m in u te s expo ­ s u re t o a tem p e ra tu re o f 6 $ ° C c a u s e s com p le te i n h i b i t i o n o f bone , l i v e r , and i n t e s t i n a l a l k a l i n e p h o s p h a ta s e . No i n h i b i t i o n o f th e p l a c e n t a l isoenzym e o c c u r s . Isoenzym e e x p o su re t o L -p eny l a l a n in e c a u se s m in im a l i n h i b i t i o n o f l i v e r and bone p h o s p h a ta s e w h i le i n t e s t i ­ n a l and p l a c e n t a l p h o s p h a ta s e show g r e a t e r i n h i b i t i o n ( S to lb a c h , 1 9 6 9 ) . E xposu re t o v a r io u s c o n c e n t r a t io n s o f u r e a a t 3 7 ° C f o r 30 m in u te s i s a n o th e r d i f f e r e n t i a l m ethod . JSn i r r e v e r s i b l e i n a c t i v a t i o n c o n c e n t r a t io n i s 8 m o la r (M) u r e a f o r p l a c e n t a l , 6 -7 M f o r i n t e s t i n a l , 3 M f o r l i v e r , and l e s s th a n 3 M f o r bone n h o sp h a ta s e (M oss, 1 9 6 9 ) . U rea e x p e r im e n ts h ave a l s o shown a d i f f e r e n c e i n 3-D s t r u c t u r e f o r p l a c e n t a l and Regan isoenzym es w h ich o th e rw is e a r e i n d i s t i n g u i s h a b l e (F ishm an , 1969) . MATERIALS AND METHODS Sou rce and M a in ten an ce o f A nim als C F l/A n l m ice w ere o b ta in e d from A rgonne N a t io n a l L a b o ra to ry . P u r in a Mouse Chow and w a te r w ere s u p p l i e d ad l i b i t u m t o th e a n im a ls . FBJ VIRUS STUDY GROUPS U n in je c te d a n im a ls , T e s t in g was begun a t 28 day s o f ag e on fem a le C F l/A n l m ic e . B lood was drawn from th e r e t r o - o r b i t a l s in u s by m eans o f h e p a r in iz e d c a p i l l a r y t u b e s . The tu b e s w ere s e a le d w i th c la y and p lasm a was s e p a r a te d from th e c e l l s i n a C lay-Adams m ic r o c e n t r i ­ f u g e . W eekly a s s a y s w ere c o n tin u e d f o r 3 m on th s . M ice w ere p a lp a te d w e e k ly f o r t h e p r e s e n c e o f tum o rs . U n in je c te d a n im a ls w i th x - r a y e x p o s u re . A t 28 d ay s o f a g e , 10 C F l/A n l fem a le m ice w ere b le d and t o t a l body x - r a y s w ere t a k e n . W eekly sam p lin g and x - r a y e x p o su re was c o n tin u e d f o r 3 m on th s . M ice w ere a l s o p a lp a t e d w eek ly t o d e t e c t tum o rs . FBJ v i r u s i n j e c t e d a n im a ls . Newborn C F l/A n l m ice from 12 l i t t e r s w ere i n j e c t e d i n t r a p e r i t o n e a l l y w i th 0 .1 m l o f FBJ v i r u s v i a th e l e g m u s c le s . A t 28 days o f a g e , 55 f em a le m ice w ere sam p led . Weekly sam p les w ere ta k e n and t e s t i n g was e x te n d e d f o r 2 m on ths on s u rv iv in g a n im a ls . T o ta l body x - r a y s w ere ta k e n w eek ly t o d e t e c t tum o rs . I n a d d i t i o n , p a lp a t io n s f o r tum ors w ere p e rfo rm ed tw ic e w e e k ly . STR0NTItM-90 STUDY GROUPS U n in fe c te d a n im a ls w i th m in im al x - r a y e x p o s u re . A c o n t r o l g roup o f 1E> fem a le C F l/Anl m ice from 77 t o 127 days o f age (mean ag e 97 days) w ere b le d once a month f o r 3 m on th s , th e n b iw e e k ly . T e s t in g was c o n tin u e d f o r 8 m on th s . T o ta l body x - r a y s w ere ta k e n a t th e b e g in n in g o f t e s t i n g , th e n once e v e ry it m on th s . U n in fe c te d a n im a ls w i th m axim al x - r a y e x p o s u re . F i f t e e n fem a le C F l/A n l m ice from 77 t o 127 d ay s o f age (mean age 97 d a y s ) .w e re b le d o n c e .a month f o r 3 m on th s , th e n b iw e e k ly . The m ice w ere exposed t o t o t a l body x - r a y s a t th e tim e o f e ach b l e e d in g . S tro n t ium -9 0 i n f e c t e d a n im a ls . A t e s t g roup o f 30 fem a le C F l/A n l m ice from 77 t o 127 days o f age (mean age 97 days) w e re b l e d . The f o l lo w in g day th e m ice w ere i n f e c t e d w i th s t r o n t iu m -9 0 ( I j iC i/g body w e ig h t) v ia th e t a i l v e in . The m ice w ere b le d a t 30 day i n t e r v a l s f o r 3 m on th s , th e n b iw e e k ly . T o ta l body x - r a y s were p e rfo rm ed a t e a ch sam p lin g to d e t e c t th e p r e s e n c e o f tu m o r s . SOURCE Am PREPARATION OF FBJ VIRUS FBJ v i r u s p ack ed i n d ry i c e was r e c e iv e d from A rgonne N a t io n a l L a b o r a to ry . The sn ec im en was p r e s e r v e d i n a REVCO f r e e z e r a t -7 0 ° C . A t th e tim e o f mouse i n o c u l a t i o n s , sp ec im en v i a l s w ere a g i t a t e d i n a 37° C w a te r b a th f o r m in u te s . A 1 :3 d i l u t i o n o f th e v i r u s was made w ith s t e r i l e p h o sp h a te b u f f e r e d s a l i n e and 0 .1 ml o f t h e s o lu t i o n was i n j e c t e d i n t r a p e r i t o n e a l l y v ia th e l e g m u sc le s i n t o newborn C F l/A n l m ic e . Tumors w ere e x c is e d from dead o r m oribund a n im a ls . A f te r w e ig h ­ i n g , tum o rs w ere g round i n t o a f i n e p a s t e , and H an k 's B a lan ced S a l t S o lu t i o n added on a 1 :1 (volum e t o w e ig h t) b a s i s . The s u sp e n s io n was th e n made c e l l - f r e e by d i f f e r e n t i a l c e n t r i f u g a t i o n u n d e r r e f r i g e r a t i o n , u s in g th e f o l lo w in g s c h e d u le (A rgonne N a t io n a l L a b o r a to ry , 1 9 7 0 ) : 1) Tumor p a s t e i n c lu d in g v i r u s - 10 m in u te s , 2 ,0 0 0 rpm ( a p p ro x im a te ly U^O g) 2) S u p e rn a ta n t f l u i d - 15> m in u te s , 2 ,0 0 0 rpm ( a p p ro x im a te ly 2,800 g) 3) S u p e rn a ta n t f l u i d - 30 m in u te s , 2 ,0 0 0 rpm (a p p ro x im a te ly 2 ,8 0 0 g) It) F in a l supernatant f lu id con ta in ing v irus was stored a t - 70o c. • • X-RAY PROCEDURE U n a n e s th e t iz e d m ice w ere a t t a c h e d i n th e s u p in e p o s i t i o n t o a 3/It in c h t h i c k plywood f ram e , c o n ta in in g a It x 2 in c h c u to u t . A t ta c h ­ m en t was made w i th I in c h padded a l l i g a t o r c lam ps f a s t e n e d w i th w ir e s t o sc rew s s e t i n th e f r am e . The a x i s o f th e mouse was a l i g n e d p a r a l l e l 16 t o th e x - r a y tu b e . R ed ip ac Kodak AA-2 i n d u s t r i a l ty p e x - r a y f i lm was u sed ( A rgonne N a t io n a l L a b o r a to ry , 1 9 7 0 ) . R o en tg enog raph s were t a k e n w i th a p o r t a b l e P i c k e r x - r a y m ach ine w ith f i l t e r s rem oved . The f o l l o w ­ in g s e t t i n g s w ere u s e d ; Sm a ll f o c u s . ’ KV - 3it MA - 65 Time e x p o su re - 3 /1 0 se co nd D is ta n c e from mouse t o x - r a y tu b e - 9 i n c h e s . ALKALINE PHOSPHATASE ANALYSIS. P r e p a r a t i o n o f r e a g e n ts and s t a n d a r d i z a t i o n o f t e s t . M a l k a l i n e b u f f e r w as p r e p a r e d by d i s s o lv in g 7 .50 g g ly c in e (an h y d ro u s ) and 0 .9 9 5 g MgClg o r ( 0.203 g MgClg.^HgO) in a p p ro x im a te ly 750 m l o f d i s t i l l e d w a te r . To b r in g th e pH t o 1 0 .5 , 85 ml o f I N NaOH was a d d ed . The r e a g e n t was t h e n d i l u t e d t o 1 ,0 0 0 ml w i th d i s t i l l e d w a te r and f o u r d ro p s o f c h lo ro fo rm ad d ed . To make th e b u f f e r e d s u b s t r a t e , 0 .2 g o f p - n i t r o p h e n y lp h o s p h a te was d i s s o lv e d i n a b o u t hO ml o f d i s t i l l e d w a te r i n a 100 m l v o lu m e tr ic f l a s k . An a l k a l i n e b u f f e r o f 50 m l was added and th e s o lu t i o n b ro u g h t t o 100 m l. A f t e r m ix in g , t h e s u b s t r a t e s o l u ­ t i o n was tu b ed i n I m l am oun ts . S u b s t r a t e tu b e s w ere c o v e re d w ith p a r a f i lm and s to r e d a t -7 0 ° C. A s to c k s ta n d a rd was p r e p a r e d by d i s ­ s o lv in g 1.3911 g o f p - n i t r o p h e n o l i n a l i t e r v o lu m e tr ic f l a s k (10 m M / l i t e r ) . The s ta n d a r d rem a in ed s t a b l e f o r I y e a r a t U0 C. To 17 make a w o rk ing s ta n d a rd ,- £ .0 ml o f th e p - n i t r o p h e n o l s to c k s ta n d a rd was p i p e t t e d i n t o a I - l i t e r v o lu m e tr ic f l a s k . The s o l u t i o n was th en d i l u t e d t o I l i t e r . The w o rk ing s ta n d a r d rem a in ed s t a b l e one day (D av id sohn and W e l l s , 1 9 6 3 ) . The f o l lo w in g d i l u t i o n s w ere made t o e s t a b l i s h a s ta n d a r d c u rv e ; W orking 0 .02N l U / l i t e r Tube S ta n d a rd W a te r ' NaOH A lk a l in e No. (rnl) (m l) (m l) P h o sp h a ta s e I I 9 1 .1 33 . 2 2 8 1 .1 66 3 U 6 1 .1 133 U 6 U 1 .1 200 5 8 2 1 .1 266 6 10 0 1 .1 33U The a b so rb a n c e o f e a ch o f th e above m ix tu re s was r e a d a t UlO mp, u s in g 0 .0 2 N NaOH i n th e r e f e r e n c e tu b e s and th e r e s u l t i n g v a lu e s w ere p l o t t e d on a c u rv e . A lk a l in e p h o s p h a ta s e v a lu e s w ere r e a d from th e c u rv e ( D av id sohn and W e l l s , 1 9 6 3 ) . P r i n c i p l e and p e rfo rm an c e o f t e s t . The p h o sp h a te g ro u p o f p - n i t r o p h e n y l p h o sp h a te i s s p l i t o f f b y a l k a l i n e p h o s p h a ta s e a t an a l k a l i n e pH t o y i e l d y e l lo w -c o lo r e d p - n i t r o p h e n o l . The amount o f p - n i t r o p h e n o l r e l e a s e d i s d i r e c t l y p r o p o r t i o n a l to th e amount o f a l k a ­ l i n e p h o s p h a ta s e p r e s e n t i n th e p lasm a (D avidsohn and W e lls , 1 9 6 3 ) . R e s u l t s a r e r e p o r t e d i n I n t e r n a t i o n a l U n i ts ( IU ) , d e f in e d a s th e number 18 o f m ic ro m o le c u le s o f s u b s t r a t e h y d ro ly z e d p e r m in u te by one l i t e r o f serum a t 37 ° C ( R i c h t e r i c h , 1 9 6 9 ) . To I ml o f an a l k a l i n e b u f f e r e d s o lu t i o n o f p - -n i tr o p h e n y l p h o sp h a te 0 .0 $ ml o f p la sm a was added (Sigma 1 0 b ) . A f t e r t u b e s w ere i n c u b a te d a t 37° C f o r 30 m in u te s , 10 m l o f 0 .0 2 N NaOH was added t o i n a c t i v a t e th e enzyme and d i l u t e th e s u b s t r a t e f o r r e a d in g . A Coleman 12b S p e c tro p h o tom e te r s e t a t b !0 mu was u s e d t o r e a d th e t e s t s . Ten ml o f NaOH was added t o a s u b s t r a t e tu b e t o s e r v e a s a r e a g e n t b la n k f o r e a ch t e s t . To e ach tu b e 0 .1 ml o f c o n c e n t r a te d h y d r o c h lo r ic a c id was added a f t e r th e f i r s t r e a d in g to re d u c e p - n i t r o p h e n o l t o a c o l o r l e s s s t a t e f o r u s e a s a serum c o lo r b la n k . A V e r s a to l E c o n t r o l was ru n w i th e ach g roup o f t e s t s . R e s u l t s g r e a t e r th a n 160 I l j / l i t e r w ere r e p e a te d u s in g 0 .0 2 $ m l o f p la sm a . I n some c a s e s , in a d d i t i o n t o d i l u t i n g th e p lasm a vo lum e, i t was n e c e s s a r y t o re d u c e th e in c u b a t io n tim e t o 10 m in u te s . R e s u l t s w ere th e n m u l t i p l i e d t im e s 6 . I n c u b a t io n tim e c o u ld be red u c ed s in c e t h e a l k a l i n e p h o s p h a ta s e a c t i v i t y i s l i n e a r ( R i c h t e r i c h , 1969) . Isoenzym e d i f f e r e n t a t i o n . H ea t l a b i l i t y was u se d t o t e n t a t i v e l y i d e n t i f y a l k a l i n e p h o s p h a ta s e iso enzym es in 10 p la sm a spec im en s w ith e l e v a t e d l e v e l s . The p la sm a was in c u b a te d a t $$° C f o r 16 m in u te s , th e n c o o le d im m ed ia te ly . R e s u l t s o f t h e B e ssey , Low ry, B rock a n a ly s i s on th e h e a t - t r e a t e d sp ec im en s was com pared w i th u n h e a te d sp e c im en s . A 90 t o 100 p e r c e n t l o s s o f a c t i v i t y o c c u rs i n th e human isoenzym e p r o ­ duced th ro u g h o s t e o b l a s t i c a c t i v i t y ( S to lb a c h , 1969) . RESULTS A " t H t e s t was u sed t o d e te rm in e t h e s i g n i f i c a n c e o f mean d i f f e r ­ e n c e s i n a l k a l i n e p h o s p h a ta s e l e v e l s o f th e v a r io u s t r e a tm e n t g ro u p s . No p o o le d e r r o r was u s e d , e r r o r b e in g c a l c u l a t e d s e p a r a t e l y f o r each g ro u p . The e s s e n t i a l n a tu r e and v a r i a t i o n o f c o n t r o l a n im a ls w ere e s t im a t e d . I n b o th t r e a tm e n t and c o n t r o l g ro u p s , mean a l k a l i n e p h o s ­ p h a ta s e l e v e l s o v e r t im e w ere exam ined a lo n g w i th v a r i a t i o n among i n d iv i d u a l a n im a ls . A lk a l in e P h o sp h a ta s e L e v e ls i n U n in je c te d G ro u p s . The a l k a l i n e p h o sp h a ta s e l e v e l s o f u n in je c te d a n im a ls i n th e g roup b e g in n in g a t 28 days o f age showed a s i g n i f i c a n t d i f f e r e n c e from a n im a ls b e g in n in g a t mean ag e o f 97 d a y s . The mean a l k a l i n e p h o s p h a ta s e v a lu e s o f th e y ounge r a n im a ls d ropped from 1U5 IU to S3 IU o v e r a 12 week p e r io d , w he reas th e mean v a lu e s f o r m ice b e g in n in g a t mean age o f 97 days d ropped from 65 IU t o 3h IU o v e r an 8 month p e r i o d . T h e r e f o r e , th e two age g ro u p s w ere t r e a t e d s e p a r a t e l y . FBJ V iru s Base L in e G roup . The w eek ly a l k a l i n e p h o sp h a ta s e v a lu e s o f non x - r a y e d and x - r a y e d a n im a ls w i th o u t v i r u s i n j e c t i o n a r e g iv e n i n T ab le s I and I I , r e s p e c t i v e l y . The a l k a l i n e p h o sp h a ta s e l e v e l s showed a l a r g e an im a l t o an im a l v a r i a t i o n i n u n i n j e c t e d , non . x - r a y e d a n im a ls s t a r t i n g a t 28 d ay s o f age a s i l l u s t r a t e d i n T ab le I I I . H ow ever, th e v a r i a n c e d ropped w i th a g e . Anim al to a n im a l v a r i a t i o n 20 T ab le I . W eekly a l k a l i n e p h o s p h a ta s e l e v e l s i n IU o f u n in je c te d m ice b e g in n in g a t it weeks o f a g e . Week o f A ssay House Number I 2 3 It 5 6 7 8 9 10 H 12 I l i l t lit? 80 95 6it 88 90 66 lt9 It? lt8 55 2 IltS 177 132 125 91 H l 130 76 80 7lt 68 56 3 129 102 133 H 5 79 77 75 75 58 55 56 27 h 122 205 103 9lt 66 57 77 80 67 56 58 65 2 193 212' 18? 237 122 102 120 107 IOlt 5 i 65 100 6 I lt l 176 150 139 90 91 Hit 85 78 8? 70 76 7 Ht7 176 87 Hit 90 100 133 85 93 61 53 32 6 Ili6 127 133 120 102 127 102 83 86 80 77 31 9 175 166 133 131 H 2 81 56 75 82 66 65 Itl 10 117 62 llt3 152 IltO 129 17lt ilt5 H 3 63 65 57 11 201 252 77 137 127 132 HO 71 85 77 75 60 12 137 122 99 113 88 72 97 53 63 51 51 U3 13 192 13U 126 IOlt H 7 100 75 90 100 82 81 88 lit l i l t 101 101 107 65 65 12lt 61 39 it? It? 57 15 lit? 126 HO 12? 79 8? 90 79 53 26 58 lt2 16 135 90 81t 106 82 72 58 71 63 66 63 57 17 118 151 9h l i l t 69 103 75 66 75 55 57 ItB 18 lltlt 138 91 82 82 76 107 ItB 32 lt9 52 lt3 19 128 65 120 79 60 5it 55 It? it9 It? 38 U6 20 lit6 119 it6 122 65 7lt 90 72 73 73 63 U5 21 Ht5 98 no 105 70 Bit 77 7lt 79 72 57 It? 22 138 llt5 IOlt 109 86 108 73 81 81 75 51; It? 23 lit5 116 105 129 78 51t 103 51t Itl lt9 It? 55 2lt 186 19lt 127 103 109 81 90 96 79 67 70 57 25 156 213 IltO 107 IOlt 121 106 106 88 58 61t 5 l 26 186 133 122 100 80 107 87 87 70 59 50 6 l 27 l 6 l 195 102 119 79 92 107 86 83 51t 70 56 28 109 22lt 133 H 5 105 93 86 81 67 60 58 29 29 l51t 122 108 101 8li Bit 68 70 59 59 57 52 30 lit9 113 103 100 77 87 79 79 80 69 U3 63 31 123 253 9lt 85 109 117 82 95 77 70 61 lt9 21 TABLE I. (Continued) Week of Assay Mouse Number I 2 3 it 5 6 7 . 8 9 10 11 12 32 93 153 125 92 90 80 71 .. -* U6 39 65 55 33 100 152 102 102 86 86 66 65 62 78 3h 66 3h lU6 157 126 96 90 85 85 81» 77 5U 67 lt3 35 18? 171 120 86. 91 65 70 77 77 Itlt 61 55 36 130 130 98 82 85 86 19 5U 68 65 5S 51 37 IO il 133 * 106 83 75 55 70 62 55 Itl 27 52 38 189 157 115 102 77 78 80 51 78 • Itl 58 53 * Insufficient quantity of plasma available for testing. TABLE I I 1 Weekly a l k a l i n e p h o s p h a ta s e l e v e l s i n IU o f u n i n j e c t e d m ice w i t h .x - r a y e x p o su re b e g in n in g a t U w eeks o f a g e . Week o f A ssay Mouse Number I 2 3 U 5 6 7 8 9 10 n 12 39 162 116 9U 113 85 100 90 79 6U 57 62 U8 Uo 117 131 no 87 137 131 121 77 69 .77 80 52 Ul 8U 171 90 101 123 63 H 2 68 52 53 55 57 U2 92 128 106 65 n5 76 90 80 70 80 76 57 U3 91 123 102 98 108 81 81 83 67 63 52 57 UU 108 iUo 96 115 100 81 119 67 55 65 53 U5 U5 165 1U8 8U 82 107 63 77 60 55 . 6U 91 67 U6 IlU 1U8 128 HS IUi no 89 90 85 83 86 59 U7 100 1U7 123 96 78 70 7U $8 5U 65 U7 U3 UB iUo 120 122 92 115 122 158 59 56 50 50 79 23 TABLE I I I , . P a t t e r n o f mean a l k a l i n e p h o sp h a ta s e v a r i a n c e i n 8 u n in je c te d m ice b e g in n in g a t It w eeks o f age w i th no x - r a y e x p o su re . S am p ling Week Mean V a rian ce 1 2 3 U 5 6 7 8 9 10 11 IUU 128 100 105 77 79 Qh 67 Sh 61 55 1*8 391 1,1*81 615 310 239 390 293 292 388 . 11*5 12 91* 21* 2k ■r> was g r e a t e r i n u n in j e c t e d a n im a ls w h ich r e c e iv e d x - r a y ex po su re (T a b le IV ) . A com pa riso n o f u n t r e a t e d a n im a ls w i th o u t x - r a y e x p o su re v and u n t r e a t e d a n im a ls w i th x - r a y e x p o su re showed s i g n i f i c a n t d i f f e r ­ e n c e s i n mean a l k a l i n e p h o s p h a ta s e v a lu e s i n 5 o u t o f 12 a s s a y p e r io d s (T ab le V ). FBJ V iru s I n j e c t e d G roup . The tum or i n c id e n c e o f m ice i n j e c t e d w i th FBJ v i r u s was 100 p e r c e n t . O s teo sa rcom as a p p e a re d from 18 t o 98 days a f t e r i n j e c t i o n , w i th th e m a jo r i t y o f tum o rs a p p e a r in g on th e 1 9 th d ay . The t i b i a was t h e m ost f r e q u e n t s i t e o f p r im a ry tum o rs , o c c u r r in g a s th e f i r s t tum or i n k7 p e r c e n t o f th e t e s t a n im a ls . D ea th s w ere d i s t r i b u t e d th ro u g h o u t t h e t e s t i n g p e r i o d , th e f i r s t ■i * d e a th o c c u r r in g on d ay one o f t e s t i n g . By th e 1 3 th day t h e r e was a I m o r t a l i t y o f $2 p e r c e n t . A t th e l a s t b le e d in g 5 a n im a ls rem a in ed a l i v e „ ' T ab le VI c o n ta i n s a l k a l i n e p h o s p h a ta s e v a lu e s f o r th e 55 m ice i n j e c t e d w i th FBJ v i r u s a t b i r t h . A com pa riso n o f mean v a lu e s b e g in ­ n in g a t I4 w eeks o f ag e and c o n t in u in g o v e r an 8 week p e r i o d showed no s i g n i f i c a n t d i f f e r e n c e i n FBJ i n j e c t e d m ice and u n i n j e c t e d , non x - r a y e d a n im a ls (T a b le V I I ) . The mean a l k a l i n e p h o s p h a ta s e l e v e l o f a n im a ls , a t th e t im e o f tum or a p p e a ra n c e on ro e n tg e n o g ra p h s was n o t ; s i g n i f i c a n t l y d i f f e r e n t from th e v a lu e s o f u n in je c te d non x - r a y e d a n im a ls a t th e same a g e . 22 TABLE IV . A com pa rison o f v a r i a n c e o f mean a l k a l i n e p h o s p h a ta s e l e v e l s b e tw een x - r a y e d and non x - r a y e d a n im a ls o f u n in je c te d g ro u p s b e g in n in g a t k w eeks o f a g e . X-Rayed Non X-Rayed Group Group Sam p ling _ Week 1 X X X X I 1U2 22 117 20 2 121 U3 137 28 3 112 19 106 16 h 13.0 23 97 19 2 88 22 111 9 6 88 36 90 11 7 88 lilt 101 19 8 76 12 72 10 9 71 10 63 8 10 29 . 9 66 2 11 28 12 62 3 12 23 8 26 2 26 TABLE V. C a lc u la te d nt " v a lu e s f o r mean a l k a l i n e p h o sp h a ta s e l e v e l s o f x - r a y e d and non x - r a y e d a n im a ls o f u n i n j e c t e d g roup b e g in n in g a t k w eeks o f age=, VJeek o f S am pling t I 2.71* 2 1 .37 NS 3 0 .92 NS U 0 .72 NS $ 3.U0* 6 1.61; NS 7 3.85* 8 1 .33 NS 9 1.U2 NS 10 2.86* 11 2.70* 12 0 .75 NS * - D i f f e r e n c e be tw een means s i g n i f i c a n t . NS - D i f f e r e n c e b e tw een means n o t s i g n i f i c a n t . 27 TABLE V I. ' "Weekly a l k a l i n e p h o sp h a ta s e l e v e l s i n IU o f FBJ v i r u s i n j e c t e d m ice b e g in n in g a t h w eeks o f a g e . Week o f A ssay Mouse Number I 2 3 k 5 6 7 8 I 98* 111 8k 73 13k _ 2 119 120* 122 162 — — «0 - 3 180* 60 W «K» U 193 199 108 9k 129* 76 80 77 5 186 lk8* 91 113 98 «o - 6 188* — -O « «. am a. *• 7 102* 168 99 78 83 6 l 69 8 162* 187 lk l lk l 131 . <= - 9 125* - — - •0» a. » 10 80* — — W 11 109* - - — - am - 12 9k 103 115 65 60 109* 88 - 13 133* - - - - - Ih 109* Ik9 - - - - - , IS 192* - - - - - - - 16 12k 116 8k 82 72 69* 79 75 17 122* - -W am * - - 18 93* — - - — - - 19 136* Ik7 - - - - - - 20 122 130* 100 88 - «o - - 21 101* - - - - - 22 122 137 121 121 120* 69 6k - 23 161* 130 90 98 - - - - 2k lk7 lk l 8k 105 8k 69* 66 53 25 lh7* - - - - - - - 26 97* 130 - - - - 27 102 106 - - - - - - 28 9k* - - - - ~ - 29 126 79* 102 70 103 mm .mm 30 18k lk l 108 90 IOk 72* 95 mm 31 88* 115 - * - - ™ - 32 119* 9k 9k 108 95 103 - - 28 TABLE V I. (C o n tin u ed ) Week o f A ssay Mouse Number I 2 3 Ii 5 6 7 8 33 . 79* _ _ _ _ 3h 187 155 107* 96 - - oa - 35 176 157 98 135 101 76 ii3 93* 36 192* 157 103 133 - - SM - 37 lii6 130 127* 116 - - *0 - 38 160* 176 - W - - - 39 126* — - — *• a. IiO 163* 12k — ■» « ■ - - ~ ill IOii* lii9 131 IilO iU5 179 169 ii2 109* 131 115 106 - - ■a - ii3 108* 136 183 108 116 - KU Iili 119* 151 - - - - - MB ii5 85 126 9k* 188 - ■ a. - ii6 187* 120 101 a - - ea - li7 250* 213 * - - - = *n Ii8 228* - «D - - ii9 81i* 123 - - - - «a •* 50 182 133 133* 9ii 91 - - - 51 170 123 HO 109 99 9ii 101 67* 52 270* - - - - - - 53 165 137 77* 8ii 6ii 122 78 5ii 167 165 133* 106 lii5 118 103 - 55 190* 175 - - - - Ba - - I n d i c a t e s a n im a l d e a d . * F i r s t a n a ly s i s w i th d e te c ta b l e tum or 29 TABLE V I I . Mean, S - , and v a lu e s c a l c u l a t e d f o r a l k a l i n e p h o s p h a ta s e l e v e l s o f non x - r a y e d a n im a ls o f u n i n j e c t e d g roup and FBJ v i r u s i n j e c t e d g roup b e g in n in g a t U w eeks o f a g e . Week of Test Sample Means sI t aFBJ Control FBJ Control I 1U2 I h S 52 20 0.33 2 138 I S l 20 58 1.U7 3 108 112 18 16 0.80 k 10U no 30. 19 0.8? S 102 88 U2 9 1.91 6 97 88 86 11 O.89 7 89 88 82 19 o .n 8 73 76 U3 10 0 .U6 a - D i f f e r e n c e be tw een means n o t s i g n i f i c a n t . S - - V a r ia n c e o f d i s t r i b u t i o n o f sam p le m eans. In O U n in je c te d m ice • U n in je c te d m ice w i th o u t x - r a y ex po su re A FBJ injected mice W e e k o f A s s a y o F ig u re I . Mean a l k a l i n e p h o sp h a ta s e l e v e l s v e r s u s tim e o f FBJ v i r u s g ro u p s . 31 Mean A lk a l in e P h o sp h a ta se L e v e ls R e la t i v e t o Time f o r FBJ Vi r u s I n j e c t e d and U n in je c te d M ice . F ig u re I i l l u s t r a t e s a d ro p o f mean a l k a l i n e p h o sp h a ta s e v a lu e s w ith tim e i n a l l a n im a l g ro u p s b e g in n in g a t 28 d ay s o f a g e . A lth o ugh no r e g r e s s i o n e q u a t io n was c a l c u l a t e d , a d e c l in e i n v a lu e s i s shown a s t h e m ice m a tu re . S tro n t ium -9 0 Base L in e G roup . The a l k a l i n e p h o s p h a ta s e v a lu e s o f a n im a ls w i th an a v e ra g e age o f 97 days w h ich r e c e iv e d m axim al and m in im a l x - r a y e x p o su re a r e r e c o rd e d i n T ab le s V I I I and IX . The two g ro up s showed no s i g n i f i c a n t d i f f e r e n c e s .in mean a l k a l i n e p h o sp h a ta s e l e v e l s (T a b le X ). T h e r e f o r e , th e a l k a l i n e p h o sp h a ta s e v a lu e s were p o o led i n com pa riso n s w i th s t r o n t iu m -90 i n j e c t e d a n im a ls . S t ro n t ium -9 0 I n j e c t e d G roup. T e s t a n im a ls d e v e lo p e d tum ors a s e a r l y a s 8it day s and a s l a t e a s 238 day s a f t e r th e s tr o n t iu m -9 0 i n ­ j e c t i o n , w i th tum o rs o c c u r r in g i n 87 p e r c e n t o f th e m ice a t th e tim e o f t e s t t e rm in a t i o n . T w en ty - th re e o f th e s t r o n t iu m -9 0 i n j e c t e d a n im a ls d ie d b e f o r e t h e end o f t e s t i n g , w i th th e f i r s t a n im a l d y ing 112 days a f t e r t e s t i n g b e g an . S u b se q u en t d e a th s w ere s c a t t e r e d th ro u g h o u t th e rem a in d e r o f th e t e s t i n g p e r io d , w i th 55 p e r c e n t o f th e d e a th s o c c u r r in g a t 182 d ay s . A lk a l in e p h o s p h a ta s e l e v e l s o f th e i n d iv i d u a l a n im a ls w hich r e c e iv e d s t r o n t iu m -9 0 a r e shown i n T ab le X I. Mean a l k a l i n e TABLE VIII. A lkaline phosphatase le v e ls in IU o f mice beginning a t mean age o f 97 days with minimal x -ray exposure. a Date : O f t e s t Mouse Number 9-29 Il-U 12-2 12-30 1-13 1-27 2-10 2-2U 3-10 3-2U U-7 U-21 5-5 5-19 6-2 31 51 3U 36 2U 31 37 32 2U 31 26 2U QNS 31 2U 21 32 Uo 35. 31 27 27 37 2U 16 QNS 23 22 28 U7 26 2U 33 U3 QNSb 33 Uo 37 26 27 2U 25 2U 3U 2U 3U 32 13 3h 73 60 58 26 UO U.2 U3 QNS QNS 37 28 U8 UU 6U U3 35 78 51 U6 39 QMS U7 QNS 56 U7 U7 2U 26 26 32 U2 36 8l 65 51 Uo 36 U8 U8 U? 5U 55 36 50 36 U6 QNS 37 52 36 U3 QMS 29 38 27 26 33 37 39 U7 35 61 2U 38 QMS 5U 56 61 QNS U9 59 UU 5U 55 37 65 39 32 QNS 39 61 UB 5U 38 3U Uo 38 U6 39 32 U3 3U Ul 35 UlUo 58 QNS 36 35 27 31 29 28 32 31 32 36 28 25 29 Ul 55 U2 QNS 38 52 U2 U2 36 39 Uo 32 36 52 38 U5 U2 66 QNS 68 33 65 62 5U 5U U2 37 5o 3U QNS 58 U7 U3 80 UO 50 36 51 QNS Ul Ul UO U8 37 U3 U8 U2 39 UU 60 30 Uo 33 50 30 UU 26 25 16 26 3U 38 31 29 U5 68 QMS U7 21 32 31 3U 27 16 29 U2 32 29 20 35 a - A n im als r e c e iv e d x - r a y e x p o s u re s a t b e g in n in g o f t e s t i n g , th e n once e v e ry b m on th s . b - I n s u f f i c i e n t q u a n t i t y o f p la sm a a v a i l a b l e f o r t e s t i n g . TABLE IX A lkaline phosphatase le v e ls in IU of mice beginning a t mean age o f 97 days ■with maximal x -ray exposure . a Late o f t e s t Mouse Number 9-29 Il-U 12-2 12-30 1-13 1-27 2-10 2-2U 3-10 3-2U U-7 U-ZL 5-5 5-19 6-2 U6 68u U5 Uo 3U U2 52 UU 25 35 36 UU 7U 35 31 U3U7 QNSb 3U Uo 35 33 37 U3 22 30 31 Ul 53 58 Ul 33 W 60 5o QNS U8 38- U2 hS 32 2U 26 2U 31 16 IU 16 h9 ■ U3 QNS 36 QNS 28 2U 27 23 29 19 28 17 20 19 18 50 li8 30 36 32 U3 3U 33 22 27 32 33 26 3U 26 30 51 78 QNS 52 UU 53 57 63 57 69 5U 62 U9 3U 53 55 52 67 U2 U8 UO 28 30 5U 37 U7 30 QNS 3U 36 20 2U 53 HS 35 QNS 3U 36 38 U3 32 33 32 29 35 31 U3 33 Sh ■ 9h 26 56 37 35 QNS 5l U8 U7 U8 UU 5U 70 62 QNS 55 79 QNS U6 32 UO 29 39 U5 U8 17 36 36 U3 28 36 56 68 U8 39 29 U3 3U 39 39 53 38 53 33 Ul 39 38 57 . 80 U7 60 U3 32 39 61 35 39 U7 51 U8 52 27 U6 58 85 Sh 55 35 U2 Uo 57 U8 Uo U9 52 3U U5 56 U9 59 77 hS U3 U2 32 3U 3U 28 32 25 3U IU 36 30 2560 58 U3 U3 27 3U 23 U5 3U 29 33 39 33 2U 16 QNS - A n im als r e c e iv e d x - r a y e x p o su re a t e a c h sam p lin g d a te . - I n s u f f i c i e n t q u a n t i t y o f p lasm a a v a i l a b l e f o r t e s t i n g . 3h TABLE X. C a lc u la te d " t " v a lu e s f o r mean a l k a l i n e p h o sp h a ta s e l e v e l s o f m ice w i th m in im a l and m axim al x -ray - e x p o su re b e g in n in g a t 97 day s o f a g e . Test Number t a I I . IS 2 2.00 3 0 .33 b 0 .52 5 1 .96 6 0 .9 1 7 1 .29 8 0 .32 9 1 .29 10 0 .31 11 0 .73 12 O M 13 0.11* IU 0 .30 l£ 0 .59 a - D i f f e r e n c e b e tw een means n o t s i g n i f i c a n t TABLE X L Alkaline phosphatase levels in IU of mice which received strontium-90 beginning at mean age of 97 days. Date of test Mouse Number 9-29 Il-It 12-2 12-30 1-13 1-27 2-10 2-2L 3-10 3-2L L-7 • L-21 5-5 5-19 6-2 I 7it 51 72 7it 9U 106 iLo# ILL 35L 36ob -C 2 53 27 39 29 itl 57 L5 65* 72 65 98 238 — 3 67 31 ItO 62 66 103 132 192* cm «. It 72 36 79 53 102 90* 15L 3L0 590 720b to aa 5 53 QNSa 30 2it 27 19 26 30 26 2L* 36 26 62 77 iLo6 3lt 25 32 31 . 32 L5 Li L2 L2 52 50 59# 96 108 23L7 Itit 2it 28 23 29 28 22 38 L7 39* 50 90 LLO IltLob8 lt5 27 28 28 35 39 33 36 31 26 31 39 35 30 12 9 70 39 26 55# 57 79 89 QNS 338 D ee10 55 32 38 39 Lit 56 3L 70 80 106* 9L 118 180 212 69611 9lt QNS 63 78 86 80 95* QNS 360b as>12 QNS 30 it2 ItO L8 36 L8* 5L 77 _ 13 it3 26 28 26 32 36 L6 3L 38 35 Li 6L 55 62 90*lit 6l 32 36 39 L7 57 6L 55 60 7L 83 178* 360 115215 70 36 33 3lt 39 Li L6 55 L7 97 66 88* L96 IitLob16 58 33 Itit 33 35 38 L8 L7 35 L3 38 Li 17 it? 32 ItO 33 LL 39 L5 70 70 63 70* 92 720b 16 25 27 35 56 78 37 62 82. 102* 360b 720b 2l60b19 79 38 70 88* 125 106 1L8 157 191 L92 - *=» aa TABLE XI. (Continued) Date of test Mouse Number 9-29 H-L 12-2 12-30 1-13 1-27 2-10 2-2L 3-10 3-2L L-7 L-21 5-5 5-19 6-2 20 1*6 3L 38 37 58 50 168 52 53 L6 65* 75 n 2 702 21 62 35 32 L2 38 L7 53 51 - - - e , 22 79 Li 68 91 no 131 QNS 168* 536 0« «*» •=» — ee e* 23 36 26 Lo 19 2L 27 no 26 2L 26* 27 3L 320 « 2h 8U 32 L3 6L 8L 6L 5L 135 2L2* •» - «0 25 70 2L 62 60 QNS 101 - - - — — - 26 5U 3L 35 32 LL Li 39 LL 58 LL 51* 35 87 188 L32 ' 27 86 57 69 97 101 1L2* 156 *» em *— — * oe 28 60 QNS 57 5L 33 5L 5o 88 89* 1$6 272 L50 ee ta Se 29 39 L7 31 LU 60 80 91 lLB QNSa lL? 986 65L OD «s 30 60 3L LL L3 60 89 5L 66 QNSa 150 610 1368 — " = * - F i r s t specim en w ith x - r a y d e te c ta b l e tum ors . a - I n s u i f i c i e n t q u a n t i ty o f p lasm a a v a i l a b l e f o r t e s t i n g , b = A c tu a l v a lu e g r e a t e r th a n re c o rd ed f i g u r e . c - Animal d ead . 37 p h o s p h a ta s e ' v a lu e s o f u n t r e a t e d a n im a ls and s t r o n t iu m -90 i n j e c t e d a n im a ls o f t h e same age w ere s i g n i f i c a n t l y d i f f e r e n t i n a l l b u t th e f i r s t and t h i r d t e s t i n g p e r io d s (T a b le X I I ) . From 2 t o 8 weeks b e fo re r o e n tg e n p g ra p h ic d e t e c t i o n o f tum o rs , mean a l k a l i n e p h o s ­ p h a ta s e v a lu e s o f i n j e c t e d a n im a ls w ere fo und t o be s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s (T ab le X I I I ) . H e a t L a b i l i t y o f P lasm a A lk a l in e P h o s p h a ta s e . A l l t e n p lasm a sam p le s w i th e l e v a t e d a l k a l i n e p h o s p h a ta s e l e v e l s from s t r o n t iu m -90 i n j e c t e d m ice t e s t e d f o r h e a t l a b i l i t y showed 88 t o 97 p e r c e n t i n a c t i v a t i o n o f t h e enzyme (T a b le XIV) Mean A lk a l in e P h o sp h a ta s e L e v e ls R e la t iv e t o Time f o r S t r o n ­ tium -9 0 I n j e c t e d and U n in je c te d M ic e . F ig u re 2 shows a s l i g h t d e c l in e i n a l k a l i n e p h o s p h a ta s e l e v e l s o f u n i n j e c t e d a n im a ls compared t o t im e , w h i le s t r o n t iu m -90 i n j e c t e d a n im a ls showed a s te e p r i s e i n th e mean enzyme v a lu e s . 38 TABLE X I I . Sample m ean, s | and nt " v a lu e s c a l c u l a t e d from mean a l k a l i n e p h o sp h a ta s e l e v e l s o f s t r o n t iu m -9 0 i n j e c t e d g ro u p s and combined c o n t r o l g ro u p s b e g in n in g a t 97 d ay s o f a g e . Number Sample Means sI tControl Sr-90 Control Sr-90 I ' 65 60 Hi 10 1 .05 NS 2 Ii3 36 9 2 3.68 * 3 l|6 Iili 6 8 0 .51 NS It 36 li9 5 15 3 .06 * . 5 38 59 7 27 S ta em n 6 38 68 6 35 li.77 * 7 1|2 79 8 73 lull * 8 3S 90 9 189 3.89 * 9 38 IllO 10 l , l l i 6 3.00 * 10 3S lli9 8 1,597 2.85 * 11 37 220 10 l i ,1|17 2,75 * 12 38 3U8 12 17 , IiOO 2.35 * 13 38 2l|0 9 U, 7U9 2.77 * 111 36 l|6 l lit 35,103 2.27 * IS 3U 363 10 11,590 3.05 * * - D i f f e r e n c e b e tw een means s i g n i f i c a n t . NS - D i f f e r e n c e b e tw een means n o t s i g n i f i c a n t . p S - - V a r ia n c e o f d i s t r i b u t i o n o f sam p le m eans. 39 TABLE X I I I . C a l c u la te d Mt n v a lu e s f o r mean a l k a l i n e p h o sp h a ta s e l e v e l s o f s t r o n t iu m -90 i n j e c t e d g roup and no rm al g roup from 2 t o 8 w eeks /b e fo re ro e n tg e n o g ra p h ic tum or d e t e c t i o n . Number o f Anim als i n G roup Weeks B e fo re D e te c t io n t a U8 2 5 .0 5 k8 h U..03 U8 6 3 .9 9 38 8 2 .UO 8 - D i f f e r e n c e be tw een means i s s i g n i f i c a n t . ? 1+0 TABLE XIV0 A com pa riso n o f p lasm a a l k a l i n e p h o sp h a ta s e l e v e l s in IIJ' b e fo r e and a f t e r a 16 m inu te e x p o su re o f $5>0 C. A lk a l in e p h o s p h a ta s e D ate o f - T e s t Mouse Number L e v e l B efo re H e a t in g A f t e r H e a t in g S-S 10 180 12 S-S lit 360 20 S-19 7 l,W+0 ISS S-19 11+ 1 ,1S2 1+6 6 -2 10 696 1+2 6 -2 20 702 20 6 -2 26 1+32 30 6 -S 18 2 ,l60a 270 " 6 -S 29 651+ "32 6 -S 30 1,368 66 a - A c tu a l v a lu e g r e a t e r th a n re c o rd e d f i g u r e . 10 0 20 0 CD H 1: 698- 7O I. F i n k e l , M .P ., B .O . B i s k is and C .A . R e i l l y . 1970 . I n t e r a c t i o n o f FBJ o s te o sa rcom a v i r u s w i th ^Ogr a n d 90gr o s te o s a rc om a s . 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