Toxicology Letters, 5 (1980) 351-362 o Elsevier/North-Holland Biomedical Press.

351

DIFFERENT INDUCIBILITY OF METALLOTHIONEIN IN VARIOUS MAMMALIAN CELLS IN VITRO

SHIZUKO KOBAYASHI and MASAMI KIMURA*

Department of Biology, Kyoritsu College Pharmacy, Minato-ku, Tokyo, and *Department of Experimental Toxicology, National Institute of Industrial Health, Nagao, Tama-ku, Kawasaki (Japan)

(Received December 7th, 1979) (Accepted December 14th, 1979)

SUMMARY

Human lymphocytes and mouse embryonic cells (A-31) have lower or slower inducibility of metallothionein (MT) synthesis on exposure to 2.5 pg of cadmium (Cd)/ml for 24 h than human liver cells, HeLa cells, rabbit kidney cells and mouse FRD cells. Differential inducibility has been observed among some mammalian cells in vitro.

INTRODUCTION

MT is a heavy metal binding protein of low molecular weight. The apo- protein has high cysteine content and no aromatic amino acids. The synthesis of MT may be induced not only in the liver and kidney of animals exposed to Cd or zinc (Zn) [l-5], but also in primary cultured cells from rat liver [ 61, pig kidney and liver [ 7-81 by exposure to Cd. It has been demonstrated that the heavy metal ion can mediate the induction of MT in established cell lines such as human skin (HE) [ 91, rat hepatocyte (RLC) [lo] and human cervical carcinoma (HeLa) [ 111. However, it is not known which cells have low inducibility of MT-synthesis by exposure to the heavy metal ion. In this paper differential inducibility of MT-synthesis among some mammalian cells exposed to Cd in vitro is described.

MATERIALS AND METHODS

Cells. Adult human hepatocyte cell (Chang liver), human cervical carcinoma cell line (HeLa) and rabbit kidney cell (LLC-RK1 ) were purchased from the Culture Center (Dainihonseiyaku Ltd., Osaka). Cell line (A-31) from a cloned

*Author to whom correspondence should be addressed. Abbreviation: MT, metallothionein.

continuous mouse embryonal line (BALB/3T3) transformed by the Kirsten strain of murine sarcoma virus in vitro [ 121, and cell line (FRD) from a BALB/c mouse with Rauscher virus-induced myelomonocytic leukemia [ 131 were supplied by Dr. Ikawa. Human lymphocytes were prepared from fresh normal blood by using Ficoll paque (Pharmacia Fine Chemicals).

Counting survived cells and incorporation of [3 H] thymidine. Cells in 20 ml of Eagle’s MEM or McCoy’s 5a medium supplemented with 10% fetal calf serum were incubated at 37°C in a humid atmosphere of 5% CO2 (v/v) in air. After 24 h preincubation, cells (1 -lo6 /ml) were exposed to Cd (as CdCl*) at 0, 1, 2.5, 5 or 10 pg/ml for 24 h. The number of surviving cells was measured by counting Trypan blue negative cells. [3 H] Thymidine was added to the medium to give a concentration of 0.005 pCi/ml together with Cd’*. The radioactivity of the precipitate obtained from 24 h-exposed cells using tri- chloroacetic acid (10%) was measured with a liquid scintillation spectrometer (Packard model 3003). Amounts of DNA were determined by the method of Schmidt and Thanhauser.

Synthesis of [‘09Cd] thionein and [3”S] thionein. Cells were incubated in the medium containing Cd (2.5 pg/ml and 0.1 pCi/ml) for 24 h. The 24 h- exposed cells collected from 5 dishes were washed 4 times in fresh medium by centrifugation. The pellets obtained were homogenized in 0.01 M Tris- HCl buffer, pH 8.6, containing 0.05 M NaCl and 0.2% Triton X-100, for 20 min at 37°C. The homogenate was centrifuged at 100 000 X g for 1 h. The supernatant obtained from the homogenate was applied to a column of Sephadex G-75 (2 X 95 cm), equilibrated with 0.01 M Tris-HCl buffer, pH 8.6, containing 0.05 M NaCl. The sample was eluted at a flow rate of 12 ml/h with the buffer. Each fraction was monitored by measuring absorp- tion at 250 nm and counting radioactivity of “‘Cd with an auto-gamma spectrometer (Packard Model 5220). Cells were incubated in the medium containing Cd (5 pg/ml) and 35S-cystine (0.005 pC!i/ml) for 24 h. The pellets obtained from the exposed cells were treated as described above, except for counting radioactivity of 35S with the liquid scintillation spectrometer.

Isotope. The radioactive “‘Cd (0.52 mCi/pg) as CdCl, , [j”S] cystine (1.0 mCi/4.7 pg) and [3H] thymidine (12.5 mCi/mM] were purchased from Radio-Chemical Center and New England Nuclear, respectively.

RESULTS

Table I presents the viability of 6 mammalian cells exposed to Cd at 0, 1, 2.5, 5 and 10 pg/ml for 24 h. Considerable numbers of the cultured cells survived in the medium containing 2.5 pg Cd/ml. Human cells appeared to be more resistant to Cd’+ than the other mammalian cells.

Uptake of [3H] thymidine into the acid-insoluble fraction obtained from the Cd-exposed cells is illustrated in Table II. The incorporation of [3H] - thymidine per DNA in mammalian cells exposed to Cd decreases depending on the concentration of the metal, except in the case of human lymphocytes.

359

TABLE I EFFECT OF CADMIUM ON CULTURED MAMMALIAN CELLS

Viability of cells is expressed as % of the number of surviving cells in Cd-containing medium to that of cells incubated without Cd. All values are the mean of 10 measure- ments.

Cells Concentration of Cd in medium (pg/ml)

0 1 2.5 5 10

Viability of cells (W)

Human liver 100 78 73 65 36 Human lymphocytes 100 93 78 61 47 Human HeLa 100 89 67 41 19

Rabbit kidney 100 75 45 26 6

Mouse A-31 100 85 58 36 3 Mouse FRD 100 88 64 23 0

TABLE II UPTAKE OF [ 3H] THYMIDINE INTO Cd-EXPOSED MAMMALIAN CELLS

Uptake of [ JH] thymidine into the acid insoluble fraction of the 24 h-exposed cells is expressed as % of the control. All values are the mean of 6 measurements

Cells Concentration of Cd in medium (fig/ml)

0 1 2.5 5 10

Uptake of [‘HI thymidine into Cd-exposed cells (%)

Human liver 100 110 99 94 26 Human lymphocytes 100 112 125 223 138 Human HeLa 100 88 63 54 17

Rabbit kidney 100 98 82 57 10

Mouse A-31 100 86 91 90 19 Mouse FRD 100 95 80 74 46

The preliminary gel filtration of rabbit liver MT [14] established the location in the elution pattern of Sephadex C-75 column chromatogram (Ve = 170 ml). The cytosol of Cd-exposed cells was eluted by the same pro- cedure. The elution patterns of log Cd from the cytosol of ‘ogCd-exposed human liver cells and mouse A-31 cells are shown in Figs. 1 and 2, respectively Both patterns had two radioactive peaks of “‘Cd. One of them [P-I] at the void volume was associated with high molecular weight proteins and the other [P-II] at the Ve = 170 ml corresponded to MT fraction, The elution

Elutm volume (ml)

Fig. 1. Elution pattern of cytosol from Cd-exposed human liver cells on Sephadex G-75.

Elutlon volume (ml)

Fig. 2. Elution pattern of cytosol from Cd-exposed mouse A-31 cells on Sephadex G-75.

patterns of % from the cytosol of Cd-exposed cells in the medium contain- ing [ 35S] cystine overlapped the ‘OgCd-elution patterns from the “‘Cd-exposed cells. Two of three 3sS-peaks were eluted at the same position of [P-I] and [P-II] , respectively.

Table III shows the incorporation of lo9Cd into cells and distribution of ro9Cd to [P-I] and [P-II] found in the cytosol of Cd-exposed cells. 2 to 6.3% of given ‘09Cd were recovered in 5 mammalian cells except mouse FRD cells after 24 h-exposure. Mouse FRD cells incorporated much more lo9Cd than the other cells. 70 to 90% of “‘Cd found in the cytosol of human liver, HeLa, rabbit kidney and mouse FRD cells were distributed in the [P-II] fraction in Sephadex G-75 gel filtration, which was recognized as MT fraction. However, 30% of “‘Cd found in both cytosols of human lymphocytes and mouse A-31 cells were in this fraction.

361

TABLE III DISTRIBUTION OF ‘“Wd IN CYTOSOL OF Cd-EXPOSED M~MAL~N CELLS

The ‘09Cd incorporation into the Cd-exposed cells is expressed as % of the radioactivity of isotope added to the medium. The distribution of ‘Wd in the eytosol of Cd-exposed cells is expressed as % of “Vd of [P-I] and [P-II] from gel filtration, respectively.

Cells ‘@Cd incorporated into Distribution of ‘Wd in cytosol of Cd-exposed cells (W) Cd-exposed cells (%)

P-I P-II

Human liver 4.7 20 80 Human lymphocytes 3.6 70 30 Human HeLa 6.3 20 80

Rabbit kidney 2.0 10 90

Mouse A-31 3.0 70 30 Mouse FRD 21.8 30 70

DISCUSSION

Human cells seem to be more resistant to Cd’+ than the other cells used in this experiment.

Utakoji [ 151 has reported that lymphoblastic transformation is induced with heavy metal ions such as Hg2+, Cd*+ and Zn2+ in human peripheral blood lymphocytes in vitro and that the ions stimulate the incorporation of thymi- dine into cells about 5 to 10 times as much as the control. Our results indi- cate that the level of [3H] thymidine-uptake into the Cd-exposed lymphocytes increases with the dose of Cd and reaches a maximum at 5 pg of Cd/ml. Such elevation of [ 3H] thymidine-uptake was not observed in the other mammalian cells.

Rudd and Herschman [ 111 have shown that Cd-binding in MT-fraction of RCC cells in culture is dependent on the time of exposure and concentration of Cd and increases in a linear fashion for 72 h at least when the cells are in- cubated in about 0.5 pg Cd/ml. In the present experiment the induction of MT-synthesis is compared among 6 mammalian cells exposed to 2.5 fig Cd/ml for 24 h. Bryan and Hidalgo [16] have stated that cytoplasmic Cd obtained from the liver of Cd-exposed rat is bound to high molecular weight protein fraction (corresponding to [P-I J) early after the administration of Cd and appears progressively in the low molecular weight fraction (co~esponding to [P-II] ) with time, They have also demonstrated that more than 90% of Cd in cytosol are associated with the low molecular weight protein fraction 24 h after the administration of Cd. As the lopCd found in [P-I] is not bound to MT in cytoplasm, the relative inducibility of MT-synthesis may be presented by the ratio of [P-I] IopCd to [P-II] ‘OgCd. The values from human liver cells, lymphocytes, HeLa cells, rabbit kidney cells, mouse A-31 cells and FRD cells are calculated to be 4,0.4,4, 9, 0.4 and 2.3, respectively. The synthesis

362

of MT is effectively induced with Cd in human liver cells, HeLa cells, rabbit kidney cells and mouse FRD cells. Though human lymphocytes and mouse A-31 cells can take up Cd from the cultured medium at a similar level to the other cells, they are not able to induce effectively the synthesis of MT with the metal under the condition of exposure used in this experiment. Lympho- cytes and embryonic cells (mouse A-31) present lower or slower inducibility of MT with Cd in vitro than the other cells. These results suggest that the in- ducibility of MT-synthesis with Cd is different among various mammalian cells.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Y. Ikawa for his gifts of cells.

REFERENCES

1 D.R. Winge, R. Premakumar and K.V. Rajagopalan, Metal-induced formation of metallo- thionein in rat liver, Arch. Biochem. Biophys., 170 (1975) 242-252.

2 M. Cempel and W. Webb, The time-course of cadmium-thionein synthesis in the rat, Biochem. Pharmacol., 25 (1976) 2067-2071.

3 Z.A. Shaikh and J.C. Smith, The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium, Chem.-Biol. Interact., 15 (1976) 327-336.

4 Z.A. Shaikh and J.C. Smith, The mechanisms of hepatic and renal metallothionein biosynthesis in cadmium-exposed rats, Chem.-Biol. Interact., 19 (1977) 161-171.

5 K.S. Squibb, R.J. Cousins and S.L. Feldman, Control of zinc-thionein synthesis in rat liver, Biochem. J., 164 (1977) 223-228.

6 H.A. Hidalgo, V. Koppa and S.E. Bryan, Induction of cadmium-thionein in isolated rat liver cells, Biochem. J., 170 (1978) 219-225.

7 M. Webb and M. Daniel, Induced synthesis of metallothionein by pig kidney cells in vitro in response to cadmium, Chem.-Biol. Interact., 10 (1975) 269-276.

8 M. Daniel, M. Webb and M. Cempel, Metallothionein synthesis as an indication of the parenchymal origin of cells cultured from liver, Chem.-Biol. Interact., 16 (1977) 101-106.

9 H.E. Rugstad and T. Norseth, Cadmium resistance and content of cadmium-binding protein in cultured human cells, Nature, 257 (1975) 136-137.

10 C.J. Rudd and H.R. Herschman, Metallothionein accumulation in response to cadmium in a clonal rat liver cell line, Toxicol. Appl. Pharmacol., 44 (1978) 511-521.

11 C.J. Rudd and H.R. Herschman, Metallothionein in a human cell line: the response of HeLa cells to cadmium and zinc, Toxicol. Appl. Pharmacol., 47 (1979) 273-278.

12 Y. Ikawa, A.F. Gazdar and H.C. Chopra, Epithelial features of nonproducer BALB/3T3 cells transformed by murine sarcoma virus, J. Natl. Cancer Inst., 49 (1972) 1449-1453.

13 Y. Ikawa, T.N. Fredrickson, H.L. Sims, H.J. Ortega and H.C. Chopra, Rauscher virus- induced myelomonocytic leukemia culture lines, Proc.. Am. Assoc. Cancer Res., 13 (1972) 41.

14 M. Kimura, N. Otaki and M. Imano, Rabbit liver metallothionein: tentative amino acid sequence of metallothionein-B, in J.H.R. KIgi and M. Nordberg (Eds.), Metallothionein, BirkhLuser Verlag, Basel, 1979.

15 T. Utakoji, Lymphoblastic transformation induced by heavy metal ions in human peri- pheral blood leukocyte culture in vitro, Symp. Cell. Biol. 23 (1972) 231-234.

16 S.E. Bryan and H.A. Hidalgo, Nuclear “%d: uptake and disappearance correlated with cadmium-binding protein synthesis, Biochem. Biophys. Res. Commun., 68 (1976) 858-866.