Imaging of Heavy Metal Distribution in Thin Tissue Sections of Itai-itai Disease Kidney and Rat Kidney by X-Ray Fluorescence Analysis

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I t a i - i t a i d i s e a s e i s a n e n d e m i c d i s e a s e o f T o y a m a p r e f e c t u r e , J a p a n , a n d o n c e w a s a b i g s o c i a l p r o b l e m . I t h a s b e e n c l a i m e d t h a t C d - p o l l u t e d J i n z u R i v e r w a t e r c a u s e d t h i s d i s e a s e b a s e d o n e p i d e m i o l o g i c a l s t u d i e s o f p a t i e n t Imaging of Heavy Metal Distribution in Thin Tissue Sections of Itai-itai Disease Kidney and Rat Kidney by X-Ray Fluorescence Analysis Itai-itai disease autopsy case *1 Cd administered rat *2 Hg administered rat *3 C A Fig. 1. Formaline fixed paraffin embedded kidney tissue sections. Section thickness is 3 μ m for HE and MT, 6 μ m for microbeam X-ray fluorescence ( XRF ) analysis ( Cd, Zn, and Hg ). HE=hematoxylin- eo si n st ai ni ng , MT =i mm un oh is to ch em is tr y wi th an ti -m et al lo th io ne in an ti bo dy . DA B wa s us ed as chromogen (the brown color is the signal). Bar = 1 mm. *1 Cd concentration is 16 ppm in the kidney cortex and 13 ppm in the medulla by AAS analysis. XRF : 32 keV X-rays were utilized for excitation. Beam size was approximately 100 μ m. The counting time was 2 sec/spot. Spatial resolution is 125 μ m /step. *2 Young male Wistar rat was used. Cd was administered by intraperitoneal injection of 2 mg Cd / ml C d C l 2 s o l u t i o n i n d i s t i l l e d w a t e r , 2 m g C d / k g b o d y w e i g h t . A f t e r e i g h t w e e k s o f b r e e d i n g w i t h standard laboratory food and water, the animal was sacrificed. Cd concentration is 43 ppm in the cortex, 3 ppm in the medulla by AAS analysis. XRF condition was same as shown in *1 above. *3 Young male Wistar rat was used. Hg was administered by intraperitoneal injection of 1 mg / ml HgCl 2 solution in distilled water. After the first injection of 1 mg HgCl 2 / kg body weight, the animal was bred for two weeks, injected with 2 mg HgCl 2 / kg body weight for the second time, bred for one week, and sacrificed. The Hg concentration is 32 ppm in the cortex, 1.2 ppm in the medulla by AAS analysis. XRF : 13 keV X-rays were utilized for excitation. The beam size was approximately 100 μ m. The counting time was 2 sec/spot. Spatial resolution is 125 μ m /step. distribution. However, the pathological contribution of Cd to itai-itai disease is still controversial. The kidney is one of the major organs damaged in itai- itai disease, and it is of great interest to investigate Cd distribution in itai-itai diseased kidneys since it is known that their Cd concentration is lower than that of non-itai-itai-disease kidneys. On the other hand, the relationship between Hg i n t o x i c a t i o n a n d M i n a m a t a d i s e a s e i s w e l l e s t a b l i s h e d . T h e p r o x i m a l u r i n a r y t u b u l e o f t h e kid ney is one of the tar get tis sue s for Hg tox ici ty, a n d t h e p r e c i s e l o c a t i o n i n t h e p r o x i m a l u r i n a r y tubules (segments [1]) sensible for Hg toxicity has 71 be en an al yz ed us in g di ss ec te d ne ph ro ns . Ho we ve r, t h e d i r e c t a n a l y s i s o f H g d i s t r i b u t i o n i n F o r m a l i n - f i x e d - p a r a f f i n - e m b e d d e d t i s s u e s e c t i o n s r o u t i n e l y u s e d i n h i s t o p a t h o l o g i c a l s t u d y h a s n o t b e e n reported. S o f a r , i t i s n o t p o s s i b l e t o v i s u a l i z e C d d i s t r i b u t i o n i n k i d n e y s e c t i o n s b y e i t h e r E P M A o r S I M S . Microprobe X-ray fluorescence analysis at beamline BL39XU [2] made this possible for the first time [3] ( Fig.1 a3 ). As a positive control, Cd-administered rat kidney was also analyzed ( Fig. 1 b3 ). Pictures o f h e m a t o x y l i n - e o s i n ( H E ) s t a i n e d s e c t i o n a n d i m m u n o h i s t o c h e m i s t r y w i t h a n t i - m e t a l l o t h i o n e i n (MT) antibody are also shown since Cd is known to exist bound to MT in the kidney ( Fig. 1 ). For Cd analysis, 32 keV X-rays were utilized for K -excitation. In Cd-administered rat kidney, Cd is d i s t r i b u t e d w i d e l y i n t h e c o r t e x ( F i g . 1 b 3 ) . T h e distribution pattern of Cd matches well with that of M T , c o m p a t i b l e w i t h t h e b i o c h e m i c a l f i n d i n g s s u g g e s t i n g t h a t C d e x i s t s b o u n d t o M T i n t h e k i d n e y . T h e d i s t r i b u t i o n o f M T i s l i m i t e d t o t h e e p i t h e l i u m o f t h e p r o x i m a l u r i n a r y t u b u l e s , suggesting that segment S1 +2 is the main portion of the Cd storage. In itai-itai diseased kidney, the Cd signal is limited to several spots in the medulla while the Cd signal in the cortex is background level ( Fig. 1 a3 ). These spots seem to be corresponding t o c o l l e c t i n g u r i n a r y d u c t s , w h i c h s h o w a s t r o n g signal in anti-MT immunohistochemistry ( Fig. 1 a2 ). Ho we ve r, th e pr ec is e id en ti fi ca ti on of th es e sp ot s should be carried out in the future since the outline o f t h e k i d n e y s t r u c t u r e i s n o t v i s i b l e a n d t h e distribution of MT is wider than that of the Cd spots. The distribution pattern of Zn partially corresponds to that of Cd, suggesting that some part of Zn also exists bound to MT. The XRF spectra measured in spots A and B (blank) are shown in Fig. 2 . A s a c o m p a r i s o n , d i s t r i b u t i o n o f H g i n H g - admi nist ered rat kidn ey was also anal yzed ( Fig. 1 c 3 ) . A l t h o u g h i t i s k n o w n t h a t t h e c a u s e o f M i n a m a ta d i se a se i s o rg a n i c H g i n cl u d i n g m e th yl - H g , H g C l 2 w a s a d m i n i s t e r e d t o t h e r a t a s a p i l o t study . In the case of Hg analy sis, 13 keV X-ray s were utilized for L -excitation. Images of Hg and Zn in pa ra ff in em be dd ed ti ss ue se ct io n ar e sh ow n in Fi g. 1 c3 an d c4 . In th is ex pe ri me nt , a st ro ng Hg de po si ti on wa s ob se rv ed in th e ar ea be tw ee n th e c o r t e x a n d t h e o u t e r m e d u l l a , s u g g e s t i n g t h a t s e g m e n t S 3 o f t h e p r o x i m a l u r i n a r y t u b u l e i s t h e m a i n p o r t i o n o f H g d e p o s i t i o n . T h e d i s t r i b u t i o n p a t t e r n o f H g d o e s n o t c o r r e s p o n d t o t h a t o f M T Fig. 2. XRF spectra measured in spots A and B (blank) in Fig. 1 a3. 0 100 200 300 400 500 600 700 0 5 10 15 20 25 30 35 X-ray energy (keV) counts Spot A Spot B (Blank) Cd Zn Compton 72 very well, which suggests the existence of ot he r bi nd in g pr ot ei ns fo r Hg . Th e XRF spectra measured in spots C and D (blank) are shown in Fig. 3 . With this X - r a y e n e r g y a n X - r a y m i c r o b e a m p r o d u c e d w i t h t h e n e w l y i n s t a l l e d K i r k p a t r i c k - B a e z m i r r o r s y s t e m [ 4 , 5 ] was also available. The beam size was 4 μ m × 4 μ m in this measurement, and Fi g. 4 sh ow s th e re su lt an t im ag e of a small area around spot C in Fig. 1 c3 . M i c r o b e a m X - r a y f l u o r e s c e n c e analysis is found to be a useful method t o a n a l y z e h e a v y m e t a l d i s t r i b u t i o n i n thin tissue sections. Distributions of Cd a n d H g i n p a r a f f i n e m b e d d e d h u m a n and rat kidneys were demonstrated for th e fi rs t ti me . Th is me th od wi ll be an im po rt an t to ol to an al yz e he av y me ta l toxicosis, including itai-itai disease and Minamata disease. References [1] W. Kriz and L. Bankir, Kidney Int. 33 (1988) 1. [ 2 ] S . H a y a k a w a e t a l . , J . S y n c h r o t r o n R a d . 5 (1998) 1114. [3] J. Kawai et al. , J. Trace Microprobe Tech. 19-4 (2001) 541. [4] S. Hayakawa, N. Ikuta, M. Suzuki M. Wakatsuki a n d T . H i r o k a w a , J . S y n c h r o t r o n R a d . 8 ( 2 0 0 1 ) 328. [ 5 ] S . H a y a k a w a , S . T o h n o , K . T a k a g a w a , A . Hamamoto, Y. Nishida, M. Suzuki, Y. Sato and T. Hirokawa, Anal. Sci. - in press. of a small area around spot C in Fi g. 1 c3 . XR F : Th e en er gy of X- ra y fo r e x c i t a t i o n w a s 1 3 k e V . T h e b e a m s i z e w a s appr oxim atel y 4 μ m × 4 μ m. The coun ting time was 2 sec/spot. Spatial resolution is 5 μ m /step. The signal seems to be located in the epithelium of the urinary tubules since the width of band-like Hg positive area ( * ) is approximately the same as the diameter of the urinary tubules. Fig. 3. XRF spectra measured in spots C and D (blank) in Fig. 1 Kiyoshi Takagawa a and Shinjiro Hayakawa b (a) Toyama Medical and Pharmaceutical University (b) Hiroshima University E-mail: takagawa @ ms.toyama-mpu.ac.jp 0 50 100 150 200 0 2 4 6 8 10 12 14 X-ray energy (keV) counts Spot C Spot D (Blank) Zn Compton Hg Fe c3. 73 Fig. 4. Hg imaging