Comp. Biocht~.. Ph)'.*iIfl. Yol 60C. pp 221 223 031)6-4492 78 (1701-0221502.00'0 C' Pergamon Press Ltd 1978 Printed in Great Britain

THE EFFECTS OF CLOFIBRATE ON CELL DIVISION OF LOG PHASE AND SYNCHRONIZED CELLS AND ON

MACROMOLECULAR SYNTHESIS IN TETRAHYMENA PYRIFORMIS, GL

HENRY Y. M. PAN and SHAO-CHIA CHOU Department of Pharmacology. University of Hawaii. Honolulu. Hawaii 96816

(Received 9 August 1977)

Abstract--1. Clofibrate. a hypolipidemic agent, inhibits cell growth and division of both log phase and synchronized Tetrahymena pyriformis, GL.

2. At doses which inhibited cell growth and division, the incorporation of precursors to DNA, RNA, protein and lipid was inhibited.

3. It is suggested that the primary effect of the agent is on the energetics and inhibitory effects on cell division and incorporation of precursors to macromolecules are secondary effects.

INTRODUCTION

Tetrahymena pyriformis has been used in studying the effects of hypocholesterolemic or hypolipidemic agents (Holz et al., 1962; Pollard et al., 1963; Shorb et al., 1965; Holmlund, 1973). The effects of clofibrate, a. hypolipidemic agent, on cell division in this organism has been demonstrated previously (Blum & Wexler, 1968; Nozawa, 1973). Using different strains of Tetrahymena (HSM & E), these investigations have illustrated differences in the dose levels which inhi- bited cell growth of . log phase cells. In this present investigation, a further examination of the dose which affects division of log phase and synchronized cells of T. pyriformis, GL, will be presented. The effects of clofibrate on the incorporation of macromolecules will also be presented.

MATERIALS AND METHODS

Organism, growth condition and heat synchronization. Tetrahymena pyriformis, GL were grown, incubated and synchronized as described previously (Pan et al., 1976b).

Effects of clofibrate on cell growth and log phase T. pyri- formis. Cells were harvested and washed as described pre- viously (Chou et al, 1968), and resuspended in Ryley's phosphate buffer (Ryley, 1952). Resuspended cells, in 100ml of buffer were then divided into five equal portions and placed in 125-ml Erlenmeyer flasks. Clofibrate at final concentrations of 0, 1 × 10 -aM, 2 x 10 -4M, 4 x 10 -4 M and 6 × 1 0 - ' M were added to each flask and cell counts were determined by removing 0.5 ml of cells from each flask at 2.5-hr intervals and fixing them in 4.5 ml of 0.7% formalin in Ryley's buffer. Cell counting was per- formed with the aid of a Sedgewick-Rafter counting chamber and an eyepiece with an ocular whipple disc.

Effects of clofibrate on synchronized cell division and growth. Ciofibrate at final concentrations of 0, 2 × 10-" M and 4 x 10-4M were added into 10-ml aliquots of cells at the end of the heat treatment (EHT). Cell counting and determination of division index were performed as de- scribed previously (Pan et aL, 1976a) at EHT + 80 rain (frst synchronized division and EHT + 180 rain (second synchronized division).

Effects of clofibrate on the incorporation of precursors into macromolecules in synchronized cells. The incorporation of

precursors into macromolecules in synchronized cells were as described previously (Pan et al., 1976a). In brief, 2-ml aliqunts of synchronized cells were removed and pipetted into 25-ml Erlenmeyer flasks containing measured amounts of radioactive thymidine, uridine, amino acids and acetate at EHT. Samples were taken at 0, 20, 40, 60 and 80 rain after EHT.

RESULTS

Effects of clofibrate on growth and division o f loo phase cells. Figure 1 shows that cell growth, followed for 15hr, was partially inhibited at 1 x 10 -4M,

i0 s

._t

: D Z

. J - J I,LI tO

10 4

1031 - I I I o ,5 IO 15

TIME, hr

Fig. 1. The effects of clofibrate on cell growth and division of log phase T. pyriformis, GL at 0 (O O), 1 x 10-" M (A A), 2 x 10 - 4 M (I-I C]) and 4 x 1 0 - ' M ( H ) . Cells counts were made in duplicates as de-

scribed in Materials and Methods.

221

222 HENRY Y. M. PAN and SHAo-CHIA CHOU

Table 1. The effects of clofibrate on cell division of synchronized T. pyriformis. GL

Division index (°,o)* Clofibrate First division Second division

0 85 50 2 x 10-* 51 22 4 x 10 -a 0 0

* The division indices (number of dividing cells/100 cells) were determined at EHT + 80 rain (first division) and EHT + 180 rain (second division).

and 2 x 10 - 4 M , and completely inhibited at 4 x 10 -'~ M. At 6 x 10 - 't M, all cells were rounded and lysis usually occurred 1 hr after the addition of clofibrate.

Effects of clofibrate on growth and dMsion of synchronized cells. Table 1 shows the effects of clofibrate on cell division at 2 x 1 0 - 4 M and 4 x 10 -4 M. The division index (number of dividing cells per I00 cells) of control cells at EHT + 80 min is usually 80--900/0 and at EHT + 180 min is usually between 40 -50° . At the lower of the two concen- trations tested, inhibition of the two synchronized divisions were 4&/~, and 559~, respectively. At the higher concentration, inhibition was 100?,g for both synchronized divisions.

Effects o f clofibrate on the incorporation o f precur- sors into macromolecules in synchronized cells. Figure 2 shows the effect of clofibrate on the incorporation of 1*C-labelled thymidine, uridine, amino acids and acetate into DNA, RNA, protein and. lipids, respect- ively, at 2 x 1 0 - * M and 4 x 10- '~M by synchron- ized cells. At 80 min after EHT, thymidine incorpor-

o / ation into DNA was inhibited by 61.0 and 90.0~o, respectively, at the two concentrations tested. Uridine incorporation into RNA was inhibited 77.8 and 96.3~o; amino acid incorporation into protein was in- hibited by 47.3 and 91,67/0; acetate incorporation into lipids was inhibited by 38.4 and 96.2°g.

DISCUSSION AND SUMMARY

The effects of clofibrate on growth and cell division of both log phase and synchronized cells have been demonstrated in T. pyriformis. GL. These results corresponded with the findings of Blum & Wexler (1968). These investigators using strain HSM, showed no inhibition at 1.24 x 10 -*M. partial inhibition at 2.43 x 10-* M and complete inhibition at 3.66 x 1 0 - * M in log phase cells. However, experi- ments using strain E (Nozawa, 1973), showed that there was only partial inhibition at 1 x 10-3 M and complete inhibition was not attained until a dose of 3 x 1 0 - a M was used. The results presented here showed that lysis of cells was demonstrated at 6" x 10 -4 M. These results may indicate that strains HSM and GL have similar susceptibility to the in- hibitory effects of clofibrate. Strain E. however, showed greater resistance and was able to tolerate much higher doses of the drug.

The results of clofibrate on synchronized cells also corresponded well with the results from log phase cells, showing partial inhibition at 2 x 1 0 - * M and complete inhibition at 4 x 10-* M.

"'~ '& 32-~ B !

1

3=

~ = ~

0 o

s I C / / o ,2~ O i / / e

i , /

/

4~ S~ / Z

/

o ,'o ,? , ; ~ o ~ ,o ,o ~o

T I M E , ra in

Fig. 2. The effects of clofibrate at 0 (O O), 1 x 10 -4 M (A A), 2 × 10 - 4 M (r"l El) and 4 x 10 -4M (0 - - - -0 ) on the incorporation of (a) [t4C]thymidine into DNA. (b) [~*C]uridine into RNA, (c) ['~'Clamino acids into protein, and (d) [t*C]acetate into lipids in synchron- ized cultures of T. pyriformis, GL. Samples were taken at 0, 20, 40, 60 and 80 rain after EHT in duplicates and

assayed by the filter paper disc procedure.

The effects of clofibrate on macromolecular syn- thesis indicated that, clofibrate, though a hypolipide- mic agent, has potent inhibitory effects on the syn- thesis of protein, DNA and RNA in addition to the inhibitory effects on lipid synthesis. These inhibitory effects may in part account for the effects on cell division of log phase and synchronized cells. This non-specific effect on macromolecular synthesis may indicate a common inhibitory pathway, that of the energetics, by this hypolipidemic agent. Recent inves- tigations have demonstrated that clofibrate has in- deed inhibitory effects on respiration and oxidative phosphorylation in liver mitochondria from different species (Kaytal et al., 1972; Mackerer et al., 1973. 1974; Cederbaum et al., 1974). Results on the effects of clofibrate on in vitro synthesis of lipids have also indicated that this agent inhibited the synthesis of lipids in the crude homogenate and the mitochondrial fraction but not in the microsomal and the soluble cell fraction (Pan et al., 1974, 1976a). These results again suggest the possibility of a direct effect on the energetics. Hence the inhibitory effects on protein, DNA, RNA and lipid synthesis, as well as on cell division may all be secondary consequences to the inhibitory effect on the energetics.

REFERENCES

BLUM J. J. • WEXLER J. P. (1968) Effect of c lof ibra te on Tetrahymena. Molec. Pharmac. 4. 155-161.

Effects of clofibrate on cell division 223

CEDERBAUM A. I. & RUBIN E. (1974) Effects of clofibrate on mitochondrial function. Biochem. Pharmac. 23, 1985-1996,

CHOL" S. C.. RAMANATHAN S. & CUTnNG W. C. (1968) Quinacrine: inhibition of synchronized cell division in Tetrahymena. Pharmacology I, 60-64.

HOLMLUND C. E. (1973) Growth inhibition of Tetrahymena pyriformis by a hypocholesteremic compound and the mechanism of its reversal by various lipids. Biochim. bio- phys. Acta 296, 221-233.

HOLZ G. G.. ERWIN J., ROSENBAUM N. & AARONSON S. (1962) Triparanol inhibition of Tetrahymena, and its pre- vention by lipids. Archs Biochem. Biophys. 98, 312-322.

KAYTAL S. L.. SAHA J. R," KABARA J. J. (1972) Effect in vitro of clofibrate and trans-l,4-(2-chlorobenzyla- minoethyl)-cyclo-hexane dihydroehloride (AY 9944) on respiration and adenosine triphosphate activity of mouse liver mitochondria. Biochem. Pharmac. 21, 747-751.

MACKERER C. R.. HAETTINGER J. R. & HUTSELL T. C. (1973) Effects of ciofibrate, in vitro, on mitochondria respiration and oxidative phosphorylation. Bioehem. Pharmac. 22, 513-519.

MACKERER C. R. ~ HAETTINGER J. R. (1974) Further studies concerning the effects of clofibrate on respiration and oxidative pbosphorylation of rat liver mitochondria. Biochem. Pharmac. 23, 3331-3345.

NOZ^WA Y. (1973) Inhibition of lipid biosynthesis by p-chlorophenoxyisobutyrate (CPIB) in Tetrahymena pyriformis. J. Biochem. Tokyo 74, 1157-1163.

PAN H. Y. M., Cnov S. C. & CONKLIN K. A. (1974) Effects of antimalarial drugs and clofibrate on in vitro lipid syn- thesis in Tetrahymena pyriformis. Pharmacology 12, 48-56.

PAN H. Y. M., CHOU S. C. & CONKLIN K. A. (1976a) The effects of hypolipidemic agents on lipid synthesis in sub- cellular fractions from Tetrahymena pyriformis. Pharma- cology 14, 499-510.

PAN H. Y. M., HOKAM^ Y. & CHOU S. C. (1976b) Effect of levamisole on the synthesis of macromolecules in log phase and synchronized cultures of Tetrahymena pyri- formis. GL. Res. Commun. chem. path. Pharmac. 14. 651-657.

POLLARD W. O., SHORB M. S., LUND P. G. & VASAITIS V. (1963) The effect of triparanol on synthesis of lipids by Tetrahymena pyriformis. J. Protozool. 10 SuFpl. 7.

RYLEY J. F. (1952) Studies on the metabolism of protozoa. 3. Metabolism of the ciliate Tetrahymena pyriformis (Glaucoma pyriformis). Biochem. J. 52, 483--492.

SnORe M. S., DUNLOP B. E. & POLLARD W. O. (1965) Effect by triparanol on synthesis of squalene and tetra- hymenol by Tetrahymena pyriformis. Proc. Soc. exp. Biol. Med. 118, 1140-1145.