Divergent effects of cyanate on amino acid and phosphate uptake by liver and hepatoma

321

Biochimica et Biophysica Acta, 474 (1977) 321--328 © Elsevier/North-Holland Biomedical Pre~

BBA 98814

D I V E R G E N T EFFECTS O F CYANATE ON AMINO ACID AND PHOSPHATE UPTAKE BY L I V E R AND HEPATOMA

MICHAEL A. LEA, MICHAEL R. KOCH, BENNETT BERES and VEENA DAYAL Department of Biochemistry, New Jersey Medical School, Newark, N.J. 07103 (U.S.A.) (Received July 23rd, 1976)

Summary The uptake of a-aminoiso[ 3H] butyric acid and 32Pi was observed to be inhibited by sodium cyanate in transplanted hepatomas b u t was increased in the livers of the t u m o r bearing rats. Incorporation of 32Pi into macromolecules in hepatomas was also inhibited by cyanate. Treatment with this drug did not influence circulating concentrations of isotope-labeled materials. There were relatively small effects on uptake of 3~C1- in cyanate-treated rats and the action was n o t tissue specific. The data were compatible with an inhibitory effect of cyanate on active transport in hepatomas which was n o t seen under the same conditions in host liver.

Introduction In a previous study, w e observed that sodium cyanate could cause a large inhibition of amino acid incorporation into protein of hepatomas under conditions in which there was no significant effect in the host liver [1]. There were less marked effects of cyanate on 3H-labeled amino acid incorporation into the t u m o r acid-soluble fraction than into protein, which suggested that the action of cyanate on protein synthesis was more important than its effect on amino acid uptake b y hepatoma cells. However, the incorporation of isotope labeled amino acids into the acid-soluble fraction of tissues represents a balance between transport into cells and further metabolism of the amino acids. By use of the non-metabolizable amino acid, a-aminoisobutyric acid [2], it was considered possible to determine the effects of cyanate on amino acid transport b y hepatomas and the livers of tumor-bearing rats. Several investigators have studied the uptake of a-aminoisobutyric acid in vitally transformed cells [2] and a number of hepatoma lines [2--5]. Since we have found that under appropriate conditions, cyanate can serve as a rather general inhibitor of macromolecular synthesis, namely protein, RNA and DNA synthesis [1,6], it was considered

322 possible that cyanate may inhibit a fundamental energy transfer process. It was assumed that this would be reflected in alterations of phosphate metabolism. In order to characterize such changes, we have examined the uptake of 32p. labeled phosphate and incorporation into macromolecules in hepatomas and livers of tumor-bearing rats. Methods Experiments were performed with male Buffalo rats. Water and food {Wayne Lab-Blox) were available ad libitum. Tumors were transplanted bilaterally in a subcutaneous position. The histology and growth properties of the tumors were described previously [7,8]. The HTC hepatoma was derived from Morris hepatoma 7288C transferred to tissue culture and is n o w maintained by us as subcutaneous transplants. It is a rapidly growing and poorly differentiated hepatoma. In studies on regenerating liver approximately 66% of the liver was removed by the technique of Higgins and Anderson [9] 24 h before injection of isotope. Rats treated with sodium cyanate were given intraperitoneal injections. All rats were illuminated from 6 a.m. to 7 p.m. and unless stated otherwise were killed 1 h after injection of isotope. In determinations of total tissue radioactivity, 10 pl blood or 50 pl t u m o r and liver homogenate (10% w/v in distilled water} were digested with 0.5 ml Protosol (New England Nuclear, Boston, Mass.}. Determinations of incorporation of isotope into cytoplasmic and nuclear fractions were performed as described previously [1] except total histones were extracted from nuclei with 0.24 M HC1 and were precipitated with 10 volumes of acetone. Radioactivity measurements were made with a Beckman LS250 liquid scintillation counter with counting efficiency of approximately 35% for 3H and 70% for 36C1.3H-Labeled samples giving a lower counting efficiency due to quenching were made equivalent to 35% counting efficiency by the external standard ratio method. Protein was assayed by the procedure of L o w r y et al. [10] and DNA was measured by the method of Burton [11]. ATP concentration was assayed in approximately 1 g portions of tissue which were rapidly excised and immediately homogenized in a preweighed and chilled glass homogenizing tube containing 3 ml 8% trichloroacetic acid solution. The exact weight of the tissue was determined after homogenizing. We found this technique gave identical results to a procedure in which the tissues were frozen with liquid nitrogen after excision and the values were very similar to those reported by Weber et al. [12] for a freeze-clamp m e t h o d with liver and hepatomas. After centrifuging the homogenate, ATP was assayed in the protein free supernatant using a coupled enzyme system with phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. The decrease in absorbance when NADH is oxidized to NAD was determined at 340 nm with the reagents and procedures supplied by Sigma Chemical Company, St. Louis, Mo. (Technical Bulletin N u m b e r 366-UV). Data are presented as means of a stated number of experiments -+ standard error. The results were subjected to statistical evaluation by the t test for small samples. Differences between means giving a probability of less than 5% were considered to be significant.

323

Sodium cyanate was purchased from K and K Laboratories, Plainview, N.Y. Carrier-free 32Pi and a4minoiso[Me-3H]butyric acid (10 Ci/mmol) were obtained from New England Nuclear, Boston, Mass. Na36Cl (418 pCi/mmol) was supplied by Amersham/Searle, Arlington Heights, Ill. Results

Influence of cyanate on tissue uptake of a-aminoisobutyric acid Initial studies on the effects of cyanate on the uptake of a-aminoiso[3H] butyric acid were performed with rats receiving 250 mg sodium cyanate per kg b o d y weight as this dose level had been found previously to cause a large inhibition of the incorporation of 3H-labeled amino acids into protein in hepatomas [1]. The data presented in Table I show that under these conditions there was a mean inhibition of a-aminoiso[3H]butyric acid uptake of 90% in hepatoma HTC and a 65% inhibition in hepatoma 7777. In contrast there was a 2--3-fold increase in the uptake of isotope in the host livers of rats treated with cyanate. There was no significant difference between control and treated animals in the levels of radioactivity present in the blood and, with the same protocol for administration of cyanate and a-aminoiso[3H]butyric acid, there was no significant effect of the drug on isotope uptake by regenerating liver 24 h after partial h e p a t e c t o m y (222 + 33 cpm per mg tissue from 5 rats} in comparison with control regenerating liver (185 +- 24 cpm per mg tissue from 7 rats). On the other hand, under these conditions the uptake of a-aminoiso[3H]butyric acid was greater in the livers of normal rats treated with cyanate (136 -+ 21 cpm per mg tissue from 4 rats} than with untreated rats (55 + 5 cpm per mg tissue from 4 rats). The inhibition of uptake of a-aminoisobutyric acid in hepatoma by cyanate is a prolonged effect and can be seen after a short time interval. Thus, when rats bearing hepatoma 9618A2 received 250 mg sodium cyanate per kg b o d y weight between 5 and 45 min before injection of a-aminoiso[3H]butyric acid (50 pCi per kg) and were killed at an equal time after injection of the isotope, there was a significant inhibition of isotope uptake in the tumors at each time point examined (Fig. 1). In the host livers of rats treated with cyanate, there

TABLE I EFFECT OF CYANATE ON UPTAKE OF ~-AMINOISO[3H]BUTYRIC AND HTC AND HOST LIVERS

A C I D IN H E P A T O M A S

7777

R a t s r e c e i v e d 2 5 0 m g s o d i u m c y a n a t e ( i n t x a p e r i t o n e a l l y ) p e r k g 1 h b e f o r e 5 0 /~Ci a - a m i n o i s o [ 3 H ] b u t y r i c acid p e r kg and w e r e killed 1 h a f t e r i n j e c t i o n o f t h e i s o t o p e . U p t a k e is e x p r e s s e d as m e a n s ± s t a n d a r d error for t h e n u m b e r o f r a t s given in paIcentheses.

Tissue

Hepatoma 7777 7 7 7 7 H o s t liver Hepatoma HTC H T C h o s t liver

c p m / m g tissue Control

Cyanate treated

112 145 133 116

39 316 13 364

± 1 2 (4) ± 27 (4) ± 1 8 (5) ± 5 (5)

± 10 (5) + 41 (5) ± 1 (4) ± 2 2 (4)

324 300

150

LIVER

~,

0 E

~

ioo

/

T LIVER

200

kx

T

LIVEF LIVER

"~V-

%-,o :3 "" r-~

~ IE

IOOi

50 ,,,",,

I.

I

/

/ ~HEPATOMA 9618"2

9618Aa ~

NoCNO

I

I 0

15

3,0

45

0

TIME AFTER INJECTION (rain)

I

I

I00 200 mg NoCNO PER kg

} 300

Fig. 1. E f f e c t of time o n the u p t a k e of a - a m i n o i s o [ 3 H ] b u t y r i c acid and the action of c y a n a t e in h e p a t o m a 9 6 1 8 A 2 a n d h o s t liver. Rats were l~lled at the stated time after i n t m p e r i t o n e a l injection o f 50 ~Ci c~-aminoiso[3H]butyrlc acid per kg. F o r rats w h i c h received injections p e r i t o n e a l l y ) per kg there was an equal and c o n s e c u t i v e time b e t w e e n i s o t o p e and b e t w e e n injection o f the i s o t o p e and death o f the animal. dard error for 4 - - 6 tissues from c o n t r o l animals (o) and cyanate-treated

of 2 5 0 m g s o d i u m c y a n a t e (intraa d m i n i s t r a t i o n o f c y a n a t e and the The p o i n t s represent m e a n s ± stanrats (e).

Fig. 2. Effect o f c y a n a t e c o n c e n t r a t i o n o n u p t a k e o f ~ - a m i n o i s o [ 3 H ] b u t y r l c acid in h e p a t o m a 9618A 2 and h o s t Liver. Rats received s o d i u m c y a n a t e (intraperitoneally) at the stated d o s e level 1 h before 50/~Ci ( ~ - a m i n o i s o [ 3 H ] b u t y r i c acid per kg and were killed 1 h after injection o f the i s o t o p e . The points represent m e a n s + standard errors for h e p a t o m a 9618A 2 (©) or h o s t liver ( e ) f r o m 4 - - 8 animals.

was an increased uptake of isotope which was statistically significant at the 15 and 45 min time intervals. The data recorded in Table II indicate that administration of sodium cyanate (250 mg per kg) caused an approximately 80% inhibition of the uptake of ~-aminoiso[3H] butyric acid by hepatoma 9618A2 which was still apparent 18 h after treatment with the drug. At the same time, the uptake of isotope by the host livers was approximately doubled. At 48 h after drug treatment, the uptake of isotope by tumor and host liver was not significantly different from that of control animals. The influence of the dose level of cyanate on'tissue uptake of ~-aminoiso[3H]butyric acid was examined in rats bearing hepatoma 9618A2 (Fig. 2). There was a significant decrease in the tumor after treatment with 75 mg sodium cyanate per kg with more pronounced inhibition being seen with 125 and 250 mg sodium cyanate per kg. The host livers showed the opposite trend but, due to greater individual variation, the increased uptake in the host livers was only statistically significant after administration of 250 mg sodium cyanate per kg.

Influence of cyanate on tissue uptake of 32P i It was observed that the effects of cyanate on tissue uptake of ~-aminoiso[3H]butyric acid were not peculiar to the latter substance but were also seen

325 T A B L E II TIME-DEPENDENT EFFECTS OF CYANATE ON UPTAKE INTO BLOOD, HEPATOMA 9618A 2 AND HOST LIVER

OF ~-AMINOISO[3H]BUTYRIC

ACID

Rats received 2 5 0 m g s o d i u m e y a n a t e (intzaper/tonealiy) per kg at the stated tLme interval before 5 0 / ~ C i ~nin0iso[3H]butyrie per kg and were k i l l e d 1 h after injection of the i s o t o p e . U p t a k e is e x p r e s s e d as m e a n ± standard error.

Time (h)

No. of animals

epm/pl blood

cpm/ms tumor

c p m / m s liver liver

Control 1 18 48

8 5 6 6

57 + 1 2 69 ± 15 51 ± 3 36± 3

141 + 17 26± 7 28± 7 142±43

116 ± 248± 210+ 96 ±

27 30 24 10

for the uptake of s2Pi by transplanted hepatomas and host livers (Table III). The incorporation of 32Pi after administration of cyanate (250 m g per kg) was decreased in hepatomas 9618A2, H T C and 7777 to 15%, 2 3 % and 4 6 % of the respective control values in untreated animals. Under the same conditions, the uptake of 3~Pi in the host livers of rats bearing these tumors were 177%, 201% and 1 7 7 % of control values in rats treated with cyanate. A small, but significant, increase in s2PI was also observed in regenerating liver 24 h after partial hepatectomy. Using groups of 3 rats, the mean uptake in cyanate-treated animals was 25% greater than in control animals. Similarly in normal animals mean 32Pi uptake was observed to be 4 5 % greater in a group of 3 rats treated with cyanate than in control animals. N o significantchange has been observed in the s2Pi level in the blood of cyanate-treated rats.The inhibitory effect of cyanate on tumor uptake of 32Pi was seen not only in radioactivity in the acid~oluble fraction, but also in the incorporation of isotope into macromolecular fractions in hepatoma 5123C (Table IV). Under the conditions examined, the mean incorporation of 32p into the cytoplasmic acid insoluble fraction,histones, nonhistone nuclear proteins and nucleic acids of the nucleus was decreased by

TABLE IIl EFFECT LIVERS

OF CYANATE

O N U P T A K E O F 32P i I N H E P A T O M A S

7777, HTC and 9618A 2 AND HOST

Rats received 2 5 0 m g s o d i u m e y a n a t e (intra]~erltoneaUy) per k g I h b e f o r e 1 0 0 ~uCi 32Pi p e r k g and were ldlled 1 h after inject/on o f the i s o t o p e . R a d i o a e t / v i t y w a s m e a s u r e d w i t h digests o f t h e tote1 tissue h o m o g e n a t e s . Means and standard errors axe p r e s e n t e d for t h e n u m b e r of animals given in p a r e n t h e s e s . Tissue

d p m / 1 0 ~g tissue Control

Cyanate treated

Hepatoma 7777 7 7 7 7 h o s t liver

50 ~ 92 ±

2 (4) 5 (4)

2 3 ± 3 (6) 163 ± 6 (6)

Hepatoma HTC H T C h o s t liver

5 6 ± 1 2 (3) 87 ± 2 (3)

1 3 ± 2 (3) 1 7 5 ± 7 (3)

Hepatoma 9618A2 9 6 1 8 A 2 h o s t liver

52 ± 86 ±

8 ± 2 (3) 1 5 3 ~ 7 (3)

3 (3) 5 (3)

326 TABLE IV EFFECTS LIVER

OF

CYANATE

ON

INCORPORATION

OF

32p i I N T O

HEPATOMA

5123C

AND

HOST

R a t s r e c e i v e d 2 5 0 m g s o d i u m c y a n a t e ( i n t r a p e r l t o n e a l l y ) p e r kg 1 h b e f o r e 2 5 0 p C i 32p i per kg and w e r e killed 1 h a f t e r i n j e c t i o n o f t h e i s o t o p e . I n c o r p o r a t i o n is e x p r e s s e d as m e a n ± standard error for 4 c o n t r o l and 5 treated animals. Tissue f r a c t i o n

Acid soluble Cytoplasmic Histone** Non-histone N u c l e i c acid

Hepatoma 5123C

*

(acid i n s o l u b l e ) ** n u c l e a r p r o t e i n ** in n u c l e u s * * *

H o s t liver

Control

Cyanate treated

Control

Cyanate treated

242 167 49 64 123

29 6 6 4 ,2

233 265 63 174 54

366 248 61 181 47

± 57 ± 22 +- 2 ± 14 ± 10

-+ 7 ± 1 ± 1 ± 1 ± 1

-+ 5 0 ÷ 63 + 9 ± 47 ± 16

+ 57 ± 46 ± 5 ± 31 ± 8

* d p m per 1 0 ~ g tissue. * * d p m per 1 0 pg p r o t e i n . * * * d p m per 1 p g D N A .

cyanate to 11% or less of that in tumors of control rats, but no significant de. crease was seen in any of the fractions from host livers. The same pattern of response was seen in two control and two cyanate-treated rats bearing hepatoma 20.

Influence of cyanate on chloride uptake by tissues In order to examine the action of cyanate on tissue uptake of a material Which is not subject to an active transport process, the uptake of chloride was studied in hepatoma and liver (Table V). 36C1- uptake was significantly inhibited by cyanate in both the HTC hepatoma and the liver of host rats and to a similar degree being 39% decreased in the tumor and 36% decreased in the livers of cyanate-treated animals. Using the conditions described in Table V a similar effect of cyanate was seen in two rats bearing hepatoma 9618A2 where the mean uptake of 36C1- was 29% less in tumors and 35% less in host livers in comparison with the tissues of two control animals. No significant change was observed in the ~C1- level in the blood of cyanate-treated rats.

A TP concentration in tissues of rats after administration of cyanate The concentration of ATP was examined in rats bearing hepatomas 7777 or HTC after treatment with sodium cyanate at a level of 250 mg/kg (Table VI).

TABLE V EFFECTS

OF CYANATE

ON UPTAKE

O F 36C1- I N T O B L O O D , H E P A T O M A

HTC AND HOST LIVER

R a t s r e c e i v e d 2 5 0 m g s o d i u m e y a n a t e ( i n t r a p e r i t o n e a l l y ) per k g 1 h b e f o r e 5 0 / ~ C i 3 6 C l - p e r kg and were killed 1 h a f t e r i n j e c t i o n o f the i s o t o p e . U p t a k e is e x p r e s s e d as m e a n -+ standard error for 5 a n i m a l s . Treatment

cpm/~l blood

cpm/mg tumor

c p m / m g liver

Control Cyanate

2 1 0 -+ 5 212 ± 7

9 5 -+ 5 5 8 -+ 6

6 7 -+ 3 43 ± 1

327 T A B L E VI E F F E C T S O F C Y A N A T E ON A T P C O N C E N T R A T I O N I N H E P A T O M A S A N D H O S T L I V E R S R a t s w h i c h were treated w i t h s o d i u m c y a n a t e received intxaperitoneal injections at a level of 2 5 0 m g pe~ kg and w e r e killed a f t e r 1 or 2 h, M e a n s a n d s t a n d a r d e r r o r s a m p r e s e n t e d for the n u m b e r of a n i m a l s given in parentheses. Tissue

A T P c o n c e n t r a t i o n ( # m o l p e r g) Control

Hepatoma 7777 7 7 7 7 h o s t liver Hepatoma HTC H T C h o s t liver

0.84 2.43 0.91 2.20

± ± ± ±

0.11 0.06 0.11 0.18

(5) (5) (7) (7)

Cyanate, 1 h

Cyanate, 2 h

0.60 2.42 0.38 2.28

0.66 2.58 0.34 2.55

+- 0 . 1 0 ± 0.10 ± 0.06 ± 0.14

(7) (7) (7) (7)

± ± ± ±

0.07 0.15 0.01 0.27

(7) (3) (4) (4)

At 1 or 2 h after the injection there was no statistically significant change in ATP concentration in the host livers and hepatoma 7777. In hepatoma HTC, the ATP concentration was decreased to 42% at 1 h and 38% at 2 h in comparison with untreated rats. Discussion

The effect of cyanate on the incorporation of amino acids into t u m o r acidsoluble fraction reported previously [1] was less than the inhibition of ~aminoisobutyric acid uptake described in the present study. Such data would be compatible with an inhibitory effect of cyanate on both the entry of amino acids into t u m o r cells and on the further metabolism of the naturally occurring amino acids. The latter effect would partially compensate for the decreased t u m o r uptake occurring with the naturally occurring amino acids. Although cyanate reacts with a variety of proteins including enzymes [13-15], the action of cyanate on macromolecular synthesis and tissue uptake of amino acids and phosphate might arise from its effect on a single critical protein required for generation of ATP. The selective effect of cyanate on hepatomas in comparison with normal liver suggests that such a protein might be different in the t u m o r cells than in normal liver. On the other hand, the effects of cyanate might be mediated through carbamylation of hemoglobin which is known to increase the oxygen affinity of the molecule [16]. This might produce a critical situation in the tumors which under most circumstances are relatively anaerobic in comparison with many normal tissues including the liver. In our studies, NaNCO administration resulted in a decreased ATP concentration in the HTC t u m o r and the work of Risser and Gelehrter [5] has shown that transport of ~-aminoisobutyric acid in HTC cells occurs by an energy~iependent process which can be inhibited by NaCN. We were n o t able to demonstrate a statistically significant decrease in ATP concentration in hepatoma 7777 after treatment with cyanate despite the large inhibition of a-aminoisobutyric acid uptake, which suggests that effects other than on ATP synthesis may also be important. Data on tissue uptake of ~-aminoisobutyric acid are relevant to one of several overlapping amino acid transport systems [2,3] b u t the fact that similar effects of cyanate were seen with phosphate uptake indi-

328 cates that these are n o t isolated phenomena. Since cyanate caused only small effects on the uptake of chloride by hepatoma and liver, the data raise the possibility of a distinction between the effects of cyanate on passive and active transport processes. We are not able to suggest a mechanism for the increased uptake of a-aminoisobutyric acid and phosphate by the normal liver after treatment of rats with cyanate. As the circulating levels of the isotope labeled materials did not appear to be changed, there was no evidence of increased availability of amino acid or phosphate although changes in blood flow cannot be excluded at the present time. Cyanate has been reported to show anti-mitotic activity [17,18]. Contributory factors may include an inhibitory effect on DNA polymerase (Lea, Waecker and Koch, unpublished results), changes in intermediary metabolism [19] and an alteration in metabolite uptake by cells. Cyanate has diverse effects on different aspects of metabolism in a variety of cell types, but at the same time it is capable of selective effects as seen in sickle cell erythrocytes [13] and in the present study on the uptake of compounds by liver and hepatomas. Acknowledgements We are grateful to Dr. I. Bernard Weinstein for providing HTC cells and to Dr. Harold P. Morris for supplying tumor-bearing rats from which the other hepatomas were transplanted in these studies. This work was supported by U.S. Public Health Service Grants CA-12933 and CA-16274, and the Alma T o o r o c k Memorial for Cancer Research. The secretarial assistance of Ms. Connie Sheffield is gratefully acknowledged. We are indebted to Dr. William R e d w o o d for helpful discussions on transport phenomena. References 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19

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