15. 5. 1977

667

Speeialia

lines d r a w n a t t h e v a l u e 13 in figure 2 were a r b i t r a r i l y e s t a b l i s h e d to a c t as a guideline for c o m p a r a t i v e p u r poses. A f t e r 12 weeks of t r a i n i n g , b o t h t h e S P T a n d E N D g r o u p s d e m o n s t r a t e d a ' r e t a r d a t i o n effect' i n m o t o r n e u r o n size w h e n c o m p a r e d to 12-week c o n t r o l distrib u t i o n (figures 1 a n d 2). F u r t h e r m o r e , t h e degree of ' r e t a r d a t i o n ' is a p p a r e n t l y d e p e n d e n t o n t h e i n t e n s i t y of exercise used. T h i s is show/1 ilz figures 1 a n d 2 w i t h t h e S P T g r o u p d e m o n s t r a t i n g a g r e a t e r n u m b e r of s m a l l e r s o m a a n d nuclei t l l a n t h e E N D group. T h e s e f i n d i n g s are in c o n t r a s t t o earlier s t u d i e s r e p o r t i n g n o c h a n g e s in m o t o r n e u r o n m o r p h o l o g y b e t w e e n c o n t r o l a n d experim e n t a l g r o u p s following c h r o n i c a c t i v i t y *, 10,11. I t is t h e o p i n i o n of t h e a u t h o r s t h a t t h e s e o b s e r v e d differences are p r i m a r i l y d u e to 2 r e a s o n s : a i t h e t r a i n i n g i n t e n s i t i e s

used in earlier s t u d i e s were n o t g r e a t e n o u g h t o p r o d u c e c h a n g e s , a n d b) t h e s t a t i s t i c a l t r e a t m e n t of t h e d a t a m a y have masked any changes - any statistical analyses c o m p a r i n g t h e m e a n s of t h e g r o u p s in t h i s s t u d y w o u l d h a v e r e v e a l e d n o s i g n i f i c a n t differences b e t w e e n t h e 3 groups. I n fact, h o w e v e r , t h e r e are s i g n i f i c a n t d i s t r i b u t i o n differences as e v i d e n c e d b y t h e figures 1 a n d 2. A p r o b a b l e m a t u r a t i o n effect c a n b e o b s e r v e d b e t w e e n t h e c o n t r o l zero-week a n i m a l s a n d t h e c o n t r o l 12-week a n i m a l s (table 2). T h a t is, t h e c o n t r o l 12-week d i s t r i b u t i o n shows a g r e a t e r n u m b e r of l a r g e r m o t o r n e u r o n s . I n conclusion, i t is e v i d e n t t h a t specific differences h a v e b e e n o b s e r v e d for t h e t r e a t m e n t s used. I t a p p e a r s t h a t m o t o r n e u r o n s in a s t a t e of ' n o r m a l i t y ' reflect p a t t e r n s of c h a n g e w h i c h are specific to v a r i o u s c h r o n i c exercise regimens.

Suppression of delayed type hypersensitivity of mice by lead S. lV[iiller, t ( . - E . Gillert, Ch. K r a u s e 1, U. Gross ~, J. L ' A g e - S t e h r 3 a n d T. D i a m a n t s t e i n 3 , l

Robert-Koch-lnstitut,

Nordu/er 20, D-IO00 Berlin (West) 65, 27 september 7976

Summary. A p p l i c a t i o n of lead s u p p r e s s e d t h e d e l a y e d t y p e h y p e r s e n s i t i v i t y ( D T H ) of mice i n d u c e d b y s h e e p r e d b l o o d cells (SRBC). I n h i b i t i o n of e l i e i t a t i o n of DTI-t in p r i m a r y as well as in s e c o n d a r y r e s p o n s e was c o r r e l a t e d w i t h t h e c o n c e n t r a t i o n of lead in t h e b l o o d of t h e mice. It has been shown that several environmental contamin a n t s 5 i n c l u d i n g s u b c l i n i c a l doses of lead 6 r e d u c e t h e r e s i s t a n c e of mice t o b a c t e r i a l infection. As r e p o r t e d recently, c h r o n i c e x p o s u r e of mice t o lead s u p p r e s s e s t h e h u m o r a l a n t i b o d y r e s p o n s e t o s h e e p r e d Mood cells (SRBC) as m e a s u r e d b y t h e n u m b e r of a n t i - S R B C prod u c i n g spleen cells 5. T h e a u t h o r s c o n c l u d e d t h a t t h e

91

;d

6.5 ..... 1'.0

1'.5

2'.0ppm lead

Fig. 1. Dose-dependent inhibition of DTH in mice exposed to lead. Levels of DTH elicited by inoculation of l0 s SRBC into the right hind footpad of balb/c mice (6-8 weeks old) sensitized 4 days previously wittl 106 SRBC i.v. DTH was measured 24 h after antigen challenge. Various amounts of lead acetate were injected i.p. in a volume of 0.5 ml saline daily for 30 days. In tile first experiment the mice received 0.25 mg, 0.10 mg and 0.025 mg lead acetate daily (9 In the second experiment the mice received 0.20 rag, 0.10 rag, 0.075 rag, 0.05 mg and 0.025 mg lead acetate daily (e). The lead content in the blood of mice was determined by means of an atomic absorption spectrophotometer immediately after DTH-measurement. Abscissa: ppm lead detected in whole blood. Ordinata: units of footpad reaction (1 unit = 0.1 ram). Each point represents the mean value detected in each group of i0 mice. In the group o1 highest lead concentration, 4 mice died in consequence of lead intoxication.

i m p a i r e d r e s i s t a n c e to t h e b a c t e r i a l i n f e c t i o n was p r o b a b l y d u e to a s u p p r e s s e d h u m o r a l a n t i b o d y f o r m a t i o n . B u t o t h e r w i s e n o t o n l y t h e h u m o r a l b u t also t h e cell m e d i a t e d i m m u n i t y (CMI), as well as n o n s p e c i f i c defense m e c h a n i s m s d u e to p h a g o c y t i c cells, are i n v o l v e d in t h e s e processes. W e i n v e s t i g a t e d t h e effect of c h r o n i c lead e x p o s u r e on t h e T - l y m p h o c y t e m e d i a t e d a n d m o n o c y t e d e p e n d e n t del a y e d t y p e h y p e r s e n s i t i v i t y ( D T H ) a g a i n s t S R B C in mice. 10 m i c e i n e a c h g r o u p were i n j e c t e d i.p. o n c e d a i l y for 30 d a y s w i t h v a r i o u s a m o u n t s of lead a c e t a t e dissolved in saline. O n t h e 3 0 t h d a y t h e mice were s e n s i t i z e d w i t h 105 S R B C i.v. To assess t h e D T H mice were c h a l l e n g e d 4 d a y s a f t e r s e n s i t i z a t i o n b y i n j e c t i o n of 10 s S R B C i n 40 [zl of saline i n t o t h e r i g h t h i n d f o o t p a d . As c o n t r o l s n o n - s e n s i t i z e d mice were i n j e c t e d i n t o t h e f o o t p a d w i t h t h e s a m e dose of a n t i g e n . F o r this, l e a d - t r e a t e d as well as n o n - t r e a t e d a n i m a l s were used. T h e f o o t p a d swelling was m e a s u r e d w i t h a dial g a u g e caliper before a n t i g e n c h a l lenge, 24 h, a n d 48 h a f t e r c h a l l e n g e a c c o r d i n g t o Miller e t al.L T h e mice were killed a n d t h e lead c o n t e n t of t h e b l o o d was d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o photometry.

1 2 3 4

5 6 7

Institut ftir Wasser-, Boden- und Lufthygiene, Corrensplatz 1, D-1000 Berlin (West) 33. Institut fiir Pathologie, Klioikum Steglitz, Freie Universit~it 13erlin, Hindenburgdamm 3if, D-1000 Berlin (West) 45. Immunologische Forschungseinheit, Klinikum Steglitz, Freie Universitgtt Berlin, Hindenburgdamm 30, D-1000 Berlin (West) 45. Acknowledgments. Supported by the 'Georg-und-Agnes-Blumenthal-Stiftung' and the 'Verein fiir Wasser-, 13oden- und Lufthygiene'. We thank Miss Sehachtsehneider for the excellent technical assistance. L.D. Koller and S. Kovacic, Nature 250, 148 (1974). F.E. Hemphill, M. L. Kaeberle and W. ]3. Buck, Science 772, 1031 (1971). T . E . Miller, G. 13. Maekaness and P. H. LaGrange, J. Nat. Cancer Inst. 51, 1669 (1973).

668

Speeialia

I t is of i n t e r e s t to c o m p a r e o u r d a t a o n lead a b s o r p t i o n w i t h t h o s e f o u n d in m e n ; b l o o d levels of lead u p to 0.2 p p m are c o n s i d e r e d n o r m a l , clinical signs of t o x i c i t y a p p e a r u p w a r d of 0.35 p p m s. I n t h e b l o o d of c o n t r o l mice 0.05 p p m lead was d e t e c t e d . T h e b l o o d levels in t h e leadt r e a t e d mice rose a c c o r d i n g t o t h e dose of a d m i n i s t e r e d lead u p t o 2 p p m . S y m p t o m s of t o x i c i t y c o u l d b e seen in t h e mice o n l y a t t h e l a t t e r h i g h b l o o d level of lead. T h e doses g i v e n were t h e s a m e as t h o s e r e p o r t e d b y H e m p h i l l e t al. 5.

~'4 E

oo l.z-

., od'

24 Timeafter challenge

48 h

Fig. 2. Inhibition of the secondary DTH-response by lead. 10 mice were injected with 10 ~ SRBC i.v. and challenged 4 days later with 108 SRBC into the right hind footpad. After 4 days DTH was elicited by inoculation of 108 SRBC into the left hind footpad. The inflammatory response was followed for 2 days. - - - , saline; . . . . , 0.25 mg lead acetate; . . . . . . . 0.10 mg lead acetate;-..-..-..-, 0.025 mg lead acetate. Each point represents the mean value detected in eaeh group of mice. Vertical bars represent the SE of the mean.

EXFERIENTIA 33/5

T h e l y m p h n o d e s a n d t h y m i of t h e l e a d - e x p o s e d a n i m a l s showed, as c o m p a r e d to t h e controls, n o s i g n i f i c a n t m o r p h o l o g i c a l changes. H o w e v e r , in some cases t h e m a r g i n a l zone of t h e w h i t e spleen p u l p of l e a d - t r e a t e d anim a l s s e e m e d to be smaller a n d c o n t a i n e d a lower n u m b e r of cells t h a n t h a t of controls. T h e r e s u l t s of 2 i n d e p e n d e n t e x p e r i m e n t s s u m m a r i z e d in figure 1 clearly d e m o n s t r a t e a d o s e - d e p e n d e n t s u p p r e s s i o n of D T H - r e s p o n s e b y lead acetate. To assess t h e effect of lead o n t h e s e c o n d a r y D T H response, g r o u p s of mice r e c e i v i n g v a r i o u s doses of lead for 30 d a y s were s e n s i t i z e d w i t h 105 S R B C i.v. ; c h a l l e n g e d 4 d a y s l a t e r w i t h 10 s S R B C i n t o t h e r i g h t h i n d f o o t p a d a n d r e c h a l l e n g e d w i t h t h e s a m e a n t i g e n dose i n t o t h e left h i n d f o o t p a d a f t e r a n o t h e r 4 d a y s (figure 2). Similar t o t h e p r i m a r y r e s p o n s e l e a d - t r e a t e d a n i m a l s s h o w e d also a suppressed secondary DTH-response. T h e q u e s t i o n arises w h e t h e r lead e x p o s u r e m a y a f f e c t t h e a f f e r e n t or e f f e r e n t arc of D T H . I n o t h e r words, does t h e e x p o s u r e of t h e a n i m a l s to lead affect t h e g e n e r a t i o n of sensitized l y m p h o c y t e s r e s p o n s i b l e for D T H , or does lead affect t h e e x p r e s s i o n of a l r e a d y e s t a b l i s h e d DTI-I ? T h e r e s u l t of a p r e l i m i n a r y e x p e r i m e n t ( d a t a n o t g i v e n here) seems t o a r g u e a g a i n s t t h e first possibility. Spleen cells of s e n s i t i z e d n o r m a l mice, as well as spleen cells d e r i v e d f r o m d o n o r s e x p o s e d p r e v i o u s l y t o lead, b o t h elicited D T H w h e n t r a n s f e r r e d i n t o n o r m a l recipients. If t h i s is t r u e , t h e m o s t p r o b a b l e t a r g e t s for lead effects seem t o b e m o n o c y t e s k n o w n t o b e i n v o l v e d in t h e e x p r e s s i o n of cellm e d i a t e d i m m u n i t y 9 a n d m a c r o p h a g e s p a r t i c i p a t i n g in i n d u c t i o n (or m o d u l a t i o n ) of h u m o r a l i m m u n e responses 10. 8

Kommission der Europ~ischen Gemeinschaft, Komm. (75) 166, Brtissel, April 1975. 9 Y.L. Turk, ill: Delayed Hypersensitivity ill Frontiers of Biology, voh 4, p. 235. Ed. A. Neuberger and E. L. Tatum. North Holland Publishing Company, Amsterdam 1975. 10 Z.A. Cohn, Adv. Immun. 9, 164 (1968).

Degradation of [SH]thymidine by a pentosyltransferase (EC 2.4.2.4) in the plasma of man and different animals 1 J. L. P a u l y , M. G. Schuller, A. A. Zelcer a n d 3/[. J. G e r m a i n

Department o/Medicine B, Roswell Park Memorial Institute, New York State Department o/Health, 666 Elm Street, Buffalo (New York 14263, USA), 18 November 7976 Summary. [ 3 H ] T h y m i d i n e is d e g r a d e d b y a n e n z y m e ( t h y m i d i n e p h o s p h o r y l a s e ; E C 2.4.2.4) w h i c h we h a v e identified in t h e p l a s m a of m a n a n d s o m e a n i m a l s . T h e p r e s e n c e of t h i s e n z y m e in p l a s m a or sera u s e d t o s u p p l e m e n t c u l t u r e m e d i a m a y , u n d e r c e r t a i n e x p e r i m e n t a l c o n d i t i o n s , l i m i t t h e v a l i d i t y of m e a s u r i n g t h e u p t a k e of r a d i o l a b e l e d t h y m i d i n e as a m e a n s of d e f i n i n g D N A s y n t h e s i s . T h y m i d i n e p h o s p h o r y l a s e (EC 2.4.2.4; t h y m i d i n e - o r t h o phosphate deoxyribosyltransferase) catalyses the reversible r e a c t i o n of t h y m i d i n e (TdR) + o r t h o p h o s p h a t e t h y m i n e (Th) + 2 - d e o x y - e - D - r i b o s e - l - p h o s p h a t e 3, 8. T h i s e n z y m e is p r e s e n t i n m a n y n o r m a l a n d n e o p l a s t i c ceils of m a n a n d d i f f e r e n t a n i m a l s 2-13 as well as in v a r i o u s p a t h o g e n i c a n d n o n p a t h o g e n i c p r o k a r y o t i c cells 2, 3,14. W e r e p o r t h e r e t h e i d e n t i f i c a t i o n of T d R p h o s p h o r y l a s e in t h e p l a s m a of m a n a n d s o m e a n i m a l s , a n d discuss t h o s e l i m i t a t i o n w h i c h t h i s e n z y m e m a y h a v e o n a s s a y s in w h i c h [aI-t]TdR u p t a k e is u s e d as a m e a n s of d e f i n i n g DNA synthesis.

Material and methods. P l a s m a a s s a y e d was f r o m fresh b l o o d w h i c h h a d b e e n collected a s e p t i c a l l y i n t o siliconized t u b e s c o n t a i n i n g E D T A a n t i c o a g u l a n t (1.5 m g / m l ) . A f t e r d e p o s i t i n g t h e cells b y c e n t r i f u g a t i o n (500 • 12 min), t h e p l a s m a was collected c a r e f u l l y and, in some i n s t a n c e s , p a s s e d t h r o u g h a m i c r o p o r e (0.22 tzm) filter; p a r t i c u l a r care was used in h a n d l i n g t h e b l o o d so as t o a v o i d cont a m i n a t i o n or cell d a m a g e . T d R p h o s p h o r y l a s e was evalu a t e d u s i n g o u r m i c r o a s s a y l ~ t h i s is a m o d i f i c a t i o n of t e c h n i q u e s w h i c h h a v e b e e n d e s c r i b e d p r e v i o u s l y ~, 5-~, 11 a n d w a s c o n s t r u c t e d t o s i m u l a t e c o n d i t i o n s p r e v a l e n t in m i c r o c u l t u r e s . I n t h i s assay, 20 [zl of p l a s m a w a s a d d e d