Subcellular distribution of 111In and 169Yb in tumor and liver

Eur J NucI Med (1981) 6:221-226 European Nuclear Journal of

Medicine © Springer-Verlag 1981

Subcellular Distribution of 111In and 169"yb in Tumor and Liver

Atsushi Ando, Itsuko Ando, Masazumi Takeshita, Tatsunosuke Hiraki, and Kinichi Hisada Schools of Paramedicine and Medicine, Kanazawa University, Kanazawa, and Medical College of Oita, Oita, Japan

Abstract. Subcellular distribution of 1,1in and 1 6 9 y b

was quantitatively determined to evaluate the role of the lysosome in accumulation of these nuclides in malignant tumor tissue and in the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of these nuclides was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of these nuclides and reached approximately 50% of the total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30% of the total radioactivity was concentrated in this fraction after 24 h. It is concluded that the lysosome does not play a major role in the tumor concentration of these nuclides, although it may play an important role in their liver concentration. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of these nuclides, hepatoma AH109A possessing some residual features of the liver.

Introduction

Edward and Hayes (1969) reported that 67Ga-citrate concentrated in soft tissue tumors in humans. Tumor affinity of 1, lin_chlorid e was first reported by Hunter et al. (Hunter and Dekock 1969; Hunter and Ricco- bono 1970), and that of 169yb-citrate was first report-

Offprint requests to: Atsushi Ando, Ph.D. , School of Paramedi- cine, Kanazawa University, 5-11-80, Kodatsuno, Kanazawa, 920 Japan

ed by us (Hisada and Ando 1973). Although the mechanism of localization of 6VGa in malignant tumors has been extensively investigated, there have been few such studies on 111In (Takeda et al. 1977; Hagan et al. 1977) and t69yb. The present study was undertaken to elucidate the mechanism of tumor affinity of 111In and 169yb.

Materials and Methods

The following animals and transplante d tumors were used: donryu rats implanted with Yoshida sarcoma and hepatoma AH109A; ddY mice implanted with Ehrlich tumor. Carrier-free l~lIn-citrate solution, pH 6.0 8.0 (approximately 1 ml containing 10 ~tCi of

~ ~In), was prepared from 111in_chloride solution and 0.08 M sodium citrate solution. ~69yb-citrate solution, pH 6.0 8.0 (approximately 1 ml containing 5 gCi of 169yb and 0.25 ~tg of Yb) was prepared from 169yb~hloride solution and sodium citrate solution.

111In-citrate solution (0.4 ml) was injected IV into the rats and IP into the mice. Ten minutes, 1 h, 3 h, 24 h, and 48 h after the administration of H~In-citrate solution, these animals were anesthetized with sodium pentobarbital injection, and tumor tissues and liver were excised. The tumor tissues and the liver were homog- enized in cold (5 ° C) 0.25 M sucrose -0.01 M Tris (10% w/v) in a Potter-Elvehjem type homogenizer. According to the modified method of Hogeboom and Schneider (Fig. 1), subcellular fractiona-

Tumor or Liver

Homogenize in 0.25M sucrose- Tris O.OIM, pH 7.6

filter(nylon mesh) and centrifuge at 2,000rpm 15 min

1 ! Nuclei 5,000Xg 15 min (AI [

I ]05,000Xg 60 min

I [ . .[

Supernatant I crosomes (D) (C)

Fig. 1. Preparation of subcellular fractions

I Mitochondria

(B)

0340-6997/81/0006/0221/$01.20

222 A. Ando et al. : Subcellular Distribution of H~In and i69yb in Tumor and Liver

tion was carried out at 4 ° C. Fractions from the centrifugation were assayed for radioactivity of 11~In by a well-type scintillation counter. 169yb citrate solution (0.4 ml) was also injected IV into the rats and IP into the mice. These animals were treated by the same procedure used for animals injected with a* ~In-citrate.

Protein in five samples of each fraction was measured by Lowry's method (Lowry et al. 1951) to determine the amoun t of protein in each fraction. Bovine serum albumin was used as a standard.

Results

When radioactivity of nuclear fraction, mitochondrial fraction, microsomal fraction, and supernatant fraction, is expressed as A (cpm), B (cpm), C (cpm), and D (cpm), respectively, radioactivity (percentage) of the nuclear fraction can be calculated by the following formula:

A x 100%.

A + B + C + D

Radioactivity of mitochondrial fraction, microsomal fraction, and supernatant fraction was calculated by

substitution of A with B, C, and D in the numerator. Radioactivity of each fraction of three different tumor and the liver samples are shown in Tables 1 and 2.

In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity from l~In-citrate was localized in the supernatant fraction, and a small amount was localized in the mitochondrial fraction (lysosome is

contained in this fraction), nuclear fraction, and microsomal fraction. But in the liver of rats and mice, l i l In in the mitochondrial fraction increased with time after the administration of l~aIn-citrate. About 50% of total radioactivity was accumulated 24 h later in this fraction. The amounts of ~alIn concentrated in this fraction reached a plateau at this time. Con- trary to data for the mitochondrial fraction, radioactivity of the supernatant fraction decreased with time until 24 h after the administration.

In the case of hepatoma AH109A, ll~In in the mitochondrial fraction increased with time until 24 h after administration and about 27% of the total radioactivity was concentrated in this fraction. Contrary to data for the mitochondrial fraction, ~ ~ ~In in supernatant decreased with time until 24 h after administration.

In the case of a69yb-citrate, radioactivity of each fraction was similar to the results obtained using

~lIn-citrate. But in Yoshida sarcoma and hepatoma AH109A, radioactivity of the microsomal fraction after ~69yb-citrate was more than that of the micro- soma1 fraction after a 1 lIn-citrate. In the case of Ehr- lich tumor, host liver, and hepatoma AH109A, the relation between these nuclides in the mitochondrial fraction and in the supernatant fraction is indicated in Fig. 2. It is shown in this figure that these nuclides were transposed from the supernatant fraction to the mitochondrial fraction. Transposition of these nu-

Table 1. Subcellular distribution (%) of l l~In and I69yb in experimental tumors. Each value is expressed as a mean of three experiments

111in_citrate ~69yb_citrate

Nuclear Mitochondrial Microsomal Supernatant Nuclear fraction fraction fraction fraction fraction

Mitochondrial Microsomal Supernatant fraction fraction fraction

Yoshida sarcoma

10 rain 14.2 9.6 15.2 61.0 16.8 60 min 16.3 14.9 15.4 53.4 19.4

3 h 16.3 12.0 9.5 62.2 25.9 24 h 17.0 11.5 14.3 57.2 21.1 48 h 15.5 11.9 15.2 57.4 24.3

Ehrlich tumor

10 min 15.3 8.0 7.3 69.4 20.6 60 min 12.5 12.4 6.8 68.3 29.0

3 h 8.8 9.0 7.5 74.7 23.0 24 h 13.2 11.3 15.5 60.0 22.8 48 h 12.7 12.6 13.5 61.2 22.2

Hepatoma AH 109A

10 min 9.8 8.1 11.4 70.7 21.8 60 rain 10.3 8.8 12.5 68.4 20.1

3 h 12.3 18.9 18.1 50.7 25.8 24 h 14.0 27.5 20.3 38.2 26.1 48 h 15.3 26.1 19.7 38.9 23.0

15.6 37.0 30.6 19.4 34.8 26.4 22.2 26.8 25.1 25.7 32.8 20.4 24.6 26.6 24.5

15.3 32.0 32.1 16.4 22.1 32.5 15.8 21.9 39.3 16.1 29.2 31.9 18.0 26.8 33.0

10.5 40.8 26.9 17.1 40.2 22.6 25.0 31.1 18.1 26.0 30.8 17.1 28.8 35.5 12.7

A. Ando et al. : Subcellular Distribution of t ~ I n and 169yb in Tumor and Liver

Table 2. Subcellular distribution (%) of l ~ I n and 169yb in host liver. Each value is expressed as a mean of three experiments

223

11 l i n _ c i t r a t e t 6 9yb_citrate

Nuclear Mitochondrial Microsomal Supernatant Nuclear Mitochondrial Microsomal Supernatant fraction fraction fraction fraction fraction fraction fraction fraction

Rats with transpIanted Yoshida sarcoma

10 rain 16.9 18.1 14.7 50.3 13.4 13.4 28.7 44.5 60 rain 14.5 24.2 24.5 36.6 11.2 23.5 18.3 47.0

3 h 18.5 37.2 14.1 30.2 25.5 27.5 14.9 32.1 24 h 12.4 54.5 13.7 19.4 20.6 43.2 16.3 19.9 48 h 16.8 54.7 11.6 16.9 22.2 55.1 13.8 8.9

Mice with transplanted Ehrlich tumor

10 rain 11.7 9.1 13.6 66.6 16.2 7.5 22.9 53.4 60 min I2.0 13.7 12.8 57.5 12.5 12.5 13.1 61.9

3 h 12.2 26.1 19.5 42.2 21.0 20.1 16.3 42.6 24 h I7.3 48.4 16.4 17.9 18.0 40.2 23.6 18.2 48 h 19.2 47.8 18.4 14.6 19.2 45.6 19,9 15,3

Rats with transplanted hepatoma AHI09A

10 rain 11.7 19.8 13.1 55.4 21.7 11.0 33.0 34.2 60 rain 7.8 24.5 14.6 53. I 15.1 16.5 30.1 38.3

3 h 10.2 30.9 15.9 43.0 29.0 26.7 21.5 22.8 24 h 12.0 45.2 15.1 26.7 25.1 42.2 17.3 15.4 48 h 13.3 47.6 i7.0 22. i 22.6 40.9 21.6 14.9

111In - c i t r a t e 1 6 9 y b - c i t r a t e

%

80

60

40

20

E h r l i c h t u m o r

S u p . f .

M i t f .

I/---------~

• I I °

~4 I t & hr~

%

80

60

40

2 0

Ehrlich tumor

Mit . f . = =

~ c o

• 4 k - ~

24 4B hrs

%

80

60

L*O

20

Hepatoma AHIO9A

~ k ~ . . S up. f.

Nit f.

J I 3 24 { } 48 h r s

%

80

60

40

1 3

Hepatoma AH 109A

N i t . f .

Sup. f. ~

24 ~/ ] 4'8 hrs

%

60

40

20

% Normal liver

/ ,0

20

" ' I I ' 1 3 24 48 hrs

Normal liver

. . . . . . I I 1 3 24 4B hrs

Fig. 2. Distribution of ~11In (%) and 169yb (%) between mitochondrial and supernatant fractions. It is shown that these nuclides were transposed from supernatant fraction to mitochondrial fraction. Transposit ion of these nuclides from supernatant to mitochondrial fraction readily occurred in the liver but only slightly in Ehrlich tumor

224 A. Ando et al. : Subcellular Distribution of 111In and 169yb in Tumor and Liver

clides from supernatant to mitochondrial fraction occurred readily in the liver but only slightly in Ehrlich tumor.

Total protein and subcellular distribution of protein in experimental tumors and host livers are shown in Table 3. In these tumors, the greater amount of protein was in the supernatant fraction, while 8.2- 13.7% of total protein was in the mitochondrial fraction. In the liver, the greater amount of protein was in the supernatant fraction, and 23.5-24.9% of total protein was in the mitochondrial fraction.

To determine the relative specific activity of the two nuclides to protein in each fraction, the radioactivity in each fraction was divided by the quantity of protein in that fraction. Results of tumor tissues are shown in Table 4 and those of the livers are shown in Table 5. In three tumors, relative specific activities of ~ ~ 1In in the nuclear fraction were lower than those in other fractions. In hepatoma AH109A, the relative

specific activity of ~ 11in in the mitochondrial fraction increased with time while that in the supernatant fraction decreased with time. The values of the relative specific activities tend to reach a plateau at 24 h after administration. In the case of 169yb-citrate, the relative specific activity of the nuclear fraction in Yoshida sarcoma and of the supernatant fraction in hepatoma AH109A was lower than in the other fraction. In hepatoma AH109A, the relative specific activity of 1 6 9 y b in mitochondrial fraction increased with time while that in the microsomal fraction decreased with time. The values of the relative specific acitivities tend to reach a plateau at 24 h after administration.

In the liver, the relative specific activities of 111In and 169yb in the mitochondrial fraction increased with time, while those in the supernatant fraction decreased with time (Table 5), these values of relative specific activity tend to reach a plateau at 24 h after administration.

Table3. Total protein (mg) and subcellular distribution (%) of the protein in experimental tumor and host liver

Nuclear Mitochondrial Microsomal Supernatant Total protein (rag) fraction fraction fraction fraction in tissues of 100 mg

Yoshida sarcoma 38.0 13.7 8.5 39.8 8.31 Ehrlich tumor 21.6 8.2 10.2 60.0 6.46 Hepatoma AH109A 15.4 10.3 20.3 54.0 7.90 Liver of rat 17.1 23.5 18.4 41.0 15.82 Liver of mouse 20.6 24.9 17.2 37.3 15.22

Table 4. Relative specific activities (%/mg) of the protein in the nuclear, mitochondrial, microsomal and supernatant fractions of experimental tumors. Relative specific activities were calculated by dividing the values of Table 1 by the protein (rag) of each fraction

111 In-citrate 169yb_citrate


Yoshida sarcoma

10 min 4.5 60 min 5.2

3 h 5.2 24 h 5.4 48 h 4.9

Ehrlich tumor

10 min 10.9 60 rain 8.9

3 h 6.3 24 h 9.4 48 h 9.1

Hepatoma AH 109A

10 min 8.0 60 rain 8.4

3 h 10.1 24 h 11.5 48 h 12.5

8.4 21.4 18.4 5.3 13.7 52.1 9.2 13.1 21.7 16.1 6.1 17.0 49.0 8.0 10.5 13.4 18.8 8.2 19.5 37.7 7.6 10.1 20.1 17.3 6.7 22.5 46.2 6.2 I0.4 21.4 17.3 7.7 21.6 37.5 7.4

15.1 11.1 17.9 14.7 28.9 48.5 8.3 23.4 10.3 17.6 20.7 30.9 33.5 8.4 17.0 11.4 19.3 16.4 29.8 33.2 10.1 21.3 23.5 15.5 16.3 30.4 44.2 8.2 23.8 20.5 15.8 15.9 34.0 40.6 8.5

10.0 7.1 16.6 17.9 13.0 25.5 6.3 10.9 7.8 16.0 16.5 21.1 25,1 5.3 23.3 11.3 11.9 21.1 30.9 19,4 4.2 24.0 12.7 8.9 21.4 32.1 19,3 4.0 32.2 12.3 9.1 18.9 35.6 22.2 3.0

A. Ando et al. : Subcellular Distribution o f 1lain and 169yb in Tumor and Liver 225

Table 5. Relative specific activities (%/mg) of the protein in the nuclear, mitochondrial, microsomal, and supernatant fractions of host livers. Relative specific activities were calculated by dividing the values of Table 2 by the protein (rag) of each fraction

111In_citrate 169yb-citrate


Rats with transplanted Yoshida sarcoma

10 min 6.2 4.9 5.1 7.8 4.9 3.6 9.9 6.9 60 min 5.4 6.5 8.4 5.6 4.1 6.3 6.3 7.2

3 h 6.8 10.0 4.8 4.7 9.4 7.4 5.1 4.9 24 h 4.6 14.7 4.7 3.0 7.6 11.6 5.6 3.1 48 h 6.2 14.7 4.0 2.6 8.2 14.8 4.7 1.4

Mice with t ransplanted Ehrlich tumor

10 min 3.7 2.4 5.2 11.7 5.2 2.0 8.7 9.4 60 min 3.8 3.6 4.9 10.1 4.0 3.3 5.0 10.9

3 h 3.9 6.9 7.4 7.4 6.7 5.3 6.2 7.5 24 h 5.5 12.8 6.3 3.2 5.7 10.6 9.0 3.2 48 h 6.1 12.6 7.0 2.6 6.1 12.0 7.6 2.7

Rats with transplanted hepatoma AH109A

10 rain 4.3 5.3 4.5 8.5 8.0 3.0 11.3 5.3 60 min 2.9 6.6 5.0 8.2 5.6 4.4 10.3 5.9

3 h 3.8 8.3 5.5 6.6 i0.7 7.2 7.4 3.5 24 h 4.4 12.2 5.2 4.1 9.3 11.3 5.9 2.4 48 h 4.9 12.8 5.8 3.4 8.3 11.0 7.4 2.3

Discussion

111In and 169yb are elements in group III of the periodic table, which includes 6 7 G a . All three elements have similar chemical properties, and likewise it is thought that these elements have similar biologi- cal properties.

Many studies have been reported about the mechanism of tumor affinity of 67Ga. There are two opin- ions about the mechanism of accumulation of 67Ga in tumor tissues. One is the view that lysosome plays an important role in the tumor concentration of 67Ga (Swartzendruber et al. 1971 ; Brown et al. 1973; Brown et al. 1976; Takeda et al. 1977; Takeda et al. 1978), and the other is the view that lysosome does not play an important role in the tumor concentration of 67Ga (Deckner et al. 1971; Ito et al. 1971; Orii 1972). Takeda et al. (1977) reported that lysosomes are the site of accumulation for 67Ga and aaaIn, whether in tumor or normal liver cells. Hagan et al. (1977) reported that viable tumor concentration of ~1~In exceeded the nonviable tumor concentration during the first 48 h after the injection of ~ ~ ~In-chloride. We reported that ~a~In and ~69yb was more predominant in connective tissue (especially inflammatory tissues) than in viable tumor tissue, regardless of the time after administration (Ando et al. 1978).

From our present study, it was clear that lysosome

did not play an important role in the tumor concentration of ii1In and 169yb in Yoshida sarcoma and in Ehrlich tumor, but the substance (which was contained in the mitochondrial fraction) played an important role in the liver concentration of these nuclides and played a considerably important role in the tumor concentration of these nuclides in hepatom a AH109A. From the paper previously published (Tak- eda et al. 1977), it was thought that the subcellular component which plays a role in liver and hepatoma AH109A concentration of these nuclides is lysosome. Brown et al. (1973) described the disruption of lysosome in some phase of the fractionation procedures. Considering Brown's view, we carried out the subcellular fractionation of the tumor tissue and the host liver by the same procedures at the same time, and the results described above were obtained. We there- fore conclude that lysosomes of Ehrlich tumor and Yoshida sarcoma are not disrupted in the fractionation procedure, and that large amounts of 1 l lIn and 169yb had not accumulated in lysosome of these tumors before the subcellular fractionation. It is presumed that there is lysosomal affinity of these nuclides in the case of the specific organ such as the liver. Considerable affinity of these nuclides to lysosome in hepatoma AH109A is due to residual nature of the liver possessed in hepatoma AH109A. The extent of the variation of hepatoma from liver may be determined by using this phenomenon as an indicator.

226 A. Ando et al. : Subcellular Distribution of H~In and 169yb in Tumor and Liver

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Brown DH, Swartzendruber DC, Carlton JE, Byrd BL, Hayes RL (1973) The isolation and characterization of gallium-binding granules from soft tissue tumors. Cancer Res 33:2063-2066

Brown DH, Byrd BL, Carlton JE, Swartzendruber DC, Hayes RL (1976) A quantitative study of the subcellular localization of 6VGa. Cancer Res. 36:956 963

Deckner K, Becker G, Langowski U, Schwering H, Hornung G, Schmidt CG (1971) Die subcellul/ire Bindung von 67-Gallium in Ascites-Tumorzellen. Z Krebsforsch 76:293 298

Edwards CL, Hayes RL (1969) Tumor scanning with 67Ga citrate. J Nucl Med 10:103-105

Hagan PL, Chauncey DM Jr, Halpern SE, McKegney ML (1977) Comparison of viable and nonviable tumor uptake of Sc-46, Mn-54, Zn-65, In - l l l and Au-195 with Ga-67 citrate in a hepatoma model. Eur J Nucl Med 2:225~30

Hisada K, Ando A (1973) Radiolanthanides as promising tumor scanning agents. J Nucl Med 14:615~17

Hunter WW Jr, Dekock HW (1969) l l l In for tumor localization (Abstract). J Nucl Med 10:343

Hunter WW Jr, Riccobono XJ (1970) Clinical evaluation of HlIn for localization of recognized neoplastic disease (Abstract). J Nucl Med 11 : 328-329

Ito Y, Okuyama S, Sato K, Takahashi K, Sato T, Kanno I (1971) 67Ga tumor scanning and its mechanisms studied in rabbits. Radiology 100 : 357-362

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275

Orii H (1972) Tumor scanning with gallium (67Ga) and its mechanism studied in rats. Strahlentherapie 144:192-200

Swartzendruber DC, Nelson B, Hayes RL (1971) Gallium-67 localization in lysosomal-like granules of leukemic and nonleukemic murine tissues. J Natl Cancer Inst 46:941-952

Takeda S, Uchida T, Matsuzawa T (1977) A comparative study on lysosomal accumulation of gallium-67 and indium-ll 1 in Morris hepatoma 7316A. J Nucl Med 18:835 839

Takeda S, Okuyama S, Takusagawa K, Matsuzawa T (1978) Lyso- somal accumulation of gallium-67 in Morris hepatoma-7316A and Shionogi mammary carcinoma-115. Gann 69:267-271

Received June 30, 1980

Documents

Subcellular distribution of 111In and 169Yb in tumor and liver